English
Tiếng Việt
(en)The technology described herein relates to methods of detecting circulating tumor cells (CTCs), e.g. by detecting changes in the expression of certain CTC marker genes. Aberrant expression of CTC marker genes, e.g. changes in expression indicative of CTCs can also be targeted in order to treat cancer.
1.ApplicationNumber: US-201415105137-A
1.PublishNumber: US-2016312298-A1
2.Date Publish: 20161027
3.Inventor: TING DAVID T.
HABER DANIEL A.
MAHESWARAN SHYAMALA
4.Inventor Harmonized: TING DAVID T(US)
HABER DANIEL A(US)
MAHESWARAN SHYAMALA(US)
5.Country: US
6.Claims:
(en)The technology described herein relates to methods of detecting circulating tumor cells (CTCs), e.g. by detecting changes in the expression of certain CTC marker genes. Aberrant expression of CTC marker genes, e.g. changes in expression indicative of CTCs can also be targeted in order to treat cancer.
7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/918,816 filed Dec. 20, 2013 and 61/937,883 filed Feb. 10, 2014, the contents of which are incorporated herein by reference in their entirety.
GOVERNMENT SUPPORT
This invention was made with federal funding under Grant Nos. 2R01CA129933 awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.
TECHNICAL FIELD
The technology described herein relates to the diagnosis and treatment of cancer.
BACKGROUND
Circulating Tumor Cells (CTCs) are shed from primary tumors into the bloodstream, mediating the spread of cancer to distant organs (metastasis). Thus, the presence of circulating tumor cells (CTCs) in the bloodstream ultimately leads to spread of cancer to distant organs. However, CTCs are rare, estimated at one to ten tumor cells among ten billion normal blood cells in a milliliter of blood. As such, their isolation and molecular analysis has posed a significant technological challenge (Pantel et al., Nat Rev Cancer 2008 8:329-340; Yu et al., J Cell Biol 2011 192:373-382).
SUMMARY
As described herein, the inventors have identified a number of genes, the expression of which is characteristic of CTCs. In particular, the expression of these genes differentiates CTCs from primary tumor cells Accordingly, provided herein are methods and assays relating to the detection of CTCs, including diagnostic and prognostic methods and assays. Further, provided herein are treatments for cancer that target these markers of CTCs, e.g., to inhibit metastasis.
In one aspect, described herein is a method of detecting circulating tumor cells (CTCs) in a sample, the method comprising: measuring the level of a PC-CTC marker gene expression product in the sample; and determining that PC-CTCs are present if the detected level of the marker gene expression product is greater than a reference level. In some embodiments, the CTCs are pancreatic cancer CTCs. In some embodiments, the method further comprises a first step of isolating the CTCs from the sample. In some embodiments, the expression product is a nucleic acid. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization. In some embodiments, the expression product is a polypeptide. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of: Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay. In some embodiments, the CTC marker gene is selected from Table 7 or Table 8. In some embodiments, the CTC marker gene is selected from the group consisting of: ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
In one aspect, described herein is a method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of a CTC marker gene-targeted therapy to the subject. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the CTC marker gene-targeted therapy comprises an inhibitor of a CTC marker gene. In some embodiments, the inhibitor is an antibody reagent. In some embodiments, the inhibitor is an inhibitory nucleic acid reagent. In some embodiments, the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent and a chemotherapeutic agent. In some embodiments, the subject is a subject determined to have an elevated level of CTCs and/or an elevated level of a CTC marker gene present in the blood and/or stroma of the cancer.
In one aspect, described herein is a method of determining if a subject is likely to respond to treatment with a CTC marker gene-targeted therapy, the method comprising measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is likely to respond to the treatment if the level of the expression product is increased relative to a reference level. In some embodiments, the method further comprises a first step of isolating the CTCs from the sample. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the expression product is a nucleic acid. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization. In some embodiments, the expression product is a polypeptide. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of: Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay. In some embodiments, the PC-CTC marker gene is selected from Table 7 or Table 8. In some embodiments, the CTC marker gene is selected from the group consisting of: ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
In one aspect, described herein is a method of monitoring the treatment of a subject, the method comprising: administering a cancer therapy to a subject in need thereof; measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is responding if the level of the CTC marker gene expression product is decreased relative to the reference level and determining that the subject is not responding to the treatment if the CTC marker gene expression product is not decreased relative to the reference level. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the reference level is the level of the gene expression product in the patient prior to the administering step. In some embodiments, the method further comprises a first step of isolating the CTCs from the sample. In some embodiments, the expression product is a nucleic acid. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization. In some embodiments, the expression product is a polypeptide. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of: Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay. In some embodiments, the PC-CTC marker gene is selected from Table 7 or Table 8. In some embodiments, the CTC marker gene is selected from the group consisting of: ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1C demonstrate the isolation and characterization of CTCs. FIG. 1A depicts a schematic of CTC-iChip negative IFD system. FIG. 1B depicts a graph of mouse WBC depletion consistency between normal and cancer mouse models. WBC depletion shown in log 10. FIG. 1C depicts a graph of CTC enumeration by immunofluorescent staining (CK+/CD45−/DAPI+) from normal and KPC mice.
FIG. 2 depicts schematics of principal component analysis of single cell samples.
FIGS. 3A-3B demonstrate that epithelial, mesenchymal, and stem cell genes are differentially expressed in CTC-c cells vs Tumors. Depicted are boxplot of genes that are A) downregulated ( FIG. 3A ) and upregulated ( FIG. 3B ) in CTC-c cells vs Tumors. Bar=median, box plot=quartiles, scale in log 10(rpm).
FIGS. 4A-4C demonstrate CTC-iChip characterization. FIG. 4A depicts a graph of the percent of WBC deflected (y-axis) as a function of the number of anti-CD45 beads per WBC (x-axis). FIG. 4B depicts a graph of the recovery of mouse PDAC cell line NB508 spiked into normal mouse blood (4 independent experiments shown). FIG. 4C depicts a graph of the captured CTCs/mL of blood from syngeneic orthotopic PDAC tumors using NB508 cell line.
FIG. 5A depicts a table of KPC mouse genotype and characteristics. FIG. 5B depicts graphs of quality metrics of single cell sequencing with % of reads aligned and total unique alignments for cell lines (NB508, MEF), CTCs, WBC, and diluted bulk RNA from matched primary tumors. FIG. 5C depicts graphs of single cell heterogeneity using mean intra-cluster correlation coefficient for each cluster (rights) and between single cell primary tumor (TuGMP3), cancer cell line (NB508), and all CTCs (Cluster 1, 3, 4, 5, 9). Circle=mean, Range=95% CI.
FIG. 6 depicts boxplot graphs of ECM protein gene enriched in CTC-c compared to bulk primary tumors and single cell primary tumors. Bar=median, boxplot—quartiles, scale in log 10 (rpm).
FIG. 7 depicts a heatmap expression profile of human pancreatic CTCs from 3 patients. Epithelial genes used to define CTCs and enriched extracellular proteins shown. Expression shown in log 10 scale.
FIG. 8 depicts a graph of quantitative RT-PCR of SPARC expression in human pancreatic cancer cell lines.
FIG. 9 depicts invasion assays. Decreases in invasion through Matrigel of PDAC2 and PDAC 3 cell lines with shRNA against SPARC (ShF1 and ShF3) were observed. shNT=Non-target shRNA
FIG. 10 depicts a graph of the number of mice with detectable metastases by in vivo luciferase imaging in non-target shRNA (NT) and SPARC shRNA (SHF1).
FIG. 11 depicts a schematic of the process of determining CTC heterogeneity.
FIGS. 12A-12C demonstrate that CTC-Enriched Genes are Found in Epithelial and Stromal Components of Primary Tumors. Depicted are expression boxplots of ( FIG. 12A ) Aldh1a2 stem cell and CTC highly enriched genes ( FIG. 12B ) Klf4 and ( FIG. 12C ) Igfbp5 genes. Bar=median, box plot=quartiles, scale in log 10(rpm).
FIG. 13 demonstrates that human and mouse CTCs across different epithelial cancer express high levels of ECM protein genes. Depicted are expression boxplot of highly expressed ecm genes in human pdac, breast (br), and prostate (pr) ctcs. bar, median; boxplot, quartiles; scale in log 10(rpm). holm-adjusted p value<0.05 (*), 0.01 (**), 0.001 (***).
FIGS. 14A-14E demonstrate that SPARC expression in human PDAC enhances invasion and metastasis. FIG. 14A depicts a graph of proliferation of PDAC3 cell lines determined by MTT. FIG. 14B depicts a graph of tumor spheres in PDAC3 shNT versus shSPARC counted per 43 field (error bars represent SD). FIG. 14C depicts a graph of invasion of shSPARC and shNT cell lines quantitated by number of nuclei/203 field. p value<0.01 (**), 0.001 (***), 0.0001 (****). Error bars represent SD. FIG. 14D depicts a graph of Percentage of detectable lung metastases by in vivo luciferase imaging after 3 weeks after tail vein inoculation of PDAC3 cell lines. Fisher's exact test p value is shown. FIG. 14E depicts a graph of normalized metastasis burden in mice with orthotopic pancreatic tumors from PDAC3 cell lines. Error bars represent SD (*p<0.05).
FIG. 15 depicts a Summary Model of the Role of Pancreatic CTCs in the Metastatic Cascade. Shown are the heterogeneous subsets of pancreatic CTCs with a focus on the most prominent classical CTC group, which are enriched for coexpression of epithelial (keratin) and stromal (Sparc) genes.
FIG. 16A depicts a graph of PDAC2 shRNA cell lines by qRT-PCR. Average shown with max and min RQ (error bars). FIG. 16B depicts a graph of proliferation rates by MTT assay similar in PDAC2 cell line between shNT and shSPARC stable lines. FIG. 16C depicts a graph of tumor sphere invasion assay (error bars=STD) formation at 2 weeks similar between shNT and shSPARC cell lines. Quantiation done per 4× magnification field (Error bars=SD). Migratory behavior reduced by shSPARC_1 & 3 as determined by ( FIG. 16D ) invasion assay at 48 hours.
DETAILED DESCRIPTION
As described herein, the inventors have discovered that circulating tumor cells (CTCs) are characterized by the expression of certain genes, i.e. CTC marker genes. The discovery of these CTC marker genes permit methods and assays for the detection and/or measurement of CTC levels, e.g. CTC levels in a sample from a subject. These methods and assays can provide improved speed and accuracy in the measurement of CTC levels. Furthermore, because the expression of these marker genes distinguishes CTCs from other cells, e.g., other circulating cells and/or normal tumor cells, therapies can be targeted against CTCs by binding to and/or inhibiting these marker gene expression products to reduce the level and/or metastatic potential of CTCs.
As used herein, “circulating tumor cell” or “CTC” refers to tumor cells which are shed from a tumor and present in the blood, i.e. in circulation. Cell markers (e.g. marker genes) that can be used to identify and/or isolate CTCs from other components of the blood are described below herein. In some embodiments, a CTC can be a pancreatic cancer CTC.
In one aspect, described herein is a method of detecting circulating tumor cells (CTCs) in a sample, the method comprising measuring the level of a CTC marker gene expression product in the sample; and determining that CTCs are present if the detected level of the marker gene expression product is greater than a reference level.
As described herein, the inventors have discovered that a number of genes are differentially regulated in CTCs, e.g. as compared to non-circulating tumor cells. Accordingly, there are provided herein methods and assays relating to the measurement of CTC levels. Elevated CTC levels can indicate a poor prognosis, e.g. an increased risk of metastatsis. Accordingly, provided herein are methods and assays related to the prognosis, risk assessment, and treatment of subjects having cancer. In certain embodiments, the assays and methods are directed to determination and/or measurement of the expression level of a gene product (e.g. protein and/or gene transcript such as mRNA) in a biological sample of a subject. In certain embodiments the assays and methods are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, i.e. at least two genes, at least three genes, at least four genes, at least five genes, at least six genes, at least seven genes, at least eight genes, at least nine genes, at least 10 genes . . . at least 15 genes, . . . at least 25 genes, . . . at least 30 genes, or more genes, or any number of genes selected from Table 7, Table 8, and/or Table 14 as described herein.
In some embodiments, the marker gene(s) is selected from the group consisting of ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or, e.g. all of the following genes: ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4.
In some embodiments, the marker gene(s) is selected from the group consisting of ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or, e.g. all of the following genes: ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2.
In some embodiments, the marker gene(s) is selected from the group consisting of ALDH1A2; IGFBP5; KLF4; DCN; and SPARC. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or, e.g. all of the following genes: ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
In some embodiments, the marker gene(s) is selected from the group consisting of ALDH1A2; IGFBP5; KLF4; and DCN. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or e.g. all of the following genes: ALDH1A2; IGFBP5; KLF4; and DCN.
In some embodiments, the marker gene(s) is selected from the group consisting of TPT1; HMGB1; SPON 2; SPARC; and ARSA. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or, e.g. all of the following genes: TPT1; HMGB1; SPON 2; SPARC; and ARSA.
In some embodiments, the marker gene(s) is selected from the group consisting of IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or at least five genes, or at least six genes, or at least seven genes, or at least eight genes or, e.g. all of the following genes: IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A. In some embodiments, the level of polypeptide expression products are determined for the marker gene(s) is selected from the group consisting of IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A, e.g. because, as described herein, RNA levels of cell surface proteins are lower than polypeptide levels.
TABLE 7
Exemplary mouse marker genes
MOUSE
GENE
SYMBOL
Gene Name
Abcb1b
ATP-binding cassette, sub-family B (MDR/TAP), member 1B
Abi3bp
ABI gene family, member 3 (NESH) binding protein
Ablim3
actin binding LIM protein family, member 3
Acad9
acyl-Coenzyme A dehydrogenase family, member 9
Acbd3
acyl-Coenzyme A binding domain containing 3
Acini
apoptotic chromatin condensation inducer 1
Actb
actin, beta
Actg1
predicted gene 8543; actin-like 8; predicted gene 7505; predicted gene
12715; predicted gene 12003; predicted gene 8399; predicted gene 6375;
actin, gamma, cytoplasmic 1; similar to gamma-actin; predicted gene
4667; similar to cytoplasmic beta-actin; predicted gene 16385
Adamts5
similar to a disintegrin-like and metalloprotease (reprolysin type) with
thrombospondin type 1 motif, 5 (aggrecanase-2); a disintegrin-like and
metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 5
(aggrecanase-2)
Adamtsl1
ADAMTS-like 1
Add3
adducin 3 (gamma)
Aebp1
AE binding protein 1
Agap1
ArfGAP with GTPase domain, ankyrin repeat and PH domain 1
Akap13
A kinase (PRKA) anchor protein 13
Akap2
A kinase (PRKA) anchor protein 2; paralemmin 2
Akr1b3
aldo-keto reductase family 1, member B3 (aldose reductase)
Akt2
similar to RAC-beta serine/threonine-protein kinase (RAC-PK-beta)
(Protein kinase Akt-2) (Protein kinase B, beta) (PKB beta); thymoma viral
proto-oncogene 2; similar to serine/threonine kinase
Aldh1a1
aldehyde dehydrogenase family 1, subfamily A1
Aldh1a2
aldehyde dehydrogenase family 1, subfamily A2
Alox12
arachidonate 12-lipoxygenase
Amfr
autocrine motility factor receptor
Amhr2
anti-Mullerian hormone type 2 receptor
Ang
angiogenin, ribonuclease, RNase A family, 5
Ankrd11
ankyrin repeat domain 11
Ankrd12
ankyrin repeat domain 12; similar to Ankrd12 protein
Ankrd17
ankyrin repeat domain 17
Ano6
anoctamin 6
Anp32a
acidic (leucine-rich) nuclear phosphoprotein 32 family, member A
Anxa7
annexin A7
Ap1s3
predicted gene 8532; similar to adaptor-related protein complex AP-1,
sigma 3; adaptor-related protein complex AP-1, sigma 3
Ap3s1
predicted gene 7603; adaptor-related protein complex 3, sigma 1 subunit;
predicted gene 5610
Ap4e1
adaptor-related protein complex AP-4, epsilon 1
Aplp1
amyloid beta (A4) precursor-like protein 1
Apol9a
apolipoprotein L 9b; apolipoprotein L 9a
App
amyloid beta (A4) precursor protein
Aqp1
aquaporin 1
Arap2
predicted gene 336; ArfGAP with RhoGAP domain, ankyrin repeat and PH
domain 2
Arf2
ADP-ribosylation factor 2
Arf3
ADP-ribosylation factor 3
Arf5
similar to ADP-ribosylation factor; ADP-ribosylation factor 5
Arhgap28
Rho GTPase activating protein 28
Arhgap29
Rho GTPase activating protein 29
Arhgap5
Rho GTPase activating protein 5
Arhgef12
predicted gene 7281; predicted gene 5831; similar to SP140 nuclear body
protein (predicted); Rho guanine nucleotide exchange factor (GEF) 12
Arid1a
similar to AT rich interactive domain 1A isoform a; AT rich interactive
domain 1A (SWI-like)
Arid4a
AT rich interactive domain 4A (RBP1-like)
Arid4b
AT rich interactive domain 4B (RBP1-like)
Arid5b
similar to modulator recognition factor 2; AT rich interactive domain 5B
(MRF1-like)
Arl3
ADP-ribosylation factor-like 3
Arl4d
ADP-ribosylation factor-like 4D; hypothetical protein LOC100044157
Arl6ip5
ADP-ribosylation factor-like 6 interacting protein 5
Armcx3
armadillo repeat containing, X-linked 3; hypothetical protein
LOC100044266; predicted gene 9299
Arpc2
predicted gene 5492; actin related protein 2/3 complex, subunit 2
Arsa
arylsulfatase A
Arsb
arylsulfatase B
Ascc3
activating signal cointegrator 1 complex subunit 3
Atf3
activating transcription factor 3
Atg3
autophagy-related 3 (yeast)
Atp1a1
ATPase, Na+/K+ transporting, alpha 1 polypeptide
Atp1b1
ATPase, Na+/K+ transporting, beta 1 polypeptide
Atp2b1
ATPase, Ca++ transporting, plasma membrane 1
Atp6v1a
ATPase, H+ transporting, lysosomal V1 subunit A
Atxn2
ataxin 2
B230120H23Rik
RIKEN cDNA B230120H23 gene
B2m
beta-2 microglobulin
BC003331
similar to odorant response abnormal 4; cDNA sequence BC003331
BC005537
cDNA sequence BC005537
BC005561
THO complex 2; cDNA sequence BC005561
BC013529
cDNA sequence BC013529
Baz2a
bromodomain adjacent to zinc finger domain, 2A
Bbs4
Bardet-Biedl syndrome 4 (human)
Bbx
bobby sox homolog ( Drosophila )
Bcam
basal cell adhesion molecule
Bcl10
B-cell leukemia/lymphoma 10; predicted gene 6141
Bdp1
B double prime 1, subunit of RNA polymerase III transcription initiation
factor IIIB
Bicc1
bicaudal C homolog 1 ( Drosophila )
Bicd1
bicaudal D homolog 1 ( Drosophila )
Birc6
baculoviral IAP repeat-containing 6
Blvrb
biliverdin reductase B (flavin reductase (NADPH))
Bnc1
basonuclin 1
Bnc2
basonuclin 2
Bod1l
biorientation of chromosomes in cell division 1-like
Bptf
bromodomain PHD finger transcription factor
Braf
Braf transforming gene
Brd2
similar to mKIAA4005 protein; bromodomain containing 2
Brd4
bromodomain containing 4
Brp44l
similar to brain protein 44-like protein; brain protein 44-like; predicted
gene 3452; predicted gene 8219
Bst2
bone marrow stromal cell antigen 2
Btbd2
BTB (POZ) domain containing 2
Btbd7
BTB (POZ) domain containing 7
Btf3
predicted gene 9308; basic transcription factor 3; predicted gene 3531;
predicted gene 7973
Btg2
B-cell translocation gene 2, anti-proliferative
Bzw1
predicted gene 11652; predicted gene 5191; basic leucine zipper and W2
domains 1
C1d
C1D nuclear receptor co-repressor
C1ra
complement component 1, r subcomponent; predicted gene 8551
C1rl
complement component 1, r subcomponent-like
C1s
similar to Complement component 1, s subcomponent; complement
component 1, s subcomponent
C2
complement component 2 (within H-2S)
C3
complement component 3; similar to complement component C3
prepropeptide, last
C4a
similar to Complement C4 precursor; complement component 4A (Rodgers
blood group); similar to complement C4; complement component 4B
(Childo blood group)
C4b
similar to Complement C4 precursor; complement component 4A (Rodgers
blood group); similar to complement C4; complement component 4B
(Childo blood group)
Calm1
predicted gene 7743; calmodulin 3; calmodulin 2; calmodulin 1; predicted
gene 7308
Calm2
predicted gene 7743; calmodulin 3; calmodulin 2; calmodulin 1; predicted
gene 7308
Cap1
CAP, adenylate cyclase-associated protein 1 (yeast)
Cast
calpastatin
Cav1
caveolin 1, caveolae protein
Ccdc109b
coiled-coil domain containing 109B
Ccdc34
coiled-coil domain containing 34
Ccdc80
coiled-coil domain containing 80
Ccdc88a
coiled coil domain containing 88A
Ccdc90a
coiled-coil domain containing 90A
Ccnl1
cyclin L1
Cd109
CD109 antigen
Cd200
CD200 antigen; similar to MRC OX-2 antigen homolog
Cd248
CD248 antigen, endosialin
Cd34
CD34 antigen
Cd55
CD55 antigen
Cd81
CD81 antigen
Cd82
CD82 antigen
Cd9
CD9 antigen
Cdc42ep3
CDC42 effector protein (Rho GTPase binding) 3
Cdh11
cadherin 11
Cdh3
cadherin 3
Cdk13
cell division cycle 2-like 5 (cholinesterase-related cell division controller)
Cdon
cell adhesion molecule-related/down-regulated by oncogenes
Celf2
CUG triplet repeat, RNA binding protein 2
Cep164
centrosomal protein 164
Cep57
centrosomal protein 57
Cfh
complement component factor h; similar to complement component
factor H
Cfl1
cofilin 1, non-muscle; similar to Cofilin-1 (Cofilin, non-muscle isoform);
predicted gene 6180
Cfl2
cofilin 2, muscle
Chd1
chromodomain helicase DNA binding protein 1
Chd2
chromodomain helicase DNA binding protein 2
Chi3l1
chitinase 3-like 1
Chst4
carbohydrate (chondroitin 6/keratan) sulfotransferase 4
Cish
cytokine inducible SH2-containing protein
Clcn3
chloride channel 3
Cldn15
claudin 15
Cldn25
predicted gene 16492
Clec1b
C-type lectin domain family 1, member b
Clec3b
C-type lectin domain family 3, member b
Clic4
chloride intracellular channel 4 (mitochondrial)
Clip1
CAP-GLY domain containing linker protein 1
Clip3
CAP-GLY domain containing linker protein 3
Cln8
ceroid-lipofuscinosis, neuronal 8
Cmah
cytidine monophospho-N-acetylneuraminic acid hydroxylase
Cmtm3
CKLF-like MARVEL transmembrane domain containing 3
Cmtm7
CKLF-like MARVEL transmembrane domain containing 7
Cnot6l
CCR4-NOT transcription complex, subunit 6-like
Cobl
cordon-bleu
Cobll1
Cobl-like 1
Col14a1
collagen, type XIV, alpha 1
Col1a2
collagen, type I, alpha 2
Col3a1
collagen, type III, alpha 1
Col4a6
collagen, type IV, alpha 6
Colec12
collectin sub-family member 12
Coq10b
hypothetical protein LOC675736; coenzyme Q10 homolog B ( S. cerevisiae );
predicted gene 4899
Creb3l1
cAMP responsive element binding protein 3-like 1
Creb5
RIKEN cDNA 9430076C15 gene; cAMP responsive element binding
protein 5
Crebbp
CREB binding protein
Creg1
cellular represser of E1A-stimulated genes 1
Crim1
cysteine rich transmembrane BMP regulator 1 (chordin like)
Crls1
cardiolipin synthase 1
Cryab
crystallin, alpha B
Cryl1
crystallin, lambda 1
Crym
crystallin, mu
Csda
cold shock domain protein A
Csf1
colony stimulating factor 1 (macrophage)
Csnk1a1
casein kinase 1, alpha 1
Csrnp1
cysteine-serine-rich nuclear protein 1
Csrp1
cysteine and glycine-rich protein 1
Cuedc1
CUE domain containing 1
Cyb5
cytochrome b-5
Cybrd1
cytochrome b reductase 1
Cyp2d22
cytochrome P450, family 2, subfamily d, polypeptide 22
Cyp2s1
cytochrome P450, family 2, subfamily s, polypeptide 1
Cyr61
cysteine rich protein 61
Dab2
disabled homolog 2 ( Drosophila )
Dag1
dystroglycan 1
Daglb
diacylglycerol lipase, beta
Dapk1
death associated protein kinase 1
Dcn
decorin
Ddr1
discoidin domain receptor family, member 1
Ddr2
discoidin domain receptor family, member 2
Ddx3x
DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked
Ddx5
DEAD (Asp-Glu-Ala-Asp) box polypeptide 5; predicted gene 12183
Dennd5a
DENN/MADD domain containing 5A; similar to Rab6 interacting protein 1
Dhx15
DEAH (Asp-Glu-Ala-His) box polypeptide 15
Diap1
diaphanous homolog 1 ( Drosophila )
Dlgap4
discs, large homolog-associated protein 4 ( Drosophila )
Dmkn
dermokine
Dnaja2
DnaJ (Hsp40) homolog, subfamily A, member 2
Dnajb9
predicted gene 6568; DnaJ (Hsp40) homolog, subfamily B, member 9
Dnajc1
DnaJ (Hsp40) homolog, subfamily C, member 1
Dnmt1
DNA methyltransferase (cytosine-5) 1
Dpp4
dipeptidylpeptidase 4
Dpysl2
dihydropyrimidinase-like 2
Dpysl3
dihydropyrimidinase-like 3
Dst
dystonin; hypothetical protein LOC100047109
Dtx2
deltex 2 homolog ( Drosophila )
Dusp1
dual specificity phosphatase 1
Dusp14
dual specificity phosphatase 14
Dusp3
dual specificity phosphatase 3 (vaccinia virus phosphatase VH1-related)
Dync1i2
dynein cytoplasmic 1 intermediate chain 2
Ecd
ecdysoneless homolog ( Drosophila )
Eea1
early endosome antigen 1
Eef1a1
predicted gene 5869; predicted gene 7161; predicted gene 7105;
predicted gene 5822; similar to eukaryotic translation elongation factor 1
alpha 1; predicted gene 6192; predicted gene 6392; predicted gene 6767;
predicted gene 6170; predicted gene 6548; predicted gene 6789;
eukaryotic translation elongation factor 1 alpha 1
Efemp1
epidermal growth factor-containing fibulin-like extracellular matrix protein 1
Efhd2
similar to EF hand domain containing 2; EF hand domain containing 2
Efna5
ephrin A5
Egr1
early growth response 1
Ehd2
EH-domain containing 2
Eif2s3x
eukaryotic translation initiation factor 2, subunit 3, structural gene X-
linked; similar to translation initiation factor elF-2 gamma subunit;
predicted gene 2223
Eif3a
eukaryotic translation initiation factor 3, subunit A
Elf1
E74-like factor 1
Elovl6
predicted gene 11295; ELOVL family member 6, elongation of long chain
fatty acids (yeast)
Emp2
epithelial membrane protein 2
Enpp2
ectonucleotide pyrophosphatase/phosphodiesterase 2
Enpp4
ectonucleotide pyrophosphatase/phosphodiesterase 4
Esam
endothelial cell-specific adhesion molecule
Esf1
ESF1, nucleolar pre-rRNA processing protein, homolog ( S. cerevisiae )
Espn
espin
Esyt3
family with sequence similarity 62 (C2 domain containing), member C
Etfa
predicted gene 2893; electron transferring flavoprotein, alpha polypeptide
Evpl
envoplakin
Exoc4
exocyst complex component 4
F11r
F11 receptor
Faim2
Fas apoptotic inhibitory molecule 2
Fam117a
family with sequence similarity 117, memberA
Fam134b
family with sequence similarity 134, member B
Fam53b
family with sequence similarity 53, member B
Fam63b
RIKEN cDNA B230380D07 gene
Fam76a
predicted gene 7527; family with sequence similarity 76, member A
Fam84b
RIKEN cDNA D330050I23 gene
Fas
Fas (TNF receptor superfamily member 6)
Fbln1
fibulin 1
Fermt2
fermitin family homolog 2 ( Drosophila )
Fgf1
fibroblast growth factor 1
Fhl1
four and a half LIM domains 1
Filip1l
filamin A interacting protein 1-like
Fkbp5
FK506 binding protein 5
Flii
flightless I homolog ( Drosophila ); similar to cytoskeletal actin-modulating
protein
Flnc
filamin C, gamma
Flrt2
fibronectin leucine rich transmembrane protein 2
Fmo2
flavin containing monooxygenase 2
Fmod
fibromodulin
Fndc1
fibronectin type III domain containing 1; similar to fibronectin type III
domain containing 1
Fos
FBJ osteosarcoma oncogene
Foxn3
forkhead box N3
Frmd4b
FERM domain containing 4B
Fth1
ferritin heavy chain 1
Fxyd1
FXYD domain-containing ion transport regulator 1
G3bp1
Ras-GTPase-activating protein SH3-domain binding protein 1
Gabarapl1
gamma-aminobutyric acid (GABA) A receptor-associated protein-like 1
Gadd45b
growth arrest and DNA-damage-inducible 45 beta
Ganab
alpha glucosidase 2 alpha neutral subunit
Gas1
growth arrest specific 1
Gas6
growth arrest specific 6
Gata6
GATA binding protein 6
Gbp2
guanylate binding protein 2
Gbp3
guanylate binding protein 3
Gcap14
granule cell antiserum positive 14
Gcsh
predicted gene 3672; similar to Glycine cleavage system H protein,
mitochondrial precursor; glycine cleavage system protein H (aminomethyl
carrier)
Gda
guanine deaminase
Gem
GTP binding protein (gene overexpressed in skeletal muscle)
Gfm2
G elongation factor, mitochondrial 2
Gfpt2
glutamine fructose-6-phosphate transaminase 2
Gja1
gap junction protein, alpha 1
Gjb5
gap junction protein, beta 5
Gm10052
predicted gene 10052
Gm13251
predicted gene 13251; predicted gene, OTTMUSG00000010657; RIKEN
cDNA 1700029I01 gene
Gm3893
similar to 4933409K07Rik protein; predicted gene, 665845; predicted gene
2490; predicted gene 10601; predicted gene 2163; predicted gene 3892;
RIKEN cDNA 4933409K07 gene; predicted gene 3893
Gm6548
predicted gene 5869; predicted gene 7161; predicted gene 7105;
predicted gene 5822; similar to eukaryotic translation elongation factor 1
alpha 1; predicted gene 6192; predicted gene 6392; predicted gene 6767;
predicted gene 6170; predicted gene 6548; predicted gene 6789;
eukaryotic translation elongation factor 1 alpha 1
Gm6578
predicted gene 6578
Gm6644
predicted gene 6644
Gm9199
predicted gene 9199
Gnb2
guanine nucleotide binding protein (G protein), beta 2
Golga4
golgi autoantigen, golgin subfamily a, 4
Golgb1
golgi autoantigen, golgin subfamily b, macrogolgin 1
Gpc3
glypican 3
Gpc4
glypican 4; similar to Glypican 4
Gpcpd1
preimplantation protein 4
Gpm6a
glycoprotein m6a
Gpr116
G protein-coupled receptor 116
Gpr133
G protein-coupled receptor 133
Gpr64
G protein-coupled receptor 64
Gprc5b
G protein-coupled receptor, family C, group 5, member B
Gpx8
glutathione peroxidase 8 (putative)
Gsr
similar to Glutathione reductase, mitochondrial precursor (GR) (GRase);
glutathione reductase
Gsta3
glutathione S-transferase, alpha 3
Gstm1
similar to Glutathione S-transferase Mu 1 (GST class-mu 1) (Glutathione S-
transferase GT8.7) (pmGT10) (GST 1-1); predicted gene 5562; glutathione
S-transferase, mu 1
Gstm4
glutathione S-transferase, mu 4
Gucy1a3
guanylate cyclase 1, soluble, alpha 3
H2-D1
histocompatibility 2, D region; histocompatibility 2, D region locus 1
H2-K1
histocompatibility 2, K1, K region; similar to H-2K(d) antigen
H2-Q6
histocompatibility 2, Q region locus 1; histocompatibility 2, Q region locus
9; similar to H-2 class I histocompatibility antigen, L-D alpha chain
precursor; histocompatibility 2, Q region locus 8; histocompatibility 2, Q
region locus 2; similar to MHC class Ib antigen; histocompatibility 2, Q
region locus 7; histocompatibility 2, Q region locus 6; hypothetical protein
LOC100044307; similar to H-2 class I histocompatibility antigen, Q7 alpha
chain precursor (QA-2 antigen); RIKEN cDNA 0610037M15 gene
H3f3a
predicted gene 14383; predicted gene 3835; predicted gene 14384;
predicted gene 12950; predicted gene, 670915; H3 histone, family 3A;
predicted gene 12657; predicted gene 6132; predicted gene 10257;
predicted gene 7227; H3 histone, family 3B; predicted gene 6128; similar
to histone; predicted gene 1986; predicted gene 6186; hypothetical
protein LOC676337; predicted gene 6421; predicted gene 2198; predicted
gene 6817; predicted gene 8095; predicted gene 12271; predicted gene
13529; predicted gene 8029; predicted gene 4938; predicted gene 7100;
predicted gene 9014; similar to Histone H3.4 (Embryonic); predicted gene
7179; similar to H3 histone, family 3B; predicted gene 7900; predicted
gene 2099; similar to H3 histone, family 3A; predicted gene 6749;
predicted gene 6485; predicted gene 4028; predicted gene 7194
Hdac3
histone deacetylase 3
Hdac5
histone deacetylase 5
Heg1
HEG homolog 1 (zebrafish)
Herpud2
HERPUD family member 2
Hes1
hairy and enhancer of split 1 ( Drosophila )
Hexb
hexosaminidase B
Hist1h1c
histone cluster 1, H1c
Hmgb1
predicted gene 13121; predicted gene 3160; high-mobility group
(nonhistone chromosomal) protein 1-like 1; predicted gene 6090;
predicted gene 3851; predicted gene 8967; predicted gene 7782;
predicted gene 4587; predicted gene 4689; predicted gene 3307;
predicted gene 13932; predicted gene 15059; predicted gene 3565;
predicted gene 15447; predicted gene 12587; predicted gene 9012;
predicted gene 6115; predicted gene 9480; high mobility group box 1;
predicted gene 8423; predicted gene 5853; predicted gene 8288;
predicted gene 7888; predicted gene 8594; predicted gene 15387;
predicted gene 5473; predicted gene 8807; similar to high mobility group
box 1; similar to 2810416G20Rik protein; predicted gene 8390; predicted
gene, OTTMUSG00000005439; predicted gene 5842; predicted gene 5527;
predicted gene 8563; predicted gene 2710; predicted gene 12331;
predicted gene 5937; predicted gene 5504; similar to high-mobility group
box 1; predicted gene 10361; predicted gene 2607; predicted gene 7422;
predicted gene 10075; predicted gene 12568; predicted gene 6589;
predicted gene 4383; predicted gene 8031; similar to High mobility group
protein 1 (HMG-1) (High mobility group protein B1) (Amphoterin)
(Heparin-binding protein p30); predicted gene 7468; predicted gene 8554
Hnrnph1
heterogeneous nuclear ribonucleoprotein H1
Hnrnph2
heterogeneous nuclear ribonucleoprotein H2
Hnrnpl
heterogeneous nuclear ribonucleoprotein L
Hnrnpm
heterogeneous nuclear ribonucleoprotein M
Hnrnpr
predicted gene 6159; heterogeneous nuclear ribonucleoprotein R
Hook3
hook homolog 3 ( Drosophila )
Hoxa5
homeo box A5
Hp1bp3
heterochromatin protein 1, binding protein 3
Hsp90aa1
predicted gene 5511; heat shock protein 90, alpha (cytosolic), class A
member 1
Hsp90ab1
heat shock protein 90 alpha (cytosolic), class B member 1
Hsp90b1
heat shock protein 90, beta (Grp94), member 1
Hspa12a
heat shock protein 12A
Hspa2
heat shock protein 2
Hspb1
heat shock protein 1
Hspb8
heat shock protein 8
Id1
inhibitor of DNA binding 1
Id2
inhibitor of DNA binding 2
Ier2
immediate early response 2
Ifi204
interferon activated gene 204
Ifi205
interferon activated gene 205
Ifi27l2a
interferon, alpha-inducible protein 27 like 2A
Ifi35
interferon-induced protein 35
Ifit3
interferon-induced protein with tetratricopeptide repeats 3
Ifitm3
interferon induced transmembrane protein 3
Ifnar2
interferon (alpha and beta) receptor 2
Ifngr1
interferon gamma receptor 1
Ifrd1
interferon-related developmental regulator 1
Ift74
intraflagellar transport 74 homolog ( Chlamydomonas )
Igf1r
insulin-like growth factor I receptor
Igfbp5
insulin-like growth factor binding protein 5
Igfbp6
insulin-like growth factor binding protein 6
Il16
interleukin 16
Il17re
interleukin 17 receptor E
Il6ra
interleukin 6 receptor, alpha
Il6st
interleukin 6 signal transducer
Ildr2
immunoglobulin-like domain containing receptor 2
Ilf3
interleukin enhancer binding factor 3
Impad1
inositol monophosphatase domain containing 1
Ints10
integrator complex subunit 10; similar to integrator complex subunit 10
Iqsec1
IQ motif and Sec7 domain 1
Irak4
interleukin-1 receptor-associated kinase 4
Irf2bp2
interferon regulatory factor 2 binding protein 2
Irf7
interferon regulatory factor 7
Irs2
insulin receptor substrate 2
Itch
itchy, E3 ubiquitin protein ligase
Itga6
integrin alpha 6
Itpr2
inositol 1,4,5-triphosphate receptor 2
Jmjd1c
jumonji domain containing 1C
Jun
Jun oncogene
Junb
Jun-B oncogene
Jund
Jun proto-oncogene related gene d
Jup
junction plakoglobin
Kank1
KN motif and ankyrin repeat domains 1
Kcnab1
potassium voltage-gated channel, shaker-related subfamily, beta
member 1
Kdelr1
KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention
receptor 1
Kdm5a
lysine (K)-specific demethylase 5A
Kdm6b
KDM1 lysine (K)-specific demethylase 6B
Kdr
kinase insert domain protein receptor
Keap1
kelch-like ECH-associated protein 1
Kif1b
kinesin family member 1B
Kif5b
kinesin family member 5B
Klf10
Kruppel-like factor 10
Klf2
Kruppel-like factor 2 (lung)
Klf4
Kruppel-like factor 4 (gut)
Klf6
Kruppel-like factor 6
Klf7
Kruppel-like factor 7 (ubiquitous)
Klf9
Kruppel-like factor 9
Kpna1
karyopherin (importin) alpha 1
Kpna3
karyopherin (importin) alpha 3
Krcc1
lysine-rich coiled-coil 1
Krt14
keratin 14
Ktn1
kinectin 1
Lama4
laminin, alpha 4
Lamp2
lysosomal-associated membrane protein 2
Lars2
leucyl-tRNA synthetase, mitochondrial
Lass2
LAG1 homolog, ceramide synthase 2
Lass4
LAG1 homolog, ceramide synthase 4
Lgals7
lectin, galactose binding, soluble 7
Limch1
LIM and calponin homology domains 1
Lims2
LIM and senescent cell antigen like domains 2
Lman1
lectin, mannose-binding, 1
Lpar2
lysophosphatidic acid receptor 2
Lrrc20
leucine rich repeat containing 20
Lrrc58
leucine rich repeat containing 58; predicted gene, OTTMUSG00000025724
Lrrc61
leucine rich repeat containing 61
Lrrn4
leucine rich repeat neuronal 4
Lrrn4cl
LRRN4 C-terminal like
Ltbp4
latent transforming growth factor beta binding protein 4
Luc7l3
RIKEN cDNA 3300001P08 gene
Maf
similar to c-Maf long form; avian musculoaponeurotic fibrosarcoma (v-
maf) AS42 oncogene homolog
Maged1
melanoma antigen, family D, 1
Magt1
magnesium transporter 1
Malat1
metastasis associated lung adenocarcinoma transcript 1 (non-coding RNA)
Man1a
mannosidase 1, alpha
Manf
mesencephalic astrocyte-derived neurotrophic factor
Maoa
monoamine oxidase A
Map3k3
mitogen-activated protein kinase kinase kinase 3
Mapk1
mitogen-activated protein kinase 1
Mapkapk3
mitogen-activated protein kinase-activated protein kinase 3
Mapre2
microtubule-associated protein, RP/EB family, member 2
Marcksl1
MARCKS-like 1; predicted gene 9106
Mat2a
methionine adenosyltransferase II, alpha
Mat2b
methionine adenosyltransferase II, beta
Matr3
matrin 3; similar to Matrin 3
Med13l
mediator complex subunit 13-like
Med21
mediator complex subunit 21
Mef2c
myocyte enhancer factor 2C
Meis2
Meis homeobox 2
Mesdc1
mesoderm development candidate 1
Metap2
methionine aminopeptidase 2
Mettl2
methyltransferase like 2
Mettl7a1
methyltransferase like 7A1
Mfap1a
similar to microfibrillar-associated protein 1A; microfibrillar-associated
protein 1A; microfibrillar-associated protein 1B
Mfhas1
malignant fibrous histiocytoma amplified sequence 1
Mgll
monoglyceride lipase
Mgst1
microsomal glutathione S-transferase 1
Mll1
myeloid/lymphoid or mixed-lineage leukemia 1
Mll3
myeloid/lymphoid or mixed-lineage leukemia 3
Morf4l2
predicted gene 5521; similar to mortality factor 4 like 2; mortality factor 4
like 2
Mpdz
multiple PDZ domain protein
Mphosph8
M-phase phosphoprotein 8
Mras
muscle and microspikes RAS
Mrgprf
MAS-related GPR, member F
Msn
moesin
Mtap1a
microtubule-associated protein 1 A
Mtdh
metadherin
Mtmr6
myotubularin related protein 6
Mut
methylmalonyl-Coenzyme A mutase
Mxd4
Max dimerization protein 4
Myh10
myosin, heavy polypeptide 10, non-muscle
Myl7
myosin, light polypeptide 7, regulatory
Mylip
myosin regulatory light chain interacting protein
Myst4
MYST histone acetyltransferase monocytic leukemia 4
Naa25
RIKEN cDNA C330023M02 gene
Naga
N-acetyl galactosaminidase, alpha
Nckap1
NCK-associated protein 1
Ncoa1
similar to Nuclear receptor coactivator 1 (NCoA-1) (Steroid receptor
coactivator 1) (SRC-1) (Nuclear receptor coactivator protein 1) (mNRC-1);
nuclear receptor coactivator 1
Ncoa4
predicted gene 6768; nuclear receptor coactivator 4
Ncor1
nuclear receptor co-repressor 1
Ndn
necdin
Ndst1
N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1
Ndufa4
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4
Nedd4
neural precursor cell expressed, developmentally down-regulated 4
Nf1
neurofibromatosis 1
Nfe2l1
nuclear factor, erythroid derived 2, -like 1
Nfia
nuclear factor I/A
Nfic
nuclear factor I/C
Nfix
nuclear factor I/X
Nfkb2
nuclear factor of kappa light polypeptide gene enhancer in B-cells 2,
p49/p100
Nfkbia
nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor, alpha
Nfkbiz
nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor, zeta
Nfyc
nuclear transcription factor-Y gamma
Nid2
nidogen 2
Ninl
ninein-like
Nipal3
NIPA-like domain containing 3; similar to NIPA-like domain containing 3
Nipbl
Nipped-B homolog ( Drosophila )
Nkain4
Na+/K+ transporting ATPase interacting 4
Nkd1
naked cuticle 1 homolog ( Drosophila ); similar to naked cuticle 1 homolog
Nnmt
nicotinamide N-methyltransferase
Nod1
nucleotide-binding oligomerization domain containing 1
Npr1
natriuretic peptide receptor 1
Nr1d1
nuclear receptor subfamily 1, group D, member 1
Nr3c1
nuclear receptor subfamily 3, group C, member 1
Nr4a1
nuclear receptor subfamily 4, group A, member 1
Nrgn
neurogranin
Nucks1
nuclear casein kinase and cyclin-dependent kinase substrate 1
Oasl2
2′-5′ oligoadenylate synthetase-like 2
Oat
ornithine aminotransferase
Ogdh
oxoglutarate dehydrogenase (lipoamide)
Ogn
osteoglycin
Olfr1033
olfactory receptor 1033
Olfr613
olfactory receptor 614; hypothetical protein LOC100044261; olfactory
receptor 613
Opa3
optic atrophy 3 (human)
Orai3
ORAI calcium release-activated calcium modulator 3
Osr1
odd-skipped related 1 ( Drosophila )
Oxct1
3-oxoacid CoA transferase 1
Oxnad1
oxidoreductase NAD-binding domain containing 1
Pard3b
par-3 partitioning defective 3 homolog B ( C. elegans )
Parp14
poly (ADP-ribose) polymerase family, member 14
Parp4
poly (ADP-ribose) polymerase family, member 4
Parvb
parvin, beta; similar to parvin, beta
Pbx1
pre B-cell leukemia transcription factor 1; region containing RIKEN cDNA
2310056B04gene; pre B-cell leukemia transcription factor 1
Pcdh15
protocadherin 15
Pcdhgb5
protocadherin gamma subfamily B, 5
Pcm1
pericentriolar material 1
Pdap1
PDGFA associated protein 1
Pdcd6ip
programmed cell death 6 interacting protein
Pde4dip
phosphodiesterase 4D interacting protein (myomegalin)
Pdia3
protein disulfide isomerase associated 3
Pdia4
protein disulfide isomerase associated 4
Pdpn
podoplanin
Pef1
penta-EF hand domain containing 1
Peli1
pellino 1
Perl
period homolog 1 ( Drosophila )
Pf4
platelet factor 4
Pfn1
profilin 1
Pgcp
plasma glutamate carboxypeptidase
Pgrmc1
progesterone receptor membrane component 1
Phf21a
PHD finger protein 21A
Phf3
PHD finger protein 3
Phip
pleckstrin homology domain interacting protein
Pigt
phosphatidylinositol glycan anchor biosynthesis, class T; similar to GPI
transamidase component PIG-T precursor (Phosphatidylinositol-glycan
biosynthesis class T protein) (Neuronal development-associated protein 7)
Pik3c2a
phosphatidylinositol 3-kinase, C2 domain containing, alpha polypeptide
Pim1
proviral integration site 1
Pitpnm2
phosphatidylinositol transfer protein, membrane-associated 2
Pkhd1l1
polycystic kidney and hepatic disease 1-like 1
Pknox1
Pbx/knotted 1 homeobox
Pla2g4a
phospholipase A2, group IVA (cytosolic, calcium-dependent)
Plat
plasminogen activator, tissue
Plce1
phospholipase C, epsilon 1
Plk1s1
non-protein coding RNA 153
Plk2
polo-like kinase 2 ( Drosophila )
Plod2
procollagen lysine, 2-oxoglutarate 5-dioxygenase 2
Plxdc1
plexin domain containing 1
Plxdc2
plexin domain containing 2
Plxna4
plexin A4
Pmp22
peripheral myelin protein 22
Pnrc1
proline-rich nuclear receptor coactivator 1
Podn
podocan
Ppap2a
phosphatidic acid phosphatase type 2A
Ppbp
pro-platelet basic protein
Ppfibp2
protein tyrosine phosphatase, receptor-type, F interacting protein, binding
protein 2
Ppig
peptidyl-prolyl isomerase G (cyclophilin G)
Ppl
periplakin
Ppp1cb
protein phosphatase 1, catalytic subunit, beta isoform
Ppp1r12a
protein phosphatase 1, regulatory (inhibitor) subunit 12A
Ppp1r15a
protein phosphatase 1, regulatory (inhibitor) subunit 15A; myeloid
differentiation primary response gene 116
Ppp3ca
protein phosphatase 3, catalytic subunit, alpha isoform
Pppde1
PPPDE peptidase domain containing 1
Pqlc3
PQ loop repeat containing
Prelp
proline arginine-rich end leucine-rich repeat
Prg4
proteoglycan 4 (megakaryocyte stimulating factor, articular superficial
zone protein)
Prkar2a
protein kinase, cAMP dependent regulatory, type II alpha
Prpf40a
PRP40 pre-mRNA processing factor 40 homolog A (yeast)
Prr13
proline rich 13
Prss23
protease, serine, 23
Psd
pleckstrin and Sec7 domain containing
Psip1
PC4 and SFRS1 interacting protein 1
Psmb2
proteasome (prosome, macropain) subunit, beta type 2
Psmd11
predicted gene 14048; proteasome (prosome, macropain) 26S subunit,
non-ATPase, 11
Psmd7
proteasome (prosome, macropain) 26S subunit, non-ATPase, 7
Ptges3
predicted gene 9769; prostaglandin E synthase 3 (cytosolic); similar to
Sid3177p; predicted gene 11893
Ptgis
prostaglandin I2 (prostacyclin) synthase
Ptgs1
prostaglandin-endoperoxide synthase 1
Ptma
predicted gene 12504; predicted gene 9800; predicted gene 4617;
predicted gene 6625; predicted gene 7614; similar to prothymosin alpha;
prothymosin alpha; predicted gene 9009
Ptp4a2
predicted gene 13422; protein tyrosine phosphatase 4a2
Ptplad2
protein tyrosine phosphatase-like A domain containing 2
Ptprd
protein tyrosine phosphatase, receptor type, D
Ptprf
protein tyrosine phosphatase, receptor type, F
Ptrf
polymerase 1 and transcript release factor
Qrich1
glutamine-rich 1
Qser1
glutamine and serine rich 1
R74862
expressed sequence R74862
Rab11fip1
RAB11 family interacting protein 1 (class I)
Rab1b
RAB1B, member RAS oncogene family
Rab5c
RAB5C, member RAS oncogene family
Rab6b
RAB6B, member RAS oncogene family
Rab7
RAB7, member RAS oncogene family
Rabgap1l
RAB GTPase activating protein 1-like
Ralbp1
ralA binding protein 1
Raly
RIKEN cDNA C130057N11 gene; hnRNP-associated with lethal yellow
Rarres2
retinoic acid receptor responder (tazarotene induced) 2
Rb1cc1
RB1-inducible coiled-coil 1
Rbbp6
retinoblastoma binding protein 6
Rbbp8
retinoblastoma binding protein 8
Rbm25
RNA binding motif protein 25
Rbm27
RNA binding motif protein 27
Rbm3
predicted gene 15453; RNA binding motif protein 3
Rbpms
RNA binding protein gene with multiple splicing
Rdx
radixin
Rest
RE1-silencing transcription factor
Rgma
RGM domain family, member A
Rgs10
regulator of G-protein signalling 10
Rhob
ras homolog gene family, member B
Rhoj
ras homolog gene family, member J
Rhou
ras homolog gene family, member U
Rnase4
ribonuclease, RNase A family 4
Rnd3
Rho family GTPase 3
Rnf167
ring finger protein 167
Rnf20
ring finger protein 20
Rock1
Rho-associated coiled-coil containing protein kinase 1
Rock2
Rho-associated coiled-coil containing protein kinase 2
Rpp25
ribonuclease P 25 subunit (human)
Rras2
related RAS viral (r-ras) oncogene homolog 2
Rspo1
R-spondin homolog ( Xenopus laevis )
Rtf1
Rtf1, Paf1/RNA polymerase II complex component, homolog ( S. cerevisiae )
Rtn1
reticulon 1
Ryk
receptor-like tyrosine kinase
Sarnp
predicted gene 6563; SAP domain containing ribonucleoprotein
Sat1
similar to spermidine/spermine N1-acetyltransferase; predicted gene
5552; spermidine/spermine N1-acetyl transferase 1
Sbsn
suprabasin
Scd1
stearoyl-Coenzyme A desaturase 1
Sdc4
syndecan 4
Sdpr
serum deprivation response
Sec62
SEC62 homolog ( S. cerevisiae )
Secisbp2
SECIS binding protein 2
Sema5a
sema domain, seven thrombospondin repeats (type 1 and type 1-like),
transmembrane domain (TM) and short cytoplasmic domain, (semaphorin)
5A
Senp6
similar to Sentrin-specific protease 6 (Sentrin/SUMO-specific protease
SENP6) (SUMO-1-specific protease 1); SUMO/sentrin specific peptidase 6
Sep15
selenoprotein
Sept9
septin 9
Serinc5
serine incorporator 5
Serpinb6b
serine (or cysteine) peptidase inhibitor, clade B, member 6b
Serping1
serine (or cysteine) peptidase inhibitor, clade G, member 1
Serpinh1
serine (or cysteine) peptidase inhibitor, clade H, member 1
Sesn1
sestrin 1
Setd2
SET domain containing 2
Sf3b1
splicing factor 3b, subunit 1
Sf3b4
predicted gene 7935; splicing factor 3b, subunit 4
Sfrs18
splicing factor, arginine/serine-rich 18
Shc1
predicted gene 5500; src homology 2 domain-containing transforming
protein C1
Shfm1
split hand/foot malformation (ectrodactyly) type 1
Siae
sialic acid acetylesterase
Siah1a
seven in absentia 1A
Sirt2
sirtuin 2 (silent mating type information regulation 2, homolog) 2
( S. cerevisiae )
Slc10a3
solute carrier family 10 (sodium/bile acid cotransporter family), member 3
Slc16a1
solute carrier family 16 (monocarboxylic acid transporters), member 1
Slc1a5
solute carrier family 1 (neutral amino acid transporter), member 5
Slc26a3
solute carrier family 26, member 3
Slc27a3
solute carrier family 27 (fatty acid transporter), member 3
Slc38a1
solute carrier family 38, member 1
Slc39a8
solute carrier family 39 (metal ion transporter), member 8
Slc43a3
solute carrier family 43, member 3
Slc4a4
solute carrier family 4 (anion exchanger), member 4
Slc6a4
solute carrier family 6 (neurotransmitter transporter, serotonin), member 4
Slc6a6
solute carrier family 6 (neurotransmitter transporter, taurine), member 6
Slc8a1
solute carrier family 8 (sodium/calcium exchanger), member 1
Slc9a3r1
solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1
Slpi
secretory leukocyte peptidase inhibitor
Sltm
SAFB-like, transcription modulator
Slu7
SLU7 splicing factor homolog ( S. cerevisiae )
Slurp1
secreted Ly6/Plaur domain containing 1
Smad4
similar to MAD homolog 4 ( Drosophila ); MAD homolog 4 ( Drosophila )
Smarca2
SWI/SNF related, matrix associated, actin dependent regulator of
chromatin, subfamily a, member 2
Smarca5
predicted gene 13034; SWI/SNF related, matrix associated, actin
dependent regulator of chromatin, subfamily a, member 5
Smc2
structural maintenance of chromosomes 2
Smc3
predicted gene 8892; structural maintenace of chromosomes 3
Smc4
structural maintenance of chromosomes 4
Smc6
structural maintenance of chromosomes 6
Smchd1
SMC hinge domain containing 1
Smpd3
sphingomyelin phosphodiesterase 3, neutral
Snrnp70
small nuclear ribonucleoprotein 70 (U1)
Sntb2
similar to beta-2-syntrophin; syntrophin, basic 2
Soat1
sterol O-acyltransferase 1
Socs3
suppressor of cytokine signaling 3
Sod3
superoxide dismutase 3, extracellular
Sorbs1
sorbin and SH3 domain containing 1
Sorbs3
sorbin and SH3 domain containing 3
Sox6
SRY-box containing gene 6
Sp100
nuclear antigen Sp100
Spag9
sperm associated antigen 9
Spare
secreted acidic cysteine rich glycoprotein; similar to Secreted acidic
cysteine rich glycoprotein
Spen
SPEN homolog, transcriptional regulator ( Drosophila )
Spint2
serine protease inhibitor, Kunitz type 2
Spnb2
spectrin beta 2
Spock2
sparc/osteonectin, cwcv and kazal-like domains proteoglycan 2
Spon2
spondin 2, extracellular matrix protein
Spop
speckle-type POZ protein
Src
Rous sarcoma oncogene
Srrm1
serine/arginine repetitive matrix 1
Ssh2
slingshot homolog 2 ( Drosophila )
Ssr3
signal sequence receptor, gamma
St3gal1
ST3 beta-galactoside alpha-2,3-sialyltransferase 1
Stag1
stromal antigen 1
Star
steroidogenic acute regulatory protein
Stard5
StAR-related lipid transfer (START) domain containing 5
Stat3
similar to Stat3B; signal transducer and activator of transcription 3
Stim1
similar to Stromal interaction molecule 1; stromal interaction molecule 1
Stk10
serine/threonine kinase 10
Stk40
serine/threonine kinase 40
Stmn2
stathmin-like 2
Stra6
stimulated by retinoic acid gene 6
Strn3
striatin, calmodulin binding protein 3
Sulf1
sulfatase 1
Sulf2
sulfatase 2
Supt16h
suppressor of Ty 16 homolog ( S. cerevisiae )
Sv2a
synaptic vesicle glycoprotein 2 a
Syne1
synaptic nuclear envelope 1
Syne2
synaptic nuclear envelope 2
Syt11
synaptotagmin XI; similar to synaptotagmin XI
Sytl1
synaptotagmin-like 1; similar to synaptotagmin-like 1
Taf3
TAF3 RNA polymerase II, TATA box binding protein (TBP)-associated factor
Taf7
TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated factor
Tapbp
TAP binding protein
Tbc1d15
TBC1 domain family, member 15
Tbcel
tubulin folding cofactor E-like
Tbl1x
transducin (beta)-like 1 X-linked
Tbx18
T-box18
Tceal8
transcription elongation factor A (SII)-like 8; similar to transcription
elongation factor A (SII)-like 8
Tcf7l1
transcription factor 3
Tfdp2
transcription factor Dp 2
Tgfb1i1
transforming growth factor beta 1 induced transcript 1
Tgfb2
transforming growth factor, beta 2
Tgfbr2
transforming growth factor, beta receptor II
Tgm2
transglutaminase 2, C polypeptide
Thbd
thrombomodulin
Thbs1
thrombospondin 1; similar to thrombospondin 1
Thoc2
THO complex 2; cDNA sequence BC005561
Thrap3
thyroid hormone receptor associated protein 3; predicted gene 5898
Thsd4
thrombospondin, type I, domain containing 4
Timp2
tissue inhibitor of metalloproteinase 2
Tirap
toll-interleukin 1 receptor (TIR) domain-containing adaptor protein
Tlr2
toll-like receptor 2
Tm4sf1
transmembrane 4 superfamily member 1
Tm4sf5
transmembrane 4 superfamily member 5
Tmcc3
transmembrane and coiled coil domains 3
Tmco1
transmembrane and coiled-coil domains 1
Tmco7
transmembrane and coiled-coil domains 7
Tmed2
transmembrane emp24 domain trafficking protein 2; predicted gene
10698; predicted gene 7318
Tmem119
transmembrane protein 119
Tmem140
transmembrane protein 140
Tmem151a
transmembrane protein 151A
Tmem221
transmembrane protein 221
Tmem50a
transmembrane protein 50A
Tmem98
transmembrane protein 98
Tmod3
tropomodulin 3
Tmpo
thymopoietin
Tmsb4x
thymosin, beta 4, X chromosome; similar to thymosin beta-4
Tnxb
tenascin XB
Tob2
transducer of ERBB2, 2
Topors
topoisomerase 1 binding, arginine/serine-rich
Tpm3
predicted gene 7848; predicted gene 7839; predicted gene 4157; similar to
tropomyosin 3, gamma; tropomyosin 3, gamma; predicted gene 4903
Tppp3
tubulin polymerization-promoting protein family member 3
Tpt1
predicted gene 1974; tumor protein, translationally-controlled 1
pseudogene; tumor protein, translationally-controlled 1; predicted gene
14456
Trafd1
TRAF type zinc finger domain containing 1
Trib1
tribbles homolog 1 ( Drosophila )
Trim8
tripartite motif protein 8
Trpm7
transient receptor potential cation channel, subfamily M, member 7
Tsc22d3
TSC22 domain family, member 3
Tshz1
teashirt zinc finger family member 1
Tsix
X (inactive)-specific transcript, antisense
Tspan31
tetraspanin 31
Tspan5
tetraspanin 5
Ttc28
tetratricopeptide repeat domain 28
Ttc38
tetratricopeptide repeat domain 38
Tuba1a
predicted gene 7172; similar to tubulin, alpha 1; tubulin, alpha 1A
Tubb2a
tubulin, beta 2A
Twsg1
twisted gastrulation homolog 1 ( Drosophila )
Txndc5
thioredoxin domain containing 5
Txnrd1
thioredoxin reductase 1
Uap1
UDP-N-acetylglucosamine pyrophosphorylase 1
Uba7
ubiquitin-activating enzyme E1-like; RIKEN cDNA D330022A01 gene
Ube2d1
ubiquitin-conjugating enzyme E2D 1, UBC4/5 homolog (yeast)
Ube2l6
ubiquitin-conjugating enzyme E2L 6
Ube2n
ubiquitin-conjugating enzyme E2N; similar to ubiquitin-conjugating
enzyme E2 UbcH-ben; similar to ubiquitin-conjugating enzyme E2N;
predicted gene 5943
Ube2v1
ubiquitin-conjugating enzyme E2 variant 1; predicted gene 7181; predicted
gene 12502; similar to ubiquitin-conjugating enzyme E2 variant 1
Ubqln2
ubiquilin 2
Ubxn2a
UBX domain protein 2A; predicted gene 6245
Ubxn4
UBX domain protein 4
Ugdh
UDP-glucose dehydrogenase
Upk1b
uroplakin 1B
Upk3b
uroplakin 3B
Usp16
ubiquitin specific peptidase 16
Usp2
ubiquitin specific peptidase 2
Usp25
ubiquitin specific peptidase 25
Usp54
ubiquitin specific peptidase 54
Usp8
ubiquitin specific peptidase 8
Utp20
UTP20, small subunit (SSU) processome component, homolog (yeast)
Vat1
vesicle amine transport protein 1 homolog ( T californica )
Vim
vimentin
Vps13a
vacuolar protein sorting 13A (yeast)
Vwa5a
von Willebrand factor A domain containing 5A
Wac
similar to WW domain-containing adapter protein with coiled-coil; WW
domain containing adaptor with coiled-coil
Wasf2
WAS protein family, member 2
Wdr26
WD repeat domain 26; similar to myocardial ischemic preconditioning
upregulated protein 2
Wdr92
WD repeat domain 92
Wfdc1
WAP four-disulfide core domain 1
Wls
G protein-coupled receptor 177
Wnt4
wingless-related MMTV integration site 4
Wrnip1
Werner helicase interacting protein 1
Wt1
similar to Wilms tumor homolog; Wilms tumor 1 homolog
Wwc2
WW, C2 and coiled-coil domain containing 2
Xdh
xanthine dehydrogenase
Xist
inactive X specific transcripts
Yipf5
Yip1 domain family, member 5; predicted gene 5738
Ywhaz
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation
protein, zeta polypeptide; predicted gene 4202
Zbed6
similar to Zinc finger BED domain containing protein 4
Zbtb16
zinc finger and BTB domain containing 16
Zbtb20
zinc finger and BTB domain containing 20
Zbtb4
zinc finger and BTB domain containing 4
Zbtb7c
zinc finger and BTB domain containing 7C
Zc3h13
zinc finger CCCH type containing 13
Zc3h18
predicted gene 5939; zinc finger CCCH-type containing 18
Zcchc11
zinc finger, CCHC domain containing 11
Zcchc3
zinc finger, CCHC domain containing 3
Zfand6
zinc finger, AN1-type domain 6
Zfhx4
zinc finger homeodomain 4
Zfp148
zinc finger protein 148
Zfp277
zinc finger protein 277
Zfp281
zinc finger protein 281
Zfp318
zinc finger protein 318
Zfp353
zinc finger protein 353
Zfp36
zinc finger protein 36
Zfp385a
zinc finger protein 385A
Zfp488
zinc finger protein 488
Zfp672
zinc finger protein 672
Zfp704
zinc finger protein 704
Zmat1
zinc finger, matrin type 1
Zrsr1
zinc finger (CCCH type), RNA binding motif and serine/arginine rich 1
Zzef1
zinc finger, ZZ-type with EF hand domain 1
1110002B05Rik
RIKEN cDNA 1110002B05 gene
1110003E01Rik
RIKEN cDNA 1110003E01 gene
1110004F10Rik
predicted gene 9169; RIKEN cDNA 1110004F10 gene; similar to small acidic
protein
1500003O03Rik
RIKEN cDNA 1500003003 gene; similar to EF-hand Ca2+ binding protein
p22
1600029D21Rik
RIKEN cDNA 1600029D21 gene
1810014B01Rik
RIKEN cDNA 1810014B01 gene
1810041L15Rik
RIKEN cDNA 1810041L15 gene
1810074P20Rik
RIKEN cDNA 1810074P20 gene
2010107G12Rik
RIKEN cDNA 2010107G12 gene
2210403K04Rik
hypothetical protein LOC100042498
2310030G06Rik
RIKEN cDNA 2310030G06 gene
2510002D24Rik
RIKEN cDNA 2510002D24 gene
2610034B18Rik
RIKEN cDNA 2610034B18 gene
2610101N10Rik
RIKEN cDNA 2610101N10 gene
2810474O19Rik
RIKEN cDNA 2810474O19 gene
2900002K06Rik
RIKEN cDNA 2900002K06 gene
3110062M04Rik
RIKEN cDNA 3110062M04 gene
4930402H24Rik
RIKEN cDNA 4930402H24 gene
4930523C07Rik
RIKEN cDNA 4930523C07 gene
5430435G22Rik
RIKEN cDNA 5430435G22 gene
6330406I15Rik
RIKEN cDNA 6330406I15 gene
A130040M12Rik
RIKEN cDNA A130040M12 gene
AI848100
expressed sequence AI848100
Gm16897
kg:uc009lxf.1
Prrc2c
kg:uc007won.1
kg:uc009ogv.1
kg:uc009iln.1
kg:uc007qca.1
Atxn7l3b
kg:uc008ewj.2
kg:uc008wkn.1
kg:uc007bgn.1
Ces2g
kg:uc009cvm.1
kg:uc008ehr.1
Tmem234
kg:uc012hdk.1
kg:uc008ajk.1
eg:245190:chr7:m
kg:uc007qse.1
kg:uc007bvx.1
Mob3c
kg:uc008dzh.1
kg:uc009okn.1
kg:uc007zts.1
kg:uc008jup.1
kg:uc008tkz.1
kg:uc007zwh.1
kg:uc008znh.1
Mau2
kg:uc009mng.1
kg:uc007ded.1
kg:uc007ctp.1
kg:uc007zak.1
eg:497210:chr14:m
kg:uc007vsr.1
Mir3064
kg:uc009ize.1
Kansl1
eg:320169:chr9:p
kg:uc009vev.1
kg:uc009acs.1
kg:uc009tuw.1
kg:uc007pff.1
kg:uc007vnc.1
kg:uc009igb.1
kg:uc008oki.1
kg:uc008tky.1
TABLE 8
Exemplary human marker genes
HUMAN
GENE
SYMBOL
Gene Name
ABI3BP
ABI family, member 3 (NESH) binding protein
ABLIM3
actin binding LIM protein family, member 3
ACAD9
acyl-Coenzyme A dehydrogenase family, member 9
ACBD3
acyl-Coenzyme A binding domain containing 3
ACIN1
apoptotic chromatin condensation inducer 1
ACTB
actin, beta
ACTG1
actin, gamma 1
ADAMTS5
ADAM metallopeptidase with thrombospondin type 1 motif, 5
ADAMTSL1
ADAMTS-like 1
ADD3
adducin 3 (gamma)
AEBP1
AE binding protein 1
AGAP1
ArfGAP with GTPase domain, ankyrin repeat and PH domain 1
AKAP13
A kinase (PRKA) anchor protein 13
AKAP2
A kinase (PRKA) anchor protein 2; paralemmin 2; PALM2-AKAP2
readthrough transcript
AKT2
v-akt murine thymoma viral oncogene homolog 2
ALDH1A1
aldehyde dehydrogenase 1 family, member A1
ALDH1A2
aldehyde dehydrogenase 1 family, member A2
ALOX12
arachidonate 12-lipoxygenase
AMFR
autocrine motility factor receptor
AMHR2
anti-Mullerian hormone receptor, type II
ANG
angiogenin, ribonuclease, RNase A family, 5
ANKRD11
ankyrin repeat domain 11; hypothetical protein LOC100128265
ANKRD12
ankyrin repeat domain 12
ANKRD17
ankyrin repeat domain 17
ANO6
anoctamin 6
ANP32A
hepatopoietin PCn127; acidic (leucine-rich) nuclear
phosphoprotein 32 family, member A
ANXA7
annexin A7
AP1S3
adaptor-related protein complex 1, sigma 3 subunit
AP3S1
adaptor-related protein complex 3, sigma 1 subunit
AP4E1
adaptor-related protein complex 4, epsilon 1 subunit
APLP1
amyloid beta (A4) precursor-like protein 1
APP
amyloid beta (A4) precursor protein
AQP1
aquaporin 1 (Colton blood group)
ARAP2
ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 2
ARF3
ADP-ribosylation factor 3
ARF5
ADP-ribosylation factor 5
ARHGAP28
Rho GTPase activating protein 28
ARHGAP29
Rho GTPase activating protein 29
ARHGAP5
Rho GTPase activating protein 5
ARHGEF12
Rho guanine nucleotide exchange factor (GEF) 12
ARID1A
AT rich interactive domain 1A (SWI-like)
ARID4A
AT rich interactive domain 4A (RBP1-like)
ARID4B
AT rich interactive domain 4B (RBP1-like)
ARID5B
AT rich interactive domain 5B (MRF1-like)
ARL3
ADP-ribosylation factor-like 3
ARL4D
ADP-ribosylation factor-like 4D
ARL6IP5
ADP-ribosylation-like factor 6 interacting protein 5
ARMCX3
armadillo repeat containing, X-linked 3
ARPC2
actin related protein 2/3 complex, subunit 2, 34 kDa
ARSA
arylsulfatase A
ARSB
arylsulfatase B
ASCC3
activating signal cointegrator 1 complex subunit 3
ATF3
activating transcription factor 3
ATG3
ATG3 autophagy related 3 homolog ( S. cerevisiae )
ATP1A1
ATPase, Na+/K+ transporting, alpha 1 polypeptide
ATP1B1
ATPase, Na+/K+ transporting, beta 1 polypeptide
ATP2B1
ATPase, Ca++ transporting, plasma membrane 1
ATP6V1A
ATPase, H+ transporting, lysosomal 70 kDa, V1 subunit A
ATXN2
ataxin 2
B2M
beta-2-microglobulin
BAZ2A
bromodomain adjacent to zinc finger domain, 2A
BBS4
Bardet-Biedl syndrome 4
BBX
bobby sox homolog ( Drosophila )
BCAM
basal cell adhesion molecule (Lutheran blood group)
BCL10
B-cell CLL/lymphoma 10; hypothetical LOC646626
BDP1
B double prime 1, subunit of RNA polymerase III transcription
initiation factor IIIB
BICC1
bicaudal C homolog 1 ( Drosophila )
BICD1
bicaudal D homolog 1 ( Drosophila )
BIRC6
baculoviral IAP repeat-containing 6
BLVRB
biliverdin reductase B (flavin reductase (NADPH))
BNC1
basonuclin 1
BNC2
basonuclin 2
BOD1L
biorientation of chromosomes in cell division 1-like
BPTF
bromodomain PHD finger transcription factor
BRAF
v-raf murine sarcoma viral oncogene homolog B1
BRD2
bromodomain containing 2
BRD4
bromodomain containing 4
BRP44L
brain protein 44-like
BST2
NPC-A-7; bone marrow stromal cell antigen 2
BTBD2
BTB (POZ) domain containing 2
BTBD7
BTB (POZ) domain containing 7
BTF3
basic transcription factor 3; basic transcription factor 3, like 1
pseudogene
BTG2
BTG family, member 2
BZW1
basic leucine zipper and W2 domains 1 pseudogene 1; basic leucine
zipper and W2 domains 1 like 1; basic leucine zipper and W2 domains 1
C1D
C1D nuclear receptor co-repressor; similar to nuclear DNA-binding
protein; similar to hCG1791993
C1RL
complement component 1, r subcomponent-like
C1S
complement component 1, s subcomponent
C2
complement component 2
C3
similar to Complement C3 precursor; complement component 3;
hypothetical protein LOC100133511
C4A
complement component 4A (Rodgers blood group)
C4B
complement component 4B (Chido blood group)
CALM1
calmodulin 3 (phosphorylase kinase, delta); calmodulin 2
(phosphorylase kinase, delta); calmodulin 1 (phosphorylase kinase,
delta)
CALM2
calmodulin 3 (phosphorylase kinase, delta); calmodulin 2
(phosphorylase kinase, delta); calmodulin 1 (phosphorylase kinase,
delta)
CAP1
CAP, adenylate cyclase-associated protein 1 (yeast)
CAST
calpastatin
CAV1
caveolin 1, caveolae protein, 22 kDa
CCDC109B
coiled-coil domain containing 109B
CCDC34
coiled-coil domain containing 34
CCDC80
coiled-coil domain containing 80
CCDC88A
coiled-coil domain containing 88A
CCDC90A
coiled-coil domain containing 90A
CCNL1
cyclin L1
CD109
CD109 molecule
CD200
CD200 molecule
CD248
CD248 molecule, endosialin
CD34
CD34 molecule
CD55
CD55 molecule, decay accelerating factor for complement (Cromer
blood group)
CD81
CD81 molecule
CD82
CD82 molecule
CD9
CD9 molecule
CDC42EP3
CDC42 effector protein (Rho GTPase binding) 3
CDH11
cadherin 11, type 2, OB-cadherin (osteoblast)
CDH3
cadherin 3, type 1, P-cadherin (placental)
CDK13
cell division cycle 2-like 5 (cholinesterase-related cell division
controller)
CDON
Cdon homolog (mouse)
CELF2
CUG triplet repeat, RNA binding protein 2
CEP164
centrosomal protein 164 kDa
CEP57
centrosomal protein 57 kDa
CFH
complement factor H
CFL1
cofilin 1 (non-muscle)
CFL2
cofilin 2 (muscle)
CHD1
chromodomain helicase DNA binding protein 1
CHD2
chromodomain helicase DNA binding protein 2
CHI3L1
chitinase 3-like 1 (cartilage glycoprotein-39)
CHST4
carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 4
CISH
cytokine inducible SH2-containing protein
CLCN3
chloride channel 3
CLDN10
claudin 10
CLDN15
claudin 15
CLDN25
claudin-like
CLEC1B
C-type lectin domain family 1, member B
CLEC3B
C-type lectin domain family 3, member B
CLIC4
chloride intracellular channel 4
CLIP1
CAP-GLY domain containing linker protein 1
CLIP3
CAP-GLY domain containing linker protein 3
CLN8
ceroid-lipofuscinosis, neuronal 8 (epilepsy, progressive with mental
retardation)
CMAH
cytidine monophosphate-N-acetylneuraminic acid hydroxylase
(CMP-N-acetylneuraminate monooxygenase) pseudogene
CMTM3
CKLF-like MARVEL transmembrane domain containing 3
CMTM7
CKLF-like MARVEL transmembrane domain containing 7
CNOT6L
CCR4-NOT transcription complex, subunit 6-like
COBL
cordon-bleu homolog (mouse)
COBLL1
COBL-like 1
COL14A1
collagen, type XIV, alpha 1
COL1A2
collagen, type I, alpha 2
COL3A1
collagen, type III, alpha 1
COL4A6
collagen, type IV, alpha 6
COLEC12
collectin sub-family member 12
COQ10B
coenzyme Q10 homolog B ( S. cerevisiae )
CREB3L1
cAMP responsive element binding protein 3-like 1
CREB5
cAMP responsive element binding protein 5
CREBBP
CREB binding protein
CREG1
cellular repressor of E1A-stimulated genes 1
CRIM1
cysteine rich transmembrane BMP regulator 1 (chordin-like)
CRLS1
cardiolipin synthase 1
CRYAB
crystallin, alpha B
CRYL1
crystallin, lambda 1
CRYM
crystallin, mu
CSDA
cold shock domain protein A; cold shock domain protein A
pseudogene 1
CSF1
colony stimulating factor 1 (macrophage)
CSNK1A1
casein kinase 1, alpha 1
CSRNP1
cysteine-serine-rich nuclear protein 1
CSRP1
cysteine and glycine-rich protein 1
CUEDC1
CUE domain containing 1
CYBRD1
cytochrome b reductase 1
CYP2S1
cytochrome P450, family 2, subfamily S, polypeptide 1
CYR61
cysteine-rich, angiogenic inducer, 61
DAB2
disabled homolog 2, mitogen-responsive phosphoprotein ( Drosophila )
DAG1
dystroglycan 1 (dystrophin-associated glycoprotein 1)
DAGLB
diacylglycerol lipase, beta
DAPK1
death-associated protein kinase 1
DCN
decorin
DDR1
discoidin domain receptor tyrosine kinase 1
DDR2
discoidin domain receptor tyrosine kinase 2
DDX3X
DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked
DDX5
DEAD (Asp-Glu-Ala-Asp) box polypeptide 5
DENND5A
DENN/MADD domain containing 5A
DHX15
DEAH (Asp-Glu-Ala-His) box polypeptide 15
DLGAP4
discs, large ( Drosophila ) homolog-associated protein 4
DMKN
dermokine
DNAJA2
DnaJ (Hsp40) homolog, subfamily A, member 2
DNAJB9
DnaJ (Hsp40) homolog, subfamily B, member 9
DNAJC1
DnaJ (Hsp40) homolog, subfamily C, member 1
DNMT1
DNA (cytosine-5-)-methyltransferase 1
DPP4
dipeptidyl-peptidase 4
DPYSL2
dihydropyrimidinase-like 2
DPYSL3
dihydropyrimidinase-like 3
DST
dystonin
DTX2
deltex homolog 2 ( Drosophila )
DUSP1
dual specificity phosphatase 1
DUSP14
dual specificity phosphatase 14
DUSP3
dual specificity phosphatase 3
DYNC1I2
similar to dynein cytoplasmic 1 intermediate chain 2; dynein,
cytoplasmic 1, intermediate chain 2
ECD
ecdysoneless homolog ( Drosophila )
EEA1
early endosome antigen 1
EEF1A1
eukaryotic translation elongation factor 1 alpha-like 7; eukaryotic
translation elongation factor 1 alpha-like 3; similar to eukaryotic
translation elongation factor 1 alpha 1; eukaryotic translation
elongation factor 1 alpha 1
EFEMP1
EGF-containing fibulin-like extracellular matrix protein 1
EFHD2
EF-hand domain family, member D2
EFNA5
ephrin-A5
EGR1
early growth response 1
EHD2
EH-domain containing 2
EIF3A
eukaryotic translation initiation factor 3, subunit A
ELF1
E74-like factor 1 (ets domain transcription factor)
ELOVL6
ELOVL family member 6, elongation of long chain fatty acids
(FEN1/Elo2, SUR4/Elo3-like, yeast)
EMP2
epithelial membrane protein 2
ENPP2
ectonucleotide pyrophosphatase/phosphodiesterase 2
ENPP4
ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative
function)
ESAM
endothelial cell adhesion molecule
ESF1
similar to ABT1-associated protein; ESF1, nucleolar pre-rRNA
processing protein, homolog ( S. cerevisiae )
ESPN
espin
ESYT3
family with sequence similarity 62 (C2 domain containing), member C
ETFA
electron-transfer-flavoprotein, alpha polypeptide
EVPL
envoplakin
EXOC4
exocyst complex component 4
F11R
F11 receptor
FAIM2
Fas apoptotic inhibitory molecule 2
FAM117A
family with sequence similarity 117, member A
FAM134B
family with sequence similarity 134, member B
FAM53B
family with sequence similarity 53, member B
FAM63B
family with sequence similarity 63, member B
FAM76A
family with sequence similarity 76, member A
FAM84B
family with sequence similarity 84, member B
FAS
Fas (TNF receptor superfamily, member 6)
FBLN1
fibulin 1
FERMT2
fermitin family homolog 2 ( Drosophila )
FGF1
fibroblast growth factor 1 (acidic)
FHL1
four and a half LIM domains 1
FILIP1L
filamin A interacting protein 1-like
FKBP5
FK506 binding protein 5
FLII
flightless I homolog ( Drosophila )
FLNC
filamin C, gamma (actin binding protein 280)
FLRT2
fibronectin leucine rich transmembrane protein 2
FMO2
flavin containing monooxygenase 2 (non-functional)
FMOD
fibromodulin
FNDC1
fibronectin type III domain containing 1
FOS
v-fos FBJ murine osteosarcoma viral oncogene homolog
FOXN3
forkhead box N3
FRMD4B
FERM domain containing 4B
FTH1
ferritin, heavy polypeptide 1; ferritin, heavy polypeptide-like 16; similar
to ferritin, heavy polypeptide 1; ferritin, heavy polypeptide-like 3
pseudogene
FXYD1
FXYD domain containing ion transport regulator 1
G3BP1
GTPase activating protein (SH3 domain) binding protein 1
GABARAPL1
GABA(A) receptors associated protein like 3 (pseudogene); GABA(A)
receptor-associated protein like 1
GADD45B
growth arrest and DNA-damage-inducible, beta
GANAB
glucosidase, alpha; neutral AB
GAS1
growth arrest-specific 1
GAS6
similar to growth arrest-specific 6; growth arrest-specific 6
GATA6
GATA binding protein 6
GBP2
guanylate binding protein 2, interferon-inducible
GBP3
guanylate binding protein 3
GBP7
guanylate binding protein 7
GCSH
similar to Glycine cleavage system H protein, mitochondrial precursor;
glycine cleavage system protein H (aminomethyl carrier); similar to
Glycine cleavage system H protein, mitochondrial
GDA
guanine deaminase
GEM
GTP binding protein overexpressed in skeletal muscle
GFM2
G elongation factor, mitochondrial 2
GFPT2
glutamine-fructose-6-phosphate transaminase 2
GJA1
gap junction protein, alpha 1, 43 kDa
GJB5
gap junction protein, beta 5, 31.1 kDa
GNB2
guanine nucleotide binding protein (G protein), beta polypeptide 2
GOLGA4
golgi autoantigen, golgin subfamily a, 4
GOLGB1
golgin B1, golgi integral membrane protein
GPC3
glypican 3
GPC4
glypican 4
GPCPD1
hypothetical protein KIAA1434
GPM6A
glycoprotein M6A
GPR116
G protein-coupled receptor 116
GPR133
G protein-coupled receptor 133
GPR64
G protein-coupled receptor 64
GPRC5B
G protein-coupled receptor, family C, group 5, member B
GPX8
glutathione peroxidase 8 (putative)
GSR
glutathione reductase
GSTA3
glutathione S-transferase alpha 3
GSTM1
glutathione S-transferase mu 1
GSTM4
glutathione S-transferase mu 4
GUCY1A3
guanylate cyclase 1, soluble, alpha 3
H3F3A
H3 histone, family 3B (H3.3B); H3 histone, family 3A pseudogene; H3
histone, family 3A; similar to H3 histone, family 3B; similar to histone
H3.3B
HDAC3
histone deacetylase 3
HDAC5
histone deacetylase 5
HEG1
HEG homolog 1 (zebrafish)
HERPUD2
HERPUD family member 2
HES1
hairy and enhancer of split 1, ( Drosophila )
HEXB
hexosaminidase B (beta polypeptide)
HIST1H1C
histone cluster 1, H1c
HMGB1
high-mobility group box 1; high-mobility group box 1-like 10
HNRNPH1
heterogeneous nuclear ribonucleoprotein H1 (H)
HNRNPH2
ribosomal protein L36a pseudogene 51; ribosomal protein L36a
pseudogene 37; ribosomal protein L36a pseudogene 49;
heterogeneous nuclear ribonucleoprotein H2 (H′); ribosomal protein
L36a
HNRNPL
similar to heterogeneous nuclear ribonucleoprotein L-like;
heterogeneous nuclear ribonucleoprotein L
HNRNPM
heterogeneous nuclear ribonucleoprotein M
HNRNPR
heterogeneous nuclear ribonucleoprotein R
HOOK3
hook homolog 3 ( Drosophila )
HOXA5
homeobox A5
HP1BP3
heterochromatin protein 1, binding protein 3
HSP90AA1
heat shock protein 90 kDa alpha (cytosolic), class A member 2; heat
shock protein 90 kDa alpha (cytosolic), class A member 1
HSP90AB1
heat shock protein 90 kDa alpha (cytosolic), class B member 1
HSP90B1
heat shock protein 90 kDa beta (Grp94), member 1
HSPA12A
heat shock 70 kDa protein 12A
HSPA2
heat shock 70 kDa protein 2
HSPB1
heat shock 27 kDa protein-like 2 pseudogene; heat shock 27 kDa
protein 1
HSPB8
heat shock 22 kDa protein 8
ID1
inhibitor of DNA binding 1, dominant negative helix-loop-helix protein
ID2
inhibitor of DNA binding 2, dominant negative helix-loop-helix protein
IER2
immediate early response 2
IFI35
interferon-induced protein 35
IFIT3
interferon-induced protein with tetratricopeptide repeats 3
IFITM3
interferon induced transmembrane protein 3 (1-8 U)
IFNAR2
interferon (alpha, beta and omega) receptor 2
IFNGR1
interferon gamma receptor 1
IFRD1
interferon-related developmental regulator 1
IFT74
intraflagellar transport 74 homolog ( Chlamydomonas )
IGF1R
insulin-like growth factor 1 receptor
IGFBP5
insulin-like growth factor binding protein 5
IGFBP6
insulin-like growth factor binding protein 6
IL16
interleukin 16 (lymphocyte chemoattractant factor)
IL17RE
interleukin 17 receptor E
IL6ST
interleukin 6 signal transducer (gp130, oncostatin M receptor)
ILDR2
immunoglobulin-like domain containing receptor 2
ILF3
interleukin enhancer binding factor 3, 90 kDa
IMPAD1
inositol monophosphatase domain containing 1
INTS10
integrator complex subunit 10
IQSEC1
IQ motif and Sec7 domain 1
IRAK4
interleukin-1 receptor-associated kinase 4
IRF2BP2
interferon regulatory factor 2 binding protein 2
IRF7
interferon regulatory factor 7
IRS2
insulin receptor substrate 2
ITCH
itchy E3 ubiquitin protein ligase homolog (mouse)
ITGA6
integrin, alpha 6
ITPR2
inositol 1,4,5-triphosphate receptor, type 2
JMJD1C
jumonji domain containing 1C
JUN
jun oncogene
JUNB
jun B proto-oncogene
JUND
jun D proto-oncogene
JUP
junction plakoglobin
KANK1
KN motif and ankyrin repeat domains 1; similar to ankyrin repeat
domain protein 15 isoform b
KCNAB1
potassium voltage-gated channel, shaker-related subfamily, beta
member 1
KDELR1
KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention
receptor 1
KDM5A
lysine (K)-specific demethylase 5A
KDM6B
lysine (K)-specific demethylase 6B
KDR
kinase insert domain receptor (a type III receptor tyrosine kinase)
KEAP1
kelch-like ECH-associated protein 1
KIF1B
kinesin family member 1B
KIF5B
kinesin family member 5B
KLF10
Kruppel-like factor 10
KLF2
Kruppel-like factor 2 (lung)
KLF4
Kruppel-like factor 4 (gut)
KLF6
Kruppel-like factor 6
KLF7
Kruppel-like factor 7 (ubiquitous)
KLF9
Kruppel-like factor 9
KPNA1
karyopherin alpha 1 (importin alpha 5)
KPNA3
karyopherin alpha 3 (importin alpha 4)
KRCC1
lysine-rich coiled-coil 1
KRT14
keratin 14
KTN1
kinectin 1 (kinesin receptor)
LAMA4
laminin, alpha 4
LAMP2
lysosomal-associated membrane protein 2
LARS2
leucyl-tRNA synthetase 2, mitochondrial
LASS2
LAG1 homolog, ceramide synthase 2
LASS4
LAG1 homolog, ceramide synthase 4
LGALS7
lectin, galactoside-binding, soluble, 7; lectin, galactoside-binding,
soluble, 7B
LIMCH1
LIM and calponin homology domains 1
LIMS2
LIM and senescent cell antigen-like domains 2
LMAN1
lectin, mannose-binding, 1
LPAR2
lysophosphatidic acid receptor 2
LRRC20
leucine rich repeat containing 20
LRRC58
leucine rich repeat containing 58
LRRC61
leucine rich repeat containing 61
LRRN4
leucine rich repeat neuronal 4
LRRN4CL
LRRN4C-terminal like
LTBP4
latent transforming growth factor beta binding protein 4
LUC7L3
cisplatin resistance-associated overexpressed protein
MAF
v-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian)
MAGED1
melanoma antigen family D, 1
MAGT1
magnesium transporter 1
MALAT1
metastasis associated lung adenocarcinoma transcript 1 (non-protein
coding)
MANF
mesencephalic astrocyte-derived neurotrophic factor
MAOA
monoamine oxidase A
MAP3K3
mitogen-activated protein kinase kinase kinase 3
MAPK1
mitogen-activated protein kinase 1
MAPKAPK3
mitogen-activated protein kinase-activated protein kinase 3
MAPRE2
microtubule-associated protein, RP/EB family, member 2
MARCKSL1
MARCKS-like 1
MAT2A
methionine adenosyltransferase II, alpha
MAT2B
methionine adenosyltransferase II, beta
MATR3
matrin 3
MED13L
mediator complex subunit 13-like
MED21
mediator complex subunit 21
MEF2C
myocyte enhancer factor 2C
MEIS2
Meis homeobox 2
MESDC1
mesoderm development candidate 1
METAP2
methionyl aminopeptidase 2
MFHAS1
malignant fibrous histiocytoma amplified sequence 1
MGLL
monoglyceride lipase
MGST1
microsomal glutathione S-transferase 1
MLL3
myeloid/lymphoid or mixed-lineage leukemia 3
MORF4L2
mortality factor 4 like 2
MPDZ
multiple PDZ domain protein
MPHOSPH8
M-phase phosphoprotein 8
MRAS
muscle RAS oncogene homolog
MRGPRF
MAS-related GPR, member F
MSN
moesin
MTDH
metadherin
MTMR6
myotubularin related protein 6
MUT
methylmalonyl Coenzyme A mutase
MXD4
MAX dimerization protein 4
MYH10
myosin, heavy chain 10, non-muscle
MYL12A
myosin, light chain 12A, regulatory, non-sarcomeric
MYL7
myosin, light chain 7, regulatory
MYLIP
myosin regulatory light chain interacting protein
MYST4
MYST histone acetyltransferase (monocytic leukemia) 4
NAA25
chromosome 12 open reading frame 30
NAGA
N-acetylgalactosaminidase, alpha-
NCKAP1
NCK-associated protein 1
NCOA1
nuclear receptor coactivator 1
NCOA4
nuclear receptor coactivator 4
NCOR1
nuclear receptor co-repressor 1
NDN
necdin homolog (mouse)
NDST1
N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1
NDUFA4
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9 kDa
NEDD4
neural precursor cell expressed, developmentally down-regulated 4
NF1
neurofibromin 1
NFE2L1
nuclear factor (erythroid-derived 2)-like 1
NFIA
nuclear factor I/A
NFIC
nuclear factor I/C (CCAAT-binding transcription factor)
NFIX
nuclear factor I/X (CCAAT-binding transcription factor)
NFKB2
nuclear factor of kappa light polypeptide gene enhancer in B-cells 2
(p49/p100)
NFKBIA
nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor, alpha
NFKBIZ
nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor, zeta
NFYC
nuclear transcription factor Y, gamma
NID2
nidogen 2 (osteonidogen)
NINL
ninein-like
NIPAL3
NIPA-like domain containing 3
NIPBL
Nipped-B homolog ( Drosophila )
NKAIN4
Na+/K+ transporting ATPase interacting 4
NKD1
naked cuticle homolog 1 ( Drosophila )
NNMT
nicotinamide N-methyltransferase
NOD1
nucleotide-binding oligomerization domain containing 1
NPR1
natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic
peptide receptor A)
NR1D1
nuclear receptor subfamily 1, group D, member 1
NR3C1
nuclear receptor subfamily 3, group C, member 1 (glucocorticoid
receptor)
NR4A1
nuclear receptor subfamily 4, group A, member 1
NRGN
neurogranin (protein kinase C substrate, RC3)
NUCKS1
nuclear casein kinase and cyclin-dependent kinase substrate 1
OAT
ornithine aminotransferase (gyrate atrophy)
OGDH
oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide)
OGN
osteoglycin
OPA3
optic atrophy 3 (autosomal recessive, with chorea and spastic
paraplegia)
ORAI3
ORAI calcium release-activated calcium modulator 3
OSR1
odd-skipped related 1 ( Drosophila )
OXCT1
3-oxoacid CoA transferase 1
OXNAD1
oxidoreductase NAD-binding domain containing 1
PARD3B
par-3 partitioning defective 3 homolog B ( C. elegans )
PARP14
poly (ADP-ribose) polymerase family, member 14
PARP4
poly (ADP-ribose) polymerase family, member 4
PARVB
parvin, beta
PBX1
pre-B-cell leukemia homeobox 1
PCDH15
protocadherin 15
PCDHGB5
protocadherin gamma subfamily B, 5
PCM1
pericentriolar material 1
PDAP1
PDGFA associated protein 1; similar to PDGFA associated protein 1
PDCD6IP
programmed cell death 6 interacting protein
PDE4DIP
hypothetical protein LOC100134230; similar to KIAA0454 protein;
similar to phosphodiesterase 4D interacting protein isoform 2;
phosphodiesterase 4D interacting protein
PDIA3
protein disulfide isomerase family A, member 3
PDIA4
protein disulfide isomerase family A, member 4
PDPN
podoplanin
PEF1
penta-EF-hand domain containing 1
PELI1
pellino homolog 1 ( Drosophila )
PER1
period homolog 1 ( Drosophila )
PF4
platelet factor 4
PFN1
profilin 1
PGCP
plasma glutamate carboxypeptidase
PGRMC1
progesterone receptor membrane component 1
PHF21A
PHD finger protein 21A
PHF3
PHD finger protein 3
PHIP
pleckstrin homology domain interacting protein
PIGT
phosphatidylinositol glycan anchor biosynthesis, class T
PIK3C2A
phosphoinositide-3-kinase, class 2, alpha polypeptide
PIM1
pim-1 oncogene
PITPNM2
phosphatidylinositol transfer protein, membrane-associated 2
PKHD1L1
polycystic kidney and hepatic disease 1 (autosomal recessive)-like 1
PKNOX1
PBX/knotted 1 homeobox 1
PLA2G4A
phospholipase A2, group IVA (cytosolic, calcium-dependent)
PLAT
plasminogen activator, tissue
PLCE1
phospholipase C, epsilon 1
PLK1S1
non-protein coding RNA 153
PLK2
polo-like kinase 2 ( Drosophila )
PLOD2
procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2
PLXDC1
plexin domain containing 1
PLXDC2
plexin domain containing 2
PLXNA4
plexin A4
PMP22
peripheral myelin protein 22
PNRC1
proline-rich nuclear receptor coactivator 1
PODN
podocan
PPAP2A
phosphatidic acid phosphatase type 2A
PPBP
pro-platelet basic protein (chemokine (C-X-C motif) ligand 7)
PPFIBP2
PTPRF interacting protein, binding protein 2 (liprin beta 2)
PPIG
peptidylprolyl isomerase G (cyclophilin G)
PPL
periplakin
PPP1CB
protein phosphatase 1, catalytic subunit, beta isoform; speedy homolog
A ( Xenopus laevis )
PPP1R12A
protein phosphatase 1, regulatory (inhibitor) subunit 12A
PPP1R15A
protein phosphatase 1, regulatory (inhibitor) subunit 15A
PPP3CA
protein phosphatase 3 (formerly 2B), catalytic subunit, alpha isoform
PPPDE1
PPPDE peptidase domain containing 1
PQLC3
PQ loop repeat containing 3
PRELP
proline/arginine-rich end leucine-rich repeat protein
PRG4
proteoglycan 4
PRKAR2A
protein kinase, cAMP-dependent, regulatory, type II, alpha
PRPF40A
PRP40 pre-mRNA processing factor 40 homolog A ( S. cerevisiae )
PRR13
proline rich 13
PRSS23
protease, serine, 23
PSD
pleckstrin and Sec7 domain containing
PSIP1
PC4 and SFRS1 interacting protein 1
PSMB2
proteasome (prosome, macropain) subunit, beta type, 2
PSMD11
proteasome (prosome, macropain) 26S subunit, non-ATPase, 11
PSMD7
proteasome (prosome, macropain) 26S subunit, non-ATPase, 7
PTGES3
prostaglandin E synthase 3 (cytosolic)
PTGIS
prostaglandin I2 (prostacyclin) synthase
PTGS1
prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase
and cyclooxygenase)
PTMA
hypothetical LOC728026; prothymosin, alpha; hypothetical gene
supported by BC013859; prothymosin, alpha pseudogene 4 (gene
sequence 112)
PTP4A2
protein tyrosine phosphatase type IVA, member 2
PTPLAD2
protein tyrosine phosphatase-like A domain containing 2
PTPRD
protein tyrosine phosphatase, receptor type, D
PTPRF
protein tyrosine phosphatase, receptor type, F
PTRF
polymerase 1 and transcript release factor
QRICH1
glutamine-rich 1
QSER1
glutamine and serine rich 1
RAB11FIP1
RAB11 family interacting protein 1 (class I)
RAB1B
RAB1B, member RAS oncogene family
RAB5C
RAB5C, member RAS oncogene family
RAB6B
RAB6B, member RAS oncogene family
RABGAP1L
RAB GTPase activating protein 1-like
RALBP1
hypothetical LOC100129773; ralA binding protein 1
RALY
RNA binding protein, autoantigenic (hnRNP-associated with lethal
yellow homolog (mouse))
RARRES2
retinoic acid receptor responder (tazarotene induced) 2
RB1CC1
RB1-inducible coiled-coil 1
RBBP6
retinoblastoma binding protein 6
RBBP8
retinoblastoma binding protein 8
RBM25
RNA binding motif protein 25
RBM27
RNA binding motif protein 27
RBM3
RNA binding motif (RNP1, RRM) protein 3
RBPMS
RNA binding protein with multiple splicing
RDX
radixin
REST
RE1-silencing transcription factor
RGMA
RGM domain family, member A
RGS10
regulator of G-protein signaling 10
RHOB
ras homolog gene family, member B
RHOJ
ras homolog gene family, member J
RHOU
ras homolog gene family, member U
RNASE4
ribonuclease, RNase A family, 4
RND3
Rho family GTPase 3
RNF167
ring finger protein 167
RNF20
ring finger protein 20
ROCK1
similar to Rho-associated, coiled-coil containing protein kinase 1; Rho-
associated, coiled-coil containing protein kinase 1
ROCK2
Rho-associated, coiled-coil containing protein kinase 2
RPP25
ribonuclease P/MRP 25 kDa subunit
RRAS2
related RAS viral (r-ras) oncogene homolog 2; similar to related RAS
viral (r-ras) oncogene homolog 2
RSPO1
R-spondin homolog ( Xenopus laevis )
RTF1
Rtf1, Paf1/RNA polymerase II complex component, homolog
( S. cerevisiae )
RTN1
reticulon 1
RYK
RYK receptor-like tyrosine kinase
SARNP
SAP domain containing ribonucleoprotein
SAT1
spermidine/spermine N1-acetyltransferase 1
SBSN
suprabasin
SDC4
syndecan 4
SDPR
serum deprivation response (phosphatidylserine binding protein)
SEC62
SEC62 homolog ( S. cerevisiae )
SECISBP2
SECIS binding protein 2
SEMA5A
sema domain, seven thrombospondin repeats (type 1 and type 1-like),
transmembrane domain (TM) and short cytoplasmic domain,
(semaphorin) 5A
SENP6
SUMO1/sentrin specific peptidase 6
SEP15
15 kDa selenoprotein
SEPT9
septin 9
SERINC5
serine incorporator 5
SERPING1
serpin peptidase inhibitor, clade G (C1 inhibitor), member 1
SERPINH1
serpin peptidase inhibitor, clade H (heat shock protein 47), member 1,
(collagen binding protein 1)
SESN1
sestrin 1
SETD2
SET domain containing 2
SF3B1
splicing factor 3b, subunit 1, 155 kDa
SF3B4
splicing factor 3b, subunit 4, 49 kDa
SFRS18
splicing factor, arginine/serine-rich 18
SHC1
SHC (Src homology 2 domain containing) transforming protein 1
SHFM1
split hand/foot malformation (ectrodactyly) type 1
SIAE
sialic acid acetylesterase
SIRT2
sirtuin (silent mating type information regulation 2 homolog) 2
( S. cerevisiae )
SLC10A3
solute carrier family 10 (sodium/bile acid cotransporter family),
member 3
SLC16A1
solute carrier family 16, member 1 (monocarboxylic acid transporter 1)
SLC1A5
solute carrier family 1 (neutral amino acid transporter), member 5
SLC26A3
solute carrier family 26, member 3
SLC27A3
solute carrier family 27 (fatty acid transporter), member 3
SLC38A1
solute carrier family 38, member 1
SLC39A8
solute carrier family 39 (zinc transporter), member 8
SLC43A3
solute carrier family 43, member 3
SLC4A4
solute carrier family 4, sodium bicarbonate cotransporter, member 4
SLC6A4
solute carrier family 6 (neurotransmitter transporter, serotonin),
member 4
SLC6A6
solute carrier family 6 (neurotransmitter transporter, taurine),
member 6
SLC8A1
solute carrier family 8 (sodium/calcium exchanger), member 1
SLC9A3R1
solute carrier family 9 (sodium/hydrogen exchanger), member 3
regulator 1
SLPI
secretory leukocyte peptidase inhibitor
SLTM
SAFB-like, transcription modulator
SLU7
SLU7 splicing factor homolog ( S. cerevisiae )
SLURP1
secreted LY6/PLAUR domain containing 1
SMAD4
SMAD family member 4
SMARCA2
SWI/SNF related, matrix associated, actin dependent regulator of
chromatin, subfamily a, member 2
SMARCA5
SWI/SNF related, matrix associated, actin dependent regulator of
chromatin, subfamily a, member 5
SMC2
structural maintenance of chromosomes 2
SMC3
structural maintenance of chromosomes 3
SMC4
structural maintenance of chromosomes 4
SMC6
structural maintenance of chromosomes 6
SMCHD1
structural maintenance of chromosomes flexible hinge domain
containing 1
SMPD3
sphingomyelin phosphodiesterase 3, neutral membrane (neutral
sphingomyelinase II)
SNRNP70
small nuclear ribonucleoprotein 70 kDa (U1)
SNTB2
syntrophin, beta 2 (dystrophin-associated protein A1, 59 kDa, basic
component 2)
SOAT1
sterol O-acyltransferase 1
SOCS3
suppressor of cytokine signaling 3
SOD3
superoxide dismutase 3, extracellular
SORBS1
sorbin and SH3 domain containing 1
SORBS3
sorbin and SH3 domain containing 3
SOX6
SRY (sex determining region Y)-box 6
SP100
SP100 nuclear antigen
SPAG9
sperm associated antigen 9
SPARC
secreted protein, acidic, cysteine-rich (osteonectin)
SPEN
spen homolog, transcriptional regulator ( Drosophila )
SPINT2
serine peptidase inhibitor, Kunitz type, 2
SPOCK2
sparc/osteonectin, cwcv and kazal-like domains proteoglycan
(testican) 2
SPON2
spondin 2, extracellular matrix protein
SPOP
speckle-type POZ protein
SRC
v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian)
SRRM1
serine/arginine repetitive matrix 1
SSH2
slingshot homolog 2 ( Drosophila )
SSR3
signal sequence receptor, gamma (translocon-associated protein
gamma)
ST3GAL1
ST3 beta-galactoside alpha-2,3-sialyltransferase 1
STAG1
stromal antigen 1
STAR
steroidogenic acute regulatory protein
STARD5
StAR-related lipid transfer (START) domain containing 5
STAT3
signal transducer and activator of transcription 3 (acute-phase
response factor)
STIM1
stromal interaction molecule 1
STK10
serine/threonine kinase 10
STK40
serine/threonine kinase 40
STMN2
stathmin-like 2
STRA6
stimulated by retinoic acid gene 6 homolog (mouse)
STRN3
striatin, calmodulin binding protein 3
SULF1
sulfatase 1
SULF2
sulfatase 2
SUPT16H
suppressor of Ty 16 homolog ( S. cerevisiae ); suppressor of Ty 16
homolog ( S. cerevisiae ) pseudogene
SV2A
synaptic vesicle glycoprotein 2A
SYNE1
spectrin repeat containing, nuclear envelope 1
SYNE2
spectrin repeat containing, nuclear envelope 2
SYT11
synaptotagmin XI
SYTL1
synaptotagmin-like 1
TAF3
TAF3 RNA polymerase II, TATA box binding protein (TBP)-associated
factor, 140 kDa
TAF7
TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated
factor, 55 kDa
TAPBP
TAP binding protein (tapasin)
TBC1D15
TBC1 domain family, member 15
TBCEL
tubulin folding cofactor E-like
TBL1X
transducin (beta)-like 1X-linked
TBX18
T-box 18
TCEAL8
transcription elongation factor A (SII)-like 8
TCF7L1
transcription factor 7-like 1 (T-cell specific, HMG-box)
TFDP2
transcription factor Dp-2 (E2F dimerization partner 2)
TGFB1I1
transforming growth factor beta 1 induced transcript 1
TGFB2
transforming growth factor, beta 2
TGFBR2
transforming growth factor, beta receptor II (70/80 kDa)
TGM2
transglutaminase 2 (C polypeptide, protein-glutamine-gamma-
glutamyltransferase)
THBD
thrombomodulin
THBS1
thrombospondin 1
THOC2
THO complex 2
THRAP3
thyroid hormone receptor associated protein 3
THSD4
thrombospondin, type I, domain containing 4
TIMP2
TIMP metallopeptidase inhibitor 2
TIRAP
toll-interleukin 1 receptor (TIR) domain containing adaptor protein
TLR2
toll-like receptor 2
TM4SF1
transmembrane 4 L six family member 1
TM4SF5
transmembrane 4 L six family member 5
TMCC3
transmembrane and coiled-coil domain family 3
TMCO1
transmembrane and coiled-coil domains 1
TMCO7
transmembrane and coiled-coil domains 7
TMED2
transmembrane emp24 domain trafficking protein 2
TMEM119
transmembrane protein 119
TMEM140
transmembrane protein 140
TMEM151A
transmembrane protein 151A
TMEM221
transmembrane protein 221
TMEM50A
transmembrane protein 50A
TMEM98
similar to transmembrane protein 98; transmembrane protein 98
TMOD3
tropomodulin 3 (ubiquitous)
TMPO
thymopoietin
TMSB4X
thymosin-like 2 (pseudogene); thymosin-like 1 (pseudogene); thymosin
beta 4, X-linked
TNXB
tenascin XB; tenascin XA pseudogene
TOB2
transducer of ERBB2, 2
TOPORS
topoisomerase 1 binding, arginine/serine-rich
TPM3
tropomyosin 3
TPPP3
tubulin polymerization-promoting protein family member 3
TPT1
similar to tumor protein, translationally-controlled 1; tumor protein,
translationally-controlled 1
TRAFD1
TRAF-type zinc finger domain containing 1
TRIB1
tribbles homolog 1 ( Drosophila )
TRIM8
tripartite motif-containing 8
TRPM7
transient receptor potential cation channel, subfamily M, member 7
TSC22D3
TSC22 domain family, member 3; GRAM domain containing 4
TSHZ1
teashirt zinc finger homeobox 1
TSIX
XIST antisense RNA (non-protein coding)
TSPAN31
tetraspanin 31
TSPAN5
tetraspanin 5
TTC28
chromosome 6 open reading frame 35; hCG1820764; tetratricopeptide
repeat domain 28
TTC38
tetratricopeptide repeat domain 38
TUBA1A
tubulin, alpha la
TUBB2A
tubulin, beta 2A
TWSG1
twisted gastrulation homolog 1 ( Drosophila )
TXNDC5
thioredoxin domain containing 5 (endoplasmic reticulum); muted
homolog (mouse)
TXNRD1
thioredoxin reductase 1; hypothetical LOC100130902
UAP1
UDP-N-acteylglucosamine pyrophosphorylase 1
UBA7
ubiquitin-like modifier activating enzyme 7
UBE2D1
ubiquitin-conjugating enzyme E2D 1 (UBC4/5 homolog, yeast)
UBE2L6
ubiquitin-conjugating enzyme E2L 6
UBE2N
ubiquitin-conjugating enzyme E2N (UBC13 homolog, yeast)
UBE2V1
ubiquitin-conjugating enzyme E2 variant 1; ubiquitin-conjugating
enzyme E2 variant 1 pseudogene 2; transmembrane protein 189;
TMEM189-UBE2V1 readthrough transcript
UBQLN2
ubiquilin 2
UBXN2A
UBX domain protein 2A
UBXN4
UBX domain protein 4
UGDH
UDP-glucose dehydrogenase
UPK1B
uroplakin 1B
UPK3B
uroplakin 3B
USP16
ubiquitin specific peptidase 16
USP2
ubiquitin specific peptidase 2
USP25
ubiquitin specific peptidase 25
USP54
ubiquitin specific peptidase 54
USP8
ubiquitin specific peptidase 8
UTP20
similar to Down-regulated in metastasis protein (Key-1A6 protein)
(Novel nucleolar protein 73) (NNP73); UTP20, small subunit (SSU)
processome component, homolog (yeast)
VAT1
vesicle amine transport protein 1 homolog ( T. californica )
VIM
vimentin
VPS13A
vacuolar protein sorting 13 homolog A ( S. cerevisiae )
VWA5A
von Willebrand factor A domain containing 5A
WAC
WW domain containing adaptor with coiled-coil
WASF2
WAS protein family, member 2
WDR26
WD repeat domain 26
WDR92
WD repeat domain 92
WFDC1
WAP four-disulfide core domain 1
WLS
G protein-coupled receptor 177
WNT4
wingless-type MMTV integration site family, member 4
WRNIP1
Werner helicase interacting protein 1
WT1
Wilms tumor 1
WWC2
WW and C2 domain containing 2
XDH
xanthine dehydrogenase
XIST
X (inactive)-specific transcript (non-protein coding)
YIPF5
Yip1 domain family, member 5
YWHAZ
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation
protein, zeta polypeptide
ZBTB16
zinc finger and BTB domain containing 16
ZBTB20
zinc finger and BTB domain containing 20
ZBTB4
zinc finger and BTB domain containing 4
ZBTB7C
zinc finger and BTB domain containing 7C
ZC3H13
zinc finger CCCH-type containing 13
ZC3H18
zinc finger CCCH-type containing 18
ZCCHC11
zinc finger, CCHC domain containing 11
ZCCHC3
zinc finger, CCHC domain containing 3
ZFAND6
zinc finger, AN1-type domain 6
ZFHX4
zinc finger homeobox 4
ZFP36
zinc finger protein 36, C3H type, homolog (mouse)
ZMAT1
zinc finger, matrin type 1
ZRSR1
zinc finger (CCCH type), RNA-binding motif and serine/arginine rich 1
ZZEF1
zinc finger, ZZ-type with EF-hand domain 1
The gene names listed in Table 7 and Table 8 are common names. NCBI Gene ID numbers for each of the genes listed in Table 7 or Table 8 can be obtained by searching the “Gene” Database of the NCBI (available on the World Wide Web at http://www.ncbi.nlm.nih.gov/) using the common name as the query and selecting the first returned Homo sapiens (for the genes in Table 8) or Mus musculus gene (for the genes in Table 7). Other genes may be obtained using the UCSC genome browser (available on the World Wide Web at http://genome.ucsc.edu) using the Gene Sorter function. Human homologs of mouse genes can be readily identified, e.g. the identified homologs in the NCBI database, or by querying databases such as BLAST. In certain embodiments, the marker gene(s) are selected from the genes listed in Table 7, Table 8, or Table 14.
In a CTC, the marker genes listed in Table 7, Table 8, or Table 14 can be upregulated, e.g. for marker genes listed in Table 7, Table 8, or Table 14, if the measured marker gene expression in a cell or sample is higher as compared to a reference level of that marker gene's expression, then the cell is identified as a CTC and/or the sample is identified as comprising CTCs. Preferably, once looks at a statistically significant change. However, even if a few genes in a group do not differ from normal, a sample can be identified as comprising CTCs if the overall change of the group shows a significant change, preferably a statistically significant change. All possible combinations of 2 or more of the indicated markers are contemplated herein.
The level of a gene expression product of a marker gene in Table 7, Table 8, or Table 14 which is higher than a reference level of that marker gene by at least about 10% than the reference amount, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, at least about 100%, at least about 200%, at least about 300%, at least about 500% or at least about 1000% or more, is indicative of the presence of a CTC.
In some embodiments, the reference can be a level of expression of the marker gene product in a cell or population of cells which are not CTCs, e.g. the average level in non-circulating tumor cells and/or circulating cells which are not cancer cells. In some embodiments, the reference can also be a level of expression of the marker gene product in a control sample, a pooled sample of control individuals or a numeric value or range of values based on the same.
In some embodiments, the methods and assays described herein include (a) transforming the gene expression product into a detectable gene target; (b) measuring the amount of the detectable gene target; and (c) comparing the amount of the detectable gene target to an amount of a reference, wherein if the amount of the detectable gene target is statistically significantly different than the amount of the reference level, the presence and/or level of CTCs is determined. In some embodiments, if the amount of the detectable gene target is not statistically significantly different than the amount of the reference level, the sample is identified as not comprising CTCs.
As used herein, the term “transforming” or “transformation” refers to changing an object or a substance, e.g., biological sample, nucleic acid or protein, into another substance. The transformation can be physical, biological or chemical. Exemplary physical transformation includes, but not limited to, pre-treatment of a biological sample, e.g., from whole blood to blood serum by differential centrifugation. A biological/chemical transformation can involve at least one enzyme and/or a chemical reagent in a reaction. For example, a DNA sample can be digested into fragments by one or more restriction enzyme, or an exogenous molecule can be attached to a fragmented DNA sample with a ligase. In some embodiments, a DNA sample can undergo enzymatic replication, e.g., by polymerase chain reaction (PCR).
Methods to measure gene expression products associated with the marker genes described herein are well known to a skilled artisan. Such methods to measure gene expression products, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, FACS, radioimmunological assay; (RIA); sandwich assay; fluorescent in situ hybridization (FISH); immunohistological staining; immunoelectrophoresis; immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents. Alternatively, a peptide can be detected in a subject by introducing into a subject a labeled anti-peptide antibody and other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in the subject is detected by standard imaging techniques.
For example, antibodies for the polypeptide expression products of the marker genes described herein are commercially available and can be used for the purposes of the invention to measure protein expression levels, e.g. anti-IGFBP5 (Cat. No. 4255; Abcam; Cambridge, Mass.). Alternatively, since the amino acid sequences for the marker genes described herein are known and publically available at NCBI website, one of skill in the art can raise their own antibodies against these proteins of interest for the purpose of the invention. The amino acid sequences of the marker genes described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat.
In some embodiments, immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) techniques can be used. IHC is the application of immunochemistry to tissue sections, whereas ICC is the application of immunochemistry to cells or tissue imprints after they have undergone specific cytological preparations such as, for example, liquid-based preparations. Immunochemistry is a family of techniques based on the use of an antibody, wherein the antibodies are used to specifically target molecules inside or on the surface of cells. The antibody typically contains a marker that will undergo a biochemical reaction, and thereby experience a change color, upon encountering the targeted molecules. In some instances, signal amplification can be integrated into the particular protocol, wherein a secondary antibody, that includes the marker stain or marker signal, follows the application of a primary specific antibody.
In some embodiments, the assay can be a Western blot analysis. Alternatively, proteins can be separated by two-dimensional gel electrophoresis systems. Two-dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. These methods also require a considerable amount of cellular material. The analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel, or can be performed using immune detection. In other embodiments, protein samples are analyzed by mass spectroscopy.
Immunological tests can be used with the methods and assays described herein and include, for example, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassay (RIA), ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence-linked immunoassay), chemiluminescence immunoassays (CLIA), electrochemiluminescence immunoassay (ECLIA, counting immunoassay (CIA), lateral flow tests or immunoassay (LFIA), magnetic immunoassay (MIA), and protein A immunoassays. Methods for performing such assays are known in the art, provided an appropriate antibody reagent is available. In some embodiment, the immunoassay can be a quantitative or a semi-quantitative immunoassay.
An immunoassay is a biochemical test that measures the concentration of a substance in a biological sample, typically a fluid sample such as serum, using the interaction of an antibody or antibodies to its antigen. The assay takes advantage of the highly specific binding of an antibody with its antigen. For the methods and assays described herein, specific binding of the target polypeptides with respective proteins or protein fragments, or an isolated peptide, or a fusion protein described herein occurs in the immunoassay to form a target protein/peptide complex. The complex is then detected by a variety of methods known in the art. An immunoassay also often involves the use of a detection antibody.
Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.
In one embodiment, an ELISA involving at least one antibody with specificity for the particular desired antigen (i.e. a marker gene polypeptide as described herein) can also be performed. A known amount of sample and/or antigen is immobilized on a solid support (usually a polystyrene micro titer plate). Immobilization can be either non-specific (e.g., by adsorption to the surface) or specific (e.g. where another antibody immobilized on the surface is used to capture antigen or a primary antibody). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bio-conjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity.
In another embodiment, a competitive ELISA is used. Purified antibodies that are directed against a target polypeptide or fragment thereof are coated on the solid phase of multi-well plate, i.e., conjugated to a solid surface. A second batch of purified antibodies that are not conjugated on any solid support is also needed. These non-conjugated purified antibodies are labeled for detection purposes, for example, labeled with horseradish peroxidase to produce a detectable signal. A sample (e.g., tumor, blood, serum or urine) from a subject is mixed with a known amount of desired antigen (e.g., a known volume or concentration of a sample comprising a target polypeptide) together with the horseradish peroxidase labeled antibodies and the mixture is then are added to coated wells to form competitive combination. After incubation, if the polypeptide level is high in the sample, a complex of labeled antibody reagent-antigen will form. This complex is free in solution and can be washed away. Washing the wells will remove the complex. Then the wells are incubated with TMB (3, 3′, 5, 5′-tetramethylbenzidene) color development substrate for localization of horseradish peroxidase-conjugated antibodies in the wells. There will be no color change or little color change if the target polypeptide level is high in the sample. If there is little or no target polypeptide present in the sample, a different complex in formed, the complex of solid support bound antibody reagents-target polypeptide. This complex is immobilized on the plate and is not washed away in the wash step. Subsequent incubation with TMB will produce much color change. Such a competitive ELSA test is specific, sensitive, reproducible and easy to operate.
There are other different forms of ELISA, which are well known to those skilled in the art. The standard techniques known in the art for ELISA are described in “Methods in Immunodiagnosis”, 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem. 22:895-904. These references are hereby incorporated by reference in their entirety.
In one embodiment, the levels of a polypeptide in a sample can be detected by a lateral flow immunoassay test (LFIA), also known as the immunochromatographic assay, or strip test. LFIAs are a simple device intended to detect the presence (or absence) of antigen, e.g. a polypeptide, in a fluid sample. There are currently many LFIA tests are used for medical diagnostics either for home testing, point of care testing, or laboratory use. LFIA tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test strip it encounters a colored reagent (generally comprising antibody specific for the test target antigen) bound to microparticles which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with another antibody or antigen. Depending upon the level of target polypeptides present in the sample the colored reagent can be captured and become bound at the test line or zone. LFIAs are essentially immunoassays adapted to operate along a single axis to suit the test strip format or a dipstick format. Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens from fluid samples such as urine, blood, water, and/or homogenized tumor samples etc. Strip tests are also known as dip stick test, the name bearing from the literal action of “dipping” the test strip into a fluid sample to be tested. LFIA strip tests are easy to use, require minimum training and can easily be included as components of point-of-care test (POCT) diagnostics to be use on site in the field. LFIA tests can be operated as either competitive or sandwich assays. Sandwich LFIAs are similar to sandwich ELISA. The sample first encounters colored particles which are labeled with antibodies raised to the target antigen. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the antigen. The test line will show as a colored band in positive samples. In some embodiments, the lateral flow immunoassay can be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof. Competitive LFIAs are similar to competitive ELISA. The sample first encounters colored particles which are labeled with the target antigen or an analogue. The test line contains antibodies to the target/its analogue. Unlabelled antigen in the sample will block the binding sites on the antibodies preventing uptake of the colored particles. The test line will show as a colored band in negative samples. There are a number of variations on lateral flow technology. It is also possible to apply multiple capture zones to create a multiplex test.
The use of “dip sticks” or LFIA test strips and other solid supports have been described in the art in the context of an immunoassay for a number of antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871; 6,565,808, U.S. patent application Ser. No. 10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No. 10/717,082, which are incorporated herein by reference in their entirety, are non-limiting examples of such lateral flow test devices. Examples of patents that describe the use of “dip stick” technology to detect soluble antigens via immunochemical assays include, but are not limited to U.S. Pat. Nos. 4,444,880; 4,305,924; and 4,135,884; which are incorporated by reference herein in their entireties. The apparatuses and methods of these three patents broadly describe a first component fixed to a solid surface on a “dip stick” which is exposed to a solution containing a soluble antigen that binds to the component fixed upon the “dip stick,” prior to detection of the component-antigen complex upon the stick. It is within the skill of one in the art to modify the teachings of this “dip stick” technology for the detection of polypeptides using antibody reagents as described herein.
Other techniques can be used to detect the level of a polypeptide in a sample. One such technique is the dot blot, an adaptation of Western blotting (Towbin et al., Proc. Nat. Acad. Sci. 76:4350 (1979)). In a Western blot, the polypeptide or fragment thereof can be dissociated with detergents and heat, and separated on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose or PVDF membrane. The membrane is incubated with an antibody reagent specific for the target polypeptide or a fragment thereof. The membrane is then washed to remove unbound proteins and proteins with non-specific binding. Detectably labeled enzyme-linked secondary or detection antibodies can then be used to detect and assess the amount of polypeptide in the sample tested. The intensity of the signal from the detectable label corresponds to the amount of enzyme present, and therefore the amount of polypeptide. Levels can be quantified, for example by densitometry.
Flow cytometry is a well-known technique for analyzing and sorting cells (or other small particles) suspended in a fluid stream. This technique allows simultaneous analysis of the physical and/or chemical characteristics of single cells flowing through an optical, electronic, or magnetic detection apparatus. As applied to FACS, the flow cytometer consists of a flow cell which carries the cells in a fluid stream in single file through a light source with excites the fluorescently labeled detection marker(s) (for example, antibody reagents) and measures the fluorescent character of the cell. The fluid stream is then ejected through a nozzle and a charging ring, under pressure, which breaks the fluid into droplets. The flow cell device and fluid stream is calibrated such that there is a relatively large distance between individual cells or bound groups of cells, resulting in a low probability that any droplet contains more than a single cell or bound group of cells. The charging ring charges the droplets based on the fluorescence characteristic of the cell which is contained therein. The charged droplets are then deflected by an electrostatically-charged deflection system which diverts the droplets into various containers based upon their charge (related to the fluorescence intensity of the cell). A FACS system (e.g. the FACSARIA™ flow cytometer (BD Biosciences) and FLOWJO™ Version 7.6.4 (TreeStar)) can detect and record the number of total cells as well as the number of cells which display one or more fluorescent characteristics, e.g. the total number of cells bound by one or more antibody reagents specific for a CTC marker gene.
In certain embodiments, the gene expression products as described herein can be instead determined by determining the level of messenger RNA (mRNA) expression of genes associated with the marker genes described herein. Such molecules can be isolated, derived, or amplified from a biological sample, such as a tumor biopsy. Detection of mRNA expression is known by persons skilled in the art, and comprise, for example but not limited to, PCR procedures, RT-PCR, quantitative PCR or RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization methods, next-generation sequencing etc. Non-limiting examples of next-generation sequencing technologies can include Ion Torrent, Illumina, SOLiD, 454; Massively Parallel Signature Sequencing solid-phase, reversible dye-terminator sequencing; and DNA nanoball sequencing.
In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes or sequences within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a thermostable DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to a strand of the genomic locus to be amplified. In an alternative embodiment, mRNA level of gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art. The nucleic acid sequences of the marker genes described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat. Accordingly, a skilled artisan can design an appropriate primer based on the known sequence for determining the mRNA level of the respective gene.
Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. For example, freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials; heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine; and proteinase K extraction can be used to obtain nucleic acid from blood (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).
In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to each strand of the nucleic acid molecule to be amplified.
In an alternative embodiment, mRNA level of gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art.
In some embodiments, one or more of the reagents (e.g. an antibody reagent and/or nucleic acid probe) described herein can comprise a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product). Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g. antibodies and nucleic acid probes) are well known in the art.
In some embodiments, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means. The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.
In other embodiments, the detection reagent is label with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments, a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3™, Cy5™, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 6-carboxyfhiorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2′,4′,7′,4,7-hexachlorofiuorescein (HEX), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfiuorescein (JOE or J), N,N,N′,N′-tetramethyl-6carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes. In some embodiments, a detectable label can be a radiolabel including, but not limited to 3 H, 125 I, 35 S, 14 C, 32 P, and 33 P. In some embodiments, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In some embodiments, a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In some embodiments, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.
In some embodiments, detection reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available, e. g. from DAKO; Carpinteria, Calif. A reagent can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
In some embodiments of any of the aspects described herein, the level of expression products of more than one gene can be determined simultaneously (e.g. a multiplex assay) or in parallel. In some embodiments, the level of expression products of no more than 200 other genes is determined. In some embodiments, the level of expression products of no more than 100 other genes is determined. In some embodiments, the level of expression products of no more than 20 other genes is determined. In some embodiments, the level of expression products of no more than 10 other genes is determined.
The term “sample” or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a tumor sample from a subject. Exemplary biological samples include, but are not limited to, a biofluid sample; serum; plasma; urine; saliva; a tumor sample; a tumor biopsy and/or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term “test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments, a test sample can comprise cells from subject. In some embodiments, a test sample can be a tumor cell test sample, e.g. the sample can comprise cancerous cells, cells from a tumor, and/or a tumor biopsy. In some embodiments, the test sample can be a blood sample.
The test sample can be obtained by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells (e.g. isolated at a prior timepoint and isolated by the same or another person). In addition, the test sample can be freshly collected or a previously collected sample.
In some embodiments, the test sample can be an untreated test sample. As used herein, the phrase “untreated test sample” refers to a test sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments, the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments, the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof. In some embodiments, the test sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. The skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for determination of the level of an expression product as described herein.
In some embodiments, the methods, assays, and systems described herein can further comprise a step of obtaining a test sample from a subject. In some embodiments, the subject can be a human subject.
In some embodiments, the methods and assays described herein can further comprise a step of isolating CTCs or potential CTCs from a sample prior to measuring the level the expression product of one or more of the marker genes described herein. By way of non-limitng example, CTCs can be isolated from, e.g. a blood sample by hydrodynamic size-based separation and/or immunodepletetion of other cell types present in blood samples. The CTC-iChip, described in the Examples herein combines these two approaches to isolate CTCs.
Subjects with high, or at least detectable, levels of CTCs are most likely to benefit from treatment with therapies that specifically target CTCs. Accordingly, provided herein is a method of determining if a subject is likely to respond to treatment with a CTC marker gene-targeted therapy, the method comprising: measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is likely to respond to the treatment if the level of the expression product is increased relative to a reference level. CTC marker gene-targeted therapies are discussed below herein.
Decreased levels of CTCs after administration of a therapy can be indicative of an improvement in the condition of the subject, e.g. the cancer is reduced in size, growth, and/or metastatic potential. Accordingly, provided herein is a method of monitoring the treatment of a subject, the method comprising administering a cancer therapy to a subject in need thereof; measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is responding if the level of the CTC marker gene expression product is decreased relative to the reference level and determining that the subject is not responding to the treatment if the CTC marker gene expression product is not decreased relative to the reference level. In some embodiments the therapy is a chemotherapy, surgical therapy, and/or radiation therapy. In some embodiments, the therapy is a CTC marker gene-targeted therapy. In some embodiments, the reference level is the level of the gene expression product in the patient prior to the administering step.
The CTC marker genes described herein can be targeted directly and/or used to physically target a chemotherapeutic agent to reduce the levels and/or pathogenic activity of CTCs (e.g. metastatic activity). Accordingly, described herein is a method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of a CTC marker gene-targeted therapy to the subject. In some embodiments, the subject is a subject determined to have an elevated level of CTCs and/or an elevated level of a CTC marker gene present in the blood and/or stroma of the cancer.
In some embodiments, the CTC marker gene-targeted therapy can comprise an inhibitor of a CTC marker gene, e.g. the CTC marker gene-targeted therapy can inhibit the level and/or activity of a CTC marker gene. As used herein, the term “inhibitor” refers to an agent which can decrease the expression and/or activity of the targeted expression product (e.g. mRNA encoding the target or a target polypeptide), e.g. by at least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more. The efficacy of an inhibitor of a CTC marker gene, e.g. its ability to decrease the level and/or activity of the CTC marker gene can be determined, e.g. by measuring the level of an expression product and/or the activity of the CTC marker gene. Methods for measuring the level of a given mRNA and/or polypeptide are known to one of skill in the art, e.g. RTPCR with primers can be used to determine the level of RNA and Western blotting with an antibody can be used to determine the level of a polypeptide. The activity of, e.g. a CTC marker gene can be determined, e.g. by measuring the levels and/or survival of CTCs using methods known in the art and described elsewhere herein. In some embodiments, the inhibitor of a CTC marker gene can be an inhibitory nucleic acid; an aptamer; an antibody reagent; an antibody; or a small molecule.
In some embodiments, the inhibitor of a CTC marker gene can be an antibody reagent. As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgE molecules or antigen-specific antibody fragments thereof (including, but not limited to, a Fab, F(ab′) 2 , Fv, disulphide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulphide-linked scfv, diabody), whether derived from any species that naturally produces an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria.
As described herein, an “antigen” is a molecule that is bound by a binding site on an antibody agent. Typically, antigens are bound by antibody ligands and are capable of raising an antibody response in vivo. An antigen can be a polypeptide, protein, nucleic acid or other molecule or portion thereof. The term “antigenic determinant” refers to an epitope on the antigen recognized by an antigen-binding molecule, and more particularly, by the antigen-binding site of said molecule.
As used herein, the term “antibody reagent” refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen. An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody reagent” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.
The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (“FR”). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated by reference herein in their entireties). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
The terms “antigen-binding fragment” or “antigen-binding domain”, which are used interchangeably herein are used to refer to one or more fragments of a full length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term “antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546; which is incorporated by reference herein in its entirety), which consists of a VH or VL domain; and (vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding functionality. As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity.
Additionally, and as described herein, a recombinant humanized antibody can be further optimized to decrease potential immunogenicity, while maintaining functional activity, for therapy in humans. In this regard, functional activity means a polypeptide capable of displaying one or more known functional activities associated with a recombinant antibody or antibody reagent thereof as described herein. Such functional activities include, e.g. the ability to bind to a given CTC marker gene.
In some embodiments, the inhibitor of a CTC marker gene can be an inhibitory nucleic acid reagent. In some embodiments, the inhibitory nucleic acid is an inhibitory RNA (iRNA). Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein can include an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part the targeted mRNA transcript. The use of these iRNAs enables the targeted degradation of mRNA transcripts, resulting in decreased expression and/or activity of the target.
As used herein, the term “iRNA” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNA as described herein effects inhibition of the expression and/or activity of the target mRNA. In certain embodiments, contacting a cell with the inhibitor (e.g. an iRNA) results in a decrease in the target mRNA level in a cell by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the cell without the presence of the iRNA.
In some embodiments, the iRNA can be a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of the target. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive. In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.
In yet another embodiment, the RNA of an iRNA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.
Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,835, 826; 6,858,715; 6,867,289; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; 7,834,171; 7,919,612; 7,960,360; 7,989,603; 8,309,707; 6,524,681; and U.S. Pat. RE39464, each of which is herein incorporated by reference
Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts. Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.
In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH 2 —NH—CH 2 —, —CH 2 —N(CH 3 )—O—CH 2 — [known as a methylene (methylimino) or MMI backbone], —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 — and —N(CH 3 )—CH 2 —CH 2 — [wherein the native phosphodiester backbone is represented as —O—P—O—CH 2 —] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH 2 ) n O] m CH 3 , O(CH 2 ). n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH 2 —O—CH 2 —N(CH 2 ) 2 , also described in examples herein below.
Other modifications include 2′-methoxy (2′-OCH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,700,920; 8,084,600; 8,124,745; 8,377,644 each of which is herein incorporated by reference.
An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is herein incorporated by reference in its entirety.
Another modification of the RNA of an iRNA featured in the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
In some embodiments the CTC marker gene-targeted therapy can comprise an agent that binds to the CTC marker gene expression product and an agent that is chemotherapeutic. In some embodiments, the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent and a chemotherapeutic agent. A CTC marker gene-binding antibody reagent can be an antibody reagent that binds, e.g. a CTC marker gene polypeptide. The binding antibody reagent can be an inhibitor or can exhibit no inhibitory effect on its own. By binding to the CTC marker gene, and thereby a CTC, it concentrates and localizes the chemotherapeutic agent at CTC cells in the circulation and/or stroma of the tumor—increasing efficacy and reducing side effects.
In some embodiments, the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent that binds a marker gene selected from Table 14. In some embodiments, the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent that binds a marker gene selected from the group consisting of: IL6ST, SULF2, and SV2A.
As used herein the term “chemotherapeutic agent” refers to any chemical or biological agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms and cancer as well as diseases characterized by hyperplastic growth. These agents can function to inhibit a cellular activity upon which the cancer cell depends for continued proliferation. In some aspect of all the embodiments, a chemotherapeutic agent is a cell cycle inhibitor or a cell division inhibitor. Categories of chemotherapeutic agents that are useful in the methods of the invention include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most of these agents are directly or indirectly toxic to cancer cells. In one embodiment, a chemotherapeutic agent is a radioactive molecule. One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed. 2000 Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). In some embodiments, the chemotherapeutic agent can be a cytotoxic chemotherapeutic. The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. At211, I1131, I1125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
Non-limiting examples of chemotherapeutic agents can include gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE.™vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb™); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
In some embodiments, the binding antibody reagent and the chemotherapeutic agent can be directly conjugated and/or bound to each other, e.g. an antibody-drug conjugate. In some embodiments, binding can be non-covalent, e.g., by hydrogen, electrostatic, or van der waals interactions, however, binding may also be covalent. By “conjugated” is meant the covalent linkage of at least two molecules. In some embodiments, the composition can be an antibody-drug conjugate.
In some embodiments, the binding antibody reagent can be bound to and/or conjugated to multiple chemotherapeutic molecules. In some embodiments, the ratio of a given chemotherapeutic molecule to the binding antibody reagent molecule can be from about 1:1 to about 1,000:1, e.g. a single antibody binding reagent molecule can be linked to, conjugated to, etc. from about 1 to about 1,000 individual chemotherapeutic molecules.
In some embodiments, the binding antibody reagent and the chemotherapeutic agent can be present in a scaffold material. Scaffold materials suitable for use in therapeutic compositions are known in the art and can include, but are not limited to, a nanoparticle; a matrix; a hydrogel; and a biomaterial, biocompatible, and/or biodegradable scaffold material. As used herein, the term “nanoparticle” refers to particles that are on the order of about 10 −9 or one billionth of a meter. The term “nanoparticle” includes nanospheres; nanorods; nanoshells; and nanoprisms; and these nanoparticles may be part of a nanonetwork.
The term “nanoparticles” also encompasses liposomes and lipid particles having the size of a nanoparticle. As used herein, the term “matrix” refers to a 3-dimensional structure comprising the components of a composition described herein (e.g. a binding reagent, kinase inhibitor, and/or EGFR inhibitor). Non-limiting examples of matrix structures include foams; hydrogels; electrospun fibers; gels; fiber mats; sponges; 3-dimensional scaffolds; non-woven mats; woven materials; knit materials; fiber bundles; and fibers and other material formats (See, e.g. Rockwood et al. Nature Protocols 2011 6:1612-1631 and US Patent Publications 2011/0167602; 2011/0009960; 2012/0296352; and U.S. Pat. No. 8,172,901; each of which is incorporated by reference herein in its entirety). The structure of the matrix can be selected by one of skill in the art depending upon the intended application of the composition, e.g. electrospun matrices can have greater surface area than foams.
In some embodiments, the scaffold is a hydrogel. As used herein, the term “hydrogel” refers to a three-dimensional polymeric structure that is insoluble in water but which is capable of absorbing and retaining large quantities of water to form a stable, often soft and pliable, structure. In some embodiments, water can penetrate in between the polymer chains of the polymer network, subsequently causing swelling and the formation of a hydrogel. In general, hydrogels are superabsorbent. Hydrogels have many desirable properties for biomedical applications. For example, they can be made nontoxic and compatible with tissue, and they are highly permeable to water, ions, and small molecules. Hydrogels are super-absorbent (they can contain over 99% water) and can be comprised of natural (e.g., silk) or synthetic polymers, e.g., PEG.
As used herein, “biomaterial” refers to a material that is biocompatible and biodegradable. As used herein, the term “biocompatible” refers to substances that are not toxic to cells. In some embodiments, a substance is considered to be “biocompatible” if its addition to cells in vitro results in less than or equal to approximately 20% cell death. In some embodiments, a substance is considered to be “biocompatible” if its addition to cells in vivo does not induce inflammation and/or other adverse effects in vivo. As used herein, the term “biodegradable” refers to substances that are degraded under physiological conditions. In some embodiments, a biodegradable substance is a substance that is broken down by cellular machinery. In some embodiments, a biodegradable substance is a substance that is broken down by chemical processes.
In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having cancer with a CTC marker-gene targeted therapy. In some embodiments, the cancer can be pancreatic cancer. Subjects having cancer can be identified by a physician using current methods of diagnosing cancer. Symptoms and/or complications of cancer, e.g. pancreatic cancer, which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, pain in the upper abdomen, heartburn, nausea, vomiting, diarrhea, cachexia, jaundice, pulmonary embolism, Trousseau syndrome, and diabetes mellitus. Tests that may aid in a diagnosis of, e.g. pancreatic cancer include, but are not limited to, liver function tests, CA19-9 tests, CT and endoscopic ultrasound. A family history of pancreatic cancer or exposure to risk factors for pancreatic cancer (e.g. smoking or drinking) can also aid in determining if a subject is likely to have cancer or in making a diagnosis of cancer.
The compositions and methods described herein can be administered to a subject having or diagnosed as having cancer, e.g. pancreatic cancer. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. a CTC marker-gene targeted therapy to a subject in order to alleviate a symptom of a cancer. As used herein, “alleviating a symptom of a cancer” is ameliorating any condition or symptom associated with the cancer. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.
The term “effective amount” as used herein refers to the amount of a CTC marker-gene targeted therapy needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term “therapeutically effective amount” therefore refers to an amount of CTC marker-gene targeted therapy that is sufficient to provide a particular anti-cancer effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of a CTC marker-gene targeted therapy, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for CTC levels, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
In some embodiments, the technology described herein relates to a pharmaceutical composition comprising a CTC marker-gene targeted therapy as described herein, and optionally a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C 2 -C 12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g. a CTC marker-gene targeted therapy as described herein.
In some embodiments, the pharmaceutical composition comprising a CTC marker-gene targeted therapy as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping.
Suitable vehicles that can be used to provide parenteral dosage forms of a CTC marker-gene targeted therapy as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a CTC marker-gene targeted therapy as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.
Pharmaceutical compositions comprising a CTC marker-gene targeted therapy can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia Pa. (2005).
Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the CTC marker-gene targeted therapy can be administered in a sustained release formulation.
Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).
Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.
The methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. Non-limiting examples of a second agent and/or treatment can include radiation therapy, surgery, and chemotherapeutic agents as described above herein.
In certain embodiments, an effective dose of a composition comprising a CTC marker gene-targeted therapy as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising a CTC marker gene-targeted therapy can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a composition comprising a CTC marker gene-targeted therapy, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.
In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. CTC levels by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more.
The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the CTC marker gene-targeted therapy. The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months. Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition comprising a CTC marker gene-targeted therapy can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
The dosage ranges for the administration of a CTC marker gene-targeted therapy, according to the methods described herein depend upon, for example, the form of the CTC marker gene-targeted therapy, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for CTC levels. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.
The efficacy of a CTC marker gene-targeted therapy in, e.g. the treatment of a condition described herein, or to induce a response as described herein (e.g. reduction of CTC levels) can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. tumor size and/or growth. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. CTC levels). It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of cancer, e.g. pancreatic cancer. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. a change in CTC levels.
For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.
For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.
The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.
As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.
Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cancer. A subject can be male or female.
A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for cancer or the one or more complications related to cancer. Alternatively, a subject can also be one who has not been previously diagnosed as having cancer or one or more complications related to cancer. For example, a subject can be one who exhibits one or more risk factors for cancer or one or more complications related to cancer or a subject who does not exhibit risk factors.
A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
As used herein, the term “cancer” or “tumor” refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems. A subject who has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
The term “agent” refers generally to any entity which is normally not present or not present at the levels being administered to a cell, tissue or subject. An agent can be selected from a group including but not limited to: polynucleotides; polypeptides; small molecules; and antibodies or antigen-binding fragments thereof. A polynucleotide can be RNA or DNA, and can be single or double stranded, and can be selected from a group including, for example, nucleic acids and nucleic acid analogues that encode a polypeptide. A polypeptide can be, but is not limited to, a naturally-occurring polypeptide, a mutated polypeptide or a fragment thereof that retains the function of interest. Further examples of agents include, but are not limited to a nucleic acid aptamer, peptide-nucleic acid (PNA), locked nucleic acid (LNA), small organic or inorganic molecules; saccharide; oligosaccharides; polysaccharides; biological macromolecules, peptidomimetics; nucleic acid analogs and derivatives; extracts made from biological materials such as bacteria, plants, fungi, or mammalian cells or tissues and naturally occurring or synthetic compositions. An agent can be applied to the media, where it contacts the cell and induces its effects. Alternatively, an agent can be intracellular as a result of introduction of a nucleic acid sequence encoding the agent into the cell and its transcription resulting in the production of the nucleic acid and/or protein environmental stimuli within the cell. In some embodiments, the agent is any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities. In certain embodiments the agent is a small molecule having a chemical moiety selected, for example, from unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds. As used herein, the term “small molecule” can refer to compounds that are “natural product-like,” however, the term “small molecule” is not limited to “natural product-like” compounds. Rather, a small molecule is typically characterized in that it contains several carbon-carbon bonds, and has a molecular weight more than about 50, but less than about 5000 Daltons (5 kD). Preferably the small molecule has a molecular weight of less than 3 kD, still more preferably less than 2 kD, and most preferably less than 1 kD. In some cases it is preferred that a small molecule have a molecular mass equal to or less than 700 Daltons.
Aptamers are short synthetic single-stranded oligonucleotides that specifically bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells and tissues. These small nucleic acid molecules can form secondary and tertiary structures capable of specifically binding proteins or other cellular targets, and are essentially a chemical equivalent of antibodies. Aptamers are highly specific, relatively small in size, and non-immunogenic. Aptamers are generally selected from a biopanning method known as SELEX (Systematic Evolution of Ligands by Exponential enrichment) (Ellington et al. Nature. 1990; 346(6287):818-822; Tuerk et al., Science. 1990; 249(4968):505-510; Ni et al., Curr Med Chem. 2011; 18(27):4206-14; which are incorporated by reference herein in their entireties). Methods of generating an apatmer for any given target are well known in the art. Preclinical studies using, e.g. aptamer-siRNA chimeras and aptamer targeted nanoparticle therapeutics have been very successful in mouse models of cancer and HIV (Ni et al., Curr Med Chem. 2011; 18(27):4206-14).
As used herein, the terms “protein” and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.
As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. cancer. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a cancer. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
As used herein, the term “administering,” refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.
As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.
The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.
The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”
Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.
Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.
Other terms are defined herein within the description of the various aspects of the invention.
All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.
Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
1. A method of detecting circulating tumor cells (CTCs) in a sample, the method comprising:
measuring the level of a PC-CTC marker gene expression product in the sample; and determining that PC-CTCs are present if the detected level of the marker gene expression product is greater than a reference level.
2. The method of paragraph 1, wherein the CTCs are pancreatic cancer CTCs. 3. The method of any of paragraphs 1-2, wherein the method further comprises a first step of isolating the CTCs from the sample. 4. The method of any of paragraphs 1-3, wherein the expression product is a nucleic acid. 5. The method of paragraph 4, wherein the level of the expression product is determined using a method selected from the group consisting of:
RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization.
6. The method of any of paragraphs 1-3, wherein the expression product is a polypeptide. 7. The method of paragraph 6, wherein the level of the expression product is determined using a method selected from the group consisting of:
Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay.
8. The method of any of paragraphs 1-7, wherein the CTC marker gene is selected from Table 7; Table 8; or Table 14. 9. The method of any of paragraphs 1-8, wherein the CTC marker gene is selected from the group consisting of:
ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4.
10. The method of any of paragraphs 1-8, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2.
11. The method of any of paragraphs 1-9, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
12. The method of any of paragraphs 1-9, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A2; IGFBP5; KLF4; and DCN.
13. The method of any of paragraphs 1-9, wherein the CTC marker gene is selected from the group consisting of:
TPT1; HMGB1; SPON 2; SPARC; and ARSA.
14. The method of any of paragraphs 1-9, wherein the CTC marker gene is selected from the group consisting of:
IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A.
15. A method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of a CTC marker gene-targeted therapy to the subject. 16. The method of paragraph 15, wherein the cancer is pancreatic cancer. 17. The method of any of paragraphs 15-16, wherein the CTC marker gene-targeted therapy comprises an inhibitor of a CTC marker gene. 18. The method of paragraph 17, wherein the inhibitor is an antibody reagent. 19. The method of paragraph 17, wherein the inhibitor is an inhibitory nucleic acid reagent. 20. The method of any of paragraphs 15-19, wherein the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent and a chemotherapeutic agent. 21. The method of any of paragraphs 15-20, wherein the subject is a subject determined to have an elevated level of CTCs and/or an elevated level of a CTC marker gene present in the blood and/or stroma of the cancer. 22. The method of any of paragraphs 15-21, wherein the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent that binds a marker gene selected from the group consisting of:
IL6ST, SULF2, and SV2A.
23. A method of determining if a subject is likely to respond to treatment with a CTC marker gene-targeted therapy, the method comprising:
measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is likely to respond to the treatment if the level of the expression product is increased relative to a reference level.
24. The method of paragraph 23, wherein the method further comprises a first step of isolating the CTCs from the sample. 25. The method of any of paragraphs 23-24, wherein the cancer is pancreatic cancer. 26. The method of any of paragraphs 23-25, wherein the expression product is a nucleic acid. 27. The method of paragraph 26, wherein the level of the expression product is determined using a method selected from the group consisting of:
RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization.
28. The method of any of paragraphs 23-26, wherein the expression product is a polypeptide. 29. The method of paragraph 28, wherein the level of the expression product is determined using a method selected from the group consisting of:
Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay.
30. The method of any of paragraphs 23-29, wherein the PC-CTC marker gene is selected from Table 7; Table 8; or Table 14. 31. The method of any of paragraphs 23-30, wherein the CTC marker gene is selected from the group consisting of:
ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4.
32. The method of any of paragraphs 23-31, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2.
33. The method of any of paragraphs 23-31, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
34. The method of any of paragraphs 23-31, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A2; IGFBP5; KLF4; and DCN.
35. The method of any of paragraphs 23-31, wherein the CTC marker gene is selected from the group consisting of:
TPT1; HMGB1; SPON 2; SPARC; and ARSA.
36. The method of any of paragraphs 23-31, wherein the CTC marker gene is selected from the group consisting of:
IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A.
37. A method of monitoring the treatment of a subject, the method comprising:
administering a cancer therapy to a subject in need thereof; measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is responding if the level of the CTC marker gene expression product is decreased relative to the reference level and determining that the subject is not responding to the treatment if the CTC marker gene expression product is not decreased relative to the reference level.
38. The method of paragraph 37, wherein the cancer is pancreatic cancer. 39. The method of any of paragraphs 37-38, wherein the reference level is the level of the gene expression product in the patient prior to the administering step. 40. The method of any of paragraphs 37-39, wherein the method further comprises a first step of isolating the CTCs from the sample. 41. The method of any of paragraphs 37-40, wherein the expression product is a nucleic acid. 42. The method of paragraph 41, wherein the level of the expression product is determined using a method selected from the group consisting of:
RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization.
43. The method of any of paragraphs 37-40, wherein the expression product is a polypeptide. 44. The method of paragraph 43, wherein the level of the expression product is determined using a method selected from the group consisting of:
Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay.
45. The method of any of paragraphs 37-44, wherein the PC-CTC marker gene is selected from Table 7; Table 8; or Table 14. 46. The method of any of paragraphs 37-45, wherein the CTC marker gene is selected from the group consisting of:
ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4.
47. The method of any of paragraphs 37-46, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2.
48. The method of any of paragraphs 37-46, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
49. The method of any of paragraphs 37-46, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A2; IGFBP5; KLF4; and DCN.
50. The method of any of paragraphs 37-46, wherein the CTC marker gene is selected from the group consisting of:
TPT1; HMGB1; SPON 2; SPARC; and ARSA.
51. The method of any of paragraphs 37-46, wherein the CTC marker gene is selected from the group consisting of:
IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A.
EXAMPLES
Example 1
Single Cell RNA-Sequencing of Mouse Pancreatic Circulating Tumor Cells Reveals their Expression of ECM Proteins
Circulating Tumor Cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. Using a pancreatic cancer mouse model, a microfluidic device was applied to isolate CTCs independently of tumor epitopes, subjecting these to single cell RNA-sequencing. CTCs clustered into multiple subsets, distinct from primary tumors and cancer cell lines. While proliferative signatures were generally low, CTCs were enriched for MAPK, as well as WNT, TGF-β, Neurotrophin, Toll-like receptor, and B-cell receptor signaling pathways. CTCs were highly enriched for expression of the stem-cell associated gene Aldh1a2. Their virtually universal expression of Igfbp5 and Klf4 was correlated with a subset of primary tumor cells localized to the epithelial/stromal boundary, consistent with the presence of both epithelial and mesenchymal markers in CTCs. The very high CTC expression of stromal-derived extracellular matrix proteins, including Dcn and Sparc, indicates microenvironmental contributions to metastasis and identifies unexpected therapeutic targets.
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the US, with a 6% overall survival at 5 years (Society, 2013). The high mortality of this cancer stems from the rapid dissemination of tumor cells leading to widespread metastasis. While local tissue and lymphatic invasion are evident even in early PDAC, the presence of circulating tumor cells (CTCs) in the bloodstream ultimately leads to spread of cancer to distant organs. CTCs are rare, estimated at one to ten tumor cells among ten billion normal blood cells in a milliliter of blood. As such, their isolation and molecular analysis has posed a significant technological challenge (Pantel et al., 2008; Yu et al., 2011). Given their role in blood-borne metastasis, CTC populations are likely to be enriched for metastatic precursors, and their analysis may identify potential therapeutic targets, as well as providing opportunities for early detection of pancreatic cancer.
Genetically engineered mouse pancreatic cancer models have provided important insight into the progression of this disease. Specifically, the genetically engineered LSL-Kras G12D , Trp53 flox/flox or + , Pdx1-Cre (KPC) mouse model recapitulates the histological progression from preneoplastic pancreatic intraepithelial neoplasia (PanlN) lesions to invasive carcinoma (Bardeesy et al., 2006). Recent studies have suggested that epithelial-to-mesenchymal transition (EMT) occurs early in this model potentially enhancing tumor invasiveness (Rhim et al., 2012). In an initial molecular characterization of mouse pancreatic CTCs, RNA sequencing of CTC-enriched populations was performed, thereby identifying activation of non-canonical WNT signaling as a recurrent event, potentially contributing to the anoikis resistance of circulating epithelial cells (Yu et al., 2012). In that study, analysis of purified CTC populations was accomplished using single molecule RNA sequencing, combined with digital subtraction of matched leukocyte RNA reads, so as to derive a CTC-enriched expression signature. However, transcriptomic analysis of such partially purified cell populations is limited by depth of coverage to the most highly differentially expressed genes, and such studies of bulk CTC populations cannot resolve the degree of heterogeneity across these poorly understood cell populations
To achieve a deep RNA sequencing profile of CTCs at the single cell level, a novel inertial focusing-enhanced device, the CTC-iChip, which allows high efficiency negative depletion of normal blood cells, leaving unattached CTCs in solution where they can be selected and analyzed as single cells (Ozkumur et al., 2013) was used. By avoiding tumor epitope-specific capture, such as targeting the epithelial marker EpCAM, the CTC-iChip is unbiased in isolating cancer cells with both epithelial and mesenchymal characteristics. Further, the high quality of RNA purified from viable, untagged CTCs is particularly well suited for detailed transcriptomic analysis. Finally, the use of a mouse model of pancreatic cancer allows for simultaneous analysis of primary tumor and CTCs, while the shared driver mutations across different animals facilitates the identification of CTC-specific heterogeneity. Described herein is a comprehensive transcriptome analysis of CTCs at the single cell level, pointing to distinct cell subsets within CTC populations, signaling pathways that are enriched in CTCs, and identifying unique CTC markers and therapeutic targets.
Results
Isolation of Mouse Pancreatic CTCs.
The CTC-iChip, an integrated microfluidic cell separation platform applied directly to whole blood specimens for isolation of CTCs (Ozkumur et al., 2013) was used in the experiments described herein. It combines initial hydrodynamic size-based separation of all nucleated cells (leukocytes (WBC) and CTCs) away from red blood cells, platelets and plasma, with subsequent inertial focusing of the nucleated cells within a single streamline to achieve high efficiency in-line magnetic sorting. While tumor epitopes are highly variable, WBC cell surface markers are well established; applying magnetic-conjugated anti-WBC antibodies to this very high throughput microfluidic cell separation device can thus exclude the vast majority of WBCs to reveal a small number of untagged CTCs ( FIG. 1A ). The CTC-iChip was adapted for depletion of murine hematopoietic cells and applied to the KPC pancreatic cancer mouse model. This PDAC model generates significant numbers of CTCs (Rhim et al., 2012; Yu et al., 2012). Whole blood labeling using 100 anti-CD45 beads per WBC achieved >10 3 depletion in normal mice, mice bearing orthotopic tumors, and the genetically engineered KPC mice ( FIGS. 1B and 4A-4C ).
CTC recovery was measured as a mean of 95% (+/−3% std), using GFP-tagged NB508 mouse pancreatic cancer cells spiked into whole mouse blood and processed through the CTC-iChip ( FIGS. 4A-4C ). NB508 cells were previously generated from a pancreatic tumor arising in the same Kras/Trp53-driven KPC mouse model (Bardeesy et al., 2006). In comparison, only 35% recovery of the same cells was achieved using an alternative microfluidic platform based on anti-EpCAM capture of mouse CTCs (Yu et al., 2012). Applying the CTC-iChip to orthotopic tumors derived from pancreatic inoculation of GFP-tagged NB508 cells generated >1000 CTCs/mL in all three mice tested ( FIGS. 4A-4C ). Finally, testing the CTC-iChip with the genetically engineered KPC model, followed by dual immunofluorescence staining of isolated cells for the epithelial marker pan-cytokeratin (CK) versus the leukocyte marker CD45, revealed a median 118 CTCs/mL (mean 429 CTCs/mL; range 0-1694) ( FIG. 1C ). No CK positive cells were isolated from 7 healthy control mice. The vast majority of CD45 positive cells that failed to be deflected in the microfluidic device retained some immunomagnetic beads on their surface. Thus, CTCs were readily distinguished from WBCs in the CTC-iChip product, enabling single cell manipulation without requiring staining for epithelial-specific cell surface epitopes, such as EpCAM.
Single CTC RNA-Sequencing.
Five tumor-bearing KPC mice generated a total of 168 single CTCs that were subjected to a modified initial cDNA amplification and library protocol (Tang et al., 2010), and screened for RNA quality (Gapdh, Actb), presence of pancreatic markers (Krt8, Krt18, Krt19, Pdx1), and absence of WBC markers (Cd45/Ptprc) ( FIGS. 5A-5C ). Of these, 75 (45%) were of sufficient quality to proceed to further amplification and library construction for next generation sequencing. It is noteworthy that a majority of candidate CTCs (55%) appeared morphologically intact but had degraded RNA. These cells likely represent tumor cells that have lost viability in the bloodstream. Given the rapid processing of blood samples from mouse models, the minimal shear condition in the microfluidic device, and the preserved RNA quality of control cells processed identically, it is unlikely that cells underwent such damage during in vitro purification. For comparison with pancreatic CTCs, single cell RNA-sequencing was also performed on 12 WBCs from a control mouse, 12 mouse embryonic fibroblasts (MEFs), and 16 single cells from the mouse NB508 pancreatic cancer cell line. Over 90% of single cells from NB508 and MEF cultures met criteria for sequencing quality, highlighting the high frequency of CTCs with compromised RNA templates under the same conditions. To compare CTC profiles to that of matched parental tumors harvested at the time of CTC isolation, bulk RNA from each primary tumor was diluted to 1 or 10 cell equivalents (10 or 100 pg RNA) and subjected to the same amplification and RNA-sequencing protocol (n=34; min 8 replicates from 4 matched tumors).
Single cell RNA sequencing performance was comparable for all samples analyzed, with a mean 4.4-8.5 million reads, of which a mean 46-61% were uniquely aligned to the genome ( FIGS. 5A-5C ). Genome aligned reads were annotated and counted using UCSC Known Gene transcriptome reference and normalized in reads per million (RPM). Normalized reads were then analyzed by unsupervised hierarchical clustering (data not shown). Single cell transcriptomes from MEFs, the NB508 pancreatic cancer cell line and normal WBCs were tightly clustered, supporting the analytic reliability of the RNA sequencing strategy. Five distinct clusters of candidate CTCs were identified, all of which were distinct from matched primary tumor sequences, as well as from cancer-derived cell lines. Principal component analysis demonstrates the clustering and inter-relationships of these different groups ( FIG. 2 ).
The uniform genetic drivers of PDAC in the KPC mouse model made it possible to quantify measures of cellular heterogeneity in CTCs derived from individual mice and across different mice. Single cell heterogeneity within each CTC cluster was assessed by calculating the intra-cluster correlation coefficients, where lower correlation coefficients reflect higher heterogeneity ( FIGS. 5A-5C ). As expected, CTC clusters showed considerably more heterogeneity (mean 0.42, 95% CI 0.36-0.47) than single cells derived from the NB508 cancer cell line (mean 0.86, 95% CI 0.80-0.91, p-value 1.2×10 −15 ). To assess heterogeneity of cells within a primary PDAC, a conditional Tomato/EGFP (mT/mG) expression marker (Muzumdar et al., 2007) was crossed with the KPC mouse to generate a lineage-tagged mouse tumor (KPC-mT/mG), which could be used to isolate individual EGFP positive primary tumor cells away from contaminating stromal cells. A primary tumor (TuGMP3) was disaggregated into single cell suspension and 20 EGFP positive cells were subjected to RNA sequencing. The single primary tumor cells clustered well within the previously analyzed bulk tumor material (data not shown), with a heterogeneity score (mean 0.38, 95% CI 0.28-0.47) similar to that of CTCs (p-value 0.49).
In summary, described herein is the single cell RNA-sequencing of mouse pancreatic CTCs isolated without positive selection bias, along with parental tumors, an established genotype-matched cancer cell line, MEFs and WBCs. CTCs clustered separately from the primary tumor (both bulk tumor and isolated single cells) and from the tumor-derived cell line, with comparable degrees of intercellular heterogeneity between CTCs and primary tumor cells.
Defining Subsets of Pancreatic CTCs.
To identify and classify candidate CTCs, gene sets for known epithelial, hematopoietic, and endothelial markers were applied across all clustered samples. As expected, epithelial markers (Krt7, Krt8, Krt18, Krt19, Epcam, Egfr, Cdh1) were highly expressed in primary pancreatic tumors and in the cancer cell line NB508, and nearly absent in the non-epithelial MEFs and in normal WBCs (data not shown). In contrast, hematopoietic markers (Ptprc/Cd45, Csf3r/Cd114, Cd14, Fcgr3/Cd16, Itga2b/Cd41, Itgb3/Cd61) were present in normal WBCs, and absent in NB508 and MEFs. Some expression of hematopoietic markers was detectable in the bulk primary tumor samples, consistent with varying degrees of leukocytic infiltrates. No specific cluster of endothelial cells was identified, based on expression of characteristic markers (Cdh5/Cd144, Vwf Thbd/Cd141, Pecaml/Cd31, Mcam/Cd146, Sele/E-selectin, Cd34) and absence of epithelial and hematopoietic markers.
Interrogation of single cells isolated by CD45-depletion from tumor-bearing mice, using the epithelial, hematopoietic and endothelial markers, revealed five major candidate CTC groupings (Clusters 1, 3, 4, 5 and 9; data not shown). Clusters 3, 4, and 5 were all part of a larger grouping, showing strong expression of epithelial markers, consistent with “classical” CTCs (denoted CTC-c). A subset of these cells expressed Cd34, an endothelial progenitor marker that is also found in mesenchymal cells including MEFs (data not shown) and stromal cells (Krause et al., 1994), but other characteristic endothelial lineage markers were absent. Clusters 1 and 9 were more complex, with the former noteworthy for enrichment of platelet markers CD41 (Itga2b) and CD61 (Itgb3) (hence denoted CTC-plt), and the latter having a prominent cellular proliferation signature (CTC-pro).
To better define the characteristics of each candidate CTC cluster, a non-parametric differential gene expression analysis including a rank product (RP) methodology adapted to variations in absolute transcript levels and differences in transcriptome representation from cell to cell was used (Breitling et al., 2004). Setting very stringent parameters (FDR≦0.01), the control comparison of primary tumors versus WBCs identified 927 genes relatively overexpressed in tumors and 293 genes high in WBCs, including the expected differential expression of epithelial tumor markers keratin 7, 8, 18, and 19, versus the leukocyte specific CD45 (data not shown). Comparing the “classical” CTC-c cluster to WBCs also showed enrichment for cytokeratin 18 and 19 in CTCs versus CD45 in WBCs, validating the RP methodology to identify relevant differentially expressed genes between single cell populations.
The most abundant CTC cluster, CTC-c, comprised 41 of 75 cells (55%) meeting established criteria for epithelial tumor cells (versus CTC-plt: 32%; CTC-pro: 13%). Of note, the only mouse with multiple gross metastases (MP7) had large numbers of CTCs within this class. Compared with matched primary tumors CTC-c cells had 878 transcripts increased in expression and 774 genes with reduced expression (Table 2). Gene Ontology (GO) analysis of CTC-c enriched genes (Table 3) indicated enrichment for signatures associated with cellular interactions with environmental signals (GO:0045785—positive regulation of cell adhesion; GO:0048584—positive regulation of response to stimulus), cell shape and structure (GO: 0030036—actin cytoskeleton organization; GO:0060429—epithelium development), and transcriptional states (GO:0045449—regulation of transcription; GO:0051276—chromosome organization). To evaluate the contribution of signaling pathways activated by external stimuli in CTC-c cells, the enriched genes were annotated using the KEGG database (Table 1). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis similarly showed enrichment for focal adhesion (odds ratio [OR]2.7, q-value 6.7 3 10.4) and regulation of actin cytoskeleton (OR 2.4, q-value 0.005). Notably, of the KEGG signaling pathways annotated, the mitogen-activated protein kinase (MAPK) pathway was most highly enriched Most highly represented was the MAPK pathway (OR 2.2, q-value 0.006); MAPK signaling is already activated in the Kras G12D driven primary tumor. However, while MSigDB Kras dependency signatures were enriched in primary tumors compared with CTCs, the latter had increased expression of Braf, Mras and Rras2, pointing to alternative paths to further activate MAPK in CTCs. This finding is consistent with another study that identified the MAPK pathway as being the most highly enriched in pancreatic CTCs using microarray based methodologies (Sergeant et al., 2012).
CTC enriched genes also had representation of well established signaling pathways involved with metastasis, including TGF-β (Ikushima and Miyazono, 2010; Siegel and Massague, 2003), WNT (Anastas and Moon, 2013; Clevers and Nusse, 2012; Katoh and Katoh, 2007), and VEGF (Carmeliet and Jain, 2011; Folkman, 1995). In this cohort of pancreatic cancer CTCs, Wnt4 and Tgfb2 were most highly enriched in CTCs relative to primary tumor, implicating autocrine signaling involving these major pathways. In addition to these well defined contributors to metastasis, CTC expression analyses also revealed activation of unexpected signaling pathways, including the neurotrophin, toll-like receptor, and B-cell receptor pathways. Neurotrophin pathway activation has been reported in pancreatic cancer, particularly in association with increased perineural invasion (Miknyoczki et al., 1996; Miknyoczki et al., 1999; Ohta et al., 1997; Wang et al., 2009; Zhang et al., 2005). Toll-like receptor and B-cell receptor pathways had less representation among CTC reads, but they suggest aberrant activation of immunomodulatory signaling components. Ultimately, the establishment of CTC-derived cultures will be required to test the functional significance of these activated signaling pathways.
While single cells within the CTC-c cluster fulfilled characteristic criteria for tumor cells, defining the identity of the non-classical CTC clusters, CTC-plt and CTC-pro, required additional analyses. Compared with CTC-c, single cells within the CTC-plt cluster had a high enrichment for wound healing and hemostasis signatures, as well as MSigDB platelet and megakaryocyte expression profiles (Table 4). This indicates that these cells are either circulating megakaryocytes/giant platelets or CTCs covered with adherent platelets. Tumor cell specific lineage tagging supports the identification of CTC-plt cells being of tumor origin. Eighteen EGFP lineage-tagged single CTCs from two KPC-mT/mG mice were subjected to single cell RNA sequencing: a total of 9 CTCs from the two mice (7/7 CTCs from mouse GMP1 and 2/11 from mouse GMP2) were included within CTC-plt, using unsupervised hierarchical clustering (data not shown). Thus, the CTC-plt cluster includes CTCs that exhibit strong platelet markers, most likely derived from transcripts encoded by adherent platelets. Interestingly, CTC-plt cells maintained their distinct segregation from CTC-c even after digital removal of all annotated platelet transcripts (data not shown). It is therefore possible that the adherence of abundant platelets may modulate the intrinsic CTC expression profile, as recently suggested by in vitro modeling experiments (Labelle et al., 2011).
The CTC-pro cluster was most similar to both the NB508 pancreatic cancer cell line and MEFs, and it was enriched for the cellular proliferation marker Mki67 when compared to CTC-c. Multiple lineages are likely to have contributed to this complex grouping: CTCs from KPC mice with tumor-restricted, lineage-tagged EGFP expression clustered with CTC-pro (data not shown), noteworthy for abundant expression of Mki67 and an annotated cell cycle signature in MSigDB (Whitfield et al., 2002) (data not shown). One single cell within the CTC-pro cluster was derived from the pancreatic cancer cell line NB508, while another (MP3-2) had high keratin/high E-cadherin expression characteristic of classical CTCs (data not shown). Nonetheless, another sub-cluster contained immune and dendritic cells, identified by their expression of antigen processing and presentation genes (GO:0019886—antigen processing and presentation of exogenous peptide antigen via MHC class II; Table 5). Taken together, the CTC-pro cluster appears to represent a grouping of highly proliferative cells, of which a subset are tumor-derived.
Together, unbiased isolation and RNA sequencing evaluation of single pancreatic CTCs indicate that over half of these are nonviable with RNA at various stages of degradation. Among the remaining viable CTCs, three major classes are distinguishable by unsupervised clustering: the classical subset (CTC-c) accounts for 55%, with a second platelet adherent group (CTC-plt; 32%) and a third heterogeneous cluster marked by proliferative signatures (CTC-pro; 13%). Given their most clearly defined tumor-derived characteristics, we selected the CTC-c cluster for detailed analysis of metastasis-associated pathways.
Pancreatic CTCs Co-Express Epithelial, Mesenchymal, and Stem Cell Markers.
The relevance of EMT to early metastasis in pancreatic cancer has been supported by lineage tracing studies in the KPC mouse model (Rhim et al., 2012). In human breast cancer CTCs, a distribution of epithelial and mesenchymal markers within individual CTCs was recently reported by the inventors, reflecting both tumor histology and response or resistance to diverse therapies (Yu et al., 2013). To directly test for EMT in the mouse pancreatic CTCs, established epithelial (E) and mesenchymal (M) markers (Kalluri and Weinberg, 2009) were used to evaluate each cell within the CTC-c cluster (data not shown). Compared with the primary tumor, CTC-c cells demonstrated clear loss of the epithelial markers E-cadherin (Cdh1) and Muc1, whereas mesenchymal transcripts were mixed, with some showing increased expression (Cdh11, Vim) and others with reduced levels (S100a4, Itga5, Sdc1) ( FIGS. 3A and 3B ). Notably, even the mesenchymal genes that were upregulated in CTCs showed a high degree of heterogeneous expression across single cells (data not shown). In contrast, loss of epithelial marks, including E-cadherin (Cdh1) was nearly universal across all classical CTCs.
CTCs are also thought to be enriched for metastatic precursors, capable of initiating metastatic tumor deposits. The relationship between such precursor cells and postulated cancer stem cells is uncertain, as is the relevance of established stem cell markers in identifying these cells. Proposed pancreatic cancer stem cell genes (Rasheed and Matsui, 2012; Rasheed et al., 2010) were evaluated in the single cell RNA sequencing reads ( FIG. 3B ). Among all candidate markers tested (Aldh1a1, Aldh1a2, Prom1/Cd133, Cd44, Met, EpCAM), only Aldh1a1 and Aldh1a2 were enriched in CTCs. Classical CTCs expressed predominantly the Aldh1a2 isoform, while CTC-plt cells were enriched for Aldh1a1, but these isoforms were also co-expressed within some single CTCs. MEFs, NB508 pancreatic cancer cells and normal WBCs also expressed Aldh1a1, but not Aldh1a2 (data not shown). Within single CTCs, there was no correlation between expression of Aldh1 isoforms and enrichment for the mesenchymal genes Cdh11 or Vim, suggesting that these two biomarkers are not intrinsically linked.
Given the identification of Aldh1a2 as a potential stem-like marker expressed by CTCs, its expression within matched primary tumors was tested using RNA in situ hybridization (RNA-ISH). Expression patterns within tumors were heterogeneous: Aldh1a2 expressing cells were primarily localized within the “stromal” or non-epithelial (i.e. keratin low) compartment of the tumor (data not shown). The origin of these non-epithelial cells, which are particularly abundant in pancreatic cancer, is likely to be mixed. Both histological evaluation and negative KRAS mutational analysis (Biankin et al., 2012; Ogino et al., 2005) in human pancreatic cancer have indicated that most of these cells represent reactive fibroblasts or stroma, rather than being of tumor origin. However, lineage tracing in KPC mice has recently shown that a small fraction of these supposedly stromal cells are in fact tumor-derived, presumably having undergone EMT to appear fibroblastic (Rhim et al., 2012). Interestingly, the mouse with the most metastases and the highest number of Aldh1a2 positive CTCs, MP7, also had the primary tumor with the highest levels of Aldh1a2. In that case, Aldh1a2-positive cells were present diffusely in the stromal compartment, as well as comprising a small subpopulation of the epithelial (keratin high) component (data not shown). Thus, classical CTCs, which are keratin-high, express the stem cell-associated gene Aldh1a2, whose expression in primary tumors is restricted to the stromal (keratin low) compartment and only a small subpopulation of epithelial cells.
Classical CTCs Share Expression of Stromal Enriched Genes.
Beside the evident diversity of CTCs, shared transcripts were sought that might provide further insight into their cell of origin within the primary tumor, the mechanisms by which they invade and survive within the bloodstream, and ultimately identify potential CTC-specific therapeutic targets. Rigorous criteria were selected to identify the most highly enriched CTC transcripts (RP score<300), expressed at very high levels (>100 RPM) in ≧90% of all classical CTCs. Three genes met these criteria: Decorin (Dcn), a extracellular matrix proteoglycan expressed in tumor stroma across a variety of different cancers (Adany et al., 1990; Bostrom et al., 2013; Henke et al., 2012; Hunzelmann et al., 1995; Iozzo and Cohen, 1994; Mu et al., 2013; Nash et al., 2002); Insulin-like growth factor binding protein 5 (Igfbp5), an extracellular growth factor binding protein expressed in human PDAC reported to have both pro and anti-proliferative properties (Johnson et al., 2006; Johnson and Haun, 2009); and Kruppel-like factor 4 (Klf4), one of the key stem cell (iPS) reprogramming factors (Takahashi and Yamanaka, 2006), which has been implicated in pancreatic cancer development (Brembeck and Rustgi, 2000; Prasad et al., 2005; Wei et al., 2010). By RNA-ISH, Dcn was expressed diffusely in the stromal elements of the tumor ( FIG. 6 ). Remarkably, both Igfbp5 and Klf4 were expressed focally, predominantly within stromal-appearing cells that border the epithelial compartments of the tumor (data not shown). RNA-ISH of EGFP lineage restricted primary tumors confirmed that the Igfbp5 positive cells at the epithelial/stromal interface are of tumor origin (data not shown). In addition to this transitional region, analysis of Klf4 in this EGFP-tagged tumor also found expression in a subset of epithelial ducts (data not shown). Of note, while they are expressed in only a small subset of primary tumor cells, both Igfbp5 and Klf4 are highly co-expressed in 85% of all classical CTCs. Together with the mixed epithelial/mesenchymal markers evident in CTCs, these observations raise the possibility that many CTCs are derived from foci at the epithelial/stromal interface, that may be defined by Igfbp5 and Klf4 expression.
In addition to the three most highly expressed transcripts, CTCs were noteworthy for high level expression of genes implicated in stromal cell matrix. Gene ontology analysis of all CTC-enriched genes (Table 3) identified 60 extracellular proteins (GO:0044421, OR 1.7, q-value 6.4×10 −3 ), of which 32 are found in proteinaceous extracellular matrix (ECM) (GO:0005578, OR 2.4, q-value 4.8×10 −3 ). Recent studies have highlighted the importance of the reactive stroma to pancreatic cancer pathogenesis and metastasis (Feig et al., 2012; Neesse et al., 2013; Neesse et al., 2011; Olive et al., 2009; Provenzano et al., 2012), however, the expression of these stroma-associated ECM genes within tumor cells in circulation was unexpected. To identify the predominant stromal enriched genes in the mouse pancreatic tumor model, we performed RP differential expression analysis between the bulk tumor samples representing tumor cells mixed with reactive stromal cells versus purified EGFP-tagged single cells from the primary tumor (TuGMP3). A total of 51 proteinaceous ECM genes were enriched in bulk tumors versus single primary tumor cells (GO:0005578, OR 4.8, q-value 3.4×10 −18 ). Of these, 6 genes (Ccdc80, Col1a2, Col3a1, Dcn, Sparc, Timp2) were shared with the previously identified CTC-enriched gene set (data not shown). Decorin (Dcn), as noted above, was identified as the most highly enriched (median 10,686 rpm) in CTCs with high level expression (>100 rpm) in 98% of CTCs. The second most abundant gene was Sparc (median 3,913 rpm) with high expression in 88% of CTCs. These two genes were co-expressed at high levels in 88% of classical CTCs. RNA-ISH of primary tumors for both Dcn ( FIG. 6 ) and Sparc (data not shown) confirmed that these genes are expressed throughout the reactive stroma and are not present in the epithelial keratin-rich regions of primary tumors.
The expression of stromal-derived ECM genes is a common feature of all classical CTCs, yet a mouse-specific bias in distribution among these genes was evident, despite their identical Kras/p53 genetic drivers. This mouse-specific clustering was evident in the unsupervised analysis (p-value<2.2×10 −16 ). For instance, sub-cluster 3 was over-represented with single CTCs from mouse MP6, while sub-cluster 4 was enriched for mouse MP7, and sub-cluster 5 for mouse MP2. Of 68 transcripts differentially expressed between the CTCs of mice MP2 and MP7 by RP analysis, gene ontology indicated significant enrichment for 11 extracellular proteins (GO:0044421, OR 3.8, q-value 0.06), 7 of which are found in proteinaceous ECM (GO:0005578, OR 6.3, q-value 0.05) (data not shown). Together, these data indicate that most CTCs derived from a mouse pancreatic cancer model express at high levels a set of ECM genes normally found in the stromal, rather than the epithelial compartment of the primary tumor. This may reflect the origin of many CTCs at the epithelial/stromal interface, consistent with their expression of uniquely restricted markers such as Igfbp5 and Klf4. The fact that individual genetically matched mouse tumors generate CTCs with both shared and unique patterns of ECM gene expression suggests tumor-specific invasion pathways that are superimposed upon fundamental characteristics of CTCs. The high levels of extracellular proteins expressed by CTCs provide unexpected opportunities for targeting these metastatic precursors.
Human Pancreatic CTCs Express the ECM Protein SPARC.
To determine the relevance of ECM protein expression to human disease, CTCs were isolated from the blood of metastatic PDAC patients and subjected to single cell RNA-sequencing. Analysis of 7 pancreatic CTCs from 3 patients revealed that the majority expressed keratins defining their epithelial origins and a total of 13 of 60 extracellular protein genes enriched in mouse CTCs were expressed at high levels (>100 rpm) in at least one human pancreatic CTC ( FIG. 7 ). Human SPARC was the only gene found at high levels in all human pancreatic CTCs. Analysis of human prostate and breast CTCs also show significant expression of extracellular proteins including SPARC highlighting that these targets are commonly shared in metastatic epithelial cancer cells (data not shown). RNA-ISH of Sparc/SPARC in both mouse and human PDAC found expression confined primarily to the stromal compartment of tumors (data not shown). SPARC expression was found in 196/198 (99%) human primary PDAC tumors and 36% of positive tumors had some detectable SPARC in epithelial tumor cells albeit the minority of the overall signal. The presence of SPARC as an extracellular protein permits antibody directed therapies that target SPARC. Together these data indicate that findings in mouse pancreatic CTCs can be found in human disease and offer both novel biomarkers and therapeutic targets.
Discussion
Described herein is a detailed analysis of CTC composition and diversity, using single cell RNA sequencing. In total, high quality transcriptomes were achieved in 93 single mouse pancreatic CTCs, which were compared with 20 single cells from matched primary tumors, as well as bulk tumor preparations, and with 16 cells from an immortalized cell line established from the same mouse pancreatic tumor model. The use of a mouse model, which closely matches human PDAC, made it possible to compare primary tumor specimens isolated simultaneously with the CTCs. Given the shared Kras/Trp53 genetic drivers in the KPC mouse model, it was also possible to examine CTC heterogeneity within individual mice and across different animals. Finally, the use of the CTC-iChip technology enabled the selection of untagged CTCs, irrespective of their cell surface epitopes, thus avoiding any bias associated with tumor marker-specific cell purification. Together, these observations include the following: 1. CTCs cluster into multiple subsets, including a major “classical CTC” group, and others that are marked by platelet-derived markers or proliferative signatures; 2. While individual mouse tumors may produce CTCs that fit into each of these clusters, there are unique patterns to CTCs derived from individual mice, despite their shared genetic drivers; 3. Common markers shared by virtually all classical CTCs include both epithelial and mesenchymal markers, the Aldh1a2 stem cell marker, and two highly expressed transcripts (Igfbp5 and Klf4) that identify foci localized to the epithelial/stromal boundary of primary tumors; and 4. The most highly enriched CTC-specific transcripts shared by almost all classical CTCs encode extracellular matrix proteins associated with the tumor stromal compartment.
Compared with previous RNA sequencing of partially purified, bulk CTC populations, the single cell analysis reported here provides considerably more depth of tumor cell-specific reads. As such, the detailed analysis of classical CTCs from the mouse pancreatic cancer model is unprecedented. It is demonstrated herein that pancreatic cancer CTCs uniformly lose expression of the epithelial marker E-cadherin (Cdh1), a key feature of epithelial-to-mesenchymal transition. However, the cells do not lose expression of other epithelial markers, such as cytokeratins, nor is there a consistent increase in classical EMT mesenchymal markers such as vimentin. As such, most classical CTCs appear arrested in a biphenotypic state. Despite their expression of cytokeratins (present in the epithelial components of the primary tumor), most other highly expressed markers in CTCs were shared with the non-epithelial or “stromal” component of the primary tumor. Among these stromal genes expressed in classical CTCs is Aldh1a2, a putative pancreatic cancer stem cell marker (Rasheed and Matsui, 2012; Rasheed et al., 2010). Whether Aldh1a2 is a functionally significant marker of cellular plasticity in metastatic precursors remains to be determined.
A provocative observation relating to the shared epithelial and mesenchymal state of classical CTCs is their virtually uniform (>85%) high level co-expression of Igfbp5 and Klf4, two genes that are only expressed in a small subpopulation of cells at the epithelial/stromal interface within primary tumors. This raises the intriguing possibility that this critical location within the tumor generates a disproportionate fraction of viable CTCs. Indeed, tumor cells that are actively undergoing EMT are presumably enriched at the epithelial-stromal function, contributing to the mixed lineage of the tumor stroma, with both tumor-derived and non-malignant reactive cell types. The potential roles of both IGF signaling and Klf4 transcriptional regulation in embryonic development and pancreatic malignancy make their unique expression pattern in both tumors and CTCs particularly noteworthy.
Finally, the most unexpected observation from this single CTC RNA sequencing study is the very high level abundance of ECM proteins on the vast majority of classical CTCs. Notably, prior evaluation of matched primary and metastatic breast tumors identified the most prevalent gene expression difference as enrichment for ECM molecules in the metastases, comprising some 18% of differentially expressed genes (Weigelt et al., 2005). While this has been interpreted as reflecting differences in the local environment of the metastatic site, the present data indicate that ECM proteins are highly expressed by CTCs themselves. By analogy with the classical “seed versus soil” debate (Fidler, 2003), CTCs may in fact be seeds carrying some of their own soil.
The ultimate goal of detailed molecular analysis of CTCs is to understand the process by which they are generated and their therapeutic vulnerabilities. In this regard, an important observation derived from the present single CTC RNA sequencing analysis is the unexpected expression of extracellular proteins with a preponderance of proteins found in ECM. Two of the most abundant and commonly shared ECM proteins in CTCs are Dcn and Sparc, both of which are established tumor stromal genes. Notably, Sparc expressing stroma appears to bind albumin-conjugated chemotherapy-containing nanoparticles (nab-paclitaxel) allowing for increased cytotoxicity and efficacy in human PDAC (Neuzillet et al., 2013; Von Hoff et al., 2011; Yardley, 2013). Indeed, considerable effort has been directed to targeting pancreatic cancer stroma as a means of improving delivery of chemotherapeutics and stripping tumor cells of their supportive microenvironment (Neesse et al., 2011; Olive et al., 2009; Provenzano et al., 2012; Rasheed et al., 2012). The finding that these gene products are also expressed by CTCs indicates that antibody-directed therapies can be used not only against primary tumor stroma, but also to target tumor cells as they transit in the blood.
As described herein, the present CTC analyses to extend from matching them to known tumor-defining markers to interrogating them for unique properties that distinguish them from most primary tumor cells and may underlie their ability to survive in the bloodstream and generate distant metastases. Such insights into the cellular process of human cancer metastasis are critical to the goal of ultimately preventing the spread of a primary tumor to distant organs.
Experimental Procedures
Mice and cell lines. Mice with pancreatic cancer used in these experiments express Cre driven by Pdx1, LSL-Kras G12D , and Trp53 lox/+ or Trp53 lox/lox as previously described (Bardeesy et al., 2006). EGFP pancreatic lineage tagged KPC mice were generated by breeding the mT/mG mouse (Jackson Laboratory—Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J) into the breeder pairs used for KPC mouse generation. Normal FVB mice were purchased from Jackson Laboratory. All mice care and procedures were done under MGH SRAC approved protocols.
Adaptation of CTC Enrichment Technology.
Given the desire for an unbiased enrichment system, the previously presented negative depletion technology was selected for this application (Ozkumur et al., 2013). All processing protocols were identical to those previously identified, except a rat anti-mouse CD45 antibody (BAM114, R&D Systems, USA) was conjugated to MyOne beads.
Single Cell Micromanipulation, Amplification, and Sequencing.
After whole blood anti-CD45 negative depletion, the product containing enriched cells was collected in a 35 mm petri dish and viewed using a Nikon Eclipse Ti™ inverted fluorescent microscope. Cells of interest were identified based on intact cellular morphology and lack of labeling with anti-CD45 magnetic beads. These target cells were individually micromanipulated with a 10 μm transfer tip on an Eppendorf TransferMan® NK 2 micromanipulator and ejected into PCR tubes containing RNA protective lysis buffer and immediately flash frozen in liquid nitrogen. Single cells were amplified with a modified protocol (Tang et al., 2010) and sequenced on the ABI 5500XL™ system.
RNA In Situ Hybridization (RNA-ISH).
RNA-ISH was performed according to the Affymetrix QuantiGene ViewRNA ISH Tissue-2 Plex Assay™.
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TABLE 1
Annotation of CTC enriched genes in KEGG defined signaling pathways.
*indicates gene found in multiple pathway gene sets.
TGF-
Toll-Like
Neurotropin
beta
Receptor
VEGF
MAPK Pathway
WNT Pathway
Pathway
Pathway
Pathway
Pathway
1500003o03rik*
Jund
1500003o03rik*
Akt2*
Amhr2
Akt2*
1500003o03rik*
Akt2*
Map3k3*
Crebbp*
Braf*
Crebbp*
Fos*
Akt2*
B230120h23rik
Mapk1*
Csnk1a1
Calm1
Dcn
Ifnar2
Hspb1*
Braf*
Mapkapk3*
Jun*
Calm2
Id1
Irak4*
Kdr
Dusp1
Mef2c
Nkd1
Irak4*
Id2
Irf7
Mapk1*
Dusp14
Mras
Ppp3ca*
Irs2
Mapk1*
Jun*
Mapkapk3*
Dusp3
Nf1
Rock1*
Jun*
Rock1*
Mapk1*
Pla2g4a*
Fas
Nfkb2
Rock2*
Maged1
Rock2*
Nfkbia*
Ppp3ca*
Fgf1
Nr4a1
Siah1a
Map3k3*
Smad4*
Tirap
Src
Flnc
Pla2g4a*
Smad4*
Mapk1*
Tgfb2*
Tlr2
Fos*
Ppp3ca*
Tbl1x
Nfkbia*
Tgfbr2*
Gadd45b
Rras2
Tcf7l1
Shc1
Thbs1
Hspa2
Tgfb2*
Wnt4
Ywhaz
Hspb1*
Tgfbr2*
Jun*
TABLE 2
Significantly Expressed Genes by Rank Product (FDR < 0.01)
CTC-c vs Primary
Primary Tumor vs
CTC-plt vs
CTC-pro vs
Count
Tumor Enriched Gene
CTC-c Enriched Gene
CTC-c
CTC-c
1
Upk3b
Tff2
Clec1b
kg:uc007pge.1
2
Ier2
Wfdc2
AU023871
kg:uc007pgd.1
3
Egr1
Lamb3
Alox12
kg:uc007pgf.1
4
Nkain4
Lad1
Itga2b
kg:uc007pgg.1
5
Igfbp5
Dmbt1
Ppbp
Igj
6
Slc6a4
Npy
Gng11
kg:uc012enb.1
7
Klf4
Pmepa1
Vwf
2010001M09Rik
8
Tmem221
Kcnn4
Pf4
kg:uc009cfw.1
9
Arl4d
Serinc2
Fcer1g
kg:uc007pgi.1
10
Lrrn4
5730559C18Rik
Tmem40
kg:uc007pgh.1
11
Cldn15
Muc1
Hba-a2
kg:uc007yos.1
12
Gpm6a
Chi3l3
Stom
Coro1a
13
Atf3
Pglyrp1
Beta-s
Pou2af1
14
Ptma
Arl4c
Plek
kg:uc011yvj.1
15
Slc9a3r1
Spp1
Srgn
Glipr1
16
Fos
Col15a1
Myl9
Cd52
17
Tmem119
C1qb
Cd84
Cd79b
18
Ptgis
Tnnt2
F5
Sec11c
19
Dcn
Gkn3
Treml1
Tnfrsf17
20
Gbp2
Onecut2
Hbb-b1
Krr1
21
Dmkn
Mmp7
Itgb3
Gmfg
22
Sdc4
Cd74
Gp9
Ccr9
23
Ildi2
Ctss
Mpl
Pycard
24
Akap2
Lamc2
Ctla2a
Derl3
25
Gfpt2
Olfml3
Tubb1
Rac2
26
Klf6
Lgals4
Mylk
Srgn
27
Btg2
Lcn2
F13a1
Cytip
28
Myl7
Ly6a
Slamf1
Edem2
29
Igfbp6
Pak1
Rgs10
Itgb7
30
Gpr133
Capn5
Mkrn1
Lsp1
31
Oasl2
Ptprn
Laptm5
Lcp1
32
Pfn1
Reg3b
1810058I24Rik
Cyfip2
33
Cap1
Fmnl3
Itgb2
Nans
34
Nfkbia
Sdc1
Slc2a3
Slamf7
35
Malat1
Prom1
Pcmt1
Ell2
36
Rarres2
Ankrd50
Gp5
H2-Eb1
37
Rspo1
Ccl6
Ube2o
Creld2
38
Espn
Slc4a11
5430417L22Rik
Cd74
39
Klf9
Oraov1
Ptpn18
Blnk
40
Zbtb7c
Aldh1l1
Lat
Fmnl1
41
Brd2
Slc20a1
Fermt3
Snrnp70
42
Olfr1033
Cldn7
Nrgn
Sec61b
43
Wt1
Acsbg1
Mrvi1
Edem1
44
Esam
Las1l
Lyz2
Tspan13
45
kg:uc009igb.1
C1qc
Epb4.1
Psmb8
46
Tmem151a
Lama5
Rasgrp2
Pim1
47
Mgll
Mgat4a
Treml2
Sept1
48
Csrnp1
Cldn2
Hist1h4i
Cd48
49
Cd9
Mcpt2
March2
Sub1
50
Gjb5
Fxyd3
Ltbp1
Lims1
51
Lrrc61
Il4ra
Nptn
Ncoa2
52
Wasf2
Itga5
Abtb1
Ctnnbl1
53
Pdpn
Porcn
Ctla2b
Fdps
54
kg:uc009ogv.1
Mast3
Prkab2
Ube2j1
55
Sdpr
Scara3
Arhgdib
Mettl1
56
Gpr64
Atox1
Alas2
Lax1
57
Flnc
Arrdc1
Odc1
Rilpl2
58
Add3
Mmp2
Ptpn11
Ctse
59
Gata6
Saa3
Dhcr24
Glrx
60
Wfdc1
Serpinf1
Mfsd2b
Fut8
61
A130040M12Rik
Sox11
Gp1bb
AI662270
62
Ankrd12
Prpsap1
Rbpms2
Gramd3
63
Adamtsl1
Mcpt1
Fyb
Il2rg
64
C2
Mfge8
Smox
Rasgrp3
65
Prss23
Col18a1
P2rx1
Impdh1
66
Ube2v1
Lyz2
Otud7b
Plek
67
Cryab
C1qa
kg:uc007ttx.1
Ints5
68
Pkhd1l1
Acp5
Samd14
Blmh
69
Rtn1
Angptl4
Clca1
Dnmt1
70
Birc6
Ccnd1
kg:uc007tty.1
Galk1
71
Xdh
Asl
Gpr56
kg:uc007hxv.1
72
Cd34
Ctxn1
Sh3bgrl2
Ccdc88b
73
Rab6b
Pgs1
Pttg1ip
Selplg
74
Dusp1
Anapc2
Nomo1
Sar1b
75
Clic4
Cp
Gnaz
Lat2
76
C3
Gpx3
Mmrn1
Slc16a6
77
Rhob
Lama3
Gp1ba
Mki67
78
Mir3064
Rbp1
Sh3bgrl3
Dnajc3
79
Thbd
Cotl1
Slc24a3
H2-Ab1
80
Dpysl2
Nek6
Sord
Ndufs6
81
Cob1
Cpxm1
Nfe2
Actr3
82
Npr1
Sfrp1
Tuba4a
Etnk1
83
Dnajb9
Ttr
Zyx
Herpud1
84
Arhgap29
Gsto1
Cnn2
Ptpn7
85
Cav1
Npepl1
Itgb5
Ctss
86
Gbp7
Usmg5
Gata1
Cs
87
Hes1
Polr2l
Hist1h1c
Fbxw7
88
Gm16897
Sphk1
Tbxas1
Ppp2r5c
89
Ppp1r12a
Asxl1
Ptplad2
Znrd1
90
Sv2a
Ctsh
Bpgm
Rfc2
91
Ang
Egfl7
Pdlim7
Preb
92
Aldh1a2
C1qtnf6
Mmd
Fcer1g
93
Cryl1
Rras
G6b
Dnajb11
94
Kank1
Lgi4
kg:uc009duo.1
Slc35b1
95
2210403K04Rik
Hmga2
Lyz1
Sin3b
96
kg:uc009okn.1
Cep250
Tacc1
Nktr
97
Osr1
B4galt3
Dap
98
kg:uc008ewj.2
Tmem223
Mast2
99
kg:uc009tuw.1
Ltbp2
Atp2a3
100
Gadd45b
Tnfrsf23
Snca
101
Ablim3
Col7a1
Stx11
102
Clec3b
Ggct
C030046I01Rik
103
Usp25
Rab25
Trpt1
104
Sntb2
Nedd8
Tsc22d1
105
Rock2
9430023L20Rik
Prkar2b
106
Col14a1
Arl2
Cd9
107
Cd200
Wbp1
Pgm2l1
108
kg:uc008ehr.1
H2-Ab1
Gp6
109
Atp2b1
Preb
Pde5a
110
Exoc4
Sgsm3
Itga6
111
Abcb1b
Sfn
Itga1
112
Nrgn
Prrx2
Edem1
113
kg:uc009cvm.1
Ptprk
Isg20
114
Ncoa4
Reg1
Cdc42ep5
115
Ndufa4
Sdcbp2
Nipal3
116
Upk1b
Pcbd1
Ccdc92
117
Jun
Slc25a1
Sort1
118
Syne2
Vamp5
Ly6g6c
119
kg:uc007bvx.1
Crlf1
Ubash3b
120
Ap4e1
Avil
Inf2
121
Spock2
2700094K13Rik
Asap1
122
Efemp1
Ctse
Sec11c
123
Prpf40a
Penk
Gas2l1
124
Tspan5
Tmc4
Parvb
125
Lgals7
Dhrs3
Tmsb4x
126
Kif5b
Ap1s1
kg:uc007xrw.1
127
Psip1
Arl6ip4
Nudt3
128
kg:uc008oki.1
9430008C03Rik
Bcl2l1
129
1810014B01Rik
Fcer1g
B230312A22Rik
130
Ptges3
Uqcr11
Cnp
131
Limch1
Nhp2
Plp1
132
Bicd1
Plbd2
Cnst
133
Rdx
Capg
Rgs18
134
Pcdh15
Pnpla6
Lsm12
135
Foxn3
Ppdpf
Alox5ap
136
Morf4l2
Hgfac
Ppif
137
Ppp1r15a
Apoe
Spnb1
138
Cdc42ep3
Fam40a
Ormdl3
139
Pard3b
Lyz1
Hpse
140
Bicc1
2200002D01Rik
Srxn1
141
Amhr2
Laptm5
2010002N04Rik
142
Gucy1a3
Qars
Hist1h2bc
143
Psmb2
Tmx2
Cyba
144
Mapkapk3
Fkbp4
Chst12
145
Ube2l6
Plin2
kg:uc009sps.1
146
kg:uc007pff.1
Fcgr3
Max
147
kg:uc007ctp.1
Gkn1
Was
148
Nedd4
Snhg1
Isca1
149
Plxna4
Lsp1
Pdzk1ip1
150
2010107G12Rik
Gm20605
Lyn
151
Ifhgr1
Ly6c1
Mob3a
152
Bcam
Aim1
H2-T24
153
Ccnl1
2310007B03Rik
Slc44a1
154
Hoxa5
Tgfbi
Derl1
155
Fhl1
Tsta3
Gclm
156
1810041L15Rik
Pafah1b3
Fech
157
2900002K06Rik
Chid1
Ywhah
158
Hspb1
Smox
Igtp
159
Podn
1500012F01Rik
Myl6
160
Fam63b
Tspan4
Thbs1
161
Hsp90b1
Agrn
Tln1
162
Dpp4
Cfp
kg:uc009apq.1
163
Gas1
Cdh1
Bcap31
164
kg:uc007zak.1
Rasgrf1
Ilk
165
Zc3h13
Nxf1
Epha1
166
Sox6
Pdrg1
2810453I06Rik
167
Arid4a
Polr2j
Rnf19b
168
Tnxb
Suds3
Gsn
169
Tsix
D0H4S114
Flna
170
Scd1
Ccl9
Arrb1
171
Jund
Neat1
kg:uc007pum.1
172
Crls1
Ccdc12
Mbnl1
173
1110003E01Rik
Prr24
Ccnd3
174
Rnase4
Impdh1
Pdlim1
175
Arhgef12
Card10
Ctse
176
Irf7
Cpsf1
Tspan17
177
Bbx
Sema4g
Gpx4
178
Sema5a
Hes6
Bnip3l
179
Mau2
C130074G19Rik
P2ry12
180
Abi3bp
Ctrb1
kg:uc009vev.1
181
Dag1
Rnaseh2a
Prkab1
182
Cyp2s1
Golm1
F2rl2
183
Sfrs18
Ctsz
Stk4
184
Hspb8
Cyb561
Fhl1
185
Cnot6l
Ndufs8
Rnf10
186
Twsg1
Atp6ap1
Rasa3
187
Gpc3
Srd5a1
Taldo1
188
Lrrn4cl
Carkd
Bysl
189
Cdh3
Cd24a
Esd
190
Cyr61
Eng
Aldh2
191
Cyp2d22
Tcirg1
Rhog
192
Hist1h1c
Slc9a3r2
kg:uc009ecr.1
193
Aplp1
0910001L09Rik
Cald1
194
Tbl1x
Cox5b
Wbp2
195
Pcm1
Adipor2
Ptprj
196
Ifi204
Scarf2
Tpm4
197
Nfix
Myo7a
Mxi1
198
Flrt2
Ppap2c
Ly6g6f
199
Heg1
Pea15a
Sla
200
Il6ra
Sh3pxd2b
Slpi
201
Ralbp1
H19
Bicd2
202
Rhoj
Tpd52
Clu
203
Ktn1
2610203C20Rik
Mtmr14
204
Arl6ip5
Naa10
Abca7
205
Crebbp
Fermt1
Ppp1r18
206
Ppig
Sap30l
Kif2a
207
Akap13
Bgn
Prdx6
208
Rab7
Timm13
kg:uc009ize.1
209
Plxdc2
Krt20
Calm3
210
Aldh1a1
Itga3
Dhrs1
211
Bnc2
Pfkl
Cfl1
212
Slc4a4
Agpat6
Glipr2
213
Tbx18
Mrpl11
Slc25a37
214
Zbtb16
Ramp1
Atox1
215
Arid4b
Hmga1
BC057079
216
Enpp2
Gpx2
Pla2g16
217
Ptplad2
0610012G03Rik
Rnf144b
218
Akr1b3
9130017N09Rik
Stk16
219
Gm6644
Cygb
Rsad2
220
Arf5
Tmprss4
Paip2
221
Chi3l1
Paox
Capzb
222
Gpr116
Endod1
Ppp1r12c
223
Cd82
Cndp2
4930412F15Rik
224
Srrm1
Suv39h1
Ninj1
225
Fmo2
Cog4
2510009E07Rik
226
Tgfb1i1
Trim27
kg:uc007vsr.1
227
Qrich1
Cyhr1
Pygb
228
Nfia
Trmt1
Tlk1
229
Pmp22
Zfyve19
Myct1
230
Cdh11
Esrp1
Rnasek
231
Arid5b
kg:uc008oow.1
Ctsd
232
Rbm3
Dync1h1
0610010K14Rik
233
Prelp
Tab1
Bcas3
234
kg:uc007qse.1
Pla2g6
Atpif1
235
Ddx3x
Timp1
Serf2
236
Sulf1
Eif3f
Becn1
237
Spnb2
Abhd11
Tspan9
238
Tspan31
Pmm2
Acer2
239
Prr13
Tyrobp
Vdac3
240
Ppp1cb
Farsb
kg:uc008kbg.1
241
Fbln1
Plod3
Oaz2
242
Gm6548
Abtb1
Serpine2
243
Uap1
Brf1
Ccdc90a
244
Mpdz
Tnk2
Ndufa1
245
Sat1
Rfc2
Tssc1
246
Stim1
Stxbp2
Mboat7
247
Mll3
Pdlim7
Cd44
248
Slurp1
A430105I19Rik
Cxx1c
249
Cd81
Vill
Ecm1
250
Emp2
Bmp1
Mff
251
Trpm7
Mpzl1
Ptpn12
252
Crym
Thy1
Mgmt
253
Enpp4
Stab1
Cox4i1
254
Raly
Aldh16a1
Tollip
255
Celf2
Eif4ebp3
Cds2
256
Ap3s1
Itpripl2
Ybx1
257
C1s
Mrpl52
Gypc
258
Frmd4b
2310002L13Rik
Dgkd
259
Nr4a1
Mcm6
Pecam1
260
Acini
Kcnk1
Ftl2
261
Plod2
Pmf1
Nt5c3
262
Id1
Cuta
1700037H04Rik
263
Creg1
Nt5dc2
Cd151
264
Zfp318
Rmnd5b
Lpin2
265
Tmem140
Araf
6430548M08Rik
266
Mras
Wwp2
Pon2
267
Vwa5a
Lamb1
Ndufa3
268
Esyt3
Kcne3
6330578E17Rik
269
Hexb
Uqcrq
Mfap31
270
Nckap1
Gps1
Mink1
271
Nipal3
Rexo4
Ston2
272
Ubxn4
Coro1c
Rac2
273
Zfp36
Hras1
Fyn
274
Hnrnpl
Spint1
Serinc3
275
C1ra
Cblc
Maged2
276
Nnmt
Fhod1
Ap2m1
277
Mut
Atp13a1
Pacsin2
278
kg:uc008jup.1
Man2c1
Ftl1
279
Pnrc1
Vsig2
Adipor1
280
Usp8
Bpgm
kg:uc009qdo.1
281
Pgcp
Bap1
Snap23
282
Junb
Smpd2
Tagln2
283
C1rl
Ubqln4
Cox6c
284
Slc6a6
Sirt7
Creg1
285
kg:uc008znh.1
Krt23
Bsg
286
Aqp1
D8Ertd738e
Cmtm6
287
Myh10
Mapk13
Cntd1
288
Slc43a3
kg:uc008bcq.1
Plekho2
289
Spint2
Polr2g
Arrb2
290
Hnrnph1
Ndufs2
Pard3b
291
Arhgap28
Dad1
Mlec
292
Cfh
Wnt7b
Taf10
293
Brd4
Fam20c
Gabarapl2
294
Fndc1
Cxxc5
Bag1
295
Star
Polr2f
Galnt2
296
Nfkbiz
Ltf
Hk1
297
Arsb
2210407C18Rik
Fbxo9
298
Rnd3
Cdipt
kg:uc009izd.1
299
Stard5
Glrx5
Pnpo
300
Thbs1
Gemin7
Fam46c
301
kg:uc008wkn.1
Man1b1
Pkm
302
Slc26a3
Heatr7a
Ap1b1
303
Phip
Arid5a
Rap1b
304
Usp2
Sumo3
Itgb1
305
Golgb1
Srm
St7
306
Rock1
Plscr3
Smap1
307
Rgma
2210010C17Rik
Rabgap11
308
Actg1
Fam102a
Tmbim4
309
BC013529
Dlst
H3f3a
310
kg:uc007zwh.1
Vps37c
Frmd8
311
3110062M04Rik
Ngfrap1
Nlrx1
312
Cast
Pold4
Oaz1
313
Mob3c
Grcc10
Fam125b
314
Slc16a1
Wnt7a
Hexa
315
Fam117a
2010111I01Rik
Tspo
316
Pdia3
Pxdn
Dcaf12
317
Trim8
Coasy
Nav1
318
kg:uc009mng.1
Dctn1
Cd24a
319
eg:245190:chr7:m
Ncor2
Uqcr11
320
Sbsn
Postn
Wipf1
321
Serpinb6b
Col4a2
F10
322
Daglb
Cib1
Erlec1
323
Smarca2
Tbc1d13
Map2k3
324
Mef2c
Ccnl2
Stk24
325
Prrc2c
Dcakd
Ldlrap1
326
BC005537
Cdc34
Ehd4
327
Hsp90ab1
Atp6v0b
Atp6v1f
328
Snrnp70
Abhd12
Gnas
329
Ppl
Flot2
Arhgap18
330
Serpinh1
Sla2
Arhgap10
331
Sorbs3
Rhbdf1
Pitpnm1
332
Golga4
Cdh17
S100a1
333
Acbd3
Psmb5
Bin1
334
Hook3
Serf1
Ttyh3
335
Map3k3
Slc15a3
Selp
336
Rhou
Sftpd
Trappc9
337
Smc2
Pop5
Aes
338
C1d
Nudc
Taok3
339
kg:uc008dzh.1
Sh2d5
Zfand3
340
Psmd7
kg:uc007fwp.1
Stim1
341
Dab2
Mrpl37
Rnf11
342
Cep164
Rin1
Sep15
343
Crim1
Podxl
kg:uc012hdk.1
344
Rtf1
Paqr5
Lgals9
345
Fxyd1
Sepx1
Cox6b1
346
H2-D1
Agr2
Riok3
347
Zfp704
Bax
Slc38a10
348
Mtap1a
Rxrb
Rtn3
349
Ascc3
Tes
B3gat2
350
Med13l
Hdac6
Ccndbp1
351
Jup
1110008F13Rik
Rsu1
352
Nid2
Mpnd
kg:uc007upr.1
353
Kdr
Gmppa
Itm2b
354
Ifnar2
Gramd1a
St3gal1
355
5430435G22Rik
Wars
Sec61g
356
Col4a6
Mtap
Ptpn1
357
Il17re
C1qtnf5
kg:uc012bhf.1
358
Gbp3
Mrpl28
B2m
359
Slc39a8
Mfrp
Rasgrp3
360
Cfl2
Kars
Memo1
361
Slc38a1
Lbp
Slc39a4
362
Cuedc1
Plxnb1
Sdcbp
363
Fgf1
2700081O15Rik
Tspan14
364
Gas6
Mrps24
Ubl7
365
Cldn25
Klc4
Nras
366
Sorbs1
Dctn3
Ssx2ip
367
Hspa12a
Kcnq1
kg:uc007zbz.1
368
kg:uc007zts.1
Smurf1
Wbp1
369
Slc1a5
Fam162a
1110003E01Rik
370
Nr3c1
Hip1r
Clip2
371
Adamts5
kg:uc007hyr.2
Gapdh
372
Gpcpd1
Gys1
Gm6578
373
Dpysl3
Sac3d1
Actn1
374
Colec12
Ndufs6
St3gal2
375
Pdcd6ip
Rgl2
3110001D03Rik
376
Dst
Atp5g1
Ctsz
377
Ifit4
Itgb4
kg:uc007vdl.1
378
Chst4
Sars
Fam73a
379
Xist
2310003F16Rik
Vcl
380
Ifi27l2a
Nhp2l1
Lims1
381
Fkbp5
D19Wsu162e
Lars2
382
Agap1
Cd320
Birc2
383
Ankrd11
Pigq
Lamp2
384
kg:uc007qca.1
Chd3
Rasl10a
385
Syt11
Zdhhc4
Mif
386
Ptrf
Eif3l
Rab10
387
Krcc1
St8sia3
Pabpc1
388
Zfp488
Rcan3
Wwp2
389
Lama4
Meg3
Nqo2
390
Aebp1
Nudt4
kg:uc007fte.1
391
Fam134b
Gss
Plxna4
392
Tppp3
Pih1d1
Gm1821
393
Maf
Limd2
Gadd45a
394
Peli1
Ap1s2
Slc25a39
395
Zfp353
BC056474
kg:uc009pet.1
396
Cdon
Mms19
Ubb
397
Sarnp
Clip2
Ppp1r2
398
Atxn7l3b
2310016M24Rik
Rab27b
399
Pef1
Itpa
Cap1
400
App
Slc25a10
Jarid2
401
Mtdh
Fibp
Rnf11
402
Lrrc20
Higd2a
Tmem50b
403
Btbd2
Snrpd2
Myh9
404
Gnb2
Eri3
Tmem128
405
Pigt
Nbeal2
Stradb
406
Efna5
Trim28
Cela1
407
Tm4sf1
S100a4
Ndrg2
408
Coq10b
Ivns1abp
Dhrs3
409
Eif2s3x
Ppp1r18
Hipk1
410
Cmah
Efemp2
Atg9a
411
Sf3b1
Med22
412
Eea1
Nelf
413
Slpi
2810428I15Rik
414
Tmod3
D2Wsu81e
415
Ppp3ca
Trappc6a
416
Tceal8
Trappc21
417
Anp32a
Antxr2
418
Actb
Rab11fip5
419
Ddx5
Ldhd
420
Cobll1
Npnt
421
Cish
Acrbp
422
Nod1
Pafah1b2
423
Psd
Angptl2
424
Gm10052
Fzr1
425
Lims2
Aaas
426
Stra6
Eif2b2
427
kg:uc007bgn.1
1190003J15Rik
428
Plxdc1
5730403B10Rik
429
Nfe2l1
Adamts13
430
Smpd3
Eif3b
431
Bcl10
Znrf1
432
Ilf3
Pkp3
433
Fam76a
Lemd2
434
Cybrd1
Rab34
435
Gm3893
Mpv17l2
436
Siae
Cdkn2b
437
Ssh2
Snrpe
438
Nfic
Gm14005
439
Btf3
Prdx4
440
Sp100
Xab2
441
Ndn
Dpp3
442
Matr3
Tyms
443
Gm13251
Leprotl1
444
Arhgap5
Uqcr10
445
Zbtb4
Cdk5rap3
446
Pgrmc1
Gorasp2
447
4930402H24Rik
Wbp7
448
Bptf
Sort1
449
Dusp3
Ddx41
450
Pla2g4a
Cct3
451
Brp44l
Mrps33
452
Oxct1
Frmd8
453
Stk40
1110049F12Rik
454
Ddr1
Fscn1
455
Ifi205
Ndufa2
456
Col3a1
Dpcd
457
Nipb1
Unc13a
458
Plk1s1
Eif1ad
459
Bdp1
Sgta
460
Smc3
Chaf1a
461
Ifitm3
Plxna1
462
Ndst1
Hspa9
463
Zbed6
1110014N23Rik
464
Rest
Cd99l2
465
kg:uc007vnc.1
Snrpa
466
Ccdc88a
Mcm7
467
Stat3
Tars2
468
Arf2
Gon4l
469
Trib1
Stk38
470
Gcap14
C1qtnf1
471
Tbc1d15
Tbrg4
472
Igf1r
Tmem132a
473
Ppbp
Cox6c
474
kg:uc008tky.1
Alcam
475
Rab1b
Phka2
476
Krt14
Trim3
477
Med21
Ppp1r14b
478
Gja1
Gpaa1
479
Klf10
Ctps2
480
Id2
Ptpn23
481
Mfap1a
Endog
482
Ogn
Mrto4
483
Gpc4
Mrps6
484
Bst2
Pvr
485
Dtx2
Phgdh
486
Wac
Itpr3
487
Kpna3
Polr2e
488
Kcnab1
Sec16a
489
Orai3
Mdp1
490
Gcsh
Fbf1
491
Wdr92
Mcpt8
492
Olfr613
Rps6ka4
493
Tcf7l1
Mical1
494
Tgfb2
Mrpl34
495
Il16
Agpat3
496
Manf
2310044H10Rik
497
Mgst1
Myo9b
498
kg:uc008tkz.1
Ndufb10
499
Creb3l1
Apex1
500
Txndc5
Elk3
501
Klf2
Cpsf3l
502
Slu7
Tnk1
503
Ttc28
Pmvk
504
1110002B05Rik
Ppp1r16a
505
Zcchc11
Arhgef5
506
Ptp4a2
Lonp1
507
Pbx1
Pla2g7
508
Clcn3
Pip5k1c
509
Tmco7
Inf2
510
Lrrc58
Pgk1
511
Eif3a
Parp6
512
Cldn10
Urm1
513
H2-Q6
Mad2l2
514
Ccdc80
Ing4
515
kg:uc009iln.1
Rbck1
516
Rab5c
Cant1
517
Tsc22d3
Sgpl1
518
Tm4sf5
Ehbp1l1
519
Hmgb1
Runx1
520
Sec62
Slc27a4
521
Maoa
Ndufa7
522
Clec1b
Mcm3ap
523
Mphosph8
1110008P14Rik
524
Oat
Rassf7
525
Ncor1
Ptpmt1
526
Cyb5
Arfgap1
527
Trafd1
Sec61a1
528
Rpp25
Rps6ka1
529
kg:uc007ded.1
Ints1
530
2610101N10Rik
Tpcn1
531
Il6st
Iffo2
532
Evpl
Trim44
533
Psmd11
kg:uc012ctw.1
534
Dync1i2
Golga2
535
Lars2
Msto1
536
Pdia4
Ppp6r3
537
Cd55
Trmt2a
538
Amfr
Appl2
539
Zcchc3
Sparcl1
540
Herpud2
Rapgef1
541
Txnrd1
Zfpl1
542
Vat1
Psmc4
543
Diap1
Mosc2
544
Tmed2
Fam101b
545
Arf3
1500010J02Rik
546
Arap2
Ccdc124
547
St3gal1
Ptges
548
Man1a
Fam189b
549
Rgs10
Th11
550
Tmsb4x
Kctd2
551
Uba7
Olfr1372-ps1
552
C4b
Hexa
553
Tmem98
Anapc5
554
Lpar2
Serpina3n
555
Gabarapl1
1810046J19Rik
556
Cmtm7
Tmem167
557
Spon2
Gm11428
558
Smarca5
Gcn1l1
559
Mxd4
Kansl3
560
Smc4
Fasn
561
Thsd4
Slc50a1
562
Gsr
Smad3
563
Ptprd
Trip6
564
Clip1
Atp6v1e1
565
Cln8
Chchd5
566
Rbm27
Adssl1
567
Zmat1
Nes
568
Smc6
Ap1b1
569
B2m
Fcgrt
570
Irf2bp2
Ltbp3
571
Ppap2a
Csf2rb
572
Zfhx4
Ssna1
573
Tob2
Mrps16
574
Rabgap1l
Cyba
575
Nfkb2
Cyth2
576
Nfyc
Igf2
577
Ube2d1
Pisd-ps1
578
Creb5
Atp13a2
579
Opa3
Mlph
580
Csnk1a1
Cyp4f16
581
Fam84b
2010107E04Rik
582
Ddr2
Gas5
583
Usp54
Eif3k
584
Akt2
Fam149a
585
Strn3
Mif
586
Hnrnpm
B230312A22Rik
587
eg:497210:chr14:m
Ppp1r12c
588
Tpt1
Tfip11
589
Naa25
Tex 10
590
Eef1a1
Slc16a3
591
Parp4
Stk16
592
Msn
Epn1
593
Zbtb20
Noc4l
594
Fermt2
Rcc2
595
Bod1l
Rgs12
596
Sltm
Shkbp1
597
Dapk1
Got2
598
Hnrnpr
Plek2
599
Baz2a
Lilrb3
600
Rnf167
Ndufb5
601
Mapk1
Tesk1
602
eg:320169:chr9:p
Rab24
603
4930523C07Rik
Atp5j2
604
Nf1
Commd9
605
Fam53b
Rtkn
606
Faim2
Prpf19
607
Tgm2
6720401G13Rik
608
Calm2
Ppa1
609
AI848100
Pgp
610
Slc10a3
Hps1
611
Ogdh
Puf60
612
Arl3
Mdm2
613
Timp2
kg:uc012cgd.1
614
Atxn2
kg:uc009uim.1
615
Mll1
Pyy
616
Ces2g
Zfp358
617
Mat2a
Timm8b
618
Esf1
Ddx39
619
Hsp90aa1
Pgm2
620
Zfp385a
kg:uc008gbp.1
621
Zfp672
Sipa1
622
Csda
Mgat1
623
Pf4
Tmem208
624
Arsa
Ruvbl2
625
F11r
8430410A17Rik
626
C4a
Bad
627
Kpna1
Pfdn5
628
Rbbp8
Eme1
629
Oxnad1
kg:uc009mzj.1
630
Rb1cc1
Igf1
631
Setd2
Prkag1
632
Kif1b
kg:uc009sua.1
633
2510002D24Rik
Uap1l1
634
Cep57
Trappc4
635
Chd2
Bola2
636
Serinc5
Usp5
637
Marcksl1
Ear2
638
Shfm1
Cars
639
Bbs4
1810027O10Rik
640
Impad1
Amdhd2
641
Tbcel
Phb
642
Kdelr1
Kcmf1
643
Ninl
Lsmd1
644
Sytl1
Sec11c
645
Tpm3
Pcbp4
646
Rbbp6
Mepce
647
Lman1
Tpd52l2
648
Ankrd17
Trf
649
Naga
Hsd17b11
650
Rbpms
Pilra
651
Magt1
Atn1
652
Tfdp2
Pgf
653
Gem
Nxn
654
Pde4dip
Inpp5k
655
Mrgprf
Actr1a
656
kg:uc008ajk.1
Cd68
657
Itch
Eef1g
658
Elf1
Fbn1
659
Meis2
Hint1
660
Arid1a
March5
661
Serping1
Usp48
662
Slc27a3
Hnf1b
663
Thoc2
Gga3
664
Gsta3
Drosha
665
Hnrnph2
Ubp1
666
Socs3
Pkn3
667
Armcx3
Tmem192
668
Siah1a
Prpf31
669
kg:uc009ize.1
Hspd1
670
Irs2
Otub1
671
Mettl7a1
Mrpl20
672
Ppfibp2
Tead2
673
Blvrb
Phpt1
674
Yipf5
Neu1
675
Plat
Pygo2
676
Gm6578
Myeov2
677
Mat2b
Cdk5
678
Tmpo
Ndor1
679
Metap2
Rbp4
680
Zfp277
Psat1
681
Wls
Mrpl41
682
Mesdc1
Snrpg
683
kg:uc009acs.1
Acot7
684
Col1a2
Vars
685
Csf1
Nono
686
Sulf2
Gtf2i
687
Ifrd1
Traf3
688
Wrnip1
Ppp2r4
689
Flii
Actg2
690
2810474O19Rik
Pi4k2a
691
Sep15
Slc35b2
692
2310030G06Rik
Ubqln1
693
Cmtm3
Ppox
694
Mylip
Bud31
695
Slc8a1
Man2b1
696
Btbd7
Nat15
697
Hdac5
Spon1
698
Zfand6
Cyc1
699
Tapbp
Mpeg1
700
Keap1
Nsun2
701
Ube2n
Rab4a
702
Ssr3
Mtmr11
703
H3f3a
BC004004
704
Myst4
B4galnt1
705
G3bp1
Atp5k
706
Ugdh
Lin37
707
Lamp2
D330041H03Rik
708
Zrsr1
Tbc1d17
709
Pim1
March6
710
Gm9199
2410015M20Rik
711
Supt16h
1810013D10Rik
712
Ano6
Eif2s1
713
Soat1
Traf7
714
Eci1
Rpl36al
715
Plce1
Psenen
716
Atg3
Aip
717
Bnc1
Cmas
718
Pik3c2a
Rpia
719
Pqlc3
Ncbp1
720
Thrap3
Mea1
721
Irak4
Timm50
722
Kdm6b
Ear12
723
Apol9a
Fkbp1a
724
Wnt4
Commd4
725
1500003O03Rik
Col5a3
726
Phf3
Fblim1
727
1110004F10Rik
Cwh43
728
Kansl1
Arl2bp
729
Fth1
Mrpl46
730
Tmem50a
Tcn2
731
Utp20
Add2
732
Smad4
Specc1l
733
Stmn2
Ppcs
734
Gstm1
Vrk3
735
Senp6
Trim25
736
Gda
Nfatc1
737
Nucks1
Rap1gap
738
Ints10
Hsd17b12
739
Syne1
Epas1
740
Itga6
Ddx1
741
Acad9
Prdx6
742
Maged1
Mmp24
743
Spen
Ndufb9
744
Chd1
Phf23
745
Taf3
Rpa2
746
Ptgs1
5031439G07Rik
747
Spare
Rrp7a
748
R74862
Arfip2
749
B230120H23Rik
Efna1
750
Tmem234
Agps
751
Ryk
Sephs1
752
Dlgap4
Apoc2
753
Atp1b1
Mrps27
754
Parp14
Snn
755
Tgfbr2
Serinc3
756
Ccdc90a
Pdcd5
757
Ncoa1
AA986860
758
Pppde1
Pitpna
759
Luc7l3
Vac14
760
Prg4
2810025M15Rik
761
Rab11fip1
Def8
762
Plk2
Hilpda
763
Ifi35
Eif6
764
Pdap1
Brd7
765
Cd248
Fes
766
Sesn1
Sbf1
767
Ecd
Ak2
768
Ap1s3
1810035L17Rik
769
H2-K1
Lime1
770
Spag9
Hspe1
771
Tshz1
Csrp2bp
772
Dennd5a
Uba5
773
Stag1
Gsta4
774
Gpx8
2900092E17Rik
775
Sod3
776
BC005561
777
kg:uc009vev.1
778
Ywhaz
779
Ganab
780
Rras2
781
Dusp14
782
kg:uc012hdk.1
783
Nr1d1
784
Wwc2
785
Ubxn2a
786
Iqsec1
787
kg:uc007vsr.1
788
Cfl1
789
Csrp1
790
Smchd1
791
Myl12a
792
Ubqln2
793
Tmcc3
794
Kdm5a
795
Rbm25
796
Wdr26
797
Vim
798
Arpc2
799
Calm1
800
Dnaja2
801
Shc1
802
Vps13a
803
Klf7
804
1810074P20Rik
805
BC003331
806
Itpr2
807
Jmjd1c
808
Pcdhgb5
809
Tubb2a
810
Ehd2
811
Ift74
812
Per1
813
Pitpnm2
814
Gstm4
815
Dnmt1
816
Tmco1
817
Lass4
818
Ptprf
819
Sirt2
820
Gfm2
821
Taf7
822
Spop
823
Zzef1
824
Ccdc34
825
Zfp281
826
Tuba1a
827
Ccdc109b
828
Cdk13
829
Dhx15
830
Src
831
Braf
832
Mapre2
833
Anxa7
834
Sept9
835
Alox12
836
Pknox1
837
2610034B18Rik
838
Topors
839
Phf21a
840
Qser1
841
Tirap
842
Fas
843
Lass2
844
6330406I15Rik
845
Parvb
846
Atp1a1
847
Mtmr6
848
Cd109
849
Dnajc1
850
Hp1bp3
851
1600029D21Rik
852
Ttc38
853
Mfhas1
854
Filip1l
855
Zfp148
856
Nkd1
857
Usp16
858
Tlr2
859
Zc3h18
860
Stk10
861
Ltbp4
862
Hdac3
863
Efhd2
864
Prkar2a
865
Atp6v1a
866
Sf3b4
867
Gprc5b
868
Clip3
869
Mettl2
870
Secisbp2
871
Fmod
872
kg:uc0091xf.1
873
Elovl6
874
Bzw1
875
Etfa
876
Hspa2
877
kg:uc007won.1
878
Rnf20
TABLE 3
Most Significant Gene Ontology Terms in CTC-c enriched genes using
BP_FAT and CC_FAT Datasets
q-value < 0.01
Odds
Benjamini
Source
Term
Count
Ratio
(q-value)
GOTERM_BP_FAT
GO: 0060429~epithelium development
35
2.92
8.72E−05
GOTERM_BP_FAT
GO: 0030029~actin filament-based
27
3.47
6.85E−05
process
GOTERM_BP_FAT
GO: 0030036~actin cytoskeleton
26
3.57
4.95E−05
organization
GOTERM_BP_FAT
GO: 0007010~cytoskeleton organization
36
2.50
6.27E−04
GOTERM_BP_FAT
GO: 0051173~positive regulation of
49
2.11
6.62E−04
nitrogen compound metabolic process
GOTERM_BP_FAT
GO: 0035295~tube development
31
2.66
7.80E−04
GOTERM_BP_FAT
GO: 0010604~positive regulation of
54
1.93
0.001727
macromolecule metabolic process
GOTERM_BP_FAT
GO: 0031328~positive regulation of
49
2.01
0.0015751
cellular biosynthetic process
GOTERM_BP_FAT
GO: 0051789~response to protein stimulus
16
4.16
0.0014484
GOTERM_BP_FAT
GO: 0035239~tube morphogenesis
23
3.05
0.0015064
GOTERM_BP_FAT
GO: 0045449~regulation of transcription
140
1.42
0.0014097
GOTERM_BP_FAT
GO: 0048729~tissue morphogenesis
28
2.66
0.0013058
GOTERM_BP_FAT
GO: 0009891~positive regulation of
49
1.99
0.0012408
biosynthetic process
GOTERM_BP_FAT
GO: 0045935~positive regulation of
46
2.04
0.0012061
nucleobase, nucleoside, nucleotide and
nucleic acid metabolic process
GOTERM_BP_FAT
GO: 0002009~morphogenesis of an
23
3.01
0.0012149
epithelium
GOTERM_BP_FAT
GO: 0048584~positive regulation of
24
2.92
0.0011396
response to stimulus
GOTERM_BP_FAT
GO: 0051276~chromosome organization
39
2.19
0.0012619
GOTERM_BP_FAT
GO: 0045637~regulation of myeloid cell
12
5.33
0.0014358
differentiation
GOTERM_BP_FAT
GO: 0045785~positive regulation of cell
11
5.79
0.0016889
adhesion
GOTERM_BP_FAT
GO: 0045941~positive regulation of
43
2.05
0.0016795
transcription
GOTERM_BP_FAT
GO: 0045893~positive regulation of
39
2.12
0.0019852
transcription, DNA-dependent
GOTERM_BP_FAT
GO: 0051254~positive regulation of RNA
39
2.11
0.0022107
metabolic process
GOTERM_BP_FAT
GO: 0006357~regulation of transcription
51
1.87
0.0022801
from RNA polymerase II promoter
GOTERM_BP_FAT
GO: 0006325~chromatin organization
32
2.30
0.0025187
GOTERM_BP_FAT
GO: 0010628~positive regulation of gene
43
2.00
0.0025252
expression
GOTERM_BP_FAT
GO: 0060562~epithelial tube
17
3.47
0.0025847
morphogenesis
GOTERM_BP_FAT
GO: 0042127~regulation of cell
45
1.89
0.0051485
proliferation
GOTERM_BP_FAT
GO: 0010557~positive regulation of
44
1.88
0.0071937
macromolecule biosynthetic process
GOTERM_BP_FAT
GO: 0002253~activation of immune
14
3.69
0.0078441
response
GOTERM_BP_FAT
GO: 0050778~positive regulation of
18
3.00
0.0080458
immune response
GOTERM_BP_FAT
GO: 0002684~positive regulation of
23
2.53
0.0088166
immune system process
GOTERM_BP_FAT
GO: 0045944~positive regulation of
33
2.09
0.0090124
transcription from RNA polymerase II
promoter
GOTERM_CC_FAT
GO: 0005578~proteinaceous extracellular
32
2.38
0.0047511
matrix
GOTERM_CC_FAT
GO: 0031012~extracellular matrix
32
2.28
0.0051923
GOTERM_CC_FAT
GO: 0044421~extracellular region part
60
1.71
0.0064365
GOTERM_CC_FAT
GO: 0031981~nuclear lumen
65
1.62
0.0102413
GOTERM_CC_FAT
GO: 0043233~organelle lumen
79
1.53
0.0085938
GOTERM_CC_FAT
GO: 0005829~cytosol
45
1.81
0.0100772
GOTERM_CC_FAT
GO: 0070013~intracellular organelle
78
1.52
0.0093866
lumen
GOTERM_CC_FAT
GO: 0031982~vesicle
43
1.83
0.0087123
GOTERM_CC_FAT
GO: 0031974~membrane-enclosed lumen
80
1.50
0.0082696
TABLE 4
Most Significant Gene Sets Enriched in CTC-plt vs CTC-c
q-value < 0.01
Odds
Benjamini
Source
Term
Count
Ratio
(q-value)
GOTERM_BP_FAT
GO: 0042060~wound healing
18
7.8
1.86E−07
GOTERM_BP_FAT
GO: 0007596~blood coagulation
15
10.4
9.31E−08
GOTERM_BP_FAT
GO: 0050817~coagulation
15
10.4
9.31E−08
GOTERM_BP_FAT
GO: 0007599~hemostasis
15
10.3
7.59E−08
GOTERM_BP_FAT
GO: 0050878~regulation of body fluid levels
15
8.2
1.30E−06
GOTERM_BP_FAT
GO: 0030029~actin filament-based process
20
5.5
1.14E−06
GOTERM_BP_FAT
GO: 0007010~cytoskeleton organization
26
3.9
3.95E−06
GOTERM_BP_FAT
GO: 0030036~actin cytoskeleton organization
18
5.3
1.11E−05
GOTERM_BP_FAT
GO: 0009611~response to wounding
26
3.6
1.02E−05
GOTERM_BP_FAT
GO: 0007155~cell adhesion
33
2.9
2.86E−05
GOTERM_BP_FAT
GO: 0022610~biological adhesion
33
2.8
2.70E−05
GOTERM_BP_FAT
GO: 0001775~cell activation
19
3.7
4.70E−04
GOTERM_BP_FAT
GO: 0030168~platelet activation
6
18.2
1.68E−03
GOTERM_BP_FAT
GO: 0007229~integrin-mediated signaling
10
6.4
2.95E−03
pathway
GOTERM_BP_FAT
GO: 0016192~vesicle-mediated transport
25
2.6
3.81E−03
MSigDBv3.1 CGP
GNATENKO PLATELET SIGNATURE
20
55.1
3.91E−24
MSigDBv3.1 CGP
TENEDINI MEGAKARYOCYTE
14
15.3
1.35E−11
MARKERS
MSigDBv3.1
REACTOME FACTORS INVOLVED IN
6
2.9
2.25E−02
CP:REACTOME
MEGAKARYOCYTE DEVELOPMENT
AND PLATELET PRODUCTION
TABLE 5
Most Significant Gene Sets Enriched in CTC-pro vs CTC-c
q-value < 0.01
Odds
Benjamini
Source
Term
Count
Ratio
(q-value)
GOTERM_BP_FAT
GO: 0002495~antigen processing and
5
59.81
6.97E−04
presentation of peptide antigen via MHC class II
GOTERM_BP_FAT
GO: 0019886~antigen processing and
5
59.81
6.97E−04
presentation of exogenous peptide antigen via
MHC class II
GOTERM_BP_FAT
GO: 0002504~antigen processing and
5
50.36
7.34E−04
presentation of peptide or polysaccharide
antigen via MHC class II
GOTERM_BP_FAT
GO: 0002478~antigen processing and
5
41.60
1.10E−03
presentation of exogenous peptide antigen
GOTERM_BP_FAT
GO: 0019884~antigen processing and
5
34.18
1.87E−03
presentation of exogenous antigen
GOTERM_BP_FAT
GO: 0048002~antigen processing and
5
27.34
3.72E−03
presentation of peptide antigen
GOTERM_BP_FAT
GO: 0001775~cell activation
9
7.00
3.82E−03
GOTERM_BP_FAT
GO: 0019882~antigen processing and
6
13.20
7.40E−03
presentation
Example 2
Supplemental Methods
Mice and cell lines. Mice with pancreatic cancer used in these experiments express Cre driven by Pdx1, LSL-Kras G12D , and Trp53 lox/+ or Trp53 lox/lox (otherwise referred to as KPC) as previously described (Bardeesy et al., 2006). EGFP pancreatic lineage tagged KPC mice were generated by breeding the mT/mG mouse (Purchased from the Jackson Laboratory—Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J) into the breeder pairs used for KPC mouse generation. Normal FVB mice were purchased from Jackson Laboratory. All mice care and procedures were done under MGH SRAC approved protocols.
For cardiocentesis, animals were sedated with isofluorane, the chest wall was sterilized with ethanol and a skin incision was made above the rib cage to expose the thoracic cavity and eliminate normal skin epithelial cell contamination. A 23-gauge needle was used to draw approximately 1 mL of blood into a 1 mL syringe primed with 100 μL of PBS-10 mM EDTA pH 7.4 (Gibco). Blood EDTA concentration was raised to 5 mM by either the addition of a concentrated bolus of 500 mM EDTA or 1:1 dilution with 10 mM EDTA. Animals were then euthanized per animal protocol guidelines.
A mouse pancreatic cell line NB508 (Pdx1-Cre/Kras G12D /Trp53 lox/+ ) previously generated from primary tumors developed in this endogenous model was GFP transfected by lentivirus (NB508-GFP). This cell line was used for spiked cell experiments and orthotopic tumor formation.
NB508-GFP Cell lines were maintained in standard culture conditions using RPMI-1640 medium+10% FBS+1% Pen/Strep (Gibco/Invitrogen).
For orthotopic experiments, NB508-GFP cells were orthotopically injected into the pancreas of healthy syngeneic (FVB background) mice. Briefly, mice were anesthesized with isofluorane and the left abdominal wall was treated with Nair® hair removal product, and sterilized with 70% ethanol. A small incision was made on the upper left lateral abdominal wall and the pancreas was mobilized. Approximately 1 million NB508-GFP cells in PBS in a total volume of 0.1 mL was injected into the pancreas. The peritoneum and abdominal wall was closed by sterile surgical staples. The tumors were allowed to grow for 2 weeks, at which time blood was obtained by cardiocentesis for CTC-iChip processing.
Adaptation of CTC Enrichment Technology.
Given the desire for an unbiased enrichment system, the negative depletion technology was selected for this application. All processing protocols were identical to those previously identified, except a rat anti-mouse CD45 antibody (BAM114, R&D Systems, USA) was conjugated to MyOne beads.
Spiked cell experiments were conducted to validate the system by spiking ˜1000 GFP expressing NB508 cells into 1 mL of healthy mouse blood and processing to determine recovery efficiency. Orthotopic models were used to validate recovery efficiency as well as initially determine expected depletion efficiency from tumor-bearing mice. In these experiments, enriched samples were evaluated for the number of GFP+ cells observed in the product.
Immunostaining of CTCs Isolated from the Endogenous Model.
Isolated CTCs were spun onto glass slides and immunostained using a primary-secondary approach. Primary antibodies were rabbit anti-wide spectrum cytokeratin (1:50, Abcam ab9377), and goat anti-mouse CD45 (1:500, R&D systems AF114). Secondary immunofluorescent-tagged antibodies were used for signal amplification. These were donkey anti-rabbit Alexa Fluor 594 (1:500, Invitrogen A-21207), and donkey anti-goat Alexa Fluor 488 (1:500, Invitrogen A-11055). Nuclei were then counterstained with DAPI and the slides were rinsed with PBS, cover slipped and stored at 4° C. They were imaged under 10× magnification using the BioView™ Ltd. automated imaging system (Billerica, Mass.) as well as an automated upright fluorescence microscope (Eclipse 90i™, Nikon, Melville, N.Y.). Positive staining for CK, without CD45 staining, was required for scoring potential CTCs, which were then manually reviewed. Threshold and baseline signals were established using specimens from non-tumor bearing mice.
Single Cell Micromanipulation.
After whole blood anti-CD45 negative depletion, the product containing enriched cells was collected in a 35 mm petri dish and viewed using a Nikon Eclipse Ti™ inverted fluorescent microscope. Cells of interest were identified based on intact cellular morphology and lack of labeling with anti-CD45 magnetic beads. These target cells were individually micromanipulated with a 10 μm transfer tip on an Eppendorf TransferMan® NK 2 micromanipulator and ejected into PCR tubes containing RNA protective lysis buffer (10×PCR Buffer II, 25 mM MgCl2, 10% NP40, 0.1 M DTT, SUPERase-In, Rnase Inhibitor, 0.5 uM UP1 Primer, 10 mM dNTP and Nuclease-free water) and immediately flash frozen in liquid nitrogen.
Single Cell Amplification and Sequencing.
Single cell amplification and sequencing were done as previously described (Tang et al., 2010) with slight modifications underlined below. RNA samples from extracted single circulating tumor cells were thawed on ice and incubated at 70° C. for 90 seconds. To generate cDNA, samples were treated with reverse transcription master mix (0.05 uL RNase inhibitor, 0.07 uL T4 gene 32 protein, and 0.33 uL SuperScript™ III Reverse Transcriptase per 1× volume) and incubated on thermocycler at 50° C. for 30 minutes and 70° C. for 15 minutes. To remove free primer, 1.0 uL of EXOSAP mix was added to each sample, which was incubated at 37° C. for 30 minutes and inactivated at 80° C. for 25 minutes. Next, a 3′-poly-A tail was added to the cDNA in each sample by incubating in master mix (0.6 uL 10×PCR Buffer II, 0.36 uL 25 mM MgCl 2 , 0.18 uL 100 mM dATP, 0.3 uL Terminal Transferase, 0.3 uL RNase H, and 4.26 uL H 2 O per 1× volume) at 37° C. for 15 minutes and inactivated at 70° C. for 10 minutes. A second strand cDNA was synthesis by dividing each sample into 4 and incubating in master mix (2.2 uL 10× High Fidelity PCR Buffer, 1.76 uL 2.5 mM each dNTP, 0.066 uL UP2 Primer at 100 uM, 0.88 uL 50 mM MgSO 4 , 0.44 uL Platinum Taq DNA Polymerase, and 13.654 uL H 2 O per 1× volume) at 95° C. for 3 minutes, 50° C. for 2 minutes, and 72° C. for 10 minutes.
PCR amplification (95° C. for 3 minutes, 20 cycles of 95° C. for 30 seconds, 67° C. for 1 minute, and 72° C. for 6 minutes 6 seconds) was performed with master mix (4.1 uL 10× High Fidelity PCR Buffer, 1.64 uL 50 mM MgSO 4 , 4.1 uL 2.5 mM each dNTP, 0.82 uL AUP1 Primer at 100 uM, 0.82 uL AUP2 Primer at 100 uM, 0.82 uL Platinum Taq DNA Polymerase, and 6.7 uL H 2 O per 1× volume). The 4 reactions of each sample were pooled and purified using the QIAGEN PCR Purification Kit (Cat. No 28106) and eluted in 50 uL EB buffer. Samples were selected by testing for genes Gapdh, ActB, Ptprc (CD45), Krt8, Krt18, Krt19, and Pdx1 using qPCR. Each sample was again divided in 4 and a second round of PCR amplification (9 cycles of 98° C. for 3 minutes, 67° C. for 1 minute, and 72° C. for 6 minutes 6 seconds) was performed with master mix (9 uL 10× High Fidelity PCR Buffer, 3.6 uL 50 mM MgSO 4 , 13.5 uL 2.5 mM each dNTP, 0.9 uL AUP1 Primer at 100 uM, 0.9 uL AUP2 Primer at 100 uM, 1.8 uL Platinum Taq DNA Polymerase, and 59.1 uL H 2 O per 1× volume). Samples were pooled and purified using Agencourt AMPure XP beads and eluted in 40 uL 1× low TE buffer.
Sequencing Library Construction.
To shear the DNA using the Covaris S2™ System, 1× low TE buffer and 1.2 uL shear buffer were added to each sample. Conditions of the shearing program include: 6 cycles, 5° C. bath temperature, 15° C. bath temperature limit, 10% duty cycle, intensity of 5, 100 cycles/burst, and 60 seconds. Then, samples were end-polished at room temperature for 30 minutes with master mix (40 uL 5× Reaction Buffer, 8 uL 10 mM dNTP, 8 uL End Polish Enzyme1, 10 uL End Polish Enzyme2, and 14 uL H 2 O per 1× volume). DNA fragments larger than 500 bp were removed with 0.5× volumes of Agencourt AMPure XP™ beads. Supernatant was transferred to separate tubes. To size-select 200-500 bp DNA products, 0.3× volumes of beads were added and samples were washed 2× with 70% EtOH. The products were eluted in 36 uL low TE buffer. A dA-tail was added to each size-selected DNA by treating with master mix (10 uL 5× Reaction Buffer, 1 uL 10 mM dATP, and 5 uL A-Tailing Enzyme I per 1× volume) and incubated at 68° C. for 30 minutes and cooled to room temperature. To label and distinguish each DNA sample for sequencing, barcode adaptors (5500 SOLiD 4464405) were ligated to DNA using the 5500 SOLiD Fragment Library Enzyme Module™ (4464413). Following barcoding, samples were purified twice using the Agencourt AMPure XP™ beads and eluted in 22 uL low TE buffer. Following a round of PCR Amplification (95° C. for 5 minutes, 12 cycles of 95° C. for 15 seconds, 62° C. for 15 seconds, and 70° C. for 1 minute, and 70° C. for 5 minutes), the libraries were purified with AMPure XP beads. Finally, to quantify the amount of ligated DNA, SOLiD Library TaqMan Quantitation Kit™ was used to perform qPCR. Completed barcoded libraries were then subjected to emulsion PCR with template beads preparation and sequenced on the ABI 5500XL™.
RNA In Situ Hybridization (RNA-ISH).
Paraffin-embedded tissue blocks were freshly cut and frozen at −80° C. Upon removal from the freezer, slides were baked for 1 hr at 60° C. and fixed in %10 formaldehyde for 1 hr at room temperature (RT). Paraffin was removed using Histo-Clear™ and RNA-ISH™ was performed according to the Affymetrix QuantiGene ViewRNA ISH Tissue-2 Plex Assay™. Tissue sections were permeabilized by pretreating in buffer solution for 10 min at 95° C. and digested with protease for 10 min, before being fixed at RT in 5% formaldehyde. Target probe sets were applied and hybridized to the tissue by incubating for 2 hr at 40° C. Type 1 probes were used at a dilution of 1:50 and included Aldh1a2 (VB1-14197), Dcn (VB1-14962), Klf4 (VB1-14988), Igfbp5 (VB1-14987), and Sparc (VB1-14196). Type 6 probes included EGFP (VF6-13336) at 1:50 and pooled Krt8 (VB6-11060) and Krt18 (VB6-11059) at 1:100 each. Signal was amplified through the sequential hybridization of PreAmplifier and Amplifer QT mixes to the target probe set. Target mRNA molecules were detected by applying Type 6 Label Probe with Fast Blue substrate and Type 1 Label Probe with Fast Red substrate. Tissue was counterstained with Gill's Hemotoxylin for 10 sec at RT. DAPI (Invitrogen, D3571; 3.0 μg/ml) staining was performed for 1 min. Fluorescence microscopy using a Nikon 90i was used to visualize target mRNAs. Type 1 probes were detected in the Cy3 channel and Type 6 probes in the Cy5 channel. Merged images were generated using NIS-Elements™ software.
Determination of Reads-Per-Million (rpm)
Color space reads were aligned using Tophat™ version 2.0.4 (Trapnell et al., 2009) and Bowtie1™ version 0.12.7 with the no-novel-juncs argument set with mouse genome version mm9 and transcriptome defined by the mm9 knownGene table from genome.ucsc.edu. Reads that did not align or aligned to multiple locations in the genome were discarded. The mm9 table knownToLocusLink from genome.ucsc.edu was used to map, if possible, each aligned read to the gene who's exons the read had aligned to. The reads count for each gene was the number of reads that were so mapped to that gene. This count was divided by the total number of reads that were mapped to any gene and multiplied by one million to form the reads-per-million (rpm) count. Rpm rather than rpkm was used because a 3′ bias was noted in the alignments.
Unsupervised Hierarchical Clustering and Principal Components Analysis.
The minimum of 1 and the smallest positive value of the rpm matrix was added to the rpm matrix to eliminate zeros. The result was then log 10 transformed, yielding what is termed the log 10(rpm) matrix. The rows (corresponding to genes) of the log 10(rpm) matrix with the top 2000 standard deviations were retained and the rest of the rows discarded. The result was then median polished. The result was clustered using agglomerative hierarchical clustering with average linkage with distance metric equal to 1 minus the Pearson correlation coefficient. The principal components of the log 10(rpm) matrix were computed and the coordinates of the samples with respect to the first three principal components were plotted.
Measures of Cellular Heterogeneity.
For a collection of clusters of samples, a statistic, M, was defined as the mean over the clusters of the mean over all the pairs of samples in the cluster of the atan h of the correlation coefficient between the two columns of the rpm matrix corresponding to the pair. The “mean intra-cluster correlation coefficient” was defined as tan h(M). The jackknife estimator was used with respect to the samples to estimate a standard deviation, s, of the statistic. The 95% CI was defined as tan h (M±sφ −1 (0.975)), where 4 is the cumulative distribution function of the standard normal distribution. To compute a p-value for the null hypothesis that the mean of the distribution of the M statistic for a cluster is the same as the mean of the distribution of the M statistic for a collection of clusters, we let p=2(1−φ(|M1−M2|/√(s 2 1 +s 2 2 ))). Of note, bootstrap was performed on the same data as an alternative to jackknife and similar results obtained (data not shown).
Supervised Differential Gene Expression Using Rank Product.
To find differentially expressed genes between two sets of samples, analysis was begin with the log 10(rpm) matrix defined above. Columns corresponding to samples not in either set of samples were removed. Then removed rows for which the 90 th percentile of the values was less than log 10(10) were removed. The RP function of the Bioconductor (Gentleman et al., 2004) RankProd™ package (version 2.28.0) was used to get FDR estimates for both up and down differential expression. Genes were considered to be differentially expressed if their FDR estimate was less than 0.01, but discarded if they were both up and down differentially expressed, if there were any.
Gene Set Enrichment.
Enrichment was considered in four gene set collections: (1) all of KEGG™, as found in DAVID™ 6.7 (Huang da et al., 2009), (2) Gene Ontology (GO) using GO_BP as found in DAVID 6.7, and (3) GO_CC as found in DAVID 6.7. Sets of genes found to be differentially expressed were tested for enrichment in the gene set collections using a hypergeometric test for each gene set in the collection. The resulting p-values for each collection were converted to FDR estimates using the Benjamini-Hochberg method (Benjamini and Hochberg, 1995).
Digital Removal of all Annotated Platelet Transcripts
The 446 genes whose expression in the log 10(rpm) matrix had an absolute value of correlation coefficient greater than 0.6 with the expression of any of the genes in the gene sets named GNATENKO_PLATELET_SIGNATURE and TENEDINI_MEGAKARYOCYTE_MARKERS in MSigDB v3.1 were removed from the log 10(rpm) matrix (defined above). Clustering was then performed as described above.
SUPPLEMENTAL METHODS REFERENCES
Bardeesy, N., Aguirre, A. J., Chu, G. C., Cheng, K. H., Lopez, L. V., Hezel, A. F., Feng, B., Brennan, C., Weissleder, R., Mahmood, U., et al. (2006). Both p16(Ink4a) and the p19(Arf)-p53 pathway constrain progression of pancreatic adenocarcinoma in the mouse. Proc Natl Acad Sci USA 103, 5947-5952.
Benjamini, Y., and Hochberg, Y. (1995). Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B (Methodological) 57, 289-300.
Gentleman, R. C., Carey, V. J., Bates, D. M., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B., Gautier, L., Ge, Y., Gentry, J., et al. (2004). Bioconductor: open software development for computational biology and bioinformatics. Genome biology 5, R80.
Huang da, W., Sherman, B. T., and Lempicki, R. A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4, 44-57.
Tang, F., Barbacioru, C., Nordman, E., Li, B., Xu, N., Bashkirov, V. I., Lao, K., and Surani, M. A. (2010). RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nat Protoc 5, 516-535.
Trapnell, C., Pachter, L., and Salzberg, S. L. (2009). TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105-1111.
Example 3
A comparative analysis of mouse pancreatic CTCs indicated an enrichment of 60 extracellular proteins (Table 6). Evaluation of these particular biomarkers and therapeutic targets was undertaken in human pancreatic circulating tumor cells and the most abundant targets in human pancreatic CTCs are shown ( FIG. 7 ). These not only represent potential biomarkers, but given their nature as proteins on the external surface of tumor cells, they are therapeutic targets. The extracellular proteins of Table 6 can be targeted, e.g. by antibody-based therapeutics (e.g. as in the cases of trastuzumab for HER2, cetuximab for EGFR, and bevacizumab for VEGF) to treat cancer.
TABLE 6
List of Pancreatic CTC enriched Extracellular Proteins.
OFFICIAL GENE
SYMBOL
Gene Name
Abi3bp
ABI gene family, member 3 (NESH) binding protein
Adamts5
similar to a disintegrin-like and metalloprotease (reprolysin type) with
thrombospondin type 1 motif, 5 (aggrecanase-2); a disintegrin-like and
metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 5
(aggrecanase-2)
Adamtsl1
ADAMTS-like 1
Ang
angiogenin, ribonuclease, RNase A family, 5
Arsa
arylsulfatase A
C1rl
complement component 1, r subcomponent-like
C3
complement component 3; similar to complement component C3
prepropeptide, last
C4a
similar to Complement C4 precursor; complement component 4A (Rodgers
blood group); similar to complement C4; complement component 4B
(Childo blood group)
C4b
similar to Complement C4 precursor; complement component 4A (Rodgers
blood group); similar to complement C4; complement component 4B
(Childo blood group)
Ccdc80
coiled-coil domain containing 80
Cd109
CD109 antigen
Chi3l1
chitinase 3-like 1
Clec3b
C-type lectin domain family 3, member b
Cmtm3
CKLF-like MARVEL transmembrane domain containing 3
Cmtm7
CKLF-like MARVEL transmembrane domain containing 7
Col14a1
collagen, type XIV, alpha 1
Col1a2
collagen, type I, alpha 2
Col3a1
collagen, type III, alpha 1
Col4a6
collagen, type IV, alpha 6
Csf1
colony stimulating factor 1 (macrophage)
Dag1
dystroglycan 1
Dcn
decorin
Dmkn
dermokine
Fbln1
fibulin 1
Fgf1
fibroblast growth factor 1
Fmod
fibromodulin
Gpc3
glypican 3
Gpc4
glypican 4; similar to Glypican 4
Hmgb1
high mobility group box 1
Ifnar2
interferon (alpha and beta) receptor 2
Igfbp5
insulin-like growth factor binding protein 5
Il16
interleukin 16
Lama4
laminin, alpha 4
Ltbp4
latent transforming growth factor beta binding protein 4
Mfap1a
similar to microfibrillar-associated protein 1A; microfibrillar-associated
protein 1A; microfibrillar-associated protein 1B
Nid2
nidogen 2
Ogn
osteoglycin
Pdap1
PDGFA associated protein 1
Pf4
platelet factor 4
Plat
plasminogen activator, tissue
Podn
podocan
Prelp
proline arginine-rich end leucine-rich repeat
Rspo1
R-spondin homolog ( Xenopus laevis )
Serping1
serine (or cysteine) peptidase inhibitor, clade G, member 1
Slurp1
secreted Ly6/Plaur domain containing 1
Sod3
superoxide dismutase 3, extracellular
Sparc
secreted acidic cysteine rich glycoprotein; similar to Secreted acidic cysteine
rich glycoprotein
Spock2
sparc/osteonectin, cwcv and kazal-like domains proteoglycan 2
Spon2
spondin 2, extracellular matrix protein
Sulf1
sulfatase 1
Sulf2
sulfatase 2
Tgfb2
transforming growth factor, beta 2
Tgm2
transglutaminase 2, C polypeptide
Thbd
thrombomodulin
Thbs1
thrombospondin 1; similar to thrombospondin 1
Thsd4
thrombospondin, type I, domain containing 4
Timp2
tissue inhibitor of metalloproteinase 2
Tnxb
tenascin XB
Tpt1
predicted gene 1974; tumor protein, translationally-controlled 1
pseudogene; tumor protein, translationally-controlled 1; predicted gene
14456
Twsg1
twisted gastrulation homolog 1 ( Drosophila )
Wnt4
wingless-related MMTV integration site 4
Extending these CTC enriched genes to human pancreatic, breast, and prostate single cell CTC data identified 5 candidate genes shown in Table 9.
TABLE 9
Percent of human single CTCs with high expression by RNA-seq
Percent of Single CTCs >50 RPM of
Expression
Pancreas
Breast
Prostate
ALL
Cancer Type
(N = 7)
(N = 29)
(N = 77)
(N = 113)
TPT1
86%
90%
90%
89%
HMGB1
43%
62%
44%
49%
SPON2
43%
7%
45%
35%
SPARC
100%
41%
9%
23%
ARSA
71%
17%
5%
12%
Focusing on pancreatic cancer, SPARC was selected as an initial gene to evaluate. SPARC RNA-ISH in mouse and human primary tumors (data not shown) demonstrated significant expression in the stromal cells of the tumor that provides essential microenvironmental signals to tumors. Much effort in the field focuses on targeting the stroma of PDAC for therapeutic efficacy [1-4] making SPARC a CTC therapeutic target as well as a stromal directed target. A total of 196/198 (99%) of human pancreatic tumors were positive for SPARC and 36% with clear epithelial tumor cell expression.
Evaluation of human pancreatic cancer cell lines identified 3 of 5 cell lines with elevated SPARC expression which correlates to increased migratory behavior, a surrogate in vitro assay that correlates with metastatic behavior ( FIG. 8 ).
Evaluation of SPARC function in human pancreatic cancer was done using short hairpin RNA interferences (shRNA) on the two cell lines with highest SPARC expression (PDAC2 and PDAC3). Multiple in vitro assays were done including proliferation, migration, invasion, scratch, and soft agar. The most profound effects of suppressing SPARC expression was on migratory behavior ( FIG. 9 and data not shown), indicating SPARC is not only present in many CTCs, but has functional consequences when inhibited in cell line models.
Given these data, in vivo tail vein inoculation was performed using PDAC-3 to determine if SPARC knockdown affected metastasis. Initial data at 2 weeks post tail vein injection indicates there is reduced metastatic potential when SPARC is inhibited by shRNA with 83% of control mice with metastases compared to 40% in cell lines with shRNA against SPARC ( FIG. 10 ).
Surface Protein Targets
Most of the targets identified in Table 9 are secreted factors and analysis of genes annotated as cell surface proteins are summarized in Table 14.
TABLE 14
Percent of human single CTCs with high expression
of surface protein genes
Percent of Single CTCs >50 RPM of Expression
Cancer
Pancreas
Breast
Prostate
type
(N = 7)
(N = 29)
(N = 77)
ALL (N = 113)
IL6ST
0%
38%
8%
15%
ARSA
71%
17%
5%
12%
TIMP2
0%
21%
4%
8%
CD55
0%
17%
4%
7%
SULF2
0%
24%
0%
6%
ITGA6
0%
14%
3%
5%
SDC4
0%
14%
3%
5%
CDON
0%
7%
5%
5%
SV2A
14%
3%
1%
3%
It is contemplated herein that these genes are targets given they would be integrated into the plasma membrane of CTCs. In general, RNA expression of cell surface markers tend to be lower than actual protein levels on cells.
Contemplated herein are antibodies to IL6ST, SULF2, and SV2A for therapeutic utility.
1. IL6ST—signal transducer for IL6, LIF, CNTF, and oncostatin M.
a. Important for STAT3 activation downstream b. Antibodies against IL6 receptor and IL6 have been developed for human disease including cancer
2. SULF2—sulfatase modifies heparin sulfate by removing 6-O-sulfate groups
a. Expression enriched in cancer progression and metastasis b. Drugs have been developed against sulfatase activity and tested with activity in liver cancer models
3. SV2A—synaptic vesicle glycoprotein elevated in neuroendocrine cells
a. A marker of neuroendocrine cells, which appear at the epithelial stromal border of human pancreatic cancer b. Neuroendocrine differentiation common feature in cancers and portends to more aggressive disease
REFERENCES
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8. Betts, B. C., E. T. St Angelo, M. Kennedy, and J. W. Young, Anti - IL 6- receptor - alpha ( tocilizumab ) does not inhibit human monocyte - derived dendritic cell maturation or alloreactive T - cell responses . Blood, 2011. 118(19): p. 5340-3.
9. Bayliss, T. J., J. T. Smith, M. Schuster, K. H. Dragnev, and J. R. Rigas, A humanized anti - IL -6 antibody ( ALD 518) in non - small cell lung cancer . Expert Opin Biol Ther, 2011. 11(12): p. 1663-8.
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11. Lui, N. S., A. van Zante, S. D. Rosen, D. M. Jablons, and H. Lemjabbar-Alaoui, SULF 2 expression by immunohistochemistry and overall survival in oesophageal cancer: a cohort study . BMJ Open, 2012. 2(6).
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Example 5
Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, genomewide expression profiles of CTCs were compared with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low proliferative signatures, enrichment for Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.
Classical CTCs expressed predominantly the Aldh1a2 isoform, while Aldh1a1 was expressed in a variety of cell types (data not shown). Within single CTCs, there was no correlation between expression of Aldh1 isoforms and either enrichment for the mesenchymal genes (Cdh11, Vim) or loss of epithelial genes (Cdh1, Muc1), indicating that stem cell and EMT markers are not intrinsically linked in CTCs. Analysis of primary pancreatic tumors for Aldh1a2 using RNA in situ hybridization (RNA-ISH) identified rare epithelial tumor cells expressing this stem cell marker, but the majority of expression was present within the cancer associated stromal cells ( FIG. 12A ), consistent with immunohistochemistry for ALDH protein in human PDAC (Rasheed et al., 2010).
Besides the evident diversity of CTCs, shared transcripts were searched for that could provide further insight into their cell of origin within the primary tumor and the mechanisms by which they invade and survive within the bloodstream and ultimately identify potential CTC-specific therapeutic targets. Rigorous criteria were selected to identify the most highly enriched CTC-c transcripts (RP score<300), expressed at very high levels (>100 rpm) in R90% of all classical CTCs. Three genes met these criteria: Kruppel-like factor 4 (Klf4), one of the key stem cell (iPS) reprogramming factors (Takahashi and Yamanaka, 2006), insulin-like growth factor binding protein 5 (Igfbp5), an extracellular growth factor binding protein and decorin (Dcn). RNA-ISH was utilized in primary tumor specimens to identify the potential colocalization of these three highly enriched CTC genes. In contrast to Aldh1a2, Klf4 is expressed in epithelial components of the primary tumor ( FIG. 12B ). Igfbp5 is of particular interest, in that it is expressed focally at the tumor epithelial-stromal interface ( FIG. 12C ). It is contemplated herein that this geographic area is enriched for cancer cells undergoing EMT, contributing to the mixed epithelial/stromal transcriptional programs evident by RNA-seq of single CTCs.
In addition to highly expressing Dcn, CTCs consistently had high levels of multiple ECM gene transcripts. GO analysis of all CTC-enriched genes (Table 3) identified 32 proteinaceous ECM genes (GO:0005578, OR 2.4, q-value 4.8 3 10.3). These genes are normally expressed in reactive stromal cells, rather than in epithelial cancer cells, and while recent studies have highlighted the importance of the stroma in supporting pancreatic cancer pathogenesis and metastasis (Feig et al., 2012; Neesse et al., 2011, 2013; Olive et al., 2009; Provenzano et al., 2012), the expression of these stroma-associated ECM genes within tumor cells in circulation was unexpected. Using RP differential expression analysis, CTCs were compared with purified EGFP-tagged primary tumor single cells (TuGMP3) and bulk tumor samples (tumor cells admixed with reactive stromal cells). Six proteinaceous ECM genes were highly expressed by CTCs and by stromal component, but not by epithelial cells within primary tumors: Dcn, Sparc, Ccdc80, Col1a2, Col3a1, and Timp2 (data not shown). RNA-ISH analysis of both Dcn and Spare confirmed diffuse expression in stromal elements of mouse primary tumors, with rare areas where these transcripts are colocalized with keratin-expressing cells at the epithelial-stromal border (data not shown).
SPARC is a ECM protein gene. RNA-ISH analysis of 198 primary human PDACs demonstrates abundant stromal cell expression of SPARC transcripts in 99% of cases, with up to a third of tumors with rare epithelial cells expressing this ECM gene product (data not shown). Consistent with these observations, RNA-seq of EGFP-tagged single primary tumor cells (data not shown) identified only 1 of 20 cells (5%) with coexpression of high levels (>100 rpm) of Spare and Krt19.
In summary, abundant expression of ECM genes is a common feature of all keratin-rich classical CTCs. This is in marked contrast to the primary tumor, where these gene products are secreted by supporting stromal cells and not by the epithelial cancer cells. However, rare cells at the epithelial-stromal interface of primary tumors do appear to express both keratins and ECM genes, consistent with the pattern observed in CTCs themselves.
To confirm the expression of proteinaceous ECM genes by human cancer cells circulating in the bloodstream, single CTCs were isolated from patients with pancreatic (n=7), breast (n=29), and prostate (n=77) cancers and subjected these to single-cell RNA-seq. Six ECM protein genes were highly expressed in human CTCs (>100 rpm in >15% of all CTC samples) ( FIG. 13 ; Table 13). Notably, three genes (SPARC, MGP, SPON2) are ECM glycoproteins, defined as part of the core matrisome (Naba et al., 2012). The core matrisome protein SPARC was particularly enriched in pancreatic CTCs being expressed at high levels (>100 rpm) in 100% of pancreatic CTCs compared to 31% of breast and 9% of prostate CTCs. The notable differences in ECM protein gene expression across human epithelial CTCs suggest microenvironment tissue specificity as well as probable redundancies in ECM protein signaling. Together, the consistent expression of ECM gene family members in human CTCs indicates that their upregulation contributes either to the generation of CTCs from primary tumors or to the survival of cancer cells deprived of microenvironmental signals as they circulate in the bloodstream.
In order to define the functional consequences of SPARC expression in pancreatic cancer cells, a panel of patient-derived, low-passage PDAC cell lines was screened for expression. Two human PDAC cell lines with relatively high SPARC expression were identified (PDAC2 and PDAC3), making it possible to test the consequences of small hairpin RNA (shRNA)-mediated knockdown ( FIG. 8, 9 , FIGS. 16A-16D ). Suppression of endogenous SPARC expression in both PDAC2 and PDAC3 cell lines using two independent shRNA constructs did not affect proliferation in 2D cultures or anchorage-independent tumor sphere formation ( FIGS. 14A-14B , FIGS. 16A-16D ). However, SPARC knockdown by both shRNAs significantly reduced pancreatic cancer cell migration in wound scratch assays and their invasive properties, as measured by in vitro Boyden assays (data not shown).
Tail vein injection of SPARC-suppressed PDAC3 cells using both shRNA constructs generated significantly fewer lung metastases than cells expressing nontargeting hairpin (shNT) controls ( FIG. 14D ). Metastases generated from orthotopic pancreatic xenografts were also significantly reduced for SPARC-suppressed PDAC3 cells, as measured by luciferase imaging and normalized for primary tumor size ( FIG. 14E ). Thus, SPARC expression by pancreatic cancer cells appears to selectively enhance their invasive and migratory properties to augment metastatic virulence. The high levels of SPARC expression evident in virtually all pancreatic CTCs thus raises the possibility that it contributes significantly to the metastatic spread of pancreatic cancer.
Discussion
Described herein is the detailed analysis of CTC composition and diversity in pancreatic cancer, using single-cell RNA-seq. High-quality transcriptomes were achieved in 93 single mouse pancreatic CTCs, which were compared with bulk and single-cell preparations from matched primary tumors and from an immortalized cell line established from the same mouse pancreatic tumor model. The use of the KPC mouse model made it possible to compare simultaneously isolated primary tumor specimens and CTCs, and it allowed measurements of CTC heterogeneityacross multiple mice sharing the same Kras/Trp53 genetic drivers. The large number of isolated CTCs and the high quality of the isolated RNA from these cells reflect the application of the CTC-iChip technology, which effectively depletes normal blood components, enriching for CTCs that are untagged and accessible for single-cell manipulation. Finally, the purification of CTCs irrespective of their cell-surface epitopes avoids any bias associated with their purification based on expression of common epithelial markers such as EpCAM.
Together, the observations made herein include the following. (1) CTC expression profiles cluster into three classes, including a major “classical CTC” group, and others that are defined by platelet derived markers or proliferative signatures. (2) Common features shared by virtually all classical CTCs include expression of both epithelial and mesenchymal markers, the stem cell-associated gene Aldh1a2, and three highly expressed transcripts, Klf4, Igfbp5, and Dcn. The specific localization of Igfbp5-expressing cells at the epithelial-stromal boundary within primary tumors may point to a region that contributes significantly to CTC generation. (3) The most highly enriched CTC-specific transcripts shared by almost all classical CTCs encode extracellular matrix proteins, such as Sparc. (4) Aberrant expression in CTCs of this ECM gene product, which is normally abundant in the tumor stromal compartment, is observed in both mouse and human pancreatic CTCs, and its knockdown attenuates cancer cell migration and invasion in reconstituted systems. ( FIG. 15 ) Compared with RNA-seq of partially purified, bulk CTC populations, which required digital subtraction of leukocyte-derived reads (Yu et al., 2012, 2013), the single-cell analysis reported here provides considerably more depth of tumor cell-specific transcript reads, and it allows measurements of CTC heterogeneity.
It is contemplated herein that in addition to the initiating mutations, somatically acquired genetic and epigenetic changes may distinguish CTCs derived from different tumors. Multiple mouse tumors contributed to each of the three distinct clusters of CTCs. Despite their atypical expression pattern, the identification of platelet-associated and proliferative CTC subsets as being tumor-derived is established by their inclusion of lineage-tagged tumor cells. The more characteristic expression pattern exhibited by the classical CTC cluster enabled detailed comparison with primary tumor cells, thereby providing further insight into the origin and properties of CTCs.
Mouse pancreatic classical CTCs uniformly lose expression of the epithelial marker E-cadherin (Cdh1), a key feature of epithelial-to-mesenchymal transition. However, the cells do not lose expression of other epithelial markers, such as cytokeratins, nor is there a consistent increase in classical mesenchymal markers such as vimentin. As such, most classical CTCs appear arrested in a biphenotypic state. Despite their expression of cytokeratins, which are present in the epithelial components of the primary tumor, most other highly expressed markers in CTCs are shared with the stromal component of the primary tumor. Among these stromal genes is Aldh1a2 (Rasheed and Matsui, 2012; Rasheed et al., 2010). A provocative observation relating to the shared epithelial and mesenchymal state of classical CTCs is their virtually universal (93%) expression of Igfbp5, which is uniquely expressed in a small subpopulation of cells at the epithelial/stromal interface within primary tumors. This raises the possibility that this critical location within the primary tumor generates a disproportionate fraction of viable CTCs.
The most unexpected observation from the single-CTC RNAseq study is the high abundance of ECM transcripts in the vast majority of classical CTCs. The coexpression of pancreatic cancer-enriched cytokeratins (Krt7 and Krt19) in single cells expressing these ECM gene products excludes the possibility that these represent circulating tumor-derived fibroblasts.
Consistent with the aberrant expression of SPARC in some pancreatic cancer cells, a subset of patient-derived tumor cell lines also coexpress it along with epithelial cytokeratins. The reduction in cell migration and metastatic potential exhibited by these pancreatic cell lines following SPARC knockdown indicates that it contributes to CTC-mediated metastasis. It is contemplated herein that Sparc expression contributes to metastasis, but inherent redundancies in ECM protein expression may mitigate this effect in some embodiments.
Considerable effort has been directed to targeting the pancreatic cancer stroma as a means of improving delivery of chemotherapeutics as well as stripping tumor cells of their supportive microenvironment (Neesse et al., 2011; Olive et al., 2009; Provenzano et al., 2012; Rasheed et al., 2012). The findings described herein, e.g., that these gene products are also expressed by CTCs themselves suggests a remarkable level of cellular plasticity. To the extent that invasive properties of CTCs are mediated in part by expression of such ECM proteins, it also raises the possibility of targeting cancer cells in the blood.
TABLE 13
Human CTC ECM Gene Expression
Percent of Samples >100 RPM
ECM Gene
Prostate
Count
Symbol
All CTCs
PDAC CTCs
Breast CTCs
CTCs
1
ANXA2
36.3%
0.0%
51.7%
33.8%
2
SPON2
29.2%
0.0%
3.4%
41.6%
3
LGALS3
22.1%
42.9%
37.9%
14.3%
4
SPARC
21.2%
100.0%
31.0%
10.4%
5
LGALS3BP
16.8%
0.0%
34.5%
11.7%
6
MGP
15.9%
57.1%
44.8%
1.3%
7
LAMC1
15.0%
0.0%
6.9%
19.5%
8
SMC3
15.0%
42.9%
17.2%
11.7%
9
CALR
14.2%
0.0%
6.9%
18.2%
10
TIMP1
13.3%
14.3%
27.6%
7.8%
11
MMP24
11.5%
0.0%
10.3%
13.0%
12
DAG1
10.6%
0.0%
20.7%
7.8%
13
ERBB2IP
10.6%
14.3%
20.7%
6.5%
14
MMP19
10.6%
0.0%
10.3%
11.7%
15
AGRN
8.8%
0.0%
6.9%
10.4%
16
CRTAP
8.8%
0.0%
6.9%
10.4%
17
COL24A1
8.0%
57.1%
17.2%
0.0%
18
ANG
7.1%
0.0%
0.0%
10.4%
19
MFAP1
7.1%
0.0%
6.9%
7.8%
20
VWF
7.1%
14.3%
17.2%
2.6%
21
VWA1
7.1%
0.0%
3.4%
9.1%
22
TIMP2
6.2%
0.0%
13.8%
3.9%
23
ECM1
6.2%
0.0%
24.1%
0.0%
24
LTBP1
6.2%
28.6%
10.3%
2.6%
25
LGALS1
6.2%
0.0%
10.3%
5.2%
26
SERPINA1
6.2%
0.0%
20.7%
1.3%
27
SPOCK1
6.2%
14.3%
0.0%
7.8%
28
TFF3
6.2%
0.0%
17.2%
2.6%
29
NPNT
5.3%
0.0%
3.4%
6.5%
30
TFIP11
5.3%
14.3%
6.9%
3.9%
31
COL9A2
4.4%
0.0%
0.0%
6.5%
32
COL6A1
4.4%
0.0%
0.0%
6.5%
33
FN1
4.4%
14.3%
10.3%
1.3%
34
LAD1
4.4%
0.0%
10.3%
2.6%
35
LAMA1
4.4%
14.3%
3.4%
3.9%
36
LAMB2
4.4%
0.0%
10.3%
2.6%
37
MATN2
4.4%
14.3%
3.4%
3.9%
38
ZP3
4.4%
0.0%
0.0%
6.5%
39
ADAMTSL3
3.5%
28.6%
3.4%
1.3%
40
FRAS1
3.5%
14.3%
0.0%
3.9%
41
TIMP3
3.5%
0.0%
3.4%
3.9%
42
DST
3.5%
0.0%
6.9%
2.6%
43
GFOD2
3.5%
14.3%
0.0%
3.9%
44
LAMA3
3.5%
14.3%
0.0%
3.9%
45
LAMB1
3.5%
14.3%
0.0%
3.9%
46
MMP7
3.5%
0.0%
0.0%
5.2%
47
ANGPTL4
2.7%
0.0%
0.0%
3.9%
48
BMP4
2.7%
0.0%
0.0%
3.9%
49
LTBP2
2.7%
28.6%
3.4%
0.0%
50
LEPRE1
2.7%
0.0%
0.0%
3.9%
51
LUM
2.7%
0.0%
0.0%
3.9%
52
NID2
2.7%
14.3%
6.9%
0.0%
53
SLC1A3
2.7%
28.6%
0.0%
1.3%
54
TECTA
2.7%
14.3%
3.4%
1.3%
55
THSD4
2.7%
0.0%
6.9%
1.3%
56
ADAMTS15
1.8%
0.0%
6.9%
0.0%
57
USH2A
1.8%
14.3%
3.4%
0.0%
58
APLP1
1.8%
0.0%
0.0%
2.6%
59
COL4A3
1.8%
14.3%
3.4%
0.0%
60
COL7A1
1.8%
0.0%
3.4%
1.3%
61
COL11A1
1.8%
0.0%
6.9%
0.0%
62
COL11A2
1.8%
0.0%
0.0%
2.6%
63
COL15A1
1.8%
28.6%
0.0%
0.0%
64
CTGF
1.8%
0.0%
0.0%
2.6%
65
CRISP3
1.8%
0.0%
0.0%
2.6%
66
DCN
1.8%
0.0%
0.0%
2.6%
67
ENTPD2
1.8%
0.0%
0.0%
2.6%
68
FMOD
1.8%
0.0%
3.4%
1.3%
69
GPC1
1.8%
0.0%
0.0%
2.6%
70
HSPG2
1.8%
0.0%
0.0%
2.6%
71
LAMA5
1.8%
0.0%
3.4%
1.3%
72
LAMC2
1.8%
14.3%
0.0%
1.3%
73
MMP10
1.8%
0.0%
3.4%
1.3%
74
MMP12
1.8%
0.0%
0.0%
2.6%
75
NTN4
1.8%
0.0%
6.9%
0.0%
76
NAV2
1.8%
0.0%
6.9%
0.0%
77
PAPLN
1.8%
0.0%
3.4%
1.3%
78
SFTPA2
1.8%
0.0%
0.0%
2.6%
79
VCAN
1.8%
14.3%
0.0%
1.3%
80
ADAMTS13
0.9%
0.0%
3.4%
0.0%
81
ADAMTS3
0.9%
14.3%
0.0%
0.0%
82
ADAMTS5
0.9%
14.3%
0.0%
0.0%
83
ADAMTSL4
0.9%
0.0%
0.0%
1.3%
84
EFEMP1
0.9%
0.0%
3.4%
0.0%
85
EFEMP2
0.9%
0.0%
3.4%
0.0%
86
EGFLAM
0.9%
14.3%
0.0%
0.0%
87
KAL1
0.9%
0.0%
0.0%
1.3%
88
KAZALD1
0.9%
0.0%
0.0%
1.3%
89
MAMDC2
0.9%
14.3%
0.0%
0.0%
90
SMOC1
0.9%
0.0%
0.0%
1.3%
91
SMOC2
0.9%
0.0%
0.0%
1.3%
92
ACHE
0.9%
0.0%
0.0%
1.3%
93
AMTN
0.9%
0.0%
3.4%
0.0%
94
ANXA2P2
0.9%
0.0%
3.4%
0.0%
95
CPZ
0.9%
0.0%
3.4%
0.0%
96
CHADL
0.9%
0.0%
0.0%
1.3%
97
COCH
0.9%
0.0%
0.0%
1.3%
98
COL6A6
0.9%
14.3%
0.0%
0.0%
99
COL1A2
0.9%
0.0%
3.4%
0.0%
100
COL2A1
0.9%
0.0%
0.0%
1.3%
101
COL4A1
0.9%
14.3%
0.0%
0.0%
102
COL4A2
0.9%
0.0%
0.0%
1.3%
103
COL4A6
0.9%
0.0%
0.0%
1.3%
104
COL5A1
0.9%
14.3%
0.0%
0.0%
105
COL6A2
0.9%
0.0%
0.0%
1.3%
106
COL8A1
0.9%
14.3%
0.0%
0.0%
107
COL12A1
0.9%
14.3%
0.0%
0.0%
108
COL14A1
0.9%
14.3%
0.0%
0.0%
109
COL19A1
0.9%
14.3%
0.0%
0.0%
110
COL17A1
0.9%
14.3%
0.0%
0.0%
111
COL22A1
0.9%
14.3%
0.0%
0.0%
112
ENTPD1
0.9%
14.3%
0.0%
0.0%
113
FBN2
0.9%
0.0%
0.0%
1.3%
114
FBN3
0.9%
0.0%
3.4%
0.0%
115
FBLN1
0.9%
14.3%
0.0%
0.0%
116
FBLN7
0.9%
0.0%
0.0%
1.3%
117
GPC4
0.9%
0.0%
3.4%
0.0%
118
HMCN1
0.9%
14.3%
0.0%
0.0%
119
IMPG1
0.9%
14.3%
0.0%
0.0%
120
IMPG2
0.9%
0.0%
3.4%
0.0%
121
LAMA2
0.9%
0.0%
3.4%
0.0%
122
LAMB3
0.9%
14.3%
0.0%
0.0%
123
MEPE
0.9%
0.0%
3.4%
0.0%
124
MMP1
0.9%
14.3%
0.0%
0.0%
125
MMP2
0.9%
0.0%
3.4%
0.0%
126
MMP25
0.9%
0.0%
0.0%
1.3%
127
MMP3
0.9%
0.0%
3.4%
0.0%
128
MMP9
0.9%
14.3%
0.0%
0.0%
129
OGN
0.9%
14.3%
0.0%
0.0%
130
PI3
0.9%
0.0%
0.0%
1.3%
131
PRELP
0.9%
14.3%
0.0%
0.0%
132
PTPRZ1
0.9%
14.3%
0.0%
0.0%
133
RELN
0.9%
0.0%
3.4%
0.0%
134
ADAMTSL2
0.9%
0.0%
0.0%
1.3%
135
TGFBI
0.9%
0.0%
3.4%
0.0%
136
UCMA
0.9%
0.0%
3.4%
0.0%
137
VIT
0.9%
0.0%
3.4%
0.0%
138
WNT10A
0.9%
14.3%
0.0%
0.0%
139
WNT10B
0.9%
0.0%
0.0%
1.3%
140
WNT11
0.9%
0.0%
3.4%
0.0%
141
WNT4
0.9%
0.0%
0.0%
1.3%
142
ZP2
0.9%
14.3%
0.0%
0.0%
143
ADAMTS1
0.0%
0.0%
0.0%
0.0%
144
ADAMTS10
0.0%
0.0%
0.0%
0.0%
145
ADAMTS12
0.0%
0.0%
0.0%
0.0%
146
ADAMTS14
0.0%
0.0%
0.0%
0.0%
147
ADAMTS16
0.0%
0.0%
0.0%
0.0%
148
ADAMTS17
0.0%
0.0%
0.0%
0.0%
149
ADAMTS18
0.0%
0.0%
0.0%
0.0%
150
ADAMTS19
0.0%
0.0%
0.0%
0.0%
151
ADAMTS2
0.0%
0.0%
0.0%
0.0%
152
ADAMTS20
0.0%
0.0%
0.0%
0.0%
153
ADAMTS4
0.0%
0.0%
0.0%
0.0%
154
ADAMTS6
0.0%
0.0%
0.0%
0.0%
155
ADAMTS8
0.0%
0.0%
0.0%
0.0%
156
ADAMTS9
0.0%
0.0%
0.0%
0.0%
157
ADAMTSL1
0.0%
0.0%
0.0%
0.0%
158
ADAMTSL5
0.0%
0.0%
0.0%
0.0%
159
CD248
0.0%
0.0%
0.0%
0.0%
160
DGCR6
0.0%
0.0%
0.0%
0.0%
161
EGFL6
0.0%
0.0%
0.0%
0.0%
162
EMID1
0.0%
0.0%
0.0%
0.0%
163
FREM1
0.0%
0.0%
0.0%
0.0%
164
FREM2
0.0%
0.0%
0.0%
0.0%
165
RELL2
0.0%
0.0%
0.0%
0.0%
166
SPARCL1
0.0%
0.0%
0.0%
0.0%
167
ACAN
0.0%
0.0%
0.0%
0.0%
168
AMBN
0.0%
0.0%
0.0%
0.0%
169
AMELX
0.0%
0.0%
0.0%
0.0%
170
AMELY
0.0%
0.0%
0.0%
0.0%
171
ASPN
0.0%
0.0%
0.0%
0.0%
172
BGN
0.0%
0.0%
0.0%
0.0%
173
BCAN
0.0%
0.0%
0.0%
0.0%
174
CRTAC1
0.0%
0.0%
0.0%
0.0%
175
CILP2
0.0%
0.0%
0.0%
0.0%
176
CILP
0.0%
0.0%
0.0%
0.0%
177
COMP
0.0%
0.0%
0.0%
0.0%
178
CHL1
0.0%
0.0%
0.0%
0.0%
179
CHI3L1
0.0%
0.0%
0.0%
0.0%
180
CHAD
0.0%
0.0%
0.0%
0.0%
181
C6orf15
0.0%
0.0%
0.0%
0.0%
182
CCDC80
0.0%
0.0%
0.0%
0.0%
183
CTHRC1
0.0%
0.0%
0.0%
0.0%
184
COL1A1
0.0%
0.0%
0.0%
0.0%
185
COL3A1
0.0%
0.0%
0.0%
0.0%
186
COL4A4
0.0%
0.0%
0.0%
0.0%
187
COL4A5
0.0%
0.0%
0.0%
0.0%
188
COL9A1
0.0%
0.0%
0.0%
0.0%
189
COL9A3
0.0%
0.0%
0.0%
0.0%
190
COL5A2
0.0%
0.0%
0.0%
0.0%
191
COL5A3
0.0%
0.0%
0.0%
0.0%
192
COL6A3
0.0%
0.0%
0.0%
0.0%
193
COL8A2
0.0%
0.0%
0.0%
0.0%
194
COL10A1
0.0%
0.0%
0.0%
0.0%
195
COL16A1
0.0%
0.0%
0.0%
0.0%
196
COL18A1
0.0%
0.0%
0.0%
0.0%
197
COL21A1
0.0%
0.0%
0.0%
0.0%
198
COL27A1
0.0%
0.0%
0.0%
0.0%
199
COL28A1
0.0%
0.0%
0.0%
0.0%
200
COLQ
0.0%
0.0%
0.0%
0.0%
201
DMP1
0.0%
0.0%
0.0%
0.0%
202
DSPP
0.0%
0.0%
0.0%
0.0%
203
DPT
0.0%
0.0%
0.0%
0.0%
204
ELN
0.0%
0.0%
0.0%
0.0%
205
EMILIN1
0.0%
0.0%
0.0%
0.0%
206
EMILIN2
0.0%
0.0%
0.0%
0.0%
207
EMILIN3
0.0%
0.0%
0.0%
0.0%
208
ENAM
0.0%
0.0%
0.0%
0.0%
209
EPYC
0.0%
0.0%
0.0%
0.0%
210
ECM2
0.0%
0.0%
0.0%
0.0%
211
FBN1
0.0%
0.0%
0.0%
0.0%
212
FGF1
0.0%
0.0%
0.0%
0.0%
213
FGF9
0.0%
0.0%
0.0%
0.0%
214
FLRT1
0.0%
0.0%
0.0%
0.0%
215
FLRT2
0.0%
0.0%
0.0%
0.0%
216
FLRT3
0.0%
0.0%
0.0%
0.0%
217
FBLN2
0.0%
0.0%
0.0%
0.0%
218
FBLN5
0.0%
0.0%
0.0%
0.0%
219
GPLD1
0.0%
0.0%
0.0%
0.0%
220
GPC2
0.0%
0.0%
0.0%
0.0%
221
GPC3
0.0%
0.0%
0.0%
0.0%
222
GPC5
0.0%
0.0%
0.0%
0.0%
223
GPC6
0.0%
0.0%
0.0%
0.0%
224
HAPLN1
0.0%
0.0%
0.0%
0.0%
225
HAPLN2
0.0%
0.0%
0.0%
0.0%
226
HAPLN3
0.0%
0.0%
0.0%
0.0%
227
HAPLN4
0.0%
0.0%
0.0%
0.0%
228
KERA
0.0%
0.0%
0.0%
0.0%
229
LAMA4
0.0%
0.0%
0.0%
0.0%
230
LAMB4
0.0%
0.0%
0.0%
0.0%
231
LAMC3
0.0%
0.0%
0.0%
0.0%
232
LTBP4
0.0%
0.0%
0.0%
0.0%
233
LOX
0.0%
0.0%
0.0%
0.0%
234
LOXL1
0.0%
0.0%
0.0%
0.0%
235
MATN1
0.0%
0.0%
0.0%
0.0%
236
MATN3
0.0%
0.0%
0.0%
0.0%
237
MMP11
0.0%
0.0%
0.0%
0.0%
238
MMP13
0.0%
0.0%
0.0%
0.0%
239
MMP16
0.0%
0.0%
0.0%
0.0%
240
MMP17
0.0%
0.0%
0.0%
0.0%
241
MMP20
0.0%
0.0%
0.0%
0.0%
242
MMP23A
0.0%
0.0%
0.0%
0.0%
243
MMP26
0.0%
0.0%
0.0%
0.0%
244
MMP27
0.0%
0.0%
0.0%
0.0%
245
MMP28
0.0%
0.0%
0.0%
0.0%
246
MMP8
0.0%
0.0%
0.0%
0.0%
247
MFAP5
0.0%
0.0%
0.0%
0.0%
248
MFAP2
0.0%
0.0%
0.0%
0.0%
249
MFAP4
0.0%
0.0%
0.0%
0.0%
250
MUC4
0.0%
0.0%
0.0%
0.0%
251
MMRN2
0.0%
0.0%
0.0%
0.0%
252
NTN1
0.0%
0.0%
0.0%
0.0%
253
NTN3
0.0%
0.0%
0.0%
0.0%
254
NID1
0.0%
0.0%
0.0%
0.0%
255
NYX
0.0%
0.0%
0.0%
0.0%
256
ODAM
0.0%
0.0%
0.0%
0.0%
257
OPTC
0.0%
0.0%
0.0%
0.0%
258
OMD
0.0%
0.0%
0.0%
0.0%
259
OTOA
0.0%
0.0%
0.0%
0.0%
260
POSTN
0.0%
0.0%
0.0%
0.0%
261
PODN
0.0%
0.0%
0.0%
0.0%
262
PODNL1
0.0%
0.0%
0.0%
0.0%
263
PRSS36
0.0%
0.0%
0.0%
0.0%
264
RPTN
0.0%
0.0%
0.0%
0.0%
265
RBP3
0.0%
0.0%
0.0%
0.0%
266
SPN
0.0%
0.0%
0.0%
0.0%
267
ADAMTS7
0.0%
0.0%
0.0%
0.0%
268
SPOCK2
0.0%
0.0%
0.0%
0.0%
269
SPOCK3
0.0%
0.0%
0.0%
0.0%
270
SPON1
0.0%
0.0%
0.0%
0.0%
271
SFTPA1
0.0%
0.0%
0.0%
0.0%
272
SFTPD
0.0%
0.0%
0.0%
0.0%
273
TECTB
0.0%
0.0%
0.0%
0.0%
274
TNC
0.0%
0.0%
0.0%
0.0%
275
TNN
0.0%
0.0%
0.0%
0.0%
276
TNR
0.0%
0.0%
0.0%
0.0%
277
TNXB
0.0%
0.0%
0.0%
0.0%
278
THBS4
0.0%
0.0%
0.0%
0.0%
279
TFPI2
0.0%
0.0%
0.0%
0.0%
280
TGFB1
0.0%
0.0%
0.0%
0.0%
281
TINAG
0.0%
0.0%
0.0%
0.0%
282
TNFRSF11B
0.0%
0.0%
0.0%
0.0%
283
VEGFA
0.0%
0.0%
0.0%
0.0%
284
VTN
0.0%
0.0%
0.0%
0.0%
285
VWC2
0.0%
0.0%
0.0%
0.0%
286
WNT2
0.0%
0.0%
0.0%
0.0%
287
WNT1
0.0%
0.0%
0.0%
0.0%
288
WNT16
0.0%
0.0%
0.0%
0.0%
289
WNT2B
0.0%
0.0%
0.0%
0.0%
290
WNT3
0.0%
0.0%
0.0%
0.0%
291
WNT3A
0.0%
0.0%
0.0%
0.0%
292
WNT5A
0.0%
0.0%
0.0%
0.0%
293
WNT5B
0.0%
0.0%
0.0%
0.0%
294
WNT6
0.0%
0.0%
0.0%
0.0%
295
WNT7A
0.0%
0.0%
0.0%
0.0%
296
WNT7B
0.0%
0.0%
0.0%
0.0%
297
WNT8A
0.0%
0.0%
0.0%
0.0%
298
WNT8B
0.0%
0.0%
0.0%
0.0%
299
WNT9A
0.0%
0.0%
0.0%
0.0%
300
WNT9B
0.0%
0.0%
0.0%
0.0%
301
ZP1
0.0%
0.0%
0.0%
0.0%
302
ZP4
0.0%
0.0%
0.0%
0.0%
TABLE 10
Most significant Gene Sets Enriched in CTC-pro vs. CTC-c
q-value < 0.01
Odds
Benjamini
Source
Term
Count
Ratio
(q-value)
GOTERM_BP_FAT
GO: 0002495~antigen processing and presentation of
5
59.81
6.97E−04
peptide antigen via MHC class II
GOTERM_BP_FAT
GO: 0019886~antigen processing and presentation of
5
59.81
6.97E−04
exogenous peptide antigen via MHC class II
GOTERM_BP_FAT
GO: 0002504~antigen processing and presentation of
5
50.36
7.34E−04
peptide or polysaccharide antigen via MHC class II
GOTERM_BP_FAT
GO: 0002478~antigen processing and presentation of
5
41.60
1.10E−03
exogenous peptide antigen
GOTERM_BP_FAT
GO: 0019884~antigen processing and presentation of
5
34.18
1.87E−03
exogenous antigen
GOTERM_BP_FAT
GO: 0048002~antigen processing and presentation of
5
27.34
3.72E−03
peptide antigen
GOTERM_BP_FAT
GO: 0001775~cell activation
9
7.00
3.82E−03
GOTERM_BP_FAT
GO: 0019882~antigen processing and presentation
6
13.20
7.40E−03
TABLE 11
Most significant Gene Sets Enriched in CTC-plt vs. CTC-c
q-value < 0.01
Odds
Benjamini
Source
Term
Count
Ratio
(q-value)
GOTERM_BP_FAT
GO: 0042060~wound healing
18
7.8
1.86E−07
GOTERM_BP_FAT
GO: 0007596~blood coagulation
15
10.4
9.31E−08
GOTERM_BP_FAT
GO: 0050817~coagulation
15
10.4
9.31E−08
GOTERM_BP_FAT
GO: 0007599~hemostasis
15
10.3
7.59E−08
GOTERM_BP_FAT
GO: 0050878~regulation of body
15
8.2
1.30E−06
fluid levels
GOTERM_BP_FAT
GO: 0030029~actin filament-based
20
5.5
1.14E−06
process
GOTERM_BP_FAT
GO: 0007010~cytoskeleton
26
3.9
3.95E−06
organization
GOTERM_BP_FAT
GO: 0030036~actin cytoskeleton
18
5.3
1.11E−05
organization
GOTERM_BP_FAT
GO: 0009611~response to
26
3.6
1.02E−05
wounding
GOTERM_BP_FAT
GO: 0007155~cell adhesion
33
2.9
2.86E−05
GOTERM_BP_FAT
GO: 0022610~biological adhesion
33
2.8
2.70E−05
GOTERM_BP_FAT
GO: 0001775~cell activation
19
3.7
4.70E−04
GOTERM_BP_FAT
GO: 0030168~platelet activation
6
18.2
1.68E−03
GOTERM_BP_FAT
GO: 0007229~integrin-mediated
10
6.4
2.95E−03
signaling pathway
GOTERM_BP_FAT
GO: 0016192~vesicle-mediated
25
2.6
3.81E−03
transport
MSigDBv3.1 CGP
GNATENKO PLATELET SIGNATURE
20
55.1
3.91E−24
MSigDBv3.1 CGP
TENEDINI MEGAKARYOCYTE
14
15.3
1.35E−11
MARKERS
MSigDBv3.1
REACTOME FACTORS INVOLVED IN
6
2.9
2.25E−02
CP: REACTOME
MEGAKARYOCYTE DEVELOPMENT
AND PLATELET PRODUCTION
TABLE 12
Significantly Expressed Genes by Rank Product (FDR <0.01)
Primary
CTC-c vs
Tumor vs
Primary
CTC-c
Tumor
Enriched
CTC-plt vs
Count
Enriched Gene
Gene
CTC-c
CTC-pro vs CTC-c
1
Upk3b
Tff2
Clec1b
kg:uc007pge.1
2
Ier2
Wfdc2
AU023871
kg:uc007pgd.1
3
Egr1
Lamb3
Alox12
kg:uc007pgf.1
4
Nkain4
Lad1
Itga2b
kg:uc007pgg.1
5
Igfbp5
Dmbt1
Ppbp
Igj
6
Slc6a4
Npy
Gng11
kg:uc012enb.1
7
Klf4
Pmepa1
Vwf
2010001M09Rik
8
Tmem221
Kcnn4
Pf4
kg:uc009cfw.1
9
Arl4d
Serinc2
Fcer1g
kg:uc007pgi.1
10
Lrrn4
5730559C18Rik
Tmem40
kg:uc007pgh.1
11
Cldn15
Muc1
Hba-a2
kg:uc007yos.1
12
Gpm6a
Chi3l3
Stom
Coro1a
13
Atf3
Pglyrp1
Beta-s
Pou2af1
14
Ptma
Arl4c
Plek
kg:uc011yvj.1
15
Slc9a3r1
Spp1
Srgn
Glipr1
16
Fos
Col15a1
Myl9
Cd52
17
Tmem119
C1qb
Cd84
Cd79b
18
Ptgis
Tnnt2
F5
Sec11c
19
Dcn
Gkn3
Treml1
Tnfrsf17
20
Gbp2
Onecut2
Hbb-b1
Krr1
21
Dmkn
Mmp7
Itgb3
Gmfg
22
Sdc4
Cd74
Gp9
Ccr9
23
Ildr2
Ctss
Mpl
Pycard
24
Akap2
Lamc2
Ctla2a
Derl3
25
Gfpt2
Olfml3
Tubb1
Rac2
26
Klf6
Lgals4
Mylk
Srgn
27
Btg2
Lcn2
F13a1
Cytip
28
Myl7
Ly6a
Slamf1
Edem2
29
Igfbp6
Pak1
Rgs10
Itgb7
30
Gpr133
Capn5
Mkrn1
Lsp1
31
Oasl2
Ptprn
Laptm5
Lcp1
32
Pfn1
Reg3b
1810058l24Rik
Cyfip2
33
Cap1
Fmnl3
Itgb2
Nans
34
Nfkbia
Sdc1
Slc2a3
Slamf7
35
Malat1
Prom1
Pcmt1
Ell2
36
Rarres2
Ankrd50
Gp5
H2-Eb1
37
Rspo1
Ccl6
Ube2o
Creld2
38
Espn
Slc4a11
5430417L22Rik
Cd74
39
Klf9
Oraov1
Ptpn18
Blnk
40
Zbtb7c
Aldh1l1
Lat
Fmnl1
41
Brd2
Slc20a1
Fermt3
Snrnp70
42
Olfr1033
Cldn7
Nrgn
Sec61b
43
Wt1
Acsbg1
Mrvi1
Edem1
44
Esam
Las1l
Lyz2
Tspan13
45
kg:uc009igb.1
C1qc
Epb4.1
Psmb8
46
Tmem151a
Lama5
Rasgrp2
Pim1
47
Mgll
Mgat4a
Treml2
Sept1
48
Csrnp1
Cldn2
Hist1h4i
Cd48
49
Cd9
Mcpt2
March2
Sub1
50
Gjb5
Fxyd3
Ltbp1
Lims1
51
Lrrc61
Il4ra
Nptn
Ncoa2
52
Wasf2
Itga5
Abtb1
Ctnnbl1
53
Pdpn
Porcn
Ctla2b
Fdps
54
kg:uc009ogv.1
Mast3
Prkab2
Ube2j1
55
Sdpr
Scara3
Arhgdib
Mettl1
56
Gpr64
Atox1
Alas2
Lax1
57
Flnc
Arrdc1
Odc1
Rilpl2
58
Add3
Mmp2
Ptpn11
Ctse
59
Gata6
Saa3
Dhcr24
Glrx
60
Wfdc1
Serpinf1
Mfsd2b
Fut8
61
A130040M12Rik
Sox11
Gp1bb
Al662270
62
Ankrd12
Prpsap1
Rbpms2
Gramd3
63
Adamtsl1
Mcpt1
Fyb
Il2rg
64
C2
Mfge8
Smox
Rasgrp3
65
Prss23
Col18a1
P2rx1
Impdh1
66
Ube2v1
Lyz2
Otud7b
Plek
67
Cryab
C1qa
kg:uc007ttx.1
Ints5
68
Pkhd1l1
Acp5
Samd14
Blmh
69
Rtn1
Angptl4
Clca1
Dnmt1
70
Birc6
Ccnd1
kg:uc007tty.1
Galk1
71
Xdh
Asl
Gpr56
kg:uc007hxv.1
72
Cd34
Ctxn1
Sh3bgrl2
Ccdc88b
73
Rab6b
Pgs1
Pttg1ip
Selplg
74
Dusp1
Anapc2
Nomo1
Sar1b
75
Clic4
Cp
Gnaz
Lat2
76
C3
Gpx3
Mmrn1
Slc16a6
77
Rhob
Lama3
Gp1ba
Mki67
78
Mir3064
Rbp1
Sh3bgrl3
Dnajc3
79
Thbd
Cotl1
Slc24a3
H2-Ab1
80
Dpysl2
Nek6
Sord
Ndufs6
81
Cobl
Cpxm1
Nfe2
Actr3
82
Npr1
Sfrp1
Tuba4a
Etnk1
83
Dnajb9
Ttr
Zyx
Herpud1
84
Arhgap29
Gsto1
Cnn2
Ptpn7
85
Cav1
Npepl1
Itgb5
Ctss
86
Gbp7
Usmg5
Gata1
Cs
87
Hes1
Polr2l
Hist1h1c
Fbxw7
88
Gm16897
Sphk1
Tbxas1
Ppp2r5c
89
Ppp1r12a
Asxl1
Ptplad2
Znrd1
90
Sv2a
Ctsh
Bpgm
Rfc2
91
Ang
Egfl7
Pdlim7
Preb
92
Aldh1a2
C1qtnf6
Mmd
Fcer1g
93
Cryl1
Rras
G6b
Dnajb11
94
Kank1
Lgi4
kg:uc009duo.1
Slc35b1
95
2210403K04Rik
Hmga2
Lyz1
Sin3b
96
kg:uc009okn.1
Cep250
Tacc1
Nktr
97
Osr1
B4galt3
Dap
98
kg:uc008ewj.2
Tmem223
Mast2
99
kg:uc009tuw.1
Ltbp2
Atp2a3
100
Gadd45b
Tnfrsf23
Snca
101
Ablim3
Col7a1
Stx11
102
Clec3b
Ggct
C030046I01Rik
103
Usp25
Rab25
Trpt1
104
Sntb2
Nedd8
Tsc22d1
105
Rock2
9430023L20Rik
Prkar2b
106
Col14a1
Arl2
Cd9
107
Cd200
Wbp1
Pgm2l1
108
kg:uc008ehr.1
H2-Ab1
Gp6
109
Atp2b1
Preb
Pde5a
110
Exoc4
Sgsm3
Itga6
111
Abcb1b
Sfn
Itgal
112
Nrgn
Prrx2
Edem1
113
kg:uc009cvm.1
Ptprk
Isg20
114
Ncoa4
Reg1
Cdc42ep5
115
Ndufa4
Sdcbp2
Nipal3
116
Upk1b
Pcbd1
Ccdc92
117
Jun
Slc25a1
Sort1
118
Syne2
Vamp5
Ly6g6c
119
kg:uc007bvx.1
Crlf1
Ubash3b
120
Ap4e1
Avil
Inf2
121
Spock2
2700094K13Rik
Asap1
122
Efemp1
Ctse
Sec11c
123
Prpf40a
Penk
Gas2l1
124
Tspan5
Tmc4
Parvb
125
Lgals7
Dhrs3
Tmsb4x
126
Kif5b
Ap1s1
kg:uc007xrw.1
127
Psip1
Arl6ip4
Nudt3
128
kg:uc008oki.1
9430008C03Rik
Bcl2l1
129
1810014B01Rik
Fcer1g
B230312A22Rik
130
Ptges3
Uqcr11
Cnp
131
Limch1
Nhp2
Plp1
132
Bicd1
Plbd2
Cnst
133
Rdx
Capg
Rgs18
134
Pcdh15
Pnpla6
Lsm12
135
Foxn3
Ppdpf
Alox5ap
136
Morf4l2
Hgfac
Ppif
137
Ppp1r15a
Apoe
Spnb1
138
Cdc42ep3
Fam40a
Ormdl3
139
Pard3b
Lyz1
Hpse
140
Bicc1
2200002D01Rik
Srxn1
141
Amhr2
Laptm5
2010002N04Rik
142
Gucy1a3
Qars
Hist1h2bc
143
Psmb2
Tmx2
Cyba
144
Mapkapk3
Fkbp4
Chst12
145
Ube2l6
Plin2
kg:uc009sps.1
146
kg:uc007pff.1
Fcgr3
Max
147
kg:uc007ctp.1
Gkn1
Was
148
Nedd4
Snhg1
Isca1
149
Plxna4
Lsp1
Pdzk1ip1
150
2010107G12Rik
Gm20605
Lyn
151
Ifngr1
Ly6c1
Mob3a
152
Bcam
Aim1
H2-T24
153
Ccnl1
2310007B03Rik
Slc44a1
154
Hoxa5
Tgfbi
Derl1
155
Fhl1
Tsta3
Gclm
156
1810041L15Rik
Pafah1b3
Fech
157
2900002K06Rik
Chid1
Ywhah
158
Hspb1
Smox
Igtp
159
Podn
1500012F01Rik
Myl6
160
Fam63b
Tspan4
Thbs1
161
Hsp90b1
Agrn
Tln1
162
Dpp4
Cfp
kg:uc009apq.1
163
Gas1
Cdh1
Bcap31
164
kg:uc007zak.1
Rasgrf1
Ilk
165
Zc3h13
Nxf1
Epha1
166
Sox6
Pdrg1
2810453I06Rik
167
Arid4a
Polr2j
Rnf19b
168
Tnxb
Suds3
Gsn
169
Tsix
D0H4S114
Flna
170
Scd1
Ccl9
Arrb1
171
Jund
Neat1
kg:uc007pum.1
172
Crls1
Ccdc12
Mbnl1
173
1110003E01Rik
Prr24
Ccnd3
174
Rnase4
Impdh1
Pdlim1
175
Arhgef12
Card10
Ctse
176
Irf7
Cpsf1
Tspan17
177
Bbx
Sema4g
Gpx4
178
Sema5a
Hes6
Bnip3l
179
Mau2
C130074G19Rik
P2ry12
180
Abi3bp
Ctrb1
kg:uc009vev.1
181
Dag1
Rnaseh2a
Prkab1
182
Cyp2s1
Golm1
F2rl2
183
Sfrs18
Ctsz
Stk4
184
Hspb8
Cyb561
Fhl1
185
Cnot6l
Ndufs8
Rnf10
186
Twsg1
Atp6ap1
Rasa3
187
Gpc3
Srd5a1
Taldo1
188
Lrrn4cl
Carkd
Bysl
189
Cdh3
Cd24a
Esd
190
Cyr61
Eng
Aldh2
191
Cyp2d22
Tcirg1
Rhog
192
Hist1h1c
Slc9a3r2
kg:uc009ecr.1
193
Aplp1
0910001L09Rik
Cald1
194
Tbl1x
Cox5b
Wbp2
195
Pcm1
Adipor2
Ptprj
196
Ifi204
Scarf2
Tpm4
197
Nfix
Myo7a
Mxi1
198
Flrt2
Ppap2c
Ly6g6f
199
Heg1
Pea15a
Sla
200
Il6ra
Sh3pxd2b
Slpi
201
Ralbp1
H19
Bicd2
202
Rhoj
Tpd52
Clu
203
Ktn1
2610203C20Rik
Mtmr14
204
Arl6ip5
Naa10
Abca7
205
Crebbp
Fermt1
Ppp1r18
206
Ppig
Sap30l
Kif2a
207
Akap13
Bgn
Prdx6
208
Rab7
Timm13
kg:uc009ize.1
209
Plxdc2
Krt20
Calm3
210
Aldh1a1
Itga3
Dhrs1
211
Bnc2
Pfkl
Cfl1
212
Slc4a4
Agpat6
Glipr2
213
Tbx18
Mrpl11
Slc25a37
214
Zbtb16
Ramp1
Atox1
215
Arid4b
Hmga1
BC057079
216
Enpp2
Gpx2
Pla2g16
217
Ptplad2
0610012G03Rik
Rnf144b
218
Akr1b3
9130017N09Rik
Stk16
219
Gm6644
Cygb
Rsad2
220
Arf5
Tmprss4
Paip2
221
Chi3l1
Paox
Capzb
222
Gpr116
Endod1
Ppp1r12c
223
Cd82
Cndp2
4930412F15Rik
224
Srrm1
Suv39h1
Ninj1
225
Fmo2
Cog4
2510009E07Rik
226
Tgfb1i1
Trim27
kg:uc007vsr.1
227
Qrich1
Cyhr1
Pygb
228
Nfia
Trmt1
Tlk1
229
Pmp22
Zfyve19
Myct1
230
Cdh11
Esrp1
Rnasek
231
Arid5b
kg:uc008oow.1
Ctsd
232
Rbm3
Dync1h1
0610010K14Rik
233
Prelp
Tab1
Bcas3
234
kg:uc007qse.1
Pla2g6
Atpif1
235
Ddx3x
Timp1
Serf2
236
Sulf1
Eif3f
Becn1
237
Spnb2
Abhd11
Tspan9
238
Tspan31
Pmm2
Acer2
239
Prr13
Tyrobp
Vdac3
240
Ppp1cb
Farsb
kg:uc008kbg.1
241
Fbln1
Plod3
Oaz2
242
Gm6548
Abtb1
Serpine2
243
Uap1
Brf1
Ccdc90a
244
Mpdz
Tnk2
Ndufa1
245
Sat1
Rfc2
Tssc1
246
Stim1
Stxbp2
Mboat7
247
Mll3
Pdlim7
Cd44
248
Slurp1
A430105I19Rik
Cxx1c
249
Cd81
Vill
Ecm1
250
Emp2
Bmp1
Mff
251
Trpm7
Mpzl1
Ptpn12
252
Crym
Thy1
Mgmt
253
Enpp4
Stab1
Cox4i1
254
Raly
Aldh16a1
Tollip
255
Celf2
Eif4ebp3
Cds2
256
Ap3s1
Itpripl2
Ybx1
257
C1s
Mrpl52
Gypc
258
Frmd4b
2310002L13Rik
Dgkd
259
Nr4a1
Mcm6
Pecam1
260
Acin1
Kcnk1
Ftl2
261
Plod2
Pmf1
Nt5c3
262
Id1
Cuta
1700037H04Rik
263
Creg1
Nt5dc2
Cd151
264
Zfp318
Rmnd5b
Lpin2
265
Tmem140
Araf
6430548M08Rik
266
Mras
Wwp2
Pon2
267
Vwa5a
Lamb1
Ndufa3
268
Esyt3
Kcne3
6330578E17Rik
269
Hexb
Uqcrq
Mfap3l
270
Nckap1
Gps1
Mink1
271
Nipal3
Rexo4
Ston2
272
Ubxn4
Coro1c
Rac2
273
Zfp36
Hras1
Fyn
274
Hnrnpl
Spint1
Serinc3
275
C1ra
Cblc
Maged2
276
Nnmt
Fhod1
Ap2m1
277
Mut
Atp13a1
Pacsin2
278
kg:uc008jup.1
Man2c1
Ftl1
279
Pnrc1
Vsig2
Adipor1
280
Usp8
Bpgm
kg:uc009qdo.1
281
Pgcp
Bap1
Snap23
282
Junb
Smpd2
Tagln2
283
C1rl
Ubqln4
Cox6c
284
Slc6a6
Sirt7
Creg1
285
kg:uc008znh.1
Krt23
Bsg
286
Aqp1
D8Ertd738e
Cmtm6
287
Myh10
Mapk13
Cntd1
288
Slc43a3
kg:uc008bcq.1
Plekho2
289
Spint2
Polr2g
Arrb2
290
Hnrnph1
Ndufs2
Pard3b
291
Arhgap28
Dad1
Mlec
292
Cfh
Wnt7b
Taf10
293
Brd4
Fam20c
Gabarapl2
294
Fndc1
Cxxc5
Bag1
295
Star
Polr2f
Galnt2
296
Nfkbiz
Ltf
Hk1
297
Arsb
2210407C18Rik
Fbxo9
298
Rnd3
Cdipt
kg:uc009izd.1
299
Stard5
Glrx5
Pnpo
300
Thbs1
Gemin7
Fam46c
301
kg:uc008wkn.1
Man1b1
Pkm
302
Slc26a3
Heatr7a
Ap1b1
303
Phip
Arid5a
Rap1b
304
Usp2
Sumo3
Itgb1
305
Golgb1
Srm
St7
306
Rock1
Plscr3
Smap1
307
Rgma
2210010C17Rik
Rabgap1l
308
Actg1
Fam102a
Tmbim4
309
BC013529
Dlst
H3f3a
310
kg:uc007zwh.1
Vps37c
Frmd8
311
3110062M04Rik
Ngfrap1
Nlrx1
312
Cast
Pold4
Oaz1
313
Mob3c
Grcc10
Fam125b
314
Slc16a1
Wnt7a
Hexa
315
Fam117a
2010111l01Rik
Tspo
316
Pdia3
Pxdn
Dcaf12
317
Trim8
Coasy
Nav1
318
kg:uc009mng.1
Dctn1
Cd24a
319
eg:245190:chr7:m
Ncor2
Uqcr11
320
Sbsn
Postn
Wipf1
321
Serpinb6b
Col4a2
F10
322
Daglb
Cib1
Erlec1
323
Smarca2
Tbc1d13
Map2k3
324
Mef2c
Ccnl2
Stk24
325
Prrc2c
Dcakd
Ldlrap1
326
BC005537
Cdc34
Ehd4
327
Hsp90ab1
Atp6v0b
Atp6v1f
328
Snrnp70
Abhd12
Gnas
329
Ppl
Flot2
Arhgap18
330
Serpinh1
Sla2
Arhgap10
331
Sorbs3
Rhbdf1
Pitpnm1
332
Golga4
Cdh17
S100a1
333
Acbd3
Psmb5
Bin1
334
Hook3
Serf1
Ttyh3
335
Map3k3
Slc15a3
Selp
336
Rhou
Sftpd
Trappc9
337
Smc2
Pop5
Aes
338
C1d
Nudc
Taok3
339
kg:uc008dzh.1
Sh2d5
Zfand3
340
Psmd7
kg:uc007fwp.1
Stim1
341
Dab2
Mrpl37
Rnf114
342
Cep164
Rin1
Sep15
343
Crim1
Podxl
kg:uc012hdk.1
344
Rtf1
Paqr5
Lgals9
345
Fxyd1
Sepx1
Cox6b1
346
H2-D1
Agr2
Riok3
347
Zfp704
Bax
Slc38a10
348
Mtap1a
Rxrb
Rtn3
349
Ascc3
Tes
B3gat2
350
Med13l
Hdac6
Ccndbp1
351
Jup
1110008F13Rik
Rsu1
352
Nid2
Mpnd
kg:uc007upr.1
353
Kdr
Gmppa
Itm2b
354
Ifnar2
Gramd1a
St3gal1
355
5430435G22Rik
Wars
Sec61g
356
Col4a6
Mtap
Ptpn1
357
Il17re
C1qtnf5
kg:uc012bhf.1
358
Gbp3
Mrpl28
B2m
359
Slc39a8
Mfrp
Rasgrp3
360
Cfl2
Kars
Memo1
361
Slc38a1
Lbp
Slc39a4
362
Cuedc1
Plxnb1
Sdcbp
363
Fgf1
2700081O15Rik
Tspan14
364
Gas6
Mrps24
Ubl7
365
Cldn25
Klc4
Nras
366
Sorbs1
Dctn3
Ssx2ip
367
Hspa12a
Kcnq1
kg:uc007zbz.1
368
kg:uc007zts.1
Smurf1
Wbp1
369
Slc1a5
Fam162a
1110003E01Rik
370
Nr3c1
Hip1r
Clip2
371
Adamts5
kg:uc007hyr.2
Gapdh
372
Gpcpd1
Gys1
Gm6578
373
Dpysl3
Sac3d1
Actn1
374
Colec12
Ndufs6
St3gal2
375
Pdcd6ip
Rgl2
3110001D03Rik
376
Dst
Atp5g1
Ctsz
377
Ifit3
Itgb4
kg:uc007vdl.1
378
Chst4
Sars
Fam73a
379
Xist
2310003F16Rik
Vcl
380
Ifi27l2a
Nhp2l1
Lims1
381
Fkbp5
D19Wsu162e
Lars2
382
Agap1
Cd320
Birc2
383
Ankrd11
Pigq
Lamp2
384
kg:uc007qca.1
Chd3
Rasl10a
385
Syt11
Zdhhc4
Mif
386
Ptrf
Eif3l
Rab10
387
Krcc1
St8sia3
Pabpc1
388
Zfp488
Rcan3
Wwp2
389
Lama4
Meg3
Nqo2
390
Aebp1
Nudt4
kg:uc007fte.1
391
Fam134b
Gss
Plxna4
392
Tppp3
Pih1d1
Gm1821
393
Maf
Limd2
Gadd45a
394
Peli1
Ap1s2
Slc25a39
395
Zfp353
BC056474
kg:uc009pet.1
396
Cdon
Mms19
Ubb
397
Sarnp
Clip2
Ppp1r2
398
Atxn7l3b
2310016M24Rik
Rab27b
399
Pef1
Itpa
Cap1
400
App
Slc25a10
Jarid2
401
Mtdh
Fibp
Rnf11
402
Lrrc20
Higd2a
Tmem50b
403
Btbd2
Snrpd2
Myh9
404
Gnb2
Eri3
Tmem128
405
Pigt
Nbeal2
Stradb
406
Efna5
Trim28
Cela1
407
Tm4sf1
S100a4
Ndrg2
408
Coq10b
Ivns1abp
Dhrs3
409
Eif2s3x
Ppp1r18
Hipk1
410
Cmah
Efemp2
Atg9a
411
Sf3b1
Med22
412
Eea1
Nelf
413
Slpi
2810428I15Rik
414
Tmod3
D2Wsu81e
415
Ppp3ca
Trappc6a
416
Tceal8
Trappc2l
417
Anp32a
Antxr2
418
Actb
Rab11fip5
419
Ddx5
Ldhd
420
Cobll1
Npnt
421
Cish
Acrbp
422
Nod1
Pafah1b2
423
Psd
Angptl2
424
Gm10052
Fzr1
425
Lims2
Aaas
426
Stra6
Eif2b2
427
kg:uc007bgn.1
1190003J15Rik
428
Plxdc1
5730403B10Rik
429
Nfe2l1
Adamts13
430
Smpd3
Eif3b
431
Bcl10
Znrf1
432
Ilf3
Pkp3
433
Fam76a
Lemd2
434
Cybrd1
Rab34
435
Gm3893
Mpv17l2
436
Siae
Cdkn2b
437
Ssh2
Snrpe
438
Nfic
Gm14005
439
Btf3
Prdx4
440
Sp100
Xab2
441
Ndn
Dpp3
442
Matr3
Tyms
443
Gm13251
Leprotl1
444
Arhgap5
Uqcr10
445
Zbtb4
Cdk5rap3
446
Pgrmc1
Gorasp2
447
4930402H24Rik
Wbp7
448
Bptf
Sort1
449
Dusp3
Ddx41
450
Pla2g4a
Cct3
451
Brp44l
Mrps33
452
Oxct1
Frmd8
453
Stk40
1110049F12Rik
454
Ddr1
Fscn1
455
Ifi205
Ndufa2
456
Col3a1
Dpcd
457
Nipbl
Unc13a
458
Plk1s1
Eif1ad
459
Bdp1
Sgta
460
Smc3
Chaf1a
461
Ifitm3
Plxna1
462
Ndst1
Hspa9
463
Zbed6
1110014N23Rik
464
Rest
Cd99l2
465
kg:uc007vnc.1
Snrpa
466
Ccdc88a
Mcm7
467
Stat3
Tars2
468
Arf2
Gon4l
469
Trib1
Stk38
470
Gcap14
C1qtnf1
471
Tbc1d15
Tbrg4
472
Igf1r
Tmem132a
473
Ppbp
Cox6c
474
kg:uc008tky.1
Alcam
475
Rab1b
Phka2
476
Krt14
Trim3
477
Med21
Ppp1r14b
478
Gja1
Gpaa1
479
Klf10
Ctps2
480
Id2
Ptpn23
481
Mfap1a
Endog
482
Ogn
Mrto4
483
Gpc4
Mrps6
484
Bst2
Pvr
485
Dtx2
Phgdh
486
Wac
Itpr3
487
Kpna3
Polr2e
488
Kcnab1
Sec16a
489
Orai3
Mdp1
490
Gcsh
Fbf1
491
Wdr92
Mcpt8
492
Olfr613
Rps6ka4
493
Tcf7l1
Mical1
494
Tgfb2
Mrpl34
495
Il16
Agpat3
496
Manf
2310044H10Rik
497
Mgst1
Myo9b
498
kg:uc008tkz.1
Ndufb10
499
Creb3l1
Apex1
500
Txndc5
Elk3
501
Klf2
Cpsf3l
502
Slu7
Tnk1
503
Ttc28
Pmvk
504
1110002B05Rik
Ppp1r16a
505
Zcchc11
Arhgef5
506
Ptp4a2
Lonp1
507
Pbx1
Pla2g7
508
Clcn3
Pip5k1c
509
Tmco7
Inf2
510
Lrrc58
Pgk1
511
Eif3a
Parp6
512
Cldn10
Urm1
513
H2-Q6
Mad2l2
514
Ccdc80
Ing4
515
kg:uc009iln.1
Rbck1
516
Rab5c
Cant1
517
Tsc22d3
Sgpl1
518
Tm4sf5
Ehbp1l1
519
Hmgb1
Runx1
520
Sec62
Slc27a4
521
Maoa
Ndufa7
522
Clec1b
Mcm3ap
523
Mphosph8
1110008P14Rik
524
Oat
Rassf7
525
Ncor1
Ptpmt1
526
Cyb5
Arfgap1
527
Trafd1
Sec61a1
528
Rpp25
Rps6ka1
529
kg:uc007ded.1
Ints1
530
2610101N10Rik
Tpcn1
531
Il6st
Iffo2
532
Evpl
Trim44
533
Psmd11
kg:uc012ctw.1
534
Dync1i2
Golga2
535
Lars2
Msto1
536
Pdia4
Ppp6r3
537
Cd55
Trmt2a
538
Amfr
Appl2
539
Zcchc3
Sparcl1
540
Herpud2
Rapgef1
541
Txnrd1
Zfpl1
542
Vat1
Psmc4
543
Diap1
Mosc2
544
Tmed2
Fam101b
545
Arf3
1500010J02Rik
546
Arap2
Ccdc124
547
St3gal1
Ptges
548
Man1a
Fam189b
549
Rgs10
Th1l
550
Tmsb4x
Kctd2
551
Uba7
Olfr1372-ps1
552
C4b
Hexa
553
Tmem98
Anapc5
554
Lpar2
Serpina3n
555
Gabarapl1
1810046J19Rik
556
Cmtm7
Tmem167
557
Spon2
Gm11428
558
Smarca5
Gcn1l1
559
Mxd4
Kansl3
560
Smc4
Fasn
561
Thsd4
Slc50a1
562
Gsr
Smad3
563
Ptprd
Trip6
564
Clip1
Atp6v1e1
565
Cln8
Chchd5
566
Rbm27
Adssl1
567
Zmat1
Nes
568
Smc6
Ap1b1
569
B2m
Fcgrt
570
Irf2bp2
Ltbp3
571
Ppap2a
Csf2rb
572
Zfhx4
Ssna1
573
Tob2
Mrps16
574
Rabgap1l
Cyba
575
Nfkb2
Cyth2
576
Nfyc
Igf2
577
Ube2d1
Pisd-ps1
578
Creb5
Atp13a2
579
Opa3
Mlph
580
Csnk1a1
Cyp4f16
581
Fam84b
2010107E04Rik
582
Ddr2
Gas5
583
Usp54
Eif3k
584
Akt2
Fam149a
585
Strn3
Mif
586
Hnrnpm
B230312A22Rik
587
eg:497210:chr14:m
Ppp1r12c
588
Tpt1
Tfip11
589
Naa25
Tex10
590
Eef1a1
Slc16a3
591
Parp4
Stk16
592
Msn
Epn1
593
Zbtb20
Noc4l
594
Fermt2
Rcc2
595
Bod1l
Rgs12
596
Sltm
Shkbp1
597
Dapk1
Got2
598
Hnrnpr
Plek2
599
Baz2a
Lilrb3
600
Rnf167
Ndufb5
601
Mapk1
Tesk1
602
eg:320169:chr9:p
Rab24
603
4930523C07Rik
Atp5j2
604
Nf1
Commd9
605
Fam53b
Rtkn
606
Faim2
Prpf19
607
Tgm2
6720401G13Rik
608
Calm2
Ppa1
609
AI848100
Pgp
610
Slc10a3
Hps1
611
Ogdh
Puf60
612
Arl3
Mdm2
613
Timp2
kg:uc012cgd.1
614
Atxn2
kg:uc009uim.1
615
Mll1
Pyy
616
Ces2g
Zfp358
617
Mat2a
Timm8b
618
Esf1
Ddx39
619
Hsp90aa1
Pgm2
620
Zfp385a
kg:uc008gbp.1
621
Zfp672
Sipa1
622
Csda
Mgat1
623
Pf4
Tmem208
624
Arsa
Ruvbl2
625
F11r
8430410A17Rik
626
C4a
Bad
627
Kpna1
Pfdn5
628
Rbbp8
Eme1
629
Oxnad1
kg:uc009mzj.1
630
Rb1cc1
Igf1
631
Setd2
Prkag1
632
Kif1b
kg:uc009sua.1
633
2510002D24Rik
Uap1l1
634
Cep57
Trappc4
635
Chd2
Bola2
636
Serinc5
Usp5
637
Marcksl1
Ear2
638
Shfm1
Cars
639
Bbs4
1810027O10Rik
640
Impad1
Amdhd2
641
Tbcel
Phb
642
Kdelr1
Kcmf1
643
Ninl
Lsmd1
644
Sytl1
Sec11c
645
Tpm3
Pcbp4
646
Rbbp6
Mepce
647
Lman1
Tpd52l2
648
Ankrd17
Trf
649
Naga
Hsd17b11
650
Rbpms
Pilra
651
Magt1
Atn1
652
Tfdp2
Pgf
653
Gem
Nxn
654
Pde4dip
Inpp5k
655
Mrgprf
Actr1a
656
kg:uc008ajk.1
Cd68
657
Itch
Eef1g
658
Elf1
Fbn1
659
Meis2
Hint1
660
Arid1a
March5
661
Serping1
Usp48
662
Slc27a3
Hnf1b
663
Thoc2
Gga3
664
Gsta3
Drosha
665
Hnrnph2
Ubp1
666
Socs3
Pkn3
667
Armcx3
Tmem192
668
Siah1a
Prpf31
669
kg:uc009ize.1
Hspd1
670
Irs2
Otub1
671
Mettl7a1
Mrpl20
672
Ppfibp2
Tead2
673
Blvrb
Phpt1
674
Yipf5
Neu1
675
Plat
Pygo2
676
Gm6578
Myeov2
677
Mat2b
Cdk5
678
Tmpo
Ndor1
679
Metap2
Rbp4
680
Zfp277
Psat1
681
Wls
Mrpl41
682
Mesdc1
Snrpg
683
kg:uc009acs.1
Acot7
684
Col1a2
Vars
685
Csf1
Nono
686
Sulf2
Gtf2i
687
Ifrd1
Traf3
688
Wrnip1
Ppp2r4
689
Flii
Actg2
690
2810474O19Rik
Pi4k2a
691
Sep15
Slc35b2
692
2310030G06Rik
Ubqln1
693
Cmtm3
Ppox
694
Mylip
Bud31
695
Slc8a1
Man2b1
696
Btbd7
Nat15
697
Hdac5
Spon1
698
Zfand6
Cyc1
699
Tapbp
Mpeg1
700
Keap1
Nsun2
701
Ube2n
Rab4a
702
Ssr3
Mtmr11
703
H3f3a
BC004004
704
Myst4
B4galnt1
705
G3bp1
Atp5k
706
Ugdh
Lin37
707
Lamp2
D330041H03Rik
708
Zrsr1
Tbc1d17
709
Pim1
March6
710
Gm9199
2410015M20Rik
711
Supt16h
1810013D10Rik
712
Ano6
Eif2s1
713
Soat1
Traf7
714
Eci1
Rpl36al
715
Plce1
Psenen
716
Atg3
Aip
717
Bnc1
Cmas
718
Pik3c2a
Rpia
719
Pqlc3
Ncbp1
720
Thrap3
Mea1
721
Irak4
Timm50
722
Kdm6b
Ear12
723
Apol9a
Fkbp1a
724
Wnt4
Commd4
725
1500003O03Rik
Col5a3
726
Phf3
Fblim1
727
1110004F10Rik
Cwh43
728
Kansl1
Arl2bp
729
Fth1
Mrpl46
730
Tmem50a
Tcn2
731
Utp20
Add2
732
Smad4
Specc1l
733
Stmn2
Ppcs
734
Gstm1
Vrk3
735
Senp6
Trim25
736
Gda
Nfatc1
737
Nucks1
Rap1gap
738
Ints10
Hsd17b12
739
Syne1
Epas1
740
Itga6
Ddx1
741
Acad9
Prdx6
742
Maged1
Mmp24
743
Spen
Ndufb9
744
Chd1
Phf23
745
Taf3
Rpa2
746
Ptgs1
5031439G07Rik
747
Sparc
Rrp7a
748
R74862
Arfip2
749
B230120H23Rik
Efna1
750
Tmem234
Agps
751
Ryk
Sephs1
752
Dlgap4
Apoc2
753
Atp1b1
Mrps27
754
Parp14
Snn
755
Tgfbr2
Serinc3
756
Ccdc90a
Pdcd5
757
Ncoa1
AA986860
758
Pppde1
Pitpna
759
Luc7l3
Vac14
760
Prg4
2810025M15Rik
761
Rab11fip1
Def8
762
Plk2
Hilpda
763
Ifi35
Eif6
764
Pdap1
Brd7
765
Cd248
Fes
766
Sesn1
Sbf1
767
Ecd
Ak2
768
Ap1s3
1810035L17Rik
769
H2-K1
Lime1
770
Spag9
Hspe1
771
Tshz1
Csrp2bp
772
Dennd5a
Uba5
773
Stag1
Gsta4
774
Gpx8
2900092E17Rik
775
Sod3
776
BC005561
777
kg:uc009vev.1
778
Ywhaz
779
Ganab
780
Rras2
781
Dusp14
782
kg:uc012hdk.1
783
Nr1d1
784
Wwc2
785
Ubxn2a
786
Iqsec1
787
kg:uc007vsr.1
788
Cfl1
789
Csrp1
790
Smchd1
791
Myl12a
792
Ubqln2
793
Tmcc3
794
Kdm5a
795
Rbm25
796
Wdr26
797
Vim
798
Arpc2
799
Calm1
800
Dnaja2
801
Shc1
802
Vps13a
803
Klf7
804
1810074P20Rik
805
BC003331
806
Itpr2
807
Jmjd1c
808
Pcdhgb5
809
Tubb2a
810
Ehd2
811
Ift74
812
Per1
813
Pitpnm2
814
Gstm4
815
Dnmt1
816
Tmco1
817
Lass4
818
Ptprf
819
Sirt2
820
Gfm2
821
Taf7
822
Spop
823
Zzef1
824
Ccdc34
825
Zfp281
826
Tuba1a
827
Ccdc109b
828
Cdk13
829
Dhx15
830
Src
831
Braf
832
Mapre2
833
Anxa7
834
Sept9
835
Alox12
836
Pknox1
837
2610034B18Rik
838
Topors
839
Phf21a
840
Qser1
841
Tirap
842
Fas
843
Lass2
844
6330406l15Rik
845
Parvb
846
Atp1a1
847
Mtmr6
848
Cd109
849
Dnajc1
850
Hp1bp3
851
1600029D21Rik
852
Ttc38
853
Mfhas1
854
Filip1l
855
Zfp148
856
Nkd1
857
Usp16
858
Tlr2
859
Zc3h18
860
Stk10
861
Ltbp4
862
Hdac3
863
Efhd2
864
Prkar2a
865
Atp6v1a
866
Sf3b4
867
Gprc5b
868
Clip3
869
Mettl2
870
Secisbp2
871
Fmod
872
kg:uc009lxf.1
873
Elovl6
874
Bzw1
875
Etfa
876
Hspa2
877
kg:uc007won.1
878
Rnf20
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
Primary
WBC vs
CTC-c
Tumor
Primary
vs
WBC vs
Count
vs WBC
Tumor
WBC
CTC
1
Wfdc2
Ppbp
Olfr1033
Beta-s
2
Spp1
Alas2
Crip1
Alas2
3
Cct3
Nrgn
Ppp1r12a
Hbb-b1
4
Itga3
Cd9
Vcp
Il1b
5
Gsto1
Csf3r
Klf9
Ppbp
6
Mmp2
Il1b
Mprip
Hba-a2
7
Mfge8
Gdpd3
Sdc4
kg:uc007pgs.1
8
Capg
Ms4a1
Gpc5a
kg:uc011yvj.1
9
Cd63
Hbb-b1
Vat1
Coro1a
10
Stub1
Beta-s
Wdr92
Cd74
11
Lad1
kg:uc007pgs.1
S100a11
Gdpd3
12
Myo1h
kg:uc011yvj.1
Clic4
Ccndbp1
13
Igfbp7
Rprl1
Dync1i2
kg:uc009cfw.1
14
Kcnn4
Pfn1
Nfkbiz
kg:uc012enb.1
15
D8Ertd738e
Clec1b
Cyp2s1
Ptprc
16
Lamb3
Ptprc
Esam
Csf3r
17
Chi3l3
Stim1
Surf4
Rac2
18
Arl4c
Ccndbp1
Krt19
Rprl1
19
Col18a1
Cap1
Bsg
H2-Ab1
20
Atox1
Cd79b
Tm4sf1
Epb4.1
21
Ly6a
Alox12
Lgals3
Lyz2
22
Dmbt1
Hba-a2
Clic1
Ctla2b
23
Dync1h1
Ube2l6
Capns1
Pld4
24
Adipor2
Cat
Igfbp6
kg:uc007pgt.1
25
Rpl37
Faim3
Rrbp1
Gng11
26
Kctd10
Dusp1
Calr
Mepce
27
Col15a1
kg:uc007pgt.1
Rtf1
Tyrobp
28
Surf4
E2f2
Ildr2
Isca1
29
Dad1
Phospho1
Mark2
2810453l06Rik
30
Col4a1
Abi3
Mt1
Slc30a9
31
Ap2s1
Sorl1
Akr1b3
Treml2
32
Sdc1
Treml2
Gm6644
Srgn
33
Rpl35
Cytip
Nkain4
Dcaf12
34
Sec61a1
B2m
Ppp2ca
Plek
35
Rras
Fyb
Akap2
Cat
36
Oraov1
Peli1
Hspb1
Alox12
37
Ndufa2
Plek
Ptgis
Fech
38
Anapc2
N4bp3
Msln
Rbm5
39
Pitpna
Fam117a
Emp2
Cd97
40
Psap
Srgn
Capn2
March8
41
Atp5j2
Sept9
Rhoc
Pnpo
42
Onecut2
kg:uc012hdk.1
Ptprf
Phospho1
43
Hmga1
kg:uc009vev.1
Bcam
Isg20
44
Pmepa1
Ptprcap
Ogdh
March2
45
S100a11
kg:uc007pgq.1
Sparc
Lsp1
46
Rbp1
kg:uc007pgr.1
Ahnak
1810058l24Rik
47
Rpl36al
kg:uc007vdl.1
Oasl2
Clec1b
48
S100a4
Ctla2b
Wt1
Btg1
49
Atp6ap1
Myl9
Klf4
Laptm5
50
Ndufs2
Itpr2
Cdkn1a
Nrgn
51
Anapc5
kg:uc012enb.1
Myl7
H2-Aa
52
Cox6b1
Isg20
Col1a2
Fyb
53
Krtcap2
Rasal3
Eif4a1
Cd24a
54
Atn1
Gng11
Rbpms
Fnbp4
55
5730559C18Rik
kg:uc009cfw.1
Emp3
Ehbp1l1
56
Pea15a
Tmsb4x
Scaf11
Ctla2a
57
Grcc10
Trem1
Col14a1
Sgk1
58
Lama5
Fech
Ptrf
Glyr1
59
Krt18
Epb4.1
Crip2
Myl9
60
Ccnd1
Sgk1
Ubxn4
Il2rg
61
Arhgef5
Dgkq
Eif2s2
Mrps17
62
Golm1
Snap23
S100a6
Cdr2
63
Tff2
Usp25
Hectd1
Mkrn1
64
Plin2
Kif21b
Zc3h15
Gart
65
H13
Irs2
Ube2d3
Lyz1
66
Rpl29
Pxk
A130040M12Rik
Vwf
67
1110034A24Rik
Cyp4f18
Cd34
Gadd45a
68
Trim28
Map4k1
Igfbp5
Mpp1
69
Ltbp3
Isca1
C1s
Stim1
70
Fkbp1a
Itga4
Upk3b
Psme3
71
Erp29
Dock2
Gpr133
Ets1
72
Muc1
Spib
Dab2
Snap23
73
Lamc2
2810453l06Rik
Serpinh1
Arhgdib
74
Plscr3
Cdr2
Upk1b
Hmha1
75
Agrn
Naa16
Sdf4
Itpr2
76
Park7
Arhgdib
Ctbp2
Ubl7
77
Ctnnb1
Cd79a
Psap
Ddx58
78
Atp5g1
Rbm27
Arhgef12
Nfkbie
79
Eef1g
Lmnb1
Copb2
Setd7
80
Nhp2
Slc25a37
Ctsl
Stk24
81
Rrbp1
Klf6
Aldh1a2
Hvcn1
82
Sumo3
Hist1h1c
Dcn
Plekha2
83
Scyl1
Phip
Timp3
Psme4
84
Cox6a1
Qrfp
Xdh
Ankrd44
85
Krt8
Fermt3
Irf7
B4galt5
86
Gsta4
Ptma
Tmem151a
Phf20
87
Ppp1r14b
Etv3
Aebp1
Zc3hav1
88
Tnk1
Apobr
C2
Rnf11
89
D19Wsu162e
kg:uc008ewj.2
Spen
Plk3
90
Ctsl
Malat1
kg:uc007pfe.1
Fbxw5
91
Timp1
March8
Krt18
Emb
92
S100a6
Coro1a
Arf4
kg:uc007vdl.1
93
Rps15
Rac2
Rab14
Taok2
94
Polr2j
Glyr1
Tmem98
Dhrs11
95
Hspe1
Btg2
Prss23
Slc25a39
96
Lgals4
Mtf2
Egr1
Csk
97
Edf1
Nfkbie
Perp
Bcl2
98
Mtch1
Cd84
Csrp1
kg:uc009vev.1
99
Rnf187
AW549877
Pdpn
Wipf1
100
Npy
March2
Pdcd6ip
Sept9
101
Cox5b
Add3
Rpl37
Rnf10
102
Pak1
Ddx50
F11r
Pml
103
Mmp7
Prkcb
Gpm6a
Cd9
104
Fxyd3
Klf2
Tuba1a
D4Wsu53e
105
Cuta
Dcaf12
Ctnna1
Traf7
106
Ndufb8
Il2rg
Anxa8
Pitpnc1
107
Gps1
Selplg
Tpm1
Mms19
108
Bud31
Cd37
S100a16
Naa16
109
Ppap2c
Fastkd2
Chmp4b
Sharpin
110
Dap
Rsad2
Tbrg1
Capza1
111
Slc25a1
Msn
C3
Rsad2
112
Chaf1a
2010321M09Rik
Ptgs1
kg:uc012hdk.1
113
Asxl1
Kif2a
Rhou
Ghitm
114
Jmjd8
Cd97
Cdc42
Csnk1g1
115
Tecr
Hvcn1
Gpx4
Dgkz
116
Mgp
Nipsnap3b
Ppib
B2m
117
Uqcrh
Uba7
Stub1
Irs2
118
Wdr38
1810058I24Rik
Dmkn
Emg1
119
Col4a2
Nfrkb
Rnh1
Impact
120
Tnnt2
Pabpc1
Pdgfa
Mylip
121
Ndufs8
Usp16
Rpl37a
Psmb8
122
Tspan4
Pde1b
Rabac1
Rfk
123
Agpat6
Ncoa4
Timp2
Map3k5
124
Timp3
Irf8
Serping1
Odc1
125
Ankrd50
Ppp1cb
Rbm39
Slc11a2
126
Ube2d3
Rgs2
Tgoln1
Eif2b1
127
Sf1
Smyd4
Nfix
kg:uc008wjd.1
128
Csnk1d
Arid3b
Brd4
Rexo1
129
Reg3b
Sh3bgrl2
Tmem234
Ddx50
130
Flot2
Lyl1
Wbp5
Nipsnap3b
131
Lmna
Prr13
Ppig
Sp100
132
2310044H10Rik
Plagl2
Cd63
Uggt1
133
H19
Nfkbia
Col1a1
kg:uc007czl.1
134
Slc20a1
Eef1a1
Mt2
Arpc5
135
6720456B07Rik
Brd2
Zbtb7c
Nfrkb
136
Mdh2
Egr1
Npr1
Nap1l4
137
Eif6
Mkrn1
Tmem119
Fam117a
138
Phf5a
Pld4
Atf3
Sipa1l1
139
Vps28
Aldh1a1
Ankhd1
Ttc1
140
Bag1
Dnajb9
Tmed10
kg:uc009vew.1
141
Cyc1
Gjb5
Slc6a4
142
Angptl4
Mtif2
Atxn7l3b
143
Lgals3
H2-
Rpl29
DMb2
144
Farsb
Sdpr
Ccar1
145
Mbd3
4932438A13Rik
Ltbp4
146
Timm13
Treml1
Scyl1
147
Tpd52l2
Nup153
Ap3d1
148
Ptprn
Mpp1
Iqgap1
149
Crip2
Dhrs11
Cldn15
150
Raver1
Lrmp
Spnb2
151
Eif2b2
Manf
Ano1
152
Psma7
Mll3
Lrrn4
153
Rps6ka4
Fam116b
Id3
154
Mgat4a
B4galt5
Eif3a
155
Ifitm2
kg:uc009vew.1
Prkcdbp
156
Wars
Ly6d
Atp1a1
157
Capn5
Dguok
Dnaja2
158
Bsg
Pnpo
Tubb4b
159
Sec16a
Tmem175
Hnrnpab
160
Cldn7
Gm6548
Mmp14
161
Cox7a2
Rsrc2
Atp1b1
162
Nek6
Ccdc88b
Psip1
163
Rpl39
Akna
Mgll
164
Itpr3
Tsc22d3
Rnase4
165
Ctnna1
Txndc5
Ywhab
166
Tpd52
Tubb4a
Clip1
167
Mlf2
Stx11
Syn3
168
Crip1
D4Wsu53e
Myl12a
169
Fkbp4
Amfr
Rbm25
170
Gprc5a
Tti1
Arf2
171
Slc4a11
Fam175b
Cav1
172
Syn3
Zfp36
Hnrnpc
173
Npc2
Ddx5
Syne2
174
Rpl32
Tlr7
Dst
175
Inf2
Rfk
176
Rps10
kg:uc007ded.1
177
Rps26
Gnb2
178
Rpl37a
Tmed5
179
Ctxn1
Thbs1
180
Lrrc59
eg:320169:chr9:p
181
Dctn1
Zfp335
182
Mtap4
Emg1
183
Uqcr10
Trmt61a
184
Suds3
Adipor1
185
Ap1s1
Vwf
186
S100a1
Aatf
187
Atp5j
Trib1
188
Aim1
Pcyt1a
189
Plec
Stx18
190
Prom1
Trp53bp2
191
Rhoc
Stk40
192
Mast3
Il18
193
Olfml3
1810014B01Rik
194
Uqcr11
Lcp2
195
Plp2
Gimap4
196
Spna2
Rabl2
197
1700017B05Rik
Ncf2
198
Anxa4
eg:497210:chr14:m
199
Nudc
Tpt1
200
Asl
Mll5
201
Prkcsh
H3f3a
202
Plod3
Tspan13
203
Ndufa9
Il10ra
204
Impdh2
Mdc1
205
Ccnl2
Stk24
206
Nedd8
Myst4
207
Atp6v1f
Zdhhc20
208
Mt1
Eif2b1
209
Il4ra
Exoc4
210
Cndp2
Wipf1
211
Aprt
Impa1
212
Preb
Tmem119
213
Ap3d1
Pml
214
Mcm6
Ubb
215
Ubr4
Zmat3
216
Pvrl2
Slc30a9
217
Snrpg
Lat
218
Cycs
Tgfb2
219
Efemp2
Ube2o
220
Cct4
Igfbp5
221
Gm20605
Tspan5
222
Smad3
Fmnl1
223
Card10
Fnbp4
224
Krt7
Extl3
225
Cct2
Adcy7
226
Coro1c
Enpp4
227
Ltbr
Sep15
228
Ric8
H2-Ab1
229
Ndufs6
Bnip3l
230
Fibp
Slc11a2
231
Pold4
Stom
232
Rpl34
Mfhas1
233
Rpl34-ps1
Mettl1
234
Clic1
Rnf10
235
Eri3
kg:uc009cfd.1
236
Ets2
Klf4
237
Unc13a
Psme4
238
Usmg5
Sema4a
239
Sh3pxd2b
Ftl2
240
Wdr6
Atad1
241
Las1l
Tspan31
242
Polr2f
Srrm2
243
Vamp5
Rab5c
244
Endod1
Capza1
245
Snrpd2
H2-Aa
246
Tpi1
Fhl1
247
Wwp2
Cryab
248
Dalrd3
Arid4b
249
Iqgap1
Gart
250
Ahsa1
1110004F10Rik
251
Trim27
Rnf11
252
Serpinf1
Zc3hav1
253
D330041H03Rik
kg:uc008btl.1
254
Ppp2r5d
Rnf34
255
Minos1
Dmkn
256
Tsta3
Btg1
257
Prpsap1
Syt11
258
Sphk1
Mtdh
259
Ldha
Med21
260
Abca3
Rnf2
261
B4galt3
Tcf12
262
Porcn
Tacstd2
263
Tmc4
Madd
264
Serinc2
D16Ertd472e
265
Akr1b8
Pias1
266
Nudt4
Taok2
267
Atp5l
Pold1
268
Psmc3
Cep110
269
Hint1
A930013F10Rik
270
Rpl41
Tcof1
271
Xpnpep1
kg:uc009bpd.1
272
Nav1
kg:u009bpr.2
273
Parva
Capza2
274
Immt
Ptp4a2
275
Pafah1b3
Fth1
276
Chid1
Mepce
277
Aldh1l1
Rexo1
278
Rpl31
Prg4
279
Wbp1
Ctla2a
280
Zfp622
Smarca5
281
2700060E02Rik
Icam2
282
Hspa9
Pbx1
283
Tceb2
Gnl3l
284
Rpl36a
Slc2a3
285
Pgs1
Nnmt
286
Mpnd
Rb1cc1
287
Cdc42
Bpgm
288
Dhrs3
Lcp1
289
Hexa
Sipa1l1
290
Cpsf1
Lilrb4
291
Mea1
Ankrd44
292
Polr2e
Specc1
293
Ddb1
Rif1
294
Ptcd1
295
Atp5f1
296
Sec61b
297
Psmc5
298
Fam89b
299
Lama3
300
Tomm6
301
Mrpl28
302
Syngr2
303
Ngfrap1
304
Kcmf1
305
Tubb4b
306
Anapc11
307
Vcp
308
Arpp19
309
Pglyrp1
310
Rrp1
311
Gkn3
312
Atpif1
313
Prickle3
314
Map4k4
315
Arrdc1
316
C1qtnf6
317
Hras1
318
Lamb1
319
Eif3d
320
Snrpa
321
Tbrg1
322
Nxf1
323
Pdlim7
324
Add1
325
Pfdn5
326
Stk16
327
Gm17821
328
Csnk1e
329
Rrp7a
330
Psmb6
331
Snhg1
332
Ssr4
333
Ergic3
334
Rnaseh2a
335
kg:uc009cut.1
336
Bgn
337
Gm5506
338
Uqcrq
339
Tmem167
340
Nasp
341
Mif
342
Acaa2
343
Fam162a
344
Eif4ebp3
345
Nhp2l1
346
Prelid1
347
Gss
348
Lonp1
349
Srsf2
350
Igsf8
351
Ndufa7
352
Neat1
353
S100a13
354
Apoa1bp
355
Fam40a
356
Rps25
357
Eno1
358
Cldn2
359
Capn2
360
Glo1
361
Atp5c1
362
Rab2a
363
Rab25
364
Ncor2
365
Lgi4
366
Ier3
367
Tmem223
368
Slc9a3r2
369
Atp13a1
370
Rpn2
371
Acp5
372
Cct5
373
Sdf4
374
Mprip
375
Pmm2
376
Snx22
377
Arl2
378
1110008F13Rik
379
Polr1d
380
Dpm2
381
Cela1
382
2310016M24Rik
383
Cep250
384
Mybbp1a
385
Polr2g
386
Bag6
387
Cpxm1
388
Eif3m
389
Prr24
390
Sra1
391
Scara3
392
Reg1
393
Gas5
394
Hnrnpab
395
Mcpt2
396
Tgfbi
397
Capns1
398
Fdx1l
399
S100a16
400
Nap1l1
401
Swi5
402
Rpl38
403
Dctn2
404
Pdlim1
405
Gemin7
406
Pnpla6
407
Nono
408
Sla2
409
Idh3b
410
Ppp2r4
411
Map2k2
412
Ndufb10
413
Atp5d
414
Arfgap1
415
Tmbim1
416
Ergic1
417
Pdgfa
418
Ppp2ca
419
Hk1
420
Ltbp2
421
Trim35
422
Gtf2i
423
C1qb
424
Ankhd1
425
Podxl
426
Rps21
427
Huwe1
428
Pomp
429
Dpp3
430
Fkbp8
431
Itga5
432
Hes6
433
Mrpl11
434
Poldip3
435
Scd2
436
Tmem55b
437
Ndufa13
438
Dcakd
439
Ubqln1
440
Gpx4
441
Cyb561
442
Gmppa
443
Ncaph2
444
Pdha1
445
Ndufs4
446
Fcer1g
447
Myof
448
Ppib
449
Mrpl52
450
Tes
451
Emp3
452
Ndufa11
453
Tor1aip2
454
Anp32b
455
Tnk2
456
Mcpt1
457
Ssr2
458
Psmb3
459
2700081O15Rik
460
Pcbd1
461
Eif1ax
462
Pmm1
463
Ptprk
464
Hadha
465
Calu
466
Fam73a
467
Atp5e
468
Hsd17b10
469
Rbm39
470
Egfl7
471
Psmc1
472
Perp
473
Lman2
474
Galnt1
475
Rbx1
476
Lemd2
477
Zglp1
478
Ing4
479
kg:uc008oow.1
480
1500012F01Rik
481
Cox4i1
482
kg:uc008bcq.1
483
Ubap2l
484
Pafah1b2
485
Mrpl13
486
Nucb1
487
Fbn1
488
Adrm1
489
Itgb4
490
Ctss
491
Plbd2
492
Ptpmt1
493
Sap30l
494
Ppp1r12c
495
Sgta
496
Acrbp
497
Higd2a
498
Higd1a
499
Tmem208
500
Cdh1
501
Ube2d2a
502
Suv39h1
503
Rabac1
504
Anxa5
505
Ubxn6
506
Tpm1
507
Hmga2
508
Cnbp
509
Rpl21
510
Ndufb5
511
Sec31a
512
Znhit1
513
Cyb5b
514
Sfn
515
Ccdc12
516
Elovl1
517
Psmb5
518
Slc25a11
519
Psmd2
520
Nsun2
521
Slc50a1
522
Eme1
523
Bnip2
524
Pxdn
525
Mad2l2
526
Pdcd6
527
2010107E04Rik
528
Abhd11
529
Carkd
530
Polr2l
531
Ppdpf
532
Cib1
533
Dgcr2
534
Timm50
535
Mrps24
536
Abhd12
537
Brf1
538
Man1b1
539
kg:uc012cgd.1
540
Gpaa1
541
Fmnl3
542
Mapk3
543
C1qc
544
Pgls
545
Cp
546
Serh1
547
2610203C20Rik
548
Hsbp1
549
Tmem214
550
Akt1
551
kg:uc007pfe.1
552
Tmed10
553
Ttll3
554
2200002D01Rik
555
Tnfrsf23
556
Sgsm3
557
Atp9a
558
Lcn2
559
Pdrg1
560
Tspan9
561
Nrd1
562
Rin1
563
Ndufv1
564
Naa10
565
Wnk1
566
Heatr7a
567
Slc4a2
568
Ggct
569
5730403B10Rik
570
Sh3glb2
571
Pfkl
572
Tspan3
573
Gns
574
Sdcbp2
575
C130074G19Rik
576
Cotl1
577
Tubb5
578
Sec11c
579
Pigq
580
Zc3h15
581
Lsmd1
582
Ppa1
583
Chmp4b
584
Sepn1
585
Angptl2
586
Itpripl2
587
Ddx1
588
Hbxip
589
Cdk2ap1
590
Clta
591
Cpsf3l
592
Apoe
593
Ift46
594
Sae1
595
Gpi1
596
Gorasp2
597
1500032L24Rik
598
Nsmce4a
599
Dlst
600
Bap1
601
Pitpnb
602
Meg3
603
Cyth2
604
Atp5o
605
Gon4l
606
Sox11
607
Cxxc5
608
Avil
609
Alcam
610
Eif3f
611
Cygb
612
Eif1ad
613
Polr3h
614
Araf
615
Gkn1
616
Rhog
617
Mtap
618
Eif4ebp1
619
Akr1a1
620
Trip6
621
Prdx6
622
2410015M20Rik
623
Rps6
624
Rps23
625
Stxbp2
626
Rps19
627
Ykt6
628
Atp5g2
629
Serpinb1a
630
Col7a1
631
Mrps6
632
Lgals9
633
Rcn3
634
Trim44
635
Surf2
636
Rps29
637
Cdipt
638
Lmf2
639
Psenen
640
Ltf
641
Mpzl1
642
Psmd6
643
Cttn
644
Tmc6
645
2500003M10Rik
646
Atp6v0a1
647
Med8
648
Prrx2
649
Atp5b
650
Smurf1
651
Carhsp1
652
Tpcn1
653
Ndufb9
654
Pih1d1
655
Hnrnpa0
656
Fn1
657
2810428I15Rik
658
0610012G03Rik
659
Ube2i
660
Anxa3
661
Msto1
662
Eng
663
0910001L09Rik
664
Rpl10
665
kg:uc007xxx.1
666
Mosc2
667
Vps37c
668
Sgpl1
669
Fam166a
670
Polr2b
671
Fam101b
672
Nupr1
673
Lsm4
674
Rpl36
675
0610007C21Rik
676
Psmc2
677
Supt6h
678
Rps13
679
5430437P03Rik
680
Dsp
681
Ddx56
682
Tsc2
683
Trmt2a
684
Vdac2
685
Cant1
686
Eif4h
687
Puf60
688
A430105I19Rik
689
Cacnb3
690
Prdx4
691
March5
692
Ccar1
693
Npepl1
694
Fermt1
695
Use1
696
Axl
697
Slc39a4
698
1110008P14Rik
699
Sema4g
700
Timm8b
701
Krt23
702
Rpl28
703
Lgals3bp
704
Hdgf
705
1110005A03Rik
706
Impdh1
707
Mtmr11
708
Msln
709
Zdhhc3
710
Znrf1
711
Aldh16a1
712
Bloc1s1
713
Prkag1
714
Plxnb1
715
Crat
716
Phpt1
717
5930434B04Rik
718
Kpnb1
719
Nme2
720
E430025E21Rik
721
Smyd2
722
Cyhr1
723
Mvp
724
Rps27l
725
Rbp4
726
Cars
727
kg:uc012ctw.1
728
Ssr1
729
Ssu72
730
Usp48
731
Atp5k
732
Lrrk1
733
BC056474
734
Epn1
735
Trappc1
736
Clk2
737
Sugt1
738
Nenf
739
kg:uc009cuu.1
740
Ubap2
741
Rps20
742
Atp5h
743
9430008C03Rik
744
Kars
745
Mrpl37
746
Aimp1
747
Trmt1
748
Hspa4
749
Cd164
750
9430023L20Rik
751
Rnf4
752
H1f0
753
C1qtnf1
754
Srd5a1
755
1500010J02Rik
756
Rpl35a
757
Cand2
758
C630004H02Rik
759
Acsbg1
760
Derl1
761
Cbx5
762
Tmem63a
763
Hgfac
764
Stx5a
765
Bri3
766
Tomm20
767
Fam20c
768
Cox6c
769
Tm2d2
770
Plekhb2
771
Ramp1
772
2410001C21Rik
773
Tardbp
774
Pebp1
775
kg:uc008gbp.1
776
Eif3b
777
Ccna2
778
Ptges
779
kg:uc007hyr.2
780
Wbp5
781
Chchd2
782
Fdft1
783
Srm
784
Gtf3a
785
D0H4S114
786
1810009A15Rik
787
Rps27
788
Tmem176b
789
Ndufc1
790
Lasp1
791
Fam108a
792
Mapk8ip3
793
Copa
794
Serpina3n
795
Rps17
796
Dnpep
797
Lbp
798
Krt19
799
Ei24
800
Ap1b1
801
Mogs
802
Uba1
803
Postn
804
Phf23
805
Paox
806
Nploc4
807
Ndufv2
808
Actr1a
809
Mxd3
810
Pfdn1
811
Ide
812
Foxp4
813
1810013D10Rik
814
2310007B03Rik
815
Xab2
816
Agr2
817
Dctn3
818
Urm1
819
H2-Ke2
820
Spint1
821
Slc38a2
822
Ube2z
823
Ctrb1
824
Fam195b
825
Suclg1
826
Ube2l3
827
Rpn1
828
Mrps7
829
Tsg101
830
Drosha
831
Arfip2
832
Mrto4
833
Grlf1
834
Sort1
835
Oaf
836
Ints1
837
Slc44a2
838
Dph3
839
Gramd1a
840
Fkbp9
841
Fam149a
842
1810035L17Rik
843
kg:uc007fte.1
844
Eif2s1
845
Smpd1
846
Eef1b2
847
Actr10
848
Rab11fip5
849
Ypel3
850
Flnb
851
Tcn2
852
Crlf1
853
Map3k15
854
Cul7
855
Atp6v1g1
856
Ncbp1
857
Atp1b3
858
Mtif3
859
Aldoa
860
Htra1
861
Rab14
862
Ppm1a
863
Ndufb11
864
Kansl3
865
Rab24
866
Bcl2l1
867
Lgals1
868
Samm50
869
Mrps33
870
Anxa1
871
Chchd1
872
Mapre1
873
Ctbp2
874
Rnps1
875
Spg7
876
Tnfrsf12a
877
H6pd
878
Myo7a
879
Mcm7
880
Psmd13
881
Mrpl54
882
Atp6v0b
883
Prdx1
884
Elof1
885
Rexo4
886
Mrps18a
887
Dpcd
888
D2Wsu81e
889
Cd99l2
890
Synpo
891
Atp2a2
892
Cdc5l
893
Stard7
894
Atp13a2
895
Sdha
896
Hdac6
897
Krt20
898
Ppp6r3
899
1700037H04Rik
900
Napa
901
PgP
902
Cnih
903
Atg4b
904
Cox8a
905
Srp68
906
St13
907
Gng12
908
Cfdp1
909
Rcc2
910
Pisd-
ps1
911
Ivns1abp
912
Mpv17l2
913
Ssna1
914
Gnl1
915
Tmem111
916
Hbs1l
917
Agpat3
918
Col6a2
919
March6
920
Usp39
921
Rps11
922
Ahnak
923
Lcmt1
924
Ddx41
925
H2afv
926
Fau
927
Tuba1c
The gene names listed in Table 13 and 12 are common names. NCBI Gene ID numbers for each of the genes listed in Table 13 and 12 can be obtained by searching the “Gene” Database of the NCBI (available on the World Wide Web at http://www.ncbi.nlm.nih.gov/) using the common name as the query and selecting the first returned Homo sapiens gene. Other genes may be obtained using the UCSC genome browser (available on the World Wide Web at http://genome.ucsc.edu) using the Gene Sorter function. In certain embodiments, the marker gene(s) are selected from the genes listed in Table 13 and/or 12.
In some embodiments, the marker gene(s) is selected from a marker gene indicated to be upregulated in at least one type of CTC in Table 13, e.g. marker genes 1-142. In some embodiments, the marker gene(s) is selected from a marker gene indicated to be upregulated in at least one type of CTC in Table 12, e.g. marker genes listed in the columns labeled “CTC-c vs. Primary Tumor Enriched Gene” or “CTC-c vs. WBC”.
In a CTC, the marker genes listed in Table 13 or 12 can be upregulated, e.g. for marker genes listed in Table 13 and/or 12, if the measured marker gene expression in a cell or sample is higher as compared to a reference level of that marker gene's expression, then the cell is identified as a CTC and/or the sample is identified as comprising CTCs. Preferably, once looks at a statistically significant change. However, even if a few genes in a group do not differ from normal, a sample can be identified as comprising CTCs if the overall change of the group shows a significant change, preferably a statistically significant change. All possible combinations of 2 or more of the indicated markers are contemplated herein.
1.PublishNumber: US-2016312298-A1
2.Date Publish: 20161027
3.Inventor: TING DAVID T.
HABER DANIEL A.
MAHESWARAN SHYAMALA
4.Inventor Harmonized: TING DAVID T(US)
HABER DANIEL A(US)
MAHESWARAN SHYAMALA(US)
5.Country: US
6.Claims:
(en)The technology described herein relates to methods of detecting circulating tumor cells (CTCs), e.g. by detecting changes in the expression of certain CTC marker genes. Aberrant expression of CTC marker genes, e.g. changes in expression indicative of CTCs can also be targeted in order to treat cancer.
7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/918,816 filed Dec. 20, 2013 and 61/937,883 filed Feb. 10, 2014, the contents of which are incorporated herein by reference in their entirety.
GOVERNMENT SUPPORT
This invention was made with federal funding under Grant Nos. 2R01CA129933 awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.
TECHNICAL FIELD
The technology described herein relates to the diagnosis and treatment of cancer.
BACKGROUND
Circulating Tumor Cells (CTCs) are shed from primary tumors into the bloodstream, mediating the spread of cancer to distant organs (metastasis). Thus, the presence of circulating tumor cells (CTCs) in the bloodstream ultimately leads to spread of cancer to distant organs. However, CTCs are rare, estimated at one to ten tumor cells among ten billion normal blood cells in a milliliter of blood. As such, their isolation and molecular analysis has posed a significant technological challenge (Pantel et al., Nat Rev Cancer 2008 8:329-340; Yu et al., J Cell Biol 2011 192:373-382).
SUMMARY
As described herein, the inventors have identified a number of genes, the expression of which is characteristic of CTCs. In particular, the expression of these genes differentiates CTCs from primary tumor cells Accordingly, provided herein are methods and assays relating to the detection of CTCs, including diagnostic and prognostic methods and assays. Further, provided herein are treatments for cancer that target these markers of CTCs, e.g., to inhibit metastasis.
In one aspect, described herein is a method of detecting circulating tumor cells (CTCs) in a sample, the method comprising: measuring the level of a PC-CTC marker gene expression product in the sample; and determining that PC-CTCs are present if the detected level of the marker gene expression product is greater than a reference level. In some embodiments, the CTCs are pancreatic cancer CTCs. In some embodiments, the method further comprises a first step of isolating the CTCs from the sample. In some embodiments, the expression product is a nucleic acid. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization. In some embodiments, the expression product is a polypeptide. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of: Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay. In some embodiments, the CTC marker gene is selected from Table 7 or Table 8. In some embodiments, the CTC marker gene is selected from the group consisting of: ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
In one aspect, described herein is a method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of a CTC marker gene-targeted therapy to the subject. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the CTC marker gene-targeted therapy comprises an inhibitor of a CTC marker gene. In some embodiments, the inhibitor is an antibody reagent. In some embodiments, the inhibitor is an inhibitory nucleic acid reagent. In some embodiments, the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent and a chemotherapeutic agent. In some embodiments, the subject is a subject determined to have an elevated level of CTCs and/or an elevated level of a CTC marker gene present in the blood and/or stroma of the cancer.
In one aspect, described herein is a method of determining if a subject is likely to respond to treatment with a CTC marker gene-targeted therapy, the method comprising measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is likely to respond to the treatment if the level of the expression product is increased relative to a reference level. In some embodiments, the method further comprises a first step of isolating the CTCs from the sample. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the expression product is a nucleic acid. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization. In some embodiments, the expression product is a polypeptide. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of: Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay. In some embodiments, the PC-CTC marker gene is selected from Table 7 or Table 8. In some embodiments, the CTC marker gene is selected from the group consisting of: ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
In one aspect, described herein is a method of monitoring the treatment of a subject, the method comprising: administering a cancer therapy to a subject in need thereof; measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is responding if the level of the CTC marker gene expression product is decreased relative to the reference level and determining that the subject is not responding to the treatment if the CTC marker gene expression product is not decreased relative to the reference level. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the reference level is the level of the gene expression product in the patient prior to the administering step. In some embodiments, the method further comprises a first step of isolating the CTCs from the sample. In some embodiments, the expression product is a nucleic acid. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization. In some embodiments, the expression product is a polypeptide. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of: Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay. In some embodiments, the PC-CTC marker gene is selected from Table 7 or Table 8. In some embodiments, the CTC marker gene is selected from the group consisting of: ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1C demonstrate the isolation and characterization of CTCs. FIG. 1A depicts a schematic of CTC-iChip negative IFD system. FIG. 1B depicts a graph of mouse WBC depletion consistency between normal and cancer mouse models. WBC depletion shown in log 10. FIG. 1C depicts a graph of CTC enumeration by immunofluorescent staining (CK+/CD45−/DAPI+) from normal and KPC mice.
FIG. 2 depicts schematics of principal component analysis of single cell samples.
FIGS. 3A-3B demonstrate that epithelial, mesenchymal, and stem cell genes are differentially expressed in CTC-c cells vs Tumors. Depicted are boxplot of genes that are A) downregulated ( FIG. 3A ) and upregulated ( FIG. 3B ) in CTC-c cells vs Tumors. Bar=median, box plot=quartiles, scale in log 10(rpm).
FIGS. 4A-4C demonstrate CTC-iChip characterization. FIG. 4A depicts a graph of the percent of WBC deflected (y-axis) as a function of the number of anti-CD45 beads per WBC (x-axis). FIG. 4B depicts a graph of the recovery of mouse PDAC cell line NB508 spiked into normal mouse blood (4 independent experiments shown). FIG. 4C depicts a graph of the captured CTCs/mL of blood from syngeneic orthotopic PDAC tumors using NB508 cell line.
FIG. 5A depicts a table of KPC mouse genotype and characteristics. FIG. 5B depicts graphs of quality metrics of single cell sequencing with % of reads aligned and total unique alignments for cell lines (NB508, MEF), CTCs, WBC, and diluted bulk RNA from matched primary tumors. FIG. 5C depicts graphs of single cell heterogeneity using mean intra-cluster correlation coefficient for each cluster (rights) and between single cell primary tumor (TuGMP3), cancer cell line (NB508), and all CTCs (Cluster 1, 3, 4, 5, 9). Circle=mean, Range=95% CI.
FIG. 6 depicts boxplot graphs of ECM protein gene enriched in CTC-c compared to bulk primary tumors and single cell primary tumors. Bar=median, boxplot—quartiles, scale in log 10 (rpm).
FIG. 7 depicts a heatmap expression profile of human pancreatic CTCs from 3 patients. Epithelial genes used to define CTCs and enriched extracellular proteins shown. Expression shown in log 10 scale.
FIG. 8 depicts a graph of quantitative RT-PCR of SPARC expression in human pancreatic cancer cell lines.
FIG. 9 depicts invasion assays. Decreases in invasion through Matrigel of PDAC2 and PDAC 3 cell lines with shRNA against SPARC (ShF1 and ShF3) were observed. shNT=Non-target shRNA
FIG. 10 depicts a graph of the number of mice with detectable metastases by in vivo luciferase imaging in non-target shRNA (NT) and SPARC shRNA (SHF1).
FIG. 11 depicts a schematic of the process of determining CTC heterogeneity.
FIGS. 12A-12C demonstrate that CTC-Enriched Genes are Found in Epithelial and Stromal Components of Primary Tumors. Depicted are expression boxplots of ( FIG. 12A ) Aldh1a2 stem cell and CTC highly enriched genes ( FIG. 12B ) Klf4 and ( FIG. 12C ) Igfbp5 genes. Bar=median, box plot=quartiles, scale in log 10(rpm).
FIG. 13 demonstrates that human and mouse CTCs across different epithelial cancer express high levels of ECM protein genes. Depicted are expression boxplot of highly expressed ecm genes in human pdac, breast (br), and prostate (pr) ctcs. bar, median; boxplot, quartiles; scale in log 10(rpm). holm-adjusted p value<0.05 (*), 0.01 (**), 0.001 (***).
FIGS. 14A-14E demonstrate that SPARC expression in human PDAC enhances invasion and metastasis. FIG. 14A depicts a graph of proliferation of PDAC3 cell lines determined by MTT. FIG. 14B depicts a graph of tumor spheres in PDAC3 shNT versus shSPARC counted per 43 field (error bars represent SD). FIG. 14C depicts a graph of invasion of shSPARC and shNT cell lines quantitated by number of nuclei/203 field. p value<0.01 (**), 0.001 (***), 0.0001 (****). Error bars represent SD. FIG. 14D depicts a graph of Percentage of detectable lung metastases by in vivo luciferase imaging after 3 weeks after tail vein inoculation of PDAC3 cell lines. Fisher's exact test p value is shown. FIG. 14E depicts a graph of normalized metastasis burden in mice with orthotopic pancreatic tumors from PDAC3 cell lines. Error bars represent SD (*p<0.05).
FIG. 15 depicts a Summary Model of the Role of Pancreatic CTCs in the Metastatic Cascade. Shown are the heterogeneous subsets of pancreatic CTCs with a focus on the most prominent classical CTC group, which are enriched for coexpression of epithelial (keratin) and stromal (Sparc) genes.
FIG. 16A depicts a graph of PDAC2 shRNA cell lines by qRT-PCR. Average shown with max and min RQ (error bars). FIG. 16B depicts a graph of proliferation rates by MTT assay similar in PDAC2 cell line between shNT and shSPARC stable lines. FIG. 16C depicts a graph of tumor sphere invasion assay (error bars=STD) formation at 2 weeks similar between shNT and shSPARC cell lines. Quantiation done per 4× magnification field (Error bars=SD). Migratory behavior reduced by shSPARC_1 & 3 as determined by ( FIG. 16D ) invasion assay at 48 hours.
DETAILED DESCRIPTION
As described herein, the inventors have discovered that circulating tumor cells (CTCs) are characterized by the expression of certain genes, i.e. CTC marker genes. The discovery of these CTC marker genes permit methods and assays for the detection and/or measurement of CTC levels, e.g. CTC levels in a sample from a subject. These methods and assays can provide improved speed and accuracy in the measurement of CTC levels. Furthermore, because the expression of these marker genes distinguishes CTCs from other cells, e.g., other circulating cells and/or normal tumor cells, therapies can be targeted against CTCs by binding to and/or inhibiting these marker gene expression products to reduce the level and/or metastatic potential of CTCs.
As used herein, “circulating tumor cell” or “CTC” refers to tumor cells which are shed from a tumor and present in the blood, i.e. in circulation. Cell markers (e.g. marker genes) that can be used to identify and/or isolate CTCs from other components of the blood are described below herein. In some embodiments, a CTC can be a pancreatic cancer CTC.
In one aspect, described herein is a method of detecting circulating tumor cells (CTCs) in a sample, the method comprising measuring the level of a CTC marker gene expression product in the sample; and determining that CTCs are present if the detected level of the marker gene expression product is greater than a reference level.
As described herein, the inventors have discovered that a number of genes are differentially regulated in CTCs, e.g. as compared to non-circulating tumor cells. Accordingly, there are provided herein methods and assays relating to the measurement of CTC levels. Elevated CTC levels can indicate a poor prognosis, e.g. an increased risk of metastatsis. Accordingly, provided herein are methods and assays related to the prognosis, risk assessment, and treatment of subjects having cancer. In certain embodiments, the assays and methods are directed to determination and/or measurement of the expression level of a gene product (e.g. protein and/or gene transcript such as mRNA) in a biological sample of a subject. In certain embodiments the assays and methods are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, i.e. at least two genes, at least three genes, at least four genes, at least five genes, at least six genes, at least seven genes, at least eight genes, at least nine genes, at least 10 genes . . . at least 15 genes, . . . at least 25 genes, . . . at least 30 genes, or more genes, or any number of genes selected from Table 7, Table 8, and/or Table 14 as described herein.
In some embodiments, the marker gene(s) is selected from the group consisting of ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or, e.g. all of the following genes: ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4.
In some embodiments, the marker gene(s) is selected from the group consisting of ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or, e.g. all of the following genes: ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2.
In some embodiments, the marker gene(s) is selected from the group consisting of ALDH1A2; IGFBP5; KLF4; DCN; and SPARC. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or, e.g. all of the following genes: ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
In some embodiments, the marker gene(s) is selected from the group consisting of ALDH1A2; IGFBP5; KLF4; and DCN. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or e.g. all of the following genes: ALDH1A2; IGFBP5; KLF4; and DCN.
In some embodiments, the marker gene(s) is selected from the group consisting of TPT1; HMGB1; SPON 2; SPARC; and ARSA. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or, e.g. all of the following genes: TPT1; HMGB1; SPON 2; SPARC; and ARSA.
In some embodiments, the marker gene(s) is selected from the group consisting of IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or at least five genes, or at least six genes, or at least seven genes, or at least eight genes or, e.g. all of the following genes: IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A. In some embodiments, the level of polypeptide expression products are determined for the marker gene(s) is selected from the group consisting of IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A, e.g. because, as described herein, RNA levels of cell surface proteins are lower than polypeptide levels.
TABLE 7
Exemplary mouse marker genes
MOUSE
GENE
SYMBOL
Gene Name
Abcb1b
ATP-binding cassette, sub-family B (MDR/TAP), member 1B
Abi3bp
ABI gene family, member 3 (NESH) binding protein
Ablim3
actin binding LIM protein family, member 3
Acad9
acyl-Coenzyme A dehydrogenase family, member 9
Acbd3
acyl-Coenzyme A binding domain containing 3
Acini
apoptotic chromatin condensation inducer 1
Actb
actin, beta
Actg1
predicted gene 8543; actin-like 8; predicted gene 7505; predicted gene
12715; predicted gene 12003; predicted gene 8399; predicted gene 6375;
actin, gamma, cytoplasmic 1; similar to gamma-actin; predicted gene
4667; similar to cytoplasmic beta-actin; predicted gene 16385
Adamts5
similar to a disintegrin-like and metalloprotease (reprolysin type) with
thrombospondin type 1 motif, 5 (aggrecanase-2); a disintegrin-like and
metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 5
(aggrecanase-2)
Adamtsl1
ADAMTS-like 1
Add3
adducin 3 (gamma)
Aebp1
AE binding protein 1
Agap1
ArfGAP with GTPase domain, ankyrin repeat and PH domain 1
Akap13
A kinase (PRKA) anchor protein 13
Akap2
A kinase (PRKA) anchor protein 2; paralemmin 2
Akr1b3
aldo-keto reductase family 1, member B3 (aldose reductase)
Akt2
similar to RAC-beta serine/threonine-protein kinase (RAC-PK-beta)
(Protein kinase Akt-2) (Protein kinase B, beta) (PKB beta); thymoma viral
proto-oncogene 2; similar to serine/threonine kinase
Aldh1a1
aldehyde dehydrogenase family 1, subfamily A1
Aldh1a2
aldehyde dehydrogenase family 1, subfamily A2
Alox12
arachidonate 12-lipoxygenase
Amfr
autocrine motility factor receptor
Amhr2
anti-Mullerian hormone type 2 receptor
Ang
angiogenin, ribonuclease, RNase A family, 5
Ankrd11
ankyrin repeat domain 11
Ankrd12
ankyrin repeat domain 12; similar to Ankrd12 protein
Ankrd17
ankyrin repeat domain 17
Ano6
anoctamin 6
Anp32a
acidic (leucine-rich) nuclear phosphoprotein 32 family, member A
Anxa7
annexin A7
Ap1s3
predicted gene 8532; similar to adaptor-related protein complex AP-1,
sigma 3; adaptor-related protein complex AP-1, sigma 3
Ap3s1
predicted gene 7603; adaptor-related protein complex 3, sigma 1 subunit;
predicted gene 5610
Ap4e1
adaptor-related protein complex AP-4, epsilon 1
Aplp1
amyloid beta (A4) precursor-like protein 1
Apol9a
apolipoprotein L 9b; apolipoprotein L 9a
App
amyloid beta (A4) precursor protein
Aqp1
aquaporin 1
Arap2
predicted gene 336; ArfGAP with RhoGAP domain, ankyrin repeat and PH
domain 2
Arf2
ADP-ribosylation factor 2
Arf3
ADP-ribosylation factor 3
Arf5
similar to ADP-ribosylation factor; ADP-ribosylation factor 5
Arhgap28
Rho GTPase activating protein 28
Arhgap29
Rho GTPase activating protein 29
Arhgap5
Rho GTPase activating protein 5
Arhgef12
predicted gene 7281; predicted gene 5831; similar to SP140 nuclear body
protein (predicted); Rho guanine nucleotide exchange factor (GEF) 12
Arid1a
similar to AT rich interactive domain 1A isoform a; AT rich interactive
domain 1A (SWI-like)
Arid4a
AT rich interactive domain 4A (RBP1-like)
Arid4b
AT rich interactive domain 4B (RBP1-like)
Arid5b
similar to modulator recognition factor 2; AT rich interactive domain 5B
(MRF1-like)
Arl3
ADP-ribosylation factor-like 3
Arl4d
ADP-ribosylation factor-like 4D; hypothetical protein LOC100044157
Arl6ip5
ADP-ribosylation factor-like 6 interacting protein 5
Armcx3
armadillo repeat containing, X-linked 3; hypothetical protein
LOC100044266; predicted gene 9299
Arpc2
predicted gene 5492; actin related protein 2/3 complex, subunit 2
Arsa
arylsulfatase A
Arsb
arylsulfatase B
Ascc3
activating signal cointegrator 1 complex subunit 3
Atf3
activating transcription factor 3
Atg3
autophagy-related 3 (yeast)
Atp1a1
ATPase, Na+/K+ transporting, alpha 1 polypeptide
Atp1b1
ATPase, Na+/K+ transporting, beta 1 polypeptide
Atp2b1
ATPase, Ca++ transporting, plasma membrane 1
Atp6v1a
ATPase, H+ transporting, lysosomal V1 subunit A
Atxn2
ataxin 2
B230120H23Rik
RIKEN cDNA B230120H23 gene
B2m
beta-2 microglobulin
BC003331
similar to odorant response abnormal 4; cDNA sequence BC003331
BC005537
cDNA sequence BC005537
BC005561
THO complex 2; cDNA sequence BC005561
BC013529
cDNA sequence BC013529
Baz2a
bromodomain adjacent to zinc finger domain, 2A
Bbs4
Bardet-Biedl syndrome 4 (human)
Bbx
bobby sox homolog ( Drosophila )
Bcam
basal cell adhesion molecule
Bcl10
B-cell leukemia/lymphoma 10; predicted gene 6141
Bdp1
B double prime 1, subunit of RNA polymerase III transcription initiation
factor IIIB
Bicc1
bicaudal C homolog 1 ( Drosophila )
Bicd1
bicaudal D homolog 1 ( Drosophila )
Birc6
baculoviral IAP repeat-containing 6
Blvrb
biliverdin reductase B (flavin reductase (NADPH))
Bnc1
basonuclin 1
Bnc2
basonuclin 2
Bod1l
biorientation of chromosomes in cell division 1-like
Bptf
bromodomain PHD finger transcription factor
Braf
Braf transforming gene
Brd2
similar to mKIAA4005 protein; bromodomain containing 2
Brd4
bromodomain containing 4
Brp44l
similar to brain protein 44-like protein; brain protein 44-like; predicted
gene 3452; predicted gene 8219
Bst2
bone marrow stromal cell antigen 2
Btbd2
BTB (POZ) domain containing 2
Btbd7
BTB (POZ) domain containing 7
Btf3
predicted gene 9308; basic transcription factor 3; predicted gene 3531;
predicted gene 7973
Btg2
B-cell translocation gene 2, anti-proliferative
Bzw1
predicted gene 11652; predicted gene 5191; basic leucine zipper and W2
domains 1
C1d
C1D nuclear receptor co-repressor
C1ra
complement component 1, r subcomponent; predicted gene 8551
C1rl
complement component 1, r subcomponent-like
C1s
similar to Complement component 1, s subcomponent; complement
component 1, s subcomponent
C2
complement component 2 (within H-2S)
C3
complement component 3; similar to complement component C3
prepropeptide, last
C4a
similar to Complement C4 precursor; complement component 4A (Rodgers
blood group); similar to complement C4; complement component 4B
(Childo blood group)
C4b
similar to Complement C4 precursor; complement component 4A (Rodgers
blood group); similar to complement C4; complement component 4B
(Childo blood group)
Calm1
predicted gene 7743; calmodulin 3; calmodulin 2; calmodulin 1; predicted
gene 7308
Calm2
predicted gene 7743; calmodulin 3; calmodulin 2; calmodulin 1; predicted
gene 7308
Cap1
CAP, adenylate cyclase-associated protein 1 (yeast)
Cast
calpastatin
Cav1
caveolin 1, caveolae protein
Ccdc109b
coiled-coil domain containing 109B
Ccdc34
coiled-coil domain containing 34
Ccdc80
coiled-coil domain containing 80
Ccdc88a
coiled coil domain containing 88A
Ccdc90a
coiled-coil domain containing 90A
Ccnl1
cyclin L1
Cd109
CD109 antigen
Cd200
CD200 antigen; similar to MRC OX-2 antigen homolog
Cd248
CD248 antigen, endosialin
Cd34
CD34 antigen
Cd55
CD55 antigen
Cd81
CD81 antigen
Cd82
CD82 antigen
Cd9
CD9 antigen
Cdc42ep3
CDC42 effector protein (Rho GTPase binding) 3
Cdh11
cadherin 11
Cdh3
cadherin 3
Cdk13
cell division cycle 2-like 5 (cholinesterase-related cell division controller)
Cdon
cell adhesion molecule-related/down-regulated by oncogenes
Celf2
CUG triplet repeat, RNA binding protein 2
Cep164
centrosomal protein 164
Cep57
centrosomal protein 57
Cfh
complement component factor h; similar to complement component
factor H
Cfl1
cofilin 1, non-muscle; similar to Cofilin-1 (Cofilin, non-muscle isoform);
predicted gene 6180
Cfl2
cofilin 2, muscle
Chd1
chromodomain helicase DNA binding protein 1
Chd2
chromodomain helicase DNA binding protein 2
Chi3l1
chitinase 3-like 1
Chst4
carbohydrate (chondroitin 6/keratan) sulfotransferase 4
Cish
cytokine inducible SH2-containing protein
Clcn3
chloride channel 3
Cldn15
claudin 15
Cldn25
predicted gene 16492
Clec1b
C-type lectin domain family 1, member b
Clec3b
C-type lectin domain family 3, member b
Clic4
chloride intracellular channel 4 (mitochondrial)
Clip1
CAP-GLY domain containing linker protein 1
Clip3
CAP-GLY domain containing linker protein 3
Cln8
ceroid-lipofuscinosis, neuronal 8
Cmah
cytidine monophospho-N-acetylneuraminic acid hydroxylase
Cmtm3
CKLF-like MARVEL transmembrane domain containing 3
Cmtm7
CKLF-like MARVEL transmembrane domain containing 7
Cnot6l
CCR4-NOT transcription complex, subunit 6-like
Cobl
cordon-bleu
Cobll1
Cobl-like 1
Col14a1
collagen, type XIV, alpha 1
Col1a2
collagen, type I, alpha 2
Col3a1
collagen, type III, alpha 1
Col4a6
collagen, type IV, alpha 6
Colec12
collectin sub-family member 12
Coq10b
hypothetical protein LOC675736; coenzyme Q10 homolog B ( S. cerevisiae );
predicted gene 4899
Creb3l1
cAMP responsive element binding protein 3-like 1
Creb5
RIKEN cDNA 9430076C15 gene; cAMP responsive element binding
protein 5
Crebbp
CREB binding protein
Creg1
cellular represser of E1A-stimulated genes 1
Crim1
cysteine rich transmembrane BMP regulator 1 (chordin like)
Crls1
cardiolipin synthase 1
Cryab
crystallin, alpha B
Cryl1
crystallin, lambda 1
Crym
crystallin, mu
Csda
cold shock domain protein A
Csf1
colony stimulating factor 1 (macrophage)
Csnk1a1
casein kinase 1, alpha 1
Csrnp1
cysteine-serine-rich nuclear protein 1
Csrp1
cysteine and glycine-rich protein 1
Cuedc1
CUE domain containing 1
Cyb5
cytochrome b-5
Cybrd1
cytochrome b reductase 1
Cyp2d22
cytochrome P450, family 2, subfamily d, polypeptide 22
Cyp2s1
cytochrome P450, family 2, subfamily s, polypeptide 1
Cyr61
cysteine rich protein 61
Dab2
disabled homolog 2 ( Drosophila )
Dag1
dystroglycan 1
Daglb
diacylglycerol lipase, beta
Dapk1
death associated protein kinase 1
Dcn
decorin
Ddr1
discoidin domain receptor family, member 1
Ddr2
discoidin domain receptor family, member 2
Ddx3x
DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked
Ddx5
DEAD (Asp-Glu-Ala-Asp) box polypeptide 5; predicted gene 12183
Dennd5a
DENN/MADD domain containing 5A; similar to Rab6 interacting protein 1
Dhx15
DEAH (Asp-Glu-Ala-His) box polypeptide 15
Diap1
diaphanous homolog 1 ( Drosophila )
Dlgap4
discs, large homolog-associated protein 4 ( Drosophila )
Dmkn
dermokine
Dnaja2
DnaJ (Hsp40) homolog, subfamily A, member 2
Dnajb9
predicted gene 6568; DnaJ (Hsp40) homolog, subfamily B, member 9
Dnajc1
DnaJ (Hsp40) homolog, subfamily C, member 1
Dnmt1
DNA methyltransferase (cytosine-5) 1
Dpp4
dipeptidylpeptidase 4
Dpysl2
dihydropyrimidinase-like 2
Dpysl3
dihydropyrimidinase-like 3
Dst
dystonin; hypothetical protein LOC100047109
Dtx2
deltex 2 homolog ( Drosophila )
Dusp1
dual specificity phosphatase 1
Dusp14
dual specificity phosphatase 14
Dusp3
dual specificity phosphatase 3 (vaccinia virus phosphatase VH1-related)
Dync1i2
dynein cytoplasmic 1 intermediate chain 2
Ecd
ecdysoneless homolog ( Drosophila )
Eea1
early endosome antigen 1
Eef1a1
predicted gene 5869; predicted gene 7161; predicted gene 7105;
predicted gene 5822; similar to eukaryotic translation elongation factor 1
alpha 1; predicted gene 6192; predicted gene 6392; predicted gene 6767;
predicted gene 6170; predicted gene 6548; predicted gene 6789;
eukaryotic translation elongation factor 1 alpha 1
Efemp1
epidermal growth factor-containing fibulin-like extracellular matrix protein 1
Efhd2
similar to EF hand domain containing 2; EF hand domain containing 2
Efna5
ephrin A5
Egr1
early growth response 1
Ehd2
EH-domain containing 2
Eif2s3x
eukaryotic translation initiation factor 2, subunit 3, structural gene X-
linked; similar to translation initiation factor elF-2 gamma subunit;
predicted gene 2223
Eif3a
eukaryotic translation initiation factor 3, subunit A
Elf1
E74-like factor 1
Elovl6
predicted gene 11295; ELOVL family member 6, elongation of long chain
fatty acids (yeast)
Emp2
epithelial membrane protein 2
Enpp2
ectonucleotide pyrophosphatase/phosphodiesterase 2
Enpp4
ectonucleotide pyrophosphatase/phosphodiesterase 4
Esam
endothelial cell-specific adhesion molecule
Esf1
ESF1, nucleolar pre-rRNA processing protein, homolog ( S. cerevisiae )
Espn
espin
Esyt3
family with sequence similarity 62 (C2 domain containing), member C
Etfa
predicted gene 2893; electron transferring flavoprotein, alpha polypeptide
Evpl
envoplakin
Exoc4
exocyst complex component 4
F11r
F11 receptor
Faim2
Fas apoptotic inhibitory molecule 2
Fam117a
family with sequence similarity 117, memberA
Fam134b
family with sequence similarity 134, member B
Fam53b
family with sequence similarity 53, member B
Fam63b
RIKEN cDNA B230380D07 gene
Fam76a
predicted gene 7527; family with sequence similarity 76, member A
Fam84b
RIKEN cDNA D330050I23 gene
Fas
Fas (TNF receptor superfamily member 6)
Fbln1
fibulin 1
Fermt2
fermitin family homolog 2 ( Drosophila )
Fgf1
fibroblast growth factor 1
Fhl1
four and a half LIM domains 1
Filip1l
filamin A interacting protein 1-like
Fkbp5
FK506 binding protein 5
Flii
flightless I homolog ( Drosophila ); similar to cytoskeletal actin-modulating
protein
Flnc
filamin C, gamma
Flrt2
fibronectin leucine rich transmembrane protein 2
Fmo2
flavin containing monooxygenase 2
Fmod
fibromodulin
Fndc1
fibronectin type III domain containing 1; similar to fibronectin type III
domain containing 1
Fos
FBJ osteosarcoma oncogene
Foxn3
forkhead box N3
Frmd4b
FERM domain containing 4B
Fth1
ferritin heavy chain 1
Fxyd1
FXYD domain-containing ion transport regulator 1
G3bp1
Ras-GTPase-activating protein SH3-domain binding protein 1
Gabarapl1
gamma-aminobutyric acid (GABA) A receptor-associated protein-like 1
Gadd45b
growth arrest and DNA-damage-inducible 45 beta
Ganab
alpha glucosidase 2 alpha neutral subunit
Gas1
growth arrest specific 1
Gas6
growth arrest specific 6
Gata6
GATA binding protein 6
Gbp2
guanylate binding protein 2
Gbp3
guanylate binding protein 3
Gcap14
granule cell antiserum positive 14
Gcsh
predicted gene 3672; similar to Glycine cleavage system H protein,
mitochondrial precursor; glycine cleavage system protein H (aminomethyl
carrier)
Gda
guanine deaminase
Gem
GTP binding protein (gene overexpressed in skeletal muscle)
Gfm2
G elongation factor, mitochondrial 2
Gfpt2
glutamine fructose-6-phosphate transaminase 2
Gja1
gap junction protein, alpha 1
Gjb5
gap junction protein, beta 5
Gm10052
predicted gene 10052
Gm13251
predicted gene 13251; predicted gene, OTTMUSG00000010657; RIKEN
cDNA 1700029I01 gene
Gm3893
similar to 4933409K07Rik protein; predicted gene, 665845; predicted gene
2490; predicted gene 10601; predicted gene 2163; predicted gene 3892;
RIKEN cDNA 4933409K07 gene; predicted gene 3893
Gm6548
predicted gene 5869; predicted gene 7161; predicted gene 7105;
predicted gene 5822; similar to eukaryotic translation elongation factor 1
alpha 1; predicted gene 6192; predicted gene 6392; predicted gene 6767;
predicted gene 6170; predicted gene 6548; predicted gene 6789;
eukaryotic translation elongation factor 1 alpha 1
Gm6578
predicted gene 6578
Gm6644
predicted gene 6644
Gm9199
predicted gene 9199
Gnb2
guanine nucleotide binding protein (G protein), beta 2
Golga4
golgi autoantigen, golgin subfamily a, 4
Golgb1
golgi autoantigen, golgin subfamily b, macrogolgin 1
Gpc3
glypican 3
Gpc4
glypican 4; similar to Glypican 4
Gpcpd1
preimplantation protein 4
Gpm6a
glycoprotein m6a
Gpr116
G protein-coupled receptor 116
Gpr133
G protein-coupled receptor 133
Gpr64
G protein-coupled receptor 64
Gprc5b
G protein-coupled receptor, family C, group 5, member B
Gpx8
glutathione peroxidase 8 (putative)
Gsr
similar to Glutathione reductase, mitochondrial precursor (GR) (GRase);
glutathione reductase
Gsta3
glutathione S-transferase, alpha 3
Gstm1
similar to Glutathione S-transferase Mu 1 (GST class-mu 1) (Glutathione S-
transferase GT8.7) (pmGT10) (GST 1-1); predicted gene 5562; glutathione
S-transferase, mu 1
Gstm4
glutathione S-transferase, mu 4
Gucy1a3
guanylate cyclase 1, soluble, alpha 3
H2-D1
histocompatibility 2, D region; histocompatibility 2, D region locus 1
H2-K1
histocompatibility 2, K1, K region; similar to H-2K(d) antigen
H2-Q6
histocompatibility 2, Q region locus 1; histocompatibility 2, Q region locus
9; similar to H-2 class I histocompatibility antigen, L-D alpha chain
precursor; histocompatibility 2, Q region locus 8; histocompatibility 2, Q
region locus 2; similar to MHC class Ib antigen; histocompatibility 2, Q
region locus 7; histocompatibility 2, Q region locus 6; hypothetical protein
LOC100044307; similar to H-2 class I histocompatibility antigen, Q7 alpha
chain precursor (QA-2 antigen); RIKEN cDNA 0610037M15 gene
H3f3a
predicted gene 14383; predicted gene 3835; predicted gene 14384;
predicted gene 12950; predicted gene, 670915; H3 histone, family 3A;
predicted gene 12657; predicted gene 6132; predicted gene 10257;
predicted gene 7227; H3 histone, family 3B; predicted gene 6128; similar
to histone; predicted gene 1986; predicted gene 6186; hypothetical
protein LOC676337; predicted gene 6421; predicted gene 2198; predicted
gene 6817; predicted gene 8095; predicted gene 12271; predicted gene
13529; predicted gene 8029; predicted gene 4938; predicted gene 7100;
predicted gene 9014; similar to Histone H3.4 (Embryonic); predicted gene
7179; similar to H3 histone, family 3B; predicted gene 7900; predicted
gene 2099; similar to H3 histone, family 3A; predicted gene 6749;
predicted gene 6485; predicted gene 4028; predicted gene 7194
Hdac3
histone deacetylase 3
Hdac5
histone deacetylase 5
Heg1
HEG homolog 1 (zebrafish)
Herpud2
HERPUD family member 2
Hes1
hairy and enhancer of split 1 ( Drosophila )
Hexb
hexosaminidase B
Hist1h1c
histone cluster 1, H1c
Hmgb1
predicted gene 13121; predicted gene 3160; high-mobility group
(nonhistone chromosomal) protein 1-like 1; predicted gene 6090;
predicted gene 3851; predicted gene 8967; predicted gene 7782;
predicted gene 4587; predicted gene 4689; predicted gene 3307;
predicted gene 13932; predicted gene 15059; predicted gene 3565;
predicted gene 15447; predicted gene 12587; predicted gene 9012;
predicted gene 6115; predicted gene 9480; high mobility group box 1;
predicted gene 8423; predicted gene 5853; predicted gene 8288;
predicted gene 7888; predicted gene 8594; predicted gene 15387;
predicted gene 5473; predicted gene 8807; similar to high mobility group
box 1; similar to 2810416G20Rik protein; predicted gene 8390; predicted
gene, OTTMUSG00000005439; predicted gene 5842; predicted gene 5527;
predicted gene 8563; predicted gene 2710; predicted gene 12331;
predicted gene 5937; predicted gene 5504; similar to high-mobility group
box 1; predicted gene 10361; predicted gene 2607; predicted gene 7422;
predicted gene 10075; predicted gene 12568; predicted gene 6589;
predicted gene 4383; predicted gene 8031; similar to High mobility group
protein 1 (HMG-1) (High mobility group protein B1) (Amphoterin)
(Heparin-binding protein p30); predicted gene 7468; predicted gene 8554
Hnrnph1
heterogeneous nuclear ribonucleoprotein H1
Hnrnph2
heterogeneous nuclear ribonucleoprotein H2
Hnrnpl
heterogeneous nuclear ribonucleoprotein L
Hnrnpm
heterogeneous nuclear ribonucleoprotein M
Hnrnpr
predicted gene 6159; heterogeneous nuclear ribonucleoprotein R
Hook3
hook homolog 3 ( Drosophila )
Hoxa5
homeo box A5
Hp1bp3
heterochromatin protein 1, binding protein 3
Hsp90aa1
predicted gene 5511; heat shock protein 90, alpha (cytosolic), class A
member 1
Hsp90ab1
heat shock protein 90 alpha (cytosolic), class B member 1
Hsp90b1
heat shock protein 90, beta (Grp94), member 1
Hspa12a
heat shock protein 12A
Hspa2
heat shock protein 2
Hspb1
heat shock protein 1
Hspb8
heat shock protein 8
Id1
inhibitor of DNA binding 1
Id2
inhibitor of DNA binding 2
Ier2
immediate early response 2
Ifi204
interferon activated gene 204
Ifi205
interferon activated gene 205
Ifi27l2a
interferon, alpha-inducible protein 27 like 2A
Ifi35
interferon-induced protein 35
Ifit3
interferon-induced protein with tetratricopeptide repeats 3
Ifitm3
interferon induced transmembrane protein 3
Ifnar2
interferon (alpha and beta) receptor 2
Ifngr1
interferon gamma receptor 1
Ifrd1
interferon-related developmental regulator 1
Ift74
intraflagellar transport 74 homolog ( Chlamydomonas )
Igf1r
insulin-like growth factor I receptor
Igfbp5
insulin-like growth factor binding protein 5
Igfbp6
insulin-like growth factor binding protein 6
Il16
interleukin 16
Il17re
interleukin 17 receptor E
Il6ra
interleukin 6 receptor, alpha
Il6st
interleukin 6 signal transducer
Ildr2
immunoglobulin-like domain containing receptor 2
Ilf3
interleukin enhancer binding factor 3
Impad1
inositol monophosphatase domain containing 1
Ints10
integrator complex subunit 10; similar to integrator complex subunit 10
Iqsec1
IQ motif and Sec7 domain 1
Irak4
interleukin-1 receptor-associated kinase 4
Irf2bp2
interferon regulatory factor 2 binding protein 2
Irf7
interferon regulatory factor 7
Irs2
insulin receptor substrate 2
Itch
itchy, E3 ubiquitin protein ligase
Itga6
integrin alpha 6
Itpr2
inositol 1,4,5-triphosphate receptor 2
Jmjd1c
jumonji domain containing 1C
Jun
Jun oncogene
Junb
Jun-B oncogene
Jund
Jun proto-oncogene related gene d
Jup
junction plakoglobin
Kank1
KN motif and ankyrin repeat domains 1
Kcnab1
potassium voltage-gated channel, shaker-related subfamily, beta
member 1
Kdelr1
KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention
receptor 1
Kdm5a
lysine (K)-specific demethylase 5A
Kdm6b
KDM1 lysine (K)-specific demethylase 6B
Kdr
kinase insert domain protein receptor
Keap1
kelch-like ECH-associated protein 1
Kif1b
kinesin family member 1B
Kif5b
kinesin family member 5B
Klf10
Kruppel-like factor 10
Klf2
Kruppel-like factor 2 (lung)
Klf4
Kruppel-like factor 4 (gut)
Klf6
Kruppel-like factor 6
Klf7
Kruppel-like factor 7 (ubiquitous)
Klf9
Kruppel-like factor 9
Kpna1
karyopherin (importin) alpha 1
Kpna3
karyopherin (importin) alpha 3
Krcc1
lysine-rich coiled-coil 1
Krt14
keratin 14
Ktn1
kinectin 1
Lama4
laminin, alpha 4
Lamp2
lysosomal-associated membrane protein 2
Lars2
leucyl-tRNA synthetase, mitochondrial
Lass2
LAG1 homolog, ceramide synthase 2
Lass4
LAG1 homolog, ceramide synthase 4
Lgals7
lectin, galactose binding, soluble 7
Limch1
LIM and calponin homology domains 1
Lims2
LIM and senescent cell antigen like domains 2
Lman1
lectin, mannose-binding, 1
Lpar2
lysophosphatidic acid receptor 2
Lrrc20
leucine rich repeat containing 20
Lrrc58
leucine rich repeat containing 58; predicted gene, OTTMUSG00000025724
Lrrc61
leucine rich repeat containing 61
Lrrn4
leucine rich repeat neuronal 4
Lrrn4cl
LRRN4 C-terminal like
Ltbp4
latent transforming growth factor beta binding protein 4
Luc7l3
RIKEN cDNA 3300001P08 gene
Maf
similar to c-Maf long form; avian musculoaponeurotic fibrosarcoma (v-
maf) AS42 oncogene homolog
Maged1
melanoma antigen, family D, 1
Magt1
magnesium transporter 1
Malat1
metastasis associated lung adenocarcinoma transcript 1 (non-coding RNA)
Man1a
mannosidase 1, alpha
Manf
mesencephalic astrocyte-derived neurotrophic factor
Maoa
monoamine oxidase A
Map3k3
mitogen-activated protein kinase kinase kinase 3
Mapk1
mitogen-activated protein kinase 1
Mapkapk3
mitogen-activated protein kinase-activated protein kinase 3
Mapre2
microtubule-associated protein, RP/EB family, member 2
Marcksl1
MARCKS-like 1; predicted gene 9106
Mat2a
methionine adenosyltransferase II, alpha
Mat2b
methionine adenosyltransferase II, beta
Matr3
matrin 3; similar to Matrin 3
Med13l
mediator complex subunit 13-like
Med21
mediator complex subunit 21
Mef2c
myocyte enhancer factor 2C
Meis2
Meis homeobox 2
Mesdc1
mesoderm development candidate 1
Metap2
methionine aminopeptidase 2
Mettl2
methyltransferase like 2
Mettl7a1
methyltransferase like 7A1
Mfap1a
similar to microfibrillar-associated protein 1A; microfibrillar-associated
protein 1A; microfibrillar-associated protein 1B
Mfhas1
malignant fibrous histiocytoma amplified sequence 1
Mgll
monoglyceride lipase
Mgst1
microsomal glutathione S-transferase 1
Mll1
myeloid/lymphoid or mixed-lineage leukemia 1
Mll3
myeloid/lymphoid or mixed-lineage leukemia 3
Morf4l2
predicted gene 5521; similar to mortality factor 4 like 2; mortality factor 4
like 2
Mpdz
multiple PDZ domain protein
Mphosph8
M-phase phosphoprotein 8
Mras
muscle and microspikes RAS
Mrgprf
MAS-related GPR, member F
Msn
moesin
Mtap1a
microtubule-associated protein 1 A
Mtdh
metadherin
Mtmr6
myotubularin related protein 6
Mut
methylmalonyl-Coenzyme A mutase
Mxd4
Max dimerization protein 4
Myh10
myosin, heavy polypeptide 10, non-muscle
Myl7
myosin, light polypeptide 7, regulatory
Mylip
myosin regulatory light chain interacting protein
Myst4
MYST histone acetyltransferase monocytic leukemia 4
Naa25
RIKEN cDNA C330023M02 gene
Naga
N-acetyl galactosaminidase, alpha
Nckap1
NCK-associated protein 1
Ncoa1
similar to Nuclear receptor coactivator 1 (NCoA-1) (Steroid receptor
coactivator 1) (SRC-1) (Nuclear receptor coactivator protein 1) (mNRC-1);
nuclear receptor coactivator 1
Ncoa4
predicted gene 6768; nuclear receptor coactivator 4
Ncor1
nuclear receptor co-repressor 1
Ndn
necdin
Ndst1
N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1
Ndufa4
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4
Nedd4
neural precursor cell expressed, developmentally down-regulated 4
Nf1
neurofibromatosis 1
Nfe2l1
nuclear factor, erythroid derived 2, -like 1
Nfia
nuclear factor I/A
Nfic
nuclear factor I/C
Nfix
nuclear factor I/X
Nfkb2
nuclear factor of kappa light polypeptide gene enhancer in B-cells 2,
p49/p100
Nfkbia
nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor, alpha
Nfkbiz
nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor, zeta
Nfyc
nuclear transcription factor-Y gamma
Nid2
nidogen 2
Ninl
ninein-like
Nipal3
NIPA-like domain containing 3; similar to NIPA-like domain containing 3
Nipbl
Nipped-B homolog ( Drosophila )
Nkain4
Na+/K+ transporting ATPase interacting 4
Nkd1
naked cuticle 1 homolog ( Drosophila ); similar to naked cuticle 1 homolog
Nnmt
nicotinamide N-methyltransferase
Nod1
nucleotide-binding oligomerization domain containing 1
Npr1
natriuretic peptide receptor 1
Nr1d1
nuclear receptor subfamily 1, group D, member 1
Nr3c1
nuclear receptor subfamily 3, group C, member 1
Nr4a1
nuclear receptor subfamily 4, group A, member 1
Nrgn
neurogranin
Nucks1
nuclear casein kinase and cyclin-dependent kinase substrate 1
Oasl2
2′-5′ oligoadenylate synthetase-like 2
Oat
ornithine aminotransferase
Ogdh
oxoglutarate dehydrogenase (lipoamide)
Ogn
osteoglycin
Olfr1033
olfactory receptor 1033
Olfr613
olfactory receptor 614; hypothetical protein LOC100044261; olfactory
receptor 613
Opa3
optic atrophy 3 (human)
Orai3
ORAI calcium release-activated calcium modulator 3
Osr1
odd-skipped related 1 ( Drosophila )
Oxct1
3-oxoacid CoA transferase 1
Oxnad1
oxidoreductase NAD-binding domain containing 1
Pard3b
par-3 partitioning defective 3 homolog B ( C. elegans )
Parp14
poly (ADP-ribose) polymerase family, member 14
Parp4
poly (ADP-ribose) polymerase family, member 4
Parvb
parvin, beta; similar to parvin, beta
Pbx1
pre B-cell leukemia transcription factor 1; region containing RIKEN cDNA
2310056B04gene; pre B-cell leukemia transcription factor 1
Pcdh15
protocadherin 15
Pcdhgb5
protocadherin gamma subfamily B, 5
Pcm1
pericentriolar material 1
Pdap1
PDGFA associated protein 1
Pdcd6ip
programmed cell death 6 interacting protein
Pde4dip
phosphodiesterase 4D interacting protein (myomegalin)
Pdia3
protein disulfide isomerase associated 3
Pdia4
protein disulfide isomerase associated 4
Pdpn
podoplanin
Pef1
penta-EF hand domain containing 1
Peli1
pellino 1
Perl
period homolog 1 ( Drosophila )
Pf4
platelet factor 4
Pfn1
profilin 1
Pgcp
plasma glutamate carboxypeptidase
Pgrmc1
progesterone receptor membrane component 1
Phf21a
PHD finger protein 21A
Phf3
PHD finger protein 3
Phip
pleckstrin homology domain interacting protein
Pigt
phosphatidylinositol glycan anchor biosynthesis, class T; similar to GPI
transamidase component PIG-T precursor (Phosphatidylinositol-glycan
biosynthesis class T protein) (Neuronal development-associated protein 7)
Pik3c2a
phosphatidylinositol 3-kinase, C2 domain containing, alpha polypeptide
Pim1
proviral integration site 1
Pitpnm2
phosphatidylinositol transfer protein, membrane-associated 2
Pkhd1l1
polycystic kidney and hepatic disease 1-like 1
Pknox1
Pbx/knotted 1 homeobox
Pla2g4a
phospholipase A2, group IVA (cytosolic, calcium-dependent)
Plat
plasminogen activator, tissue
Plce1
phospholipase C, epsilon 1
Plk1s1
non-protein coding RNA 153
Plk2
polo-like kinase 2 ( Drosophila )
Plod2
procollagen lysine, 2-oxoglutarate 5-dioxygenase 2
Plxdc1
plexin domain containing 1
Plxdc2
plexin domain containing 2
Plxna4
plexin A4
Pmp22
peripheral myelin protein 22
Pnrc1
proline-rich nuclear receptor coactivator 1
Podn
podocan
Ppap2a
phosphatidic acid phosphatase type 2A
Ppbp
pro-platelet basic protein
Ppfibp2
protein tyrosine phosphatase, receptor-type, F interacting protein, binding
protein 2
Ppig
peptidyl-prolyl isomerase G (cyclophilin G)
Ppl
periplakin
Ppp1cb
protein phosphatase 1, catalytic subunit, beta isoform
Ppp1r12a
protein phosphatase 1, regulatory (inhibitor) subunit 12A
Ppp1r15a
protein phosphatase 1, regulatory (inhibitor) subunit 15A; myeloid
differentiation primary response gene 116
Ppp3ca
protein phosphatase 3, catalytic subunit, alpha isoform
Pppde1
PPPDE peptidase domain containing 1
Pqlc3
PQ loop repeat containing
Prelp
proline arginine-rich end leucine-rich repeat
Prg4
proteoglycan 4 (megakaryocyte stimulating factor, articular superficial
zone protein)
Prkar2a
protein kinase, cAMP dependent regulatory, type II alpha
Prpf40a
PRP40 pre-mRNA processing factor 40 homolog A (yeast)
Prr13
proline rich 13
Prss23
protease, serine, 23
Psd
pleckstrin and Sec7 domain containing
Psip1
PC4 and SFRS1 interacting protein 1
Psmb2
proteasome (prosome, macropain) subunit, beta type 2
Psmd11
predicted gene 14048; proteasome (prosome, macropain) 26S subunit,
non-ATPase, 11
Psmd7
proteasome (prosome, macropain) 26S subunit, non-ATPase, 7
Ptges3
predicted gene 9769; prostaglandin E synthase 3 (cytosolic); similar to
Sid3177p; predicted gene 11893
Ptgis
prostaglandin I2 (prostacyclin) synthase
Ptgs1
prostaglandin-endoperoxide synthase 1
Ptma
predicted gene 12504; predicted gene 9800; predicted gene 4617;
predicted gene 6625; predicted gene 7614; similar to prothymosin alpha;
prothymosin alpha; predicted gene 9009
Ptp4a2
predicted gene 13422; protein tyrosine phosphatase 4a2
Ptplad2
protein tyrosine phosphatase-like A domain containing 2
Ptprd
protein tyrosine phosphatase, receptor type, D
Ptprf
protein tyrosine phosphatase, receptor type, F
Ptrf
polymerase 1 and transcript release factor
Qrich1
glutamine-rich 1
Qser1
glutamine and serine rich 1
R74862
expressed sequence R74862
Rab11fip1
RAB11 family interacting protein 1 (class I)
Rab1b
RAB1B, member RAS oncogene family
Rab5c
RAB5C, member RAS oncogene family
Rab6b
RAB6B, member RAS oncogene family
Rab7
RAB7, member RAS oncogene family
Rabgap1l
RAB GTPase activating protein 1-like
Ralbp1
ralA binding protein 1
Raly
RIKEN cDNA C130057N11 gene; hnRNP-associated with lethal yellow
Rarres2
retinoic acid receptor responder (tazarotene induced) 2
Rb1cc1
RB1-inducible coiled-coil 1
Rbbp6
retinoblastoma binding protein 6
Rbbp8
retinoblastoma binding protein 8
Rbm25
RNA binding motif protein 25
Rbm27
RNA binding motif protein 27
Rbm3
predicted gene 15453; RNA binding motif protein 3
Rbpms
RNA binding protein gene with multiple splicing
Rdx
radixin
Rest
RE1-silencing transcription factor
Rgma
RGM domain family, member A
Rgs10
regulator of G-protein signalling 10
Rhob
ras homolog gene family, member B
Rhoj
ras homolog gene family, member J
Rhou
ras homolog gene family, member U
Rnase4
ribonuclease, RNase A family 4
Rnd3
Rho family GTPase 3
Rnf167
ring finger protein 167
Rnf20
ring finger protein 20
Rock1
Rho-associated coiled-coil containing protein kinase 1
Rock2
Rho-associated coiled-coil containing protein kinase 2
Rpp25
ribonuclease P 25 subunit (human)
Rras2
related RAS viral (r-ras) oncogene homolog 2
Rspo1
R-spondin homolog ( Xenopus laevis )
Rtf1
Rtf1, Paf1/RNA polymerase II complex component, homolog ( S. cerevisiae )
Rtn1
reticulon 1
Ryk
receptor-like tyrosine kinase
Sarnp
predicted gene 6563; SAP domain containing ribonucleoprotein
Sat1
similar to spermidine/spermine N1-acetyltransferase; predicted gene
5552; spermidine/spermine N1-acetyl transferase 1
Sbsn
suprabasin
Scd1
stearoyl-Coenzyme A desaturase 1
Sdc4
syndecan 4
Sdpr
serum deprivation response
Sec62
SEC62 homolog ( S. cerevisiae )
Secisbp2
SECIS binding protein 2
Sema5a
sema domain, seven thrombospondin repeats (type 1 and type 1-like),
transmembrane domain (TM) and short cytoplasmic domain, (semaphorin)
5A
Senp6
similar to Sentrin-specific protease 6 (Sentrin/SUMO-specific protease
SENP6) (SUMO-1-specific protease 1); SUMO/sentrin specific peptidase 6
Sep15
selenoprotein
Sept9
septin 9
Serinc5
serine incorporator 5
Serpinb6b
serine (or cysteine) peptidase inhibitor, clade B, member 6b
Serping1
serine (or cysteine) peptidase inhibitor, clade G, member 1
Serpinh1
serine (or cysteine) peptidase inhibitor, clade H, member 1
Sesn1
sestrin 1
Setd2
SET domain containing 2
Sf3b1
splicing factor 3b, subunit 1
Sf3b4
predicted gene 7935; splicing factor 3b, subunit 4
Sfrs18
splicing factor, arginine/serine-rich 18
Shc1
predicted gene 5500; src homology 2 domain-containing transforming
protein C1
Shfm1
split hand/foot malformation (ectrodactyly) type 1
Siae
sialic acid acetylesterase
Siah1a
seven in absentia 1A
Sirt2
sirtuin 2 (silent mating type information regulation 2, homolog) 2
( S. cerevisiae )
Slc10a3
solute carrier family 10 (sodium/bile acid cotransporter family), member 3
Slc16a1
solute carrier family 16 (monocarboxylic acid transporters), member 1
Slc1a5
solute carrier family 1 (neutral amino acid transporter), member 5
Slc26a3
solute carrier family 26, member 3
Slc27a3
solute carrier family 27 (fatty acid transporter), member 3
Slc38a1
solute carrier family 38, member 1
Slc39a8
solute carrier family 39 (metal ion transporter), member 8
Slc43a3
solute carrier family 43, member 3
Slc4a4
solute carrier family 4 (anion exchanger), member 4
Slc6a4
solute carrier family 6 (neurotransmitter transporter, serotonin), member 4
Slc6a6
solute carrier family 6 (neurotransmitter transporter, taurine), member 6
Slc8a1
solute carrier family 8 (sodium/calcium exchanger), member 1
Slc9a3r1
solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1
Slpi
secretory leukocyte peptidase inhibitor
Sltm
SAFB-like, transcription modulator
Slu7
SLU7 splicing factor homolog ( S. cerevisiae )
Slurp1
secreted Ly6/Plaur domain containing 1
Smad4
similar to MAD homolog 4 ( Drosophila ); MAD homolog 4 ( Drosophila )
Smarca2
SWI/SNF related, matrix associated, actin dependent regulator of
chromatin, subfamily a, member 2
Smarca5
predicted gene 13034; SWI/SNF related, matrix associated, actin
dependent regulator of chromatin, subfamily a, member 5
Smc2
structural maintenance of chromosomes 2
Smc3
predicted gene 8892; structural maintenace of chromosomes 3
Smc4
structural maintenance of chromosomes 4
Smc6
structural maintenance of chromosomes 6
Smchd1
SMC hinge domain containing 1
Smpd3
sphingomyelin phosphodiesterase 3, neutral
Snrnp70
small nuclear ribonucleoprotein 70 (U1)
Sntb2
similar to beta-2-syntrophin; syntrophin, basic 2
Soat1
sterol O-acyltransferase 1
Socs3
suppressor of cytokine signaling 3
Sod3
superoxide dismutase 3, extracellular
Sorbs1
sorbin and SH3 domain containing 1
Sorbs3
sorbin and SH3 domain containing 3
Sox6
SRY-box containing gene 6
Sp100
nuclear antigen Sp100
Spag9
sperm associated antigen 9
Spare
secreted acidic cysteine rich glycoprotein; similar to Secreted acidic
cysteine rich glycoprotein
Spen
SPEN homolog, transcriptional regulator ( Drosophila )
Spint2
serine protease inhibitor, Kunitz type 2
Spnb2
spectrin beta 2
Spock2
sparc/osteonectin, cwcv and kazal-like domains proteoglycan 2
Spon2
spondin 2, extracellular matrix protein
Spop
speckle-type POZ protein
Src
Rous sarcoma oncogene
Srrm1
serine/arginine repetitive matrix 1
Ssh2
slingshot homolog 2 ( Drosophila )
Ssr3
signal sequence receptor, gamma
St3gal1
ST3 beta-galactoside alpha-2,3-sialyltransferase 1
Stag1
stromal antigen 1
Star
steroidogenic acute regulatory protein
Stard5
StAR-related lipid transfer (START) domain containing 5
Stat3
similar to Stat3B; signal transducer and activator of transcription 3
Stim1
similar to Stromal interaction molecule 1; stromal interaction molecule 1
Stk10
serine/threonine kinase 10
Stk40
serine/threonine kinase 40
Stmn2
stathmin-like 2
Stra6
stimulated by retinoic acid gene 6
Strn3
striatin, calmodulin binding protein 3
Sulf1
sulfatase 1
Sulf2
sulfatase 2
Supt16h
suppressor of Ty 16 homolog ( S. cerevisiae )
Sv2a
synaptic vesicle glycoprotein 2 a
Syne1
synaptic nuclear envelope 1
Syne2
synaptic nuclear envelope 2
Syt11
synaptotagmin XI; similar to synaptotagmin XI
Sytl1
synaptotagmin-like 1; similar to synaptotagmin-like 1
Taf3
TAF3 RNA polymerase II, TATA box binding protein (TBP)-associated factor
Taf7
TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated factor
Tapbp
TAP binding protein
Tbc1d15
TBC1 domain family, member 15
Tbcel
tubulin folding cofactor E-like
Tbl1x
transducin (beta)-like 1 X-linked
Tbx18
T-box18
Tceal8
transcription elongation factor A (SII)-like 8; similar to transcription
elongation factor A (SII)-like 8
Tcf7l1
transcription factor 3
Tfdp2
transcription factor Dp 2
Tgfb1i1
transforming growth factor beta 1 induced transcript 1
Tgfb2
transforming growth factor, beta 2
Tgfbr2
transforming growth factor, beta receptor II
Tgm2
transglutaminase 2, C polypeptide
Thbd
thrombomodulin
Thbs1
thrombospondin 1; similar to thrombospondin 1
Thoc2
THO complex 2; cDNA sequence BC005561
Thrap3
thyroid hormone receptor associated protein 3; predicted gene 5898
Thsd4
thrombospondin, type I, domain containing 4
Timp2
tissue inhibitor of metalloproteinase 2
Tirap
toll-interleukin 1 receptor (TIR) domain-containing adaptor protein
Tlr2
toll-like receptor 2
Tm4sf1
transmembrane 4 superfamily member 1
Tm4sf5
transmembrane 4 superfamily member 5
Tmcc3
transmembrane and coiled coil domains 3
Tmco1
transmembrane and coiled-coil domains 1
Tmco7
transmembrane and coiled-coil domains 7
Tmed2
transmembrane emp24 domain trafficking protein 2; predicted gene
10698; predicted gene 7318
Tmem119
transmembrane protein 119
Tmem140
transmembrane protein 140
Tmem151a
transmembrane protein 151A
Tmem221
transmembrane protein 221
Tmem50a
transmembrane protein 50A
Tmem98
transmembrane protein 98
Tmod3
tropomodulin 3
Tmpo
thymopoietin
Tmsb4x
thymosin, beta 4, X chromosome; similar to thymosin beta-4
Tnxb
tenascin XB
Tob2
transducer of ERBB2, 2
Topors
topoisomerase 1 binding, arginine/serine-rich
Tpm3
predicted gene 7848; predicted gene 7839; predicted gene 4157; similar to
tropomyosin 3, gamma; tropomyosin 3, gamma; predicted gene 4903
Tppp3
tubulin polymerization-promoting protein family member 3
Tpt1
predicted gene 1974; tumor protein, translationally-controlled 1
pseudogene; tumor protein, translationally-controlled 1; predicted gene
14456
Trafd1
TRAF type zinc finger domain containing 1
Trib1
tribbles homolog 1 ( Drosophila )
Trim8
tripartite motif protein 8
Trpm7
transient receptor potential cation channel, subfamily M, member 7
Tsc22d3
TSC22 domain family, member 3
Tshz1
teashirt zinc finger family member 1
Tsix
X (inactive)-specific transcript, antisense
Tspan31
tetraspanin 31
Tspan5
tetraspanin 5
Ttc28
tetratricopeptide repeat domain 28
Ttc38
tetratricopeptide repeat domain 38
Tuba1a
predicted gene 7172; similar to tubulin, alpha 1; tubulin, alpha 1A
Tubb2a
tubulin, beta 2A
Twsg1
twisted gastrulation homolog 1 ( Drosophila )
Txndc5
thioredoxin domain containing 5
Txnrd1
thioredoxin reductase 1
Uap1
UDP-N-acetylglucosamine pyrophosphorylase 1
Uba7
ubiquitin-activating enzyme E1-like; RIKEN cDNA D330022A01 gene
Ube2d1
ubiquitin-conjugating enzyme E2D 1, UBC4/5 homolog (yeast)
Ube2l6
ubiquitin-conjugating enzyme E2L 6
Ube2n
ubiquitin-conjugating enzyme E2N; similar to ubiquitin-conjugating
enzyme E2 UbcH-ben; similar to ubiquitin-conjugating enzyme E2N;
predicted gene 5943
Ube2v1
ubiquitin-conjugating enzyme E2 variant 1; predicted gene 7181; predicted
gene 12502; similar to ubiquitin-conjugating enzyme E2 variant 1
Ubqln2
ubiquilin 2
Ubxn2a
UBX domain protein 2A; predicted gene 6245
Ubxn4
UBX domain protein 4
Ugdh
UDP-glucose dehydrogenase
Upk1b
uroplakin 1B
Upk3b
uroplakin 3B
Usp16
ubiquitin specific peptidase 16
Usp2
ubiquitin specific peptidase 2
Usp25
ubiquitin specific peptidase 25
Usp54
ubiquitin specific peptidase 54
Usp8
ubiquitin specific peptidase 8
Utp20
UTP20, small subunit (SSU) processome component, homolog (yeast)
Vat1
vesicle amine transport protein 1 homolog ( T californica )
Vim
vimentin
Vps13a
vacuolar protein sorting 13A (yeast)
Vwa5a
von Willebrand factor A domain containing 5A
Wac
similar to WW domain-containing adapter protein with coiled-coil; WW
domain containing adaptor with coiled-coil
Wasf2
WAS protein family, member 2
Wdr26
WD repeat domain 26; similar to myocardial ischemic preconditioning
upregulated protein 2
Wdr92
WD repeat domain 92
Wfdc1
WAP four-disulfide core domain 1
Wls
G protein-coupled receptor 177
Wnt4
wingless-related MMTV integration site 4
Wrnip1
Werner helicase interacting protein 1
Wt1
similar to Wilms tumor homolog; Wilms tumor 1 homolog
Wwc2
WW, C2 and coiled-coil domain containing 2
Xdh
xanthine dehydrogenase
Xist
inactive X specific transcripts
Yipf5
Yip1 domain family, member 5; predicted gene 5738
Ywhaz
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation
protein, zeta polypeptide; predicted gene 4202
Zbed6
similar to Zinc finger BED domain containing protein 4
Zbtb16
zinc finger and BTB domain containing 16
Zbtb20
zinc finger and BTB domain containing 20
Zbtb4
zinc finger and BTB domain containing 4
Zbtb7c
zinc finger and BTB domain containing 7C
Zc3h13
zinc finger CCCH type containing 13
Zc3h18
predicted gene 5939; zinc finger CCCH-type containing 18
Zcchc11
zinc finger, CCHC domain containing 11
Zcchc3
zinc finger, CCHC domain containing 3
Zfand6
zinc finger, AN1-type domain 6
Zfhx4
zinc finger homeodomain 4
Zfp148
zinc finger protein 148
Zfp277
zinc finger protein 277
Zfp281
zinc finger protein 281
Zfp318
zinc finger protein 318
Zfp353
zinc finger protein 353
Zfp36
zinc finger protein 36
Zfp385a
zinc finger protein 385A
Zfp488
zinc finger protein 488
Zfp672
zinc finger protein 672
Zfp704
zinc finger protein 704
Zmat1
zinc finger, matrin type 1
Zrsr1
zinc finger (CCCH type), RNA binding motif and serine/arginine rich 1
Zzef1
zinc finger, ZZ-type with EF hand domain 1
1110002B05Rik
RIKEN cDNA 1110002B05 gene
1110003E01Rik
RIKEN cDNA 1110003E01 gene
1110004F10Rik
predicted gene 9169; RIKEN cDNA 1110004F10 gene; similar to small acidic
protein
1500003O03Rik
RIKEN cDNA 1500003003 gene; similar to EF-hand Ca2+ binding protein
p22
1600029D21Rik
RIKEN cDNA 1600029D21 gene
1810014B01Rik
RIKEN cDNA 1810014B01 gene
1810041L15Rik
RIKEN cDNA 1810041L15 gene
1810074P20Rik
RIKEN cDNA 1810074P20 gene
2010107G12Rik
RIKEN cDNA 2010107G12 gene
2210403K04Rik
hypothetical protein LOC100042498
2310030G06Rik
RIKEN cDNA 2310030G06 gene
2510002D24Rik
RIKEN cDNA 2510002D24 gene
2610034B18Rik
RIKEN cDNA 2610034B18 gene
2610101N10Rik
RIKEN cDNA 2610101N10 gene
2810474O19Rik
RIKEN cDNA 2810474O19 gene
2900002K06Rik
RIKEN cDNA 2900002K06 gene
3110062M04Rik
RIKEN cDNA 3110062M04 gene
4930402H24Rik
RIKEN cDNA 4930402H24 gene
4930523C07Rik
RIKEN cDNA 4930523C07 gene
5430435G22Rik
RIKEN cDNA 5430435G22 gene
6330406I15Rik
RIKEN cDNA 6330406I15 gene
A130040M12Rik
RIKEN cDNA A130040M12 gene
AI848100
expressed sequence AI848100
Gm16897
kg:uc009lxf.1
Prrc2c
kg:uc007won.1
kg:uc009ogv.1
kg:uc009iln.1
kg:uc007qca.1
Atxn7l3b
kg:uc008ewj.2
kg:uc008wkn.1
kg:uc007bgn.1
Ces2g
kg:uc009cvm.1
kg:uc008ehr.1
Tmem234
kg:uc012hdk.1
kg:uc008ajk.1
eg:245190:chr7:m
kg:uc007qse.1
kg:uc007bvx.1
Mob3c
kg:uc008dzh.1
kg:uc009okn.1
kg:uc007zts.1
kg:uc008jup.1
kg:uc008tkz.1
kg:uc007zwh.1
kg:uc008znh.1
Mau2
kg:uc009mng.1
kg:uc007ded.1
kg:uc007ctp.1
kg:uc007zak.1
eg:497210:chr14:m
kg:uc007vsr.1
Mir3064
kg:uc009ize.1
Kansl1
eg:320169:chr9:p
kg:uc009vev.1
kg:uc009acs.1
kg:uc009tuw.1
kg:uc007pff.1
kg:uc007vnc.1
kg:uc009igb.1
kg:uc008oki.1
kg:uc008tky.1
TABLE 8
Exemplary human marker genes
HUMAN
GENE
SYMBOL
Gene Name
ABI3BP
ABI family, member 3 (NESH) binding protein
ABLIM3
actin binding LIM protein family, member 3
ACAD9
acyl-Coenzyme A dehydrogenase family, member 9
ACBD3
acyl-Coenzyme A binding domain containing 3
ACIN1
apoptotic chromatin condensation inducer 1
ACTB
actin, beta
ACTG1
actin, gamma 1
ADAMTS5
ADAM metallopeptidase with thrombospondin type 1 motif, 5
ADAMTSL1
ADAMTS-like 1
ADD3
adducin 3 (gamma)
AEBP1
AE binding protein 1
AGAP1
ArfGAP with GTPase domain, ankyrin repeat and PH domain 1
AKAP13
A kinase (PRKA) anchor protein 13
AKAP2
A kinase (PRKA) anchor protein 2; paralemmin 2; PALM2-AKAP2
readthrough transcript
AKT2
v-akt murine thymoma viral oncogene homolog 2
ALDH1A1
aldehyde dehydrogenase 1 family, member A1
ALDH1A2
aldehyde dehydrogenase 1 family, member A2
ALOX12
arachidonate 12-lipoxygenase
AMFR
autocrine motility factor receptor
AMHR2
anti-Mullerian hormone receptor, type II
ANG
angiogenin, ribonuclease, RNase A family, 5
ANKRD11
ankyrin repeat domain 11; hypothetical protein LOC100128265
ANKRD12
ankyrin repeat domain 12
ANKRD17
ankyrin repeat domain 17
ANO6
anoctamin 6
ANP32A
hepatopoietin PCn127; acidic (leucine-rich) nuclear
phosphoprotein 32 family, member A
ANXA7
annexin A7
AP1S3
adaptor-related protein complex 1, sigma 3 subunit
AP3S1
adaptor-related protein complex 3, sigma 1 subunit
AP4E1
adaptor-related protein complex 4, epsilon 1 subunit
APLP1
amyloid beta (A4) precursor-like protein 1
APP
amyloid beta (A4) precursor protein
AQP1
aquaporin 1 (Colton blood group)
ARAP2
ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 2
ARF3
ADP-ribosylation factor 3
ARF5
ADP-ribosylation factor 5
ARHGAP28
Rho GTPase activating protein 28
ARHGAP29
Rho GTPase activating protein 29
ARHGAP5
Rho GTPase activating protein 5
ARHGEF12
Rho guanine nucleotide exchange factor (GEF) 12
ARID1A
AT rich interactive domain 1A (SWI-like)
ARID4A
AT rich interactive domain 4A (RBP1-like)
ARID4B
AT rich interactive domain 4B (RBP1-like)
ARID5B
AT rich interactive domain 5B (MRF1-like)
ARL3
ADP-ribosylation factor-like 3
ARL4D
ADP-ribosylation factor-like 4D
ARL6IP5
ADP-ribosylation-like factor 6 interacting protein 5
ARMCX3
armadillo repeat containing, X-linked 3
ARPC2
actin related protein 2/3 complex, subunit 2, 34 kDa
ARSA
arylsulfatase A
ARSB
arylsulfatase B
ASCC3
activating signal cointegrator 1 complex subunit 3
ATF3
activating transcription factor 3
ATG3
ATG3 autophagy related 3 homolog ( S. cerevisiae )
ATP1A1
ATPase, Na+/K+ transporting, alpha 1 polypeptide
ATP1B1
ATPase, Na+/K+ transporting, beta 1 polypeptide
ATP2B1
ATPase, Ca++ transporting, plasma membrane 1
ATP6V1A
ATPase, H+ transporting, lysosomal 70 kDa, V1 subunit A
ATXN2
ataxin 2
B2M
beta-2-microglobulin
BAZ2A
bromodomain adjacent to zinc finger domain, 2A
BBS4
Bardet-Biedl syndrome 4
BBX
bobby sox homolog ( Drosophila )
BCAM
basal cell adhesion molecule (Lutheran blood group)
BCL10
B-cell CLL/lymphoma 10; hypothetical LOC646626
BDP1
B double prime 1, subunit of RNA polymerase III transcription
initiation factor IIIB
BICC1
bicaudal C homolog 1 ( Drosophila )
BICD1
bicaudal D homolog 1 ( Drosophila )
BIRC6
baculoviral IAP repeat-containing 6
BLVRB
biliverdin reductase B (flavin reductase (NADPH))
BNC1
basonuclin 1
BNC2
basonuclin 2
BOD1L
biorientation of chromosomes in cell division 1-like
BPTF
bromodomain PHD finger transcription factor
BRAF
v-raf murine sarcoma viral oncogene homolog B1
BRD2
bromodomain containing 2
BRD4
bromodomain containing 4
BRP44L
brain protein 44-like
BST2
NPC-A-7; bone marrow stromal cell antigen 2
BTBD2
BTB (POZ) domain containing 2
BTBD7
BTB (POZ) domain containing 7
BTF3
basic transcription factor 3; basic transcription factor 3, like 1
pseudogene
BTG2
BTG family, member 2
BZW1
basic leucine zipper and W2 domains 1 pseudogene 1; basic leucine
zipper and W2 domains 1 like 1; basic leucine zipper and W2 domains 1
C1D
C1D nuclear receptor co-repressor; similar to nuclear DNA-binding
protein; similar to hCG1791993
C1RL
complement component 1, r subcomponent-like
C1S
complement component 1, s subcomponent
C2
complement component 2
C3
similar to Complement C3 precursor; complement component 3;
hypothetical protein LOC100133511
C4A
complement component 4A (Rodgers blood group)
C4B
complement component 4B (Chido blood group)
CALM1
calmodulin 3 (phosphorylase kinase, delta); calmodulin 2
(phosphorylase kinase, delta); calmodulin 1 (phosphorylase kinase,
delta)
CALM2
calmodulin 3 (phosphorylase kinase, delta); calmodulin 2
(phosphorylase kinase, delta); calmodulin 1 (phosphorylase kinase,
delta)
CAP1
CAP, adenylate cyclase-associated protein 1 (yeast)
CAST
calpastatin
CAV1
caveolin 1, caveolae protein, 22 kDa
CCDC109B
coiled-coil domain containing 109B
CCDC34
coiled-coil domain containing 34
CCDC80
coiled-coil domain containing 80
CCDC88A
coiled-coil domain containing 88A
CCDC90A
coiled-coil domain containing 90A
CCNL1
cyclin L1
CD109
CD109 molecule
CD200
CD200 molecule
CD248
CD248 molecule, endosialin
CD34
CD34 molecule
CD55
CD55 molecule, decay accelerating factor for complement (Cromer
blood group)
CD81
CD81 molecule
CD82
CD82 molecule
CD9
CD9 molecule
CDC42EP3
CDC42 effector protein (Rho GTPase binding) 3
CDH11
cadherin 11, type 2, OB-cadherin (osteoblast)
CDH3
cadherin 3, type 1, P-cadherin (placental)
CDK13
cell division cycle 2-like 5 (cholinesterase-related cell division
controller)
CDON
Cdon homolog (mouse)
CELF2
CUG triplet repeat, RNA binding protein 2
CEP164
centrosomal protein 164 kDa
CEP57
centrosomal protein 57 kDa
CFH
complement factor H
CFL1
cofilin 1 (non-muscle)
CFL2
cofilin 2 (muscle)
CHD1
chromodomain helicase DNA binding protein 1
CHD2
chromodomain helicase DNA binding protein 2
CHI3L1
chitinase 3-like 1 (cartilage glycoprotein-39)
CHST4
carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 4
CISH
cytokine inducible SH2-containing protein
CLCN3
chloride channel 3
CLDN10
claudin 10
CLDN15
claudin 15
CLDN25
claudin-like
CLEC1B
C-type lectin domain family 1, member B
CLEC3B
C-type lectin domain family 3, member B
CLIC4
chloride intracellular channel 4
CLIP1
CAP-GLY domain containing linker protein 1
CLIP3
CAP-GLY domain containing linker protein 3
CLN8
ceroid-lipofuscinosis, neuronal 8 (epilepsy, progressive with mental
retardation)
CMAH
cytidine monophosphate-N-acetylneuraminic acid hydroxylase
(CMP-N-acetylneuraminate monooxygenase) pseudogene
CMTM3
CKLF-like MARVEL transmembrane domain containing 3
CMTM7
CKLF-like MARVEL transmembrane domain containing 7
CNOT6L
CCR4-NOT transcription complex, subunit 6-like
COBL
cordon-bleu homolog (mouse)
COBLL1
COBL-like 1
COL14A1
collagen, type XIV, alpha 1
COL1A2
collagen, type I, alpha 2
COL3A1
collagen, type III, alpha 1
COL4A6
collagen, type IV, alpha 6
COLEC12
collectin sub-family member 12
COQ10B
coenzyme Q10 homolog B ( S. cerevisiae )
CREB3L1
cAMP responsive element binding protein 3-like 1
CREB5
cAMP responsive element binding protein 5
CREBBP
CREB binding protein
CREG1
cellular repressor of E1A-stimulated genes 1
CRIM1
cysteine rich transmembrane BMP regulator 1 (chordin-like)
CRLS1
cardiolipin synthase 1
CRYAB
crystallin, alpha B
CRYL1
crystallin, lambda 1
CRYM
crystallin, mu
CSDA
cold shock domain protein A; cold shock domain protein A
pseudogene 1
CSF1
colony stimulating factor 1 (macrophage)
CSNK1A1
casein kinase 1, alpha 1
CSRNP1
cysteine-serine-rich nuclear protein 1
CSRP1
cysteine and glycine-rich protein 1
CUEDC1
CUE domain containing 1
CYBRD1
cytochrome b reductase 1
CYP2S1
cytochrome P450, family 2, subfamily S, polypeptide 1
CYR61
cysteine-rich, angiogenic inducer, 61
DAB2
disabled homolog 2, mitogen-responsive phosphoprotein ( Drosophila )
DAG1
dystroglycan 1 (dystrophin-associated glycoprotein 1)
DAGLB
diacylglycerol lipase, beta
DAPK1
death-associated protein kinase 1
DCN
decorin
DDR1
discoidin domain receptor tyrosine kinase 1
DDR2
discoidin domain receptor tyrosine kinase 2
DDX3X
DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked
DDX5
DEAD (Asp-Glu-Ala-Asp) box polypeptide 5
DENND5A
DENN/MADD domain containing 5A
DHX15
DEAH (Asp-Glu-Ala-His) box polypeptide 15
DLGAP4
discs, large ( Drosophila ) homolog-associated protein 4
DMKN
dermokine
DNAJA2
DnaJ (Hsp40) homolog, subfamily A, member 2
DNAJB9
DnaJ (Hsp40) homolog, subfamily B, member 9
DNAJC1
DnaJ (Hsp40) homolog, subfamily C, member 1
DNMT1
DNA (cytosine-5-)-methyltransferase 1
DPP4
dipeptidyl-peptidase 4
DPYSL2
dihydropyrimidinase-like 2
DPYSL3
dihydropyrimidinase-like 3
DST
dystonin
DTX2
deltex homolog 2 ( Drosophila )
DUSP1
dual specificity phosphatase 1
DUSP14
dual specificity phosphatase 14
DUSP3
dual specificity phosphatase 3
DYNC1I2
similar to dynein cytoplasmic 1 intermediate chain 2; dynein,
cytoplasmic 1, intermediate chain 2
ECD
ecdysoneless homolog ( Drosophila )
EEA1
early endosome antigen 1
EEF1A1
eukaryotic translation elongation factor 1 alpha-like 7; eukaryotic
translation elongation factor 1 alpha-like 3; similar to eukaryotic
translation elongation factor 1 alpha 1; eukaryotic translation
elongation factor 1 alpha 1
EFEMP1
EGF-containing fibulin-like extracellular matrix protein 1
EFHD2
EF-hand domain family, member D2
EFNA5
ephrin-A5
EGR1
early growth response 1
EHD2
EH-domain containing 2
EIF3A
eukaryotic translation initiation factor 3, subunit A
ELF1
E74-like factor 1 (ets domain transcription factor)
ELOVL6
ELOVL family member 6, elongation of long chain fatty acids
(FEN1/Elo2, SUR4/Elo3-like, yeast)
EMP2
epithelial membrane protein 2
ENPP2
ectonucleotide pyrophosphatase/phosphodiesterase 2
ENPP4
ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative
function)
ESAM
endothelial cell adhesion molecule
ESF1
similar to ABT1-associated protein; ESF1, nucleolar pre-rRNA
processing protein, homolog ( S. cerevisiae )
ESPN
espin
ESYT3
family with sequence similarity 62 (C2 domain containing), member C
ETFA
electron-transfer-flavoprotein, alpha polypeptide
EVPL
envoplakin
EXOC4
exocyst complex component 4
F11R
F11 receptor
FAIM2
Fas apoptotic inhibitory molecule 2
FAM117A
family with sequence similarity 117, member A
FAM134B
family with sequence similarity 134, member B
FAM53B
family with sequence similarity 53, member B
FAM63B
family with sequence similarity 63, member B
FAM76A
family with sequence similarity 76, member A
FAM84B
family with sequence similarity 84, member B
FAS
Fas (TNF receptor superfamily, member 6)
FBLN1
fibulin 1
FERMT2
fermitin family homolog 2 ( Drosophila )
FGF1
fibroblast growth factor 1 (acidic)
FHL1
four and a half LIM domains 1
FILIP1L
filamin A interacting protein 1-like
FKBP5
FK506 binding protein 5
FLII
flightless I homolog ( Drosophila )
FLNC
filamin C, gamma (actin binding protein 280)
FLRT2
fibronectin leucine rich transmembrane protein 2
FMO2
flavin containing monooxygenase 2 (non-functional)
FMOD
fibromodulin
FNDC1
fibronectin type III domain containing 1
FOS
v-fos FBJ murine osteosarcoma viral oncogene homolog
FOXN3
forkhead box N3
FRMD4B
FERM domain containing 4B
FTH1
ferritin, heavy polypeptide 1; ferritin, heavy polypeptide-like 16; similar
to ferritin, heavy polypeptide 1; ferritin, heavy polypeptide-like 3
pseudogene
FXYD1
FXYD domain containing ion transport regulator 1
G3BP1
GTPase activating protein (SH3 domain) binding protein 1
GABARAPL1
GABA(A) receptors associated protein like 3 (pseudogene); GABA(A)
receptor-associated protein like 1
GADD45B
growth arrest and DNA-damage-inducible, beta
GANAB
glucosidase, alpha; neutral AB
GAS1
growth arrest-specific 1
GAS6
similar to growth arrest-specific 6; growth arrest-specific 6
GATA6
GATA binding protein 6
GBP2
guanylate binding protein 2, interferon-inducible
GBP3
guanylate binding protein 3
GBP7
guanylate binding protein 7
GCSH
similar to Glycine cleavage system H protein, mitochondrial precursor;
glycine cleavage system protein H (aminomethyl carrier); similar to
Glycine cleavage system H protein, mitochondrial
GDA
guanine deaminase
GEM
GTP binding protein overexpressed in skeletal muscle
GFM2
G elongation factor, mitochondrial 2
GFPT2
glutamine-fructose-6-phosphate transaminase 2
GJA1
gap junction protein, alpha 1, 43 kDa
GJB5
gap junction protein, beta 5, 31.1 kDa
GNB2
guanine nucleotide binding protein (G protein), beta polypeptide 2
GOLGA4
golgi autoantigen, golgin subfamily a, 4
GOLGB1
golgin B1, golgi integral membrane protein
GPC3
glypican 3
GPC4
glypican 4
GPCPD1
hypothetical protein KIAA1434
GPM6A
glycoprotein M6A
GPR116
G protein-coupled receptor 116
GPR133
G protein-coupled receptor 133
GPR64
G protein-coupled receptor 64
GPRC5B
G protein-coupled receptor, family C, group 5, member B
GPX8
glutathione peroxidase 8 (putative)
GSR
glutathione reductase
GSTA3
glutathione S-transferase alpha 3
GSTM1
glutathione S-transferase mu 1
GSTM4
glutathione S-transferase mu 4
GUCY1A3
guanylate cyclase 1, soluble, alpha 3
H3F3A
H3 histone, family 3B (H3.3B); H3 histone, family 3A pseudogene; H3
histone, family 3A; similar to H3 histone, family 3B; similar to histone
H3.3B
HDAC3
histone deacetylase 3
HDAC5
histone deacetylase 5
HEG1
HEG homolog 1 (zebrafish)
HERPUD2
HERPUD family member 2
HES1
hairy and enhancer of split 1, ( Drosophila )
HEXB
hexosaminidase B (beta polypeptide)
HIST1H1C
histone cluster 1, H1c
HMGB1
high-mobility group box 1; high-mobility group box 1-like 10
HNRNPH1
heterogeneous nuclear ribonucleoprotein H1 (H)
HNRNPH2
ribosomal protein L36a pseudogene 51; ribosomal protein L36a
pseudogene 37; ribosomal protein L36a pseudogene 49;
heterogeneous nuclear ribonucleoprotein H2 (H′); ribosomal protein
L36a
HNRNPL
similar to heterogeneous nuclear ribonucleoprotein L-like;
heterogeneous nuclear ribonucleoprotein L
HNRNPM
heterogeneous nuclear ribonucleoprotein M
HNRNPR
heterogeneous nuclear ribonucleoprotein R
HOOK3
hook homolog 3 ( Drosophila )
HOXA5
homeobox A5
HP1BP3
heterochromatin protein 1, binding protein 3
HSP90AA1
heat shock protein 90 kDa alpha (cytosolic), class A member 2; heat
shock protein 90 kDa alpha (cytosolic), class A member 1
HSP90AB1
heat shock protein 90 kDa alpha (cytosolic), class B member 1
HSP90B1
heat shock protein 90 kDa beta (Grp94), member 1
HSPA12A
heat shock 70 kDa protein 12A
HSPA2
heat shock 70 kDa protein 2
HSPB1
heat shock 27 kDa protein-like 2 pseudogene; heat shock 27 kDa
protein 1
HSPB8
heat shock 22 kDa protein 8
ID1
inhibitor of DNA binding 1, dominant negative helix-loop-helix protein
ID2
inhibitor of DNA binding 2, dominant negative helix-loop-helix protein
IER2
immediate early response 2
IFI35
interferon-induced protein 35
IFIT3
interferon-induced protein with tetratricopeptide repeats 3
IFITM3
interferon induced transmembrane protein 3 (1-8 U)
IFNAR2
interferon (alpha, beta and omega) receptor 2
IFNGR1
interferon gamma receptor 1
IFRD1
interferon-related developmental regulator 1
IFT74
intraflagellar transport 74 homolog ( Chlamydomonas )
IGF1R
insulin-like growth factor 1 receptor
IGFBP5
insulin-like growth factor binding protein 5
IGFBP6
insulin-like growth factor binding protein 6
IL16
interleukin 16 (lymphocyte chemoattractant factor)
IL17RE
interleukin 17 receptor E
IL6ST
interleukin 6 signal transducer (gp130, oncostatin M receptor)
ILDR2
immunoglobulin-like domain containing receptor 2
ILF3
interleukin enhancer binding factor 3, 90 kDa
IMPAD1
inositol monophosphatase domain containing 1
INTS10
integrator complex subunit 10
IQSEC1
IQ motif and Sec7 domain 1
IRAK4
interleukin-1 receptor-associated kinase 4
IRF2BP2
interferon regulatory factor 2 binding protein 2
IRF7
interferon regulatory factor 7
IRS2
insulin receptor substrate 2
ITCH
itchy E3 ubiquitin protein ligase homolog (mouse)
ITGA6
integrin, alpha 6
ITPR2
inositol 1,4,5-triphosphate receptor, type 2
JMJD1C
jumonji domain containing 1C
JUN
jun oncogene
JUNB
jun B proto-oncogene
JUND
jun D proto-oncogene
JUP
junction plakoglobin
KANK1
KN motif and ankyrin repeat domains 1; similar to ankyrin repeat
domain protein 15 isoform b
KCNAB1
potassium voltage-gated channel, shaker-related subfamily, beta
member 1
KDELR1
KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention
receptor 1
KDM5A
lysine (K)-specific demethylase 5A
KDM6B
lysine (K)-specific demethylase 6B
KDR
kinase insert domain receptor (a type III receptor tyrosine kinase)
KEAP1
kelch-like ECH-associated protein 1
KIF1B
kinesin family member 1B
KIF5B
kinesin family member 5B
KLF10
Kruppel-like factor 10
KLF2
Kruppel-like factor 2 (lung)
KLF4
Kruppel-like factor 4 (gut)
KLF6
Kruppel-like factor 6
KLF7
Kruppel-like factor 7 (ubiquitous)
KLF9
Kruppel-like factor 9
KPNA1
karyopherin alpha 1 (importin alpha 5)
KPNA3
karyopherin alpha 3 (importin alpha 4)
KRCC1
lysine-rich coiled-coil 1
KRT14
keratin 14
KTN1
kinectin 1 (kinesin receptor)
LAMA4
laminin, alpha 4
LAMP2
lysosomal-associated membrane protein 2
LARS2
leucyl-tRNA synthetase 2, mitochondrial
LASS2
LAG1 homolog, ceramide synthase 2
LASS4
LAG1 homolog, ceramide synthase 4
LGALS7
lectin, galactoside-binding, soluble, 7; lectin, galactoside-binding,
soluble, 7B
LIMCH1
LIM and calponin homology domains 1
LIMS2
LIM and senescent cell antigen-like domains 2
LMAN1
lectin, mannose-binding, 1
LPAR2
lysophosphatidic acid receptor 2
LRRC20
leucine rich repeat containing 20
LRRC58
leucine rich repeat containing 58
LRRC61
leucine rich repeat containing 61
LRRN4
leucine rich repeat neuronal 4
LRRN4CL
LRRN4C-terminal like
LTBP4
latent transforming growth factor beta binding protein 4
LUC7L3
cisplatin resistance-associated overexpressed protein
MAF
v-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian)
MAGED1
melanoma antigen family D, 1
MAGT1
magnesium transporter 1
MALAT1
metastasis associated lung adenocarcinoma transcript 1 (non-protein
coding)
MANF
mesencephalic astrocyte-derived neurotrophic factor
MAOA
monoamine oxidase A
MAP3K3
mitogen-activated protein kinase kinase kinase 3
MAPK1
mitogen-activated protein kinase 1
MAPKAPK3
mitogen-activated protein kinase-activated protein kinase 3
MAPRE2
microtubule-associated protein, RP/EB family, member 2
MARCKSL1
MARCKS-like 1
MAT2A
methionine adenosyltransferase II, alpha
MAT2B
methionine adenosyltransferase II, beta
MATR3
matrin 3
MED13L
mediator complex subunit 13-like
MED21
mediator complex subunit 21
MEF2C
myocyte enhancer factor 2C
MEIS2
Meis homeobox 2
MESDC1
mesoderm development candidate 1
METAP2
methionyl aminopeptidase 2
MFHAS1
malignant fibrous histiocytoma amplified sequence 1
MGLL
monoglyceride lipase
MGST1
microsomal glutathione S-transferase 1
MLL3
myeloid/lymphoid or mixed-lineage leukemia 3
MORF4L2
mortality factor 4 like 2
MPDZ
multiple PDZ domain protein
MPHOSPH8
M-phase phosphoprotein 8
MRAS
muscle RAS oncogene homolog
MRGPRF
MAS-related GPR, member F
MSN
moesin
MTDH
metadherin
MTMR6
myotubularin related protein 6
MUT
methylmalonyl Coenzyme A mutase
MXD4
MAX dimerization protein 4
MYH10
myosin, heavy chain 10, non-muscle
MYL12A
myosin, light chain 12A, regulatory, non-sarcomeric
MYL7
myosin, light chain 7, regulatory
MYLIP
myosin regulatory light chain interacting protein
MYST4
MYST histone acetyltransferase (monocytic leukemia) 4
NAA25
chromosome 12 open reading frame 30
NAGA
N-acetylgalactosaminidase, alpha-
NCKAP1
NCK-associated protein 1
NCOA1
nuclear receptor coactivator 1
NCOA4
nuclear receptor coactivator 4
NCOR1
nuclear receptor co-repressor 1
NDN
necdin homolog (mouse)
NDST1
N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1
NDUFA4
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9 kDa
NEDD4
neural precursor cell expressed, developmentally down-regulated 4
NF1
neurofibromin 1
NFE2L1
nuclear factor (erythroid-derived 2)-like 1
NFIA
nuclear factor I/A
NFIC
nuclear factor I/C (CCAAT-binding transcription factor)
NFIX
nuclear factor I/X (CCAAT-binding transcription factor)
NFKB2
nuclear factor of kappa light polypeptide gene enhancer in B-cells 2
(p49/p100)
NFKBIA
nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor, alpha
NFKBIZ
nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor, zeta
NFYC
nuclear transcription factor Y, gamma
NID2
nidogen 2 (osteonidogen)
NINL
ninein-like
NIPAL3
NIPA-like domain containing 3
NIPBL
Nipped-B homolog ( Drosophila )
NKAIN4
Na+/K+ transporting ATPase interacting 4
NKD1
naked cuticle homolog 1 ( Drosophila )
NNMT
nicotinamide N-methyltransferase
NOD1
nucleotide-binding oligomerization domain containing 1
NPR1
natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic
peptide receptor A)
NR1D1
nuclear receptor subfamily 1, group D, member 1
NR3C1
nuclear receptor subfamily 3, group C, member 1 (glucocorticoid
receptor)
NR4A1
nuclear receptor subfamily 4, group A, member 1
NRGN
neurogranin (protein kinase C substrate, RC3)
NUCKS1
nuclear casein kinase and cyclin-dependent kinase substrate 1
OAT
ornithine aminotransferase (gyrate atrophy)
OGDH
oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide)
OGN
osteoglycin
OPA3
optic atrophy 3 (autosomal recessive, with chorea and spastic
paraplegia)
ORAI3
ORAI calcium release-activated calcium modulator 3
OSR1
odd-skipped related 1 ( Drosophila )
OXCT1
3-oxoacid CoA transferase 1
OXNAD1
oxidoreductase NAD-binding domain containing 1
PARD3B
par-3 partitioning defective 3 homolog B ( C. elegans )
PARP14
poly (ADP-ribose) polymerase family, member 14
PARP4
poly (ADP-ribose) polymerase family, member 4
PARVB
parvin, beta
PBX1
pre-B-cell leukemia homeobox 1
PCDH15
protocadherin 15
PCDHGB5
protocadherin gamma subfamily B, 5
PCM1
pericentriolar material 1
PDAP1
PDGFA associated protein 1; similar to PDGFA associated protein 1
PDCD6IP
programmed cell death 6 interacting protein
PDE4DIP
hypothetical protein LOC100134230; similar to KIAA0454 protein;
similar to phosphodiesterase 4D interacting protein isoform 2;
phosphodiesterase 4D interacting protein
PDIA3
protein disulfide isomerase family A, member 3
PDIA4
protein disulfide isomerase family A, member 4
PDPN
podoplanin
PEF1
penta-EF-hand domain containing 1
PELI1
pellino homolog 1 ( Drosophila )
PER1
period homolog 1 ( Drosophila )
PF4
platelet factor 4
PFN1
profilin 1
PGCP
plasma glutamate carboxypeptidase
PGRMC1
progesterone receptor membrane component 1
PHF21A
PHD finger protein 21A
PHF3
PHD finger protein 3
PHIP
pleckstrin homology domain interacting protein
PIGT
phosphatidylinositol glycan anchor biosynthesis, class T
PIK3C2A
phosphoinositide-3-kinase, class 2, alpha polypeptide
PIM1
pim-1 oncogene
PITPNM2
phosphatidylinositol transfer protein, membrane-associated 2
PKHD1L1
polycystic kidney and hepatic disease 1 (autosomal recessive)-like 1
PKNOX1
PBX/knotted 1 homeobox 1
PLA2G4A
phospholipase A2, group IVA (cytosolic, calcium-dependent)
PLAT
plasminogen activator, tissue
PLCE1
phospholipase C, epsilon 1
PLK1S1
non-protein coding RNA 153
PLK2
polo-like kinase 2 ( Drosophila )
PLOD2
procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2
PLXDC1
plexin domain containing 1
PLXDC2
plexin domain containing 2
PLXNA4
plexin A4
PMP22
peripheral myelin protein 22
PNRC1
proline-rich nuclear receptor coactivator 1
PODN
podocan
PPAP2A
phosphatidic acid phosphatase type 2A
PPBP
pro-platelet basic protein (chemokine (C-X-C motif) ligand 7)
PPFIBP2
PTPRF interacting protein, binding protein 2 (liprin beta 2)
PPIG
peptidylprolyl isomerase G (cyclophilin G)
PPL
periplakin
PPP1CB
protein phosphatase 1, catalytic subunit, beta isoform; speedy homolog
A ( Xenopus laevis )
PPP1R12A
protein phosphatase 1, regulatory (inhibitor) subunit 12A
PPP1R15A
protein phosphatase 1, regulatory (inhibitor) subunit 15A
PPP3CA
protein phosphatase 3 (formerly 2B), catalytic subunit, alpha isoform
PPPDE1
PPPDE peptidase domain containing 1
PQLC3
PQ loop repeat containing 3
PRELP
proline/arginine-rich end leucine-rich repeat protein
PRG4
proteoglycan 4
PRKAR2A
protein kinase, cAMP-dependent, regulatory, type II, alpha
PRPF40A
PRP40 pre-mRNA processing factor 40 homolog A ( S. cerevisiae )
PRR13
proline rich 13
PRSS23
protease, serine, 23
PSD
pleckstrin and Sec7 domain containing
PSIP1
PC4 and SFRS1 interacting protein 1
PSMB2
proteasome (prosome, macropain) subunit, beta type, 2
PSMD11
proteasome (prosome, macropain) 26S subunit, non-ATPase, 11
PSMD7
proteasome (prosome, macropain) 26S subunit, non-ATPase, 7
PTGES3
prostaglandin E synthase 3 (cytosolic)
PTGIS
prostaglandin I2 (prostacyclin) synthase
PTGS1
prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase
and cyclooxygenase)
PTMA
hypothetical LOC728026; prothymosin, alpha; hypothetical gene
supported by BC013859; prothymosin, alpha pseudogene 4 (gene
sequence 112)
PTP4A2
protein tyrosine phosphatase type IVA, member 2
PTPLAD2
protein tyrosine phosphatase-like A domain containing 2
PTPRD
protein tyrosine phosphatase, receptor type, D
PTPRF
protein tyrosine phosphatase, receptor type, F
PTRF
polymerase 1 and transcript release factor
QRICH1
glutamine-rich 1
QSER1
glutamine and serine rich 1
RAB11FIP1
RAB11 family interacting protein 1 (class I)
RAB1B
RAB1B, member RAS oncogene family
RAB5C
RAB5C, member RAS oncogene family
RAB6B
RAB6B, member RAS oncogene family
RABGAP1L
RAB GTPase activating protein 1-like
RALBP1
hypothetical LOC100129773; ralA binding protein 1
RALY
RNA binding protein, autoantigenic (hnRNP-associated with lethal
yellow homolog (mouse))
RARRES2
retinoic acid receptor responder (tazarotene induced) 2
RB1CC1
RB1-inducible coiled-coil 1
RBBP6
retinoblastoma binding protein 6
RBBP8
retinoblastoma binding protein 8
RBM25
RNA binding motif protein 25
RBM27
RNA binding motif protein 27
RBM3
RNA binding motif (RNP1, RRM) protein 3
RBPMS
RNA binding protein with multiple splicing
RDX
radixin
REST
RE1-silencing transcription factor
RGMA
RGM domain family, member A
RGS10
regulator of G-protein signaling 10
RHOB
ras homolog gene family, member B
RHOJ
ras homolog gene family, member J
RHOU
ras homolog gene family, member U
RNASE4
ribonuclease, RNase A family, 4
RND3
Rho family GTPase 3
RNF167
ring finger protein 167
RNF20
ring finger protein 20
ROCK1
similar to Rho-associated, coiled-coil containing protein kinase 1; Rho-
associated, coiled-coil containing protein kinase 1
ROCK2
Rho-associated, coiled-coil containing protein kinase 2
RPP25
ribonuclease P/MRP 25 kDa subunit
RRAS2
related RAS viral (r-ras) oncogene homolog 2; similar to related RAS
viral (r-ras) oncogene homolog 2
RSPO1
R-spondin homolog ( Xenopus laevis )
RTF1
Rtf1, Paf1/RNA polymerase II complex component, homolog
( S. cerevisiae )
RTN1
reticulon 1
RYK
RYK receptor-like tyrosine kinase
SARNP
SAP domain containing ribonucleoprotein
SAT1
spermidine/spermine N1-acetyltransferase 1
SBSN
suprabasin
SDC4
syndecan 4
SDPR
serum deprivation response (phosphatidylserine binding protein)
SEC62
SEC62 homolog ( S. cerevisiae )
SECISBP2
SECIS binding protein 2
SEMA5A
sema domain, seven thrombospondin repeats (type 1 and type 1-like),
transmembrane domain (TM) and short cytoplasmic domain,
(semaphorin) 5A
SENP6
SUMO1/sentrin specific peptidase 6
SEP15
15 kDa selenoprotein
SEPT9
septin 9
SERINC5
serine incorporator 5
SERPING1
serpin peptidase inhibitor, clade G (C1 inhibitor), member 1
SERPINH1
serpin peptidase inhibitor, clade H (heat shock protein 47), member 1,
(collagen binding protein 1)
SESN1
sestrin 1
SETD2
SET domain containing 2
SF3B1
splicing factor 3b, subunit 1, 155 kDa
SF3B4
splicing factor 3b, subunit 4, 49 kDa
SFRS18
splicing factor, arginine/serine-rich 18
SHC1
SHC (Src homology 2 domain containing) transforming protein 1
SHFM1
split hand/foot malformation (ectrodactyly) type 1
SIAE
sialic acid acetylesterase
SIRT2
sirtuin (silent mating type information regulation 2 homolog) 2
( S. cerevisiae )
SLC10A3
solute carrier family 10 (sodium/bile acid cotransporter family),
member 3
SLC16A1
solute carrier family 16, member 1 (monocarboxylic acid transporter 1)
SLC1A5
solute carrier family 1 (neutral amino acid transporter), member 5
SLC26A3
solute carrier family 26, member 3
SLC27A3
solute carrier family 27 (fatty acid transporter), member 3
SLC38A1
solute carrier family 38, member 1
SLC39A8
solute carrier family 39 (zinc transporter), member 8
SLC43A3
solute carrier family 43, member 3
SLC4A4
solute carrier family 4, sodium bicarbonate cotransporter, member 4
SLC6A4
solute carrier family 6 (neurotransmitter transporter, serotonin),
member 4
SLC6A6
solute carrier family 6 (neurotransmitter transporter, taurine),
member 6
SLC8A1
solute carrier family 8 (sodium/calcium exchanger), member 1
SLC9A3R1
solute carrier family 9 (sodium/hydrogen exchanger), member 3
regulator 1
SLPI
secretory leukocyte peptidase inhibitor
SLTM
SAFB-like, transcription modulator
SLU7
SLU7 splicing factor homolog ( S. cerevisiae )
SLURP1
secreted LY6/PLAUR domain containing 1
SMAD4
SMAD family member 4
SMARCA2
SWI/SNF related, matrix associated, actin dependent regulator of
chromatin, subfamily a, member 2
SMARCA5
SWI/SNF related, matrix associated, actin dependent regulator of
chromatin, subfamily a, member 5
SMC2
structural maintenance of chromosomes 2
SMC3
structural maintenance of chromosomes 3
SMC4
structural maintenance of chromosomes 4
SMC6
structural maintenance of chromosomes 6
SMCHD1
structural maintenance of chromosomes flexible hinge domain
containing 1
SMPD3
sphingomyelin phosphodiesterase 3, neutral membrane (neutral
sphingomyelinase II)
SNRNP70
small nuclear ribonucleoprotein 70 kDa (U1)
SNTB2
syntrophin, beta 2 (dystrophin-associated protein A1, 59 kDa, basic
component 2)
SOAT1
sterol O-acyltransferase 1
SOCS3
suppressor of cytokine signaling 3
SOD3
superoxide dismutase 3, extracellular
SORBS1
sorbin and SH3 domain containing 1
SORBS3
sorbin and SH3 domain containing 3
SOX6
SRY (sex determining region Y)-box 6
SP100
SP100 nuclear antigen
SPAG9
sperm associated antigen 9
SPARC
secreted protein, acidic, cysteine-rich (osteonectin)
SPEN
spen homolog, transcriptional regulator ( Drosophila )
SPINT2
serine peptidase inhibitor, Kunitz type, 2
SPOCK2
sparc/osteonectin, cwcv and kazal-like domains proteoglycan
(testican) 2
SPON2
spondin 2, extracellular matrix protein
SPOP
speckle-type POZ protein
SRC
v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian)
SRRM1
serine/arginine repetitive matrix 1
SSH2
slingshot homolog 2 ( Drosophila )
SSR3
signal sequence receptor, gamma (translocon-associated protein
gamma)
ST3GAL1
ST3 beta-galactoside alpha-2,3-sialyltransferase 1
STAG1
stromal antigen 1
STAR
steroidogenic acute regulatory protein
STARD5
StAR-related lipid transfer (START) domain containing 5
STAT3
signal transducer and activator of transcription 3 (acute-phase
response factor)
STIM1
stromal interaction molecule 1
STK10
serine/threonine kinase 10
STK40
serine/threonine kinase 40
STMN2
stathmin-like 2
STRA6
stimulated by retinoic acid gene 6 homolog (mouse)
STRN3
striatin, calmodulin binding protein 3
SULF1
sulfatase 1
SULF2
sulfatase 2
SUPT16H
suppressor of Ty 16 homolog ( S. cerevisiae ); suppressor of Ty 16
homolog ( S. cerevisiae ) pseudogene
SV2A
synaptic vesicle glycoprotein 2A
SYNE1
spectrin repeat containing, nuclear envelope 1
SYNE2
spectrin repeat containing, nuclear envelope 2
SYT11
synaptotagmin XI
SYTL1
synaptotagmin-like 1
TAF3
TAF3 RNA polymerase II, TATA box binding protein (TBP)-associated
factor, 140 kDa
TAF7
TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated
factor, 55 kDa
TAPBP
TAP binding protein (tapasin)
TBC1D15
TBC1 domain family, member 15
TBCEL
tubulin folding cofactor E-like
TBL1X
transducin (beta)-like 1X-linked
TBX18
T-box 18
TCEAL8
transcription elongation factor A (SII)-like 8
TCF7L1
transcription factor 7-like 1 (T-cell specific, HMG-box)
TFDP2
transcription factor Dp-2 (E2F dimerization partner 2)
TGFB1I1
transforming growth factor beta 1 induced transcript 1
TGFB2
transforming growth factor, beta 2
TGFBR2
transforming growth factor, beta receptor II (70/80 kDa)
TGM2
transglutaminase 2 (C polypeptide, protein-glutamine-gamma-
glutamyltransferase)
THBD
thrombomodulin
THBS1
thrombospondin 1
THOC2
THO complex 2
THRAP3
thyroid hormone receptor associated protein 3
THSD4
thrombospondin, type I, domain containing 4
TIMP2
TIMP metallopeptidase inhibitor 2
TIRAP
toll-interleukin 1 receptor (TIR) domain containing adaptor protein
TLR2
toll-like receptor 2
TM4SF1
transmembrane 4 L six family member 1
TM4SF5
transmembrane 4 L six family member 5
TMCC3
transmembrane and coiled-coil domain family 3
TMCO1
transmembrane and coiled-coil domains 1
TMCO7
transmembrane and coiled-coil domains 7
TMED2
transmembrane emp24 domain trafficking protein 2
TMEM119
transmembrane protein 119
TMEM140
transmembrane protein 140
TMEM151A
transmembrane protein 151A
TMEM221
transmembrane protein 221
TMEM50A
transmembrane protein 50A
TMEM98
similar to transmembrane protein 98; transmembrane protein 98
TMOD3
tropomodulin 3 (ubiquitous)
TMPO
thymopoietin
TMSB4X
thymosin-like 2 (pseudogene); thymosin-like 1 (pseudogene); thymosin
beta 4, X-linked
TNXB
tenascin XB; tenascin XA pseudogene
TOB2
transducer of ERBB2, 2
TOPORS
topoisomerase 1 binding, arginine/serine-rich
TPM3
tropomyosin 3
TPPP3
tubulin polymerization-promoting protein family member 3
TPT1
similar to tumor protein, translationally-controlled 1; tumor protein,
translationally-controlled 1
TRAFD1
TRAF-type zinc finger domain containing 1
TRIB1
tribbles homolog 1 ( Drosophila )
TRIM8
tripartite motif-containing 8
TRPM7
transient receptor potential cation channel, subfamily M, member 7
TSC22D3
TSC22 domain family, member 3; GRAM domain containing 4
TSHZ1
teashirt zinc finger homeobox 1
TSIX
XIST antisense RNA (non-protein coding)
TSPAN31
tetraspanin 31
TSPAN5
tetraspanin 5
TTC28
chromosome 6 open reading frame 35; hCG1820764; tetratricopeptide
repeat domain 28
TTC38
tetratricopeptide repeat domain 38
TUBA1A
tubulin, alpha la
TUBB2A
tubulin, beta 2A
TWSG1
twisted gastrulation homolog 1 ( Drosophila )
TXNDC5
thioredoxin domain containing 5 (endoplasmic reticulum); muted
homolog (mouse)
TXNRD1
thioredoxin reductase 1; hypothetical LOC100130902
UAP1
UDP-N-acteylglucosamine pyrophosphorylase 1
UBA7
ubiquitin-like modifier activating enzyme 7
UBE2D1
ubiquitin-conjugating enzyme E2D 1 (UBC4/5 homolog, yeast)
UBE2L6
ubiquitin-conjugating enzyme E2L 6
UBE2N
ubiquitin-conjugating enzyme E2N (UBC13 homolog, yeast)
UBE2V1
ubiquitin-conjugating enzyme E2 variant 1; ubiquitin-conjugating
enzyme E2 variant 1 pseudogene 2; transmembrane protein 189;
TMEM189-UBE2V1 readthrough transcript
UBQLN2
ubiquilin 2
UBXN2A
UBX domain protein 2A
UBXN4
UBX domain protein 4
UGDH
UDP-glucose dehydrogenase
UPK1B
uroplakin 1B
UPK3B
uroplakin 3B
USP16
ubiquitin specific peptidase 16
USP2
ubiquitin specific peptidase 2
USP25
ubiquitin specific peptidase 25
USP54
ubiquitin specific peptidase 54
USP8
ubiquitin specific peptidase 8
UTP20
similar to Down-regulated in metastasis protein (Key-1A6 protein)
(Novel nucleolar protein 73) (NNP73); UTP20, small subunit (SSU)
processome component, homolog (yeast)
VAT1
vesicle amine transport protein 1 homolog ( T. californica )
VIM
vimentin
VPS13A
vacuolar protein sorting 13 homolog A ( S. cerevisiae )
VWA5A
von Willebrand factor A domain containing 5A
WAC
WW domain containing adaptor with coiled-coil
WASF2
WAS protein family, member 2
WDR26
WD repeat domain 26
WDR92
WD repeat domain 92
WFDC1
WAP four-disulfide core domain 1
WLS
G protein-coupled receptor 177
WNT4
wingless-type MMTV integration site family, member 4
WRNIP1
Werner helicase interacting protein 1
WT1
Wilms tumor 1
WWC2
WW and C2 domain containing 2
XDH
xanthine dehydrogenase
XIST
X (inactive)-specific transcript (non-protein coding)
YIPF5
Yip1 domain family, member 5
YWHAZ
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation
protein, zeta polypeptide
ZBTB16
zinc finger and BTB domain containing 16
ZBTB20
zinc finger and BTB domain containing 20
ZBTB4
zinc finger and BTB domain containing 4
ZBTB7C
zinc finger and BTB domain containing 7C
ZC3H13
zinc finger CCCH-type containing 13
ZC3H18
zinc finger CCCH-type containing 18
ZCCHC11
zinc finger, CCHC domain containing 11
ZCCHC3
zinc finger, CCHC domain containing 3
ZFAND6
zinc finger, AN1-type domain 6
ZFHX4
zinc finger homeobox 4
ZFP36
zinc finger protein 36, C3H type, homolog (mouse)
ZMAT1
zinc finger, matrin type 1
ZRSR1
zinc finger (CCCH type), RNA-binding motif and serine/arginine rich 1
ZZEF1
zinc finger, ZZ-type with EF-hand domain 1
The gene names listed in Table 7 and Table 8 are common names. NCBI Gene ID numbers for each of the genes listed in Table 7 or Table 8 can be obtained by searching the “Gene” Database of the NCBI (available on the World Wide Web at http://www.ncbi.nlm.nih.gov/) using the common name as the query and selecting the first returned Homo sapiens (for the genes in Table 8) or Mus musculus gene (for the genes in Table 7). Other genes may be obtained using the UCSC genome browser (available on the World Wide Web at http://genome.ucsc.edu) using the Gene Sorter function. Human homologs of mouse genes can be readily identified, e.g. the identified homologs in the NCBI database, or by querying databases such as BLAST. In certain embodiments, the marker gene(s) are selected from the genes listed in Table 7, Table 8, or Table 14.
In a CTC, the marker genes listed in Table 7, Table 8, or Table 14 can be upregulated, e.g. for marker genes listed in Table 7, Table 8, or Table 14, if the measured marker gene expression in a cell or sample is higher as compared to a reference level of that marker gene's expression, then the cell is identified as a CTC and/or the sample is identified as comprising CTCs. Preferably, once looks at a statistically significant change. However, even if a few genes in a group do not differ from normal, a sample can be identified as comprising CTCs if the overall change of the group shows a significant change, preferably a statistically significant change. All possible combinations of 2 or more of the indicated markers are contemplated herein.
The level of a gene expression product of a marker gene in Table 7, Table 8, or Table 14 which is higher than a reference level of that marker gene by at least about 10% than the reference amount, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, at least about 100%, at least about 200%, at least about 300%, at least about 500% or at least about 1000% or more, is indicative of the presence of a CTC.
In some embodiments, the reference can be a level of expression of the marker gene product in a cell or population of cells which are not CTCs, e.g. the average level in non-circulating tumor cells and/or circulating cells which are not cancer cells. In some embodiments, the reference can also be a level of expression of the marker gene product in a control sample, a pooled sample of control individuals or a numeric value or range of values based on the same.
In some embodiments, the methods and assays described herein include (a) transforming the gene expression product into a detectable gene target; (b) measuring the amount of the detectable gene target; and (c) comparing the amount of the detectable gene target to an amount of a reference, wherein if the amount of the detectable gene target is statistically significantly different than the amount of the reference level, the presence and/or level of CTCs is determined. In some embodiments, if the amount of the detectable gene target is not statistically significantly different than the amount of the reference level, the sample is identified as not comprising CTCs.
As used herein, the term “transforming” or “transformation” refers to changing an object or a substance, e.g., biological sample, nucleic acid or protein, into another substance. The transformation can be physical, biological or chemical. Exemplary physical transformation includes, but not limited to, pre-treatment of a biological sample, e.g., from whole blood to blood serum by differential centrifugation. A biological/chemical transformation can involve at least one enzyme and/or a chemical reagent in a reaction. For example, a DNA sample can be digested into fragments by one or more restriction enzyme, or an exogenous molecule can be attached to a fragmented DNA sample with a ligase. In some embodiments, a DNA sample can undergo enzymatic replication, e.g., by polymerase chain reaction (PCR).
Methods to measure gene expression products associated with the marker genes described herein are well known to a skilled artisan. Such methods to measure gene expression products, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, FACS, radioimmunological assay; (RIA); sandwich assay; fluorescent in situ hybridization (FISH); immunohistological staining; immunoelectrophoresis; immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents. Alternatively, a peptide can be detected in a subject by introducing into a subject a labeled anti-peptide antibody and other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in the subject is detected by standard imaging techniques.
For example, antibodies for the polypeptide expression products of the marker genes described herein are commercially available and can be used for the purposes of the invention to measure protein expression levels, e.g. anti-IGFBP5 (Cat. No. 4255; Abcam; Cambridge, Mass.). Alternatively, since the amino acid sequences for the marker genes described herein are known and publically available at NCBI website, one of skill in the art can raise their own antibodies against these proteins of interest for the purpose of the invention. The amino acid sequences of the marker genes described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat.
In some embodiments, immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) techniques can be used. IHC is the application of immunochemistry to tissue sections, whereas ICC is the application of immunochemistry to cells or tissue imprints after they have undergone specific cytological preparations such as, for example, liquid-based preparations. Immunochemistry is a family of techniques based on the use of an antibody, wherein the antibodies are used to specifically target molecules inside or on the surface of cells. The antibody typically contains a marker that will undergo a biochemical reaction, and thereby experience a change color, upon encountering the targeted molecules. In some instances, signal amplification can be integrated into the particular protocol, wherein a secondary antibody, that includes the marker stain or marker signal, follows the application of a primary specific antibody.
In some embodiments, the assay can be a Western blot analysis. Alternatively, proteins can be separated by two-dimensional gel electrophoresis systems. Two-dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. These methods also require a considerable amount of cellular material. The analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel, or can be performed using immune detection. In other embodiments, protein samples are analyzed by mass spectroscopy.
Immunological tests can be used with the methods and assays described herein and include, for example, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassay (RIA), ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence-linked immunoassay), chemiluminescence immunoassays (CLIA), electrochemiluminescence immunoassay (ECLIA, counting immunoassay (CIA), lateral flow tests or immunoassay (LFIA), magnetic immunoassay (MIA), and protein A immunoassays. Methods for performing such assays are known in the art, provided an appropriate antibody reagent is available. In some embodiment, the immunoassay can be a quantitative or a semi-quantitative immunoassay.
An immunoassay is a biochemical test that measures the concentration of a substance in a biological sample, typically a fluid sample such as serum, using the interaction of an antibody or antibodies to its antigen. The assay takes advantage of the highly specific binding of an antibody with its antigen. For the methods and assays described herein, specific binding of the target polypeptides with respective proteins or protein fragments, or an isolated peptide, or a fusion protein described herein occurs in the immunoassay to form a target protein/peptide complex. The complex is then detected by a variety of methods known in the art. An immunoassay also often involves the use of a detection antibody.
Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.
In one embodiment, an ELISA involving at least one antibody with specificity for the particular desired antigen (i.e. a marker gene polypeptide as described herein) can also be performed. A known amount of sample and/or antigen is immobilized on a solid support (usually a polystyrene micro titer plate). Immobilization can be either non-specific (e.g., by adsorption to the surface) or specific (e.g. where another antibody immobilized on the surface is used to capture antigen or a primary antibody). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bio-conjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity.
In another embodiment, a competitive ELISA is used. Purified antibodies that are directed against a target polypeptide or fragment thereof are coated on the solid phase of multi-well plate, i.e., conjugated to a solid surface. A second batch of purified antibodies that are not conjugated on any solid support is also needed. These non-conjugated purified antibodies are labeled for detection purposes, for example, labeled with horseradish peroxidase to produce a detectable signal. A sample (e.g., tumor, blood, serum or urine) from a subject is mixed with a known amount of desired antigen (e.g., a known volume or concentration of a sample comprising a target polypeptide) together with the horseradish peroxidase labeled antibodies and the mixture is then are added to coated wells to form competitive combination. After incubation, if the polypeptide level is high in the sample, a complex of labeled antibody reagent-antigen will form. This complex is free in solution and can be washed away. Washing the wells will remove the complex. Then the wells are incubated with TMB (3, 3′, 5, 5′-tetramethylbenzidene) color development substrate for localization of horseradish peroxidase-conjugated antibodies in the wells. There will be no color change or little color change if the target polypeptide level is high in the sample. If there is little or no target polypeptide present in the sample, a different complex in formed, the complex of solid support bound antibody reagents-target polypeptide. This complex is immobilized on the plate and is not washed away in the wash step. Subsequent incubation with TMB will produce much color change. Such a competitive ELSA test is specific, sensitive, reproducible and easy to operate.
There are other different forms of ELISA, which are well known to those skilled in the art. The standard techniques known in the art for ELISA are described in “Methods in Immunodiagnosis”, 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem. 22:895-904. These references are hereby incorporated by reference in their entirety.
In one embodiment, the levels of a polypeptide in a sample can be detected by a lateral flow immunoassay test (LFIA), also known as the immunochromatographic assay, or strip test. LFIAs are a simple device intended to detect the presence (or absence) of antigen, e.g. a polypeptide, in a fluid sample. There are currently many LFIA tests are used for medical diagnostics either for home testing, point of care testing, or laboratory use. LFIA tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test strip it encounters a colored reagent (generally comprising antibody specific for the test target antigen) bound to microparticles which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with another antibody or antigen. Depending upon the level of target polypeptides present in the sample the colored reagent can be captured and become bound at the test line or zone. LFIAs are essentially immunoassays adapted to operate along a single axis to suit the test strip format or a dipstick format. Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens from fluid samples such as urine, blood, water, and/or homogenized tumor samples etc. Strip tests are also known as dip stick test, the name bearing from the literal action of “dipping” the test strip into a fluid sample to be tested. LFIA strip tests are easy to use, require minimum training and can easily be included as components of point-of-care test (POCT) diagnostics to be use on site in the field. LFIA tests can be operated as either competitive or sandwich assays. Sandwich LFIAs are similar to sandwich ELISA. The sample first encounters colored particles which are labeled with antibodies raised to the target antigen. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the antigen. The test line will show as a colored band in positive samples. In some embodiments, the lateral flow immunoassay can be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof. Competitive LFIAs are similar to competitive ELISA. The sample first encounters colored particles which are labeled with the target antigen or an analogue. The test line contains antibodies to the target/its analogue. Unlabelled antigen in the sample will block the binding sites on the antibodies preventing uptake of the colored particles. The test line will show as a colored band in negative samples. There are a number of variations on lateral flow technology. It is also possible to apply multiple capture zones to create a multiplex test.
The use of “dip sticks” or LFIA test strips and other solid supports have been described in the art in the context of an immunoassay for a number of antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871; 6,565,808, U.S. patent application Ser. No. 10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No. 10/717,082, which are incorporated herein by reference in their entirety, are non-limiting examples of such lateral flow test devices. Examples of patents that describe the use of “dip stick” technology to detect soluble antigens via immunochemical assays include, but are not limited to U.S. Pat. Nos. 4,444,880; 4,305,924; and 4,135,884; which are incorporated by reference herein in their entireties. The apparatuses and methods of these three patents broadly describe a first component fixed to a solid surface on a “dip stick” which is exposed to a solution containing a soluble antigen that binds to the component fixed upon the “dip stick,” prior to detection of the component-antigen complex upon the stick. It is within the skill of one in the art to modify the teachings of this “dip stick” technology for the detection of polypeptides using antibody reagents as described herein.
Other techniques can be used to detect the level of a polypeptide in a sample. One such technique is the dot blot, an adaptation of Western blotting (Towbin et al., Proc. Nat. Acad. Sci. 76:4350 (1979)). In a Western blot, the polypeptide or fragment thereof can be dissociated with detergents and heat, and separated on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose or PVDF membrane. The membrane is incubated with an antibody reagent specific for the target polypeptide or a fragment thereof. The membrane is then washed to remove unbound proteins and proteins with non-specific binding. Detectably labeled enzyme-linked secondary or detection antibodies can then be used to detect and assess the amount of polypeptide in the sample tested. The intensity of the signal from the detectable label corresponds to the amount of enzyme present, and therefore the amount of polypeptide. Levels can be quantified, for example by densitometry.
Flow cytometry is a well-known technique for analyzing and sorting cells (or other small particles) suspended in a fluid stream. This technique allows simultaneous analysis of the physical and/or chemical characteristics of single cells flowing through an optical, electronic, or magnetic detection apparatus. As applied to FACS, the flow cytometer consists of a flow cell which carries the cells in a fluid stream in single file through a light source with excites the fluorescently labeled detection marker(s) (for example, antibody reagents) and measures the fluorescent character of the cell. The fluid stream is then ejected through a nozzle and a charging ring, under pressure, which breaks the fluid into droplets. The flow cell device and fluid stream is calibrated such that there is a relatively large distance between individual cells or bound groups of cells, resulting in a low probability that any droplet contains more than a single cell or bound group of cells. The charging ring charges the droplets based on the fluorescence characteristic of the cell which is contained therein. The charged droplets are then deflected by an electrostatically-charged deflection system which diverts the droplets into various containers based upon their charge (related to the fluorescence intensity of the cell). A FACS system (e.g. the FACSARIA™ flow cytometer (BD Biosciences) and FLOWJO™ Version 7.6.4 (TreeStar)) can detect and record the number of total cells as well as the number of cells which display one or more fluorescent characteristics, e.g. the total number of cells bound by one or more antibody reagents specific for a CTC marker gene.
In certain embodiments, the gene expression products as described herein can be instead determined by determining the level of messenger RNA (mRNA) expression of genes associated with the marker genes described herein. Such molecules can be isolated, derived, or amplified from a biological sample, such as a tumor biopsy. Detection of mRNA expression is known by persons skilled in the art, and comprise, for example but not limited to, PCR procedures, RT-PCR, quantitative PCR or RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization methods, next-generation sequencing etc. Non-limiting examples of next-generation sequencing technologies can include Ion Torrent, Illumina, SOLiD, 454; Massively Parallel Signature Sequencing solid-phase, reversible dye-terminator sequencing; and DNA nanoball sequencing.
In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes or sequences within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a thermostable DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to a strand of the genomic locus to be amplified. In an alternative embodiment, mRNA level of gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art. The nucleic acid sequences of the marker genes described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat. Accordingly, a skilled artisan can design an appropriate primer based on the known sequence for determining the mRNA level of the respective gene.
Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. For example, freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials; heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine; and proteinase K extraction can be used to obtain nucleic acid from blood (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).
In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to each strand of the nucleic acid molecule to be amplified.
In an alternative embodiment, mRNA level of gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art.
In some embodiments, one or more of the reagents (e.g. an antibody reagent and/or nucleic acid probe) described herein can comprise a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product). Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g. antibodies and nucleic acid probes) are well known in the art.
In some embodiments, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means. The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.
In other embodiments, the detection reagent is label with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments, a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3™, Cy5™, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 6-carboxyfhiorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2′,4′,7′,4,7-hexachlorofiuorescein (HEX), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfiuorescein (JOE or J), N,N,N′,N′-tetramethyl-6carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes. In some embodiments, a detectable label can be a radiolabel including, but not limited to 3 H, 125 I, 35 S, 14 C, 32 P, and 33 P. In some embodiments, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In some embodiments, a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In some embodiments, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.
In some embodiments, detection reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available, e. g. from DAKO; Carpinteria, Calif. A reagent can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
In some embodiments of any of the aspects described herein, the level of expression products of more than one gene can be determined simultaneously (e.g. a multiplex assay) or in parallel. In some embodiments, the level of expression products of no more than 200 other genes is determined. In some embodiments, the level of expression products of no more than 100 other genes is determined. In some embodiments, the level of expression products of no more than 20 other genes is determined. In some embodiments, the level of expression products of no more than 10 other genes is determined.
The term “sample” or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a tumor sample from a subject. Exemplary biological samples include, but are not limited to, a biofluid sample; serum; plasma; urine; saliva; a tumor sample; a tumor biopsy and/or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term “test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments, a test sample can comprise cells from subject. In some embodiments, a test sample can be a tumor cell test sample, e.g. the sample can comprise cancerous cells, cells from a tumor, and/or a tumor biopsy. In some embodiments, the test sample can be a blood sample.
The test sample can be obtained by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells (e.g. isolated at a prior timepoint and isolated by the same or another person). In addition, the test sample can be freshly collected or a previously collected sample.
In some embodiments, the test sample can be an untreated test sample. As used herein, the phrase “untreated test sample” refers to a test sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments, the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments, the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof. In some embodiments, the test sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. The skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for determination of the level of an expression product as described herein.
In some embodiments, the methods, assays, and systems described herein can further comprise a step of obtaining a test sample from a subject. In some embodiments, the subject can be a human subject.
In some embodiments, the methods and assays described herein can further comprise a step of isolating CTCs or potential CTCs from a sample prior to measuring the level the expression product of one or more of the marker genes described herein. By way of non-limitng example, CTCs can be isolated from, e.g. a blood sample by hydrodynamic size-based separation and/or immunodepletetion of other cell types present in blood samples. The CTC-iChip, described in the Examples herein combines these two approaches to isolate CTCs.
Subjects with high, or at least detectable, levels of CTCs are most likely to benefit from treatment with therapies that specifically target CTCs. Accordingly, provided herein is a method of determining if a subject is likely to respond to treatment with a CTC marker gene-targeted therapy, the method comprising: measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is likely to respond to the treatment if the level of the expression product is increased relative to a reference level. CTC marker gene-targeted therapies are discussed below herein.
Decreased levels of CTCs after administration of a therapy can be indicative of an improvement in the condition of the subject, e.g. the cancer is reduced in size, growth, and/or metastatic potential. Accordingly, provided herein is a method of monitoring the treatment of a subject, the method comprising administering a cancer therapy to a subject in need thereof; measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is responding if the level of the CTC marker gene expression product is decreased relative to the reference level and determining that the subject is not responding to the treatment if the CTC marker gene expression product is not decreased relative to the reference level. In some embodiments the therapy is a chemotherapy, surgical therapy, and/or radiation therapy. In some embodiments, the therapy is a CTC marker gene-targeted therapy. In some embodiments, the reference level is the level of the gene expression product in the patient prior to the administering step.
The CTC marker genes described herein can be targeted directly and/or used to physically target a chemotherapeutic agent to reduce the levels and/or pathogenic activity of CTCs (e.g. metastatic activity). Accordingly, described herein is a method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of a CTC marker gene-targeted therapy to the subject. In some embodiments, the subject is a subject determined to have an elevated level of CTCs and/or an elevated level of a CTC marker gene present in the blood and/or stroma of the cancer.
In some embodiments, the CTC marker gene-targeted therapy can comprise an inhibitor of a CTC marker gene, e.g. the CTC marker gene-targeted therapy can inhibit the level and/or activity of a CTC marker gene. As used herein, the term “inhibitor” refers to an agent which can decrease the expression and/or activity of the targeted expression product (e.g. mRNA encoding the target or a target polypeptide), e.g. by at least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more. The efficacy of an inhibitor of a CTC marker gene, e.g. its ability to decrease the level and/or activity of the CTC marker gene can be determined, e.g. by measuring the level of an expression product and/or the activity of the CTC marker gene. Methods for measuring the level of a given mRNA and/or polypeptide are known to one of skill in the art, e.g. RTPCR with primers can be used to determine the level of RNA and Western blotting with an antibody can be used to determine the level of a polypeptide. The activity of, e.g. a CTC marker gene can be determined, e.g. by measuring the levels and/or survival of CTCs using methods known in the art and described elsewhere herein. In some embodiments, the inhibitor of a CTC marker gene can be an inhibitory nucleic acid; an aptamer; an antibody reagent; an antibody; or a small molecule.
In some embodiments, the inhibitor of a CTC marker gene can be an antibody reagent. As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgE molecules or antigen-specific antibody fragments thereof (including, but not limited to, a Fab, F(ab′) 2 , Fv, disulphide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulphide-linked scfv, diabody), whether derived from any species that naturally produces an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria.
As described herein, an “antigen” is a molecule that is bound by a binding site on an antibody agent. Typically, antigens are bound by antibody ligands and are capable of raising an antibody response in vivo. An antigen can be a polypeptide, protein, nucleic acid or other molecule or portion thereof. The term “antigenic determinant” refers to an epitope on the antigen recognized by an antigen-binding molecule, and more particularly, by the antigen-binding site of said molecule.
As used herein, the term “antibody reagent” refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen. An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody reagent” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.
The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (“FR”). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated by reference herein in their entireties). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
The terms “antigen-binding fragment” or “antigen-binding domain”, which are used interchangeably herein are used to refer to one or more fragments of a full length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term “antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546; which is incorporated by reference herein in its entirety), which consists of a VH or VL domain; and (vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding functionality. As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity.
Additionally, and as described herein, a recombinant humanized antibody can be further optimized to decrease potential immunogenicity, while maintaining functional activity, for therapy in humans. In this regard, functional activity means a polypeptide capable of displaying one or more known functional activities associated with a recombinant antibody or antibody reagent thereof as described herein. Such functional activities include, e.g. the ability to bind to a given CTC marker gene.
In some embodiments, the inhibitor of a CTC marker gene can be an inhibitory nucleic acid reagent. In some embodiments, the inhibitory nucleic acid is an inhibitory RNA (iRNA). Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein can include an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part the targeted mRNA transcript. The use of these iRNAs enables the targeted degradation of mRNA transcripts, resulting in decreased expression and/or activity of the target.
As used herein, the term “iRNA” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNA as described herein effects inhibition of the expression and/or activity of the target mRNA. In certain embodiments, contacting a cell with the inhibitor (e.g. an iRNA) results in a decrease in the target mRNA level in a cell by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the cell without the presence of the iRNA.
In some embodiments, the iRNA can be a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of the target. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive. In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.
In yet another embodiment, the RNA of an iRNA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.
Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,835, 826; 6,858,715; 6,867,289; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; 7,834,171; 7,919,612; 7,960,360; 7,989,603; 8,309,707; 6,524,681; and U.S. Pat. RE39464, each of which is herein incorporated by reference
Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts. Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.
In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH 2 —NH—CH 2 —, —CH 2 —N(CH 3 )—O—CH 2 — [known as a methylene (methylimino) or MMI backbone], —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 — and —N(CH 3 )—CH 2 —CH 2 — [wherein the native phosphodiester backbone is represented as —O—P—O—CH 2 —] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH 2 ) n O] m CH 3 , O(CH 2 ). n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH 2 —O—CH 2 —N(CH 2 ) 2 , also described in examples herein below.
Other modifications include 2′-methoxy (2′-OCH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,700,920; 8,084,600; 8,124,745; 8,377,644 each of which is herein incorporated by reference.
An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is herein incorporated by reference in its entirety.
Another modification of the RNA of an iRNA featured in the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
In some embodiments the CTC marker gene-targeted therapy can comprise an agent that binds to the CTC marker gene expression product and an agent that is chemotherapeutic. In some embodiments, the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent and a chemotherapeutic agent. A CTC marker gene-binding antibody reagent can be an antibody reagent that binds, e.g. a CTC marker gene polypeptide. The binding antibody reagent can be an inhibitor or can exhibit no inhibitory effect on its own. By binding to the CTC marker gene, and thereby a CTC, it concentrates and localizes the chemotherapeutic agent at CTC cells in the circulation and/or stroma of the tumor—increasing efficacy and reducing side effects.
In some embodiments, the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent that binds a marker gene selected from Table 14. In some embodiments, the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent that binds a marker gene selected from the group consisting of: IL6ST, SULF2, and SV2A.
As used herein the term “chemotherapeutic agent” refers to any chemical or biological agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms and cancer as well as diseases characterized by hyperplastic growth. These agents can function to inhibit a cellular activity upon which the cancer cell depends for continued proliferation. In some aspect of all the embodiments, a chemotherapeutic agent is a cell cycle inhibitor or a cell division inhibitor. Categories of chemotherapeutic agents that are useful in the methods of the invention include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most of these agents are directly or indirectly toxic to cancer cells. In one embodiment, a chemotherapeutic agent is a radioactive molecule. One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed. 2000 Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). In some embodiments, the chemotherapeutic agent can be a cytotoxic chemotherapeutic. The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. At211, I1131, I1125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
Non-limiting examples of chemotherapeutic agents can include gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE.™vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb™); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
In some embodiments, the binding antibody reagent and the chemotherapeutic agent can be directly conjugated and/or bound to each other, e.g. an antibody-drug conjugate. In some embodiments, binding can be non-covalent, e.g., by hydrogen, electrostatic, or van der waals interactions, however, binding may also be covalent. By “conjugated” is meant the covalent linkage of at least two molecules. In some embodiments, the composition can be an antibody-drug conjugate.
In some embodiments, the binding antibody reagent can be bound to and/or conjugated to multiple chemotherapeutic molecules. In some embodiments, the ratio of a given chemotherapeutic molecule to the binding antibody reagent molecule can be from about 1:1 to about 1,000:1, e.g. a single antibody binding reagent molecule can be linked to, conjugated to, etc. from about 1 to about 1,000 individual chemotherapeutic molecules.
In some embodiments, the binding antibody reagent and the chemotherapeutic agent can be present in a scaffold material. Scaffold materials suitable for use in therapeutic compositions are known in the art and can include, but are not limited to, a nanoparticle; a matrix; a hydrogel; and a biomaterial, biocompatible, and/or biodegradable scaffold material. As used herein, the term “nanoparticle” refers to particles that are on the order of about 10 −9 or one billionth of a meter. The term “nanoparticle” includes nanospheres; nanorods; nanoshells; and nanoprisms; and these nanoparticles may be part of a nanonetwork.
The term “nanoparticles” also encompasses liposomes and lipid particles having the size of a nanoparticle. As used herein, the term “matrix” refers to a 3-dimensional structure comprising the components of a composition described herein (e.g. a binding reagent, kinase inhibitor, and/or EGFR inhibitor). Non-limiting examples of matrix structures include foams; hydrogels; electrospun fibers; gels; fiber mats; sponges; 3-dimensional scaffolds; non-woven mats; woven materials; knit materials; fiber bundles; and fibers and other material formats (See, e.g. Rockwood et al. Nature Protocols 2011 6:1612-1631 and US Patent Publications 2011/0167602; 2011/0009960; 2012/0296352; and U.S. Pat. No. 8,172,901; each of which is incorporated by reference herein in its entirety). The structure of the matrix can be selected by one of skill in the art depending upon the intended application of the composition, e.g. electrospun matrices can have greater surface area than foams.
In some embodiments, the scaffold is a hydrogel. As used herein, the term “hydrogel” refers to a three-dimensional polymeric structure that is insoluble in water but which is capable of absorbing and retaining large quantities of water to form a stable, often soft and pliable, structure. In some embodiments, water can penetrate in between the polymer chains of the polymer network, subsequently causing swelling and the formation of a hydrogel. In general, hydrogels are superabsorbent. Hydrogels have many desirable properties for biomedical applications. For example, they can be made nontoxic and compatible with tissue, and they are highly permeable to water, ions, and small molecules. Hydrogels are super-absorbent (they can contain over 99% water) and can be comprised of natural (e.g., silk) or synthetic polymers, e.g., PEG.
As used herein, “biomaterial” refers to a material that is biocompatible and biodegradable. As used herein, the term “biocompatible” refers to substances that are not toxic to cells. In some embodiments, a substance is considered to be “biocompatible” if its addition to cells in vitro results in less than or equal to approximately 20% cell death. In some embodiments, a substance is considered to be “biocompatible” if its addition to cells in vivo does not induce inflammation and/or other adverse effects in vivo. As used herein, the term “biodegradable” refers to substances that are degraded under physiological conditions. In some embodiments, a biodegradable substance is a substance that is broken down by cellular machinery. In some embodiments, a biodegradable substance is a substance that is broken down by chemical processes.
In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having cancer with a CTC marker-gene targeted therapy. In some embodiments, the cancer can be pancreatic cancer. Subjects having cancer can be identified by a physician using current methods of diagnosing cancer. Symptoms and/or complications of cancer, e.g. pancreatic cancer, which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, pain in the upper abdomen, heartburn, nausea, vomiting, diarrhea, cachexia, jaundice, pulmonary embolism, Trousseau syndrome, and diabetes mellitus. Tests that may aid in a diagnosis of, e.g. pancreatic cancer include, but are not limited to, liver function tests, CA19-9 tests, CT and endoscopic ultrasound. A family history of pancreatic cancer or exposure to risk factors for pancreatic cancer (e.g. smoking or drinking) can also aid in determining if a subject is likely to have cancer or in making a diagnosis of cancer.
The compositions and methods described herein can be administered to a subject having or diagnosed as having cancer, e.g. pancreatic cancer. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. a CTC marker-gene targeted therapy to a subject in order to alleviate a symptom of a cancer. As used herein, “alleviating a symptom of a cancer” is ameliorating any condition or symptom associated with the cancer. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.
The term “effective amount” as used herein refers to the amount of a CTC marker-gene targeted therapy needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term “therapeutically effective amount” therefore refers to an amount of CTC marker-gene targeted therapy that is sufficient to provide a particular anti-cancer effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of a CTC marker-gene targeted therapy, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for CTC levels, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
In some embodiments, the technology described herein relates to a pharmaceutical composition comprising a CTC marker-gene targeted therapy as described herein, and optionally a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C 2 -C 12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g. a CTC marker-gene targeted therapy as described herein.
In some embodiments, the pharmaceutical composition comprising a CTC marker-gene targeted therapy as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping.
Suitable vehicles that can be used to provide parenteral dosage forms of a CTC marker-gene targeted therapy as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a CTC marker-gene targeted therapy as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.
Pharmaceutical compositions comprising a CTC marker-gene targeted therapy can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia Pa. (2005).
Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the CTC marker-gene targeted therapy can be administered in a sustained release formulation.
Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).
Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.
The methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. Non-limiting examples of a second agent and/or treatment can include radiation therapy, surgery, and chemotherapeutic agents as described above herein.
In certain embodiments, an effective dose of a composition comprising a CTC marker gene-targeted therapy as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising a CTC marker gene-targeted therapy can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a composition comprising a CTC marker gene-targeted therapy, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.
In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. CTC levels by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more.
The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the CTC marker gene-targeted therapy. The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months. Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition comprising a CTC marker gene-targeted therapy can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
The dosage ranges for the administration of a CTC marker gene-targeted therapy, according to the methods described herein depend upon, for example, the form of the CTC marker gene-targeted therapy, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for CTC levels. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.
The efficacy of a CTC marker gene-targeted therapy in, e.g. the treatment of a condition described herein, or to induce a response as described herein (e.g. reduction of CTC levels) can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. tumor size and/or growth. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. CTC levels). It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of cancer, e.g. pancreatic cancer. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. a change in CTC levels.
For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.
For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.
The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.
As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.
Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cancer. A subject can be male or female.
A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for cancer or the one or more complications related to cancer. Alternatively, a subject can also be one who has not been previously diagnosed as having cancer or one or more complications related to cancer. For example, a subject can be one who exhibits one or more risk factors for cancer or one or more complications related to cancer or a subject who does not exhibit risk factors.
A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
As used herein, the term “cancer” or “tumor” refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems. A subject who has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
The term “agent” refers generally to any entity which is normally not present or not present at the levels being administered to a cell, tissue or subject. An agent can be selected from a group including but not limited to: polynucleotides; polypeptides; small molecules; and antibodies or antigen-binding fragments thereof. A polynucleotide can be RNA or DNA, and can be single or double stranded, and can be selected from a group including, for example, nucleic acids and nucleic acid analogues that encode a polypeptide. A polypeptide can be, but is not limited to, a naturally-occurring polypeptide, a mutated polypeptide or a fragment thereof that retains the function of interest. Further examples of agents include, but are not limited to a nucleic acid aptamer, peptide-nucleic acid (PNA), locked nucleic acid (LNA), small organic or inorganic molecules; saccharide; oligosaccharides; polysaccharides; biological macromolecules, peptidomimetics; nucleic acid analogs and derivatives; extracts made from biological materials such as bacteria, plants, fungi, or mammalian cells or tissues and naturally occurring or synthetic compositions. An agent can be applied to the media, where it contacts the cell and induces its effects. Alternatively, an agent can be intracellular as a result of introduction of a nucleic acid sequence encoding the agent into the cell and its transcription resulting in the production of the nucleic acid and/or protein environmental stimuli within the cell. In some embodiments, the agent is any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities. In certain embodiments the agent is a small molecule having a chemical moiety selected, for example, from unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds. As used herein, the term “small molecule” can refer to compounds that are “natural product-like,” however, the term “small molecule” is not limited to “natural product-like” compounds. Rather, a small molecule is typically characterized in that it contains several carbon-carbon bonds, and has a molecular weight more than about 50, but less than about 5000 Daltons (5 kD). Preferably the small molecule has a molecular weight of less than 3 kD, still more preferably less than 2 kD, and most preferably less than 1 kD. In some cases it is preferred that a small molecule have a molecular mass equal to or less than 700 Daltons.
Aptamers are short synthetic single-stranded oligonucleotides that specifically bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells and tissues. These small nucleic acid molecules can form secondary and tertiary structures capable of specifically binding proteins or other cellular targets, and are essentially a chemical equivalent of antibodies. Aptamers are highly specific, relatively small in size, and non-immunogenic. Aptamers are generally selected from a biopanning method known as SELEX (Systematic Evolution of Ligands by Exponential enrichment) (Ellington et al. Nature. 1990; 346(6287):818-822; Tuerk et al., Science. 1990; 249(4968):505-510; Ni et al., Curr Med Chem. 2011; 18(27):4206-14; which are incorporated by reference herein in their entireties). Methods of generating an apatmer for any given target are well known in the art. Preclinical studies using, e.g. aptamer-siRNA chimeras and aptamer targeted nanoparticle therapeutics have been very successful in mouse models of cancer and HIV (Ni et al., Curr Med Chem. 2011; 18(27):4206-14).
As used herein, the terms “protein” and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.
As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. cancer. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a cancer. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
As used herein, the term “administering,” refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.
As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.
The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.
The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”
Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.
Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.
Other terms are defined herein within the description of the various aspects of the invention.
All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.
Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
1. A method of detecting circulating tumor cells (CTCs) in a sample, the method comprising:
measuring the level of a PC-CTC marker gene expression product in the sample; and determining that PC-CTCs are present if the detected level of the marker gene expression product is greater than a reference level.
2. The method of paragraph 1, wherein the CTCs are pancreatic cancer CTCs. 3. The method of any of paragraphs 1-2, wherein the method further comprises a first step of isolating the CTCs from the sample. 4. The method of any of paragraphs 1-3, wherein the expression product is a nucleic acid. 5. The method of paragraph 4, wherein the level of the expression product is determined using a method selected from the group consisting of:
RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization.
6. The method of any of paragraphs 1-3, wherein the expression product is a polypeptide. 7. The method of paragraph 6, wherein the level of the expression product is determined using a method selected from the group consisting of:
Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay.
8. The method of any of paragraphs 1-7, wherein the CTC marker gene is selected from Table 7; Table 8; or Table 14. 9. The method of any of paragraphs 1-8, wherein the CTC marker gene is selected from the group consisting of:
ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4.
10. The method of any of paragraphs 1-8, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2.
11. The method of any of paragraphs 1-9, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
12. The method of any of paragraphs 1-9, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A2; IGFBP5; KLF4; and DCN.
13. The method of any of paragraphs 1-9, wherein the CTC marker gene is selected from the group consisting of:
TPT1; HMGB1; SPON 2; SPARC; and ARSA.
14. The method of any of paragraphs 1-9, wherein the CTC marker gene is selected from the group consisting of:
IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A.
15. A method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of a CTC marker gene-targeted therapy to the subject. 16. The method of paragraph 15, wherein the cancer is pancreatic cancer. 17. The method of any of paragraphs 15-16, wherein the CTC marker gene-targeted therapy comprises an inhibitor of a CTC marker gene. 18. The method of paragraph 17, wherein the inhibitor is an antibody reagent. 19. The method of paragraph 17, wherein the inhibitor is an inhibitory nucleic acid reagent. 20. The method of any of paragraphs 15-19, wherein the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent and a chemotherapeutic agent. 21. The method of any of paragraphs 15-20, wherein the subject is a subject determined to have an elevated level of CTCs and/or an elevated level of a CTC marker gene present in the blood and/or stroma of the cancer. 22. The method of any of paragraphs 15-21, wherein the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent that binds a marker gene selected from the group consisting of:
IL6ST, SULF2, and SV2A.
23. A method of determining if a subject is likely to respond to treatment with a CTC marker gene-targeted therapy, the method comprising:
measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is likely to respond to the treatment if the level of the expression product is increased relative to a reference level.
24. The method of paragraph 23, wherein the method further comprises a first step of isolating the CTCs from the sample. 25. The method of any of paragraphs 23-24, wherein the cancer is pancreatic cancer. 26. The method of any of paragraphs 23-25, wherein the expression product is a nucleic acid. 27. The method of paragraph 26, wherein the level of the expression product is determined using a method selected from the group consisting of:
RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization.
28. The method of any of paragraphs 23-26, wherein the expression product is a polypeptide. 29. The method of paragraph 28, wherein the level of the expression product is determined using a method selected from the group consisting of:
Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay.
30. The method of any of paragraphs 23-29, wherein the PC-CTC marker gene is selected from Table 7; Table 8; or Table 14. 31. The method of any of paragraphs 23-30, wherein the CTC marker gene is selected from the group consisting of:
ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4.
32. The method of any of paragraphs 23-31, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2.
33. The method of any of paragraphs 23-31, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
34. The method of any of paragraphs 23-31, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A2; IGFBP5; KLF4; and DCN.
35. The method of any of paragraphs 23-31, wherein the CTC marker gene is selected from the group consisting of:
TPT1; HMGB1; SPON 2; SPARC; and ARSA.
36. The method of any of paragraphs 23-31, wherein the CTC marker gene is selected from the group consisting of:
IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A.
37. A method of monitoring the treatment of a subject, the method comprising:
administering a cancer therapy to a subject in need thereof; measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is responding if the level of the CTC marker gene expression product is decreased relative to the reference level and determining that the subject is not responding to the treatment if the CTC marker gene expression product is not decreased relative to the reference level.
38. The method of paragraph 37, wherein the cancer is pancreatic cancer. 39. The method of any of paragraphs 37-38, wherein the reference level is the level of the gene expression product in the patient prior to the administering step. 40. The method of any of paragraphs 37-39, wherein the method further comprises a first step of isolating the CTCs from the sample. 41. The method of any of paragraphs 37-40, wherein the expression product is a nucleic acid. 42. The method of paragraph 41, wherein the level of the expression product is determined using a method selected from the group consisting of:
RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization.
43. The method of any of paragraphs 37-40, wherein the expression product is a polypeptide. 44. The method of paragraph 43, wherein the level of the expression product is determined using a method selected from the group consisting of:
Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay.
45. The method of any of paragraphs 37-44, wherein the PC-CTC marker gene is selected from Table 7; Table 8; or Table 14. 46. The method of any of paragraphs 37-45, wherein the CTC marker gene is selected from the group consisting of:
ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4.
47. The method of any of paragraphs 37-46, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2.
48. The method of any of paragraphs 37-46, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
49. The method of any of paragraphs 37-46, wherein the CTC marker gene is selected from the group consisting of:
ALDH1A2; IGFBP5; KLF4; and DCN.
50. The method of any of paragraphs 37-46, wherein the CTC marker gene is selected from the group consisting of:
TPT1; HMGB1; SPON 2; SPARC; and ARSA.
51. The method of any of paragraphs 37-46, wherein the CTC marker gene is selected from the group consisting of:
IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A.
EXAMPLES
Example 1
Single Cell RNA-Sequencing of Mouse Pancreatic Circulating Tumor Cells Reveals their Expression of ECM Proteins
Circulating Tumor Cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. Using a pancreatic cancer mouse model, a microfluidic device was applied to isolate CTCs independently of tumor epitopes, subjecting these to single cell RNA-sequencing. CTCs clustered into multiple subsets, distinct from primary tumors and cancer cell lines. While proliferative signatures were generally low, CTCs were enriched for MAPK, as well as WNT, TGF-β, Neurotrophin, Toll-like receptor, and B-cell receptor signaling pathways. CTCs were highly enriched for expression of the stem-cell associated gene Aldh1a2. Their virtually universal expression of Igfbp5 and Klf4 was correlated with a subset of primary tumor cells localized to the epithelial/stromal boundary, consistent with the presence of both epithelial and mesenchymal markers in CTCs. The very high CTC expression of stromal-derived extracellular matrix proteins, including Dcn and Sparc, indicates microenvironmental contributions to metastasis and identifies unexpected therapeutic targets.
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the US, with a 6% overall survival at 5 years (Society, 2013). The high mortality of this cancer stems from the rapid dissemination of tumor cells leading to widespread metastasis. While local tissue and lymphatic invasion are evident even in early PDAC, the presence of circulating tumor cells (CTCs) in the bloodstream ultimately leads to spread of cancer to distant organs. CTCs are rare, estimated at one to ten tumor cells among ten billion normal blood cells in a milliliter of blood. As such, their isolation and molecular analysis has posed a significant technological challenge (Pantel et al., 2008; Yu et al., 2011). Given their role in blood-borne metastasis, CTC populations are likely to be enriched for metastatic precursors, and their analysis may identify potential therapeutic targets, as well as providing opportunities for early detection of pancreatic cancer.
Genetically engineered mouse pancreatic cancer models have provided important insight into the progression of this disease. Specifically, the genetically engineered LSL-Kras G12D , Trp53 flox/flox or + , Pdx1-Cre (KPC) mouse model recapitulates the histological progression from preneoplastic pancreatic intraepithelial neoplasia (PanlN) lesions to invasive carcinoma (Bardeesy et al., 2006). Recent studies have suggested that epithelial-to-mesenchymal transition (EMT) occurs early in this model potentially enhancing tumor invasiveness (Rhim et al., 2012). In an initial molecular characterization of mouse pancreatic CTCs, RNA sequencing of CTC-enriched populations was performed, thereby identifying activation of non-canonical WNT signaling as a recurrent event, potentially contributing to the anoikis resistance of circulating epithelial cells (Yu et al., 2012). In that study, analysis of purified CTC populations was accomplished using single molecule RNA sequencing, combined with digital subtraction of matched leukocyte RNA reads, so as to derive a CTC-enriched expression signature. However, transcriptomic analysis of such partially purified cell populations is limited by depth of coverage to the most highly differentially expressed genes, and such studies of bulk CTC populations cannot resolve the degree of heterogeneity across these poorly understood cell populations
To achieve a deep RNA sequencing profile of CTCs at the single cell level, a novel inertial focusing-enhanced device, the CTC-iChip, which allows high efficiency negative depletion of normal blood cells, leaving unattached CTCs in solution where they can be selected and analyzed as single cells (Ozkumur et al., 2013) was used. By avoiding tumor epitope-specific capture, such as targeting the epithelial marker EpCAM, the CTC-iChip is unbiased in isolating cancer cells with both epithelial and mesenchymal characteristics. Further, the high quality of RNA purified from viable, untagged CTCs is particularly well suited for detailed transcriptomic analysis. Finally, the use of a mouse model of pancreatic cancer allows for simultaneous analysis of primary tumor and CTCs, while the shared driver mutations across different animals facilitates the identification of CTC-specific heterogeneity. Described herein is a comprehensive transcriptome analysis of CTCs at the single cell level, pointing to distinct cell subsets within CTC populations, signaling pathways that are enriched in CTCs, and identifying unique CTC markers and therapeutic targets.
Results
Isolation of Mouse Pancreatic CTCs.
The CTC-iChip, an integrated microfluidic cell separation platform applied directly to whole blood specimens for isolation of CTCs (Ozkumur et al., 2013) was used in the experiments described herein. It combines initial hydrodynamic size-based separation of all nucleated cells (leukocytes (WBC) and CTCs) away from red blood cells, platelets and plasma, with subsequent inertial focusing of the nucleated cells within a single streamline to achieve high efficiency in-line magnetic sorting. While tumor epitopes are highly variable, WBC cell surface markers are well established; applying magnetic-conjugated anti-WBC antibodies to this very high throughput microfluidic cell separation device can thus exclude the vast majority of WBCs to reveal a small number of untagged CTCs ( FIG. 1A ). The CTC-iChip was adapted for depletion of murine hematopoietic cells and applied to the KPC pancreatic cancer mouse model. This PDAC model generates significant numbers of CTCs (Rhim et al., 2012; Yu et al., 2012). Whole blood labeling using 100 anti-CD45 beads per WBC achieved >10 3 depletion in normal mice, mice bearing orthotopic tumors, and the genetically engineered KPC mice ( FIGS. 1B and 4A-4C ).
CTC recovery was measured as a mean of 95% (+/−3% std), using GFP-tagged NB508 mouse pancreatic cancer cells spiked into whole mouse blood and processed through the CTC-iChip ( FIGS. 4A-4C ). NB508 cells were previously generated from a pancreatic tumor arising in the same Kras/Trp53-driven KPC mouse model (Bardeesy et al., 2006). In comparison, only 35% recovery of the same cells was achieved using an alternative microfluidic platform based on anti-EpCAM capture of mouse CTCs (Yu et al., 2012). Applying the CTC-iChip to orthotopic tumors derived from pancreatic inoculation of GFP-tagged NB508 cells generated >1000 CTCs/mL in all three mice tested ( FIGS. 4A-4C ). Finally, testing the CTC-iChip with the genetically engineered KPC model, followed by dual immunofluorescence staining of isolated cells for the epithelial marker pan-cytokeratin (CK) versus the leukocyte marker CD45, revealed a median 118 CTCs/mL (mean 429 CTCs/mL; range 0-1694) ( FIG. 1C ). No CK positive cells were isolated from 7 healthy control mice. The vast majority of CD45 positive cells that failed to be deflected in the microfluidic device retained some immunomagnetic beads on their surface. Thus, CTCs were readily distinguished from WBCs in the CTC-iChip product, enabling single cell manipulation without requiring staining for epithelial-specific cell surface epitopes, such as EpCAM.
Single CTC RNA-Sequencing.
Five tumor-bearing KPC mice generated a total of 168 single CTCs that were subjected to a modified initial cDNA amplification and library protocol (Tang et al., 2010), and screened for RNA quality (Gapdh, Actb), presence of pancreatic markers (Krt8, Krt18, Krt19, Pdx1), and absence of WBC markers (Cd45/Ptprc) ( FIGS. 5A-5C ). Of these, 75 (45%) were of sufficient quality to proceed to further amplification and library construction for next generation sequencing. It is noteworthy that a majority of candidate CTCs (55%) appeared morphologically intact but had degraded RNA. These cells likely represent tumor cells that have lost viability in the bloodstream. Given the rapid processing of blood samples from mouse models, the minimal shear condition in the microfluidic device, and the preserved RNA quality of control cells processed identically, it is unlikely that cells underwent such damage during in vitro purification. For comparison with pancreatic CTCs, single cell RNA-sequencing was also performed on 12 WBCs from a control mouse, 12 mouse embryonic fibroblasts (MEFs), and 16 single cells from the mouse NB508 pancreatic cancer cell line. Over 90% of single cells from NB508 and MEF cultures met criteria for sequencing quality, highlighting the high frequency of CTCs with compromised RNA templates under the same conditions. To compare CTC profiles to that of matched parental tumors harvested at the time of CTC isolation, bulk RNA from each primary tumor was diluted to 1 or 10 cell equivalents (10 or 100 pg RNA) and subjected to the same amplification and RNA-sequencing protocol (n=34; min 8 replicates from 4 matched tumors).
Single cell RNA sequencing performance was comparable for all samples analyzed, with a mean 4.4-8.5 million reads, of which a mean 46-61% were uniquely aligned to the genome ( FIGS. 5A-5C ). Genome aligned reads were annotated and counted using UCSC Known Gene transcriptome reference and normalized in reads per million (RPM). Normalized reads were then analyzed by unsupervised hierarchical clustering (data not shown). Single cell transcriptomes from MEFs, the NB508 pancreatic cancer cell line and normal WBCs were tightly clustered, supporting the analytic reliability of the RNA sequencing strategy. Five distinct clusters of candidate CTCs were identified, all of which were distinct from matched primary tumor sequences, as well as from cancer-derived cell lines. Principal component analysis demonstrates the clustering and inter-relationships of these different groups ( FIG. 2 ).
The uniform genetic drivers of PDAC in the KPC mouse model made it possible to quantify measures of cellular heterogeneity in CTCs derived from individual mice and across different mice. Single cell heterogeneity within each CTC cluster was assessed by calculating the intra-cluster correlation coefficients, where lower correlation coefficients reflect higher heterogeneity ( FIGS. 5A-5C ). As expected, CTC clusters showed considerably more heterogeneity (mean 0.42, 95% CI 0.36-0.47) than single cells derived from the NB508 cancer cell line (mean 0.86, 95% CI 0.80-0.91, p-value 1.2×10 −15 ). To assess heterogeneity of cells within a primary PDAC, a conditional Tomato/EGFP (mT/mG) expression marker (Muzumdar et al., 2007) was crossed with the KPC mouse to generate a lineage-tagged mouse tumor (KPC-mT/mG), which could be used to isolate individual EGFP positive primary tumor cells away from contaminating stromal cells. A primary tumor (TuGMP3) was disaggregated into single cell suspension and 20 EGFP positive cells were subjected to RNA sequencing. The single primary tumor cells clustered well within the previously analyzed bulk tumor material (data not shown), with a heterogeneity score (mean 0.38, 95% CI 0.28-0.47) similar to that of CTCs (p-value 0.49).
In summary, described herein is the single cell RNA-sequencing of mouse pancreatic CTCs isolated without positive selection bias, along with parental tumors, an established genotype-matched cancer cell line, MEFs and WBCs. CTCs clustered separately from the primary tumor (both bulk tumor and isolated single cells) and from the tumor-derived cell line, with comparable degrees of intercellular heterogeneity between CTCs and primary tumor cells.
Defining Subsets of Pancreatic CTCs.
To identify and classify candidate CTCs, gene sets for known epithelial, hematopoietic, and endothelial markers were applied across all clustered samples. As expected, epithelial markers (Krt7, Krt8, Krt18, Krt19, Epcam, Egfr, Cdh1) were highly expressed in primary pancreatic tumors and in the cancer cell line NB508, and nearly absent in the non-epithelial MEFs and in normal WBCs (data not shown). In contrast, hematopoietic markers (Ptprc/Cd45, Csf3r/Cd114, Cd14, Fcgr3/Cd16, Itga2b/Cd41, Itgb3/Cd61) were present in normal WBCs, and absent in NB508 and MEFs. Some expression of hematopoietic markers was detectable in the bulk primary tumor samples, consistent with varying degrees of leukocytic infiltrates. No specific cluster of endothelial cells was identified, based on expression of characteristic markers (Cdh5/Cd144, Vwf Thbd/Cd141, Pecaml/Cd31, Mcam/Cd146, Sele/E-selectin, Cd34) and absence of epithelial and hematopoietic markers.
Interrogation of single cells isolated by CD45-depletion from tumor-bearing mice, using the epithelial, hematopoietic and endothelial markers, revealed five major candidate CTC groupings (Clusters 1, 3, 4, 5 and 9; data not shown). Clusters 3, 4, and 5 were all part of a larger grouping, showing strong expression of epithelial markers, consistent with “classical” CTCs (denoted CTC-c). A subset of these cells expressed Cd34, an endothelial progenitor marker that is also found in mesenchymal cells including MEFs (data not shown) and stromal cells (Krause et al., 1994), but other characteristic endothelial lineage markers were absent. Clusters 1 and 9 were more complex, with the former noteworthy for enrichment of platelet markers CD41 (Itga2b) and CD61 (Itgb3) (hence denoted CTC-plt), and the latter having a prominent cellular proliferation signature (CTC-pro).
To better define the characteristics of each candidate CTC cluster, a non-parametric differential gene expression analysis including a rank product (RP) methodology adapted to variations in absolute transcript levels and differences in transcriptome representation from cell to cell was used (Breitling et al., 2004). Setting very stringent parameters (FDR≦0.01), the control comparison of primary tumors versus WBCs identified 927 genes relatively overexpressed in tumors and 293 genes high in WBCs, including the expected differential expression of epithelial tumor markers keratin 7, 8, 18, and 19, versus the leukocyte specific CD45 (data not shown). Comparing the “classical” CTC-c cluster to WBCs also showed enrichment for cytokeratin 18 and 19 in CTCs versus CD45 in WBCs, validating the RP methodology to identify relevant differentially expressed genes between single cell populations.
The most abundant CTC cluster, CTC-c, comprised 41 of 75 cells (55%) meeting established criteria for epithelial tumor cells (versus CTC-plt: 32%; CTC-pro: 13%). Of note, the only mouse with multiple gross metastases (MP7) had large numbers of CTCs within this class. Compared with matched primary tumors CTC-c cells had 878 transcripts increased in expression and 774 genes with reduced expression (Table 2). Gene Ontology (GO) analysis of CTC-c enriched genes (Table 3) indicated enrichment for signatures associated with cellular interactions with environmental signals (GO:0045785—positive regulation of cell adhesion; GO:0048584—positive regulation of response to stimulus), cell shape and structure (GO: 0030036—actin cytoskeleton organization; GO:0060429—epithelium development), and transcriptional states (GO:0045449—regulation of transcription; GO:0051276—chromosome organization). To evaluate the contribution of signaling pathways activated by external stimuli in CTC-c cells, the enriched genes were annotated using the KEGG database (Table 1). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis similarly showed enrichment for focal adhesion (odds ratio [OR]2.7, q-value 6.7 3 10.4) and regulation of actin cytoskeleton (OR 2.4, q-value 0.005). Notably, of the KEGG signaling pathways annotated, the mitogen-activated protein kinase (MAPK) pathway was most highly enriched Most highly represented was the MAPK pathway (OR 2.2, q-value 0.006); MAPK signaling is already activated in the Kras G12D driven primary tumor. However, while MSigDB Kras dependency signatures were enriched in primary tumors compared with CTCs, the latter had increased expression of Braf, Mras and Rras2, pointing to alternative paths to further activate MAPK in CTCs. This finding is consistent with another study that identified the MAPK pathway as being the most highly enriched in pancreatic CTCs using microarray based methodologies (Sergeant et al., 2012).
CTC enriched genes also had representation of well established signaling pathways involved with metastasis, including TGF-β (Ikushima and Miyazono, 2010; Siegel and Massague, 2003), WNT (Anastas and Moon, 2013; Clevers and Nusse, 2012; Katoh and Katoh, 2007), and VEGF (Carmeliet and Jain, 2011; Folkman, 1995). In this cohort of pancreatic cancer CTCs, Wnt4 and Tgfb2 were most highly enriched in CTCs relative to primary tumor, implicating autocrine signaling involving these major pathways. In addition to these well defined contributors to metastasis, CTC expression analyses also revealed activation of unexpected signaling pathways, including the neurotrophin, toll-like receptor, and B-cell receptor pathways. Neurotrophin pathway activation has been reported in pancreatic cancer, particularly in association with increased perineural invasion (Miknyoczki et al., 1996; Miknyoczki et al., 1999; Ohta et al., 1997; Wang et al., 2009; Zhang et al., 2005). Toll-like receptor and B-cell receptor pathways had less representation among CTC reads, but they suggest aberrant activation of immunomodulatory signaling components. Ultimately, the establishment of CTC-derived cultures will be required to test the functional significance of these activated signaling pathways.
While single cells within the CTC-c cluster fulfilled characteristic criteria for tumor cells, defining the identity of the non-classical CTC clusters, CTC-plt and CTC-pro, required additional analyses. Compared with CTC-c, single cells within the CTC-plt cluster had a high enrichment for wound healing and hemostasis signatures, as well as MSigDB platelet and megakaryocyte expression profiles (Table 4). This indicates that these cells are either circulating megakaryocytes/giant platelets or CTCs covered with adherent platelets. Tumor cell specific lineage tagging supports the identification of CTC-plt cells being of tumor origin. Eighteen EGFP lineage-tagged single CTCs from two KPC-mT/mG mice were subjected to single cell RNA sequencing: a total of 9 CTCs from the two mice (7/7 CTCs from mouse GMP1 and 2/11 from mouse GMP2) were included within CTC-plt, using unsupervised hierarchical clustering (data not shown). Thus, the CTC-plt cluster includes CTCs that exhibit strong platelet markers, most likely derived from transcripts encoded by adherent platelets. Interestingly, CTC-plt cells maintained their distinct segregation from CTC-c even after digital removal of all annotated platelet transcripts (data not shown). It is therefore possible that the adherence of abundant platelets may modulate the intrinsic CTC expression profile, as recently suggested by in vitro modeling experiments (Labelle et al., 2011).
The CTC-pro cluster was most similar to both the NB508 pancreatic cancer cell line and MEFs, and it was enriched for the cellular proliferation marker Mki67 when compared to CTC-c. Multiple lineages are likely to have contributed to this complex grouping: CTCs from KPC mice with tumor-restricted, lineage-tagged EGFP expression clustered with CTC-pro (data not shown), noteworthy for abundant expression of Mki67 and an annotated cell cycle signature in MSigDB (Whitfield et al., 2002) (data not shown). One single cell within the CTC-pro cluster was derived from the pancreatic cancer cell line NB508, while another (MP3-2) had high keratin/high E-cadherin expression characteristic of classical CTCs (data not shown). Nonetheless, another sub-cluster contained immune and dendritic cells, identified by their expression of antigen processing and presentation genes (GO:0019886—antigen processing and presentation of exogenous peptide antigen via MHC class II; Table 5). Taken together, the CTC-pro cluster appears to represent a grouping of highly proliferative cells, of which a subset are tumor-derived.
Together, unbiased isolation and RNA sequencing evaluation of single pancreatic CTCs indicate that over half of these are nonviable with RNA at various stages of degradation. Among the remaining viable CTCs, three major classes are distinguishable by unsupervised clustering: the classical subset (CTC-c) accounts for 55%, with a second platelet adherent group (CTC-plt; 32%) and a third heterogeneous cluster marked by proliferative signatures (CTC-pro; 13%). Given their most clearly defined tumor-derived characteristics, we selected the CTC-c cluster for detailed analysis of metastasis-associated pathways.
Pancreatic CTCs Co-Express Epithelial, Mesenchymal, and Stem Cell Markers.
The relevance of EMT to early metastasis in pancreatic cancer has been supported by lineage tracing studies in the KPC mouse model (Rhim et al., 2012). In human breast cancer CTCs, a distribution of epithelial and mesenchymal markers within individual CTCs was recently reported by the inventors, reflecting both tumor histology and response or resistance to diverse therapies (Yu et al., 2013). To directly test for EMT in the mouse pancreatic CTCs, established epithelial (E) and mesenchymal (M) markers (Kalluri and Weinberg, 2009) were used to evaluate each cell within the CTC-c cluster (data not shown). Compared with the primary tumor, CTC-c cells demonstrated clear loss of the epithelial markers E-cadherin (Cdh1) and Muc1, whereas mesenchymal transcripts were mixed, with some showing increased expression (Cdh11, Vim) and others with reduced levels (S100a4, Itga5, Sdc1) ( FIGS. 3A and 3B ). Notably, even the mesenchymal genes that were upregulated in CTCs showed a high degree of heterogeneous expression across single cells (data not shown). In contrast, loss of epithelial marks, including E-cadherin (Cdh1) was nearly universal across all classical CTCs.
CTCs are also thought to be enriched for metastatic precursors, capable of initiating metastatic tumor deposits. The relationship between such precursor cells and postulated cancer stem cells is uncertain, as is the relevance of established stem cell markers in identifying these cells. Proposed pancreatic cancer stem cell genes (Rasheed and Matsui, 2012; Rasheed et al., 2010) were evaluated in the single cell RNA sequencing reads ( FIG. 3B ). Among all candidate markers tested (Aldh1a1, Aldh1a2, Prom1/Cd133, Cd44, Met, EpCAM), only Aldh1a1 and Aldh1a2 were enriched in CTCs. Classical CTCs expressed predominantly the Aldh1a2 isoform, while CTC-plt cells were enriched for Aldh1a1, but these isoforms were also co-expressed within some single CTCs. MEFs, NB508 pancreatic cancer cells and normal WBCs also expressed Aldh1a1, but not Aldh1a2 (data not shown). Within single CTCs, there was no correlation between expression of Aldh1 isoforms and enrichment for the mesenchymal genes Cdh11 or Vim, suggesting that these two biomarkers are not intrinsically linked.
Given the identification of Aldh1a2 as a potential stem-like marker expressed by CTCs, its expression within matched primary tumors was tested using RNA in situ hybridization (RNA-ISH). Expression patterns within tumors were heterogeneous: Aldh1a2 expressing cells were primarily localized within the “stromal” or non-epithelial (i.e. keratin low) compartment of the tumor (data not shown). The origin of these non-epithelial cells, which are particularly abundant in pancreatic cancer, is likely to be mixed. Both histological evaluation and negative KRAS mutational analysis (Biankin et al., 2012; Ogino et al., 2005) in human pancreatic cancer have indicated that most of these cells represent reactive fibroblasts or stroma, rather than being of tumor origin. However, lineage tracing in KPC mice has recently shown that a small fraction of these supposedly stromal cells are in fact tumor-derived, presumably having undergone EMT to appear fibroblastic (Rhim et al., 2012). Interestingly, the mouse with the most metastases and the highest number of Aldh1a2 positive CTCs, MP7, also had the primary tumor with the highest levels of Aldh1a2. In that case, Aldh1a2-positive cells were present diffusely in the stromal compartment, as well as comprising a small subpopulation of the epithelial (keratin high) component (data not shown). Thus, classical CTCs, which are keratin-high, express the stem cell-associated gene Aldh1a2, whose expression in primary tumors is restricted to the stromal (keratin low) compartment and only a small subpopulation of epithelial cells.
Classical CTCs Share Expression of Stromal Enriched Genes.
Beside the evident diversity of CTCs, shared transcripts were sought that might provide further insight into their cell of origin within the primary tumor, the mechanisms by which they invade and survive within the bloodstream, and ultimately identify potential CTC-specific therapeutic targets. Rigorous criteria were selected to identify the most highly enriched CTC transcripts (RP score<300), expressed at very high levels (>100 RPM) in ≧90% of all classical CTCs. Three genes met these criteria: Decorin (Dcn), a extracellular matrix proteoglycan expressed in tumor stroma across a variety of different cancers (Adany et al., 1990; Bostrom et al., 2013; Henke et al., 2012; Hunzelmann et al., 1995; Iozzo and Cohen, 1994; Mu et al., 2013; Nash et al., 2002); Insulin-like growth factor binding protein 5 (Igfbp5), an extracellular growth factor binding protein expressed in human PDAC reported to have both pro and anti-proliferative properties (Johnson et al., 2006; Johnson and Haun, 2009); and Kruppel-like factor 4 (Klf4), one of the key stem cell (iPS) reprogramming factors (Takahashi and Yamanaka, 2006), which has been implicated in pancreatic cancer development (Brembeck and Rustgi, 2000; Prasad et al., 2005; Wei et al., 2010). By RNA-ISH, Dcn was expressed diffusely in the stromal elements of the tumor ( FIG. 6 ). Remarkably, both Igfbp5 and Klf4 were expressed focally, predominantly within stromal-appearing cells that border the epithelial compartments of the tumor (data not shown). RNA-ISH of EGFP lineage restricted primary tumors confirmed that the Igfbp5 positive cells at the epithelial/stromal interface are of tumor origin (data not shown). In addition to this transitional region, analysis of Klf4 in this EGFP-tagged tumor also found expression in a subset of epithelial ducts (data not shown). Of note, while they are expressed in only a small subset of primary tumor cells, both Igfbp5 and Klf4 are highly co-expressed in 85% of all classical CTCs. Together with the mixed epithelial/mesenchymal markers evident in CTCs, these observations raise the possibility that many CTCs are derived from foci at the epithelial/stromal interface, that may be defined by Igfbp5 and Klf4 expression.
In addition to the three most highly expressed transcripts, CTCs were noteworthy for high level expression of genes implicated in stromal cell matrix. Gene ontology analysis of all CTC-enriched genes (Table 3) identified 60 extracellular proteins (GO:0044421, OR 1.7, q-value 6.4×10 −3 ), of which 32 are found in proteinaceous extracellular matrix (ECM) (GO:0005578, OR 2.4, q-value 4.8×10 −3 ). Recent studies have highlighted the importance of the reactive stroma to pancreatic cancer pathogenesis and metastasis (Feig et al., 2012; Neesse et al., 2013; Neesse et al., 2011; Olive et al., 2009; Provenzano et al., 2012), however, the expression of these stroma-associated ECM genes within tumor cells in circulation was unexpected. To identify the predominant stromal enriched genes in the mouse pancreatic tumor model, we performed RP differential expression analysis between the bulk tumor samples representing tumor cells mixed with reactive stromal cells versus purified EGFP-tagged single cells from the primary tumor (TuGMP3). A total of 51 proteinaceous ECM genes were enriched in bulk tumors versus single primary tumor cells (GO:0005578, OR 4.8, q-value 3.4×10 −18 ). Of these, 6 genes (Ccdc80, Col1a2, Col3a1, Dcn, Sparc, Timp2) were shared with the previously identified CTC-enriched gene set (data not shown). Decorin (Dcn), as noted above, was identified as the most highly enriched (median 10,686 rpm) in CTCs with high level expression (>100 rpm) in 98% of CTCs. The second most abundant gene was Sparc (median 3,913 rpm) with high expression in 88% of CTCs. These two genes were co-expressed at high levels in 88% of classical CTCs. RNA-ISH of primary tumors for both Dcn ( FIG. 6 ) and Sparc (data not shown) confirmed that these genes are expressed throughout the reactive stroma and are not present in the epithelial keratin-rich regions of primary tumors.
The expression of stromal-derived ECM genes is a common feature of all classical CTCs, yet a mouse-specific bias in distribution among these genes was evident, despite their identical Kras/p53 genetic drivers. This mouse-specific clustering was evident in the unsupervised analysis (p-value<2.2×10 −16 ). For instance, sub-cluster 3 was over-represented with single CTCs from mouse MP6, while sub-cluster 4 was enriched for mouse MP7, and sub-cluster 5 for mouse MP2. Of 68 transcripts differentially expressed between the CTCs of mice MP2 and MP7 by RP analysis, gene ontology indicated significant enrichment for 11 extracellular proteins (GO:0044421, OR 3.8, q-value 0.06), 7 of which are found in proteinaceous ECM (GO:0005578, OR 6.3, q-value 0.05) (data not shown). Together, these data indicate that most CTCs derived from a mouse pancreatic cancer model express at high levels a set of ECM genes normally found in the stromal, rather than the epithelial compartment of the primary tumor. This may reflect the origin of many CTCs at the epithelial/stromal interface, consistent with their expression of uniquely restricted markers such as Igfbp5 and Klf4. The fact that individual genetically matched mouse tumors generate CTCs with both shared and unique patterns of ECM gene expression suggests tumor-specific invasion pathways that are superimposed upon fundamental characteristics of CTCs. The high levels of extracellular proteins expressed by CTCs provide unexpected opportunities for targeting these metastatic precursors.
Human Pancreatic CTCs Express the ECM Protein SPARC.
To determine the relevance of ECM protein expression to human disease, CTCs were isolated from the blood of metastatic PDAC patients and subjected to single cell RNA-sequencing. Analysis of 7 pancreatic CTCs from 3 patients revealed that the majority expressed keratins defining their epithelial origins and a total of 13 of 60 extracellular protein genes enriched in mouse CTCs were expressed at high levels (>100 rpm) in at least one human pancreatic CTC ( FIG. 7 ). Human SPARC was the only gene found at high levels in all human pancreatic CTCs. Analysis of human prostate and breast CTCs also show significant expression of extracellular proteins including SPARC highlighting that these targets are commonly shared in metastatic epithelial cancer cells (data not shown). RNA-ISH of Sparc/SPARC in both mouse and human PDAC found expression confined primarily to the stromal compartment of tumors (data not shown). SPARC expression was found in 196/198 (99%) human primary PDAC tumors and 36% of positive tumors had some detectable SPARC in epithelial tumor cells albeit the minority of the overall signal. The presence of SPARC as an extracellular protein permits antibody directed therapies that target SPARC. Together these data indicate that findings in mouse pancreatic CTCs can be found in human disease and offer both novel biomarkers and therapeutic targets.
Discussion
Described herein is a detailed analysis of CTC composition and diversity, using single cell RNA sequencing. In total, high quality transcriptomes were achieved in 93 single mouse pancreatic CTCs, which were compared with 20 single cells from matched primary tumors, as well as bulk tumor preparations, and with 16 cells from an immortalized cell line established from the same mouse pancreatic tumor model. The use of a mouse model, which closely matches human PDAC, made it possible to compare primary tumor specimens isolated simultaneously with the CTCs. Given the shared Kras/Trp53 genetic drivers in the KPC mouse model, it was also possible to examine CTC heterogeneity within individual mice and across different animals. Finally, the use of the CTC-iChip technology enabled the selection of untagged CTCs, irrespective of their cell surface epitopes, thus avoiding any bias associated with tumor marker-specific cell purification. Together, these observations include the following: 1. CTCs cluster into multiple subsets, including a major “classical CTC” group, and others that are marked by platelet-derived markers or proliferative signatures; 2. While individual mouse tumors may produce CTCs that fit into each of these clusters, there are unique patterns to CTCs derived from individual mice, despite their shared genetic drivers; 3. Common markers shared by virtually all classical CTCs include both epithelial and mesenchymal markers, the Aldh1a2 stem cell marker, and two highly expressed transcripts (Igfbp5 and Klf4) that identify foci localized to the epithelial/stromal boundary of primary tumors; and 4. The most highly enriched CTC-specific transcripts shared by almost all classical CTCs encode extracellular matrix proteins associated with the tumor stromal compartment.
Compared with previous RNA sequencing of partially purified, bulk CTC populations, the single cell analysis reported here provides considerably more depth of tumor cell-specific reads. As such, the detailed analysis of classical CTCs from the mouse pancreatic cancer model is unprecedented. It is demonstrated herein that pancreatic cancer CTCs uniformly lose expression of the epithelial marker E-cadherin (Cdh1), a key feature of epithelial-to-mesenchymal transition. However, the cells do not lose expression of other epithelial markers, such as cytokeratins, nor is there a consistent increase in classical EMT mesenchymal markers such as vimentin. As such, most classical CTCs appear arrested in a biphenotypic state. Despite their expression of cytokeratins (present in the epithelial components of the primary tumor), most other highly expressed markers in CTCs were shared with the non-epithelial or “stromal” component of the primary tumor. Among these stromal genes expressed in classical CTCs is Aldh1a2, a putative pancreatic cancer stem cell marker (Rasheed and Matsui, 2012; Rasheed et al., 2010). Whether Aldh1a2 is a functionally significant marker of cellular plasticity in metastatic precursors remains to be determined.
A provocative observation relating to the shared epithelial and mesenchymal state of classical CTCs is their virtually uniform (>85%) high level co-expression of Igfbp5 and Klf4, two genes that are only expressed in a small subpopulation of cells at the epithelial/stromal interface within primary tumors. This raises the intriguing possibility that this critical location within the tumor generates a disproportionate fraction of viable CTCs. Indeed, tumor cells that are actively undergoing EMT are presumably enriched at the epithelial-stromal function, contributing to the mixed lineage of the tumor stroma, with both tumor-derived and non-malignant reactive cell types. The potential roles of both IGF signaling and Klf4 transcriptional regulation in embryonic development and pancreatic malignancy make their unique expression pattern in both tumors and CTCs particularly noteworthy.
Finally, the most unexpected observation from this single CTC RNA sequencing study is the very high level abundance of ECM proteins on the vast majority of classical CTCs. Notably, prior evaluation of matched primary and metastatic breast tumors identified the most prevalent gene expression difference as enrichment for ECM molecules in the metastases, comprising some 18% of differentially expressed genes (Weigelt et al., 2005). While this has been interpreted as reflecting differences in the local environment of the metastatic site, the present data indicate that ECM proteins are highly expressed by CTCs themselves. By analogy with the classical “seed versus soil” debate (Fidler, 2003), CTCs may in fact be seeds carrying some of their own soil.
The ultimate goal of detailed molecular analysis of CTCs is to understand the process by which they are generated and their therapeutic vulnerabilities. In this regard, an important observation derived from the present single CTC RNA sequencing analysis is the unexpected expression of extracellular proteins with a preponderance of proteins found in ECM. Two of the most abundant and commonly shared ECM proteins in CTCs are Dcn and Sparc, both of which are established tumor stromal genes. Notably, Sparc expressing stroma appears to bind albumin-conjugated chemotherapy-containing nanoparticles (nab-paclitaxel) allowing for increased cytotoxicity and efficacy in human PDAC (Neuzillet et al., 2013; Von Hoff et al., 2011; Yardley, 2013). Indeed, considerable effort has been directed to targeting pancreatic cancer stroma as a means of improving delivery of chemotherapeutics and stripping tumor cells of their supportive microenvironment (Neesse et al., 2011; Olive et al., 2009; Provenzano et al., 2012; Rasheed et al., 2012). The finding that these gene products are also expressed by CTCs indicates that antibody-directed therapies can be used not only against primary tumor stroma, but also to target tumor cells as they transit in the blood.
As described herein, the present CTC analyses to extend from matching them to known tumor-defining markers to interrogating them for unique properties that distinguish them from most primary tumor cells and may underlie their ability to survive in the bloodstream and generate distant metastases. Such insights into the cellular process of human cancer metastasis are critical to the goal of ultimately preventing the spread of a primary tumor to distant organs.
Experimental Procedures
Mice and cell lines. Mice with pancreatic cancer used in these experiments express Cre driven by Pdx1, LSL-Kras G12D , and Trp53 lox/+ or Trp53 lox/lox as previously described (Bardeesy et al., 2006). EGFP pancreatic lineage tagged KPC mice were generated by breeding the mT/mG mouse (Jackson Laboratory—Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J) into the breeder pairs used for KPC mouse generation. Normal FVB mice were purchased from Jackson Laboratory. All mice care and procedures were done under MGH SRAC approved protocols.
Adaptation of CTC Enrichment Technology.
Given the desire for an unbiased enrichment system, the previously presented negative depletion technology was selected for this application (Ozkumur et al., 2013). All processing protocols were identical to those previously identified, except a rat anti-mouse CD45 antibody (BAM114, R&D Systems, USA) was conjugated to MyOne beads.
Single Cell Micromanipulation, Amplification, and Sequencing.
After whole blood anti-CD45 negative depletion, the product containing enriched cells was collected in a 35 mm petri dish and viewed using a Nikon Eclipse Ti™ inverted fluorescent microscope. Cells of interest were identified based on intact cellular morphology and lack of labeling with anti-CD45 magnetic beads. These target cells were individually micromanipulated with a 10 μm transfer tip on an Eppendorf TransferMan® NK 2 micromanipulator and ejected into PCR tubes containing RNA protective lysis buffer and immediately flash frozen in liquid nitrogen. Single cells were amplified with a modified protocol (Tang et al., 2010) and sequenced on the ABI 5500XL™ system.
RNA In Situ Hybridization (RNA-ISH).
RNA-ISH was performed according to the Affymetrix QuantiGene ViewRNA ISH Tissue-2 Plex Assay™.
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TABLE 1
Annotation of CTC enriched genes in KEGG defined signaling pathways.
*indicates gene found in multiple pathway gene sets.
TGF-
Toll-Like
Neurotropin
beta
Receptor
VEGF
MAPK Pathway
WNT Pathway
Pathway
Pathway
Pathway
Pathway
1500003o03rik*
Jund
1500003o03rik*
Akt2*
Amhr2
Akt2*
1500003o03rik*
Akt2*
Map3k3*
Crebbp*
Braf*
Crebbp*
Fos*
Akt2*
B230120h23rik
Mapk1*
Csnk1a1
Calm1
Dcn
Ifnar2
Hspb1*
Braf*
Mapkapk3*
Jun*
Calm2
Id1
Irak4*
Kdr
Dusp1
Mef2c
Nkd1
Irak4*
Id2
Irf7
Mapk1*
Dusp14
Mras
Ppp3ca*
Irs2
Mapk1*
Jun*
Mapkapk3*
Dusp3
Nf1
Rock1*
Jun*
Rock1*
Mapk1*
Pla2g4a*
Fas
Nfkb2
Rock2*
Maged1
Rock2*
Nfkbia*
Ppp3ca*
Fgf1
Nr4a1
Siah1a
Map3k3*
Smad4*
Tirap
Src
Flnc
Pla2g4a*
Smad4*
Mapk1*
Tgfb2*
Tlr2
Fos*
Ppp3ca*
Tbl1x
Nfkbia*
Tgfbr2*
Gadd45b
Rras2
Tcf7l1
Shc1
Thbs1
Hspa2
Tgfb2*
Wnt4
Ywhaz
Hspb1*
Tgfbr2*
Jun*
TABLE 2
Significantly Expressed Genes by Rank Product (FDR < 0.01)
CTC-c vs Primary
Primary Tumor vs
CTC-plt vs
CTC-pro vs
Count
Tumor Enriched Gene
CTC-c Enriched Gene
CTC-c
CTC-c
1
Upk3b
Tff2
Clec1b
kg:uc007pge.1
2
Ier2
Wfdc2
AU023871
kg:uc007pgd.1
3
Egr1
Lamb3
Alox12
kg:uc007pgf.1
4
Nkain4
Lad1
Itga2b
kg:uc007pgg.1
5
Igfbp5
Dmbt1
Ppbp
Igj
6
Slc6a4
Npy
Gng11
kg:uc012enb.1
7
Klf4
Pmepa1
Vwf
2010001M09Rik
8
Tmem221
Kcnn4
Pf4
kg:uc009cfw.1
9
Arl4d
Serinc2
Fcer1g
kg:uc007pgi.1
10
Lrrn4
5730559C18Rik
Tmem40
kg:uc007pgh.1
11
Cldn15
Muc1
Hba-a2
kg:uc007yos.1
12
Gpm6a
Chi3l3
Stom
Coro1a
13
Atf3
Pglyrp1
Beta-s
Pou2af1
14
Ptma
Arl4c
Plek
kg:uc011yvj.1
15
Slc9a3r1
Spp1
Srgn
Glipr1
16
Fos
Col15a1
Myl9
Cd52
17
Tmem119
C1qb
Cd84
Cd79b
18
Ptgis
Tnnt2
F5
Sec11c
19
Dcn
Gkn3
Treml1
Tnfrsf17
20
Gbp2
Onecut2
Hbb-b1
Krr1
21
Dmkn
Mmp7
Itgb3
Gmfg
22
Sdc4
Cd74
Gp9
Ccr9
23
Ildi2
Ctss
Mpl
Pycard
24
Akap2
Lamc2
Ctla2a
Derl3
25
Gfpt2
Olfml3
Tubb1
Rac2
26
Klf6
Lgals4
Mylk
Srgn
27
Btg2
Lcn2
F13a1
Cytip
28
Myl7
Ly6a
Slamf1
Edem2
29
Igfbp6
Pak1
Rgs10
Itgb7
30
Gpr133
Capn5
Mkrn1
Lsp1
31
Oasl2
Ptprn
Laptm5
Lcp1
32
Pfn1
Reg3b
1810058I24Rik
Cyfip2
33
Cap1
Fmnl3
Itgb2
Nans
34
Nfkbia
Sdc1
Slc2a3
Slamf7
35
Malat1
Prom1
Pcmt1
Ell2
36
Rarres2
Ankrd50
Gp5
H2-Eb1
37
Rspo1
Ccl6
Ube2o
Creld2
38
Espn
Slc4a11
5430417L22Rik
Cd74
39
Klf9
Oraov1
Ptpn18
Blnk
40
Zbtb7c
Aldh1l1
Lat
Fmnl1
41
Brd2
Slc20a1
Fermt3
Snrnp70
42
Olfr1033
Cldn7
Nrgn
Sec61b
43
Wt1
Acsbg1
Mrvi1
Edem1
44
Esam
Las1l
Lyz2
Tspan13
45
kg:uc009igb.1
C1qc
Epb4.1
Psmb8
46
Tmem151a
Lama5
Rasgrp2
Pim1
47
Mgll
Mgat4a
Treml2
Sept1
48
Csrnp1
Cldn2
Hist1h4i
Cd48
49
Cd9
Mcpt2
March2
Sub1
50
Gjb5
Fxyd3
Ltbp1
Lims1
51
Lrrc61
Il4ra
Nptn
Ncoa2
52
Wasf2
Itga5
Abtb1
Ctnnbl1
53
Pdpn
Porcn
Ctla2b
Fdps
54
kg:uc009ogv.1
Mast3
Prkab2
Ube2j1
55
Sdpr
Scara3
Arhgdib
Mettl1
56
Gpr64
Atox1
Alas2
Lax1
57
Flnc
Arrdc1
Odc1
Rilpl2
58
Add3
Mmp2
Ptpn11
Ctse
59
Gata6
Saa3
Dhcr24
Glrx
60
Wfdc1
Serpinf1
Mfsd2b
Fut8
61
A130040M12Rik
Sox11
Gp1bb
AI662270
62
Ankrd12
Prpsap1
Rbpms2
Gramd3
63
Adamtsl1
Mcpt1
Fyb
Il2rg
64
C2
Mfge8
Smox
Rasgrp3
65
Prss23
Col18a1
P2rx1
Impdh1
66
Ube2v1
Lyz2
Otud7b
Plek
67
Cryab
C1qa
kg:uc007ttx.1
Ints5
68
Pkhd1l1
Acp5
Samd14
Blmh
69
Rtn1
Angptl4
Clca1
Dnmt1
70
Birc6
Ccnd1
kg:uc007tty.1
Galk1
71
Xdh
Asl
Gpr56
kg:uc007hxv.1
72
Cd34
Ctxn1
Sh3bgrl2
Ccdc88b
73
Rab6b
Pgs1
Pttg1ip
Selplg
74
Dusp1
Anapc2
Nomo1
Sar1b
75
Clic4
Cp
Gnaz
Lat2
76
C3
Gpx3
Mmrn1
Slc16a6
77
Rhob
Lama3
Gp1ba
Mki67
78
Mir3064
Rbp1
Sh3bgrl3
Dnajc3
79
Thbd
Cotl1
Slc24a3
H2-Ab1
80
Dpysl2
Nek6
Sord
Ndufs6
81
Cob1
Cpxm1
Nfe2
Actr3
82
Npr1
Sfrp1
Tuba4a
Etnk1
83
Dnajb9
Ttr
Zyx
Herpud1
84
Arhgap29
Gsto1
Cnn2
Ptpn7
85
Cav1
Npepl1
Itgb5
Ctss
86
Gbp7
Usmg5
Gata1
Cs
87
Hes1
Polr2l
Hist1h1c
Fbxw7
88
Gm16897
Sphk1
Tbxas1
Ppp2r5c
89
Ppp1r12a
Asxl1
Ptplad2
Znrd1
90
Sv2a
Ctsh
Bpgm
Rfc2
91
Ang
Egfl7
Pdlim7
Preb
92
Aldh1a2
C1qtnf6
Mmd
Fcer1g
93
Cryl1
Rras
G6b
Dnajb11
94
Kank1
Lgi4
kg:uc009duo.1
Slc35b1
95
2210403K04Rik
Hmga2
Lyz1
Sin3b
96
kg:uc009okn.1
Cep250
Tacc1
Nktr
97
Osr1
B4galt3
Dap
98
kg:uc008ewj.2
Tmem223
Mast2
99
kg:uc009tuw.1
Ltbp2
Atp2a3
100
Gadd45b
Tnfrsf23
Snca
101
Ablim3
Col7a1
Stx11
102
Clec3b
Ggct
C030046I01Rik
103
Usp25
Rab25
Trpt1
104
Sntb2
Nedd8
Tsc22d1
105
Rock2
9430023L20Rik
Prkar2b
106
Col14a1
Arl2
Cd9
107
Cd200
Wbp1
Pgm2l1
108
kg:uc008ehr.1
H2-Ab1
Gp6
109
Atp2b1
Preb
Pde5a
110
Exoc4
Sgsm3
Itga6
111
Abcb1b
Sfn
Itga1
112
Nrgn
Prrx2
Edem1
113
kg:uc009cvm.1
Ptprk
Isg20
114
Ncoa4
Reg1
Cdc42ep5
115
Ndufa4
Sdcbp2
Nipal3
116
Upk1b
Pcbd1
Ccdc92
117
Jun
Slc25a1
Sort1
118
Syne2
Vamp5
Ly6g6c
119
kg:uc007bvx.1
Crlf1
Ubash3b
120
Ap4e1
Avil
Inf2
121
Spock2
2700094K13Rik
Asap1
122
Efemp1
Ctse
Sec11c
123
Prpf40a
Penk
Gas2l1
124
Tspan5
Tmc4
Parvb
125
Lgals7
Dhrs3
Tmsb4x
126
Kif5b
Ap1s1
kg:uc007xrw.1
127
Psip1
Arl6ip4
Nudt3
128
kg:uc008oki.1
9430008C03Rik
Bcl2l1
129
1810014B01Rik
Fcer1g
B230312A22Rik
130
Ptges3
Uqcr11
Cnp
131
Limch1
Nhp2
Plp1
132
Bicd1
Plbd2
Cnst
133
Rdx
Capg
Rgs18
134
Pcdh15
Pnpla6
Lsm12
135
Foxn3
Ppdpf
Alox5ap
136
Morf4l2
Hgfac
Ppif
137
Ppp1r15a
Apoe
Spnb1
138
Cdc42ep3
Fam40a
Ormdl3
139
Pard3b
Lyz1
Hpse
140
Bicc1
2200002D01Rik
Srxn1
141
Amhr2
Laptm5
2010002N04Rik
142
Gucy1a3
Qars
Hist1h2bc
143
Psmb2
Tmx2
Cyba
144
Mapkapk3
Fkbp4
Chst12
145
Ube2l6
Plin2
kg:uc009sps.1
146
kg:uc007pff.1
Fcgr3
Max
147
kg:uc007ctp.1
Gkn1
Was
148
Nedd4
Snhg1
Isca1
149
Plxna4
Lsp1
Pdzk1ip1
150
2010107G12Rik
Gm20605
Lyn
151
Ifhgr1
Ly6c1
Mob3a
152
Bcam
Aim1
H2-T24
153
Ccnl1
2310007B03Rik
Slc44a1
154
Hoxa5
Tgfbi
Derl1
155
Fhl1
Tsta3
Gclm
156
1810041L15Rik
Pafah1b3
Fech
157
2900002K06Rik
Chid1
Ywhah
158
Hspb1
Smox
Igtp
159
Podn
1500012F01Rik
Myl6
160
Fam63b
Tspan4
Thbs1
161
Hsp90b1
Agrn
Tln1
162
Dpp4
Cfp
kg:uc009apq.1
163
Gas1
Cdh1
Bcap31
164
kg:uc007zak.1
Rasgrf1
Ilk
165
Zc3h13
Nxf1
Epha1
166
Sox6
Pdrg1
2810453I06Rik
167
Arid4a
Polr2j
Rnf19b
168
Tnxb
Suds3
Gsn
169
Tsix
D0H4S114
Flna
170
Scd1
Ccl9
Arrb1
171
Jund
Neat1
kg:uc007pum.1
172
Crls1
Ccdc12
Mbnl1
173
1110003E01Rik
Prr24
Ccnd3
174
Rnase4
Impdh1
Pdlim1
175
Arhgef12
Card10
Ctse
176
Irf7
Cpsf1
Tspan17
177
Bbx
Sema4g
Gpx4
178
Sema5a
Hes6
Bnip3l
179
Mau2
C130074G19Rik
P2ry12
180
Abi3bp
Ctrb1
kg:uc009vev.1
181
Dag1
Rnaseh2a
Prkab1
182
Cyp2s1
Golm1
F2rl2
183
Sfrs18
Ctsz
Stk4
184
Hspb8
Cyb561
Fhl1
185
Cnot6l
Ndufs8
Rnf10
186
Twsg1
Atp6ap1
Rasa3
187
Gpc3
Srd5a1
Taldo1
188
Lrrn4cl
Carkd
Bysl
189
Cdh3
Cd24a
Esd
190
Cyr61
Eng
Aldh2
191
Cyp2d22
Tcirg1
Rhog
192
Hist1h1c
Slc9a3r2
kg:uc009ecr.1
193
Aplp1
0910001L09Rik
Cald1
194
Tbl1x
Cox5b
Wbp2
195
Pcm1
Adipor2
Ptprj
196
Ifi204
Scarf2
Tpm4
197
Nfix
Myo7a
Mxi1
198
Flrt2
Ppap2c
Ly6g6f
199
Heg1
Pea15a
Sla
200
Il6ra
Sh3pxd2b
Slpi
201
Ralbp1
H19
Bicd2
202
Rhoj
Tpd52
Clu
203
Ktn1
2610203C20Rik
Mtmr14
204
Arl6ip5
Naa10
Abca7
205
Crebbp
Fermt1
Ppp1r18
206
Ppig
Sap30l
Kif2a
207
Akap13
Bgn
Prdx6
208
Rab7
Timm13
kg:uc009ize.1
209
Plxdc2
Krt20
Calm3
210
Aldh1a1
Itga3
Dhrs1
211
Bnc2
Pfkl
Cfl1
212
Slc4a4
Agpat6
Glipr2
213
Tbx18
Mrpl11
Slc25a37
214
Zbtb16
Ramp1
Atox1
215
Arid4b
Hmga1
BC057079
216
Enpp2
Gpx2
Pla2g16
217
Ptplad2
0610012G03Rik
Rnf144b
218
Akr1b3
9130017N09Rik
Stk16
219
Gm6644
Cygb
Rsad2
220
Arf5
Tmprss4
Paip2
221
Chi3l1
Paox
Capzb
222
Gpr116
Endod1
Ppp1r12c
223
Cd82
Cndp2
4930412F15Rik
224
Srrm1
Suv39h1
Ninj1
225
Fmo2
Cog4
2510009E07Rik
226
Tgfb1i1
Trim27
kg:uc007vsr.1
227
Qrich1
Cyhr1
Pygb
228
Nfia
Trmt1
Tlk1
229
Pmp22
Zfyve19
Myct1
230
Cdh11
Esrp1
Rnasek
231
Arid5b
kg:uc008oow.1
Ctsd
232
Rbm3
Dync1h1
0610010K14Rik
233
Prelp
Tab1
Bcas3
234
kg:uc007qse.1
Pla2g6
Atpif1
235
Ddx3x
Timp1
Serf2
236
Sulf1
Eif3f
Becn1
237
Spnb2
Abhd11
Tspan9
238
Tspan31
Pmm2
Acer2
239
Prr13
Tyrobp
Vdac3
240
Ppp1cb
Farsb
kg:uc008kbg.1
241
Fbln1
Plod3
Oaz2
242
Gm6548
Abtb1
Serpine2
243
Uap1
Brf1
Ccdc90a
244
Mpdz
Tnk2
Ndufa1
245
Sat1
Rfc2
Tssc1
246
Stim1
Stxbp2
Mboat7
247
Mll3
Pdlim7
Cd44
248
Slurp1
A430105I19Rik
Cxx1c
249
Cd81
Vill
Ecm1
250
Emp2
Bmp1
Mff
251
Trpm7
Mpzl1
Ptpn12
252
Crym
Thy1
Mgmt
253
Enpp4
Stab1
Cox4i1
254
Raly
Aldh16a1
Tollip
255
Celf2
Eif4ebp3
Cds2
256
Ap3s1
Itpripl2
Ybx1
257
C1s
Mrpl52
Gypc
258
Frmd4b
2310002L13Rik
Dgkd
259
Nr4a1
Mcm6
Pecam1
260
Acini
Kcnk1
Ftl2
261
Plod2
Pmf1
Nt5c3
262
Id1
Cuta
1700037H04Rik
263
Creg1
Nt5dc2
Cd151
264
Zfp318
Rmnd5b
Lpin2
265
Tmem140
Araf
6430548M08Rik
266
Mras
Wwp2
Pon2
267
Vwa5a
Lamb1
Ndufa3
268
Esyt3
Kcne3
6330578E17Rik
269
Hexb
Uqcrq
Mfap31
270
Nckap1
Gps1
Mink1
271
Nipal3
Rexo4
Ston2
272
Ubxn4
Coro1c
Rac2
273
Zfp36
Hras1
Fyn
274
Hnrnpl
Spint1
Serinc3
275
C1ra
Cblc
Maged2
276
Nnmt
Fhod1
Ap2m1
277
Mut
Atp13a1
Pacsin2
278
kg:uc008jup.1
Man2c1
Ftl1
279
Pnrc1
Vsig2
Adipor1
280
Usp8
Bpgm
kg:uc009qdo.1
281
Pgcp
Bap1
Snap23
282
Junb
Smpd2
Tagln2
283
C1rl
Ubqln4
Cox6c
284
Slc6a6
Sirt7
Creg1
285
kg:uc008znh.1
Krt23
Bsg
286
Aqp1
D8Ertd738e
Cmtm6
287
Myh10
Mapk13
Cntd1
288
Slc43a3
kg:uc008bcq.1
Plekho2
289
Spint2
Polr2g
Arrb2
290
Hnrnph1
Ndufs2
Pard3b
291
Arhgap28
Dad1
Mlec
292
Cfh
Wnt7b
Taf10
293
Brd4
Fam20c
Gabarapl2
294
Fndc1
Cxxc5
Bag1
295
Star
Polr2f
Galnt2
296
Nfkbiz
Ltf
Hk1
297
Arsb
2210407C18Rik
Fbxo9
298
Rnd3
Cdipt
kg:uc009izd.1
299
Stard5
Glrx5
Pnpo
300
Thbs1
Gemin7
Fam46c
301
kg:uc008wkn.1
Man1b1
Pkm
302
Slc26a3
Heatr7a
Ap1b1
303
Phip
Arid5a
Rap1b
304
Usp2
Sumo3
Itgb1
305
Golgb1
Srm
St7
306
Rock1
Plscr3
Smap1
307
Rgma
2210010C17Rik
Rabgap11
308
Actg1
Fam102a
Tmbim4
309
BC013529
Dlst
H3f3a
310
kg:uc007zwh.1
Vps37c
Frmd8
311
3110062M04Rik
Ngfrap1
Nlrx1
312
Cast
Pold4
Oaz1
313
Mob3c
Grcc10
Fam125b
314
Slc16a1
Wnt7a
Hexa
315
Fam117a
2010111I01Rik
Tspo
316
Pdia3
Pxdn
Dcaf12
317
Trim8
Coasy
Nav1
318
kg:uc009mng.1
Dctn1
Cd24a
319
eg:245190:chr7:m
Ncor2
Uqcr11
320
Sbsn
Postn
Wipf1
321
Serpinb6b
Col4a2
F10
322
Daglb
Cib1
Erlec1
323
Smarca2
Tbc1d13
Map2k3
324
Mef2c
Ccnl2
Stk24
325
Prrc2c
Dcakd
Ldlrap1
326
BC005537
Cdc34
Ehd4
327
Hsp90ab1
Atp6v0b
Atp6v1f
328
Snrnp70
Abhd12
Gnas
329
Ppl
Flot2
Arhgap18
330
Serpinh1
Sla2
Arhgap10
331
Sorbs3
Rhbdf1
Pitpnm1
332
Golga4
Cdh17
S100a1
333
Acbd3
Psmb5
Bin1
334
Hook3
Serf1
Ttyh3
335
Map3k3
Slc15a3
Selp
336
Rhou
Sftpd
Trappc9
337
Smc2
Pop5
Aes
338
C1d
Nudc
Taok3
339
kg:uc008dzh.1
Sh2d5
Zfand3
340
Psmd7
kg:uc007fwp.1
Stim1
341
Dab2
Mrpl37
Rnf11
342
Cep164
Rin1
Sep15
343
Crim1
Podxl
kg:uc012hdk.1
344
Rtf1
Paqr5
Lgals9
345
Fxyd1
Sepx1
Cox6b1
346
H2-D1
Agr2
Riok3
347
Zfp704
Bax
Slc38a10
348
Mtap1a
Rxrb
Rtn3
349
Ascc3
Tes
B3gat2
350
Med13l
Hdac6
Ccndbp1
351
Jup
1110008F13Rik
Rsu1
352
Nid2
Mpnd
kg:uc007upr.1
353
Kdr
Gmppa
Itm2b
354
Ifnar2
Gramd1a
St3gal1
355
5430435G22Rik
Wars
Sec61g
356
Col4a6
Mtap
Ptpn1
357
Il17re
C1qtnf5
kg:uc012bhf.1
358
Gbp3
Mrpl28
B2m
359
Slc39a8
Mfrp
Rasgrp3
360
Cfl2
Kars
Memo1
361
Slc38a1
Lbp
Slc39a4
362
Cuedc1
Plxnb1
Sdcbp
363
Fgf1
2700081O15Rik
Tspan14
364
Gas6
Mrps24
Ubl7
365
Cldn25
Klc4
Nras
366
Sorbs1
Dctn3
Ssx2ip
367
Hspa12a
Kcnq1
kg:uc007zbz.1
368
kg:uc007zts.1
Smurf1
Wbp1
369
Slc1a5
Fam162a
1110003E01Rik
370
Nr3c1
Hip1r
Clip2
371
Adamts5
kg:uc007hyr.2
Gapdh
372
Gpcpd1
Gys1
Gm6578
373
Dpysl3
Sac3d1
Actn1
374
Colec12
Ndufs6
St3gal2
375
Pdcd6ip
Rgl2
3110001D03Rik
376
Dst
Atp5g1
Ctsz
377
Ifit4
Itgb4
kg:uc007vdl.1
378
Chst4
Sars
Fam73a
379
Xist
2310003F16Rik
Vcl
380
Ifi27l2a
Nhp2l1
Lims1
381
Fkbp5
D19Wsu162e
Lars2
382
Agap1
Cd320
Birc2
383
Ankrd11
Pigq
Lamp2
384
kg:uc007qca.1
Chd3
Rasl10a
385
Syt11
Zdhhc4
Mif
386
Ptrf
Eif3l
Rab10
387
Krcc1
St8sia3
Pabpc1
388
Zfp488
Rcan3
Wwp2
389
Lama4
Meg3
Nqo2
390
Aebp1
Nudt4
kg:uc007fte.1
391
Fam134b
Gss
Plxna4
392
Tppp3
Pih1d1
Gm1821
393
Maf
Limd2
Gadd45a
394
Peli1
Ap1s2
Slc25a39
395
Zfp353
BC056474
kg:uc009pet.1
396
Cdon
Mms19
Ubb
397
Sarnp
Clip2
Ppp1r2
398
Atxn7l3b
2310016M24Rik
Rab27b
399
Pef1
Itpa
Cap1
400
App
Slc25a10
Jarid2
401
Mtdh
Fibp
Rnf11
402
Lrrc20
Higd2a
Tmem50b
403
Btbd2
Snrpd2
Myh9
404
Gnb2
Eri3
Tmem128
405
Pigt
Nbeal2
Stradb
406
Efna5
Trim28
Cela1
407
Tm4sf1
S100a4
Ndrg2
408
Coq10b
Ivns1abp
Dhrs3
409
Eif2s3x
Ppp1r18
Hipk1
410
Cmah
Efemp2
Atg9a
411
Sf3b1
Med22
412
Eea1
Nelf
413
Slpi
2810428I15Rik
414
Tmod3
D2Wsu81e
415
Ppp3ca
Trappc6a
416
Tceal8
Trappc21
417
Anp32a
Antxr2
418
Actb
Rab11fip5
419
Ddx5
Ldhd
420
Cobll1
Npnt
421
Cish
Acrbp
422
Nod1
Pafah1b2
423
Psd
Angptl2
424
Gm10052
Fzr1
425
Lims2
Aaas
426
Stra6
Eif2b2
427
kg:uc007bgn.1
1190003J15Rik
428
Plxdc1
5730403B10Rik
429
Nfe2l1
Adamts13
430
Smpd3
Eif3b
431
Bcl10
Znrf1
432
Ilf3
Pkp3
433
Fam76a
Lemd2
434
Cybrd1
Rab34
435
Gm3893
Mpv17l2
436
Siae
Cdkn2b
437
Ssh2
Snrpe
438
Nfic
Gm14005
439
Btf3
Prdx4
440
Sp100
Xab2
441
Ndn
Dpp3
442
Matr3
Tyms
443
Gm13251
Leprotl1
444
Arhgap5
Uqcr10
445
Zbtb4
Cdk5rap3
446
Pgrmc1
Gorasp2
447
4930402H24Rik
Wbp7
448
Bptf
Sort1
449
Dusp3
Ddx41
450
Pla2g4a
Cct3
451
Brp44l
Mrps33
452
Oxct1
Frmd8
453
Stk40
1110049F12Rik
454
Ddr1
Fscn1
455
Ifi205
Ndufa2
456
Col3a1
Dpcd
457
Nipb1
Unc13a
458
Plk1s1
Eif1ad
459
Bdp1
Sgta
460
Smc3
Chaf1a
461
Ifitm3
Plxna1
462
Ndst1
Hspa9
463
Zbed6
1110014N23Rik
464
Rest
Cd99l2
465
kg:uc007vnc.1
Snrpa
466
Ccdc88a
Mcm7
467
Stat3
Tars2
468
Arf2
Gon4l
469
Trib1
Stk38
470
Gcap14
C1qtnf1
471
Tbc1d15
Tbrg4
472
Igf1r
Tmem132a
473
Ppbp
Cox6c
474
kg:uc008tky.1
Alcam
475
Rab1b
Phka2
476
Krt14
Trim3
477
Med21
Ppp1r14b
478
Gja1
Gpaa1
479
Klf10
Ctps2
480
Id2
Ptpn23
481
Mfap1a
Endog
482
Ogn
Mrto4
483
Gpc4
Mrps6
484
Bst2
Pvr
485
Dtx2
Phgdh
486
Wac
Itpr3
487
Kpna3
Polr2e
488
Kcnab1
Sec16a
489
Orai3
Mdp1
490
Gcsh
Fbf1
491
Wdr92
Mcpt8
492
Olfr613
Rps6ka4
493
Tcf7l1
Mical1
494
Tgfb2
Mrpl34
495
Il16
Agpat3
496
Manf
2310044H10Rik
497
Mgst1
Myo9b
498
kg:uc008tkz.1
Ndufb10
499
Creb3l1
Apex1
500
Txndc5
Elk3
501
Klf2
Cpsf3l
502
Slu7
Tnk1
503
Ttc28
Pmvk
504
1110002B05Rik
Ppp1r16a
505
Zcchc11
Arhgef5
506
Ptp4a2
Lonp1
507
Pbx1
Pla2g7
508
Clcn3
Pip5k1c
509
Tmco7
Inf2
510
Lrrc58
Pgk1
511
Eif3a
Parp6
512
Cldn10
Urm1
513
H2-Q6
Mad2l2
514
Ccdc80
Ing4
515
kg:uc009iln.1
Rbck1
516
Rab5c
Cant1
517
Tsc22d3
Sgpl1
518
Tm4sf5
Ehbp1l1
519
Hmgb1
Runx1
520
Sec62
Slc27a4
521
Maoa
Ndufa7
522
Clec1b
Mcm3ap
523
Mphosph8
1110008P14Rik
524
Oat
Rassf7
525
Ncor1
Ptpmt1
526
Cyb5
Arfgap1
527
Trafd1
Sec61a1
528
Rpp25
Rps6ka1
529
kg:uc007ded.1
Ints1
530
2610101N10Rik
Tpcn1
531
Il6st
Iffo2
532
Evpl
Trim44
533
Psmd11
kg:uc012ctw.1
534
Dync1i2
Golga2
535
Lars2
Msto1
536
Pdia4
Ppp6r3
537
Cd55
Trmt2a
538
Amfr
Appl2
539
Zcchc3
Sparcl1
540
Herpud2
Rapgef1
541
Txnrd1
Zfpl1
542
Vat1
Psmc4
543
Diap1
Mosc2
544
Tmed2
Fam101b
545
Arf3
1500010J02Rik
546
Arap2
Ccdc124
547
St3gal1
Ptges
548
Man1a
Fam189b
549
Rgs10
Th11
550
Tmsb4x
Kctd2
551
Uba7
Olfr1372-ps1
552
C4b
Hexa
553
Tmem98
Anapc5
554
Lpar2
Serpina3n
555
Gabarapl1
1810046J19Rik
556
Cmtm7
Tmem167
557
Spon2
Gm11428
558
Smarca5
Gcn1l1
559
Mxd4
Kansl3
560
Smc4
Fasn
561
Thsd4
Slc50a1
562
Gsr
Smad3
563
Ptprd
Trip6
564
Clip1
Atp6v1e1
565
Cln8
Chchd5
566
Rbm27
Adssl1
567
Zmat1
Nes
568
Smc6
Ap1b1
569
B2m
Fcgrt
570
Irf2bp2
Ltbp3
571
Ppap2a
Csf2rb
572
Zfhx4
Ssna1
573
Tob2
Mrps16
574
Rabgap1l
Cyba
575
Nfkb2
Cyth2
576
Nfyc
Igf2
577
Ube2d1
Pisd-ps1
578
Creb5
Atp13a2
579
Opa3
Mlph
580
Csnk1a1
Cyp4f16
581
Fam84b
2010107E04Rik
582
Ddr2
Gas5
583
Usp54
Eif3k
584
Akt2
Fam149a
585
Strn3
Mif
586
Hnrnpm
B230312A22Rik
587
eg:497210:chr14:m
Ppp1r12c
588
Tpt1
Tfip11
589
Naa25
Tex 10
590
Eef1a1
Slc16a3
591
Parp4
Stk16
592
Msn
Epn1
593
Zbtb20
Noc4l
594
Fermt2
Rcc2
595
Bod1l
Rgs12
596
Sltm
Shkbp1
597
Dapk1
Got2
598
Hnrnpr
Plek2
599
Baz2a
Lilrb3
600
Rnf167
Ndufb5
601
Mapk1
Tesk1
602
eg:320169:chr9:p
Rab24
603
4930523C07Rik
Atp5j2
604
Nf1
Commd9
605
Fam53b
Rtkn
606
Faim2
Prpf19
607
Tgm2
6720401G13Rik
608
Calm2
Ppa1
609
AI848100
Pgp
610
Slc10a3
Hps1
611
Ogdh
Puf60
612
Arl3
Mdm2
613
Timp2
kg:uc012cgd.1
614
Atxn2
kg:uc009uim.1
615
Mll1
Pyy
616
Ces2g
Zfp358
617
Mat2a
Timm8b
618
Esf1
Ddx39
619
Hsp90aa1
Pgm2
620
Zfp385a
kg:uc008gbp.1
621
Zfp672
Sipa1
622
Csda
Mgat1
623
Pf4
Tmem208
624
Arsa
Ruvbl2
625
F11r
8430410A17Rik
626
C4a
Bad
627
Kpna1
Pfdn5
628
Rbbp8
Eme1
629
Oxnad1
kg:uc009mzj.1
630
Rb1cc1
Igf1
631
Setd2
Prkag1
632
Kif1b
kg:uc009sua.1
633
2510002D24Rik
Uap1l1
634
Cep57
Trappc4
635
Chd2
Bola2
636
Serinc5
Usp5
637
Marcksl1
Ear2
638
Shfm1
Cars
639
Bbs4
1810027O10Rik
640
Impad1
Amdhd2
641
Tbcel
Phb
642
Kdelr1
Kcmf1
643
Ninl
Lsmd1
644
Sytl1
Sec11c
645
Tpm3
Pcbp4
646
Rbbp6
Mepce
647
Lman1
Tpd52l2
648
Ankrd17
Trf
649
Naga
Hsd17b11
650
Rbpms
Pilra
651
Magt1
Atn1
652
Tfdp2
Pgf
653
Gem
Nxn
654
Pde4dip
Inpp5k
655
Mrgprf
Actr1a
656
kg:uc008ajk.1
Cd68
657
Itch
Eef1g
658
Elf1
Fbn1
659
Meis2
Hint1
660
Arid1a
March5
661
Serping1
Usp48
662
Slc27a3
Hnf1b
663
Thoc2
Gga3
664
Gsta3
Drosha
665
Hnrnph2
Ubp1
666
Socs3
Pkn3
667
Armcx3
Tmem192
668
Siah1a
Prpf31
669
kg:uc009ize.1
Hspd1
670
Irs2
Otub1
671
Mettl7a1
Mrpl20
672
Ppfibp2
Tead2
673
Blvrb
Phpt1
674
Yipf5
Neu1
675
Plat
Pygo2
676
Gm6578
Myeov2
677
Mat2b
Cdk5
678
Tmpo
Ndor1
679
Metap2
Rbp4
680
Zfp277
Psat1
681
Wls
Mrpl41
682
Mesdc1
Snrpg
683
kg:uc009acs.1
Acot7
684
Col1a2
Vars
685
Csf1
Nono
686
Sulf2
Gtf2i
687
Ifrd1
Traf3
688
Wrnip1
Ppp2r4
689
Flii
Actg2
690
2810474O19Rik
Pi4k2a
691
Sep15
Slc35b2
692
2310030G06Rik
Ubqln1
693
Cmtm3
Ppox
694
Mylip
Bud31
695
Slc8a1
Man2b1
696
Btbd7
Nat15
697
Hdac5
Spon1
698
Zfand6
Cyc1
699
Tapbp
Mpeg1
700
Keap1
Nsun2
701
Ube2n
Rab4a
702
Ssr3
Mtmr11
703
H3f3a
BC004004
704
Myst4
B4galnt1
705
G3bp1
Atp5k
706
Ugdh
Lin37
707
Lamp2
D330041H03Rik
708
Zrsr1
Tbc1d17
709
Pim1
March6
710
Gm9199
2410015M20Rik
711
Supt16h
1810013D10Rik
712
Ano6
Eif2s1
713
Soat1
Traf7
714
Eci1
Rpl36al
715
Plce1
Psenen
716
Atg3
Aip
717
Bnc1
Cmas
718
Pik3c2a
Rpia
719
Pqlc3
Ncbp1
720
Thrap3
Mea1
721
Irak4
Timm50
722
Kdm6b
Ear12
723
Apol9a
Fkbp1a
724
Wnt4
Commd4
725
1500003O03Rik
Col5a3
726
Phf3
Fblim1
727
1110004F10Rik
Cwh43
728
Kansl1
Arl2bp
729
Fth1
Mrpl46
730
Tmem50a
Tcn2
731
Utp20
Add2
732
Smad4
Specc1l
733
Stmn2
Ppcs
734
Gstm1
Vrk3
735
Senp6
Trim25
736
Gda
Nfatc1
737
Nucks1
Rap1gap
738
Ints10
Hsd17b12
739
Syne1
Epas1
740
Itga6
Ddx1
741
Acad9
Prdx6
742
Maged1
Mmp24
743
Spen
Ndufb9
744
Chd1
Phf23
745
Taf3
Rpa2
746
Ptgs1
5031439G07Rik
747
Spare
Rrp7a
748
R74862
Arfip2
749
B230120H23Rik
Efna1
750
Tmem234
Agps
751
Ryk
Sephs1
752
Dlgap4
Apoc2
753
Atp1b1
Mrps27
754
Parp14
Snn
755
Tgfbr2
Serinc3
756
Ccdc90a
Pdcd5
757
Ncoa1
AA986860
758
Pppde1
Pitpna
759
Luc7l3
Vac14
760
Prg4
2810025M15Rik
761
Rab11fip1
Def8
762
Plk2
Hilpda
763
Ifi35
Eif6
764
Pdap1
Brd7
765
Cd248
Fes
766
Sesn1
Sbf1
767
Ecd
Ak2
768
Ap1s3
1810035L17Rik
769
H2-K1
Lime1
770
Spag9
Hspe1
771
Tshz1
Csrp2bp
772
Dennd5a
Uba5
773
Stag1
Gsta4
774
Gpx8
2900092E17Rik
775
Sod3
776
BC005561
777
kg:uc009vev.1
778
Ywhaz
779
Ganab
780
Rras2
781
Dusp14
782
kg:uc012hdk.1
783
Nr1d1
784
Wwc2
785
Ubxn2a
786
Iqsec1
787
kg:uc007vsr.1
788
Cfl1
789
Csrp1
790
Smchd1
791
Myl12a
792
Ubqln2
793
Tmcc3
794
Kdm5a
795
Rbm25
796
Wdr26
797
Vim
798
Arpc2
799
Calm1
800
Dnaja2
801
Shc1
802
Vps13a
803
Klf7
804
1810074P20Rik
805
BC003331
806
Itpr2
807
Jmjd1c
808
Pcdhgb5
809
Tubb2a
810
Ehd2
811
Ift74
812
Per1
813
Pitpnm2
814
Gstm4
815
Dnmt1
816
Tmco1
817
Lass4
818
Ptprf
819
Sirt2
820
Gfm2
821
Taf7
822
Spop
823
Zzef1
824
Ccdc34
825
Zfp281
826
Tuba1a
827
Ccdc109b
828
Cdk13
829
Dhx15
830
Src
831
Braf
832
Mapre2
833
Anxa7
834
Sept9
835
Alox12
836
Pknox1
837
2610034B18Rik
838
Topors
839
Phf21a
840
Qser1
841
Tirap
842
Fas
843
Lass2
844
6330406I15Rik
845
Parvb
846
Atp1a1
847
Mtmr6
848
Cd109
849
Dnajc1
850
Hp1bp3
851
1600029D21Rik
852
Ttc38
853
Mfhas1
854
Filip1l
855
Zfp148
856
Nkd1
857
Usp16
858
Tlr2
859
Zc3h18
860
Stk10
861
Ltbp4
862
Hdac3
863
Efhd2
864
Prkar2a
865
Atp6v1a
866
Sf3b4
867
Gprc5b
868
Clip3
869
Mettl2
870
Secisbp2
871
Fmod
872
kg:uc0091xf.1
873
Elovl6
874
Bzw1
875
Etfa
876
Hspa2
877
kg:uc007won.1
878
Rnf20
TABLE 3
Most Significant Gene Ontology Terms in CTC-c enriched genes using
BP_FAT and CC_FAT Datasets
q-value < 0.01
Odds
Benjamini
Source
Term
Count
Ratio
(q-value)
GOTERM_BP_FAT
GO: 0060429~epithelium development
35
2.92
8.72E−05
GOTERM_BP_FAT
GO: 0030029~actin filament-based
27
3.47
6.85E−05
process
GOTERM_BP_FAT
GO: 0030036~actin cytoskeleton
26
3.57
4.95E−05
organization
GOTERM_BP_FAT
GO: 0007010~cytoskeleton organization
36
2.50
6.27E−04
GOTERM_BP_FAT
GO: 0051173~positive regulation of
49
2.11
6.62E−04
nitrogen compound metabolic process
GOTERM_BP_FAT
GO: 0035295~tube development
31
2.66
7.80E−04
GOTERM_BP_FAT
GO: 0010604~positive regulation of
54
1.93
0.001727
macromolecule metabolic process
GOTERM_BP_FAT
GO: 0031328~positive regulation of
49
2.01
0.0015751
cellular biosynthetic process
GOTERM_BP_FAT
GO: 0051789~response to protein stimulus
16
4.16
0.0014484
GOTERM_BP_FAT
GO: 0035239~tube morphogenesis
23
3.05
0.0015064
GOTERM_BP_FAT
GO: 0045449~regulation of transcription
140
1.42
0.0014097
GOTERM_BP_FAT
GO: 0048729~tissue morphogenesis
28
2.66
0.0013058
GOTERM_BP_FAT
GO: 0009891~positive regulation of
49
1.99
0.0012408
biosynthetic process
GOTERM_BP_FAT
GO: 0045935~positive regulation of
46
2.04
0.0012061
nucleobase, nucleoside, nucleotide and
nucleic acid metabolic process
GOTERM_BP_FAT
GO: 0002009~morphogenesis of an
23
3.01
0.0012149
epithelium
GOTERM_BP_FAT
GO: 0048584~positive regulation of
24
2.92
0.0011396
response to stimulus
GOTERM_BP_FAT
GO: 0051276~chromosome organization
39
2.19
0.0012619
GOTERM_BP_FAT
GO: 0045637~regulation of myeloid cell
12
5.33
0.0014358
differentiation
GOTERM_BP_FAT
GO: 0045785~positive regulation of cell
11
5.79
0.0016889
adhesion
GOTERM_BP_FAT
GO: 0045941~positive regulation of
43
2.05
0.0016795
transcription
GOTERM_BP_FAT
GO: 0045893~positive regulation of
39
2.12
0.0019852
transcription, DNA-dependent
GOTERM_BP_FAT
GO: 0051254~positive regulation of RNA
39
2.11
0.0022107
metabolic process
GOTERM_BP_FAT
GO: 0006357~regulation of transcription
51
1.87
0.0022801
from RNA polymerase II promoter
GOTERM_BP_FAT
GO: 0006325~chromatin organization
32
2.30
0.0025187
GOTERM_BP_FAT
GO: 0010628~positive regulation of gene
43
2.00
0.0025252
expression
GOTERM_BP_FAT
GO: 0060562~epithelial tube
17
3.47
0.0025847
morphogenesis
GOTERM_BP_FAT
GO: 0042127~regulation of cell
45
1.89
0.0051485
proliferation
GOTERM_BP_FAT
GO: 0010557~positive regulation of
44
1.88
0.0071937
macromolecule biosynthetic process
GOTERM_BP_FAT
GO: 0002253~activation of immune
14
3.69
0.0078441
response
GOTERM_BP_FAT
GO: 0050778~positive regulation of
18
3.00
0.0080458
immune response
GOTERM_BP_FAT
GO: 0002684~positive regulation of
23
2.53
0.0088166
immune system process
GOTERM_BP_FAT
GO: 0045944~positive regulation of
33
2.09
0.0090124
transcription from RNA polymerase II
promoter
GOTERM_CC_FAT
GO: 0005578~proteinaceous extracellular
32
2.38
0.0047511
matrix
GOTERM_CC_FAT
GO: 0031012~extracellular matrix
32
2.28
0.0051923
GOTERM_CC_FAT
GO: 0044421~extracellular region part
60
1.71
0.0064365
GOTERM_CC_FAT
GO: 0031981~nuclear lumen
65
1.62
0.0102413
GOTERM_CC_FAT
GO: 0043233~organelle lumen
79
1.53
0.0085938
GOTERM_CC_FAT
GO: 0005829~cytosol
45
1.81
0.0100772
GOTERM_CC_FAT
GO: 0070013~intracellular organelle
78
1.52
0.0093866
lumen
GOTERM_CC_FAT
GO: 0031982~vesicle
43
1.83
0.0087123
GOTERM_CC_FAT
GO: 0031974~membrane-enclosed lumen
80
1.50
0.0082696
TABLE 4
Most Significant Gene Sets Enriched in CTC-plt vs CTC-c
q-value < 0.01
Odds
Benjamini
Source
Term
Count
Ratio
(q-value)
GOTERM_BP_FAT
GO: 0042060~wound healing
18
7.8
1.86E−07
GOTERM_BP_FAT
GO: 0007596~blood coagulation
15
10.4
9.31E−08
GOTERM_BP_FAT
GO: 0050817~coagulation
15
10.4
9.31E−08
GOTERM_BP_FAT
GO: 0007599~hemostasis
15
10.3
7.59E−08
GOTERM_BP_FAT
GO: 0050878~regulation of body fluid levels
15
8.2
1.30E−06
GOTERM_BP_FAT
GO: 0030029~actin filament-based process
20
5.5
1.14E−06
GOTERM_BP_FAT
GO: 0007010~cytoskeleton organization
26
3.9
3.95E−06
GOTERM_BP_FAT
GO: 0030036~actin cytoskeleton organization
18
5.3
1.11E−05
GOTERM_BP_FAT
GO: 0009611~response to wounding
26
3.6
1.02E−05
GOTERM_BP_FAT
GO: 0007155~cell adhesion
33
2.9
2.86E−05
GOTERM_BP_FAT
GO: 0022610~biological adhesion
33
2.8
2.70E−05
GOTERM_BP_FAT
GO: 0001775~cell activation
19
3.7
4.70E−04
GOTERM_BP_FAT
GO: 0030168~platelet activation
6
18.2
1.68E−03
GOTERM_BP_FAT
GO: 0007229~integrin-mediated signaling
10
6.4
2.95E−03
pathway
GOTERM_BP_FAT
GO: 0016192~vesicle-mediated transport
25
2.6
3.81E−03
MSigDBv3.1 CGP
GNATENKO PLATELET SIGNATURE
20
55.1
3.91E−24
MSigDBv3.1 CGP
TENEDINI MEGAKARYOCYTE
14
15.3
1.35E−11
MARKERS
MSigDBv3.1
REACTOME FACTORS INVOLVED IN
6
2.9
2.25E−02
CP:REACTOME
MEGAKARYOCYTE DEVELOPMENT
AND PLATELET PRODUCTION
TABLE 5
Most Significant Gene Sets Enriched in CTC-pro vs CTC-c
q-value < 0.01
Odds
Benjamini
Source
Term
Count
Ratio
(q-value)
GOTERM_BP_FAT
GO: 0002495~antigen processing and
5
59.81
6.97E−04
presentation of peptide antigen via MHC class II
GOTERM_BP_FAT
GO: 0019886~antigen processing and
5
59.81
6.97E−04
presentation of exogenous peptide antigen via
MHC class II
GOTERM_BP_FAT
GO: 0002504~antigen processing and
5
50.36
7.34E−04
presentation of peptide or polysaccharide
antigen via MHC class II
GOTERM_BP_FAT
GO: 0002478~antigen processing and
5
41.60
1.10E−03
presentation of exogenous peptide antigen
GOTERM_BP_FAT
GO: 0019884~antigen processing and
5
34.18
1.87E−03
presentation of exogenous antigen
GOTERM_BP_FAT
GO: 0048002~antigen processing and
5
27.34
3.72E−03
presentation of peptide antigen
GOTERM_BP_FAT
GO: 0001775~cell activation
9
7.00
3.82E−03
GOTERM_BP_FAT
GO: 0019882~antigen processing and
6
13.20
7.40E−03
presentation
Example 2
Supplemental Methods
Mice and cell lines. Mice with pancreatic cancer used in these experiments express Cre driven by Pdx1, LSL-Kras G12D , and Trp53 lox/+ or Trp53 lox/lox (otherwise referred to as KPC) as previously described (Bardeesy et al., 2006). EGFP pancreatic lineage tagged KPC mice were generated by breeding the mT/mG mouse (Purchased from the Jackson Laboratory—Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J) into the breeder pairs used for KPC mouse generation. Normal FVB mice were purchased from Jackson Laboratory. All mice care and procedures were done under MGH SRAC approved protocols.
For cardiocentesis, animals were sedated with isofluorane, the chest wall was sterilized with ethanol and a skin incision was made above the rib cage to expose the thoracic cavity and eliminate normal skin epithelial cell contamination. A 23-gauge needle was used to draw approximately 1 mL of blood into a 1 mL syringe primed with 100 μL of PBS-10 mM EDTA pH 7.4 (Gibco). Blood EDTA concentration was raised to 5 mM by either the addition of a concentrated bolus of 500 mM EDTA or 1:1 dilution with 10 mM EDTA. Animals were then euthanized per animal protocol guidelines.
A mouse pancreatic cell line NB508 (Pdx1-Cre/Kras G12D /Trp53 lox/+ ) previously generated from primary tumors developed in this endogenous model was GFP transfected by lentivirus (NB508-GFP). This cell line was used for spiked cell experiments and orthotopic tumor formation.
NB508-GFP Cell lines were maintained in standard culture conditions using RPMI-1640 medium+10% FBS+1% Pen/Strep (Gibco/Invitrogen).
For orthotopic experiments, NB508-GFP cells were orthotopically injected into the pancreas of healthy syngeneic (FVB background) mice. Briefly, mice were anesthesized with isofluorane and the left abdominal wall was treated with Nair® hair removal product, and sterilized with 70% ethanol. A small incision was made on the upper left lateral abdominal wall and the pancreas was mobilized. Approximately 1 million NB508-GFP cells in PBS in a total volume of 0.1 mL was injected into the pancreas. The peritoneum and abdominal wall was closed by sterile surgical staples. The tumors were allowed to grow for 2 weeks, at which time blood was obtained by cardiocentesis for CTC-iChip processing.
Adaptation of CTC Enrichment Technology.
Given the desire for an unbiased enrichment system, the negative depletion technology was selected for this application. All processing protocols were identical to those previously identified, except a rat anti-mouse CD45 antibody (BAM114, R&D Systems, USA) was conjugated to MyOne beads.
Spiked cell experiments were conducted to validate the system by spiking ˜1000 GFP expressing NB508 cells into 1 mL of healthy mouse blood and processing to determine recovery efficiency. Orthotopic models were used to validate recovery efficiency as well as initially determine expected depletion efficiency from tumor-bearing mice. In these experiments, enriched samples were evaluated for the number of GFP+ cells observed in the product.
Immunostaining of CTCs Isolated from the Endogenous Model.
Isolated CTCs were spun onto glass slides and immunostained using a primary-secondary approach. Primary antibodies were rabbit anti-wide spectrum cytokeratin (1:50, Abcam ab9377), and goat anti-mouse CD45 (1:500, R&D systems AF114). Secondary immunofluorescent-tagged antibodies were used for signal amplification. These were donkey anti-rabbit Alexa Fluor 594 (1:500, Invitrogen A-21207), and donkey anti-goat Alexa Fluor 488 (1:500, Invitrogen A-11055). Nuclei were then counterstained with DAPI and the slides were rinsed with PBS, cover slipped and stored at 4° C. They were imaged under 10× magnification using the BioView™ Ltd. automated imaging system (Billerica, Mass.) as well as an automated upright fluorescence microscope (Eclipse 90i™, Nikon, Melville, N.Y.). Positive staining for CK, without CD45 staining, was required for scoring potential CTCs, which were then manually reviewed. Threshold and baseline signals were established using specimens from non-tumor bearing mice.
Single Cell Micromanipulation.
After whole blood anti-CD45 negative depletion, the product containing enriched cells was collected in a 35 mm petri dish and viewed using a Nikon Eclipse Ti™ inverted fluorescent microscope. Cells of interest were identified based on intact cellular morphology and lack of labeling with anti-CD45 magnetic beads. These target cells were individually micromanipulated with a 10 μm transfer tip on an Eppendorf TransferMan® NK 2 micromanipulator and ejected into PCR tubes containing RNA protective lysis buffer (10×PCR Buffer II, 25 mM MgCl2, 10% NP40, 0.1 M DTT, SUPERase-In, Rnase Inhibitor, 0.5 uM UP1 Primer, 10 mM dNTP and Nuclease-free water) and immediately flash frozen in liquid nitrogen.
Single Cell Amplification and Sequencing.
Single cell amplification and sequencing were done as previously described (Tang et al., 2010) with slight modifications underlined below. RNA samples from extracted single circulating tumor cells were thawed on ice and incubated at 70° C. for 90 seconds. To generate cDNA, samples were treated with reverse transcription master mix (0.05 uL RNase inhibitor, 0.07 uL T4 gene 32 protein, and 0.33 uL SuperScript™ III Reverse Transcriptase per 1× volume) and incubated on thermocycler at 50° C. for 30 minutes and 70° C. for 15 minutes. To remove free primer, 1.0 uL of EXOSAP mix was added to each sample, which was incubated at 37° C. for 30 minutes and inactivated at 80° C. for 25 minutes. Next, a 3′-poly-A tail was added to the cDNA in each sample by incubating in master mix (0.6 uL 10×PCR Buffer II, 0.36 uL 25 mM MgCl 2 , 0.18 uL 100 mM dATP, 0.3 uL Terminal Transferase, 0.3 uL RNase H, and 4.26 uL H 2 O per 1× volume) at 37° C. for 15 minutes and inactivated at 70° C. for 10 minutes. A second strand cDNA was synthesis by dividing each sample into 4 and incubating in master mix (2.2 uL 10× High Fidelity PCR Buffer, 1.76 uL 2.5 mM each dNTP, 0.066 uL UP2 Primer at 100 uM, 0.88 uL 50 mM MgSO 4 , 0.44 uL Platinum Taq DNA Polymerase, and 13.654 uL H 2 O per 1× volume) at 95° C. for 3 minutes, 50° C. for 2 minutes, and 72° C. for 10 minutes.
PCR amplification (95° C. for 3 minutes, 20 cycles of 95° C. for 30 seconds, 67° C. for 1 minute, and 72° C. for 6 minutes 6 seconds) was performed with master mix (4.1 uL 10× High Fidelity PCR Buffer, 1.64 uL 50 mM MgSO 4 , 4.1 uL 2.5 mM each dNTP, 0.82 uL AUP1 Primer at 100 uM, 0.82 uL AUP2 Primer at 100 uM, 0.82 uL Platinum Taq DNA Polymerase, and 6.7 uL H 2 O per 1× volume). The 4 reactions of each sample were pooled and purified using the QIAGEN PCR Purification Kit (Cat. No 28106) and eluted in 50 uL EB buffer. Samples were selected by testing for genes Gapdh, ActB, Ptprc (CD45), Krt8, Krt18, Krt19, and Pdx1 using qPCR. Each sample was again divided in 4 and a second round of PCR amplification (9 cycles of 98° C. for 3 minutes, 67° C. for 1 minute, and 72° C. for 6 minutes 6 seconds) was performed with master mix (9 uL 10× High Fidelity PCR Buffer, 3.6 uL 50 mM MgSO 4 , 13.5 uL 2.5 mM each dNTP, 0.9 uL AUP1 Primer at 100 uM, 0.9 uL AUP2 Primer at 100 uM, 1.8 uL Platinum Taq DNA Polymerase, and 59.1 uL H 2 O per 1× volume). Samples were pooled and purified using Agencourt AMPure XP beads and eluted in 40 uL 1× low TE buffer.
Sequencing Library Construction.
To shear the DNA using the Covaris S2™ System, 1× low TE buffer and 1.2 uL shear buffer were added to each sample. Conditions of the shearing program include: 6 cycles, 5° C. bath temperature, 15° C. bath temperature limit, 10% duty cycle, intensity of 5, 100 cycles/burst, and 60 seconds. Then, samples were end-polished at room temperature for 30 minutes with master mix (40 uL 5× Reaction Buffer, 8 uL 10 mM dNTP, 8 uL End Polish Enzyme1, 10 uL End Polish Enzyme2, and 14 uL H 2 O per 1× volume). DNA fragments larger than 500 bp were removed with 0.5× volumes of Agencourt AMPure XP™ beads. Supernatant was transferred to separate tubes. To size-select 200-500 bp DNA products, 0.3× volumes of beads were added and samples were washed 2× with 70% EtOH. The products were eluted in 36 uL low TE buffer. A dA-tail was added to each size-selected DNA by treating with master mix (10 uL 5× Reaction Buffer, 1 uL 10 mM dATP, and 5 uL A-Tailing Enzyme I per 1× volume) and incubated at 68° C. for 30 minutes and cooled to room temperature. To label and distinguish each DNA sample for sequencing, barcode adaptors (5500 SOLiD 4464405) were ligated to DNA using the 5500 SOLiD Fragment Library Enzyme Module™ (4464413). Following barcoding, samples were purified twice using the Agencourt AMPure XP™ beads and eluted in 22 uL low TE buffer. Following a round of PCR Amplification (95° C. for 5 minutes, 12 cycles of 95° C. for 15 seconds, 62° C. for 15 seconds, and 70° C. for 1 minute, and 70° C. for 5 minutes), the libraries were purified with AMPure XP beads. Finally, to quantify the amount of ligated DNA, SOLiD Library TaqMan Quantitation Kit™ was used to perform qPCR. Completed barcoded libraries were then subjected to emulsion PCR with template beads preparation and sequenced on the ABI 5500XL™.
RNA In Situ Hybridization (RNA-ISH).
Paraffin-embedded tissue blocks were freshly cut and frozen at −80° C. Upon removal from the freezer, slides were baked for 1 hr at 60° C. and fixed in %10 formaldehyde for 1 hr at room temperature (RT). Paraffin was removed using Histo-Clear™ and RNA-ISH™ was performed according to the Affymetrix QuantiGene ViewRNA ISH Tissue-2 Plex Assay™. Tissue sections were permeabilized by pretreating in buffer solution for 10 min at 95° C. and digested with protease for 10 min, before being fixed at RT in 5% formaldehyde. Target probe sets were applied and hybridized to the tissue by incubating for 2 hr at 40° C. Type 1 probes were used at a dilution of 1:50 and included Aldh1a2 (VB1-14197), Dcn (VB1-14962), Klf4 (VB1-14988), Igfbp5 (VB1-14987), and Sparc (VB1-14196). Type 6 probes included EGFP (VF6-13336) at 1:50 and pooled Krt8 (VB6-11060) and Krt18 (VB6-11059) at 1:100 each. Signal was amplified through the sequential hybridization of PreAmplifier and Amplifer QT mixes to the target probe set. Target mRNA molecules were detected by applying Type 6 Label Probe with Fast Blue substrate and Type 1 Label Probe with Fast Red substrate. Tissue was counterstained with Gill's Hemotoxylin for 10 sec at RT. DAPI (Invitrogen, D3571; 3.0 μg/ml) staining was performed for 1 min. Fluorescence microscopy using a Nikon 90i was used to visualize target mRNAs. Type 1 probes were detected in the Cy3 channel and Type 6 probes in the Cy5 channel. Merged images were generated using NIS-Elements™ software.
Determination of Reads-Per-Million (rpm)
Color space reads were aligned using Tophat™ version 2.0.4 (Trapnell et al., 2009) and Bowtie1™ version 0.12.7 with the no-novel-juncs argument set with mouse genome version mm9 and transcriptome defined by the mm9 knownGene table from genome.ucsc.edu. Reads that did not align or aligned to multiple locations in the genome were discarded. The mm9 table knownToLocusLink from genome.ucsc.edu was used to map, if possible, each aligned read to the gene who's exons the read had aligned to. The reads count for each gene was the number of reads that were so mapped to that gene. This count was divided by the total number of reads that were mapped to any gene and multiplied by one million to form the reads-per-million (rpm) count. Rpm rather than rpkm was used because a 3′ bias was noted in the alignments.
Unsupervised Hierarchical Clustering and Principal Components Analysis.
The minimum of 1 and the smallest positive value of the rpm matrix was added to the rpm matrix to eliminate zeros. The result was then log 10 transformed, yielding what is termed the log 10(rpm) matrix. The rows (corresponding to genes) of the log 10(rpm) matrix with the top 2000 standard deviations were retained and the rest of the rows discarded. The result was then median polished. The result was clustered using agglomerative hierarchical clustering with average linkage with distance metric equal to 1 minus the Pearson correlation coefficient. The principal components of the log 10(rpm) matrix were computed and the coordinates of the samples with respect to the first three principal components were plotted.
Measures of Cellular Heterogeneity.
For a collection of clusters of samples, a statistic, M, was defined as the mean over the clusters of the mean over all the pairs of samples in the cluster of the atan h of the correlation coefficient between the two columns of the rpm matrix corresponding to the pair. The “mean intra-cluster correlation coefficient” was defined as tan h(M). The jackknife estimator was used with respect to the samples to estimate a standard deviation, s, of the statistic. The 95% CI was defined as tan h (M±sφ −1 (0.975)), where 4 is the cumulative distribution function of the standard normal distribution. To compute a p-value for the null hypothesis that the mean of the distribution of the M statistic for a cluster is the same as the mean of the distribution of the M statistic for a collection of clusters, we let p=2(1−φ(|M1−M2|/√(s 2 1 +s 2 2 ))). Of note, bootstrap was performed on the same data as an alternative to jackknife and similar results obtained (data not shown).
Supervised Differential Gene Expression Using Rank Product.
To find differentially expressed genes between two sets of samples, analysis was begin with the log 10(rpm) matrix defined above. Columns corresponding to samples not in either set of samples were removed. Then removed rows for which the 90 th percentile of the values was less than log 10(10) were removed. The RP function of the Bioconductor (Gentleman et al., 2004) RankProd™ package (version 2.28.0) was used to get FDR estimates for both up and down differential expression. Genes were considered to be differentially expressed if their FDR estimate was less than 0.01, but discarded if they were both up and down differentially expressed, if there were any.
Gene Set Enrichment.
Enrichment was considered in four gene set collections: (1) all of KEGG™, as found in DAVID™ 6.7 (Huang da et al., 2009), (2) Gene Ontology (GO) using GO_BP as found in DAVID 6.7, and (3) GO_CC as found in DAVID 6.7. Sets of genes found to be differentially expressed were tested for enrichment in the gene set collections using a hypergeometric test for each gene set in the collection. The resulting p-values for each collection were converted to FDR estimates using the Benjamini-Hochberg method (Benjamini and Hochberg, 1995).
Digital Removal of all Annotated Platelet Transcripts
The 446 genes whose expression in the log 10(rpm) matrix had an absolute value of correlation coefficient greater than 0.6 with the expression of any of the genes in the gene sets named GNATENKO_PLATELET_SIGNATURE and TENEDINI_MEGAKARYOCYTE_MARKERS in MSigDB v3.1 were removed from the log 10(rpm) matrix (defined above). Clustering was then performed as described above.
SUPPLEMENTAL METHODS REFERENCES
Bardeesy, N., Aguirre, A. J., Chu, G. C., Cheng, K. H., Lopez, L. V., Hezel, A. F., Feng, B., Brennan, C., Weissleder, R., Mahmood, U., et al. (2006). Both p16(Ink4a) and the p19(Arf)-p53 pathway constrain progression of pancreatic adenocarcinoma in the mouse. Proc Natl Acad Sci USA 103, 5947-5952.
Benjamini, Y., and Hochberg, Y. (1995). Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B (Methodological) 57, 289-300.
Gentleman, R. C., Carey, V. J., Bates, D. M., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B., Gautier, L., Ge, Y., Gentry, J., et al. (2004). Bioconductor: open software development for computational biology and bioinformatics. Genome biology 5, R80.
Huang da, W., Sherman, B. T., and Lempicki, R. A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4, 44-57.
Tang, F., Barbacioru, C., Nordman, E., Li, B., Xu, N., Bashkirov, V. I., Lao, K., and Surani, M. A. (2010). RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nat Protoc 5, 516-535.
Trapnell, C., Pachter, L., and Salzberg, S. L. (2009). TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105-1111.
Example 3
A comparative analysis of mouse pancreatic CTCs indicated an enrichment of 60 extracellular proteins (Table 6). Evaluation of these particular biomarkers and therapeutic targets was undertaken in human pancreatic circulating tumor cells and the most abundant targets in human pancreatic CTCs are shown ( FIG. 7 ). These not only represent potential biomarkers, but given their nature as proteins on the external surface of tumor cells, they are therapeutic targets. The extracellular proteins of Table 6 can be targeted, e.g. by antibody-based therapeutics (e.g. as in the cases of trastuzumab for HER2, cetuximab for EGFR, and bevacizumab for VEGF) to treat cancer.
TABLE 6
List of Pancreatic CTC enriched Extracellular Proteins.
OFFICIAL GENE
SYMBOL
Gene Name
Abi3bp
ABI gene family, member 3 (NESH) binding protein
Adamts5
similar to a disintegrin-like and metalloprotease (reprolysin type) with
thrombospondin type 1 motif, 5 (aggrecanase-2); a disintegrin-like and
metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 5
(aggrecanase-2)
Adamtsl1
ADAMTS-like 1
Ang
angiogenin, ribonuclease, RNase A family, 5
Arsa
arylsulfatase A
C1rl
complement component 1, r subcomponent-like
C3
complement component 3; similar to complement component C3
prepropeptide, last
C4a
similar to Complement C4 precursor; complement component 4A (Rodgers
blood group); similar to complement C4; complement component 4B
(Childo blood group)
C4b
similar to Complement C4 precursor; complement component 4A (Rodgers
blood group); similar to complement C4; complement component 4B
(Childo blood group)
Ccdc80
coiled-coil domain containing 80
Cd109
CD109 antigen
Chi3l1
chitinase 3-like 1
Clec3b
C-type lectin domain family 3, member b
Cmtm3
CKLF-like MARVEL transmembrane domain containing 3
Cmtm7
CKLF-like MARVEL transmembrane domain containing 7
Col14a1
collagen, type XIV, alpha 1
Col1a2
collagen, type I, alpha 2
Col3a1
collagen, type III, alpha 1
Col4a6
collagen, type IV, alpha 6
Csf1
colony stimulating factor 1 (macrophage)
Dag1
dystroglycan 1
Dcn
decorin
Dmkn
dermokine
Fbln1
fibulin 1
Fgf1
fibroblast growth factor 1
Fmod
fibromodulin
Gpc3
glypican 3
Gpc4
glypican 4; similar to Glypican 4
Hmgb1
high mobility group box 1
Ifnar2
interferon (alpha and beta) receptor 2
Igfbp5
insulin-like growth factor binding protein 5
Il16
interleukin 16
Lama4
laminin, alpha 4
Ltbp4
latent transforming growth factor beta binding protein 4
Mfap1a
similar to microfibrillar-associated protein 1A; microfibrillar-associated
protein 1A; microfibrillar-associated protein 1B
Nid2
nidogen 2
Ogn
osteoglycin
Pdap1
PDGFA associated protein 1
Pf4
platelet factor 4
Plat
plasminogen activator, tissue
Podn
podocan
Prelp
proline arginine-rich end leucine-rich repeat
Rspo1
R-spondin homolog ( Xenopus laevis )
Serping1
serine (or cysteine) peptidase inhibitor, clade G, member 1
Slurp1
secreted Ly6/Plaur domain containing 1
Sod3
superoxide dismutase 3, extracellular
Sparc
secreted acidic cysteine rich glycoprotein; similar to Secreted acidic cysteine
rich glycoprotein
Spock2
sparc/osteonectin, cwcv and kazal-like domains proteoglycan 2
Spon2
spondin 2, extracellular matrix protein
Sulf1
sulfatase 1
Sulf2
sulfatase 2
Tgfb2
transforming growth factor, beta 2
Tgm2
transglutaminase 2, C polypeptide
Thbd
thrombomodulin
Thbs1
thrombospondin 1; similar to thrombospondin 1
Thsd4
thrombospondin, type I, domain containing 4
Timp2
tissue inhibitor of metalloproteinase 2
Tnxb
tenascin XB
Tpt1
predicted gene 1974; tumor protein, translationally-controlled 1
pseudogene; tumor protein, translationally-controlled 1; predicted gene
14456
Twsg1
twisted gastrulation homolog 1 ( Drosophila )
Wnt4
wingless-related MMTV integration site 4
Extending these CTC enriched genes to human pancreatic, breast, and prostate single cell CTC data identified 5 candidate genes shown in Table 9.
TABLE 9
Percent of human single CTCs with high expression by RNA-seq
Percent of Single CTCs >50 RPM of
Expression
Pancreas
Breast
Prostate
ALL
Cancer Type
(N = 7)
(N = 29)
(N = 77)
(N = 113)
TPT1
86%
90%
90%
89%
HMGB1
43%
62%
44%
49%
SPON2
43%
7%
45%
35%
SPARC
100%
41%
9%
23%
ARSA
71%
17%
5%
12%
Focusing on pancreatic cancer, SPARC was selected as an initial gene to evaluate. SPARC RNA-ISH in mouse and human primary tumors (data not shown) demonstrated significant expression in the stromal cells of the tumor that provides essential microenvironmental signals to tumors. Much effort in the field focuses on targeting the stroma of PDAC for therapeutic efficacy [1-4] making SPARC a CTC therapeutic target as well as a stromal directed target. A total of 196/198 (99%) of human pancreatic tumors were positive for SPARC and 36% with clear epithelial tumor cell expression.
Evaluation of human pancreatic cancer cell lines identified 3 of 5 cell lines with elevated SPARC expression which correlates to increased migratory behavior, a surrogate in vitro assay that correlates with metastatic behavior ( FIG. 8 ).
Evaluation of SPARC function in human pancreatic cancer was done using short hairpin RNA interferences (shRNA) on the two cell lines with highest SPARC expression (PDAC2 and PDAC3). Multiple in vitro assays were done including proliferation, migration, invasion, scratch, and soft agar. The most profound effects of suppressing SPARC expression was on migratory behavior ( FIG. 9 and data not shown), indicating SPARC is not only present in many CTCs, but has functional consequences when inhibited in cell line models.
Given these data, in vivo tail vein inoculation was performed using PDAC-3 to determine if SPARC knockdown affected metastasis. Initial data at 2 weeks post tail vein injection indicates there is reduced metastatic potential when SPARC is inhibited by shRNA with 83% of control mice with metastases compared to 40% in cell lines with shRNA against SPARC ( FIG. 10 ).
Surface Protein Targets
Most of the targets identified in Table 9 are secreted factors and analysis of genes annotated as cell surface proteins are summarized in Table 14.
TABLE 14
Percent of human single CTCs with high expression
of surface protein genes
Percent of Single CTCs >50 RPM of Expression
Cancer
Pancreas
Breast
Prostate
type
(N = 7)
(N = 29)
(N = 77)
ALL (N = 113)
IL6ST
0%
38%
8%
15%
ARSA
71%
17%
5%
12%
TIMP2
0%
21%
4%
8%
CD55
0%
17%
4%
7%
SULF2
0%
24%
0%
6%
ITGA6
0%
14%
3%
5%
SDC4
0%
14%
3%
5%
CDON
0%
7%
5%
5%
SV2A
14%
3%
1%
3%
It is contemplated herein that these genes are targets given they would be integrated into the plasma membrane of CTCs. In general, RNA expression of cell surface markers tend to be lower than actual protein levels on cells.
Contemplated herein are antibodies to IL6ST, SULF2, and SV2A for therapeutic utility.
1. IL6ST—signal transducer for IL6, LIF, CNTF, and oncostatin M.
a. Important for STAT3 activation downstream b. Antibodies against IL6 receptor and IL6 have been developed for human disease including cancer
2. SULF2—sulfatase modifies heparin sulfate by removing 6-O-sulfate groups
a. Expression enriched in cancer progression and metastasis b. Drugs have been developed against sulfatase activity and tested with activity in liver cancer models
3. SV2A—synaptic vesicle glycoprotein elevated in neuroendocrine cells
a. A marker of neuroendocrine cells, which appear at the epithelial stromal border of human pancreatic cancer b. Neuroendocrine differentiation common feature in cancers and portends to more aggressive disease
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Example 5
Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, genomewide expression profiles of CTCs were compared with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low proliferative signatures, enrichment for Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.
Classical CTCs expressed predominantly the Aldh1a2 isoform, while Aldh1a1 was expressed in a variety of cell types (data not shown). Within single CTCs, there was no correlation between expression of Aldh1 isoforms and either enrichment for the mesenchymal genes (Cdh11, Vim) or loss of epithelial genes (Cdh1, Muc1), indicating that stem cell and EMT markers are not intrinsically linked in CTCs. Analysis of primary pancreatic tumors for Aldh1a2 using RNA in situ hybridization (RNA-ISH) identified rare epithelial tumor cells expressing this stem cell marker, but the majority of expression was present within the cancer associated stromal cells ( FIG. 12A ), consistent with immunohistochemistry for ALDH protein in human PDAC (Rasheed et al., 2010).
Besides the evident diversity of CTCs, shared transcripts were searched for that could provide further insight into their cell of origin within the primary tumor and the mechanisms by which they invade and survive within the bloodstream and ultimately identify potential CTC-specific therapeutic targets. Rigorous criteria were selected to identify the most highly enriched CTC-c transcripts (RP score<300), expressed at very high levels (>100 rpm) in R90% of all classical CTCs. Three genes met these criteria: Kruppel-like factor 4 (Klf4), one of the key stem cell (iPS) reprogramming factors (Takahashi and Yamanaka, 2006), insulin-like growth factor binding protein 5 (Igfbp5), an extracellular growth factor binding protein and decorin (Dcn). RNA-ISH was utilized in primary tumor specimens to identify the potential colocalization of these three highly enriched CTC genes. In contrast to Aldh1a2, Klf4 is expressed in epithelial components of the primary tumor ( FIG. 12B ). Igfbp5 is of particular interest, in that it is expressed focally at the tumor epithelial-stromal interface ( FIG. 12C ). It is contemplated herein that this geographic area is enriched for cancer cells undergoing EMT, contributing to the mixed epithelial/stromal transcriptional programs evident by RNA-seq of single CTCs.
In addition to highly expressing Dcn, CTCs consistently had high levels of multiple ECM gene transcripts. GO analysis of all CTC-enriched genes (Table 3) identified 32 proteinaceous ECM genes (GO:0005578, OR 2.4, q-value 4.8 3 10.3). These genes are normally expressed in reactive stromal cells, rather than in epithelial cancer cells, and while recent studies have highlighted the importance of the stroma in supporting pancreatic cancer pathogenesis and metastasis (Feig et al., 2012; Neesse et al., 2011, 2013; Olive et al., 2009; Provenzano et al., 2012), the expression of these stroma-associated ECM genes within tumor cells in circulation was unexpected. Using RP differential expression analysis, CTCs were compared with purified EGFP-tagged primary tumor single cells (TuGMP3) and bulk tumor samples (tumor cells admixed with reactive stromal cells). Six proteinaceous ECM genes were highly expressed by CTCs and by stromal component, but not by epithelial cells within primary tumors: Dcn, Sparc, Ccdc80, Col1a2, Col3a1, and Timp2 (data not shown). RNA-ISH analysis of both Dcn and Spare confirmed diffuse expression in stromal elements of mouse primary tumors, with rare areas where these transcripts are colocalized with keratin-expressing cells at the epithelial-stromal border (data not shown).
SPARC is a ECM protein gene. RNA-ISH analysis of 198 primary human PDACs demonstrates abundant stromal cell expression of SPARC transcripts in 99% of cases, with up to a third of tumors with rare epithelial cells expressing this ECM gene product (data not shown). Consistent with these observations, RNA-seq of EGFP-tagged single primary tumor cells (data not shown) identified only 1 of 20 cells (5%) with coexpression of high levels (>100 rpm) of Spare and Krt19.
In summary, abundant expression of ECM genes is a common feature of all keratin-rich classical CTCs. This is in marked contrast to the primary tumor, where these gene products are secreted by supporting stromal cells and not by the epithelial cancer cells. However, rare cells at the epithelial-stromal interface of primary tumors do appear to express both keratins and ECM genes, consistent with the pattern observed in CTCs themselves.
To confirm the expression of proteinaceous ECM genes by human cancer cells circulating in the bloodstream, single CTCs were isolated from patients with pancreatic (n=7), breast (n=29), and prostate (n=77) cancers and subjected these to single-cell RNA-seq. Six ECM protein genes were highly expressed in human CTCs (>100 rpm in >15% of all CTC samples) ( FIG. 13 ; Table 13). Notably, three genes (SPARC, MGP, SPON2) are ECM glycoproteins, defined as part of the core matrisome (Naba et al., 2012). The core matrisome protein SPARC was particularly enriched in pancreatic CTCs being expressed at high levels (>100 rpm) in 100% of pancreatic CTCs compared to 31% of breast and 9% of prostate CTCs. The notable differences in ECM protein gene expression across human epithelial CTCs suggest microenvironment tissue specificity as well as probable redundancies in ECM protein signaling. Together, the consistent expression of ECM gene family members in human CTCs indicates that their upregulation contributes either to the generation of CTCs from primary tumors or to the survival of cancer cells deprived of microenvironmental signals as they circulate in the bloodstream.
In order to define the functional consequences of SPARC expression in pancreatic cancer cells, a panel of patient-derived, low-passage PDAC cell lines was screened for expression. Two human PDAC cell lines with relatively high SPARC expression were identified (PDAC2 and PDAC3), making it possible to test the consequences of small hairpin RNA (shRNA)-mediated knockdown ( FIG. 8, 9 , FIGS. 16A-16D ). Suppression of endogenous SPARC expression in both PDAC2 and PDAC3 cell lines using two independent shRNA constructs did not affect proliferation in 2D cultures or anchorage-independent tumor sphere formation ( FIGS. 14A-14B , FIGS. 16A-16D ). However, SPARC knockdown by both shRNAs significantly reduced pancreatic cancer cell migration in wound scratch assays and their invasive properties, as measured by in vitro Boyden assays (data not shown).
Tail vein injection of SPARC-suppressed PDAC3 cells using both shRNA constructs generated significantly fewer lung metastases than cells expressing nontargeting hairpin (shNT) controls ( FIG. 14D ). Metastases generated from orthotopic pancreatic xenografts were also significantly reduced for SPARC-suppressed PDAC3 cells, as measured by luciferase imaging and normalized for primary tumor size ( FIG. 14E ). Thus, SPARC expression by pancreatic cancer cells appears to selectively enhance their invasive and migratory properties to augment metastatic virulence. The high levels of SPARC expression evident in virtually all pancreatic CTCs thus raises the possibility that it contributes significantly to the metastatic spread of pancreatic cancer.
Discussion
Described herein is the detailed analysis of CTC composition and diversity in pancreatic cancer, using single-cell RNA-seq. High-quality transcriptomes were achieved in 93 single mouse pancreatic CTCs, which were compared with bulk and single-cell preparations from matched primary tumors and from an immortalized cell line established from the same mouse pancreatic tumor model. The use of the KPC mouse model made it possible to compare simultaneously isolated primary tumor specimens and CTCs, and it allowed measurements of CTC heterogeneityacross multiple mice sharing the same Kras/Trp53 genetic drivers. The large number of isolated CTCs and the high quality of the isolated RNA from these cells reflect the application of the CTC-iChip technology, which effectively depletes normal blood components, enriching for CTCs that are untagged and accessible for single-cell manipulation. Finally, the purification of CTCs irrespective of their cell-surface epitopes avoids any bias associated with their purification based on expression of common epithelial markers such as EpCAM.
Together, the observations made herein include the following. (1) CTC expression profiles cluster into three classes, including a major “classical CTC” group, and others that are defined by platelet derived markers or proliferative signatures. (2) Common features shared by virtually all classical CTCs include expression of both epithelial and mesenchymal markers, the stem cell-associated gene Aldh1a2, and three highly expressed transcripts, Klf4, Igfbp5, and Dcn. The specific localization of Igfbp5-expressing cells at the epithelial-stromal boundary within primary tumors may point to a region that contributes significantly to CTC generation. (3) The most highly enriched CTC-specific transcripts shared by almost all classical CTCs encode extracellular matrix proteins, such as Sparc. (4) Aberrant expression in CTCs of this ECM gene product, which is normally abundant in the tumor stromal compartment, is observed in both mouse and human pancreatic CTCs, and its knockdown attenuates cancer cell migration and invasion in reconstituted systems. ( FIG. 15 ) Compared with RNA-seq of partially purified, bulk CTC populations, which required digital subtraction of leukocyte-derived reads (Yu et al., 2012, 2013), the single-cell analysis reported here provides considerably more depth of tumor cell-specific transcript reads, and it allows measurements of CTC heterogeneity.
It is contemplated herein that in addition to the initiating mutations, somatically acquired genetic and epigenetic changes may distinguish CTCs derived from different tumors. Multiple mouse tumors contributed to each of the three distinct clusters of CTCs. Despite their atypical expression pattern, the identification of platelet-associated and proliferative CTC subsets as being tumor-derived is established by their inclusion of lineage-tagged tumor cells. The more characteristic expression pattern exhibited by the classical CTC cluster enabled detailed comparison with primary tumor cells, thereby providing further insight into the origin and properties of CTCs.
Mouse pancreatic classical CTCs uniformly lose expression of the epithelial marker E-cadherin (Cdh1), a key feature of epithelial-to-mesenchymal transition. However, the cells do not lose expression of other epithelial markers, such as cytokeratins, nor is there a consistent increase in classical mesenchymal markers such as vimentin. As such, most classical CTCs appear arrested in a biphenotypic state. Despite their expression of cytokeratins, which are present in the epithelial components of the primary tumor, most other highly expressed markers in CTCs are shared with the stromal component of the primary tumor. Among these stromal genes is Aldh1a2 (Rasheed and Matsui, 2012; Rasheed et al., 2010). A provocative observation relating to the shared epithelial and mesenchymal state of classical CTCs is their virtually universal (93%) expression of Igfbp5, which is uniquely expressed in a small subpopulation of cells at the epithelial/stromal interface within primary tumors. This raises the possibility that this critical location within the primary tumor generates a disproportionate fraction of viable CTCs.
The most unexpected observation from the single-CTC RNAseq study is the high abundance of ECM transcripts in the vast majority of classical CTCs. The coexpression of pancreatic cancer-enriched cytokeratins (Krt7 and Krt19) in single cells expressing these ECM gene products excludes the possibility that these represent circulating tumor-derived fibroblasts.
Consistent with the aberrant expression of SPARC in some pancreatic cancer cells, a subset of patient-derived tumor cell lines also coexpress it along with epithelial cytokeratins. The reduction in cell migration and metastatic potential exhibited by these pancreatic cell lines following SPARC knockdown indicates that it contributes to CTC-mediated metastasis. It is contemplated herein that Sparc expression contributes to metastasis, but inherent redundancies in ECM protein expression may mitigate this effect in some embodiments.
Considerable effort has been directed to targeting the pancreatic cancer stroma as a means of improving delivery of chemotherapeutics as well as stripping tumor cells of their supportive microenvironment (Neesse et al., 2011; Olive et al., 2009; Provenzano et al., 2012; Rasheed et al., 2012). The findings described herein, e.g., that these gene products are also expressed by CTCs themselves suggests a remarkable level of cellular plasticity. To the extent that invasive properties of CTCs are mediated in part by expression of such ECM proteins, it also raises the possibility of targeting cancer cells in the blood.
TABLE 13
Human CTC ECM Gene Expression
Percent of Samples >100 RPM
ECM Gene
Prostate
Count
Symbol
All CTCs
PDAC CTCs
Breast CTCs
CTCs
1
ANXA2
36.3%
0.0%
51.7%
33.8%
2
SPON2
29.2%
0.0%
3.4%
41.6%
3
LGALS3
22.1%
42.9%
37.9%
14.3%
4
SPARC
21.2%
100.0%
31.0%
10.4%
5
LGALS3BP
16.8%
0.0%
34.5%
11.7%
6
MGP
15.9%
57.1%
44.8%
1.3%
7
LAMC1
15.0%
0.0%
6.9%
19.5%
8
SMC3
15.0%
42.9%
17.2%
11.7%
9
CALR
14.2%
0.0%
6.9%
18.2%
10
TIMP1
13.3%
14.3%
27.6%
7.8%
11
MMP24
11.5%
0.0%
10.3%
13.0%
12
DAG1
10.6%
0.0%
20.7%
7.8%
13
ERBB2IP
10.6%
14.3%
20.7%
6.5%
14
MMP19
10.6%
0.0%
10.3%
11.7%
15
AGRN
8.8%
0.0%
6.9%
10.4%
16
CRTAP
8.8%
0.0%
6.9%
10.4%
17
COL24A1
8.0%
57.1%
17.2%
0.0%
18
ANG
7.1%
0.0%
0.0%
10.4%
19
MFAP1
7.1%
0.0%
6.9%
7.8%
20
VWF
7.1%
14.3%
17.2%
2.6%
21
VWA1
7.1%
0.0%
3.4%
9.1%
22
TIMP2
6.2%
0.0%
13.8%
3.9%
23
ECM1
6.2%
0.0%
24.1%
0.0%
24
LTBP1
6.2%
28.6%
10.3%
2.6%
25
LGALS1
6.2%
0.0%
10.3%
5.2%
26
SERPINA1
6.2%
0.0%
20.7%
1.3%
27
SPOCK1
6.2%
14.3%
0.0%
7.8%
28
TFF3
6.2%
0.0%
17.2%
2.6%
29
NPNT
5.3%
0.0%
3.4%
6.5%
30
TFIP11
5.3%
14.3%
6.9%
3.9%
31
COL9A2
4.4%
0.0%
0.0%
6.5%
32
COL6A1
4.4%
0.0%
0.0%
6.5%
33
FN1
4.4%
14.3%
10.3%
1.3%
34
LAD1
4.4%
0.0%
10.3%
2.6%
35
LAMA1
4.4%
14.3%
3.4%
3.9%
36
LAMB2
4.4%
0.0%
10.3%
2.6%
37
MATN2
4.4%
14.3%
3.4%
3.9%
38
ZP3
4.4%
0.0%
0.0%
6.5%
39
ADAMTSL3
3.5%
28.6%
3.4%
1.3%
40
FRAS1
3.5%
14.3%
0.0%
3.9%
41
TIMP3
3.5%
0.0%
3.4%
3.9%
42
DST
3.5%
0.0%
6.9%
2.6%
43
GFOD2
3.5%
14.3%
0.0%
3.9%
44
LAMA3
3.5%
14.3%
0.0%
3.9%
45
LAMB1
3.5%
14.3%
0.0%
3.9%
46
MMP7
3.5%
0.0%
0.0%
5.2%
47
ANGPTL4
2.7%
0.0%
0.0%
3.9%
48
BMP4
2.7%
0.0%
0.0%
3.9%
49
LTBP2
2.7%
28.6%
3.4%
0.0%
50
LEPRE1
2.7%
0.0%
0.0%
3.9%
51
LUM
2.7%
0.0%
0.0%
3.9%
52
NID2
2.7%
14.3%
6.9%
0.0%
53
SLC1A3
2.7%
28.6%
0.0%
1.3%
54
TECTA
2.7%
14.3%
3.4%
1.3%
55
THSD4
2.7%
0.0%
6.9%
1.3%
56
ADAMTS15
1.8%
0.0%
6.9%
0.0%
57
USH2A
1.8%
14.3%
3.4%
0.0%
58
APLP1
1.8%
0.0%
0.0%
2.6%
59
COL4A3
1.8%
14.3%
3.4%
0.0%
60
COL7A1
1.8%
0.0%
3.4%
1.3%
61
COL11A1
1.8%
0.0%
6.9%
0.0%
62
COL11A2
1.8%
0.0%
0.0%
2.6%
63
COL15A1
1.8%
28.6%
0.0%
0.0%
64
CTGF
1.8%
0.0%
0.0%
2.6%
65
CRISP3
1.8%
0.0%
0.0%
2.6%
66
DCN
1.8%
0.0%
0.0%
2.6%
67
ENTPD2
1.8%
0.0%
0.0%
2.6%
68
FMOD
1.8%
0.0%
3.4%
1.3%
69
GPC1
1.8%
0.0%
0.0%
2.6%
70
HSPG2
1.8%
0.0%
0.0%
2.6%
71
LAMA5
1.8%
0.0%
3.4%
1.3%
72
LAMC2
1.8%
14.3%
0.0%
1.3%
73
MMP10
1.8%
0.0%
3.4%
1.3%
74
MMP12
1.8%
0.0%
0.0%
2.6%
75
NTN4
1.8%
0.0%
6.9%
0.0%
76
NAV2
1.8%
0.0%
6.9%
0.0%
77
PAPLN
1.8%
0.0%
3.4%
1.3%
78
SFTPA2
1.8%
0.0%
0.0%
2.6%
79
VCAN
1.8%
14.3%
0.0%
1.3%
80
ADAMTS13
0.9%
0.0%
3.4%
0.0%
81
ADAMTS3
0.9%
14.3%
0.0%
0.0%
82
ADAMTS5
0.9%
14.3%
0.0%
0.0%
83
ADAMTSL4
0.9%
0.0%
0.0%
1.3%
84
EFEMP1
0.9%
0.0%
3.4%
0.0%
85
EFEMP2
0.9%
0.0%
3.4%
0.0%
86
EGFLAM
0.9%
14.3%
0.0%
0.0%
87
KAL1
0.9%
0.0%
0.0%
1.3%
88
KAZALD1
0.9%
0.0%
0.0%
1.3%
89
MAMDC2
0.9%
14.3%
0.0%
0.0%
90
SMOC1
0.9%
0.0%
0.0%
1.3%
91
SMOC2
0.9%
0.0%
0.0%
1.3%
92
ACHE
0.9%
0.0%
0.0%
1.3%
93
AMTN
0.9%
0.0%
3.4%
0.0%
94
ANXA2P2
0.9%
0.0%
3.4%
0.0%
95
CPZ
0.9%
0.0%
3.4%
0.0%
96
CHADL
0.9%
0.0%
0.0%
1.3%
97
COCH
0.9%
0.0%
0.0%
1.3%
98
COL6A6
0.9%
14.3%
0.0%
0.0%
99
COL1A2
0.9%
0.0%
3.4%
0.0%
100
COL2A1
0.9%
0.0%
0.0%
1.3%
101
COL4A1
0.9%
14.3%
0.0%
0.0%
102
COL4A2
0.9%
0.0%
0.0%
1.3%
103
COL4A6
0.9%
0.0%
0.0%
1.3%
104
COL5A1
0.9%
14.3%
0.0%
0.0%
105
COL6A2
0.9%
0.0%
0.0%
1.3%
106
COL8A1
0.9%
14.3%
0.0%
0.0%
107
COL12A1
0.9%
14.3%
0.0%
0.0%
108
COL14A1
0.9%
14.3%
0.0%
0.0%
109
COL19A1
0.9%
14.3%
0.0%
0.0%
110
COL17A1
0.9%
14.3%
0.0%
0.0%
111
COL22A1
0.9%
14.3%
0.0%
0.0%
112
ENTPD1
0.9%
14.3%
0.0%
0.0%
113
FBN2
0.9%
0.0%
0.0%
1.3%
114
FBN3
0.9%
0.0%
3.4%
0.0%
115
FBLN1
0.9%
14.3%
0.0%
0.0%
116
FBLN7
0.9%
0.0%
0.0%
1.3%
117
GPC4
0.9%
0.0%
3.4%
0.0%
118
HMCN1
0.9%
14.3%
0.0%
0.0%
119
IMPG1
0.9%
14.3%
0.0%
0.0%
120
IMPG2
0.9%
0.0%
3.4%
0.0%
121
LAMA2
0.9%
0.0%
3.4%
0.0%
122
LAMB3
0.9%
14.3%
0.0%
0.0%
123
MEPE
0.9%
0.0%
3.4%
0.0%
124
MMP1
0.9%
14.3%
0.0%
0.0%
125
MMP2
0.9%
0.0%
3.4%
0.0%
126
MMP25
0.9%
0.0%
0.0%
1.3%
127
MMP3
0.9%
0.0%
3.4%
0.0%
128
MMP9
0.9%
14.3%
0.0%
0.0%
129
OGN
0.9%
14.3%
0.0%
0.0%
130
PI3
0.9%
0.0%
0.0%
1.3%
131
PRELP
0.9%
14.3%
0.0%
0.0%
132
PTPRZ1
0.9%
14.3%
0.0%
0.0%
133
RELN
0.9%
0.0%
3.4%
0.0%
134
ADAMTSL2
0.9%
0.0%
0.0%
1.3%
135
TGFBI
0.9%
0.0%
3.4%
0.0%
136
UCMA
0.9%
0.0%
3.4%
0.0%
137
VIT
0.9%
0.0%
3.4%
0.0%
138
WNT10A
0.9%
14.3%
0.0%
0.0%
139
WNT10B
0.9%
0.0%
0.0%
1.3%
140
WNT11
0.9%
0.0%
3.4%
0.0%
141
WNT4
0.9%
0.0%
0.0%
1.3%
142
ZP2
0.9%
14.3%
0.0%
0.0%
143
ADAMTS1
0.0%
0.0%
0.0%
0.0%
144
ADAMTS10
0.0%
0.0%
0.0%
0.0%
145
ADAMTS12
0.0%
0.0%
0.0%
0.0%
146
ADAMTS14
0.0%
0.0%
0.0%
0.0%
147
ADAMTS16
0.0%
0.0%
0.0%
0.0%
148
ADAMTS17
0.0%
0.0%
0.0%
0.0%
149
ADAMTS18
0.0%
0.0%
0.0%
0.0%
150
ADAMTS19
0.0%
0.0%
0.0%
0.0%
151
ADAMTS2
0.0%
0.0%
0.0%
0.0%
152
ADAMTS20
0.0%
0.0%
0.0%
0.0%
153
ADAMTS4
0.0%
0.0%
0.0%
0.0%
154
ADAMTS6
0.0%
0.0%
0.0%
0.0%
155
ADAMTS8
0.0%
0.0%
0.0%
0.0%
156
ADAMTS9
0.0%
0.0%
0.0%
0.0%
157
ADAMTSL1
0.0%
0.0%
0.0%
0.0%
158
ADAMTSL5
0.0%
0.0%
0.0%
0.0%
159
CD248
0.0%
0.0%
0.0%
0.0%
160
DGCR6
0.0%
0.0%
0.0%
0.0%
161
EGFL6
0.0%
0.0%
0.0%
0.0%
162
EMID1
0.0%
0.0%
0.0%
0.0%
163
FREM1
0.0%
0.0%
0.0%
0.0%
164
FREM2
0.0%
0.0%
0.0%
0.0%
165
RELL2
0.0%
0.0%
0.0%
0.0%
166
SPARCL1
0.0%
0.0%
0.0%
0.0%
167
ACAN
0.0%
0.0%
0.0%
0.0%
168
AMBN
0.0%
0.0%
0.0%
0.0%
169
AMELX
0.0%
0.0%
0.0%
0.0%
170
AMELY
0.0%
0.0%
0.0%
0.0%
171
ASPN
0.0%
0.0%
0.0%
0.0%
172
BGN
0.0%
0.0%
0.0%
0.0%
173
BCAN
0.0%
0.0%
0.0%
0.0%
174
CRTAC1
0.0%
0.0%
0.0%
0.0%
175
CILP2
0.0%
0.0%
0.0%
0.0%
176
CILP
0.0%
0.0%
0.0%
0.0%
177
COMP
0.0%
0.0%
0.0%
0.0%
178
CHL1
0.0%
0.0%
0.0%
0.0%
179
CHI3L1
0.0%
0.0%
0.0%
0.0%
180
CHAD
0.0%
0.0%
0.0%
0.0%
181
C6orf15
0.0%
0.0%
0.0%
0.0%
182
CCDC80
0.0%
0.0%
0.0%
0.0%
183
CTHRC1
0.0%
0.0%
0.0%
0.0%
184
COL1A1
0.0%
0.0%
0.0%
0.0%
185
COL3A1
0.0%
0.0%
0.0%
0.0%
186
COL4A4
0.0%
0.0%
0.0%
0.0%
187
COL4A5
0.0%
0.0%
0.0%
0.0%
188
COL9A1
0.0%
0.0%
0.0%
0.0%
189
COL9A3
0.0%
0.0%
0.0%
0.0%
190
COL5A2
0.0%
0.0%
0.0%
0.0%
191
COL5A3
0.0%
0.0%
0.0%
0.0%
192
COL6A3
0.0%
0.0%
0.0%
0.0%
193
COL8A2
0.0%
0.0%
0.0%
0.0%
194
COL10A1
0.0%
0.0%
0.0%
0.0%
195
COL16A1
0.0%
0.0%
0.0%
0.0%
196
COL18A1
0.0%
0.0%
0.0%
0.0%
197
COL21A1
0.0%
0.0%
0.0%
0.0%
198
COL27A1
0.0%
0.0%
0.0%
0.0%
199
COL28A1
0.0%
0.0%
0.0%
0.0%
200
COLQ
0.0%
0.0%
0.0%
0.0%
201
DMP1
0.0%
0.0%
0.0%
0.0%
202
DSPP
0.0%
0.0%
0.0%
0.0%
203
DPT
0.0%
0.0%
0.0%
0.0%
204
ELN
0.0%
0.0%
0.0%
0.0%
205
EMILIN1
0.0%
0.0%
0.0%
0.0%
206
EMILIN2
0.0%
0.0%
0.0%
0.0%
207
EMILIN3
0.0%
0.0%
0.0%
0.0%
208
ENAM
0.0%
0.0%
0.0%
0.0%
209
EPYC
0.0%
0.0%
0.0%
0.0%
210
ECM2
0.0%
0.0%
0.0%
0.0%
211
FBN1
0.0%
0.0%
0.0%
0.0%
212
FGF1
0.0%
0.0%
0.0%
0.0%
213
FGF9
0.0%
0.0%
0.0%
0.0%
214
FLRT1
0.0%
0.0%
0.0%
0.0%
215
FLRT2
0.0%
0.0%
0.0%
0.0%
216
FLRT3
0.0%
0.0%
0.0%
0.0%
217
FBLN2
0.0%
0.0%
0.0%
0.0%
218
FBLN5
0.0%
0.0%
0.0%
0.0%
219
GPLD1
0.0%
0.0%
0.0%
0.0%
220
GPC2
0.0%
0.0%
0.0%
0.0%
221
GPC3
0.0%
0.0%
0.0%
0.0%
222
GPC5
0.0%
0.0%
0.0%
0.0%
223
GPC6
0.0%
0.0%
0.0%
0.0%
224
HAPLN1
0.0%
0.0%
0.0%
0.0%
225
HAPLN2
0.0%
0.0%
0.0%
0.0%
226
HAPLN3
0.0%
0.0%
0.0%
0.0%
227
HAPLN4
0.0%
0.0%
0.0%
0.0%
228
KERA
0.0%
0.0%
0.0%
0.0%
229
LAMA4
0.0%
0.0%
0.0%
0.0%
230
LAMB4
0.0%
0.0%
0.0%
0.0%
231
LAMC3
0.0%
0.0%
0.0%
0.0%
232
LTBP4
0.0%
0.0%
0.0%
0.0%
233
LOX
0.0%
0.0%
0.0%
0.0%
234
LOXL1
0.0%
0.0%
0.0%
0.0%
235
MATN1
0.0%
0.0%
0.0%
0.0%
236
MATN3
0.0%
0.0%
0.0%
0.0%
237
MMP11
0.0%
0.0%
0.0%
0.0%
238
MMP13
0.0%
0.0%
0.0%
0.0%
239
MMP16
0.0%
0.0%
0.0%
0.0%
240
MMP17
0.0%
0.0%
0.0%
0.0%
241
MMP20
0.0%
0.0%
0.0%
0.0%
242
MMP23A
0.0%
0.0%
0.0%
0.0%
243
MMP26
0.0%
0.0%
0.0%
0.0%
244
MMP27
0.0%
0.0%
0.0%
0.0%
245
MMP28
0.0%
0.0%
0.0%
0.0%
246
MMP8
0.0%
0.0%
0.0%
0.0%
247
MFAP5
0.0%
0.0%
0.0%
0.0%
248
MFAP2
0.0%
0.0%
0.0%
0.0%
249
MFAP4
0.0%
0.0%
0.0%
0.0%
250
MUC4
0.0%
0.0%
0.0%
0.0%
251
MMRN2
0.0%
0.0%
0.0%
0.0%
252
NTN1
0.0%
0.0%
0.0%
0.0%
253
NTN3
0.0%
0.0%
0.0%
0.0%
254
NID1
0.0%
0.0%
0.0%
0.0%
255
NYX
0.0%
0.0%
0.0%
0.0%
256
ODAM
0.0%
0.0%
0.0%
0.0%
257
OPTC
0.0%
0.0%
0.0%
0.0%
258
OMD
0.0%
0.0%
0.0%
0.0%
259
OTOA
0.0%
0.0%
0.0%
0.0%
260
POSTN
0.0%
0.0%
0.0%
0.0%
261
PODN
0.0%
0.0%
0.0%
0.0%
262
PODNL1
0.0%
0.0%
0.0%
0.0%
263
PRSS36
0.0%
0.0%
0.0%
0.0%
264
RPTN
0.0%
0.0%
0.0%
0.0%
265
RBP3
0.0%
0.0%
0.0%
0.0%
266
SPN
0.0%
0.0%
0.0%
0.0%
267
ADAMTS7
0.0%
0.0%
0.0%
0.0%
268
SPOCK2
0.0%
0.0%
0.0%
0.0%
269
SPOCK3
0.0%
0.0%
0.0%
0.0%
270
SPON1
0.0%
0.0%
0.0%
0.0%
271
SFTPA1
0.0%
0.0%
0.0%
0.0%
272
SFTPD
0.0%
0.0%
0.0%
0.0%
273
TECTB
0.0%
0.0%
0.0%
0.0%
274
TNC
0.0%
0.0%
0.0%
0.0%
275
TNN
0.0%
0.0%
0.0%
0.0%
276
TNR
0.0%
0.0%
0.0%
0.0%
277
TNXB
0.0%
0.0%
0.0%
0.0%
278
THBS4
0.0%
0.0%
0.0%
0.0%
279
TFPI2
0.0%
0.0%
0.0%
0.0%
280
TGFB1
0.0%
0.0%
0.0%
0.0%
281
TINAG
0.0%
0.0%
0.0%
0.0%
282
TNFRSF11B
0.0%
0.0%
0.0%
0.0%
283
VEGFA
0.0%
0.0%
0.0%
0.0%
284
VTN
0.0%
0.0%
0.0%
0.0%
285
VWC2
0.0%
0.0%
0.0%
0.0%
286
WNT2
0.0%
0.0%
0.0%
0.0%
287
WNT1
0.0%
0.0%
0.0%
0.0%
288
WNT16
0.0%
0.0%
0.0%
0.0%
289
WNT2B
0.0%
0.0%
0.0%
0.0%
290
WNT3
0.0%
0.0%
0.0%
0.0%
291
WNT3A
0.0%
0.0%
0.0%
0.0%
292
WNT5A
0.0%
0.0%
0.0%
0.0%
293
WNT5B
0.0%
0.0%
0.0%
0.0%
294
WNT6
0.0%
0.0%
0.0%
0.0%
295
WNT7A
0.0%
0.0%
0.0%
0.0%
296
WNT7B
0.0%
0.0%
0.0%
0.0%
297
WNT8A
0.0%
0.0%
0.0%
0.0%
298
WNT8B
0.0%
0.0%
0.0%
0.0%
299
WNT9A
0.0%
0.0%
0.0%
0.0%
300
WNT9B
0.0%
0.0%
0.0%
0.0%
301
ZP1
0.0%
0.0%
0.0%
0.0%
302
ZP4
0.0%
0.0%
0.0%
0.0%
TABLE 10
Most significant Gene Sets Enriched in CTC-pro vs. CTC-c
q-value < 0.01
Odds
Benjamini
Source
Term
Count
Ratio
(q-value)
GOTERM_BP_FAT
GO: 0002495~antigen processing and presentation of
5
59.81
6.97E−04
peptide antigen via MHC class II
GOTERM_BP_FAT
GO: 0019886~antigen processing and presentation of
5
59.81
6.97E−04
exogenous peptide antigen via MHC class II
GOTERM_BP_FAT
GO: 0002504~antigen processing and presentation of
5
50.36
7.34E−04
peptide or polysaccharide antigen via MHC class II
GOTERM_BP_FAT
GO: 0002478~antigen processing and presentation of
5
41.60
1.10E−03
exogenous peptide antigen
GOTERM_BP_FAT
GO: 0019884~antigen processing and presentation of
5
34.18
1.87E−03
exogenous antigen
GOTERM_BP_FAT
GO: 0048002~antigen processing and presentation of
5
27.34
3.72E−03
peptide antigen
GOTERM_BP_FAT
GO: 0001775~cell activation
9
7.00
3.82E−03
GOTERM_BP_FAT
GO: 0019882~antigen processing and presentation
6
13.20
7.40E−03
TABLE 11
Most significant Gene Sets Enriched in CTC-plt vs. CTC-c
q-value < 0.01
Odds
Benjamini
Source
Term
Count
Ratio
(q-value)
GOTERM_BP_FAT
GO: 0042060~wound healing
18
7.8
1.86E−07
GOTERM_BP_FAT
GO: 0007596~blood coagulation
15
10.4
9.31E−08
GOTERM_BP_FAT
GO: 0050817~coagulation
15
10.4
9.31E−08
GOTERM_BP_FAT
GO: 0007599~hemostasis
15
10.3
7.59E−08
GOTERM_BP_FAT
GO: 0050878~regulation of body
15
8.2
1.30E−06
fluid levels
GOTERM_BP_FAT
GO: 0030029~actin filament-based
20
5.5
1.14E−06
process
GOTERM_BP_FAT
GO: 0007010~cytoskeleton
26
3.9
3.95E−06
organization
GOTERM_BP_FAT
GO: 0030036~actin cytoskeleton
18
5.3
1.11E−05
organization
GOTERM_BP_FAT
GO: 0009611~response to
26
3.6
1.02E−05
wounding
GOTERM_BP_FAT
GO: 0007155~cell adhesion
33
2.9
2.86E−05
GOTERM_BP_FAT
GO: 0022610~biological adhesion
33
2.8
2.70E−05
GOTERM_BP_FAT
GO: 0001775~cell activation
19
3.7
4.70E−04
GOTERM_BP_FAT
GO: 0030168~platelet activation
6
18.2
1.68E−03
GOTERM_BP_FAT
GO: 0007229~integrin-mediated
10
6.4
2.95E−03
signaling pathway
GOTERM_BP_FAT
GO: 0016192~vesicle-mediated
25
2.6
3.81E−03
transport
MSigDBv3.1 CGP
GNATENKO PLATELET SIGNATURE
20
55.1
3.91E−24
MSigDBv3.1 CGP
TENEDINI MEGAKARYOCYTE
14
15.3
1.35E−11
MARKERS
MSigDBv3.1
REACTOME FACTORS INVOLVED IN
6
2.9
2.25E−02
CP: REACTOME
MEGAKARYOCYTE DEVELOPMENT
AND PLATELET PRODUCTION
TABLE 12
Significantly Expressed Genes by Rank Product (FDR <0.01)
Primary
CTC-c vs
Tumor vs
Primary
CTC-c
Tumor
Enriched
CTC-plt vs
Count
Enriched Gene
Gene
CTC-c
CTC-pro vs CTC-c
1
Upk3b
Tff2
Clec1b
kg:uc007pge.1
2
Ier2
Wfdc2
AU023871
kg:uc007pgd.1
3
Egr1
Lamb3
Alox12
kg:uc007pgf.1
4
Nkain4
Lad1
Itga2b
kg:uc007pgg.1
5
Igfbp5
Dmbt1
Ppbp
Igj
6
Slc6a4
Npy
Gng11
kg:uc012enb.1
7
Klf4
Pmepa1
Vwf
2010001M09Rik
8
Tmem221
Kcnn4
Pf4
kg:uc009cfw.1
9
Arl4d
Serinc2
Fcer1g
kg:uc007pgi.1
10
Lrrn4
5730559C18Rik
Tmem40
kg:uc007pgh.1
11
Cldn15
Muc1
Hba-a2
kg:uc007yos.1
12
Gpm6a
Chi3l3
Stom
Coro1a
13
Atf3
Pglyrp1
Beta-s
Pou2af1
14
Ptma
Arl4c
Plek
kg:uc011yvj.1
15
Slc9a3r1
Spp1
Srgn
Glipr1
16
Fos
Col15a1
Myl9
Cd52
17
Tmem119
C1qb
Cd84
Cd79b
18
Ptgis
Tnnt2
F5
Sec11c
19
Dcn
Gkn3
Treml1
Tnfrsf17
20
Gbp2
Onecut2
Hbb-b1
Krr1
21
Dmkn
Mmp7
Itgb3
Gmfg
22
Sdc4
Cd74
Gp9
Ccr9
23
Ildr2
Ctss
Mpl
Pycard
24
Akap2
Lamc2
Ctla2a
Derl3
25
Gfpt2
Olfml3
Tubb1
Rac2
26
Klf6
Lgals4
Mylk
Srgn
27
Btg2
Lcn2
F13a1
Cytip
28
Myl7
Ly6a
Slamf1
Edem2
29
Igfbp6
Pak1
Rgs10
Itgb7
30
Gpr133
Capn5
Mkrn1
Lsp1
31
Oasl2
Ptprn
Laptm5
Lcp1
32
Pfn1
Reg3b
1810058l24Rik
Cyfip2
33
Cap1
Fmnl3
Itgb2
Nans
34
Nfkbia
Sdc1
Slc2a3
Slamf7
35
Malat1
Prom1
Pcmt1
Ell2
36
Rarres2
Ankrd50
Gp5
H2-Eb1
37
Rspo1
Ccl6
Ube2o
Creld2
38
Espn
Slc4a11
5430417L22Rik
Cd74
39
Klf9
Oraov1
Ptpn18
Blnk
40
Zbtb7c
Aldh1l1
Lat
Fmnl1
41
Brd2
Slc20a1
Fermt3
Snrnp70
42
Olfr1033
Cldn7
Nrgn
Sec61b
43
Wt1
Acsbg1
Mrvi1
Edem1
44
Esam
Las1l
Lyz2
Tspan13
45
kg:uc009igb.1
C1qc
Epb4.1
Psmb8
46
Tmem151a
Lama5
Rasgrp2
Pim1
47
Mgll
Mgat4a
Treml2
Sept1
48
Csrnp1
Cldn2
Hist1h4i
Cd48
49
Cd9
Mcpt2
March2
Sub1
50
Gjb5
Fxyd3
Ltbp1
Lims1
51
Lrrc61
Il4ra
Nptn
Ncoa2
52
Wasf2
Itga5
Abtb1
Ctnnbl1
53
Pdpn
Porcn
Ctla2b
Fdps
54
kg:uc009ogv.1
Mast3
Prkab2
Ube2j1
55
Sdpr
Scara3
Arhgdib
Mettl1
56
Gpr64
Atox1
Alas2
Lax1
57
Flnc
Arrdc1
Odc1
Rilpl2
58
Add3
Mmp2
Ptpn11
Ctse
59
Gata6
Saa3
Dhcr24
Glrx
60
Wfdc1
Serpinf1
Mfsd2b
Fut8
61
A130040M12Rik
Sox11
Gp1bb
Al662270
62
Ankrd12
Prpsap1
Rbpms2
Gramd3
63
Adamtsl1
Mcpt1
Fyb
Il2rg
64
C2
Mfge8
Smox
Rasgrp3
65
Prss23
Col18a1
P2rx1
Impdh1
66
Ube2v1
Lyz2
Otud7b
Plek
67
Cryab
C1qa
kg:uc007ttx.1
Ints5
68
Pkhd1l1
Acp5
Samd14
Blmh
69
Rtn1
Angptl4
Clca1
Dnmt1
70
Birc6
Ccnd1
kg:uc007tty.1
Galk1
71
Xdh
Asl
Gpr56
kg:uc007hxv.1
72
Cd34
Ctxn1
Sh3bgrl2
Ccdc88b
73
Rab6b
Pgs1
Pttg1ip
Selplg
74
Dusp1
Anapc2
Nomo1
Sar1b
75
Clic4
Cp
Gnaz
Lat2
76
C3
Gpx3
Mmrn1
Slc16a6
77
Rhob
Lama3
Gp1ba
Mki67
78
Mir3064
Rbp1
Sh3bgrl3
Dnajc3
79
Thbd
Cotl1
Slc24a3
H2-Ab1
80
Dpysl2
Nek6
Sord
Ndufs6
81
Cobl
Cpxm1
Nfe2
Actr3
82
Npr1
Sfrp1
Tuba4a
Etnk1
83
Dnajb9
Ttr
Zyx
Herpud1
84
Arhgap29
Gsto1
Cnn2
Ptpn7
85
Cav1
Npepl1
Itgb5
Ctss
86
Gbp7
Usmg5
Gata1
Cs
87
Hes1
Polr2l
Hist1h1c
Fbxw7
88
Gm16897
Sphk1
Tbxas1
Ppp2r5c
89
Ppp1r12a
Asxl1
Ptplad2
Znrd1
90
Sv2a
Ctsh
Bpgm
Rfc2
91
Ang
Egfl7
Pdlim7
Preb
92
Aldh1a2
C1qtnf6
Mmd
Fcer1g
93
Cryl1
Rras
G6b
Dnajb11
94
Kank1
Lgi4
kg:uc009duo.1
Slc35b1
95
2210403K04Rik
Hmga2
Lyz1
Sin3b
96
kg:uc009okn.1
Cep250
Tacc1
Nktr
97
Osr1
B4galt3
Dap
98
kg:uc008ewj.2
Tmem223
Mast2
99
kg:uc009tuw.1
Ltbp2
Atp2a3
100
Gadd45b
Tnfrsf23
Snca
101
Ablim3
Col7a1
Stx11
102
Clec3b
Ggct
C030046I01Rik
103
Usp25
Rab25
Trpt1
104
Sntb2
Nedd8
Tsc22d1
105
Rock2
9430023L20Rik
Prkar2b
106
Col14a1
Arl2
Cd9
107
Cd200
Wbp1
Pgm2l1
108
kg:uc008ehr.1
H2-Ab1
Gp6
109
Atp2b1
Preb
Pde5a
110
Exoc4
Sgsm3
Itga6
111
Abcb1b
Sfn
Itgal
112
Nrgn
Prrx2
Edem1
113
kg:uc009cvm.1
Ptprk
Isg20
114
Ncoa4
Reg1
Cdc42ep5
115
Ndufa4
Sdcbp2
Nipal3
116
Upk1b
Pcbd1
Ccdc92
117
Jun
Slc25a1
Sort1
118
Syne2
Vamp5
Ly6g6c
119
kg:uc007bvx.1
Crlf1
Ubash3b
120
Ap4e1
Avil
Inf2
121
Spock2
2700094K13Rik
Asap1
122
Efemp1
Ctse
Sec11c
123
Prpf40a
Penk
Gas2l1
124
Tspan5
Tmc4
Parvb
125
Lgals7
Dhrs3
Tmsb4x
126
Kif5b
Ap1s1
kg:uc007xrw.1
127
Psip1
Arl6ip4
Nudt3
128
kg:uc008oki.1
9430008C03Rik
Bcl2l1
129
1810014B01Rik
Fcer1g
B230312A22Rik
130
Ptges3
Uqcr11
Cnp
131
Limch1
Nhp2
Plp1
132
Bicd1
Plbd2
Cnst
133
Rdx
Capg
Rgs18
134
Pcdh15
Pnpla6
Lsm12
135
Foxn3
Ppdpf
Alox5ap
136
Morf4l2
Hgfac
Ppif
137
Ppp1r15a
Apoe
Spnb1
138
Cdc42ep3
Fam40a
Ormdl3
139
Pard3b
Lyz1
Hpse
140
Bicc1
2200002D01Rik
Srxn1
141
Amhr2
Laptm5
2010002N04Rik
142
Gucy1a3
Qars
Hist1h2bc
143
Psmb2
Tmx2
Cyba
144
Mapkapk3
Fkbp4
Chst12
145
Ube2l6
Plin2
kg:uc009sps.1
146
kg:uc007pff.1
Fcgr3
Max
147
kg:uc007ctp.1
Gkn1
Was
148
Nedd4
Snhg1
Isca1
149
Plxna4
Lsp1
Pdzk1ip1
150
2010107G12Rik
Gm20605
Lyn
151
Ifngr1
Ly6c1
Mob3a
152
Bcam
Aim1
H2-T24
153
Ccnl1
2310007B03Rik
Slc44a1
154
Hoxa5
Tgfbi
Derl1
155
Fhl1
Tsta3
Gclm
156
1810041L15Rik
Pafah1b3
Fech
157
2900002K06Rik
Chid1
Ywhah
158
Hspb1
Smox
Igtp
159
Podn
1500012F01Rik
Myl6
160
Fam63b
Tspan4
Thbs1
161
Hsp90b1
Agrn
Tln1
162
Dpp4
Cfp
kg:uc009apq.1
163
Gas1
Cdh1
Bcap31
164
kg:uc007zak.1
Rasgrf1
Ilk
165
Zc3h13
Nxf1
Epha1
166
Sox6
Pdrg1
2810453I06Rik
167
Arid4a
Polr2j
Rnf19b
168
Tnxb
Suds3
Gsn
169
Tsix
D0H4S114
Flna
170
Scd1
Ccl9
Arrb1
171
Jund
Neat1
kg:uc007pum.1
172
Crls1
Ccdc12
Mbnl1
173
1110003E01Rik
Prr24
Ccnd3
174
Rnase4
Impdh1
Pdlim1
175
Arhgef12
Card10
Ctse
176
Irf7
Cpsf1
Tspan17
177
Bbx
Sema4g
Gpx4
178
Sema5a
Hes6
Bnip3l
179
Mau2
C130074G19Rik
P2ry12
180
Abi3bp
Ctrb1
kg:uc009vev.1
181
Dag1
Rnaseh2a
Prkab1
182
Cyp2s1
Golm1
F2rl2
183
Sfrs18
Ctsz
Stk4
184
Hspb8
Cyb561
Fhl1
185
Cnot6l
Ndufs8
Rnf10
186
Twsg1
Atp6ap1
Rasa3
187
Gpc3
Srd5a1
Taldo1
188
Lrrn4cl
Carkd
Bysl
189
Cdh3
Cd24a
Esd
190
Cyr61
Eng
Aldh2
191
Cyp2d22
Tcirg1
Rhog
192
Hist1h1c
Slc9a3r2
kg:uc009ecr.1
193
Aplp1
0910001L09Rik
Cald1
194
Tbl1x
Cox5b
Wbp2
195
Pcm1
Adipor2
Ptprj
196
Ifi204
Scarf2
Tpm4
197
Nfix
Myo7a
Mxi1
198
Flrt2
Ppap2c
Ly6g6f
199
Heg1
Pea15a
Sla
200
Il6ra
Sh3pxd2b
Slpi
201
Ralbp1
H19
Bicd2
202
Rhoj
Tpd52
Clu
203
Ktn1
2610203C20Rik
Mtmr14
204
Arl6ip5
Naa10
Abca7
205
Crebbp
Fermt1
Ppp1r18
206
Ppig
Sap30l
Kif2a
207
Akap13
Bgn
Prdx6
208
Rab7
Timm13
kg:uc009ize.1
209
Plxdc2
Krt20
Calm3
210
Aldh1a1
Itga3
Dhrs1
211
Bnc2
Pfkl
Cfl1
212
Slc4a4
Agpat6
Glipr2
213
Tbx18
Mrpl11
Slc25a37
214
Zbtb16
Ramp1
Atox1
215
Arid4b
Hmga1
BC057079
216
Enpp2
Gpx2
Pla2g16
217
Ptplad2
0610012G03Rik
Rnf144b
218
Akr1b3
9130017N09Rik
Stk16
219
Gm6644
Cygb
Rsad2
220
Arf5
Tmprss4
Paip2
221
Chi3l1
Paox
Capzb
222
Gpr116
Endod1
Ppp1r12c
223
Cd82
Cndp2
4930412F15Rik
224
Srrm1
Suv39h1
Ninj1
225
Fmo2
Cog4
2510009E07Rik
226
Tgfb1i1
Trim27
kg:uc007vsr.1
227
Qrich1
Cyhr1
Pygb
228
Nfia
Trmt1
Tlk1
229
Pmp22
Zfyve19
Myct1
230
Cdh11
Esrp1
Rnasek
231
Arid5b
kg:uc008oow.1
Ctsd
232
Rbm3
Dync1h1
0610010K14Rik
233
Prelp
Tab1
Bcas3
234
kg:uc007qse.1
Pla2g6
Atpif1
235
Ddx3x
Timp1
Serf2
236
Sulf1
Eif3f
Becn1
237
Spnb2
Abhd11
Tspan9
238
Tspan31
Pmm2
Acer2
239
Prr13
Tyrobp
Vdac3
240
Ppp1cb
Farsb
kg:uc008kbg.1
241
Fbln1
Plod3
Oaz2
242
Gm6548
Abtb1
Serpine2
243
Uap1
Brf1
Ccdc90a
244
Mpdz
Tnk2
Ndufa1
245
Sat1
Rfc2
Tssc1
246
Stim1
Stxbp2
Mboat7
247
Mll3
Pdlim7
Cd44
248
Slurp1
A430105I19Rik
Cxx1c
249
Cd81
Vill
Ecm1
250
Emp2
Bmp1
Mff
251
Trpm7
Mpzl1
Ptpn12
252
Crym
Thy1
Mgmt
253
Enpp4
Stab1
Cox4i1
254
Raly
Aldh16a1
Tollip
255
Celf2
Eif4ebp3
Cds2
256
Ap3s1
Itpripl2
Ybx1
257
C1s
Mrpl52
Gypc
258
Frmd4b
2310002L13Rik
Dgkd
259
Nr4a1
Mcm6
Pecam1
260
Acin1
Kcnk1
Ftl2
261
Plod2
Pmf1
Nt5c3
262
Id1
Cuta
1700037H04Rik
263
Creg1
Nt5dc2
Cd151
264
Zfp318
Rmnd5b
Lpin2
265
Tmem140
Araf
6430548M08Rik
266
Mras
Wwp2
Pon2
267
Vwa5a
Lamb1
Ndufa3
268
Esyt3
Kcne3
6330578E17Rik
269
Hexb
Uqcrq
Mfap3l
270
Nckap1
Gps1
Mink1
271
Nipal3
Rexo4
Ston2
272
Ubxn4
Coro1c
Rac2
273
Zfp36
Hras1
Fyn
274
Hnrnpl
Spint1
Serinc3
275
C1ra
Cblc
Maged2
276
Nnmt
Fhod1
Ap2m1
277
Mut
Atp13a1
Pacsin2
278
kg:uc008jup.1
Man2c1
Ftl1
279
Pnrc1
Vsig2
Adipor1
280
Usp8
Bpgm
kg:uc009qdo.1
281
Pgcp
Bap1
Snap23
282
Junb
Smpd2
Tagln2
283
C1rl
Ubqln4
Cox6c
284
Slc6a6
Sirt7
Creg1
285
kg:uc008znh.1
Krt23
Bsg
286
Aqp1
D8Ertd738e
Cmtm6
287
Myh10
Mapk13
Cntd1
288
Slc43a3
kg:uc008bcq.1
Plekho2
289
Spint2
Polr2g
Arrb2
290
Hnrnph1
Ndufs2
Pard3b
291
Arhgap28
Dad1
Mlec
292
Cfh
Wnt7b
Taf10
293
Brd4
Fam20c
Gabarapl2
294
Fndc1
Cxxc5
Bag1
295
Star
Polr2f
Galnt2
296
Nfkbiz
Ltf
Hk1
297
Arsb
2210407C18Rik
Fbxo9
298
Rnd3
Cdipt
kg:uc009izd.1
299
Stard5
Glrx5
Pnpo
300
Thbs1
Gemin7
Fam46c
301
kg:uc008wkn.1
Man1b1
Pkm
302
Slc26a3
Heatr7a
Ap1b1
303
Phip
Arid5a
Rap1b
304
Usp2
Sumo3
Itgb1
305
Golgb1
Srm
St7
306
Rock1
Plscr3
Smap1
307
Rgma
2210010C17Rik
Rabgap1l
308
Actg1
Fam102a
Tmbim4
309
BC013529
Dlst
H3f3a
310
kg:uc007zwh.1
Vps37c
Frmd8
311
3110062M04Rik
Ngfrap1
Nlrx1
312
Cast
Pold4
Oaz1
313
Mob3c
Grcc10
Fam125b
314
Slc16a1
Wnt7a
Hexa
315
Fam117a
2010111l01Rik
Tspo
316
Pdia3
Pxdn
Dcaf12
317
Trim8
Coasy
Nav1
318
kg:uc009mng.1
Dctn1
Cd24a
319
eg:245190:chr7:m
Ncor2
Uqcr11
320
Sbsn
Postn
Wipf1
321
Serpinb6b
Col4a2
F10
322
Daglb
Cib1
Erlec1
323
Smarca2
Tbc1d13
Map2k3
324
Mef2c
Ccnl2
Stk24
325
Prrc2c
Dcakd
Ldlrap1
326
BC005537
Cdc34
Ehd4
327
Hsp90ab1
Atp6v0b
Atp6v1f
328
Snrnp70
Abhd12
Gnas
329
Ppl
Flot2
Arhgap18
330
Serpinh1
Sla2
Arhgap10
331
Sorbs3
Rhbdf1
Pitpnm1
332
Golga4
Cdh17
S100a1
333
Acbd3
Psmb5
Bin1
334
Hook3
Serf1
Ttyh3
335
Map3k3
Slc15a3
Selp
336
Rhou
Sftpd
Trappc9
337
Smc2
Pop5
Aes
338
C1d
Nudc
Taok3
339
kg:uc008dzh.1
Sh2d5
Zfand3
340
Psmd7
kg:uc007fwp.1
Stim1
341
Dab2
Mrpl37
Rnf114
342
Cep164
Rin1
Sep15
343
Crim1
Podxl
kg:uc012hdk.1
344
Rtf1
Paqr5
Lgals9
345
Fxyd1
Sepx1
Cox6b1
346
H2-D1
Agr2
Riok3
347
Zfp704
Bax
Slc38a10
348
Mtap1a
Rxrb
Rtn3
349
Ascc3
Tes
B3gat2
350
Med13l
Hdac6
Ccndbp1
351
Jup
1110008F13Rik
Rsu1
352
Nid2
Mpnd
kg:uc007upr.1
353
Kdr
Gmppa
Itm2b
354
Ifnar2
Gramd1a
St3gal1
355
5430435G22Rik
Wars
Sec61g
356
Col4a6
Mtap
Ptpn1
357
Il17re
C1qtnf5
kg:uc012bhf.1
358
Gbp3
Mrpl28
B2m
359
Slc39a8
Mfrp
Rasgrp3
360
Cfl2
Kars
Memo1
361
Slc38a1
Lbp
Slc39a4
362
Cuedc1
Plxnb1
Sdcbp
363
Fgf1
2700081O15Rik
Tspan14
364
Gas6
Mrps24
Ubl7
365
Cldn25
Klc4
Nras
366
Sorbs1
Dctn3
Ssx2ip
367
Hspa12a
Kcnq1
kg:uc007zbz.1
368
kg:uc007zts.1
Smurf1
Wbp1
369
Slc1a5
Fam162a
1110003E01Rik
370
Nr3c1
Hip1r
Clip2
371
Adamts5
kg:uc007hyr.2
Gapdh
372
Gpcpd1
Gys1
Gm6578
373
Dpysl3
Sac3d1
Actn1
374
Colec12
Ndufs6
St3gal2
375
Pdcd6ip
Rgl2
3110001D03Rik
376
Dst
Atp5g1
Ctsz
377
Ifit3
Itgb4
kg:uc007vdl.1
378
Chst4
Sars
Fam73a
379
Xist
2310003F16Rik
Vcl
380
Ifi27l2a
Nhp2l1
Lims1
381
Fkbp5
D19Wsu162e
Lars2
382
Agap1
Cd320
Birc2
383
Ankrd11
Pigq
Lamp2
384
kg:uc007qca.1
Chd3
Rasl10a
385
Syt11
Zdhhc4
Mif
386
Ptrf
Eif3l
Rab10
387
Krcc1
St8sia3
Pabpc1
388
Zfp488
Rcan3
Wwp2
389
Lama4
Meg3
Nqo2
390
Aebp1
Nudt4
kg:uc007fte.1
391
Fam134b
Gss
Plxna4
392
Tppp3
Pih1d1
Gm1821
393
Maf
Limd2
Gadd45a
394
Peli1
Ap1s2
Slc25a39
395
Zfp353
BC056474
kg:uc009pet.1
396
Cdon
Mms19
Ubb
397
Sarnp
Clip2
Ppp1r2
398
Atxn7l3b
2310016M24Rik
Rab27b
399
Pef1
Itpa
Cap1
400
App
Slc25a10
Jarid2
401
Mtdh
Fibp
Rnf11
402
Lrrc20
Higd2a
Tmem50b
403
Btbd2
Snrpd2
Myh9
404
Gnb2
Eri3
Tmem128
405
Pigt
Nbeal2
Stradb
406
Efna5
Trim28
Cela1
407
Tm4sf1
S100a4
Ndrg2
408
Coq10b
Ivns1abp
Dhrs3
409
Eif2s3x
Ppp1r18
Hipk1
410
Cmah
Efemp2
Atg9a
411
Sf3b1
Med22
412
Eea1
Nelf
413
Slpi
2810428I15Rik
414
Tmod3
D2Wsu81e
415
Ppp3ca
Trappc6a
416
Tceal8
Trappc2l
417
Anp32a
Antxr2
418
Actb
Rab11fip5
419
Ddx5
Ldhd
420
Cobll1
Npnt
421
Cish
Acrbp
422
Nod1
Pafah1b2
423
Psd
Angptl2
424
Gm10052
Fzr1
425
Lims2
Aaas
426
Stra6
Eif2b2
427
kg:uc007bgn.1
1190003J15Rik
428
Plxdc1
5730403B10Rik
429
Nfe2l1
Adamts13
430
Smpd3
Eif3b
431
Bcl10
Znrf1
432
Ilf3
Pkp3
433
Fam76a
Lemd2
434
Cybrd1
Rab34
435
Gm3893
Mpv17l2
436
Siae
Cdkn2b
437
Ssh2
Snrpe
438
Nfic
Gm14005
439
Btf3
Prdx4
440
Sp100
Xab2
441
Ndn
Dpp3
442
Matr3
Tyms
443
Gm13251
Leprotl1
444
Arhgap5
Uqcr10
445
Zbtb4
Cdk5rap3
446
Pgrmc1
Gorasp2
447
4930402H24Rik
Wbp7
448
Bptf
Sort1
449
Dusp3
Ddx41
450
Pla2g4a
Cct3
451
Brp44l
Mrps33
452
Oxct1
Frmd8
453
Stk40
1110049F12Rik
454
Ddr1
Fscn1
455
Ifi205
Ndufa2
456
Col3a1
Dpcd
457
Nipbl
Unc13a
458
Plk1s1
Eif1ad
459
Bdp1
Sgta
460
Smc3
Chaf1a
461
Ifitm3
Plxna1
462
Ndst1
Hspa9
463
Zbed6
1110014N23Rik
464
Rest
Cd99l2
465
kg:uc007vnc.1
Snrpa
466
Ccdc88a
Mcm7
467
Stat3
Tars2
468
Arf2
Gon4l
469
Trib1
Stk38
470
Gcap14
C1qtnf1
471
Tbc1d15
Tbrg4
472
Igf1r
Tmem132a
473
Ppbp
Cox6c
474
kg:uc008tky.1
Alcam
475
Rab1b
Phka2
476
Krt14
Trim3
477
Med21
Ppp1r14b
478
Gja1
Gpaa1
479
Klf10
Ctps2
480
Id2
Ptpn23
481
Mfap1a
Endog
482
Ogn
Mrto4
483
Gpc4
Mrps6
484
Bst2
Pvr
485
Dtx2
Phgdh
486
Wac
Itpr3
487
Kpna3
Polr2e
488
Kcnab1
Sec16a
489
Orai3
Mdp1
490
Gcsh
Fbf1
491
Wdr92
Mcpt8
492
Olfr613
Rps6ka4
493
Tcf7l1
Mical1
494
Tgfb2
Mrpl34
495
Il16
Agpat3
496
Manf
2310044H10Rik
497
Mgst1
Myo9b
498
kg:uc008tkz.1
Ndufb10
499
Creb3l1
Apex1
500
Txndc5
Elk3
501
Klf2
Cpsf3l
502
Slu7
Tnk1
503
Ttc28
Pmvk
504
1110002B05Rik
Ppp1r16a
505
Zcchc11
Arhgef5
506
Ptp4a2
Lonp1
507
Pbx1
Pla2g7
508
Clcn3
Pip5k1c
509
Tmco7
Inf2
510
Lrrc58
Pgk1
511
Eif3a
Parp6
512
Cldn10
Urm1
513
H2-Q6
Mad2l2
514
Ccdc80
Ing4
515
kg:uc009iln.1
Rbck1
516
Rab5c
Cant1
517
Tsc22d3
Sgpl1
518
Tm4sf5
Ehbp1l1
519
Hmgb1
Runx1
520
Sec62
Slc27a4
521
Maoa
Ndufa7
522
Clec1b
Mcm3ap
523
Mphosph8
1110008P14Rik
524
Oat
Rassf7
525
Ncor1
Ptpmt1
526
Cyb5
Arfgap1
527
Trafd1
Sec61a1
528
Rpp25
Rps6ka1
529
kg:uc007ded.1
Ints1
530
2610101N10Rik
Tpcn1
531
Il6st
Iffo2
532
Evpl
Trim44
533
Psmd11
kg:uc012ctw.1
534
Dync1i2
Golga2
535
Lars2
Msto1
536
Pdia4
Ppp6r3
537
Cd55
Trmt2a
538
Amfr
Appl2
539
Zcchc3
Sparcl1
540
Herpud2
Rapgef1
541
Txnrd1
Zfpl1
542
Vat1
Psmc4
543
Diap1
Mosc2
544
Tmed2
Fam101b
545
Arf3
1500010J02Rik
546
Arap2
Ccdc124
547
St3gal1
Ptges
548
Man1a
Fam189b
549
Rgs10
Th1l
550
Tmsb4x
Kctd2
551
Uba7
Olfr1372-ps1
552
C4b
Hexa
553
Tmem98
Anapc5
554
Lpar2
Serpina3n
555
Gabarapl1
1810046J19Rik
556
Cmtm7
Tmem167
557
Spon2
Gm11428
558
Smarca5
Gcn1l1
559
Mxd4
Kansl3
560
Smc4
Fasn
561
Thsd4
Slc50a1
562
Gsr
Smad3
563
Ptprd
Trip6
564
Clip1
Atp6v1e1
565
Cln8
Chchd5
566
Rbm27
Adssl1
567
Zmat1
Nes
568
Smc6
Ap1b1
569
B2m
Fcgrt
570
Irf2bp2
Ltbp3
571
Ppap2a
Csf2rb
572
Zfhx4
Ssna1
573
Tob2
Mrps16
574
Rabgap1l
Cyba
575
Nfkb2
Cyth2
576
Nfyc
Igf2
577
Ube2d1
Pisd-ps1
578
Creb5
Atp13a2
579
Opa3
Mlph
580
Csnk1a1
Cyp4f16
581
Fam84b
2010107E04Rik
582
Ddr2
Gas5
583
Usp54
Eif3k
584
Akt2
Fam149a
585
Strn3
Mif
586
Hnrnpm
B230312A22Rik
587
eg:497210:chr14:m
Ppp1r12c
588
Tpt1
Tfip11
589
Naa25
Tex10
590
Eef1a1
Slc16a3
591
Parp4
Stk16
592
Msn
Epn1
593
Zbtb20
Noc4l
594
Fermt2
Rcc2
595
Bod1l
Rgs12
596
Sltm
Shkbp1
597
Dapk1
Got2
598
Hnrnpr
Plek2
599
Baz2a
Lilrb3
600
Rnf167
Ndufb5
601
Mapk1
Tesk1
602
eg:320169:chr9:p
Rab24
603
4930523C07Rik
Atp5j2
604
Nf1
Commd9
605
Fam53b
Rtkn
606
Faim2
Prpf19
607
Tgm2
6720401G13Rik
608
Calm2
Ppa1
609
AI848100
Pgp
610
Slc10a3
Hps1
611
Ogdh
Puf60
612
Arl3
Mdm2
613
Timp2
kg:uc012cgd.1
614
Atxn2
kg:uc009uim.1
615
Mll1
Pyy
616
Ces2g
Zfp358
617
Mat2a
Timm8b
618
Esf1
Ddx39
619
Hsp90aa1
Pgm2
620
Zfp385a
kg:uc008gbp.1
621
Zfp672
Sipa1
622
Csda
Mgat1
623
Pf4
Tmem208
624
Arsa
Ruvbl2
625
F11r
8430410A17Rik
626
C4a
Bad
627
Kpna1
Pfdn5
628
Rbbp8
Eme1
629
Oxnad1
kg:uc009mzj.1
630
Rb1cc1
Igf1
631
Setd2
Prkag1
632
Kif1b
kg:uc009sua.1
633
2510002D24Rik
Uap1l1
634
Cep57
Trappc4
635
Chd2
Bola2
636
Serinc5
Usp5
637
Marcksl1
Ear2
638
Shfm1
Cars
639
Bbs4
1810027O10Rik
640
Impad1
Amdhd2
641
Tbcel
Phb
642
Kdelr1
Kcmf1
643
Ninl
Lsmd1
644
Sytl1
Sec11c
645
Tpm3
Pcbp4
646
Rbbp6
Mepce
647
Lman1
Tpd52l2
648
Ankrd17
Trf
649
Naga
Hsd17b11
650
Rbpms
Pilra
651
Magt1
Atn1
652
Tfdp2
Pgf
653
Gem
Nxn
654
Pde4dip
Inpp5k
655
Mrgprf
Actr1a
656
kg:uc008ajk.1
Cd68
657
Itch
Eef1g
658
Elf1
Fbn1
659
Meis2
Hint1
660
Arid1a
March5
661
Serping1
Usp48
662
Slc27a3
Hnf1b
663
Thoc2
Gga3
664
Gsta3
Drosha
665
Hnrnph2
Ubp1
666
Socs3
Pkn3
667
Armcx3
Tmem192
668
Siah1a
Prpf31
669
kg:uc009ize.1
Hspd1
670
Irs2
Otub1
671
Mettl7a1
Mrpl20
672
Ppfibp2
Tead2
673
Blvrb
Phpt1
674
Yipf5
Neu1
675
Plat
Pygo2
676
Gm6578
Myeov2
677
Mat2b
Cdk5
678
Tmpo
Ndor1
679
Metap2
Rbp4
680
Zfp277
Psat1
681
Wls
Mrpl41
682
Mesdc1
Snrpg
683
kg:uc009acs.1
Acot7
684
Col1a2
Vars
685
Csf1
Nono
686
Sulf2
Gtf2i
687
Ifrd1
Traf3
688
Wrnip1
Ppp2r4
689
Flii
Actg2
690
2810474O19Rik
Pi4k2a
691
Sep15
Slc35b2
692
2310030G06Rik
Ubqln1
693
Cmtm3
Ppox
694
Mylip
Bud31
695
Slc8a1
Man2b1
696
Btbd7
Nat15
697
Hdac5
Spon1
698
Zfand6
Cyc1
699
Tapbp
Mpeg1
700
Keap1
Nsun2
701
Ube2n
Rab4a
702
Ssr3
Mtmr11
703
H3f3a
BC004004
704
Myst4
B4galnt1
705
G3bp1
Atp5k
706
Ugdh
Lin37
707
Lamp2
D330041H03Rik
708
Zrsr1
Tbc1d17
709
Pim1
March6
710
Gm9199
2410015M20Rik
711
Supt16h
1810013D10Rik
712
Ano6
Eif2s1
713
Soat1
Traf7
714
Eci1
Rpl36al
715
Plce1
Psenen
716
Atg3
Aip
717
Bnc1
Cmas
718
Pik3c2a
Rpia
719
Pqlc3
Ncbp1
720
Thrap3
Mea1
721
Irak4
Timm50
722
Kdm6b
Ear12
723
Apol9a
Fkbp1a
724
Wnt4
Commd4
725
1500003O03Rik
Col5a3
726
Phf3
Fblim1
727
1110004F10Rik
Cwh43
728
Kansl1
Arl2bp
729
Fth1
Mrpl46
730
Tmem50a
Tcn2
731
Utp20
Add2
732
Smad4
Specc1l
733
Stmn2
Ppcs
734
Gstm1
Vrk3
735
Senp6
Trim25
736
Gda
Nfatc1
737
Nucks1
Rap1gap
738
Ints10
Hsd17b12
739
Syne1
Epas1
740
Itga6
Ddx1
741
Acad9
Prdx6
742
Maged1
Mmp24
743
Spen
Ndufb9
744
Chd1
Phf23
745
Taf3
Rpa2
746
Ptgs1
5031439G07Rik
747
Sparc
Rrp7a
748
R74862
Arfip2
749
B230120H23Rik
Efna1
750
Tmem234
Agps
751
Ryk
Sephs1
752
Dlgap4
Apoc2
753
Atp1b1
Mrps27
754
Parp14
Snn
755
Tgfbr2
Serinc3
756
Ccdc90a
Pdcd5
757
Ncoa1
AA986860
758
Pppde1
Pitpna
759
Luc7l3
Vac14
760
Prg4
2810025M15Rik
761
Rab11fip1
Def8
762
Plk2
Hilpda
763
Ifi35
Eif6
764
Pdap1
Brd7
765
Cd248
Fes
766
Sesn1
Sbf1
767
Ecd
Ak2
768
Ap1s3
1810035L17Rik
769
H2-K1
Lime1
770
Spag9
Hspe1
771
Tshz1
Csrp2bp
772
Dennd5a
Uba5
773
Stag1
Gsta4
774
Gpx8
2900092E17Rik
775
Sod3
776
BC005561
777
kg:uc009vev.1
778
Ywhaz
779
Ganab
780
Rras2
781
Dusp14
782
kg:uc012hdk.1
783
Nr1d1
784
Wwc2
785
Ubxn2a
786
Iqsec1
787
kg:uc007vsr.1
788
Cfl1
789
Csrp1
790
Smchd1
791
Myl12a
792
Ubqln2
793
Tmcc3
794
Kdm5a
795
Rbm25
796
Wdr26
797
Vim
798
Arpc2
799
Calm1
800
Dnaja2
801
Shc1
802
Vps13a
803
Klf7
804
1810074P20Rik
805
BC003331
806
Itpr2
807
Jmjd1c
808
Pcdhgb5
809
Tubb2a
810
Ehd2
811
Ift74
812
Per1
813
Pitpnm2
814
Gstm4
815
Dnmt1
816
Tmco1
817
Lass4
818
Ptprf
819
Sirt2
820
Gfm2
821
Taf7
822
Spop
823
Zzef1
824
Ccdc34
825
Zfp281
826
Tuba1a
827
Ccdc109b
828
Cdk13
829
Dhx15
830
Src
831
Braf
832
Mapre2
833
Anxa7
834
Sept9
835
Alox12
836
Pknox1
837
2610034B18Rik
838
Topors
839
Phf21a
840
Qser1
841
Tirap
842
Fas
843
Lass2
844
6330406l15Rik
845
Parvb
846
Atp1a1
847
Mtmr6
848
Cd109
849
Dnajc1
850
Hp1bp3
851
1600029D21Rik
852
Ttc38
853
Mfhas1
854
Filip1l
855
Zfp148
856
Nkd1
857
Usp16
858
Tlr2
859
Zc3h18
860
Stk10
861
Ltbp4
862
Hdac3
863
Efhd2
864
Prkar2a
865
Atp6v1a
866
Sf3b4
867
Gprc5b
868
Clip3
869
Mettl2
870
Secisbp2
871
Fmod
872
kg:uc009lxf.1
873
Elovl6
874
Bzw1
875
Etfa
876
Hspa2
877
kg:uc007won.1
878
Rnf20
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
Primary
WBC vs
CTC-c
Tumor
Primary
vs
WBC vs
Count
vs WBC
Tumor
WBC
CTC
1
Wfdc2
Ppbp
Olfr1033
Beta-s
2
Spp1
Alas2
Crip1
Alas2
3
Cct3
Nrgn
Ppp1r12a
Hbb-b1
4
Itga3
Cd9
Vcp
Il1b
5
Gsto1
Csf3r
Klf9
Ppbp
6
Mmp2
Il1b
Mprip
Hba-a2
7
Mfge8
Gdpd3
Sdc4
kg:uc007pgs.1
8
Capg
Ms4a1
Gpc5a
kg:uc011yvj.1
9
Cd63
Hbb-b1
Vat1
Coro1a
10
Stub1
Beta-s
Wdr92
Cd74
11
Lad1
kg:uc007pgs.1
S100a11
Gdpd3
12
Myo1h
kg:uc011yvj.1
Clic4
Ccndbp1
13
Igfbp7
Rprl1
Dync1i2
kg:uc009cfw.1
14
Kcnn4
Pfn1
Nfkbiz
kg:uc012enb.1
15
D8Ertd738e
Clec1b
Cyp2s1
Ptprc
16
Lamb3
Ptprc
Esam
Csf3r
17
Chi3l3
Stim1
Surf4
Rac2
18
Arl4c
Ccndbp1
Krt19
Rprl1
19
Col18a1
Cap1
Bsg
H2-Ab1
20
Atox1
Cd79b
Tm4sf1
Epb4.1
21
Ly6a
Alox12
Lgals3
Lyz2
22
Dmbt1
Hba-a2
Clic1
Ctla2b
23
Dync1h1
Ube2l6
Capns1
Pld4
24
Adipor2
Cat
Igfbp6
kg:uc007pgt.1
25
Rpl37
Faim3
Rrbp1
Gng11
26
Kctd10
Dusp1
Calr
Mepce
27
Col15a1
kg:uc007pgt.1
Rtf1
Tyrobp
28
Surf4
E2f2
Ildr2
Isca1
29
Dad1
Phospho1
Mark2
2810453l06Rik
30
Col4a1
Abi3
Mt1
Slc30a9
31
Ap2s1
Sorl1
Akr1b3
Treml2
32
Sdc1
Treml2
Gm6644
Srgn
33
Rpl35
Cytip
Nkain4
Dcaf12
34
Sec61a1
B2m
Ppp2ca
Plek
35
Rras
Fyb
Akap2
Cat
36
Oraov1
Peli1
Hspb1
Alox12
37
Ndufa2
Plek
Ptgis
Fech
38
Anapc2
N4bp3
Msln
Rbm5
39
Pitpna
Fam117a
Emp2
Cd97
40
Psap
Srgn
Capn2
March8
41
Atp5j2
Sept9
Rhoc
Pnpo
42
Onecut2
kg:uc012hdk.1
Ptprf
Phospho1
43
Hmga1
kg:uc009vev.1
Bcam
Isg20
44
Pmepa1
Ptprcap
Ogdh
March2
45
S100a11
kg:uc007pgq.1
Sparc
Lsp1
46
Rbp1
kg:uc007pgr.1
Ahnak
1810058l24Rik
47
Rpl36al
kg:uc007vdl.1
Oasl2
Clec1b
48
S100a4
Ctla2b
Wt1
Btg1
49
Atp6ap1
Myl9
Klf4
Laptm5
50
Ndufs2
Itpr2
Cdkn1a
Nrgn
51
Anapc5
kg:uc012enb.1
Myl7
H2-Aa
52
Cox6b1
Isg20
Col1a2
Fyb
53
Krtcap2
Rasal3
Eif4a1
Cd24a
54
Atn1
Gng11
Rbpms
Fnbp4
55
5730559C18Rik
kg:uc009cfw.1
Emp3
Ehbp1l1
56
Pea15a
Tmsb4x
Scaf11
Ctla2a
57
Grcc10
Trem1
Col14a1
Sgk1
58
Lama5
Fech
Ptrf
Glyr1
59
Krt18
Epb4.1
Crip2
Myl9
60
Ccnd1
Sgk1
Ubxn4
Il2rg
61
Arhgef5
Dgkq
Eif2s2
Mrps17
62
Golm1
Snap23
S100a6
Cdr2
63
Tff2
Usp25
Hectd1
Mkrn1
64
Plin2
Kif21b
Zc3h15
Gart
65
H13
Irs2
Ube2d3
Lyz1
66
Rpl29
Pxk
A130040M12Rik
Vwf
67
1110034A24Rik
Cyp4f18
Cd34
Gadd45a
68
Trim28
Map4k1
Igfbp5
Mpp1
69
Ltbp3
Isca1
C1s
Stim1
70
Fkbp1a
Itga4
Upk3b
Psme3
71
Erp29
Dock2
Gpr133
Ets1
72
Muc1
Spib
Dab2
Snap23
73
Lamc2
2810453l06Rik
Serpinh1
Arhgdib
74
Plscr3
Cdr2
Upk1b
Hmha1
75
Agrn
Naa16
Sdf4
Itpr2
76
Park7
Arhgdib
Ctbp2
Ubl7
77
Ctnnb1
Cd79a
Psap
Ddx58
78
Atp5g1
Rbm27
Arhgef12
Nfkbie
79
Eef1g
Lmnb1
Copb2
Setd7
80
Nhp2
Slc25a37
Ctsl
Stk24
81
Rrbp1
Klf6
Aldh1a2
Hvcn1
82
Sumo3
Hist1h1c
Dcn
Plekha2
83
Scyl1
Phip
Timp3
Psme4
84
Cox6a1
Qrfp
Xdh
Ankrd44
85
Krt8
Fermt3
Irf7
B4galt5
86
Gsta4
Ptma
Tmem151a
Phf20
87
Ppp1r14b
Etv3
Aebp1
Zc3hav1
88
Tnk1
Apobr
C2
Rnf11
89
D19Wsu162e
kg:uc008ewj.2
Spen
Plk3
90
Ctsl
Malat1
kg:uc007pfe.1
Fbxw5
91
Timp1
March8
Krt18
Emb
92
S100a6
Coro1a
Arf4
kg:uc007vdl.1
93
Rps15
Rac2
Rab14
Taok2
94
Polr2j
Glyr1
Tmem98
Dhrs11
95
Hspe1
Btg2
Prss23
Slc25a39
96
Lgals4
Mtf2
Egr1
Csk
97
Edf1
Nfkbie
Perp
Bcl2
98
Mtch1
Cd84
Csrp1
kg:uc009vev.1
99
Rnf187
AW549877
Pdpn
Wipf1
100
Npy
March2
Pdcd6ip
Sept9
101
Cox5b
Add3
Rpl37
Rnf10
102
Pak1
Ddx50
F11r
Pml
103
Mmp7
Prkcb
Gpm6a
Cd9
104
Fxyd3
Klf2
Tuba1a
D4Wsu53e
105
Cuta
Dcaf12
Ctnna1
Traf7
106
Ndufb8
Il2rg
Anxa8
Pitpnc1
107
Gps1
Selplg
Tpm1
Mms19
108
Bud31
Cd37
S100a16
Naa16
109
Ppap2c
Fastkd2
Chmp4b
Sharpin
110
Dap
Rsad2
Tbrg1
Capza1
111
Slc25a1
Msn
C3
Rsad2
112
Chaf1a
2010321M09Rik
Ptgs1
kg:uc012hdk.1
113
Asxl1
Kif2a
Rhou
Ghitm
114
Jmjd8
Cd97
Cdc42
Csnk1g1
115
Tecr
Hvcn1
Gpx4
Dgkz
116
Mgp
Nipsnap3b
Ppib
B2m
117
Uqcrh
Uba7
Stub1
Irs2
118
Wdr38
1810058I24Rik
Dmkn
Emg1
119
Col4a2
Nfrkb
Rnh1
Impact
120
Tnnt2
Pabpc1
Pdgfa
Mylip
121
Ndufs8
Usp16
Rpl37a
Psmb8
122
Tspan4
Pde1b
Rabac1
Rfk
123
Agpat6
Ncoa4
Timp2
Map3k5
124
Timp3
Irf8
Serping1
Odc1
125
Ankrd50
Ppp1cb
Rbm39
Slc11a2
126
Ube2d3
Rgs2
Tgoln1
Eif2b1
127
Sf1
Smyd4
Nfix
kg:uc008wjd.1
128
Csnk1d
Arid3b
Brd4
Rexo1
129
Reg3b
Sh3bgrl2
Tmem234
Ddx50
130
Flot2
Lyl1
Wbp5
Nipsnap3b
131
Lmna
Prr13
Ppig
Sp100
132
2310044H10Rik
Plagl2
Cd63
Uggt1
133
H19
Nfkbia
Col1a1
kg:uc007czl.1
134
Slc20a1
Eef1a1
Mt2
Arpc5
135
6720456B07Rik
Brd2
Zbtb7c
Nfrkb
136
Mdh2
Egr1
Npr1
Nap1l4
137
Eif6
Mkrn1
Tmem119
Fam117a
138
Phf5a
Pld4
Atf3
Sipa1l1
139
Vps28
Aldh1a1
Ankhd1
Ttc1
140
Bag1
Dnajb9
Tmed10
kg:uc009vew.1
141
Cyc1
Gjb5
Slc6a4
142
Angptl4
Mtif2
Atxn7l3b
143
Lgals3
H2-
Rpl29
DMb2
144
Farsb
Sdpr
Ccar1
145
Mbd3
4932438A13Rik
Ltbp4
146
Timm13
Treml1
Scyl1
147
Tpd52l2
Nup153
Ap3d1
148
Ptprn
Mpp1
Iqgap1
149
Crip2
Dhrs11
Cldn15
150
Raver1
Lrmp
Spnb2
151
Eif2b2
Manf
Ano1
152
Psma7
Mll3
Lrrn4
153
Rps6ka4
Fam116b
Id3
154
Mgat4a
B4galt5
Eif3a
155
Ifitm2
kg:uc009vew.1
Prkcdbp
156
Wars
Ly6d
Atp1a1
157
Capn5
Dguok
Dnaja2
158
Bsg
Pnpo
Tubb4b
159
Sec16a
Tmem175
Hnrnpab
160
Cldn7
Gm6548
Mmp14
161
Cox7a2
Rsrc2
Atp1b1
162
Nek6
Ccdc88b
Psip1
163
Rpl39
Akna
Mgll
164
Itpr3
Tsc22d3
Rnase4
165
Ctnna1
Txndc5
Ywhab
166
Tpd52
Tubb4a
Clip1
167
Mlf2
Stx11
Syn3
168
Crip1
D4Wsu53e
Myl12a
169
Fkbp4
Amfr
Rbm25
170
Gprc5a
Tti1
Arf2
171
Slc4a11
Fam175b
Cav1
172
Syn3
Zfp36
Hnrnpc
173
Npc2
Ddx5
Syne2
174
Rpl32
Tlr7
Dst
175
Inf2
Rfk
176
Rps10
kg:uc007ded.1
177
Rps26
Gnb2
178
Rpl37a
Tmed5
179
Ctxn1
Thbs1
180
Lrrc59
eg:320169:chr9:p
181
Dctn1
Zfp335
182
Mtap4
Emg1
183
Uqcr10
Trmt61a
184
Suds3
Adipor1
185
Ap1s1
Vwf
186
S100a1
Aatf
187
Atp5j
Trib1
188
Aim1
Pcyt1a
189
Plec
Stx18
190
Prom1
Trp53bp2
191
Rhoc
Stk40
192
Mast3
Il18
193
Olfml3
1810014B01Rik
194
Uqcr11
Lcp2
195
Plp2
Gimap4
196
Spna2
Rabl2
197
1700017B05Rik
Ncf2
198
Anxa4
eg:497210:chr14:m
199
Nudc
Tpt1
200
Asl
Mll5
201
Prkcsh
H3f3a
202
Plod3
Tspan13
203
Ndufa9
Il10ra
204
Impdh2
Mdc1
205
Ccnl2
Stk24
206
Nedd8
Myst4
207
Atp6v1f
Zdhhc20
208
Mt1
Eif2b1
209
Il4ra
Exoc4
210
Cndp2
Wipf1
211
Aprt
Impa1
212
Preb
Tmem119
213
Ap3d1
Pml
214
Mcm6
Ubb
215
Ubr4
Zmat3
216
Pvrl2
Slc30a9
217
Snrpg
Lat
218
Cycs
Tgfb2
219
Efemp2
Ube2o
220
Cct4
Igfbp5
221
Gm20605
Tspan5
222
Smad3
Fmnl1
223
Card10
Fnbp4
224
Krt7
Extl3
225
Cct2
Adcy7
226
Coro1c
Enpp4
227
Ltbr
Sep15
228
Ric8
H2-Ab1
229
Ndufs6
Bnip3l
230
Fibp
Slc11a2
231
Pold4
Stom
232
Rpl34
Mfhas1
233
Rpl34-ps1
Mettl1
234
Clic1
Rnf10
235
Eri3
kg:uc009cfd.1
236
Ets2
Klf4
237
Unc13a
Psme4
238
Usmg5
Sema4a
239
Sh3pxd2b
Ftl2
240
Wdr6
Atad1
241
Las1l
Tspan31
242
Polr2f
Srrm2
243
Vamp5
Rab5c
244
Endod1
Capza1
245
Snrpd2
H2-Aa
246
Tpi1
Fhl1
247
Wwp2
Cryab
248
Dalrd3
Arid4b
249
Iqgap1
Gart
250
Ahsa1
1110004F10Rik
251
Trim27
Rnf11
252
Serpinf1
Zc3hav1
253
D330041H03Rik
kg:uc008btl.1
254
Ppp2r5d
Rnf34
255
Minos1
Dmkn
256
Tsta3
Btg1
257
Prpsap1
Syt11
258
Sphk1
Mtdh
259
Ldha
Med21
260
Abca3
Rnf2
261
B4galt3
Tcf12
262
Porcn
Tacstd2
263
Tmc4
Madd
264
Serinc2
D16Ertd472e
265
Akr1b8
Pias1
266
Nudt4
Taok2
267
Atp5l
Pold1
268
Psmc3
Cep110
269
Hint1
A930013F10Rik
270
Rpl41
Tcof1
271
Xpnpep1
kg:uc009bpd.1
272
Nav1
kg:u009bpr.2
273
Parva
Capza2
274
Immt
Ptp4a2
275
Pafah1b3
Fth1
276
Chid1
Mepce
277
Aldh1l1
Rexo1
278
Rpl31
Prg4
279
Wbp1
Ctla2a
280
Zfp622
Smarca5
281
2700060E02Rik
Icam2
282
Hspa9
Pbx1
283
Tceb2
Gnl3l
284
Rpl36a
Slc2a3
285
Pgs1
Nnmt
286
Mpnd
Rb1cc1
287
Cdc42
Bpgm
288
Dhrs3
Lcp1
289
Hexa
Sipa1l1
290
Cpsf1
Lilrb4
291
Mea1
Ankrd44
292
Polr2e
Specc1
293
Ddb1
Rif1
294
Ptcd1
295
Atp5f1
296
Sec61b
297
Psmc5
298
Fam89b
299
Lama3
300
Tomm6
301
Mrpl28
302
Syngr2
303
Ngfrap1
304
Kcmf1
305
Tubb4b
306
Anapc11
307
Vcp
308
Arpp19
309
Pglyrp1
310
Rrp1
311
Gkn3
312
Atpif1
313
Prickle3
314
Map4k4
315
Arrdc1
316
C1qtnf6
317
Hras1
318
Lamb1
319
Eif3d
320
Snrpa
321
Tbrg1
322
Nxf1
323
Pdlim7
324
Add1
325
Pfdn5
326
Stk16
327
Gm17821
328
Csnk1e
329
Rrp7a
330
Psmb6
331
Snhg1
332
Ssr4
333
Ergic3
334
Rnaseh2a
335
kg:uc009cut.1
336
Bgn
337
Gm5506
338
Uqcrq
339
Tmem167
340
Nasp
341
Mif
342
Acaa2
343
Fam162a
344
Eif4ebp3
345
Nhp2l1
346
Prelid1
347
Gss
348
Lonp1
349
Srsf2
350
Igsf8
351
Ndufa7
352
Neat1
353
S100a13
354
Apoa1bp
355
Fam40a
356
Rps25
357
Eno1
358
Cldn2
359
Capn2
360
Glo1
361
Atp5c1
362
Rab2a
363
Rab25
364
Ncor2
365
Lgi4
366
Ier3
367
Tmem223
368
Slc9a3r2
369
Atp13a1
370
Rpn2
371
Acp5
372
Cct5
373
Sdf4
374
Mprip
375
Pmm2
376
Snx22
377
Arl2
378
1110008F13Rik
379
Polr1d
380
Dpm2
381
Cela1
382
2310016M24Rik
383
Cep250
384
Mybbp1a
385
Polr2g
386
Bag6
387
Cpxm1
388
Eif3m
389
Prr24
390
Sra1
391
Scara3
392
Reg1
393
Gas5
394
Hnrnpab
395
Mcpt2
396
Tgfbi
397
Capns1
398
Fdx1l
399
S100a16
400
Nap1l1
401
Swi5
402
Rpl38
403
Dctn2
404
Pdlim1
405
Gemin7
406
Pnpla6
407
Nono
408
Sla2
409
Idh3b
410
Ppp2r4
411
Map2k2
412
Ndufb10
413
Atp5d
414
Arfgap1
415
Tmbim1
416
Ergic1
417
Pdgfa
418
Ppp2ca
419
Hk1
420
Ltbp2
421
Trim35
422
Gtf2i
423
C1qb
424
Ankhd1
425
Podxl
426
Rps21
427
Huwe1
428
Pomp
429
Dpp3
430
Fkbp8
431
Itga5
432
Hes6
433
Mrpl11
434
Poldip3
435
Scd2
436
Tmem55b
437
Ndufa13
438
Dcakd
439
Ubqln1
440
Gpx4
441
Cyb561
442
Gmppa
443
Ncaph2
444
Pdha1
445
Ndufs4
446
Fcer1g
447
Myof
448
Ppib
449
Mrpl52
450
Tes
451
Emp3
452
Ndufa11
453
Tor1aip2
454
Anp32b
455
Tnk2
456
Mcpt1
457
Ssr2
458
Psmb3
459
2700081O15Rik
460
Pcbd1
461
Eif1ax
462
Pmm1
463
Ptprk
464
Hadha
465
Calu
466
Fam73a
467
Atp5e
468
Hsd17b10
469
Rbm39
470
Egfl7
471
Psmc1
472
Perp
473
Lman2
474
Galnt1
475
Rbx1
476
Lemd2
477
Zglp1
478
Ing4
479
kg:uc008oow.1
480
1500012F01Rik
481
Cox4i1
482
kg:uc008bcq.1
483
Ubap2l
484
Pafah1b2
485
Mrpl13
486
Nucb1
487
Fbn1
488
Adrm1
489
Itgb4
490
Ctss
491
Plbd2
492
Ptpmt1
493
Sap30l
494
Ppp1r12c
495
Sgta
496
Acrbp
497
Higd2a
498
Higd1a
499
Tmem208
500
Cdh1
501
Ube2d2a
502
Suv39h1
503
Rabac1
504
Anxa5
505
Ubxn6
506
Tpm1
507
Hmga2
508
Cnbp
509
Rpl21
510
Ndufb5
511
Sec31a
512
Znhit1
513
Cyb5b
514
Sfn
515
Ccdc12
516
Elovl1
517
Psmb5
518
Slc25a11
519
Psmd2
520
Nsun2
521
Slc50a1
522
Eme1
523
Bnip2
524
Pxdn
525
Mad2l2
526
Pdcd6
527
2010107E04Rik
528
Abhd11
529
Carkd
530
Polr2l
531
Ppdpf
532
Cib1
533
Dgcr2
534
Timm50
535
Mrps24
536
Abhd12
537
Brf1
538
Man1b1
539
kg:uc012cgd.1
540
Gpaa1
541
Fmnl3
542
Mapk3
543
C1qc
544
Pgls
545
Cp
546
Serh1
547
2610203C20Rik
548
Hsbp1
549
Tmem214
550
Akt1
551
kg:uc007pfe.1
552
Tmed10
553
Ttll3
554
2200002D01Rik
555
Tnfrsf23
556
Sgsm3
557
Atp9a
558
Lcn2
559
Pdrg1
560
Tspan9
561
Nrd1
562
Rin1
563
Ndufv1
564
Naa10
565
Wnk1
566
Heatr7a
567
Slc4a2
568
Ggct
569
5730403B10Rik
570
Sh3glb2
571
Pfkl
572
Tspan3
573
Gns
574
Sdcbp2
575
C130074G19Rik
576
Cotl1
577
Tubb5
578
Sec11c
579
Pigq
580
Zc3h15
581
Lsmd1
582
Ppa1
583
Chmp4b
584
Sepn1
585
Angptl2
586
Itpripl2
587
Ddx1
588
Hbxip
589
Cdk2ap1
590
Clta
591
Cpsf3l
592
Apoe
593
Ift46
594
Sae1
595
Gpi1
596
Gorasp2
597
1500032L24Rik
598
Nsmce4a
599
Dlst
600
Bap1
601
Pitpnb
602
Meg3
603
Cyth2
604
Atp5o
605
Gon4l
606
Sox11
607
Cxxc5
608
Avil
609
Alcam
610
Eif3f
611
Cygb
612
Eif1ad
613
Polr3h
614
Araf
615
Gkn1
616
Rhog
617
Mtap
618
Eif4ebp1
619
Akr1a1
620
Trip6
621
Prdx6
622
2410015M20Rik
623
Rps6
624
Rps23
625
Stxbp2
626
Rps19
627
Ykt6
628
Atp5g2
629
Serpinb1a
630
Col7a1
631
Mrps6
632
Lgals9
633
Rcn3
634
Trim44
635
Surf2
636
Rps29
637
Cdipt
638
Lmf2
639
Psenen
640
Ltf
641
Mpzl1
642
Psmd6
643
Cttn
644
Tmc6
645
2500003M10Rik
646
Atp6v0a1
647
Med8
648
Prrx2
649
Atp5b
650
Smurf1
651
Carhsp1
652
Tpcn1
653
Ndufb9
654
Pih1d1
655
Hnrnpa0
656
Fn1
657
2810428I15Rik
658
0610012G03Rik
659
Ube2i
660
Anxa3
661
Msto1
662
Eng
663
0910001L09Rik
664
Rpl10
665
kg:uc007xxx.1
666
Mosc2
667
Vps37c
668
Sgpl1
669
Fam166a
670
Polr2b
671
Fam101b
672
Nupr1
673
Lsm4
674
Rpl36
675
0610007C21Rik
676
Psmc2
677
Supt6h
678
Rps13
679
5430437P03Rik
680
Dsp
681
Ddx56
682
Tsc2
683
Trmt2a
684
Vdac2
685
Cant1
686
Eif4h
687
Puf60
688
A430105I19Rik
689
Cacnb3
690
Prdx4
691
March5
692
Ccar1
693
Npepl1
694
Fermt1
695
Use1
696
Axl
697
Slc39a4
698
1110008P14Rik
699
Sema4g
700
Timm8b
701
Krt23
702
Rpl28
703
Lgals3bp
704
Hdgf
705
1110005A03Rik
706
Impdh1
707
Mtmr11
708
Msln
709
Zdhhc3
710
Znrf1
711
Aldh16a1
712
Bloc1s1
713
Prkag1
714
Plxnb1
715
Crat
716
Phpt1
717
5930434B04Rik
718
Kpnb1
719
Nme2
720
E430025E21Rik
721
Smyd2
722
Cyhr1
723
Mvp
724
Rps27l
725
Rbp4
726
Cars
727
kg:uc012ctw.1
728
Ssr1
729
Ssu72
730
Usp48
731
Atp5k
732
Lrrk1
733
BC056474
734
Epn1
735
Trappc1
736
Clk2
737
Sugt1
738
Nenf
739
kg:uc009cuu.1
740
Ubap2
741
Rps20
742
Atp5h
743
9430008C03Rik
744
Kars
745
Mrpl37
746
Aimp1
747
Trmt1
748
Hspa4
749
Cd164
750
9430023L20Rik
751
Rnf4
752
H1f0
753
C1qtnf1
754
Srd5a1
755
1500010J02Rik
756
Rpl35a
757
Cand2
758
C630004H02Rik
759
Acsbg1
760
Derl1
761
Cbx5
762
Tmem63a
763
Hgfac
764
Stx5a
765
Bri3
766
Tomm20
767
Fam20c
768
Cox6c
769
Tm2d2
770
Plekhb2
771
Ramp1
772
2410001C21Rik
773
Tardbp
774
Pebp1
775
kg:uc008gbp.1
776
Eif3b
777
Ccna2
778
Ptges
779
kg:uc007hyr.2
780
Wbp5
781
Chchd2
782
Fdft1
783
Srm
784
Gtf3a
785
D0H4S114
786
1810009A15Rik
787
Rps27
788
Tmem176b
789
Ndufc1
790
Lasp1
791
Fam108a
792
Mapk8ip3
793
Copa
794
Serpina3n
795
Rps17
796
Dnpep
797
Lbp
798
Krt19
799
Ei24
800
Ap1b1
801
Mogs
802
Uba1
803
Postn
804
Phf23
805
Paox
806
Nploc4
807
Ndufv2
808
Actr1a
809
Mxd3
810
Pfdn1
811
Ide
812
Foxp4
813
1810013D10Rik
814
2310007B03Rik
815
Xab2
816
Agr2
817
Dctn3
818
Urm1
819
H2-Ke2
820
Spint1
821
Slc38a2
822
Ube2z
823
Ctrb1
824
Fam195b
825
Suclg1
826
Ube2l3
827
Rpn1
828
Mrps7
829
Tsg101
830
Drosha
831
Arfip2
832
Mrto4
833
Grlf1
834
Sort1
835
Oaf
836
Ints1
837
Slc44a2
838
Dph3
839
Gramd1a
840
Fkbp9
841
Fam149a
842
1810035L17Rik
843
kg:uc007fte.1
844
Eif2s1
845
Smpd1
846
Eef1b2
847
Actr10
848
Rab11fip5
849
Ypel3
850
Flnb
851
Tcn2
852
Crlf1
853
Map3k15
854
Cul7
855
Atp6v1g1
856
Ncbp1
857
Atp1b3
858
Mtif3
859
Aldoa
860
Htra1
861
Rab14
862
Ppm1a
863
Ndufb11
864
Kansl3
865
Rab24
866
Bcl2l1
867
Lgals1
868
Samm50
869
Mrps33
870
Anxa1
871
Chchd1
872
Mapre1
873
Ctbp2
874
Rnps1
875
Spg7
876
Tnfrsf12a
877
H6pd
878
Myo7a
879
Mcm7
880
Psmd13
881
Mrpl54
882
Atp6v0b
883
Prdx1
884
Elof1
885
Rexo4
886
Mrps18a
887
Dpcd
888
D2Wsu81e
889
Cd99l2
890
Synpo
891
Atp2a2
892
Cdc5l
893
Stard7
894
Atp13a2
895
Sdha
896
Hdac6
897
Krt20
898
Ppp6r3
899
1700037H04Rik
900
Napa
901
PgP
902
Cnih
903
Atg4b
904
Cox8a
905
Srp68
906
St13
907
Gng12
908
Cfdp1
909
Rcc2
910
Pisd-
ps1
911
Ivns1abp
912
Mpv17l2
913
Ssna1
914
Gnl1
915
Tmem111
916
Hbs1l
917
Agpat3
918
Col6a2
919
March6
920
Usp39
921
Rps11
922
Ahnak
923
Lcmt1
924
Ddx41
925
H2afv
926
Fau
927
Tuba1c
The gene names listed in Table 13 and 12 are common names. NCBI Gene ID numbers for each of the genes listed in Table 13 and 12 can be obtained by searching the “Gene” Database of the NCBI (available on the World Wide Web at http://www.ncbi.nlm.nih.gov/) using the common name as the query and selecting the first returned Homo sapiens gene. Other genes may be obtained using the UCSC genome browser (available on the World Wide Web at http://genome.ucsc.edu) using the Gene Sorter function. In certain embodiments, the marker gene(s) are selected from the genes listed in Table 13 and/or 12.
In some embodiments, the marker gene(s) is selected from a marker gene indicated to be upregulated in at least one type of CTC in Table 13, e.g. marker genes 1-142. In some embodiments, the marker gene(s) is selected from a marker gene indicated to be upregulated in at least one type of CTC in Table 12, e.g. marker genes listed in the columns labeled “CTC-c vs. Primary Tumor Enriched Gene” or “CTC-c vs. WBC”.
In a CTC, the marker genes listed in Table 13 or 12 can be upregulated, e.g. for marker genes listed in Table 13 and/or 12, if the measured marker gene expression in a cell or sample is higher as compared to a reference level of that marker gene's expression, then the cell is identified as a CTC and/or the sample is identified as comprising CTCs. Preferably, once looks at a statistically significant change. However, even if a few genes in a group do not differ from normal, a sample can be identified as comprising CTCs if the overall change of the group shows a significant change, preferably a statistically significant change. All possible combinations of 2 or more of the indicated markers are contemplated herein.
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