Tumor vasculature markers and methods of use thereof

FIELD OF INVENTION

This invention provides methods of detecting and localizing tumor vasculature cells (TVC) and solid tumors; and treating, impeding vascularization of, and determining the stage of solid tumors in a subject, comprising the step of contacting a subject or a TVC with a ligand that binds to a nucleic acid molecule of the present invention, or binds to a protein encoded by the nucleic acid molecule.

BACKGROUND OF THE INVENTION

Markers of solid tumors and their vasculature have been difficult to identify because of difficulty in isolating these cells in contaminant-free preparations. Such markers are urgently need for use in a variety of therapeutic and diagnostic applications.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds a DR6-encoding nucleic acid molecule or that binds a DR6 protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds an ADAM12-encoding nucleic acid molecule or that binds an ADAM12 protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. In another embodiment, the ADAM12-encoding nucleic acid molecule or ADAM12 protein is a short isoform thereof. In another embodiment, the nucleotide or protein is any other isoform known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds a long isoform of an ADAM12-encoding nucleic acid molecule or an ADAM12 protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds a CDCP1-CUB-encoding nucleic acid molecule or a CDCP1-CUB protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. In another embodiment, the CDCP1-CUB-encoding nucleic acid molecule or CDCP1-CUB protein is a long isoform thereof. In another embodiment, the nucleotide or protein is any other isoform known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds a short isoform of a CDCP1-CUB-encoding nucleic acid molecule or a CDCP1-CUB protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds a SLC11A1-NRAMP-encoding nucleic acid molecule or that binds a SLC11A1-NRAMP protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds a BLAME/SLAMF8-encoding nucleic acid molecule or that binds a BLAME/SLAMF8 protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds an ESM1-encoding nucleic acid molecule or that binds an ESM1 protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is a renal tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds an EGFL6-encoding nucleic acid molecule or that binds an EGFL6 protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds an FZD10-encoding nucleic acid molecule or that binds an FZD10 protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds a COL11A1-encoding nucleic acid molecule or that binds a COL11A1 protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is a breast cancer tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds a TNFAIP6-encoding nucleic acid molecule or that binds a TNFAIP6 protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds an Adlican-encoding nucleic acid molecule or that binds an Adlican protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds a nucleic acid molecule selected from DSG2, c6orf55, SCGB2A1, EPSTI1, SEC23B, MS4A6A, LOC51136, KCNE3, KCNE4, c14orf100, C2orf6, KCNK5, C14orf28, PCDHB2, ST14, OLFML2B, GPR105, SDC1, FLJ46072, IVNS1ABP, SPP1, DKFZp762e1312, WFDC2, KIAA1892, C6orf69, and KIBRA, and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds a nucleic acid molecule that encodes a protein selected from DSG2, c6orf55, SCGB2A1, EPSTI1, SEC23B, MS4A6A, LOC51136, KCNE3, KCNE4, c14orf100, C2orf6, KCNK5, C14orf28, PCDHB2, ST14, OLFML2B, GPR105, SDC1, FLJ46072, IVNS1ABP, SPP1, DKFZp762e1312, WFDC2, KIAA1892, C6orf69, and KIBRA, and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds a protein selected from DSG2, c6orf55, SCGB2A1, EPSTI1, SEC23B, MS4A6A, LOC51136, KCNE3, KCNE4, c14orf100, C2orf6, KCNK5, C14orf28, PCDHB2, ST14, OLFML2B, GPR105, SDC1, FLJ46072, IVNS1ABP, SPP1, DKFZp762e1312, WFDC2, KIAA1892, C6orf69, and KIBRA, and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a solid tumor in a subject, the method comprising the step of contacting the subject with an antibody or ligand that is conjugated to a cytotoxic agent or cytotoxic drug and that binds a nucleic acid molecule selected from DR6, ADAM12, CDCP1-CUB, SLC11A1-NRAMP, BLAME/SLAMF8, EGFL6, FZD10, TNFAIP6, Adlican, DSG2, c6orf55, SCGB2A1, EPSTI1, SEC23B, MS4A6A, LOC51136, KCNE3, KCNE4, c14orf100, C2orf6, KCNK5, C14orf28, PCDHB2, ST14, OLFML2B, GPR105, SDC1, FLJ46072, IVNS1ABP, SPP1, DKFZp762e1312, WFDC2, KIAA1892, C6orf69, and KIBRA, thereby treating a solid tumor in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a solid tumor in a subject, the method comprising the step of contacting the subject with an antibody or ligand that is conjugated to a cytotoxic agent or cytotoxic drug and that binds a nucleic acid molecule encoding a protein selected from DR6, ADAM12, CDCP1-CUB, SLC11AI-NRAMP, BLAME/SLAMF8, EGFL6, FZD10, TNFAIP6, Adlican, DSG2, c6orf55, SCGB2A1, EPSTI1, SEC23B, MS4A6A, LOC51136, KCNE3, KCNE4, c14orf100, C2orf6, KCNK5, C14orf28, PCDHB2, ST14, OLFML2B, GPR105, SDC1, FLJ46072, IVNS1ABP, SPP1, DKFZp762e1312, WFDC2, KIAA1892, C6orf69, and KIBRA, thereby treating a solid tumor in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a solid tumor in a subject, the method comprising the step of contacting the subject with an antibody or ligand that is conjugated to a cytotoxic agent or cytotoxic drug and that binds a protein selected from DR6, ADAM12, CDCP1-CUB, SLC11A1-NRAMP, BLAME/SLAMF8, EGFL6, FZD10, TNFAIP6, Adlican, DSG2, c6orf55, SCGB2A1, EPSTI1, SEC23B, MS4A6A, LOC51136, KCNE3, KCNE4, c14orf100, C2orf6, KCNK5, C14orf28, PCDHB2, ST14, OLFML2B, GPR105, SDC1, FLJ46072, IVNS1ABP, SPP1, DKFZp762e1312, WFDC2, KIAA1892, C6orf69, and KIBRA, thereby treating a solid tumor in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of impeding a vascularization of a solid tumor in a subject, the method comprising the step of contacting the subject with a ligand capable of inhibiting an activity of a nucleic acid molecule selected from DR6, ADAM12, CDCP1-CUB, SLC11A1-NRAMP, BLAME/SLAMF8, EGFL6, FZD10, TNFAIP6, Adlican, DSG2, c6orf55, SCGB2A1, EPSTI1, SEC23B, MS4A6A, LOC51136, KCNE3, KCNE4, c14orf100, C2orf6, KCNK5, C14orf28, PCDHB2, ST14, OLFML2B, GPR105, SDC1, FLJ46072, IVNS1ABP, SPP1, DKFZp762e1312, WFDC2, KIAA1892, C6orf69, and KIBRA, whereby the ligand is taken up by a vasculature cell of the solid tumor, thereby impeding a vascularization of a solid tumor in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of impeding a vascularization of a solid tumor in a subject, the method comprising the step of contacting the subject with a ligand capable of inhibiting an activity of a nucleic acid molecule encoding a protein selected from DR6, ADAM12, CDCP1-CUB, SLC11A1-NRAMP, BLAME/SLAMF8, EGFL6, FZD10, TNFAIP6, Adlican, DSG2, c6orf55, SCGB2A1, EPSTI1, SEC23B, MS4A6A, LOC51136, KCNE3, KCNE4, c14orf100, C2orf6, KCNK5, C14orf28, PCDHB2, ST14, OLFML2B, GPR105, SDC1, FLJ46072, IVNS1ABP, SPP1, DKFZp762e1312, WFDC2, KIAA1892, C6orf69, and KIBRA, whereby the ligand is taken up by a vasculature cell of the solid tumor, thereby impeding a vascularization of a solid tumor in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of impeding a vascularization of a solid tumor in a subject, the method comprising the step of contacting the subject with a ligand capable of inhibiting an activity of a protein selected from DR6, ADAM12, CDCP1-CUB, SLC11A1-NRAMP, BLAME/SLAMF8, EGFL6, FZD10, TNFAIP6, Adlican, DSG2, c6orf55, SCGB2A1, EPSTI1, SEC23B, MS4A6A, LOC51136, KCNE3, KCNE4, c14orf100, C2orf6, KCNK5, C14orf28, PCDHB2, ST14, OLFML2B, GPR105, SDC1, FLJ46072, IVNS1ABP, SPP1, DKFZp762e1312, WFDC2, KIAA1892, C6orf69, and KIBRA, whereby the ligand is taken up by a vasculature cell of the solid tumor, thereby impeding a vascularization of a solid tumor in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of impeding a vascularization of a solid tumor in a subject, the method comprising the step of contacting the subject with a ligand capable of inhibiting an activity of an ESM1-encoding nucleic acid molecule or an ESM1 protein, whereby the ligand is taken up by a vasculature cell of the solid tumor, thereby impeding a vascularization of a solid tumor in a subject. In another embodiment, the solid tumor is a renal tumor.

In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of impeding a vascularization of a solid tumor in a subject, the method comprising the step of contacting the subject with a ligand capable of inhibiting an activity of a COL11A1-encoding nucleic acid molecule or a COL11A1 protein, whereby the ligand is taken up by a vasculature cell of the solid tumor, thereby impeding a vascularization of a solid tumor in a subject. In another embodiment, the solid tumor is a breast cancer tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of detecting a presence of a solid tumor in a subject, the method comprising the step of detecting a presence of an Adlican protein in a body fluid of the subject, whereby the presence in a body fluid of the protein indicates the presence of a solid tumor in the subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of detecting a presence of a solid tumor in a subject, the method comprising the step of detecting a presence of an Adlican-encoding nucleotide molecule in a body fluid of the subject, whereby the presence in a body fluid of the nucleotide molecule indicates the presence of a solid tumor in the subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of detecting a presence of a solid tumor in a subject, the method comprising the step of detecting a presence of a DR6 protein in a body fluid of the subject, whereby the presence in a body fluid of the protein indicates the presence of a solid tumor in the subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of detecting a presence of a solid tumor in a subject, the method comprising the step of detecting a presence of a DR6-encoding nucleotide molecule in a body fluid of the subject, whereby the presence in a body fluid of the nucleotide molecule indicates the presence of a solid tumor in the subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of detecting a presence of a solid tumor vasculature cell in a subject, the method comprising the step of detecting a presence, in a body fluid of the subject, of a protein selected from FLJ46072, IVNS1ABP, SPP1, TNPAIP6, DKFZp762e1312, WFDC2, KIAA1892, C6orf69, and KIBRA, whereby the presence in a body fluid of the protein indicates the presence of a solid tumor vasculature cell in the subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of detecting a presence of a solid tumor vasculature cell in a subject, the method comprising the step of detecting a presence, in a body fluid of the subject, of a nucleotide molecule that encodes a protein selected from FLJ46072, IVNS1ABP, SPP1, TNPAIP6, DKFZp762e1312, WFDC2, KIAA1892, C6orf69, and KIBRA, whereby the presence in a body fluid of the nucleotide molecule indicates the presence of a solid tumor vasculature cell in the subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of detecting a presence of a solid tumor vasculature cell in a subject, the method comprising the step of detecting a presence of a COL11A1 protein in a body fluid of the subject, whereby the presence in a body fluid of the protein indicates the presence of a solid tumor vasculature cell in the subject. In another embodiment, the solid tumor is a breast cancer tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of detecting a presence of a solid tumor vasculature cell in a subject, the method comprising the step of detecting a presence, in a body fluid of the subject, of a nucleotide molecule encoding a COL11A1 protein, whereby the presence in a body fluid of the nucleotide molecule indicates the presence of a solid tumor vasculature cell in the subject. In another embodiment, the solid tumor is a breast cancer tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a peptide having the amino acid sequence CPGAKALSRVREDIVEDE (SEQ ID No: 88).

In another embodiment, the present invention provides an antibody that recognizes a peptide of the present invention.

In another embodiment, the present invention provides a method of treating a solid tumor in a subject, the method comprising the steps of localizing the solid tumor by a method of the present invention, and irradiating the solid tumor, thereby treating a solid tumor in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is a breast cancer tumor. In another embodiment, the solid tumor is a renal tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of treating a solid tumor in a subject, the method comprising the steps of: (a) contacting the subject with a photo-activatable cytotoxic drug or pharmaceutical composition; (b) localizing the solid tumor by a method of the present invention; and (c) contacting the solid tumor with a concentrated light source, thereby treating a solid tumor in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is a breast cancer tumor. In another embodiment, the solid tumor is a renal tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: A. Immuno-LCM steps. Left panel: Rapid IHC for CD31 allows prompt identification of vasculature in ovarian cancer frozen sections. Middle panel: Tissue section after LCM of CD31⁺ cells. Right panel: captured tumor vascular cells. B. RT-PCR analysis of lineage-specific markers in RNA from tumor vascular cells (TVC) isolated with immuno-LCM, whole tumor (Tum) or a no template control (NTC). C. Scatter plot and correlation value of amplified RNA from unstained tissue (Pre IHC) versus RNA amplified after IHC optimized as in Table 1 (Post IHC). D. Part 1. Heat map condition tree developed using a hierarchical clustering algorithm, excluding all genes where the difference between the means of the tumor and normal vascular samples was less than its standard error. D, part 2: Red color shown alone. D, part 3: Green color shown alone. E. Archived Gene Expression Datasets. Data used for expression of the TVM in normal and tumor tissue samples; also available in the Gene Expression Omnibus (GEO; National Center for Biotechnology Information [NCBI]) with series numbers GSE3526 and GSE2109, respectively. All CEL-files were similarly processed using the Robust Multi-array Average (RMA) algorithm.

FIG. 2. Quality of total RNA isolated from 8 μm frozen ovarian cancer tissue sections through different methodologies and analyzed by Agilent Bioanalyzer (A-E), quantitative real-time PCR (F) or Affymetrix U133A arrays (G). A. RNA distribution profiles following fixation with different fixatives. (Lanes: 1, ethanol; 2, methanol; 3, acetone; 4, acetic acid+ethanol; 5, paraformaldehyde). B. RNA profiles after isolation without immunostaining (Lane 1) or following IHC with or without RNAse inhibitor. (Lanes: 2, RNA Protector; 3, Placental RNAse inhibitor; 4, SuperRNASin; 5, RNA Protector+SuperRNASin; 6, no RNAse inhibitor. C. RNA profiles after different immunostaining procedures. (Lanes: 1, IHC using DAB; 2, IHC using AEC; 3, immunofluorescence). D. Time course demonstrating RNA stability after IHC performed with procedure optimized as in Table 1. (Lanes: 1, 0 min; 2, 30 min; 3, one hr; 4, two hrs; 5, three hrs). E. RNA profiles following different RNA isolation methods. (Lanes: 1, Arcturus kit; 2, Stratagene kit; 3, modified Trizol; 4, Zymo kit). F. qRT-PCR for β-actin transcripts with RNA purified with the indicated RNA purification protocols. G. Scatter plots and correlation values of amplified RNA (y-axis) to unamplified total tumor RNA (x-axis).

FIG. 3: Expression of novel TVM. A. qRT-PCR analysis of tumor vascular markers (TVM) in whole epithelial ovarian cancer (Ovarian Ca, n=20) and normal ovaries (Nml Ovary, n=5). Results are expressed as relative expression units against β-actin. B. Expression of select TVM by qRT-PCR in a panel of normal human tissues (Sm Intest, small intestine; Sk Muscle, skeletal muscle); expression in ovarian cancer (Ov Ca) is defined as 100%. C. Expression of select TVM by qRT-PCR in different tumor types; expression in ovarian cancer is defined as 100%. Data were normalized against CD31. D. Expression of TVM in tissues with physiologic angiogenesis including corpus luteum (Corp Lut); proliferative endometrium (Prolif Endomet); and placenta, as well as ovarian cancer (Ov Ca). Data were normalized against CD31. E. Expression of TVM in normal human tissues by digital northern derived from the NIH SAGE expression database. Sp Ch (spinal cord), Kid (kidney, Ov (ovary), Lym (lymph node), BM (bone marrow), Vasc (vasculature). Squares with 32-63 tags are indicated by a double arrow to the left. Squares with 16-31 tags have a single arrow to the left. The remainder of the light-colored squares had 15 or fewer tags. “EPB41L3” refers to EPB41L3. This figure also includes additional markers that do not appear in Table 5; selection of these additional markers is described below in Example 11.

FIG. 4. Part 1. Immuno-localization of identified markers to tumor vasculature. IHC is shown for CD24, endothelial lipase (Endo Lipase), FolH1 and AML-1. Immunofluorescent localization of SPON-1 to vascular structures and co-staining of PAR2, STC2 and AML-1 (red) with CD31 (green) in microvascular structures in human ovarian tumors. Merge includes nuclear staining with DAPI. Red fluorescence is shown alone in part 2. Green fluorescence is shown alone in part 3. Blue fluorescence from DAPI is shown alone in part 4.

FIG. 5. A. In-situ hybridization demonstrates expression of mRNA for the indicated tumor vascular markers in association with vascular structures in ovarian cancer. Negative controls included hybridization of tumor section with sense strand (Sense), hybridization of normal ovary with antisense probe (Nml Ov) and hybridization of RNAse-pretreated tumor section with antisense probe (RNAse Tx) for all TVM. Representative controls are shown for adlican, but were identical for all TVM. Merge is shown with DAPI nuclear staining. Red fluorescence is shown alone in part 2. Blue fluorescence from DAPI is shown alone in part 3.

FIG. 6. Expression of select TVM by qRT-PCR in FACS-purified VE-cadherin⁺ CD146⁺ CD45⁻ tumor endothelial cells (TEC) and VE-cadherin⁺ CD146⁺ CD45⁺ vascular leukocytes (VLC) compared with peripheral blood mononuclear cells (PBMC). Data were normalized against β-actin.

FIG. 7, part 1. Expression of novel human TVM by myeloid monocytic cells. A. Flow cytometry analysis; Top, Expression of CD45 (x-axis) and VE-cadherin (y-axis) in DC derived from PBMC prior to exposure to rhVEGF₁₆₅(Pre VEGF-A) and following 7-day treatment with rhVEGF₁₆₅(Post VEGF-A); VE-cadherin⁺ CD45⁺ cells appear after VEGF165 treatment. Middle, Expression of CD45 and VE-cadherin in DC derived from enriched monocytes prior to (Pre VEGF-A) and following 7-day treatment with rhVEGF₁₆₅(Post VEGF-A); 91% of output cells after VEGF₁₆₅treatment were VE-cadherin⁺ CD45⁺, indicating that double positive cells derived from the monocyte fraction. Bottom, No significant CFSE dilution was noted in monocyte-derived DC after VEGF₁₆₅treatment, indicating no cell proliferation in these cultures. B. Expression of markers by PBMC-derived DC at baseline (Day 0) and following exposure to rhVEGF₁₆₅, with or without antibody against VEGF-A for 2 weeks. Induction of several tumor vascular markers and suppression of AML-1 and LZTS1 are observed after VEGF₁₆₅. This was abrogated by VEGF blockade, except for AML-1 and LTZS1. Data were normalized against β-actin. C. Expression of TVM in human 2008 ovarian cancer xenografts injected alone (2008) or enriched with human PBMC-derived DC pretreated with VEGF₁₆₅ex vivo (DC), FACS-purified human tumor endothelial cells (TEC) or FACS-purified human tumor vascular leukocytes (VLC). TVM are expressed by xenografts enriched with human myeloid cells or TEC but not by xenografts generated with human tumor cells only. Data were normalized against human β-actin. D. Immunofluorescent detection of human CD34⁺ vascular structures within 2008 tumors supplemented with human PBMC-derived DC pretreated with VEGF₁₆₅: 1: One CD34-PE positive vessel was noted in the early tumor xenograft. 2 and 3: Merge of CD34-PE and DAPI depicts vascular structures at higher magnification. 4: Control tumor generated with 2008 cells alone without VEGF-pretreated DC, TEC or VLC shows no expression of human CD34. Similar results were obtained with immunostaining for human CD31. E. Raw data-described in legend for FIG. 7C.

FIG. 7, parts 2-3. Red fluorescence is shown alone in part 2. Blue fluorescence from DAPI is shown alone in part 3.

FIG. 8, part 1. Secreted (sec) and transmembrane (TM) TVM. Column 1: expression rank (described in Example 2). Column 2: “Scnt”: The number of normal samples that ranked in the bottom 4 positions. (e.g. if snt=4, the 4 lowest expressing samples are all normal samples). Column 3: Gene symbol. Column 4: Number of normal samples (4 total) in which the transcript was classified as present. Column 5: Number of tumor samples (21 total) in which the transcript was classified as present. Column 6: Mean signal from the tumor samples. Column 7: Mean signal from the normal samples. Column 8: Fold increase of expression in tumor samples. Column 9: foldc3: Value of 1 means that the fold change was (>3). 2 fold change was (<3). Column 10: srank25. 1 means that the sum of ranks <25. Criteria for this list was a value of “1” in at least one of column 9 and column 10.

FIG. 8, part 2. Column 1: Gene symbol. Column 2: Localization. Column 3: Description. Column 4: Available antibodies. Abbreviations: Pred: predicted; Mb: membrane; EC: extracellular; ECM extracellular matrix; PM: plasma membrane.

FIG. 9. A. Real-time PCR analysis of Adlican in normal tissues and ovarian cancer. B. In situ hybridization of adlican in ovarian cancer. C. Real-time PCR analysis of adlican in ovarian tumors. D. Western blot shows adlican protein in tumor tissue lysate (T1-3) but not normal ovary (Nml) (left) and in serum (S1-2) and ascites (A1-2) of patients with ovarian cancer but not normal human serum (NHS).

FIG. 10. Model of step-wise vascular differentiation of monocyte-derived DC precursors in tumors. VEGF-A alone induces in monocytes downregulation of transcriptional regulators AML-1 and LZTS1, while newly identified markers (collagen 11α1, DR6, FZD10, GPM6B, SCP1 and SPON-1) as well as known endothelial markers (VE-cadherin, TEM-1) are upregulated, yielding VLC. Additional factors from the tumor microenvironment cooperate with VEGF-A to induce additional TVM found in TEC, as myeloid cells structurally participate in the assembly of vasculature.

FIG. 11. Additional TVM.

FIG. 12. List of additional TVM from another analysis.

FIG. 13. A. DR6 mRNA expression by qPCR in normal tissues and ovarian tumor. B-D. IHC of DR6 in ovarian tumor (B), high-magnification image thereof (C), and normal ovary tissue (D).

FIG. 14. DR6 protein is detected at higher levels in the serum of patients with ovarian tumors compared to healthy individuals. A—Western. B—quantitation.

FIG. 15. Quantitation of ADAM12 mRNA relative expression by qPCR in normal tissues and ov tumor. A. Long isoform. B. Short isoform.

FIG. 16. Staining with anti-ADAM12 Ab in frozen ovarian tumor sections

FIG. 17. Double fluorescent staining with anti-ADAM12 Ab (red in original) and anti-CD31 Ab (green in original) in frozen ovarian tumor sections. Nuclei are stained blue. Top panel: green color alone (appears as white). Bottom: red color alone (appears as white).

FIG. 18. Double fluorescent staining with anti-ADAM12 Ab (red in original) and anti-CD45 Ab (green in original) in frozen ovarian tumor sections. Nuclei are stained blue. Top panel: green color alone (appears as white). Bottom: orange color alone, representing overlap of green and red staining (appears as white).

FIG. 19. CDCP1 mRNA relative expression in normal tissues and ovarian tumor (qPCR). A. Long isoform. B. Short isoform. Bars represent, from left to right: Ovarian tumor, bladder, esophagus, colon, liver, lung, brain, cervix, kidney, heart, adipose, testes, thymus, skeletal muscle, small intestine, spleen, trachea, ovary, placenta, thyroid, prostate.

FIG. 20. Staining with anti-CDCP1 Ab in frozen ovarian tumor sections.

FIG. 21. SLC11A1 mRNA expression by qPCR in normal tissues and ovarian tumor.

FIG. 22. Staining with anti-SLC11A1 Ab in frozen ovarian tumor sections. Left panels: sample from first patient, at low (top) and high (bottom) magnification. Right panels: sample from second patient, at low (top) and high (bottom) magnification.

FIG. 23. Double fluorescent staining with anti-SLC11A1 Ab (red in original) and anti-CD31 Ab (green in original) in frozen ovarian tumor sections. Nuclei are stained blue. A: green color alone (appears as white). B: red color alone (appears as white).

FIG. 24. Double fluorescent staining with anti-SLC11A1 Ab (red in original) and anti-CD45 Ab (green in original) in frozen ovarian tumor sections. Nuclei are stained blue. A: green color alone (appears as white). B: orange color alone, representing overlap of green and red staining (appears as white).

FIG. 25. BLAME mRNA expression by qPCR in normal tissues and ovarian tumor.

FIG. 26. Staining with anti-BLAME Ab in frozen ovarian tumor sections. A: samples from 2 patients at low magnification (40×). B: samples from 2 patients at high magnification (60×).

FIG. 27. A-B. Double fluorescent staining with anti-BLAME Ab (red in original) and anti-CD31 Ab (green in original) in frozen ovarian tumor sections. Nuclei are stained blue. A: green color alone. B: red color alone. C-E. Double fluorescent staining with anti-BLAME Ab (red in original) and anti-CD45 Ab (green in original) in frozen ovarian tumor sections. Nuclei are stained blue. C: green color alone. D: red color alone. E. orange color alone, representing overlap of green and red staining. Selected color (green, red, or orange) appears as white in each panel.

DETAILED DESCRIPTION OF THE INVENTION

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds a long isoform of an ADAM12-encoding nucleic acid molecule or an ADAM12 protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method of localizing a solid tumor vasculature in a subject, the method comprising the steps of: (a) contacting the subject with a ligand that binds a CDCP1-CUB-encoding nucleic acid molecule or that binds a CDCP1-CUB protein; and (b) localizing the ligand, thereby localizing a solid tumor vasculature in a subject. In another embodiment, the solid tumor is an ovarian tumor. In another embodiment, the solid tumor is any other type of solid tumor known in the art. In another embodiment, the CDCP1-CUB-encoding nucleic acid molecule or CDCP1-CUB protein is a long isoform thereof. In another embodiment, the nucleotide or protein is any other isoform known in the art. Each possibility represents a separate embodiment of the present invention.