US20110287034A1 - Therapeutic and diagnostic methods relating to cancer stem cells

Info

Abstract

Description

Claims

US20110287034A1

Publication number: US20110287034A1
Application number: US13/128,915
Authority: US
Inventors: Markus H. Frank; Natasha Y. Frank
Original assignee: Brigham and Womens Hospital Inc
Current assignee: Childrens Medical Center Corp
Priority date: 2008-11-14
Filing date: 2009-11-13
Publication date: 2011-11-24
Also published as: DK3130923T3; EP3130923A1; WO2010056332A1; AU2016253648A1; PT3130923T; US10316085B2; US20160009804A1; EP3978928A1; EP3130923B1; EP2356446A4; EP3734281A3; EP2356446A1; CA2743464A1; ES2796111T3; AU2016253648B2; EP3734281A2; AU2009314556A1; AU2009314556B2; CY1123187T1

Genes that are deregulated in cancer stem cells (e.g., melanoma stem cells) are disclosed. Methods which involve modulating (e.g., inducing, inhibiting, etc.) the activity of the cancer stem cell associated genes are used to treat individuals having melanoma. Cell surface genes that are upregulated in melanoma stem cells are targeted for the selective isolation, detection, and killing of cancer stem cells in melanoma.

RELATED APPLICATION

This application claims priority under 35 U.S.C. §119 from U.S. provisional application Ser. No. 61/114,490, filed Nov. 14, 2008, the contents of which are incorporated herein in their entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant 5R01CA113796-03 from the National Cancer Institute. The Government has certain rights to this invention.

FIELD OF INVENTION

The present invention relates in part to methods for treating individuals having cancer. The methods involve modulating, e.g., inducing or inhibiting, the activity of genes that are deregulated in cancer stem cells. In some aspects, cell surface genes that are upregulated in cancer stem cells are targeted for selective isolation, detection, or killing of cancer stem cells in melanoma. Other aspects of the invention relate to reagents, arrays, compositions, and kits that are useful for diagnosing and treating cancer.

BACKGROUND OF INVENTION

Self-renewing cancer stem cells (CSCs) initiate tumours and drive malignant progression by generating and supporting replication of more differentiated non-stem cell progeny. (M. Al-Hajj, et al., Proc Natl Acad Sci USA 100 (7), 3983 (2003); D. Bonnet and J. E. Dick, Nat Med 3 (7), 730 (1997); C. A. O'Brien, et al., Nature 445 (7123), 106 (2007); L. Ricci-Vitiani, et al., Nature 445 (7123), 111 (2007); S. K. Singh, et al., Nature 432 (7015), 396 (2004); T. Schatton and M. H. Frank, Pigment cell & melanoma research 21 (1), 39 (2008)). The mechanisms by which CSCs cause tumour formation and growth and the potential role of CSC-specific differentiation plasticity in tumourigenicity are currently unknown. We recently identified a subpopulation of CSC based on expression of the chemoresistance mediator ABCB5 (ATP-binding cassette, sub-family B (MDR/TAP), member 5) (N. Y. Frank, A et al., Cancer Res 65 (10), 4320 (2005); Y. Huang, et al., Cancer Res 64 (12), 4294 (2004)) within human malignant melanoma (T. Schatton, et al., Nature 451 (7176), 345 (2008)), a highly aggressive and drug-resistant cancer. (T. Schatton and M. H. Frank, Pigment cell & melanoma research 21 (1), 39 (2008); L. Chin, L. A. Garraway, and D. E. Fisher, Genes Dev 20 (16), 2149 (2006).) ABCB5⁺ Malignant Melanoma Initiating Cells (MMIC) correlate with clinical disease progression and can be specifically targeted to abrogate tumour growth. (T. Schatton, et al., Nature 451 (7176), 345 (2008)). Consistent with these findings, the ABCB5 gene is also preferentially expressed by in vitro self-renewing melanoma minority populations (G. I. Keshet, et al., Biochem Biophys Res Commun 368 (4), 930 (2008)) and by melanoma cell lines of metastatic as opposed to primary, radial growth phase tumour origin (J. F. Sousa and E. M. Espreafico, BMC cancer 8, 19 (2008)).

SUMMARY OF INVENTION

The present invention relates in part to the discovery that a number of genes (referred to herein as CSC-associated genes) are deregulated in cancer stem cells. In some aspects, the invention relates to diagnostic arrays and methods for detecting cancer in an individual based on the expression of CSC-associated genes. In other aspects, the invention relates to methods useful for treating individuals having melanoma based on modulating the expression and/or activity of CSC-associated genes. Compositions and kits that are useful for the foregoing methods are also disclosed.
The invention, in some aspects, provides methods for diagnosing cancer in an individual. In some aspects, the methods involve determining an expression level of a cancer stem cell (CSC)-associated gene in Table 5 in a test sample from the individual and comparing the expression level of the CSC-associated gene to a reference value, wherein results of the comparison are diagnostic of cancer. In some embodiments, the cancer is melanoma, breast cancer, prostate cancer, colon cancer or renal cancer. In some embodiments, the test sample is a tissue biopsy. In some embodiments, the test sample is a skin biopsy. In some embodiments, the test sample is a sample of the cancer, such as a tumor biopsy. In some embodiments, the methods involve updating a patient record for the individual to indicate the diagnostic result of the comparison. In some embodiments, determining comprises detecting in the test sample a mRNA that is encoded by the CSC-associated gene. In some embodiments, determining comprises detecting in the test sample a polypeptide that is encoded by the CSC-associated gene. In certain embodiments, detecting comprises nucleic acid hybridization or nucleic acid amplification. In specific embodiments, the nucleic acid amplification is real-time RT-PCR or RT-PCR. In one embodiment, the nucleic acid hybridization is performed using a nucleic acid array. In certain other embodiments, detecting comprises immunodetection of the polypeptide.
In one embodiment, the immunodetection comprises an Enzyme-Linked Immunosorbent Assay (ELISA). In one embodiment, the immunodetection comprises an antibody array. In one embodiment, the immunodetection comprises immunohistochemistry.
In some embodiments of the methods, the reference value is the expression level of the CSC-associated gene in a non-cancer reference sample, and if the expression level of the CSC-associated gene in the test sample is about equal to the expression level of the CSC-associated gene in the non-cancer reference sample, then the comparison does not indicate cancer.
In some embodiments of the methods, the reference value is the expression level of the CSC-associated gene in a cancer reference sample, and if the expression level of the CSC-associated gene is about equal to the expression level of the CSC-associated gene in the cancer reference sample, then the comparison indicates cancer.
In some embodiments of the methods, the CSC-associated gene is in Table 1 or 8 and the reference value is the expression level of the CSC-associated gene in a non-cancer reference sample, and if the expression level of the CSC-associated gene in the test sample is significantly higher than the expression level of the CSC-associated gene in the non-cancer reference sample, the comparison indicates cancer.
In some embodiments of the methods, the CSC-associated gene is in Table 1 or 8 and the reference value is the expression level of the CSC-associated gene in a cancer reference sample, and if the expression level of the CSC-associated gene in the test sample is significantly lower than the expression level of the CSC-associated gene in the cancer reference sample, the comparison does not indicate cancer.
In some embodiments of the methods, the CSC-associated gene is in Table 1 or 8 and the reference value is the expression level of the CSC-associated gene in a non-cancer reference sample, and if the expression level of the CSC-associated gene in the test sample is at least 10% higher than the expression level of the CSC-associated gene in the non-cancer reference sample, the comparison indicates cancer.
In some embodiments of the methods, the CSC-associated gene is in Table 2 or 7 and the reference value is the expression level of the CSC-associated gene in a non-cancer reference sample, and if the expression level of the CSC-associated gene in the test sample is significantly lower than the expression level of the CSC-associated gene in the non-cancer reference sample, the comparison indicates cancer.
In some embodiments of the methods, the CSC-associated gene is in Table 2 or 7 and the reference value is the expression level of the CSC-associated gene in a cancer reference sample, and if the expression level of the CSC-associated gene in the test sample is significantly higher than the expression level of the CSC-associated gene in the cancer reference sample, the comparison does not indicate cancer.
In some embodiments of the methods, the CSC-associated gene is in Table 2 or 7 and the reference value is the expression level of the CSC-associated gene in a non-cancer reference sample, and if the expression level of the CSC-associated gene in the test sample is at least 10% lower than the expression level of the CSC-associated gene in the non-cancer reference sample, the comparison indicates cancer.
The invention, in some aspects, provides methods for isolating a cancer stem cell. In some aspects, the methods involve contacting a sample with an agent that binds a polypeptide, which is encoded by a CSC-associated gene in Table 4 and expressed on the surface of the cancer stem cell, and isolating the agent from the sample. If the sample contains the cancer stem cell, the agent binds to the polypeptide on the surface of the cancer stem cell such that isolation of the agent from the sample results in isolation of the cancer stem cell. In some embodiments, the CSC-associated gene is selected from the group consisting of: ANK2, NCKAPIL, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8. In some embodiments, the agent is an isolated peptide that specifically binds the polypeptide on the surface of the cancer stem cell. In certain embodiments, the isolated peptide is an antibody or antigen-binding fragment. In specific embodiments, the antibody or antigen-binding fragment is a monoclonal antibody, polyclonal antibody, human antibody, chimeric antibody, humanized antibody, single-chain antibody, F(ab)₂, Fab, Fd, Fv, or single-chain Fv fragment. In some embodiments, the isolated peptide is bound to a solid support. In some embodiments, the isolated peptide is conjugated to a detectable label. In some embodiments, the detectable label is a fluorophore which may be selected from: FITC, TRITC, Cy3, Cy5, Alexa Fluorescent Dyes, and Quantum Dots. In some embodiments, the isolating comprises performing fluorescent activated cell sorting to isolate a cancer stem cell bound to a detectable label. In some embodiments, the cancer stem cell is from a melanoma, breast cancer, prostate cancer, colon cancer or renal cancer.
The invention, in some aspects, provides methods for treating an individual having, or at risk of having, cancer. In some aspects, the methods involve administering a therapeutically effective amount of a composition that induces the expression of a CSC-associated gene selected from the group set forth in Table 2 or 7. In some embodiments, the cancer is melanoma, breast cancer, prostate cancer, colon cancer or renal cancer.
In some embodiments, the composition that induces the expression of a CSC-associated gene comprises an isolated plasmid that expresses the CSC-associated gene. In some embodiments, the isolated plasmid is in a virus capable of infecting the individual. In certain embodiments, the virus is selected from adenovirus, retrovirus, lentivirus, and adeno-associated virus. In some embodiments, the isolated plasmid comprises a cancer specific promoter operably linked to the CSC-associated gene.
The invention, in other aspects, provides methods for treating an individual having, or at risk of having, cancer that involve administering a therapeutically effective amount of a composition that targets a product of a CSC-associated gene selected from the group set forth in Table 1 or 8. In some embodiments, the cancer is melanoma, breast cancer, prostate cancer, colon cancer or renal cancer. In some embodiments, the CSC-associated gene is selected from the group set forth in Table 4. In certain embodiments, the CSC-associated gene is selected from the group consisting of: ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8.
In some embodiments, the composition that targets a product of a CSC-associated gene comprises a small interfering nucleic acid that inhibits expression of the CSC-associated gene. In some embodiments, the composition comprises an isolated plasmid that expresses the small interfering nucleic acid. In certain embodiments, the plasmid is in a virus. In specific embodiments, the virus is selected from adenovirus, retrovirus, lentivirus, and adeno-associated virus. In certain embodiments, the isolated plasmid comprises a cancer-specific promoter operably linked to a gene encoding the small interfering nucleic acid.
In some embodiments, the composition that targets a product (e.g., protein or RNA) of a CSC-associated gene comprises an isolated molecule that selectively binds to a polypeptide encoded by the CSC-associated gene. In certain embodiments, the isolated molecule is conjugated to a therapeutic agent. In specific embodiments, the isolated molecule is an antibody or antigen-binding fragment. In particular embodiments, the antibody or antigen-binding fragment is a monoclonal antibody, polyclonal antibody, human antibody, chimeric antibody, humanized antibody, a single-chain antibody, F(ab)₂, Fab, Fd, Fv, or single-chain Fv fragment. In specific embodiments, the therapeutic agent is selected from: a toxin, a small-interfering nucleic acid, and a chemotherapeutic agent. In one embodiment, the toxin is a radioisotope. In particular embodiments, the radioisotope is selected from the group consisting of: ²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ¹⁸⁶Rh, ¹⁸⁸Rh, ¹⁷⁷Lu, ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ¹²⁵I, ¹²³I, ⁷⁷Br, ¹⁵³Sm, ¹⁶⁶Bo, ⁶⁴Cu, ²¹²Pb, ²²⁴Ra and ²²³Ra. In some embodiments, the therapeutic agent is a small interfering nucleic acid that inhibits expression of a CSC-associated gene. In some embodiments, the isolated molecule binds to the polypeptide and enters an intracellular compartment of a cancer stem cell of the cancer.
In some embodiments, the treatment methods involve determining the expression level of the CSC-associated gene in the individual. In certain embodiments, the methods involve comparing the expression level of the CSC-associated gene to a reference value, wherein results of the comparison are diagnostic of cancer in the individual. In specific embodiments, if the comparison results in a diagnosis of cancer in the individual, the administering is performed. In one embodiment, the determining and the comparing are repeated at one or more intervals after the administering. In some embodiments, the administering is orally, intravenously, intrapleurally, intranasally, intramuscularly, subcutaneously, intraperitoneally, or as an aerosol.
The invention, in some aspects, provides methods of delivering a therapeutic agent to a cancer stem cell that involve contacting a cancer stem cell with an isolated molecule, which selectively binds to a polypeptide encoded by a CSC-associated gene selected from the group set forth in Table 4 and which is conjugated to a therapeutic agent, in an effective amount to deliver the therapeutic agent to the cancer stem cell. In some embodiments, the CSC-associated gene is selected from the group consisting of: ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8. In some embodiments, the isolated molecule is an antibody or antigen-binding fragment that selectively binds the polypeptide. In some embodiments, the therapeutic agent is selected from: a toxin, a small-interfering nucleic acid, and a chemotherapeutic agent. In one embodiment, the toxin is a radioisotope. In particular embodiments, the radioisotope is selected from the group consisting of: ²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ¹⁸⁶Rh, ¹⁸⁸Rh, ¹⁷⁷Lu, ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ¹²⁵I, ¹²³I, ⁷⁷Br, ¹⁵³Sm, ¹⁶⁶Bo, ⁶⁴Cu, ²¹²Pb, ²²⁴Ra and ²²³Ra. In some embodiments, the therapeutic agent is a small interfering nucleic acid that inhibits expression of a CSC-associated gene. In some embodiments, the cancer stem cell is in vitro. In other embodiments, the cancer stem cell is in vivo.
In some aspects, the invention provides nucleic acid arrays consisting essentially of at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, or more CSC-associated genes set forth in Table 5.
In some aspects, the invention provides polypeptide arrays consisting essentially of at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, or more polypeptides or immunogenic fragments thereof encoded by CSC-associated genes set forth in Table 1 or 8.
In some aspects, the invention provides antibody arrays consisting essentially of at least 2 or more different antibodies or antigen-binding fragments that selectively bind polypeptides encoded by CSC-associated genes set forth in Table 1 or 8.
In some aspects, the invention provides methods for stratifying a population comprising individuals having cancer. The methods involve determining expression levels of at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, or more CSC-associated genes set forth in Table 5 and stratifying the population based on the expression levels.
In some aspects, the invention provides an isolated molecule that selectively binds to a polypeptide encoded by a CSC-associated gene set forth in Table 4, and that is conjugated to a therapeutic agent. In some embodiments, the CSC-associated gene is selected from the group consisting of: ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8. In some embodiments, the therapeutic agent is selected from: a toxin, a small-interfering nucleic acid, and a chemotherapeutic agent.
In certain embodiments, the isolated molecule is an antibody or antigen-binding fragment. In certain embodiments, the antibody or antigen-binding fragment is a monoclonal antibody, polyclonal antibody, human antibody, chimeric antibody, humanized antibody, single-chain antibody, a F(ab′)₂, Fab, Fd, or Fv fragment. In certain embodiments, the isolated molecule is an isolated receptor ligand of the polypeptide.
The invention, in some aspects, provides compositions comprising any of the foregoing isolated molecules. In some embodiments, the compositions include a pharmaceutically acceptable carrier.
The invention, in some aspects, provides pharmaceutical kits that include a container housing any of the foregoing compositions and instructions for administering the composition to an individual having cancer.
Use of a composition of the invention for treating cancer is also provided as an aspect of the invention.
A method for manufacturing a medicament of a composition of the invention for treating cancer is also provided.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts an analysis of vasculogenic/angiogenic pathways in human melanoma. FIG. 1 a. is a graphical representation of pathway activation across ABCB5⁺ MMIC. Genes represented by nodes (dark gray circles, TRIO, MET, FLT1, PSEN1, NRP2, RHOA, PTK2, PIP5K3, KIAA1267, MLL, GABPA, ETS1, and CHD8) are overexpressed in ABCB5⁺ relative to ABCB5⁻ human melanoma cells and those represented by black nodes are expressed at lower levels, respectively. Black lines between genes show known interactions. Known gene functions in vasculogenesis and angiogenesis, and genes known as relevant drug targets are annotated (dark gray lines). Gene relationships and figure layout are based on Ingenuity Pathway Analysis and references are provided elsewhere in the text. FIG. 1 b. shows detection of vasculogenic/angiogenic pathway members by RT-PCR in ABCB5⁺ MMIC. FIG. 1 c. shows FLT1 (VEGFR-1) protein expression on ABCB5⁺ MMIC (top) and ABCB5⁻ melanoma cells (bottom) as determined by dual color flow cytometry using ABCB5 phenotype-specific cell gating, with mean percentages (mean±s.e.m., n=6 replicate experiments) shown on the right. FIG. 1 d. depicts representative immunofluorescence staining for CD144 expression (Texas red staining) by ABCB5⁺ MMIC or ABCB5⁻ melanoma cell subpopulations prior to (t=0 h) and upon 48 h of culture (t=48 h) in the presence of 100 ng/ml VEGF¹¹, with nuclei counterstained with DAPI. Mean percentages (mean±s.e.m., n=3 replicate experiments) of cells staining positively for CD144 in each sample are shown on the right. FIG. 1 e. shows representative immunofluorescence staining for CD144 expression (Texas red staining) by melanoma cells cultured for 48 h (t=48 h) in the presence of 100 ng/ml VEGF as in above, but in the presence or absence of anti-FLT1 (VEGFR-1) blocking mAb or isotype control mAb. Nuclei are counterstained with DAPI. Mean percentages (mean±s.e.m., n=3 replicate experiments) of cells staining positively for CD144 in each sample are shown in the far right panel. FIG. 1 f. shows tube formation detected by phase contrast light microscopy of melanoma cells cultured for 24 h (t=24 h) in the presence of 100 ng/ml VEGF and the presence or absence of anti-FLT1 (VEGFR-1) blocking mAb or isotype control mAb. Number of tubes/microscopy field (mean±s.e.m., n=3 replicate experiments) and tube length (μm) (mean±s.e.m., n=3 replicate experiments) are illustrated for the different experimental conditions on the far right panels, respectively. FIG. 1 g. shows the adipogenic differentiation potential of ABCB5⁺ and ABCB5⁻ human melanoma cells (Oil Red O staining, nuclei are counterstained with hematoxylin). FIG. 1 h. shows the osteogenic differentiation potential of ABCB5⁺ and ABCB5⁻ human melanoma cells (Alizarin Red staining). FIG. 1 i shows the myogenic differentiation potential of ABCB5⁺and ABCB5⁻ human melanoma cells. Absence of myogenin staining (FITC, green) is detected in ABCB5⁺ or ABCB5⁻ human melanoma cells (nuclei are counterstained with DAPI).

FIG. 2 depicts an analysis of MMIC-driven in vivo vasculogenesis. FIG. 2 a. shows conventionally-stained (H&E) sections of human melanoma growing at melanoma cell injection site within human dermis of skin xenograft to NOD/SCID mouse. FIG. 2 b. shows immunohistochemistry for human CD31 indicating angiogenic response at perimeter of melanoma within human xenograft; broken line represents interface of tumour nodule with dermal connective tissue. FIG. 2 c. shows PAS (with diastase) immunochemical staining of CD31-negative interior regions of melanoma xenograft revealing numerous anastomosing channels (inset is laminin immunohistochemistry indicating identical pattern). FIG. 2 d. shows transmission electron micrographs of interior regions of melanoma xenograft; lumenal spaces containing blood products (erythrocytes) are lined by melanoma cells and associated basement membrane-like extracellular matrix. FIG. 2 e. shows the interior zone of melanoma xenograft derived from cells expressing GFP transgene and immunohistochemically stained for endothelial marker CD144 (FAST RED Chromogen (SIGNET) from Covance Research Products, Inc); CD144 expression is confined to cells forming lumen-like spaces lined by cells that co-express GFP and CD144 (indicated by dual staining). FIGS. 2 f and g. show low (f) and high (g) magnifications of immunohistochemistry for ABCB5 protein; reactivity is restricted to anastomosing channels identical to those seen in panel c. The inset in panel f depicts similar formation of ABCB5-reactive channels in a patient-derived melanoma biopsy. FIG. 2 h. depicts in situ hybridization for ABCB5 mRNA revealing a channel pattern corresponding to that of ABCB5 protein expression (compare with panel f; inset is sense control). FIG. 2 i. shows the detection of anti-ABCB5 mAb using anti-mouse Ig immunohistochemistry in melanoma xenografts after intravenous administration in vivo; note localization to channels (inset represents anti-mouse Ig staining after intravenous administration of irrelevant isotype-matched control mAb). FIG. 2 j. shows dual-labeling immunofluorescence microscopy for ABCB5 (left panel), CD144 (middle panel), and ABCB5 & CD144 (right panel). FIG. 2 k shows dual-labeling immunofluorescence microscopy for ABCB5 (left panel), TIE-1 (middle panel), and ABCB5 & TIE-1 (right panel).

FIG. 3 depicts the interdependency of MMIC-driven vasculogenesis and tumourigenesis. FIG. 3 a. shows representative flow cytometric ABCB5 expression or control staining (FITC, Fl1) plotted against forward scatter (FSC) for human A375, MUM-2B, and MUM-2C melanoma cell inocula. FIG. 3 b. shows representative histologic sections of melanomas that developed from three unsegregated and ABCB5-depleted melanoma cell lines injected intradermally into human skin xenografts. FIG. 3 c. shows histologically determined tumour formation rate (%) 3 weeks following intradermal transplantation of unsegregated vs. ABCB5⁺-depleted human A375, MUM-2B or MUM-2C melanoma cells (2×10⁶/inoculum) into human skin/Rag2^−/− chimeric mice (n=5, respectively). FIG. 2 d. shows histological tumour volumes (mean±s.e.m.) 3 weeks following intradermal transplantation of unsegregated vs. ABCB5⁺-depleted human A375, MUM-2B or MUM-2C melanoma cells (2×10⁶/inoculum) into human skin/Rag2^−/− chimeric mice. FIG. 3 e shows immunohistochemistry for laminin revealing extent of channel formation in melanomas that developed from unsegregated or ABCB5⁺-depleted melanoma cell inocula derived from A375, MUM-2B or MUM-2C lines injected intradermally into human skin xenografts (arrows=necrosis). FIG. 3 f depicts image analysis of laminin immunoreactivity for melanomas derived from unsegregated and ABCB5⁺-depleted cell inocula; y-axis is percent of pixelated area with reactivity (mean±s.e.m.); solid bar represents tumours derived from unsegregated melanoma cells, open bars represent tumours derived from ABCB5⁺-depleted cells (A375, P<0.0032; MUM-2B, P<0.0005; MUM-2C, P<0.0059).

FIG. 4 depicts an analysis of the correlation of ABCB5 protein and mRNA expression across human melanoma cell lines. FIG. 4 a. shows western blots of ABCB5 and tubulin expression of a panel of human melanoma cell lines. FIG. 4 b shows relative ABCB5 mRNA expression (log₂) in a panel of human melanoma cell lines plotted against ABCB5 protein expression as determined by ratios of ABCB5 89 kD western blot band intensity and tubulin western blot band intensity for each human melanoma cell line. Data points in FIG. 4 b are: 1, SK-MEL-2; 2, SK-MEL-5; 3, SK-MEL-28; 4, MDA-MB-435; 5, UACC-62; 6, UACC-257; 7, M14; 8, MALME-3M. r, Spearman Rank Correlation r (corrected for ties).

DETAILED DESCRIPTION

The present invention relates in part to the discovery that numerous CSC-associated genes have altered expression or function in cancer stem cells, e.g., melanoma stem cells. In some aspects, the invention relates to diagnostic arrays and methods for detecting cancer, e.g., melanoma, in an individual based on the expression of CSC-associated genes. In other aspects, the invention relates to compositions, kits, and methods useful for treating individuals having cancer. In some embodiments, the treatment methods involve modulating e.g., inducing or inhibiting, the activity of CSC-associated genes. The CSC-associated genes can be modulated by any one of a number of ways known in the art and described herein e.g., overexpression, RNAi-based inhibition, etc. In some cases, the CSC-associated genes encode cell surface proteins which, when upregulated in cancer stem cells, may be selectively targeted for isolating, e.g., by flow cytometry, identifying, e.g., by immunolabeling, and killing of cancer stem cells, e.g., melanoma stem cells.
The mechanism by which CSCs cause tumor formation and growth and the potential role of CSC-specific differentiation plasticity in tumorigenicity are currently unknown. It has been demonstrated according to the invention that CSC play an important role in providing nutritional support to growing tumors. For instance we have shown herein (Examples) a selective capacity of ABCB5⁺ malignant melanoma initiating cells (MMIC)³to undergo vasculogenic differentiation and to generate blood-perfused vessel-like channels in vivo. A repertoire of genes differentially expressed in MMIC compared to tumour bulk populations were identified by microarray analyses on purified ABCB5⁺ and ABCB5⁻ cell subsets derived from the established human melanoma cell lines and from three separate patient-derived melanoma specimens. Using this approach, 399 genes were identified that were differentially expressed between ABCB5⁺ MMIC and ABCB5⁻ melanoma bulk populations. The genes, which are outlined in Tables 1-8, are referred to herein as CSC-associated genes. Of the CSC-associated genes, 265 were upregulated (Table 1; Table 1 includes Table 1.1 and Table 1.2) and 150 were downregulated (Table 2). For certain CSC-associated genes, subcellular location, e.g., plasma membrane, nucleus, etc., gene type, e.g., enzyme, complex, transporter, etc., and drugs that affect, e.g., target, their activity are identified (Table 3). A summary of those annotations and networks is provide in Table 3. Genes that function share a common pathway have a common “network”) designation in Table 3. Some CSC-associated genes, e.g., those which have “plasma membrane” annotations, encode proteins that are associated with the cell surface. Such cell surface proteins are useful in a variety ways. For example, cell surface proteins that are upregulated in cancer stem cells, may be selectively targeted, e.g., using the methods disclosed herein, for isolating, identifying, and killing of cancer stem cells. A listing of exemplary cell surface proteins encoded by CSC-associated genes is provided in Table 4.

TABLE 1.1

Upregulated CSC-associated genes (p < 0.05)

	GENESYMBOL	ID	Fold Change

HECW1	237295_at	11.843
RP11-139H14.4	1569124_at	11.472
CDC16	242359_at	6.261
ANK2	202921_s_at	4.162
LOC146325	1553826_a_at	3.943
UGT1A6	206094_x_at	3.86
C12ORF51	1557529_at	3.632
SNRPA1	242146_at	3.54
PDE4B	215671_at	3.457
PAPD4	222282_at	3.39
ZNF536	233890_at	3.303
KSR2	230551_at	3.211
BUB1	233445_at	3.209
ZNF292	236435_at	3.201
CABIN1	1557581_x_at	3.052
SDAD1	242190_at	3.009
ASCC3L1	214982_at	3.009
ZNF224	216983_s_at	2.986
KIDINS220	1557246_at	2.97
WIPF2	216006_at	2.916
C12ORF51	230216_at	2.874
VPS37B	236889_at	2.85
NARG1	1556381_at	2.827
LOC145757	1558649_at	2.779
SDCCAG8	243963_at	2.67
ZNF154	242170_at	2.667
ZFR	238970_at	2.655
TRPV1	1556229_at	2.636
ANAPC5	235926_at	2.631
CUL4A	232466_at	2.607
TRIO	240773_at	2.607
LOC283888	1559443_s_at	2.56
RAB11FIP3	228613_at	2.546
PTK2	234211_at	2.539
MYO10	243159_x_at	2.528
NAT8B	206964_at	2.513
CDC14B	234605_at	2.512
TRIM33	239716_at	2.496
SF1	210172_at	2.452
SGCA	1562729_at	2.395
LOC285147	236166_at	2.377
N4BP2L2	242576_x_at	2.349
HNRPH1	213472_at	2.332
FLJ10357	241627_x_at	2.31
PHF20L1	219606_at	2.3
ANKRD28	241063_at	2.297
TRNT1	243236_at	2.295
GOLGA8A	213650_at	2.289
KIAA1618	231956_at	2.27
RBM5	209936_at	2.249
LOC645513	239556_at	2.24
LOC729397	236899_at	2.231
PABPN1	213046_at	2.228
SVIL	215279_at	2.228
PIP5K3	1557719_at	2.227
STRAP	1558002_at	2.189
KIAA2013	1555933_at	2.18
NUPL1	241425_at	2.179
IFNGR1	242903_at	2.171
AKAP9	215483_at	2.168
LOC254128	1557059_at	2.164
IRS2	236338_at	2.162
RHOA	240337_at	2.143
JARID2	232835_at	2.139
GPD2	243598_at	2.13
RADIL	223693_s_at	2.126
CROP	242389_at	2.121
EXT1	242126_at	2.116
XRCC5	232633_at	2.106
PDXDC1	1560014_s_at	2.105
MEF2C	236395_at	2.104
ZNF567	242429_at	2.103
ZNF337	1565614_at	2.096
TTLL4	1557611_at	2.092
FUBP1	240307_at	2.087
NPTN	228723_at	2.086
TPM4	235094_at	2.079
NCKAP1L	209734_at	2.071
KRTAP19-1	1556410_a_at	2.07
SLC30A9	237051_at	2.063
HDAC3	240482_at	2.062
C10ORF18	244165_at	2.046
SMA4	238446_at	2.035
GBF1	233114_at	2.03
GABPA	243498_at	2.03
SFRS15	243759_at	2.028
CREB3L2	237952_at	2.013
SLC2A8	239426_at	2.012
N4BP2L1	213375_s_at	2.01
IDS	1559136_s_at	2.001
COBRA1	1556434_at	1.985
TXNL1	243664_at	1.98
LOC388135	230475_at	1.979
MTUS1	239576_at	1.975
TAF15	227891_s_at	1.971
HNRPD	241702_at	1.962
TRIM46	238147_at	1.96
NBR1	1568856_at	1.957
WDR68	233782_at	1.924
HNRPD	235999_at	1.92
BLID	239672_at	1.91
LOC145786	229178_at	1.907
HOXD3	206601_s_at	1.897
AOC3	204894_s_at	1.894
PRPF38B	230270_at	1.888
SLC20A1	230494_at	1.884
SEC16B	1552880_at	1.877
FLT1	232809_s_at	1.861
HUWE1	214673_s_at	1.858
BUB1	216277_at	1.856
GPR135	241085_at	1.851
PSEN1	242875_at	1.851
KIAA0907	230028_at	1.83
POLR2J2	1552622_s_at	1.828
SFRS15	222311_s_at	1.818
CBS	240517_at	1.818
ETS1	241435_at	1.797
LRRFIP1	239379_at	1.796
OCIAD1	235537_at	1.794
LRCH3	229387_at	1.793
CCDC14	240884_at	1.771
HNRNPC	235500_at	1.769
DCUN1D2	240478_at	1.76
NPAS2	1557690_x_at	1.76
POFUT2	207448_at	1.759
CHD2	244443_at	1.757
TMEM165	1560622_at	1.756
FLJ31306	239432_at	1.753
HPS1	239382_at	1.749
WTAP	1560274_at	1.747
TNPO1	1556116_s_at	1.739
ZFHX3	215828_at	1.737
AKR1CL2	1559982_s_at	1.732
C20ORF4	234654_at	1.731
CCDC57	214818_at	1.703
MALAT1	224568_x_at	1.699
EWSR1	229966_at	1.686
MYO10	244350_at	1.677
MALAT1	223940_x_at	1.659
ATXN2L	207798_s_at	1.656
PDK1	239798_at	1.654
POLR2J2	1552621_at	1.652
CENPJ	220885_s_at	1.64
PDSS1	236298_at	1.64
UNK	1562434_at	1.637
BDP1	224227_s_at	1.632
N4BP2L2	235547_at	1.631
MDM4	235589_s_at	1.629
SNORA28	241843_at	1.628
ZFX	207920_x_at	1.625
NAPA	239362_at	1.624
PRO1073	228582_x_at	1.607
MLL	212079_s_at	1.599
SGOL2	235425_at	1.591
RBM25	1557081_at	1.57
BARD1	205345_at	1.559
LOC388969	232145_at	1.555
GGT1	211417_x_at	1.555
FAM62C	239770_at	1.551
TTC9C	1569189_at	1.55
TCAG7.907	238678_at	1.546
OSGEP	242930_at	1.541
RHOBTB2	1556645_s_at	1.538
C5ORF24	229098_s_at	1.531
RBM4	213718_at	1.53
SLC2A11	232167_at	1.529
DDX17	213998_s_at	1.528
C22ORF30	216555_at	1.521
C9ORF85	244160_at	1.52
DNM1L	236032_at	1.503
SQLE	213577_at	1.502
CRIPAK	228318_s_at	1.486
ZNF800	227101_at	1.484
RAD54L	204558_at	1.483
TAF1B	239046_at	1.468
THRAP3	217847_s_at	1.464
CNIH3	232758_s_at	1.451
UQCC	229672_at	1.451
HOXA2	228642_at	1.44
RBM26	229433_at	1.43
RFT1	240281_at	1.426
MTERFD3	225341_at	1.422
LOC641298	208118_x_at	1.419
ZNF326	241720_at	1.418
NBPF16	201104_x_at	1.411
ASPM	232238_at	1.411
RNF43	228826_at	1.401
IPW	213447_at	1.399
TTC3	208664_s_at	1.396
USP36	224979_s_at	1.393
KIAA0841	36888_at	1.389
NEK1	213328_at	1.381
AMZ2	227567_at	1.377
TBC1D8	204526_s_at	1.373
STK36	231806_s_at	1.362
SF3B1	214305_s_at	1.359
HELLS	242890_at	1.359
SYNE2	202761_s_at	1.356
KIAA1267	224489_at	1.355
C14ORF135	1563259_at	1.353
SF3B1	201070_x_at	1.35
CLN8	229958_at	1.344
STK36	234005_x_at	1.335
ZNF226	219603_s_at	1.332
COQ4	218328_at	1.328
DTX3	49051_g_at	1.32
WFS1	1555270_a_at	1.315
ZNF251	226754_at	1.313
ARS2	201679_at	1.307
ATAD2	235266_at	1.304
CCDC73	239848_at	1.294
BCL9L	227616_at	1.291
MET	213816_s_at	1.283
NFATC2IP	217527_s_at	1.272
CHD8	212571_at	1.27
TNRC6A	234734_s_at	1.268
OSBPL5	233734_s_at	1.261
COIL	203653_s_at	1.259
CPEB2	226939_at	1.251
TBC1D8	221592_at	1.246
RUNX3	204198_s_at	1.233
LBA1	213261_at	1.225
CENPJ	234023_s_at	1.22
MARCH6	201737_s_at	1.219
ANKRD44	226641_at	1.218
NAPE-PLD	242635_s_at	1.216
C12ORF48	220060_s_at	1.216
CCDC93	219774_at	1.208
ZUFSP	228330_at	1.205
SMC6	218781_at	1.203
TAOK3	220761_s_at	1.195
JARID1A	226367_at	1.192
DCLRE1C	242927_at	1.187
TTC26	233999_s_at	1.184
EIF4G3	201935_s_at	1.174
ORMDL1	223187_s_at	1.171
TCOF1	202385_s_at	1.169
CCDC52	234995_at	1.166
PMS2L3	214473_x_at	1.159
HERC5	219863_at	1.156
CASC5	228323_at	1.144
SON	201085_s_at	1.144
APBB2	40148_at	1.139
LOC338799	226369_at	1.137
PHC1	218338_at	1.123
DEPDC1	232278_s_at	1.119
NRP2	210841_s_at	1.106
ZMYND8	209049_s_at	1.102
CEP55	218542_at	1.096

TABLE 1.2

Highly upregulated genes as detected by RT-PCR

		ABCB5+/
		ABCB5−
Description	Gname	Fold change

Angiopoietin-like 3	ANGPT5	3.0596
Brain-specific angiogenesis	FLJ41988	3.0596
inhibitor 1
Cadherin 5, type 2, VE-cadherin	7B4/CD144	3.0596
(vascular epithelium)
Epidermal growth factor (beta-	HOMG4/URG	187.8365
urogastrone)
C-fos induced growth factor	VEGF-D/VEGFD	3.5884
(vascular endothelial growth factor
D)
Hepatocyte growth factor	F-TCF/HGFB	4.542
(hepapoietin A; scatter factor)
Heparanase	HPA/HPR1	286.6871
Insulin-like growth factor 1	IGFI	4.7022
(somatomedin C)
Jagged 1 (Alagille syndrome)	AGS/AHD	1566.5046
Laminin, alpha 5	KIAA1907	3.8727
Platelet/endothelial cell adhesion	CD31/PECAM-1	11.9037
molecule (CD31 antigen)
Plexin domain containing 1	DKFZp686F0937/	3.4184
	TEM3
Stabilin
1	CLEVER-1/FEEL-1	4.357
Transforming growth factor, alpha	TFGA	3549.3357
Tumor necrosis factor (TNF	DIF/TNF-alpha	4.0652
superfamily, member 2)
Vascular endothelial growth factor C	Flt4-L/VRP	446.7529

TABLE 2

Downregulated CSC-associated genes (p < 0.05)

		Fold
GENESYMBOL	ID	Change

ECHDC1	233124_s_at	0.943
DARS	201624_at	0.928
GALNT1	201722_s_at	0.926
CGGBP1	224600_at	0.913
CSE1L	201112_s_at	0.911
GMFB	202544_at	0.904
RPL7L1	225515_s_at	0.899
SKP1	200718_s_at	0.898
IGHMBP2	215980_s_at	0.893
LOC137886	212934_at	0.886
CSE1L	210766_s_at	0.885
ERRFI1	224657_at	0.881
MAP2K4	203266_s_at	0.881
TNFAIP1	201207_at	0.88
TBXA2R	207554_x_at	0.877
SEPHS1	208940_at	0.875
IPO7	200993_at	0.875
C16ORF63	225087_at	0.872
INSIG2	209566_at	0.872
TFB1M	228075_x_at	0.87
PAK1	226507_at	0.869
C14ORF156	221434_s_at	0.867
SMYD2	212922_s_at	0.867
ENTPD5	231676_s_at	0.867
PPP3CA	202457_s_at	0.867
MBNL1	201152_s_at	0.867
MRPL42	217919_s_at	0.866
SUPT7L	201838_s_at	0.865
PMP22	210139_s_at	0.865
GABARAPL2	209046_s_at	0.863
PITPNA	201190_s_at	0.863
C2ORF30	224630_at	0.851
TXNDC12	223017_at	0.849
POP4	202868_s_at	0.847
MRPL51	224334_s_at	0.846
AK3	224655_at	0.845
GPR107	211979_at	0.843
TMEM126B	221622_s_at	0.843
PSMA2	201316_at	0.839
KIAA1737	225623_at	0.837
TRAPPC2L	218354_at	0.837
RLBP1L1	224996_at	0.835
CCDC127	226515_at	0.835
CPNE3	202119_s_at	0.833
HIAT1	225222_at	0.832
MECR	218664_at	0.832
ACBD6	225317_at	0.83
SLC16A1	202235_at	0.83
ANXA4	201302_at	0.83
DNAJC21	230893_at	0.829
C22ORF28	200042_at	0.829
SPOPL	225659_at	0.828
PDHB	211023_at	0.827
EIF2S1	201144_s_at	0.824
LOC645166	228158_at	0.823
CAMK2D	225019_at	0.823
LIMS1	212687_at	0.822
VTI1B	209452_s_at	0.821
YY1	224711_at	0.821
TRAPPC2	219351_at	0.821
LOC126917	225615_at	0.819
STX8	204690_at	0.819
NANP	228073_at	0.817
NDFIP1	217800_s_at	0.815
UBE3C	1560739_a_at	0.815
KPNA6	226976_at	0.814
C19ORF42	219097_x_at	0.813
DHX40	218277_s_at	0.812
NUCB2	203675_at	0.812
RAB1A	213440_at	0.81
USP8	229501_s_at	0.808
MAP1LC3B	208785_s_at	0.808
PDHB	208911_s_at	0.807
SH2B3	203320_at	0.806
PPP1R3D	204554_at	0.805
DEGS1	209250_at	0.804
HSDL2	209513_s_at	0.803
LOC203547	225556_at	0.802
CANX	238034_at	0.8
PSMA3	201532_at	0.798
PIGY	224660_at	0.793
CYB5R3	1554574_a_at	0.793
BRI3	223376_s_at	0.792
CREB1	204313_s_at	0.791
LOC389203	225014_at	0.79
WDR41	218055_s_at	0.789
C9ORF78	218116_at	0.789
GNPDA1	202382_s_at	0.787
RPE	225039_at	0.787
HSPA4L	205543_at	0.786
SEPT11	201307_at	0.784
HEATR2	241352_at	0.784
ENAH	222433_at	0.783
MED19	226300_at	0.782
TBC1D5	201814_at	0.782
EMP2	225079_at	0.781
STX11	235670_at	0.778
ANKH	229176_at	0.776
ENDOD1	212573_at	0.775
IL13RA1	201887_at	0.775
RAB14	200927_s_at	0.772
TMEM30A	232591_s_at	0.771
DDX52	212834_at	0.771
PTPMT1	229535_at	0.769
SRPRB	218140_x_at	0.767
FAM98A	212333_at	0.767
SRP72	208803_s_at	0.766
RPE	221770_at	0.766
HOXB9	216417_x_at	0.766
MAEA	207922_s_at	0.765
GHITM	1554510_s_at	0.764
CAPZB	201949_x_at	0.764
ANKRD52	228257_at	0.762
MOBKL1B	214812_s_at	0.762
MIA3	1569057_s_at	0.759
UBE2E3	210024_s_at	0.758
CAMK2D	228555_at	0.758
UBXD7	212840_at	0.754
C18ORF10	213617_s_at	0.754
HSD17B1	228595_at	0.753
PDLIM5	212412_at	0.752
SRP72	208801_at	0.751
ZNF618	226590_at	0.75
TSPAN31	203227_s_at	0.744
MAP3K15	200979_at	0.741
C18ORF10	212055_at	0.737
ATP5I	207335_x_at	0.737
TOX4	201685_s_at	0.73
TBXA2R	336_at	0.73
COL4A2	211966_at	0.729
TIMM23	218119_at	0.723
NDUFAF2	228355_s_at	0.722
FOXN3	218031_s_at	0.721
EIF2S1	201142_at	0.717
NDUFB6	203613_s_at	0.712
TM6SF1	1558102_at	0.704
ELOVL2	213712_at	0.699
PPP1R7	201213_at	0.698
BAT3	230513_at	0.697
ZNF668	219047_s_at	0.691
ERBB3	1563253_s_at	0.691
C12ORF45	226349_at	0.688
PGRMC2	213227_at	0.686
NUDT4	212183_at	0.685
AABHD7	239579_at	0.661
CEP27	228744_at	0.651
RAB11FIP3	216043_x_at	0.551
FHL3	218818_at	0.546
NAALAD2	1554506_x_at	0.464
LOC219731	1557208_at	0.419

TABLE 3

CSC-genes annotations

	Entrez
	Gene ID for		Fold
Name	Human	Affymetrix	Change	Networks	Location	Type	Drugs

Actin		—		1	Unknown	group
ADA	100	—		8	Cytoplasm	enzyme	pentostatin,
							vidarabine
Adaptor protein 2		—		8	Unknown	complex
AFP	174	—		5	Extracellular	transporter
					Space
AGT	183	—		8	Extracellular	other
					Space
AHR	196	—		7	Nucleus	ligand-
						dependent
						nuclear
						receptor
AKAP9	10142	215483_at	2.168	1	Cytoplasm	other
Akt		—		2	Unknown	group
amino acids		—		6	Unknown	chemical -
						endogenous
						mammalian
AMPH	273	—		8	Plasma	other
					Membrane
AMZ2	51321	227567_at	1.377	8	Unknown	other
ANAPC1	64682	—		4	Nucleus	other
ANAPC10	10393	—		4	Nucleus	enzyme
ANAPC11	51529	—		4	Unknown	enzyme
ANAPC13	25847	—		4	Unknown	other
ANAPC2	29882	—		4	Nucleus	enzyme
ANAPC4	29945	—		4	Unknown	enzyme
ANAPC5	51433	235926_at	2.631	4	Nucleus	enzyme
ANAPC7	51434	—		4	Unknown	other
ANK2	287	202921_s_at	4.162	4	Plasma	other
					Membrane
ANKRD28	23243	241063_at	2.297	13	Unknown	other
AOC3	8639	204894_s_at	1.894	2	Plasma	enzyme
					Membrane
AP2A2	161	—		8	Cytoplasm	transporter
APBB2	323	40148_at	1.139	9	Cytoplasm	other
APP	351	—		9	Plasma	other	AAB-001
					Membrane
ARD1A	8260	—		8	Nucleus	enzyme
Arf		—		8	Unknown	group
ARF5	381	—		8	Cytoplasm	transporter
ARHGDIB	397	—		7	Cytoplasm	other
ASCC3L1	23020	214982_at	3.009	9	Nucleus	enzyme
(includes
EG: 23020)
ASCL1	429	—		9	Nucleus	transcription
						regulator
ASPM	259266	232238_at	1.411	3	Nucleus	other
ATAD2	29028	235266_at	1.304	7	Unknown	other
ATP		—		9	Unknown	chemical -
						endogenous
						mammalian
ATXN2L	11273	207798_s_at	1.656	9	Unknown	other
BARD1	580	205345_at	1.559	1	Nucleus	transcription
						regulator
BCL2	596	—		6	Cytoplasm	other	oblimersen, (−)-
							gossypol
BCL9L	283149	227616_at	1.291	6	Cytoplasm	other
BDP1	55814	224227_s_at	1.632	9	Nucleus	transcription
						regulator
beta-estradiol		—		3	Unknown	chemical -
						endogenous
						mammalian
BRF1	2972	—		9	Nucleus	transcription
						regulator
BUB1 (includes	699	233445_at	3.209	5	Nucleus	kinase
EG: 699)
BUB1B	701	—		4	Nucleus	kinase
C12ORF48	55010	220060_s_at	1.216		Unknown	other
C12ORF51	283450	1557529_at	3.632	4	Unknown	other
CABIN1	23523	1557581_x_at	3.052	1	Nucleus	other
Calmodulin		—		1	Unknown	group
CASC5	57082	228323_at	1.144	3	Nucleus	other
CASP3	836	—		4	Cytoplasm	peptidase	IDN-6556
CASP6	839	—		9	Cytoplasm	peptidase
CBS	875	240517_at	1.818	1	Cytoplasm	enzyme
CD151	977	—		7	Plasma	other
					Membrane
CDC14B	8555	234605_at	2.512	5	Nucleus	phosphatase
CDC16	8881	242359_at	6.261	4	Nucleus	other
CDC20	991	—		3	Nucleus	other
CDC23 (includes	8697	—		4	Nucleus	enzyme
EG: 8697)
CDC26	246184	—		4	Nucleus	other
CDC27	996	—		4	Nucleus	other
CDC5L	988	222179_at	1.292	9	Nucleus	other
CDK2	1017	—		7	Nucleus	kinase	BMS-387032,
							flavopiridol
CDKN1A	1026	—		7	Nucleus	kinase
CDT1	81620	—		7	Nucleus	other
CDX1	1044	—		9	Nucleus	transcription
						regulator
CENPJ	55835	220885_s_at	1.64	4	Nucleus	transcription
						regulator
CEP55	55165	218542_at	1.096	5	Unknown	other
CHD8	57680	212571_at	1.27	1	Nucleus	enzyme
CHEK2	11200	—		5	Nucleus	kinase
CHRM3	1131	—		8	Plasma	G-protein	fesoterodine, ABT-
					Membrane	coupled	089,
						receptor	atropine/edrophonium,
							cyclopentolate/phenylephrine,
							ipratropium/albuterol,
							trihexyphenidyl,
							carbamylcholine,
							darifenacin,
							methacholine,
							diphenhydramine,
							quinidine,
							procyclidine,
							trospium, atropine
							sulfate/benzoic
							acid/hyoscyamine/methenamine/
							methylene
							blue/phenyl
							salicylate,
							homatropine,
							dicyclomine,
							methantheline,
							orphenadrine,
							fluoxetine/olanzapine,
							doxacurium,
							aspirin/caffeine/orphenadrine,
							propantheline,
							tridihexethyl,
							biperiden,
							anisotropine
							methylbromide,
							glycopyrrolate,
							diphenhydramine/8-
							chlorotheophylline,
							atropine/hyoscyamine/
							phenobarbital/scopolamine,
							atropine
							sulfate/diphenoxylate
							hydrochloride,
							pipecuronium,
							flavoxate,
							chlorpheniramine/methscopolamine/
							phenylephrine,
							mepenzolic
							acid, atropine
							sulfate/difenoxin
							hydrochloride,
							homatropine
							methylbromide,
							hydroxyamphetamine/
							tropicamide,
							cisatracurium,
							hyoscyamine/phenobarbital,
							bethanechol,
							olanzapine,
							oxybutynin,
							tropicamide,
							solifenacin,
							cyclopentolate,
							tolterodine,
							cevimeline,
							acetylcholine,
							ipratropium, atropine,
							pilocarpine,
							benztropine,
							hyoscyamine,
							arecoline,
							scopolamine, N-
							methylscopolamine,
							tiotropium,
							carbinoxamine,
							buclizine,
							diphenhydramine/phenylephrine,
							brompheniramine
CIB1	10519	—		7	Nucleus	other
Ck2		—		1	Unknown	complex
CKM	1158	—		5	Cytoplasm	kinase
CLIC1	1192	—		9	Nucleus	ion channel
CLIC4	25932	—		6	Cytoplasm	ion channel
CLIC5	53405	—		1	Cytoplasm	ion channel
CLN8	2055	229958_at	1.344		Cytoplasm	other
COIL	8161	203653_s_at	1.259	6	Nucleus	other
COL4A1	1282	—		5	Extracellular	other	collagenase
					Space
COPB1	1315	—		8	Cytoplasm	transporter
Creb		—		2	Unknown	group
CREB3L2	64764	237952_at	2.013	3	Unknown	other
CRIPAK	285464	228318_s_at	1.486		Cytoplasm	other
CROP	51747	242389_at	2.121	7	Nucleus	other
CRY1	1407	—		7	Nucleus	enzyme
CTNNA1	1495	—		6	Plasma	other
					Membrane
CTNNAL1	8727	—		6	Plasma	other
					Membrane
CTNNB1	1499	—		6	Nucleus	transcription
						regulator
CUL4A	8451	232466_at	2.607	1	Nucleus	other
DAPK1	1612	—		6	Cytoplasm	kinase
DCLRE1C	64421	242927_at	1.187		Nucleus	enzyme
DDX17	10521	213998_s_at	1.528	6	Nucleus	enzyme
DENND4A	10260	230607_at	2.368	1	Nucleus	other
DMD	1756	—		8	Plasma	other
					Membrane
DNM1L	10059	236032_at	1.503	2	Cytoplasm	enzyme
DSN1	79980	—		3	Nucleus	other
DTX		—		3	Unknown	group
DTX1	1840	—		3	Nucleus	transcription
						regulator
DTX2	113878	—		3	Nucleus	other
DTX3	196403	49051_g_at	1.32	3	Cytoplasm	other
DUB		—		18	Unknown	group
DVL1	1855	—		6	Cytoplasm	other
DVL2	1856	—		6	Cytoplasm	other
Dynamin		—		2	Unknown	group
EGFR	1956	—		7	Plasma	kinase	cetuximab, AEE
					Membrane		788, panitumumab,
							BMS-599626,
							ARRY-334543,
							XL647, canertinib,
							gefitinib, HKI-272,
							PD 153035, lapatinib,
							vandetanib, erlotinib
EIF4G3	8672	201935_s_at	1.174	4	Cytoplasm	translation
						regulator
EPOR	2057	—		9	Plasma	transmembrane	erythropoietin,
					Membrane	receptor	darbepoetin alfa,
							continuous
							erythropoietin
							receptor activator
ERBB2	2064	—		3	Plasma	kinase	trastuzumab, BMS-
					Membrane		599626, ARRY-
							334543, XL647, CP-
							724, 714, HKI-272,
							lapatinib, erlotinib
ETS1	2113	241435_at	1.797	2	Nucleus	transcription
						regulator
EWSR1	2130	229966_at	1.686	1	Nucleus	other
EXT1	2131	242126_at	2.116	4	Cytoplasm	enzyme
FLOT1	10211	—		3	Plasma	other
					Membrane
FLT1	2321	232809_s_at	1.861	2	Plasma	kinase	sunitinib, axitinib,
					Membrane		CEP 7055
FMR1	2332	—		7	Nucleus	other
FRK	2444	—		9	Nucleus	kinase
FUBP1	8880	240307_at	2.087	1	Nucleus	transcription
						regulator
FZR1	51343	—		4	Nucleus	other
GABPA	2551	243498_at	2.03	2	Nucleus	transcription
						regulator
GBF1	8729	233114_at	2.03	8	Cytoplasm	other
GGT1	2678	211417_x_at	1.555	6	Cytoplasm	enzyme
GPD2	2820	243598_at	2.13	3	Cytoplasm	enzyme
HDAC3	8841	240482_at	2.062	1	Nucleus	transcription	tributyrin, PXD101,
						regulator	pyroxamide,
							MGCD0103,
							vorinostat, FR901228
HECW1	23072	237295_at	11.843	6	Cytoplasm	enzyme
HELLS	3070	242890_at	1.359	3	Nucleus	enzyme
HERC5	51191	219863_at	1.156	6	Cytoplasm	enzyme
Histone h3		—		1	Unknown	group
HNRNPC	3183	235500_at	1.769	1	Nucleus	other
HNRPD	3184	241702_at	1.962	4	Nucleus	transcription
						regulator
HNRPH1	3187	213472_at	2.332	8	Nucleus	other
HOXA2	3199	228642_at	1.44	8	Nucleus	transcription
						regulator
HOXD3	3232	206601_s_at	1.897	7	Nucleus	transcription
						regulator
HPS1	3257	239382_at	1.749	14	Cytoplasm	other
HPS4	89781	—		14	Cytoplasm	other
HSPA5	3309	—		3	Cytoplasm	other
HUWE1	10075	214673_s_at	1.858	6	Nucleus	transcription
						regulator
IFNG	3458	—		9	Extracellular	cytokine
					Space
IFNGR1	3459	242903_at	2.171	4	Plasma	transmembrane	interferon gamma-1b
					Membrane	receptor
IL1B	3553	—		4	Extracellular	cytokine	IL-1 trap
					Space
Insulin		—		2	Unknown	group
IRS2	8660	236338_at	2.162	2	Cytoplasm	other
ITGB3	3690	—		7	Plasma	transmembrane	TP 9201,
					Membrane	receptor	EMD121974,
							tirofiban
ITPR1	3708	—		4	Cytoplasm	ion channel
JARID1A	5927	226367_at	1.192	9	Nucleus	transcription
						regulator
JARID2	3720	232835_at	2.139	4	Nucleus	transcription
						regulator
Jnk		—		2	Unknown	group
KIAA1267	284058	224489_at	1.355	1	Nucleus	other
KIDINS220	57498	1557246_at	2.97	6	Nucleus	transcription
						regulator
KIR2DL3	3804	—		9	Plasma	other
					Membrane
KITLG (includes	4254	—		9	Extracellular	growth factor
EG: 4254)					Space
KLF6	1316	—		5, 9	Nucleus	transcription
						regulator
LCN2	3934	—		9	Extracellular	transporter
					Space
LMO2	4005	—		9	Nucleus	other
LOC388135	388135	230475_at	1.979	5	Unknown	other
LRRFIP1	9208	239379_at	1.796	3	Nucleus	transcription
						regulator
MALAT1	378938	224568_x_at	1.699		Unknown	other
Mapk		—		2	Unknown	group
MEF2C	4208	236395_at	2.104	2	Nucleus	transcription
						regulator
MET	4233	213816_s_at	1.283	2	Plasma	kinase
					Membrane
mGluR		—		8	Unknown	group
MIS12	79003	—		3	Nucleus	other
MLL	4297	212079_s_at	1.599	1	Nucleus	transcription
						regulator
MPL	4352	—		9	Plasma	transmembrane	SB-497115
					Membrane	receptor
MTUS1	57509	239576_at	1.975	1	Unknown	other
MYC	4609	—		6	Nucleus	transcription
						regulator
MYF6	4618	—		5	Nucleus	transcription
						regulator
MYO10	4651	243159_x_at	2.528	3	Cytoplasm	other
MYOD1	4654	—		5	Nucleus	transcription
						regulator
N4BP2L1	90634	213375_s_at	2.01		Unknown	other
Nap125		—		16	Unknown	group
NAPA	8775	239362_at	1.624	2	Cytoplasm	transporter
NAPE-PLD	222236	242635_s_at	1.216	8	Cytoplasm	enzyme
NARG1	80155	1556381_at	2.827	8	Nucleus	transcription
						regulator
NAT13	80218	—		8	Cytoplasm	enzyme
NBPF15	284565	201104_x_at	1.411	1	Unknown	other
NBR1	4077	1568856_at	1.957	5	Unknown	other
NCKAP1L	3071	209734_at	2.071	16	Plasma	other
					Membrane
NCOA3	8202	—		7	Nucleus	transcription
						regulator
NEK1	4750	213328_at	1.381	6	Nucleus	kinase
NES	10763	—		5	Cytoplasm	other
NFATC2IP	84901	217527_s_at	1.272	1	Nucleus	other
NFkB		—		2	Unknown	complex
NFKBIE (includes	4794	—		13	Nucleus	transcription
EG: 4794)						regulator
NMB	4828	—		8	Extracellular	other
					Space
NPAS2	4862	1557690_x_at	1.76	7	Nucleus	transcription
						regulator
NPTN	27020	228723_at	2.086	1	Plasma	other
					Membrane
NRP2	8828	210841_s_at	1.106	2, 3	Plasma	kinase
					Membrane
NUPL1	9818	241425_at	2.179	17	Nucleus	transporter
OGG1	4968	—		9	Nucleus	enzyme
OSBPL5	114879	233734_s_at	1.261	3	Cytoplasm	other
OSGEP	55644	242930_at	1.541	3	Unknown	peptidase
P38 MAPK		—		2	Unknown	group
PABPN1	8106	213046_at	2.228	5	Nucleus	other
PAX3	5077	—		7	Nucleus	transcription
						regulator
PCBP1 (includes	5093	—		6	Nucleus	translation
EG: 5093)						regulator
PDE4B	5142	215671_at	3.457	2	Cytoplasm	enzyme	dyphylline,
							nitroglycerin,
							arofylline, tetomilast,
							L 869298,
							aminophylline,
							anagrelide,
							cilomilast, milrinone,
							rolipram,
							dipyridamole, L-
							826, 141, roflumilast,
							tolbutamide,
							theophylline,
							pentoxifylline,
							caffeine
PDE5A	8654	239556_at	2.24	4	Cytoplasm	enzyme	dyphylline,
							nitroglycerin, DA-
							8159, aminophylline,
							sildenafil,
							dipyridamole,
							aspirin/dipyridamole,
							vardenafil,
							tolbutamide,
							tadalafil,
							theophylline,
							pentoxifylline
PDGF BB		—		2	Unknown	complex
PDK1	5163	239798_at	1.654	1	Cytoplasm	kinase
PDSS1	23590	236298_at	1.64	15	Unknown	enzyme
PDXDC1	23042	1560014_s_at	2.105	8	Unknown	other
PHC1	1911	218338_at	1.123	5	Nucleus	other
PI3K		—		2	Unknown	complex
PIP5K1C	23396	—		7	Plasma	kinase
					Membrane
PIP5K3	200576	1557719_at	2.227	2	Cytoplasm	kinase
Pka		—		1	Unknown	complex
Pkc(s)		—		2	Unknown	group
PLAA	9373	—		4	Cytoplasm	other
PLC gamma		—		2	Unknown	group
Pld		—		8	Unknown	group
PLK1	5347	—		7	Nucleus	kinase	BI 2536
PMS2L3	5387	214473_x_at	1.159	3	Unknown	other
POLR2J2	246721	1552622_s_at	1.828	1	Nucleus	transcription
						regulator
POU4F2	5458	—		6	Nucleus	transcription
						regulator
PP2A		—		6	Unknown	complex
PRDM5	11107	—		5	Nucleus	other
PRKCB1	5579	—		7	Cytoplasm	kinase	enzastaurin,
							ruboxistaurin
progesterone		—		8	Unknown	chemical-
						endogenous
						mammalian
PSEN1	5663	242875_at	1.851	2	Plasma	peptidase	(R)-flurbiprofen
					Membrane
PTEN	5728	—		3	Cytoplasm	phosphatase
PTK2	5747	234211_at	2.539	2	Cytoplasm	kinase
PTPN12	5782	—		7	Cytoplasm	phosphatase
PTPN14	5784	—		6	Cytoplasm	phosphatase
PTPRA	5786	—		7	Plasma	phosphatase
					Membrane
PTPRD	5789	—		6	Plasma	phosphatase
					Membrane
PTPRE	5791	—		7	Plasma	phosphatase
					Membrane
PTPRS (includes	5802	1556116_s_at	1.739	7	Plasma	phosphatase
EG: 5802)					Membrane
RAB11FIP3	9727	228613_at	2.546	8	Cytoplasm	other
RAB11FIP4	84440	—		8	Cytoplasm	other
Rac		—		2	Unknown	group
RAD50	10111	—		5	Nucleus	enzyme
RAD54L	8438	204558_at	1.483	5	Nucleus	enzyme
RB1	5925	—		9	Nucleus	transcription
						regulator
RBM25	58517	1557081_at	1.57	7	Nucleus	other
RBM4	5936	213718_at	1.53	7	Nucleus	other
RBM5	10181	209936_at	2.249	6	Nucleus	other
RDBP	7936	—		3	Nucleus	other
RHOA	387	240337_at	2.143	2	Cytoplasm	enzyme
RHOBTB2	23221	1556645_s_at	1.538		Unknown	enzyme
RNA polymerase II		—		1	Unknown	complex
RNU1A	6060	—		1	Unknown	other
RP13-122B23.3	25920	1556434_at	1.985	3	Nucleus	other
RPL10	6134	—		6	Cytoplasm	other
RUNX3	864	204198_s_at	1.233	7	Nucleus	transcription
						regulator
SBF1	6305	—		3	Plasma	phosphatase
					Membrane
SCMH1	22955	—		5	Nucleus	transcription
						regulator
SCN3A	6328	—		4	Plasma	ion channel	riluzole
					Membrane
SEC16A	9919	—		10	Cytoplasm	phosphatase
SEC16B	89866	1552880_at	1.877	10	Nucleus	other
Secretase gamma		—		9	Unknown	complex
SF1	7536	210172_at	2.452	1, 4	Nucleus	transcription
						regulator
SF3B1	23451	214305_s_at	1.359	1	Nucleus	other
SFRS15	57466	243759_at	2.028		Nucleus	other
SGCA	6442	1562729_at	2.395	8	Plasma	other
					Membrane
SGCB	6443	—		8	Plasma	other
					Membrane
SGCD	6444	—		8	Cytoplasm	other
SGCG	6445	—		8	Plasma	other
					Membrane
SH2D1A (includes	4068	—		5	Cytoplasm	other
EG: 4068)
SKIL	6498	—		4	Nucleus	transcription
						regulator
SLC29A1	2030	—		9	Plasma	transporter
					Membrane
SLC2A11	66035	232167_at	1.529	9	Plasma	other
					Membrane
SLC2A8	29988	239426_at	2.012		Plasma	transporter
					Membrane
SLC30A9	10463	237051_at	2.063	7	Nucleus	transporter
SLC4A1	6521	—		9	Plasma	transporter
					Membrane
SMAD4	4089	—		6	Nucleus	transcription
						regulator
SMARCA5	8467	—		9	Nucleus	transcription
						regulator
SMC5	23137	—		12	Nucleus	other
SMC6	79677	218781_at	1.203	12	Nucleus	other
SMN1	6606	—		6	Nucleus	other
SNRPA1	6627	242146_at	3.54	8	Nucleus	other
SNW1	22938	—		5	Nucleus	transcription
						regulator
SON	6651	201085_s_at	1.144	5	Nucleus	other
SP4	6671	—		3	Nucleus	transcription
						regulator
sphingomyelin		—		9	Unknown	chemical-
						endogenous
						mammalian
SPN	6693	—		3	Plasma	transmembrane
					Membrane	receptor
SPTBN1	6711	—		4, 6, 8	Plasma	other
					Membrane
SQLE	6713	213577_at	1.502	3	Cytoplasm	enzyme
SQSTM1	8878	—		5	Cytoplasm	transcription
						regulator
SRC	6714	—		6	Cytoplasm	kinase	dasatinib, AZM-
							475271
STK36	27148	231806_s_at	1.362	6	Unknown	kinase
STRAP	11171	1558002_at	2.189	2	Plasma	other
					Membrane
STX3	6809	—		7	Plasma	transporter
					Membrane
SUMO1	7341	—		8	Nucleus	enzyme
SUMO2	6613	—		9	Nucleus	other
SVIL	6840	215279_at	2.228	4	Plasma	other
					Membrane
SYNE2	23224	202761_s_at	1.356	1	Nucleus	other
TAF15	8148	227891_s_at	1.971	1	Nucleus	transcription
						regulator
TAF1A	9015	—		5	Nucleus	transcription
						regulator
TAF1B	9014	239046_at	1.468	5	Nucleus	transcription
						regulator
TAF1C	9013	—		5	Nucleus	transcription
						regulator
TAOK3	51347	220761_s_at	1.195	2	Cytoplasm	kinase
Tap		—		17	Unknown	complex
TBC1D8	11138	204526_s_at	1.373	3	Plasma	other
					Membrane
TCERG1	10915	—		8	Nucleus	transcription
						regulator
TCF7L2	6934	—		6	Nucleus	transcription
						regulator
TCOF1 (includes	6949	202385_s_at	1.169	1	Nucleus	transporter
EG: 6949)
TCR		—		2	Unknown	complex
TERF2	7014	—		5	Nucleus	other
TH1L	51497	—		3	Nucleus	other
THAP7	80764	—		1	Nucleus	other
THRAP3	9967	217847_s_at	1.464	1	Nucleus	transcription
						regulator
TIMP1	7076	—		9	Extracellular	other
					Space
TNF	7124	—		4	Extracellular	cytokine	adalimumab,
					Space		etanercept,
							infliximab, CDP870,
							golimumab,
							thalidomide
TNRC6A	27327	234734_s_at	1.268	9	Nucleus	other
TP53	7157	—		5	Nucleus	transcription
						regulator
TP53BP1	7158	—		5	Nucleus	transcription
						regulator
TPM4	7171	235094_at	2.079	8	Cytoplasm	other
Trans-		—		15	Unknown	group
hexaprenyltranstransferase
TRIM33	51592	239716_at	2.496	6	Nucleus	transcription
						regulator
TRIO	7204	240773_at	2.607	2	Plasma	kinase
					Membrane
tRNA		—		19	Unknown	group
adenylyltransferase
tRNA		—		19	Unknown	group
cytidylyltransferase
TRNT1	51095	243236_at	2.295	19	Cytoplasm	enzyme
TRPV1	7442	1556229_at	2.636	2	Plasma	ion channel	SB-705498,
					Membrane		capsaicin
TSG101	7251	—		5, 7	Nucleus	transcription
						regulator
TSPAN7	7102	—		7	Plasma	other
					Membrane
TTC3	7267	208664_s_at	1.396	7	Cytoplasm	other
TXNL1	9352	243664_at	1.98	9	Cytoplasm	enzyme
Ubiquitin		—		1	Unknown	group
UGT		—		7	Unknown	group
UGT1A6	54578	206094_x_at	3.86	7	Cytoplasm	enzyme
USP36	57602	224979_s_at	1.393	18	Nucleus	peptidase
Vegf		—		2	Unknown	group
VEGFA	7422	—		3	Extracellular	growth factor	bevacizumab,
					Space		ranibizumab,
							pegaptanib
VEGFB (includes	7423	—		3	Extracellular	growth factor
EG: 7423)					Space
VPS28	51160	—		5	Cytoplasm	transporter
VPS37B	79720	236889_at	2.85	5	Nucleus	other
WAS	7454	—		11	Cytoplasm	other
WDR68	10238	233782_at	1.924	4	Cytoplasm	other
WFS1	7466	1555270_a_at	1.315	3	Cytoplasm	enzyme
WIPF2	147179	216006_at	2.916	11	Unknown	other
WT1	7490	—		6	Nucleus	transcription
						regulator
WTAP	9589	1560274_at	1.747	2	Nucleus	other
XRCC5	7520	32633_at	2.106	5	Nucleus	enzyme
YWHAG	7532	—		4	Cytoplasm	other
ZEB1	6935	—		5	Nucleus	transcription
						regulator
ZFHX3	463	215828_at	1.737	5	Nucleus	transcription
						regulator
ZFR	51663	238970_at	2.655		Nucleus	other
ZFX	7543	207920_x_at	1.625	9	Nucleus	transcription
						regulator
ZMYND8	23613	209049_s_at	1.102	7	Nucleus	transcription
						regulator
ZNF224	7767	216983_s_at	2.986	6	Nucleus	other
ZNF226	7769	219603_s_at	1.332	8	Nucleus	transcription
						regulator
ZNF326	284695	241720_at	1.418		Nucleus	transcription
						regulator
ZNF536	9745	233890_at	3.303		Unknown	other
ZWINT (includes	11130	—		3	Nucleus	other
EG: 11130)

TABLE 4

Cell Surface Genes

Entrez
Gene ID
for
Human	Name	Location

7204	TRIO	Plasma Membrane
1956	EGFR	Plasma Membrane
7102	TSPAN7	Plasma Membrane
977	CD151	Plasma Membrane
2064	ERBB2	Plasma Membrane
2321	FLT1	Plasma Membrane
2030	SLC29A1	Plasma Membrane
11171	STRAP	Plasma Membrane
8828	NRP2	Plasma Membrane
4233	MET	Plasma Membrane
273	AMPH	Plasma Membrane
351	APP	Plasma Membrane
1756	DMD	Plasma Membrane
1495	CTNNA1	Plasma Membrane
8727	CTNNAL1	Plasma Membrane
10211	FLOT1	Plasma Membrane
3459	IFNGR1	Plasma Membrane
23396	PIP5K1C	Plasma Membrane
5663	PSEN1	Plasma Membrane
6445	SGCG	Plasma Membrane
6693	SPN	Plasma Membrane
6711	SPTBN1	Plasma Membrane
6840	SVIL	Plasma Membrane
2057	EPOR	Plasma Membrane
5789	PTPRD	Plasma Membrane
4352	MPL	Plasma Membrane
5786	PTPRA	Plasma Membrane
27020	NPTN	Plasma Membrane
3690	ITGB3	Plasma Membrane
7442	TRPV1	Plasma Membrane
8639	AOC3	Plasma Membrane
1131	CHRM3	Plasma Membrane
3804	KIR2DL3	Plasma Membrane
287	ANK2	Plasma Membrane
3071	NCKAP1L	Plasma Membrane
5791	PTPRE	Plasma Membrane
5802	PTPRS	Plasma Membrane
6305	SBF1	Plasma Membrane
6328	SCN3A	Plasma Membrane
6442	SGCA	Plasma Membrane
6443	SGCB	Plasma Membrane
66035	SLC2A11	Plasma Membrane
29988	SLC2A8	Plasma Membrane
6521	SLC4A1	Plasma Membrane
6809	STX3	Plasma Membrane
11138	TBC1D8	Plasma Membrane

A used herein “CSC-associated gene” refers to a gene whose expression or function is altered in cancer stem cells. CSC-associated genes include genes whose expression is significantly altered, e.g., significantly upregulated or significantly downregulated, in cancer stem cells compared with non-cancer stem cells, e.g., cancer cells that are not stem cells, normal cells, etc. In some embodiments, genes that have significantly altered expression levels in cancer stem cells are identified by using an appropriate statistical test for establishing the significance of differences between expression levels in a cancer stem cell and a non-cancer stem cell. Tests for statistical significance are well known in the art and are exemplified in Applied Statistics for Engineers and Scientists by Petruccelli, Chen and Nandram 1999 Reprint Ed. The magnitude of up-, or down-, regulated expression of a CSC-associated gene in a cancer stem cell compared with a non-cancer stem cell may vary. In some embodiments, the expression level of a CSC-associated gene is at least 10%, at least 25%, at least 50%, at least 100%, at least 250%, at least 500%, or at least 1000% higher, or lower, than its expression level in a non-cancer stem cell. In other embodiments, the expression level of a CSC-associated gene is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, or more higher, or lower, than its expression level a non-cancer stem cell.
CSC-associated genes are not limited to genes which are upregulated or downregulated in cancer stem cells. In some embodiments, a CSC-associated gene is a gene that may or may not have altered expression in a cancer stem cell, but that nevertheless functions in a pathway that is deregulated in cancer stem cells. Typically, deregulated pathways in cancer stem cells involve the product(s) of one or more genes whose expression is upregulated or downregulated and/or the product(s) of one or more genes with altered functionality, e.g., due to a mutation, thereby resulting in altered function of the pathway, e.g., overactivity or underactivity of the pathway.
In some embodiments, CSC-associated genes are identified in cancer stem cells of a breast cancer, prostate cancer, colon cancer, lung cancer, renal cancer or melanoma. In some instances, CSC-associated genes are identified in cancer stem cells of a melanoma, which are also referred to as malignant melanoma initiating cells (MMIC). Other cancer stem cells (e.g., non-ABCB5⁺ CSCs) are known in the art.
Exemplary CSC-associated genes are disclosed in Tables 1-8. In some embodiments, a CSC-associated gene is selected from the group consisting of: ANK2, NCKAPIL, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8. In some embodiments, the CSC-associated gene is one that is not a gene of the group consisting of EGFR, CD151, ERBB2, FLT1, SLC29A1, NRP2, MET, AMPH, APP, DMD, and ITGB3. In some embodiments, the CSC-associated gene is one that is not a gene of the group consisting of: TRIO, TSPAN7, STRAP, CTNNA1, CTNNAL1, FLOT1, IFNGR1, PIP5K1C, PSEN1, SGCG, SPN, SPTBN1, SVIL, EPOR, PTPRD, MPL, PTPRA, NPTN, TRPV1, AOC3, CHRM3, and KIR2□L3. In some embodiments, the CSC-associated gene is one that is not a gene that has previously been indicated as a tumor suppressor or oncogene. In some embodiments, the CSC-associated gene is one that is not a gene of the group consisting of EWSR1, TP53, EGFR, ITPR1, NBR1, MLL, PTK2, PTPN14, RBI, JARID1A, SKIL, TNF, TP53BP1, TRIO, SF1, TAF15, NCOA3, RAD54L, CUL4A, SMARCA5, RAD50, AKAP9, DENND4A, DDX17, HECW1, ZMYND8, ANAPC13, ANAPC5, TH1L, TRIM33, and CHD8. In some embodiments, the CSC-associated gene is one that is not a gene of the group consisting of: BARD1, BCL2, CBS, CTNNB1, ERBB2, EWSR1, HPS1, IFNG, IL1B, PTEN, TP53, VEGFA, CHEK2, and HPS4.
In part, the disclosure relates to CSC-associated genes as well as the RNAs and polypeptides (CSC-associated RNA and polypeptides) that they encode and antibodies and antigen-binding fragments that specifically bind them. The CSC-associated genes, RNAs and polypeptides, encompass variants, homologues, and fragments. Variants may result from alternative splicing or allelic variation of certain genes provided in Tables 5. In general, homologues and alleles typically will share at least 90% nucleotide identity and/or at least 95% amino acid identity to the sequences of the cancer antigen nucleic acids and polypeptides, respectively, in some instances will share at least 95% nucleotide identity and/or at least 97% amino acid identity, in other instances will share at least 97% nucleotide identity and/or at least 98% amino acid identity, in other instances will share at least 99% nucleotide identity and/or at least 99% amino acid identity, and in other instances will share at least 99.5% nucleotide identity and/or at least 99.5% amino acid identity. Homology can be calculated using various, publicly available software tools known in the art, such as those developed by NCBI (Bethesda, Md.) that are available through the interne. Exemplary tools include the BLAST system (e.g., using the default nucleic acid (Blastn) or protein (Blastp) search parameters) available from the website of the National Center for Biotechnology Information (NCBI) at the National Institutes of Health.
The CSC-associated genes are, among other things, useful for diagnosing cancer, such as breast cancer, prostate cancer, colon cancer, lung cancer, renal cancer or melanoma. Because CSC-associated gene expression is altered in cancer (e.g., upregulated or downregulated), the expression level of CSC-associated gene(s), e.g., a gene listed in Table 5 or 7, in an individual is diagnostic of cancer in that individual. Accordingly, the diagnostic methods disclosed herein can involve determining the CSC-associated RNA or protein (polypeptide) levels.
The term “individual” as used herein means any mammalian individual or subject, including, e.g., humans and non-human mammals, such as primates, rodents, and dogs. Individuals specifically intended for diagnosis and treatment using the methods described herein are preferably humans.
The expression level of CSC-associated gene(s) may be determined by using any of a number of methods known in the art. In some embodiments, the expression levels are determined from a biological sample (e.g., a test sample) obtained from a individual (e.g., a human). Exemplary, biological samples include an isolated cell, an isolated tissue, saliva, gingival secretions, cerebrospinal fluid (spinal fluid), gastrointestinal fluid, mucus, urogenital secretions, synovial fluid, blood, serum, plasma, urine, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, intracellular fluid, ocular fluids, seminal fluid, mammary secretions, vitreal fluid, and nasal secretions. However, biological samples are not so limited and other exemplary biological specimens will be readily apparent to one of ordinary skill in the art. For the purposes of diagnosing melanoma, for example, the biological sample is preferably a skin tissue sample, e.g., a skin biopsy containing a suspicious lesion.
Expression levels of CSC-associated genes may be determined for diagnostic purposes using nucleic acid hybridization or nucleic acid amplification to detect the mRNAs that they encode. Methods for nucleic acid hybridization or amplification are well known in the art. In some embodiments, the nucleic acid amplification is real-time RT-PCR or RT-PCR. Other methods known to one of ordinary skill in the art could be employed to analyze mRNA levels, for example nucleic acid arrays, cDNA analysis, Northern analysis, and RNase Protection Assays. Nucleic acid arrays may be used to assay (e.g., for diagnostic purposes) the expression levels of multiple CSC-associated genes in parallel. Other suitable nucleic acid detection methods will be apparent to the skilled artisan.
Expression levels of CSC-associated genes may be determined for diagnostic purposes by detecting the polypeptides that they encode. Methods for detecting polypeptides are well known in the art. Exemplary polypeptide detection methods include, but are not limited to, Enzyme Linked Immunosorbent Assays (ELISA), radioimmunoassays (RIA), sandwich immunometric assays, flow cytometry, western blot assays, immunoprecipitation assays, immunohistochemistry, immunomicroscopy, lateral flow immuno-chromatographic assays, BIACORE technology, and proteomics methods, such as mass spectroscopy. Antibody arrays may be used to assay (e.g., for diagnostic purposes) the expression levels of multiple CSC-associated genes in parallel. Other suitable polypeptide detection methods will be apparent to the skilled artisan.
In some embodiments, e.g., where polypeptide, antibody or nucleic acid arrays are used, expression levels of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, or more CSC-associated genes may be tested in parallel.
The diagnostic methods of the invention involve a comparison between expression levels of CSC-associated genes in a test sample and a reference value. The results of the comparison are diagnostic of cancer, e.g., melanoma. In some embodiments, the reference value is the expression level of the gene in a reference sample. A reference value may be a predetermined value and may also be determined from reference samples (e.g., control biological samples) tested in parallel with the test samples. A reference value may be a positive or negative control level. A reference value can be a single cut-off value, such as a median or mean or a range of values, such as a confidence interval. Reference values can be established for various subgroups of individuals, such as individuals predisposed to cancer, individuals having early or late stage cancer, male and/or female individuals, or individuals undergoing cancer therapy. The level of the reference value will depend upon the particular population or subgroup selected. For example, an apparently healthy population will have a different “normal” value than will a population which has cancer, or a population that has a predisposition for cancer. Appropriate ranges and categories for reference values can be selected with no more than routine experimentation by those of ordinary skill in the art.
The reference sample can be any of a variety of biological samples against which a diagnostic assessment may be made. Examples of reference samples include biological samples from control populations or control samples. Reference samples may be generated through manufacture to be supplied for testing in parallel with the test samples, e.g., reference sample may be supplied in diagnostic kits. When the reference sample is from a cancer, e.g., tumor tissue, the expression level of the reference sample (the reference value) is the expression level of the CSC-associated gene in the cancer. Similarly, when the reference sample is a normal sample, e.g., non-tumor tissue, the expression level of the reference sample (the reference value) is the expression level of the CSC-associated gene in the non-tumor tissue. Similarly, when the reference sample is a cancer stem cell sample, the expression level of the reference sample (the reference value) is the expression level of the CSC-associated gene in the cancer stem cell sample. In some embodiments, the reference sample is of a melanoma and the expression level of the reference sample is the expression level of the CSC-associated gene in melanoma. In some embodiments, the reference sample is of a non-melanoma tissue and the expression level of the reference sample is the expression level of the CSC-associated gene in non-melanoma tissue. Other appropriate reference samples will be apparent to the skilled artisan.
The diagnostic methods are based in part on a comparison of expression levels of CSC-associated genes between test samples and reference sample. In some embodiments, if the expression level of the CSC-associated gene in the test sample is about equal to the expression level of the CSC-associated gene in the reference sample, then the test sample and reference sample are likely of a similar origin, category or class. For example, if expression levels in a test sample and reference sample are about the same (e.g., not statistically significantly different), and the reference sample is from a normal tissue, then the test sample is likely a normal tissue sample, and a normal diagnosis could be indicated. Alternatively, if expression levels in a test sample and reference sample are about the same, and the reference sample is from a cancer tissue, then the test sample is likely a cancer sample, and a diagnosis of cancer could be indicated. In certain embodiments, if the expression level in a test sample and reference sample are about the same, and the reference sample is from a melanoma, then the test sample is likely a melanoma sample, and a diagnosis of melanoma could be indicated.
In some cases, depending on factors such as the particular CSC-associated gene(s) being evaluated, the condition being diagnosed, and the type of reference sample, an expression level of a CSC-associated gene in a test sample that is statistically significantly higher or statistically significantly lower than its expression level in a reference sample indicates a diagnosis. For example, when the CSC-associated gene is among those listed in Table 1 or 8 and the reference value is the expression level of the CSC-associated gene in a normal (e.g., non-cancerous) reference sample, if the expression level of the CSC-associated gene in the test sample is significantly higher than the expression level of the CSC-associated gene in the normal reference sample, the comparison indicates cancer, e.g., melanoma. Similarly, when the CSC-associated gene is among those listed in Table 1 or 8 and the reference value is the expression level of the CSC-associated gene in a cancer, e.g., melanoma, reference sample, if the expression level of the CSC-associated gene in the test sample is significantly lower than the expression level of the CSC-associated gene in the cancer reference sample, the comparison does not indicate cancer. Alternatively, when the CSC-associated gene is among those listed in Table 2 or 7 and the reference value is the expression level of the CSC-associated gene in a normal reference sample, if the expression level of the CSC-associated gene in the test sample is significantly lower than the expression level of the CSC-associated gene in the normal reference sample, the comparison indicates cancer. Similarly, when the CSC-associated gene is in Table 2 or 7 and the reference value is the expression level of the CSC-associated gene in a cancer, e.g., melanoma, reference sample, if the expression level of the CSC-associated gene in the test sample is significantly higher than the expression level of the CSC-associated gene in the cancer reference sample, the comparison does not indicate melanoma. Appropriate combinations of particular CSC-associated gene(s), conditions to be diagnosed, and types of reference samples, can be selected with no more than routine experimentation by those of ordinary skill in the art for use in the diagnostic methods disclosed herein.
The magnitude of the difference between the test sample and reference sample that is sufficient to indicate a diagnosis will depend on a variety of factors such as the particular CSC-associated gene(s) being evaluated, the condition being diagnosed, heterogeneity in healthy or disease populations from which samples are drawn, the type of reference sample, the magnitude of expression level of a CSC-associated gene, the assay being used, etc. It is well within the purview of the skilled artisan to determine the appropriate magnitude of difference between the test sample and reference sample that is sufficient to indicate a diagnosis. In some embodiments, the expression level of the CSC-associated gene in the test sample is at least 10%, at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000% or more higher than the expression level of the gene in the reference sample. In other embodiments, the expression level of the CSC-associated gene in the test sample is at least 10%, at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000% or more lower than the expression level of the gene in the reference sample.
Some CSC-associated genes that are normally produced in very low quantities but whose production is dramatically increased in tumor cells, e.g., a CSC-associated gene in Table 1 or 8, can trigger an immune response. Thus, in some instances, specific immunoreactivity against a CSC-associated polypeptide, e.g., a polypeptide encoded by a gene listed in Table 1 or 8, in a individual may be diagnostic of cancer in the individual. Immunoreactivity against CSC-associated polypeptides may be humoral or cellular, and is associated with a specific immune response to a CSC-associated polypeptide that is upregulated in a cancer stem cell in an individual. In the case of a humoral response the diagnostic methods may involve detecting the presence of one or more antibodies in an individual that specifically bind CSC-associated polypeptides. Methods for detecting antibodies are disclosed herein (e.g., ELISA, peptide arrays, etc.) and are well known in the art. In some cases, the presence of CSC-polypeptide specific effector cells is diagnostic of an immune response specific to that CSC-polypeptide.
T Lymphocytes recognize complexes of peptide ligands (e.g., CSC-associated polypeptides) and Major Histocompatibility Complex (MHC) molecules presented at the surface of Antigen Presenting Cells (APC). Class I tetramers bind to a distinct set of T cell receptors (TCRs) on a subset of CD8+ T cells, and Class II tetramers bind to a distinct population of CD4+ T cells. Methods for detecting antigen-specific T cells using MHC tetramers are well known in the art (e.g., New Microarray Detects Antigen-Specific T Cells and Immune Responses. PLoS Biol 1(3): e76 2003) and can be used to detect CSC-polypeptide specific T cells which may be diagnostic of cancer in an individual. ITAG reagents, for example, from Beckman Coulter provide a convenient way to measure the cellular response directed toward a single CSC-associated peptide using MHC tetramers.
The methods for evaluating expression of CSC-associated genes, e.g., diagnostic methods, disclosed herein may be combined with methods for treating an individual having or suspected of having cancer. The treatment may comprise a step of determining the expression level of the CSC-associated gene in the individual. The treatment may also comprise a step of comparing the expression level of the CSC-associated gene to a reference value, such that the results of the comparison are diagnostic of cancer in the individual. In certain cases, if the comparison results in a diagnosis of cancer in the individual, the administering step is performed. In some cases, after a diagnosis is made using the methods disclosed herein, a treatment plan is selected. For example, if a diagnostic assay reveals that the expression of a particular CSC-associated gene is altered, e.g., increased or decreased, compared to a normal reference sample, then a treatment directed at that particular CSC-associated gene may be implemented. The diagnostic methods can also be used to evaluate the response to a treatment. For example, the determining and the comparing may be repeated at one or more intervals after the administering step to evaluate the response to the treatment.
CSC-associated polypeptide arrays and arrays of antibodies that bind CSC-associated polypeptides may be constructed by immobilizing large numbers of isolated CSC associated polypeptides or antibodies, or antigen binding fragments, to a solid support. Methods for producing polypeptide and antibody arrays are well known in the art. The methods typically involve production of proteins (CSC-associated polypeptides or antibodies) from an expression library, cloned into E. coli, yeast, or mammalian cells, or similar system, e.g., hybridomas etc., from which the expressed proteins are then purified, for example via His, GST tag, or Protein A/G affinity purification. Cell free protein transcription/translation is an alternative for synthesis of proteins which do not express well in bacterial or other in vivo systems. The purified isolated CSC associated polypeptides or antibodies are immobilized on the array surface (solid support surface) using art known methods. For example, proteins can be immobilized by adsorption, covalent (e.g., aldehydes) and non-covalent (e.g., biotin-streptavidin) interactions. Other methods of conjugation will be readily apparent to one of ordinary skill in the art. In some embodiments, the polypeptide arrays of the invention consist essentially of at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, or more polypeptides or immunogenic fragments thereof encoded by a CSC-associated gene set forth in Table 1, 5, 7, or 8. In some embodiments, the antibody arrays of the invention consist essentially of at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, or more different antibodies or antigen-binding fragments that specifically bind polypeptides (CSC-associated polypeptides) encoded by a CSC-associated gene set forth in Table 1, 5, 7, or 8.
Methods for producing nucleic acid arrays are well known in the art. Nucleic acid arrays may be constructed by, e.g., immobilizing to a solid support large numbers oligonucleotides, polynucleotides, or cDNAs having sequences complementary to CSC-associated mRNAs. The skilled artisan is also referred to Chapter 22 “Nucleic Acid Arrays” of Current Protocols In Molecular Biology (Eds. Ausubel et al. John Wiley and #38; Sons NY, 2000), International Publication WO00/58516, U.S. Pat. No. 5,677,195 and U.S. Pat. No. 5,445,934 which provide exemplary methods relating to nucleic acid array construction and use in detection of nucleic acids of interest. In some embodiments, the nucleic acid arrays consist essentially of at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, or more of the CSC-associated genes set forth in Table 1, 5, 7, or 8.
In some embodiments, the expression levels of multiple CSC-associated genes may be combined to produce an expression profile. As used herein, “expression profile” refers to a set of expression levels of a plurality (e.g., 2 or more) of CSC-associated genes. Expression profiles have a variety of uses. For example, expression profiles may be used to classify (or sub-classify) a sample, preferably a clinical sample. In some embodiments, reference samples, for which a classification, e.g., a disease category, e.g., breast cancer, prostate cancer, melanoma, etc., has already been ascertained, are used to produce known expression profiles. In some embodiments, the similarity of an expression profile of a test sample and a known expression profile, is assessed by comparing the level of the same CSC-associated gene in the test and known expression profiles (i.e., expression level pairs). In some cases, a test expression profile is compared with one or more members of a plurality of known expression profiles, and a known expression profile that most closely resembles (i.e., is most similar to) the test profile is identified. In certain cases, the classification of a known expression profile that is identified as similar to a test expression profile is assigned to the test expression profile, thereby classifying the clinical sample associated with the test expression profile. The methods are useful for classifying samples across a range of phenotypes, e.g., disease status, risk status, etc., based on expression profiles. In some embodiments, a classification model (e.g., discriminant function, naïve bayes, support vector machine, logistic regression, and others known in the art) may be built based on the reference expression profiles from various samples from individuals known to have different disorders (e.g., cancers) and/or from healthy individuals, and used to classify subsequently obtained samples (e.g., clinical samples).
The invention also provides methods for stratifying a population comprising individuals having cancer. In some embodiments, methods involve determining expression levels of at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, or more of the CSC-associated genes set forth in Table 5, 7, or 8, for example, by using the arrays of the invention, and stratifying the population based on the expression levels. The stratification methods are useful in epidemiological studies, for example, to identify subpopulations of individuals at risk of cancer. The methods are also useful in clinical studies to identify patient subpopulations that respond better or worse to a particular treatment.
In some aspects, CSC-associated genes provide a basis for identifying, isolating, cloning, propagating, and expanding CSC populations in vitro. The present invention contemplates any suitable method of employing agents, e.g., isolated peptides, e.g., antibodies, that bind to CSC-associated polypeptides to separate CSCs from other cells. Accordingly, included in the present invention is a method of producing a population of isolated CSCs. The methods involve contacting a sample, e.g., a cell suspension, with one or a combination of agents, e.g., antibodies or antigen binding fragments or ligands, which recognize and bind to an epitope, e.g., a cell surface protein, including CSC-associated polypeptides (e.g., polypeptides encoded by the genes listed in Table 4), on the CSC and separating and recovering from the sample the cells bound by the agents. The CSC-associated polypeptide may be encoded by a CSC-associated gene that is selected from the group consisting of: ANK2, NCKAPIL, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8.
In some instances, commerically available antibodies or antibody fragments that bind CSC-associated polypeptides may be used in the methods disclosed herein. For example, antibodies against ANK2 include, e.g., rabbit anti-human Ankyrin brain polyclonal antibody from Abcam; mouse anti-human ANK2 monoclonal antibody clone 2.2 from Genway Biotech, Inc.; and mouse anti-human Ankyrin, brain variant 2 (ANK2) monoclonal, clone 2.20 and rabbit anti-human ankyrin, brain variant 2 (ank2) polyclonal from Lifespan Biosciences. Antibodies against NCKAPIL include, e.g., rabbit anti-human HEM1 polyclonal antibody from Proteintech Group, Inc. and are described in Weiner O D, et al., (2006) Hem-1 Complexes Are Essential for Rac Activation, Actin Polymerization, and Myosin Regulation during Neutrophil Chemotaxis. PLoS Biol 4(2): e38. Antibodies against PTPRE include, e.g., rabbit anti-PTPepsilon C-term RB0551-0552 polyclonal antibody from Abgent, mouse anti-human PTPRE polyclonal antibody from Abnova Corporation; mouse anti-PTPRE monoclonal antibody Clone 2D10 from Abnova Corporation; chicken anti-human PTPRE polyclonal antibody from Thermo Scientific; and Rabbit Anti-Protein Tyrosine phosphatase epsilon (PTPRE) antibody from Acris Antibodies GmbH. Antibodies against PTPRS include, e.g., mouse anti-PTPRS monoclonal antibody from Abcam; mouse anti-human PTPRS monoclonal antibody Clone 1H6 from Abnova Corporation; chicken anti-human PTPRS polyclonal antibody from ABR-Affinity Bioreagents, now sold as Thermo Scientific; chicken anti-human PTPRS polyclonal antibody from GeneTex; and mouse anti-human PTPRS monoclonal antibody, Clone 1H6 from GeneTex. Antibodies against SCN3A include, e.g., rabbit anti-SCN3A polyclonal antibody from Abcam; mouse anti-human SCN3A monoclonal antibody Clone 3F3 from Abnova Corporation; and mouse Anti-human SCN3A monoclonal antibody Clone 3F3 from GeneTex. Antibodies against SCNB include, e.g., mouse anti-beta Sarcoglycan Monoclonal Antibody from Abcam; mouse anti-human SGCB monoclonal antibody Clone 1C10 from Abnova Corporation; mouse anti-human SGCB monoclonal antibody Clone 1C10 from GeneTex; and rabbit anti-human Beta-sarcoglycan (SGCB) Polyclonal from Lifespan Biosciences. Antibodies against SLC2A8 include, e.g., rabbit anti-human Glucose Transporter 8 Polyclonal Antibody from Abcam; goat anti-GLUT8/SLC2A8 polyclonal antibody from Everest Biotech; rabbit anti-GLUCOSE TRANSPORTER 8 Polyclonal Antibody from GenWay Biotech, Inc.; rabbit Anti-Human GLUCOSE TRANSPORTER 5, C Terminus Polyclonal Antibody from GenWay Biotech, Inc.; goat anti-SLC2A8 polyclonal antibody from IMGENEX; and rabbit anti-Human Solute Carrier Family 2 (Facilitated Glucose Transporter) Member 8 (Slc2a8) polyclonal from Lifespan Biosciences. Antibodies against SBF1 include, e.g., rabbit Anti-MTMR5C-term RB0717 polyclonal antibody from Abgent. Antibodies against SGCA include, e.g., mouse Anti-SGCA monoclonal antibody Clone 3C4 from Abnova Corporation; rabbit Anti-Human SGCA polyclonal antibody from Atlas Antibodies; mouse Anti-SGCA monoclonal antibody clone 3C4 from Novus Biologicals; rabbit Anti-Human SGCA PRESTIGE ANTIBODIES from Sigma-Aldrich. Antibodies against SLC2A11 include, e.g., rabbit anti-human Solute Carrier Family 2 (Facilitated Glucose Transporter), Member 11 (Slc2a11) polyclonal from Lifespan Biosciences. Antibodies against SLC4A1 include, e.g., rabbit anti-human Band 3, N Terminus polyclonal antibody from Abcam; mouse anti-human SLC4A1 MaxPab® polyclonal antibody from Abnova Corporation; rabbit anti-human SLC4A1 polyclonal antibody from Aviva Systems Biology; mouse anti-Band 3 Monoclonal Antibody Clone BIII 136 from GenWay Biotech, Inc.; and mouse anti-human Solute Carrier Family 4, Anion Exchanger, Member 1 (SLC4A1) monoclonal Clone 3h3 from Lifespan Biosciences. Antibodies against STX3 include, e.g., rabbit anti-human STX3 Polyclonal Antibody from Atlas Antibodies and rabbit anti-human STX3 from Sigma-Aldrich. Antibodies against TBC1D8 include, e.g., mouse anti-human TBC1D8 monoclonal antibody Clone 1A12 from Abnova Corporation; mouse anti-human TBC1 D8 monoclonal antibody Clone 1A12 from GeneTex; mouse anti-human TBC1D8 monoclonal antibody Clone SS-18 from Santa Cruz Biotechnology, Inc.; and rabbit anti-human TBC1D8, aa 132-231 polyclonal antibody from Strategic Diagnostics, Inc.
Agents may be linked to a solid-phase and utilized to capture CSCs from a sample. The bound cells may then be separated from the solid phase by known methods depending on the nature of the agent, e.g., antibody, and solid phase. Alternatively, the agents may be conjugated to a detectable label, e.g., a fluorophore, that can be utilized to separate cells in a liquid phase, such as by fluorescent activated cell sorting. Exemplary fluorophores are well known in the art (e.g., Invitrogen's MOLECULAR PROBES technologies) and include FITC, TRITC, Cy3, Cy5, Alexa Fluorescent Dyes, and Quantum Dots.
Systems appropriate for preparing the desired cell population include magnetic bead/paramagnetic particle column utilizing isolated peptides that bind CSC-associated polypeptides for either positive or negative selection; separation based on biotin or streptavidin affinity; and high speed flow cytometric sorting of immunofluorescent-stained CSCs mixed in a suspension of other cells. Thus, the methods of the present invention include the isolation of a population of CSCs.
Isolated CSCs may be prepared as substantially pure preparations. The term “substantially pure” means that a preparation is substantially free of other cells. For example, an isolated CSC should constitute at least 70 percent of the total cells present with greater percentages, e.g., at least 85, 90, 95 or 99 percent, being preferred. The cells may be packaged in a finished container such as a cryovial along with any other components that may be desired, e.g., agents for preserving cells, or reducing bacterial growth. The CSCs are useful for a variety of purposes. The isolated cells may be used in basic research setting and in screening assays to identify compounds or compositions that affect growth of CSCs.
Isolated CSCs, prepared according to the methods disclosed herein, may be useful in a drug discovery context for lead compound identification and optimization in cell-based screens. For example, the effect of a compound on the growth and/or survival of a CSC may be determined in a cell-based screen that uses an assay selected from: a cell counting assay, a replication labeling assay, a cell membrane integrity assay, a cellular ATP-based viability assay, a mitochondrial reductase activity assay, a caspase activity assay, an Annexin V staining assay, a DNA content assay, a DNA degradation assay, and a nuclear fragmentation assay. Other exemplary assays include BrdU, EdU, or H3-Thymidine incorporation assays; DNA content assays using a nucleic acid dye, such as Hoechst Dye, DAPI, Actinomycin D, 7-aminoactinomycin D or Propidium Iodide; Cellular metabolism assays such as AlamarBlue, MTT, XTT, and CellTitre Glo; Nuclear Fragmentation Assays; Cytoplasmic Histone Associated DNA Fragmentation Assay; PARP Cleavage Assay; TUNEL staining; and Annexin staining.
In some aspects, the agents for isolating CSCs are antibody or antigen-binding fragments. The antibodies and antigen binding fragments of the invention include monoclonal antibodies, polyclonal antibodies, human antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, F(ab′)₂, Fab, Fd, Fv, or single-chain Fv fragments.
Other aspects of the invention relate to treatment methods. In some embodiments, the methods involve modulating, e.g., inducing or inhibiting, the activity of CSC-associated genes (RNA or protein) and, thereby, inhibiting the growth and survival of cancer stem cells. In some embodiments, the treatment methods involve selective delivery, e.g., by antibodies or antigen-binding fragments, of therapeutic agents to cancer stem cells. The methods are useful for inhibiting the proliferation and/or survival of cancer stem cells and, therefore, are useful for treating cancer, e.g., melanoma. The level of modulation, e.g., inducing or inhibiting, of the activity of a CSC-associated gene that is sufficient to affect the growth and/or survival of a cancer stem cell compared with a control level depends on a variety of factors, including the particular CSC-associated gene being modulated, the cancer stem cell within which the modulation occurs, and the level of expression in the control sample. It is well within the purview of the skilled artisan to determine the appropriate level of modulation that is sufficient to sufficient to inhibit the growth and/or survival of a cancer stem cell.
The term “inhibiting” refers to any decrease in expression level or activity. As used herein, “inhibit”, “suppress”, or “reduce” may, or may not, be complete. For example, cell proliferation, may, or may not, be decreased to a state of complete arrest for an effect to be considered one of suppression, inhibition or reduction of cell proliferation. Moreover, “suppress”, “inhibit”, or “reduce” may comprise the maintenance of an existing state and the process of effecting a change in state. For example, inhibition of cell proliferation may refer to the prevention of proliferation of a non-proliferating cell (maintenance of a non-proliferating state) and the process of inhibiting the proliferation of a proliferating cell (process of affecting a proliferation state change). Similarly, inhibition of cell survival may refer to killing of a cell, or cells, such as by necrosis or apoptosis, and the process of rendering a cell susceptible to death, such as by inhibiting the expression or activity of an anti-apoptotic regulatory factor. The suppression, inhibition, or reduction may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% of control level (e.g., an untreated state). In some cases, the level of modulation (e.g., suppression, inhibition, or reduction) compared with a control level is statistically significant. “Statistically significant” is a term well known in the art and, for example, may refer to a p-value of less than 0.05, e.g., a p-value of less than 0.025 or a p-value of less than 0.01, using an appropriate statistical test (e.g., ANOVA, MANOVA, t-test, multiple comparison test, etc.).
The methods involve treating an individual having, or at risk of having, cancer. As used herein an “individual at risk of having cancer” is an individual, e.g., a human, with an increased likelihood of having cancer compared with a control population, e.g., a general population. Any one of a number of risk factors known in the art may be evaluated to determine whether or not an individual is at risk of having cancer. For example, factors that render an individual at risk of having melanoma include, for example, UV exposure, family history of melanoma, personal history of melanoma, fair skin, freckles, high numbers of nevi (moles), light hair, age, gender, and Xeroderma pigmentosum.
The treatment methods disclosed herein may involve administering a therapeutically effective amount of a composition that induces the expression of a CSC-associated gene which is downregulated in cancer (e.g., a gene in Table 2 or 7). In some instances, the composition that induces expression of a CSC-associated gene comprises a vector, such as an isolated plasmid, that expresses the CSC-associated gene.
As used herein, a “vector” may be any of a number of nucleic acid molecules into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids and virus genomes or portions thereof.
An expression vector is one into which a desired sequence may be inserted, e.g., by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells that have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes that encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase or alkaline phosphatase), and genes that visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein).
Methods for identifying and obtaining coding sequences for use in the methods disclosed herein are routine in the art. For example, the skilled artisan may search Entrez Gene database using a GeneID or GeneAlias of a CSC-associated gene, e.g., a GeneID listed in Table 5, 7 or 8, to identify transcripts associated with CSC-associated genes. In most cases, links to commercial suppliers (e.g., Open Biosystems) of cDNA's containing the transcripts are provided in the Entrez Gene webinterface, which can be utilized to procure a copy cDNA clone. In other cases, commercial sources (e.g., Sigma Aldrich) can be contacted directly.
As used herein, a coding sequence and regulatory sequences are said to be “operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5′ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. It will be appreciated that a coding sequence need not encode a protein but may instead, for example, encode a functional RNA such as an shRNA.
The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5′ non-transcribed and 5′ non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Such 5′ non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5′ leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art. One of skill in the art will be aware of appropriate regulatory sequences for expression of interfering RNA, e.g., shRNA, miRNA, etc.
In some embodiments, a virus vector for delivering a nucleic acid molecule, an isolated plasmid, is selected from the group consisting of adenoviruses, adeno-associated viruses, poxviruses including vaccinia viruses and attenuated poxviruses, Semliki Forest virus, Venezuelan equine encephalitis virus, retroviruses, Sindbis virus, and Ty virus-like particle. Examples of viruses and virus-like particles which have been used to deliver exogenous nucleic acids include: replication-defective adenoviruses (e.g., Xiang et al., Virology 219:220-227, 1996; Eloit et al., J. Virol. 7:5375-5381, 1997; Chengalvala et al., Vaccine 15:335-339, 1997), a modified retrovirus (Townsend et al., J. Virol. 71:3365-3374, 1997), a nonreplicating retrovirus (Irwin et al., J. Virol. 68:5036-5044, 1994), a replication defective Semliki Forest virus (Zhao et al., Proc. Natl. Acad. Sci. USA 92:3009-3013, 1995), canarypox virus and highly attenuated vaccinia virus derivative (Paoletti, Proc. Natl. Acad. Sci. USA 93:11349-11353, 1996), non-replicative vaccinia virus (Moss, Proc. Natl. Acad. Sci. USA 93:11341-11348, 1996), replicative vaccinia virus (Moss, Dev. Biol. Stand. 82:55-63, 1994), Venzuelan equine encephalitis virus (Davis et al., J. Virol. 70:3781-3787, 1996), Sindbis virus (Pugachev et al., Virology 212:587-594, 1995), lentiviral vectors (Naldini L, et al., Proc Natl Acad Sci USA. 1996 Oct. 15; 93(21):11382-8) and Ty virus-like particle (Allsopp et al., Eur. I Immunol 26:1951-1959, 1996).
Another virus useful for certain applications is the adeno-associated virus, a double-stranded DNA virus. The adeno-associated virus is capable of infecting a wide range of cell types and species and can be engineered to be replication-deficient. It further has advantages, such as heat and lipid solvent stability, high transduction frequencies in cells of diverse lineages, including hematopoietic cells, and lack of superinfection inhibition thus allowing multiple series of transductions. The adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.
Other useful viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include certain retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. In general, the retroviruses are replication-deficient (i.e., capable of directing synthesis of the desired transcripts, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M., “Gene Transfer and Expression, A Laboratory Manual,” W.H. Freeman Co., New York (1990) and Murry, E. J. Ed. “Methods in Molecular Biology,” vol. 7, Humana Press, Inc., Clifton, N.J. (1991).
In some embodiments, isolated plasmid vectors comprises a tumor-specific, e.g., melanoma-specific, e.g., a tyrosinase promoter, operably linked to the CSC-associated gene (See, e.g., Lillehammer, T. et al., Cancer Gene Therapy (2005) 12, 864-872). Other exemplary tumor-specific promoters are known in the art and will be apparent to the skilled artisan.
The treatment methods may involve administering a therapeutically effective amount of a composition that targets a product of a CSC-associated gene (i.e., Table 1), which are CSC-associated genes that upregulated in cancer stem cells. The composition may target a product of a CSC-associated gene selected from the group set forth in Table 4, which are upregulated in cancer stem cells and are associated with the cell surface.
The product, e.g., mRNA or protein, of a CSC-associated gene can be targeted by any one of a number of methods known in the art. For example, the composition may comprise a gene knockdown reagent, e.g., siRNA, that is complementary to a CSC-associated mRNA and inhibits its expression. In other embodiments, the composition may comprise an isolated molecule, e.g., antibody or antigen binding fragment, that is conjugated to a siRNA and that specifically binds to a CSC-associated polypeptide. Such antibody conjugated siRNAs (or similar gene suppression agents) may target a CSC-associated mRNA such as any of those encoded by the genes set forth in Table 1.
The CSC-associated gene may be selected from the following group ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8 which are upregulated in cancer stem cells and associated with the cell surface. The siRNA may target another gene in the cell that is useful for inhibiting the growth and/or survival of the cell, for example an oncogene. For example, oncogenes that may be targeted include FOS, JUN, MYB, RAS, and ABL. Other exemplary oncogenes are well known in the art and several such examples are described in, for example, The Genetic Basis of Human Cancer (Vogelstein, B. and Kinzler, K. W. eds. McGraw-Hill, New York, N.Y., 1998). Other upregulated genes include Epidermal growth factor (beta-urogastrone, HOMG4/URG); Heparanase (HPA/HPR1); Jagged 1 (Alagille syndrome, AGS/AHD); Platelet/endothelial cell adhesion molecule (CD31 antigen, CD31/PECAM-1); Transforming growth factor, alpha (TFGA); and Vascular endothelial growth factor C (Flt4-L/VRP). Homologues of such genes can also be used.
Various strategies for gene knockdown known in the art can be used to inhibit gene expression (e.g., expression of CSC-associated genes). For example, gene knockdown strategies may be used that make use of RNA interference (RNAi) and/or microRNA (miRNA) pathways including small interfering RNA (siRNA), short hairpin RNA (shRNA), double-stranded RNA (dsRNA), miRNAs, and other small interfering nucleic acid-based molecules known in the art. In one embodiment, vector-based RNAi modalities (e.g., shRNA or shRNA-mir expression constructs) are used to reduce expression of a gene (e.g., a CSC-associated) in a cell. In some embodiments, therapeutic compositions of the invention comprise an isolated plasmid vector (e.g., any isolated plasmid vector known in the art or disclosed herein) that expresses a small interfering nucleic acid such as an shRNA. The isolated plasmid may comprise a tumor-specific, e.g., melanoma-specific, promoter operably linked to a gene encoding the small interfering nucleic acid, e.g., an shRNA. In some cases, the isolated plasmid vector is packaged in a virus capable of infecting the individual. Exemplary viruses include adenovirus, retrovirus, lentivirus, adeno-associated virus, and others that are known in the art and disclosed herein.
A broad range of RNAi-based modalities could be employed to inhibit expression of a gene in a cell, such as siRNA-based oligonucleotides and/or altered siRNA-based oligonucleotides. Altered siRNA based oligonucleotides are those modified to alter potency, target affinity, safety profile and/or stability, for example, to render them resistant or partially resistant to intracellular degradation. Modifications, such as phosphorothioates, for example, can be made to oligonucleotides to increase resistance to nuclease degradation, binding affinity and/or uptake. In addition, hydrophobization and bioconjugation enhances siRNA delivery and targeting (De Paula et al., RNA. 13(4):431-56, 2007) and siRNAs with ribo-difluorotoluoyl nucleotides maintain gene silencing activity (Xia et al., ASC Chem. Biol. 1(3):176-83, (2006)). siRNAs with amide-linked oligoribonucleosides have been generated that are more resistant to S1 nuclease degradation than unmodified siRNAs (Iwase R et al. 2006 Nucleic Acids Symp Ser 50: 175-176). In addition, modification of siRNAs at the 2′-sugar position and phosphodiester linkage confers improved serum stability without loss of efficacy (Choung et al., Biochem. Biophys. Res. Commun. 342(3):919-26, 2006). Other molecules that can be used to inhibit expression of a gene (e.g., a CSC-associated gene) include sense and antisense nucleic acids (single or double stranded), ribozymes, peptides, DNAzymes, peptide nucleic acids (PNAs), triple helix forming oligonucleotides, antibodies, and aptamers and modified form(s) thereof directed to sequences in gene(s), RNA transcripts, or proteins. Antisense and ribozyme suppression strategies have led to the reversal of a tumor phenotype by reducing expression of a gene product or by cleaving a mutant transcript at the site of the mutation (Carter and Lemoine Br. J. Cancer. 67(5):869-76, 1993; Lange et al., Leukemia. 6(11):1786-94, 1993; Valera et al., J. Biol. Chem. 269(46):28543-6, 1994; Dosaka-Akita et al., Am. J. Clin. Pathol. 102(5):660-4, 1994; Feng et al., Cancer Res. 55(10):2024-8, 1995; Quattrone et al., Cancer Res. 55(1):90-5, 1995; Lewin et al., Nat. Med. 4(8):967-71, 1998). Ribozymes have also been proposed as a means of both inhibiting gene expression of a mutant gene and of correcting the mutant by targeted trans-splicing (Sullenger and Cech Nature 371(6498):619-22, 1994; Jones et al., Nat. Med. 2(6):643-8, 1996). Ribozyme activity may be augmented by the use of, for example, non-specific nucleic acid binding proteins or facilitator oligonucleotides (Herschlag et al., Embo J. 13(12):2913-24, 1994; Jankowsky and Schwenzer Nucleic Acids Res. 24(3):423-9, 1996). Multitarget ribozymes (connected or shotgun) have been suggested as a means of improving efficiency of ribozymes for gene suppression (Ohkawa et al., Nucleic Acids Symp Ser. (29):121-2, 1993).
Triple helix approaches have also been investigated for sequence-specific gene suppression. Triple helix forming oligonucleotides have been found in some cases to bind in a sequence-specific manner (Postel et al., Proc. Natl. Acad. Sci. U.S.A. 88(18):8227-31, 1991; Duval-Valentin et al., Proc. Natl. Acad. Sci. U.S.A. 89(2):504-8, 1992; Hardenbol and Van Dyke Proc. Natl. Acad. Sci. U.S.A. 93(7):2811-6, 1996; Porumb et al., Cancer Res. 56(3):515-22, 1996). Similarly, peptide nucleic acids have been shown to inhibit gene expression (Hanvey et al., Antisense Res. Dev. 1(4):307-17, 1991; Knudsen and Nielson Nucleic Acids Res. 24(3):494-500, 1996; Taylor et al., Arch. Surg. 132(11):1177-83, 1997). Minor-groove binding polyamides can bind in a sequence-specific manner to DNA targets and hence may represent useful small molecules for suppression at the DNA level (Trauger et al., Chem. Biol. 3(5):369-77, 1996). In addition, suppression has been obtained by interference at the protein level using dominant negative mutant peptides and antibodies (Herskowitz Nature 329(6136):219-22, 1987; Rimsky et al., Nature 341(6241):453-6, 1989; Wright et al., Proc. Natl. Acad. Sci. U.S.A. 86(9):3199-203, 1989). In some cases suppression strategies have led to a reduction in RNA levels without a concomitant reduction in proteins, whereas in others, reductions in RNA have been mirrored by reductions in protein. The diverse array of suppression strategies that can be employed includes the use of DNA and/or RNA aptamers that can be selected to target a protein of interest (e.g, a CSC-associated polypeptide).
Methods of delivering a therapeutic agent to a cancer stem cell are also provided. The methods involve a step of contacting a cancer stem cell with an isolated molecule that selectively binds to a cell surface polypeptide encoded by a CSC-associated gene, such as those selected from the group set forth in Table 4. The CSC-associated gene may be selected from the group consisting of: ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8. The cancer stem cell may be in vivo or in vitro. Isolated molecules that bind to CSC-associated polypeptides on the surface of a cancer stem cell may be taken up into an intracellular compartment of the cancer stem cell.
Cancer stem cells include stem cells of a colon carcinoma, a pancreatic cancer, a breast cancer, an ovarian cancer, a prostate cancer, a squamous cell carcinoma, a cervical cancer, a lung carcinoma, a small cell lung carcinoma, a bladder carcinoma, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinoma, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, a renal cancer, e.g., renal cell carcinoma, a hepatocellular carcinoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, a embryonal carcinoma, a Wilms' tumor, or a testicular tumor. In specific embodiments, the cancer stem cells are stem cells of melanoma. Cancer stem cells include ABCB5⁺ cells and ABCB5⁻cells. Stem cells of other cancers will be known to one of ordinary skill in the art.
The treatment methods of the invention involve administering compositions that comprise isolated molecules, or combinations of different isolated molecules, that specifically bind to CSC-associated polypeptides (polypeptides encoded by the CSC-associated gene) to treat cancer, e.g., melanoma, in an individual. When the CSC-associated polypeptide is associated with the extracellular surface of a cell, e.g., a cancer stem cell, e.g., a melanoma stem cell, the isolated molecule can bind the CSC-associated polypeptide and, for example, serve as an vehicle for specifically targeting therapeutic agents (therapeutic molecules) to the cell. In some embodiments, isolated molecules that binds to a CSC-associated polypeptide on the surface of a cell are taken up into an intracellular compartment of the cell bind to a secreted molecule, such as a growth factor that may assist the tumor (such as those listed in Table 1.2). The isolated molecules that interact with unregulated proteins may be used alone as therapeutics or in combination with other therapeutics.
As used herein treatment of or treating cancer includes preventing the development of a cancer, reducing the symptoms of a cancer and/or inhibiting, slowing the growth of or preventing further growth of an existing cancer. Treatment may include amelioration, cure, and/or maintenance of a cure (i.e., prevention or delay of relapse) of a disorder, e.g., cancer. Treatment after a disorder has started aims to reduce, ameliorate or altogether eliminate the disorder, and/or its associated symptoms, to prevent it from becoming worse, to slow the rate of progression, or to prevent the disorder from re-occurring once it has been initially eliminated (i.e., to prevent a relapse).
Cancers include for instance a colon carcinoma, a pancreatic cancer, a breast cancer, an ovarian cancer, a prostate cancer, a squamous cell carcinoma, a cervical cancer, a lung carcinoma, a small cell lung carcinoma, a bladder carcinoma, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinoma, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, a renal cell carcinoma, a hepatocellular carcinoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, a Wilms' tumor, or a testicular tumor. In certain embodiments, the cancer is melanoma.
The invention, in some aspects, relates to an isolated molecule that selectively binds to a polypeptide encoded by a CSC-associated gene set forth in Table 4 and that is conjugated to a therapeutic agent. In some instances, the CSC-associated gene is selected from the group consisting of: ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8. Compositions comprising the foregoing isolated molecules are also disclosed.
As used herein an “isolated molecule” is a molecule such as a polypeptide, nucleic acid, polysaccharide, drug, nucleoprotein, lipoprotein, glycoprotein, steroid, and lipid that is isolated from its natural environment or produced synthetically. In some embodiments, the isolated molecule is a ligand of a CSC-associated polypeptide. In other embodiments, the isolated molecule is an antibody or antigen-binding fragment. As disclosed herein, antibody or antigen-binding fragments include monoclonal antibodies, polyclonal antibodies, human antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, F(ab′)₂, Fab, Fd, Fv, or single-chain Fv fragments. In some embodiments, an isolated molecule may have therapeutic utility alone and need not be conjugated to a therapeutic agent. For example, an isolated molecule may bind to a cell surface receptor that is a CSC-associated polypeptide and function as an antagonist or competitive inhibitor of the receptor (e.g., to inhibit a signaling pathway).
In some embodiments, isolated molecules are conjugated to therapeutic agents. As used herein, a “therapeutic agent” is a molecule such as a polypeptide, nucleic acid, polysaccharide, drug, nucleoprotein, lipoprotein, glycoprotein, steroid, and lipid that is capable of altering the state of a cell (e.g., killing a cell, inhibiting growth of a cell) for therapeutic purposes. A therapeutic agent may be, for instance, a toxin, a small-interfering nucleic acid, or a chemotherapeutic agent. Alternatively the therapeutic may be administered in conjunction with the molecule. In conjunction refers to delivery to the same subject. The actual administration may be at the same or a different time or in the same or a different delivery vehicle.
Toxins include for example, radioisotopes such as ²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ¹⁸⁶Rh, ¹⁸⁸Rh, ¹⁷⁷Lu, ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ¹²⁵I, ¹²³I, ⁷⁷Br, ¹⁵³Sm, ¹⁶⁶Bo, ⁶⁴Cu, ²¹²Pb, ²²⁴Ra, ²²³Ra, and others known in the art. Suitable chemical toxins include members of the enediyne family of molecules, such as calicheamicin and esperamicin as well as poisonous lectins, plant toxins such as ricin, abrin, modeccin, botulina and diphtheria toxins. Of course, combinations of the various toxins could also be coupled to one isolated molecule, e.g., an antibody, thereby accommodating variable cytotoxicity. The coupling of one or more toxin molecules to the isolated molecule, e.g., antibody, is envisioned to include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding, and complexation.
Chemotherapeutic agents include the following compounds or classes of compounds: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Buniodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorombucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; DACA (N-[2-(Dimethyl-amino)ethyl]acridine-4-carboxamide); Dactinomycin; Daunorubicin Hydrochloride; Daunomycin; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Ifesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; 5-FdUMP; Fluorocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-1a; Interferon Gamma-1b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin, Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine; Tomudex; TOP-53; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine; Vinblastine Sulfate; Vincristine; Vincristine Sulfate, Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride; 2-Chlorodeoxyadenosine; 2′-Deoxyformycin; 9-aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid, 2-chloro-2′-arabino-fluoro-2′-deoxyadenosine; 2-chloro-2′-deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R; CEP-751; linomide; Piritrexim Isethionate; Sitogluside; Tamsulosin Hydrochloride and Pentomone.
The invention, in some aspects, provides kits comprising one or more containers housing one or more of the compositions of the invention. The kit may be designed to facilitate use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit. The kits may also include reference samples. As used herein, “instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the invention. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflects approval by the agency of manufacture, use or sale for animal administration.
The kit may contain any one or more of the components described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a individual. The kit may include a container housing agents described herein. The agents may be in the form of a liquid, gel or solid (powder). The agents may be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other agents prepared sterilely. Alternatively the kit may include the active agents premixed and shipped in a syringe, vial, tube, or other container. The kit may have one or more or all of the components required to administer the agents to an animal, such as a syringe, topical application devices, or iv needle tubing and bag, particularly in the case of the kits for treating individuals with cancer.
The compositions and therapeutic agents may be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time. The other therapeutic agents are administered sequentially with one another and with the modulators, when the administration of the other therapeutic agents and the modulators is temporally separated. The separation in time between the administration of these compounds may be a matter of minutes or it may be longer.
The compositions of the present invention preferably contain a pharmaceutically acceptable carrier or excipient suitable for rendering the compound or mixture administrable orally as a tablet, capsule or pill, or parenterally, intravenously, intradermally, intramuscularly or subcutaneously, or transdermally. The active ingredients may be admixed or compounded with any conventional, pharmaceutically acceptable carrier or excipient. The compositions may be sterile.
As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compositions of this invention, its use in the therapeutic formulation is contemplated. Supplementary active ingredients can also be incorporated into the pharmaceutical formulations. A composition is said to be a “pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well-known in the art.
It will be understood by those skilled in the art that any mode of administration, vehicle or carrier conventionally employed and which is inert with respect to the active agent may be utilized for preparing and administering the pharmaceutical compositions of the present invention. Illustrative of such methods, vehicles and carriers are those described, for example, in Remington's Pharmaceutical Sciences, 18th ed. (1990), the disclosure of which is incorporated herein by reference. Those skilled in the art, having been exposed to the principles of the invention, will experience no difficulty in determining suitable and appropriate vehicles, excipients and carriers or in compounding the active ingredients therewith to form the pharmaceutical compositions of the invention.
An effective amount, also referred to as a therapeutically effective amount is an amount sufficient to ameliorate at least one adverse effect associated with expression, or reduced expression, of a CSC-associated gene in a cell or in an individual in need of such inhibition or supplementation. The therapeutically effective amount to be included in pharmaceutical compositions depends, in each case, upon several factors, e.g., the type, size and condition of the patient to be treated, the intended mode of administration, the capacity of the patient to incorporate the intended dosage form, etc. Generally, an amount of active agent is included in each dosage form to provide from about 0.1 to about 250 mg/kg, and preferably from about 0.1 to about 100 mg/kg. One of ordinary skill in the art would be able to determine empirically an appropriate therapeutically effective amount.
Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular individual. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular therapeutic agent being administered, the size of the individual, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular nucleic acid and/or other therapeutic agent without necessitating undue experimentation.
The pharmaceutical compositions can be administered by any suitable route for administering medications. A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular agent or agents selected, the particular condition being treated, and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of an immune response without causing clinically unacceptable adverse effects. Preferred modes of administration are discussed herein. For use in therapy, an effective amount of the nucleic acid and/or other therapeutic agent can be administered to an individual by any mode that delivers the agent to the desired surface, e.g., mucosal, systemic.
Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to oral, parenteral, intravenous, intramuscular, intraperitoneal, intranasal, sublingual, intratracheal, inhalation, subcutaneous, ocular, vaginal, and rectal. Systemic routes include oral and parenteral. Several types of devices are regularly used for administration by inhalation. These types of devices include metered dose inhalers (MDI), breath-actuated MDI, dry powder inhaler (DPI), spacer/holding chambers in combination with MDI, and nebulizers.
In some cases, compounds of the invention are prepared in a colloidal dispersion system. Colloidal dispersion systems include lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system of the invention is a liposome. Liposomes are artificial membrane vessels which are useful as a delivery vector in vivo or in vitro. It has been shown that large unilamellar vesicles (LUVs), which range in size from 0.2-4.0 μm can encapsulate large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form. Fraley et al. (1981) Trends Biochem Sci 6:77.
Liposomes may be targeted to a particular tissue by coupling the liposome to a specific binding molecule such as one that binds to a CSC-associated polypeptide. Binding molecules which may be useful for targeting a liposome to, for example, a cancer stem cell include, but are not limited to intact or fragments of molecules, e.g., antibodies or antigen binding fragments, which interact with CSC-associated polypeptides on the surface of cancer stem cells. Such binding molecules may easily be identified by binding assays well known to those of skill in the art.
Lipid formulations for transfection are commercially available from QIAGEN, for example, as EFFECTENE™ (a non-liposomal lipid with a special DNA condensing enhancer) and SUPERFECT™ (a novel acting dendrimeric technology).
Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids such as N-[1-(2,3 dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods for making liposomes are well known in the art and have been described in many publications. Liposomes also have been reviewed by Gregoriadis G (1985) Trends Biotechnol 3:235-241.
Certain cationic lipids, including in particular N-[1-(2,3 dioleoyloxy)-propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), appear to be especially advantageous when combined with the modified oligonucleotide analogs of the invention.
In one embodiment, the vehicle is a biocompatible microparticle or implant that is suitable for implantation or administration to the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. PCT/US/03307 (Publication No. WO95/24929, entitled “Polymeric Gene Delivery System”. PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promoter. The polymeric matrix can be used to achieve sustained release of the therapeutic agent in the individual.
The polymeric matrix preferably is in the form of a microparticle such as a microsphere (e.g., wherein a therapeutic agent is dispersed throughout a solid polymeric matrix) or a microcapsule (e.g., wherein a therapeutic agent is stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the therapeutic agent include films, coatings, gels, implants, and stents. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix is introduced. The size of the polymeric matrix further is selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas. Preferably when an aerosol route is used the polymeric matrix and the therapeutic agent are encompassed in a surfactant vehicle. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the matrix is administered to a nasal and/or pulmonary surface that has sustained an injury. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time. Biocompatible microspheres that are suitable for delivery, such as oral or mucosal delivery, are disclosed in Chickering et al. (1996) Biotech Bioeng 52:96-101 and Mathiowitz E et al. (1997) Nature 386:410-414 and PCT Pat. Application WO97/03702.
Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the therapeutic agents to an individual. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable, particularly for the nucleic acid agents. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.
Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, (1993) 26:581-587, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
If the therapeutic agent is a nucleic acid, the use of compaction agents may also be desirable. Compaction agents also can be used alone, or in combination with, a biological or chemical/physical vector. A “compaction agent”, as used herein, refers to an agent, such as a histone, that neutralizes the negative charges on the nucleic acid and thereby permits compaction of the nucleic acid into a fine granule. Compaction of the nucleic acid facilitates the uptake of the nucleic acid by the target cell. The compaction agents can be used alone, i.e., to deliver a nucleic acid in a form that is more efficiently taken up by the cell or, more preferably, in combination with one or more of the above-described vectors.
Other exemplary compositions that can be used to facilitate uptake of a nucleic acid include calcium phosphate and other chemical mediators of intracellular transport, microinjection compositions, electroporation and homologous recombination compositions (e.g., for integrating a nucleic acid into a preselected location within the target cell chromosome).
The compounds may be administered alone (e.g., in saline or buffer) or using any delivery vehicle known in the art. For instance the following delivery vehicles have been described: cochleates; Emulsomes; ISCOMs; liposomes; live bacterial vectors (e.g., Salmonella, Escherichia coli, Bacillus Calmette-Guérin, Shigella, Lactobacillus); live viral vectors (e.g., Vaccinia, adenovirus, Herpes Simplex); microspheres; nucleic acid vaccines; polymers (e.g. carboxymethylcellulose, chitosan); polymer rings; proteosomes; sodium fluoride; transgenic plants; virosomes; and, virus-like particles.
The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
The term pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
For oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by an individual to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long-acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R (1990) Science 249:1527-1533, which is incorporated herein by reference.
The compounds may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Liquid dose units are vials or ampoules. Solid dose units are tablets, capsules and suppositories.
Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compounds, increasing convenience to the individual and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di-, and tri-glycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

EXAMPLES

Among the numerous CSC-associated genes are genes involved in vasculogenesis and angiogenesis. For example, global gene expression analyses validated by mRNA and protein determinations revealed preferential display of genes for vascular endothelial growth factor receptor-1 (VEGFR-1) and related members of signaling cascades involved in vasculogenesis and angiogenesis in ABCB5⁺ MMIC. In vitro, vascular endothelial growth factor (VEGF) induced expression of the endothelial-associated marker CD144 (VE-cadherin) in VEGFR-1-expressing ABCB5⁺ MMIC but not VEGFR-1-negative ABCB5⁻ melanoma bulk populations, indicating a unique capacity of CSC for VEGF/VEGFR-1 signaling-dependent vasculogenic differentiation. In vivo, tumors initiated from patient-derived melanoma cells or established melanoma cultures by xenotransplantation into the murine subcutis or by intradermal injection into human skin in chimeric murine recipients formed perfused ABCB5 mRNA- and protein-expressing vessel-like channels also detected in clinical melanoma specimens that co-expressed CD144 and the vasculogenic mimicry marker TIE-1⁴. Tumour initiation in human skin by fluorescent transgene-expressing human melanoma cells confirmed CD144⁺ channels to be of melanoma origin. Moreover, MMIC depletion in tumour grafts to human skin significantly reduced channel formation and resulted in attenuated tumour growth. Our results identify melanoma vasculogenesis driven by ABCB5⁺ MMIC as a novel mechanism by which CSC may promote tumor growth. Furthermore, they suggest that MMIC-dependent vasculogenesis represents a novel CSC target for VEGF/VEGFR-1-directed inhibitors of angiogenesis.

Example 1

Materials and Methods

Melanoma cells and culture methods. The established human cutaneous melanoma cell lines G3361, A375, MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL-28, UACC-62, UACC-257, M14 and MDA-MB-435 were cultured as described^3,5,23. Clinical cutaneous melanoma cells were derived from surgical specimen according to human subjects research protocols approved by the IRBs of the University of Würzburg Medical School or the Wistar Institute, Philadelphia, Pa. as described previously³. The established human uveal melanoma cell lines MUM-2B and MUM-2C were a gift of Dr. Mary J. Hendrix, Northwestern University, and were cultured as described¹⁶.
Cell isolation. ABCB5⁺-purified (ABCB5⁺) cells were isolated by positive selection and ABCB5⁺-depleted (ABCB5⁻) cell populations were generated by removing ABCB5⁺ cells using anti-ABCB5 mAb (clone 3C2-1D12²³) labeling and magnetic bead cell sorting as described³. Briefly, human G3361, A375, MUM-2B or MUM-2C melanoma cells or single cell suspensions derived from clinical melanoma samples were labeled with anti-ABCB5 mAb for 30 min at 4° C., washed twice for excess antibody removal, followed by incubation with secondary anti-mouse IgG mAb-coated magnetic microbeads for 30 min at 4° C. Subsequently, cells were washed twice for excess magnetic microbead removal and then sorted into ABCB5⁺ and ABCB5⁻ cell fractions by dual-passage cell separation in MidiMACS or MiniMACS separation columns (depending on cell number) according to the manufacturer's recommendations (Miltenyi Biotec, Auburn, Calif.). Assessment of purity of ABCB5⁺ and ABCB5⁻ (ABCB5⁺ cell-depleted) melanoma cell isolates and determination of cell viability following magnetic cell sorting were performed and yielded similar results as described previously³.
Global gene expression microarray analyses. Microarray analyses were performed on purified ABCB5⁺ (n=5) and ABCB5⁻ (n=5) cell subsets derived from the established human melanoma cell lines G3361 and A375 and from three distinct clinical melanoma specimen previously characterized in our laboratory with regards to ABCB5 expression and MMIC phenotype in human melanoma xenotransplantation assays³. Total RNA was extracted, processed and hybridized as described previously¹⁰onto Affymetrix human HG-U133Plus2 GeneChip microarrays (Affymetrix, Santa Clara, Calif.). Statistical analysis of microarray results was performed as described previously¹⁰. The expression data set in its entirety will be made available through GEO (gene expression omnibus). Functional gene networks were generated using Ingenuity Pathways Analysis (Ingenuity® Systems, ingenuity.com), by mapping each gene identifier to its corresponding gene object in the Ingenuity Pathways Knowledge Base. These focus genes were overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. Focus gene networks were then algorithmically generated based on their connectivity and subsequently analyzed to identify the biological functions that were most significant to the genes in the network.
RNA extraction and reverse transcriptase-PCR. Total RNA was isolated from ABCB5⁺ and ABCB5⁻ human melanoma cells using RNeasy columns (QIAGEN, Valencia, Calif.). Standard cDNA synthesis reactions were performed using 5 μg RNA and the SuperScript First-Strand Synthesis System for reverse transcriptase-PCR (Invitrogen, Carlsbad, Calif.) as per the manufacturer's instructions. For PCR analysis, 5 μl of diluted first strand product (−100 ng of cDNA) was added to 45 μl of PCR reaction mixture containing 5 units of Superscript II (Invitrogen) according to the manufacturer's protocol. The following PCR program was performed: denaturation at 95° C. for 5 min, then cycled 35 times at 94° C. for 1 min, 53° C. for 30 s, and 72° C. for 30 s, and subsequently extended at 72° C. for 10 min. The primer sequences were as follows: PTK2 forward primer, 5′-TGCCTTTTACTTTCGTGTGG-3′ (SEQ ID NO:1); PTK2 reverse primer 5′-CCAAATTCCTGTTTTGCTTCA-3′ (SEQ ID NO:2); MET forward primer 5′-CCCCACCTTATCCTGACGTA-3′ (SEQ ID NO:3); MET reverse primer 5′-CGTGTGTCCACCTCATCATC-3′ (SEQ ID NO:4); NRP2 forward primer 5′-GAGGCAGGGGAAAATAGAGG-3′ (SEQ ID NO:5); NRP2 reverse primer 5′-TCTCCCGAAAGGTTGAAATG-3′ (SEQ ID NO:6); ETS1 forward primer 5′-CGCTTACTCTGTTGGGGTCT-3′ (SEQ ID NO:7); ETS1 reverse primer 5′-TCTCCAGCAAAATGATGTGC-3′ (SEQ ID NO:8); FLT1 forward primer 5′-TGGCAACTGCTTTTATGTTCTG-3′ (SEQ ID NO:9); FLT1 reverse primer 5′-TCCATAGGGTGATGGTCAAA-3′ (SEQ ID NO: 10). The reaction products were resolved on a 1% LE agarose gel (Ambion, Austin, Tex.) and photographed. β-Actin primers were used as controls to ensure RNA integrity.
RNA extraction and real time quantitative PCR. Total RNA was isolated from unsegregated or sorted human melanoma cells using the RT²qPCR Grade RNA isolation kit (SABiosciences, Frederick, Md.). Standard cDNA synthesis reactions were performed using 1 RNA and the RT²First Strand Kit for reverse transcriptase-PCR (SABiosciences) as per the manufacturer's instructions. The reverse transcriptase product (1 μl) was amplified by primer pairs specific for ABCB5⁵. β-actin was used as a normalizing control. The primers for ABCB5 (Genebank accession no. AY234788) detection were 5′-GCTGAGGAATCCACCCAATCT-3′ (forward) (SEQ ID NO:11) and 5′-AGCCTGAATGGCCTTTTGTG-3′ (reverse) (SEQ ID NO:12). The primers for β-actin detection were 5′-CCTGGCACCCAGCACAAT-3′ (SEQ ID NO:13) (forward) and 5′-AGTACTCCGTGTGGATCGGC-3′ (reverse) (SEQ ID NO: 14). Samples were assayed using Sybergreen chemistry and kinetic PCR (ABI 7300 Sequence Detector; Applied Biosystems, Foster City, Calif.). Samples were amplified using the Sybergreen PCR reagent kit (Applied Biosystems) according to the manufacturer's protocol. Sense and antisense primers were used at a final concentration of 10 nM. The cDNA samples were amplified under following conditions: 50° C. for 2 min and 95° C. for 10 min, followed by 40 cycles of amplification at 94° C. for 15 s and 60° C. for 1 min. All samples were run in triplicate. The relative amounts of transcripts were analyzed using the 2(-Delta Delta C(T)) method as described previously^3,5,10. Statistical differences between mRNA expression levels were determined using the nonparametric Mann-Whitney test. A two-sided P value of P<0.05 was considered significant.
Western analysis. Total cell lysates were harvested from logarithmically growing cultures of the human melanoma cell lines MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL-28, UACC-62, UACC-257, M14, and MDA-MB-435 and analyzed by 8% SDS-PAGE and Western assay to detect relative levels of ABCB5 (mAb 3C2-1D12²³) and alpha-tubulin (mAb clone DM1A, Sigma-Aldrich, St. Louis, Mo.), using LI-COR Odyssey IR imaging system densitometry.
Flow cytometry. G3361, A375, MUM-2B, or MUM-2C melanoma cells were analyzed for surface ABCB5 expression by incubation with anti-ABCB5 mAb or isotype control mAb for 30 min at 4° C., followed by counterstaining with FITC-conjugated goat anti-mouse Ig or with APC-conjugated donkey anti-mouse IgG secondary Abs and single color flow cytometry at the Fl1 (FITC) or F14 (APC) emission spectrum on a Becton Dickinson FACScan as described^3,5,23. Washing was performed twice between each step. Analysis of coexpression of ABCB5 with the VEGFR-1 surface marker in G3361 melanoma cells was performed by dual-color flow cytometry as described³. Briefly, melanoma cells were incubated for 30 min at 4° C. with anti-ABCB5 mAb or isotype control mAb, followed by counterstaining with APC-conjugated donkey anti-mouse IgG secondary Ab as above. Subsequently, cells were fixed at 4° C. and then incubated for 30 min at 4° C. with PE-conjugated anti-VEGFR-1 mAb (R&D Systems, Minneapolis, Minn.) or PE-conjugated isotype control mAb (BD PharMingen, San Diego, Calif.). Dual color flow cytometry was subsequently performed with acquisition of fluorescence emission at the F14 (APC) and F12 (PE) spectra on a Becton Dickinson FACScan. Washing was performed twice between each step. Statistical differences between expression levels of markers were determined using the nonparametric Mann-Whitney test. A two-sided P value of P<0.05 was considered significant.
In vitro vasculogenic differentiation and tube formation assays. VEGF-dependent induction of CD144 expression and formation of capillary-like tube structures by human G3361 melanoma cells was assayed on growth factor reduced Matrigel, a basement membrane matrix preparation (BD Biosciences, San Jose, Calif.). Growth factor reduced Matrigel was added to eight-chamber polystyrene vessel tissue culture-treated glass slides and allowed to gelatinize for 20 min at 37° C. Purified ABCB5⁺ or ABCB5⁻ or unsegregated human melanoma cells were seeded into culture slide wells at densities of 5×10⁴cells/cm²in medium 199 containing 5% FCS¹¹in the presence or absence of VEGF (100 ng/ml). After 48-hour incubation, cells were fixed with 4% paraformaldehyde/PBS for 20 min at room temperature, and after extensive washing with PBS the cells were blocked in 5% donkey serum/0.01% Tween 20/PBS for 1 hr at room temperature. Cells were then incubated with rabbit anti-CD144 polyclonal Ab (diluted 1:100; Bethyl Laboratories, Montgomery, Tex.) overnight at 4° C. After extensive washing with 0.01% Tween 20/PBS, the cells were incubated with goat anti-rabbit Texas red-conjugated secondary Ab (diluted 1:250; Jackson ImmunoResearch Laboratories, West Grove, Pa.) for 1 hr at room temperature. Following washing with 1×PBS/0.01% Tween 20, cells were then mounted in Vectashield (Vecta Laboratories, Burlingame, Calif.) supplemented with 100 ng/ml DAPI to visualize nuclei. Cells were analyzed by fluorescent microscopy using a Mercury-100 Watts fluorescent light source (Microvideo Instruments, Avon, Mass.) attached to a Nikon Eclipse TE 300 microscope (Nikon Instruments, Melville, N.Y.) with the use of separate filters for each fluorochrome. The images were obtained using a Spot digital camera (Diagnostic Instruments Inc., Sterling Heights, Mich.), and the Spot 3.3.2. software package was imported into Adobe Photoshop (Adobe Systems, Mountain View, Calif.). For tube formation assays, unsegregated human melanoma cells were seeded into culture slide wells at densities of 5×10⁴cells/cm²in medium 199 containing 5% FCS¹¹. Immediately, cells were pretreated with medium alone, rabbit anti-VEGFR1 Ab (10 μg/ml; Santa Cruz Biotechnology, Santa Cruz, Calif.) or rabbit isotype control Ab (10 μg/ml; BD Biosciences) at 37° C. for 2 hrs prior to stimulation with VEGF (100 ng/ml). Tube formation was detected by phase contrast microscope (Nikon Eclipse TE 300 microscope) after 24 hrs of incubation. For quantitative analysis of tube formation and length and for determination of CD144 expression at 48 hrs, n=3 three randomly selected microscopy fields were photographed per experimental condition. Images were acquired as described above and tube formation was analyzed using Image J software available from the National Institutes of Health web site as described previously²⁴. For quantification of CD144 expression, positive cells were counted using Neurolucida 8.10 software (MBF Bioscience, Williston, Vt.). Differences among groups were analyzed by one-way ANOVA followed by Bonferroni post hoc tests. Differences with P values<0.05 were considered statistically significant.
In vitro adipogenic, osteogenic and myogenic differentiation assays. For adipogenic, osteogenic and myogenic differentiation assays purified ABCB5⁺ and ABCB5⁻ G3361 melanoma cells were seeded in triplicate at 3×10³cells/well in 96-well culture plates. Adipogenic and osteogenic differentiation was assessed using commercially available differentiation kits and Oil Red O and Alizarin Red staining, respectively, according to the manufacturer's instructions (Chemicon International, Temecula, Calif.). Myogenic differentiation assays were performed as described previously¹⁰. Briefly, melanoma subpopulations were incubated in growth medium consisting of DMEM with 4% glucose, 20% fetal bovine serum (vol/vol), 1% (vol/vol) penicillin-streptomycin (10,000 UI/ml-10,000 μg/ml, Invitrogen) for 10 days. The medium was exchanged every 2 days. At day 14, cells were fixed in ice cold methanol for 3 min on ice and incubated with 1:50 diluted anti-myogenin mouse monoclonal Ab (Dako, Carpinteria, Calif.) overnight at 4° C. Plates were then washed, incubated with goat anti-mouse FITC-conjugated secondary Ab (Jackson ImmunoResearch Laboratories, diluted 1:100) and then mounted with Vectashield mounting medium (Vector) supplemented with 100 ng/ml DAPI to visualize nuclei. Slides were visualized with the 20× and 40× objective on a Nikon Eclipse TE2000-S microscope, photographed using the Spot 7.4 slider camera and images processed using Spot software version 4.0.9. (Diagnostic Instruments, Sterling Heights, Mich.). For quantification of differentiation marker expression, positive cells were manually counted and statistical differences between expression levels of markers were determined using the nonparametric Mann-Whitney test. A two-sided P value of P<0.05 was considered significant.
Immunohistochemistry and immunofluorescence double labeling. The following primary Abs were used: rat anti-Laminin B2, (Abcam, Cambridge, Mass.), rabbit anti-CD144, (Cell Signaling, Beverly, Mass.), goat anti-Tie-1, (Neuromics, Edina, Minn.), and mouse anti-ABCB^3,5,23. Isotype matched irrelevant Abs served as negative control. The secondary Abs were horse anti-mouse IgG-HRP, horse anti-goat IgG-HRP, goat anti-rabbit IgG-HRP (VECTOR Laboratories, Burlingame, Calif.) and goat anti-rat IgG-HRP (Biolegend, San Diego, Calif.), and donkey anti-mouse IgG-AF488, donkey anti-rabbit IgG-AF594, and donkey anti-goat IgG-AF594 (Invitrogen, Carlsbad, Calif.). Immunohistochemistry was performed using the 2-step horseradish peroxidase method. Briefly, frozen tissue sections were fixed with −20° C. acetone for 5 min, then incubated with primary Ab at 4° C. overnight. After washing out unbound primary Ab with phosphate-buffered saline (PBS), the tissue sections were incubated with secondary Ab at room temperature for 30 min, then washed with PBS 3×5 min. Immunoreactivity was detected using NovaRed peroxidase substrate (VECTOR Laboratories, Burlingame, Calif.). For immunofluorescence double labeling, the frozen tissue sections were fixed with −20° C. acetone for 5 min, then incubated with the mix of 2 primary Abs (for example ABCB5 Ab+CD144 Ab) at 40° C. overnight. After washing with PBS, the tissue sections were incubated with the mix of 2 secondary Abs (for example donkey anti-mouse IgG-AF488+donkey anti-rabbit IgG-AF594) at room temperature for 1 hour, then washed with PBS 3×5 min, and the sections were then mounted with ProLong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, Calif.). The sections were viewed under a Olympus BX51 System fluorescence microscope (Olympus Corporation, Tokyo, Japan). For HLA-2A immunohistochemistry of SK-MEL-5 melanoma xenografts to chimeric mouse/human skin, frozen sections were incubated with 5 μg/ml mouse anti-human HLA-2A Ab (BD Pharmingen, San Jose, Calif.) at 4° C. overnight. After washing out unbound primary Ab with phosphate buffered saline (PBS), sections were incubated with 1:200 peroxidase-conjugated horse anti-mouse IgG Ab (Vector Laboratories, Burlingame, Calif.) at room temperature for 30 min. Unbound secondary Ab was washed out with PBS. Color was developed using the NovRed peroxidase substrate kit (Vector Laboratories) and sections were counterstained with hematoxylin Gill's No. 1 (Fisher Scientific, Pittsburgh, Pa.).
In situ hybridization. RNA probes were prepared as follows: PCR-derived RNA probe templates were synthesized by introducing the T7 promoter into the antisense strand and the SP6 promoter into the sense strand. The primer pair, AB5T7AS (5′-TAATACGACTCACTATAGGGATGTCTGGCTTTTTCCCTTCTTGAC-3′) (SEQ ID NO:15) and AB5SP6S (5′-GATTTAGGTGACACTATAGAAATTCAAGCTGGACGAATGACCCCA-3′) (SEQ ID NO:16), was used to generate the DNA template for antisense and sense RNA probes spanning 200 base pairs of human ABCB5 cDNA. This sequence encodes ABCB5 amino acids 499-564 (GI:34539755). The primer pair CD133T7AS (5′-TAATACGACTCACTATAGGGAGCAGCCCCAGGACACAGCATA-3′) (SEQ ID NO:17) and CD133SP6S (5′-GATTTAGGTGACACTATAGAGACCCAAGACTCCCATAAAGC-3′) (SEQ ID NO:18) was used to generate the DNA template for antisense and sense RNA probes spanning 200 base pairs of human CD133 cDNA, wherein this sequence encodes CD133 amino acid 42-108 (GI: 5174386). The sequence specificities for ABCB5 and CD133 were confirmed using the Genbank database BLAST program. The RNA probes were labeled with digoxigenin (DIG) using the DIG RNA labeling kit (Roche Applied Science, Indianapolis, Ind.). For in situ hybridization, 8 μM frozen tissue sections were baked at 50° C. for 15 min, then fixed in 4% paraformaldehyde at room temperature (RT) for 20 min. The sections were treated with 1 μg/ml proteinase K/PBS at 37° C. for 20 min and inactivated proteinase K with 0.2% glucine/PBS at RT for 5 min. Upon washes with PBS 2×2 min, the tissue sections were fixed in 4% paraformadehyde at RT for 15 min, washed with PBS 2×5 min, and then treated with 0.25% acetic anhydride/0.1M triethanolamine at RT for 10 min. The tissue sections were placed in 2×SSC and then hybridized with 500 ng/ml antisense or sense probe in hybridization buffer (0.3M NaCl, 10 mM Tris-HCl pH 7.6, 5 mM EDTA, 1×Denharts, 50% formamide, 100 μg/ml tRNA and 10% dectran sulphate) at 42° C. overnight. Post hybridization sections were treated with 0.2×SSC at 55° C. for 2×20 min, 20 □g/ml RNaseA in 0.5M NaCl, 10 mM Tris-HCl pH 7.5 at 37° C. for 30 min, and 0.2×SSC at 55° C. for 20 min. The hybridized probes were immunodetected using the DIG detection kit (Roche) and the Tyramide Signal Amplification (TSA) kit (PerkinElmer, Boston, Mass.) as follows: 1×DIG block buffer for 30 min, 1:100 anti-DIG Ab peroxidase conjugate at RT for 1 hour, 1×DIG wash 3×5 min, TSA reagent 10 min at RT, PBS 2×5 min, 1:100 streptavidin-horseradish peroxidase 30 min at RT, PBS 3×5 min. The labeling was visualized with NovaRed substrate (Vector Laboratories).
Animals. BALB/c nude mice and NOD/SCID mice were purchased from The Jackson Laboratory (Bar Harbor, Me.). SCID mice (C.B-17) and BALB/c Rag2^−/−mice were purchased from Taconic (Germantown, N.Y.). The animals were housed in autoclaved microisolator cages and were fed sterilized food and water. Mice were maintained in accordance with the institutional guidelines of Children's Hospital Boston and Harvard Medical School and experiments were performed according to approved experimental protocols.
Human to Mouse Melanoma Xenotransplantation and Human to Chimeric Mouse/Human Skin Melanoma Xenotransplantation.
Human to mouse melanoma xenografts were established by subcutaneous injection of human G3361, A375, SK-MEL-5 or clinical patient-derived human melanoma cells in BALB/c nude or NOD/SCID mice as described previously³. For human to chimeric mouse/human skin melanoma xenotransplantation, single donor-derived split human skin was obtained in accordance with the Partners HealthCare Research Management Institutional Review Board by cutting abdominal skin with a 0.016-inch gauge dermatome. Human skin was subsequently xenografted onto immunodeficient Rag2^−/−mice as described previously¹³, under a protocol approved by the institutional animal committee. Briefly, two circular graft beds, each, 1.5 cm²were prepared on bilateral dorsa of 4-8 week old Rag2^−/−mice treated with antibiotics (1 tablet of additional food per week containing Amoxicillin (3 mg), Flagyl (0.69 mg) and Bismuth (0.185 mg)) to prevent Helicobacter pylorii infection. Human donor skin was trimmed to conform to the bed and held in place with staples until 10 days following surgery. Unsegregated or ABCB5⁺-depleted A375, MUM-2B or MUM-2C melanoma cells (2×10⁶in 20 μl PBS) were intradermally microinjected into grafts after 6 weeks stabilization. All skin grafts were harvested in their entirety 3 weeks after tumour cell inoculation, fixed in formalin, serially sectioned and stained with H&E using standard methods for histological analysis of tumour formation. Sections representing maximum cross-sectional tumour area and thus best approximating the size of the generally spherical to ovoid tumour nodules were evaluated. Tumour volume (TV) was histologically determined and calculated as described³. Statistically significant differences in histological tumour formation were assessed using the Fisher's Exact test. Differences in tumour volume were statistically compared using the nonparametric Mann-Whitney Test, with a two-sided P value of P<0.05 considered significant.
Stable Green Fluorescence Protein (GFP)-Transfected Melanoma Xenografts to Human-SCID Chimeras.
Recombinant lentiviral vectors harboring GFP cDNA were obtained from Dr. M. Herlyn at the Wistar Institute and used to infect human A375 melanoma cells by lentiviral gene transfer. Two days after infection, cells were selected with puromycin (1 μg/ml) for a period of 7 days. Transgene expression was verified by fluorescence microscopy and flow cytometry. Melanoma xenografts were generated in human-SCID chimeras according to the protocol previously described¹³. SCID mice (C.B-17) between 4-6 weeks of age were purchased from Taconic (Germantown, N.Y.). Mice were anesthetized and prepared for transplantation by shaving the hair from a 2 cm²area on the dorsal torso followed by removal of full thickness skin down to the fascia. Full thickness human foreskin grafts of the same size were placed onto the wound beds. The skin grafts were then covered by Vaseline-saturated gauze and secured with band aids and 3M sports tapes. After 10 days, the dressings were removed and the mice allowed to recover for approximately 4-5 weeks before melanoma inoculation. GFP-labeled A375 melanoma cells were harvested and suspended in PBS at a density of 10⁸cells/ml. One hundred μl each of cell suspension were injected intradermally into the human skin grafts. The tumour xenografts were then harvested in 3 weeks or when the tumour reaches 1 cm³in size, and processed for frozen section. For double immunofluorescence, frozen sections (5 μm thick) of melanoma xenografts were fixed in 4% paraformaldehyde, blocked with donkey serum, and incubated sequentially with anti-CD144 (Cell Signaling, Danvers, Mass.), Texas red-conjugated donkey anti-rabbit (Invitrogen, Carlsbad, Calif.), anti-GFP (Novus Biologicals, Littleton, Colo.), and FITC-conjugated donkey anti-goat Abs (Jackson ImmunoResearch, West Grove, Pa.). After washing in PBS, the sections were coverslipped using an antiquench mountant containing DAPI (VectaShield, Vector Laboratories, Burlingame, Calif.). Irrelevant isotype-matched primary Abs were included as controls.

Example 2

The mechanisms through which ABCB5⁺ MMIC or CSC in other cancers trigger and promote neoplastic progression are currently unknown. We hypothesized that ABCB5⁺ MMIC possess vasculogenic differentiation plasticity and selectively drive melanoma growth through a specific role in providing nutritional support to growing tumours based on preferential co-expression in vivo of the vasculogenic differentiation markers CD144 (VE-cadherin) and TIE-1³by the ABCB5⁺ tumourigenic minority population.
To further characterize the repertoire of genes differentially expressed in MMIC compared to tumour bulk populations, we first performed microarray analyses on purified ABCB5⁺ (n=5) and ABCB5⁻ (n=5) cell subsets derived from the established human melanoma cell lines G3361 and A375 and from three separate patient-derived melanoma specimens, all previously characterized in our laboratory with regard to ABCB5 expression and MMIC phenotype in human melanoma xenotransplantation assays³. Using this approach¹⁰, 399 genes were identified that were differentially expressed (P<0.05) between ABCB5⁺ MMIC and ABCB5⁻ melanoma bulk populations (Table 5), in addition to ABCB5 shown overexpressed in ABCB5⁺ purified populations by real-time PCR (P<0.05). One identified functional gene network, validated by PCR-based gene expression analyses in ABCB5⁺ melanoma cell subsets, showed key molecules of vasculogenesis (the ability to differentiate along endothelial lines), FLT1 (VEGFR-1) and PTK2 (FAK), and of angiogenesis (the ability to induce ingrowth and proliferation of mature stromal blood vessels), FLT1 (VEGFR-1), PTK2 (FAK), MET (HGFR), NRP2, and ETS1, to be specifically overexpressed in ABCB5⁺ MMIC (FIG. 1 a,b).
Another set of genes differentially expressed in ABCB+ melanoma stem cells vs. ABCB5-melanoma bulk population cells was identified using RT-PCR. The data is shown in Table 6. The fold-expression levels are shown in the 7^thcolumn and can be compared to the control values shown in the last few rows of the table. A positive value indicates that the gene had higher expression levels in ABCB5+ cells and a negative value indicates that the gene had higher expression levels in ABCB5− cells. Some of the genes exhibited a greater than 100-fold and some even greater 1000-fold increase in expression in ABCB5+ versus ABCB5− cells. The highly expressed genes include factors that are likely secreted by the stem cells which may act on cells in a tumor either in an autocrine fashion on tumor stem cells, or in a paracrine fashion also on bulk population cancer cells. Appropriate therapies can be designed to treat cancers by inhibiting the expression or activity of such factors.
Preferential expression of VEGFR-1 by ABCB5⁺ MMIC vs. ABCB5⁻ subpopulations was also demonstrated by dual-color flow cytometry at the protein level (15.6±5.3% vs. 4.4±2.0% of cells, respectively, mean±s.e.m., n=6, P<0.05) (FIG. 1 c). To determine whether VEGF/VEGFR-1 interaction in MMIC influenced expression of the vasculogenic differentiation marker CD144, we evaluated functionally the effects of VEGF signaling in purified ABCB5⁺ MMIC or ABCB5⁻melanoma subpopulations. VEGF (100 ng/ml¹¹) selectively induced expression of CD144 at high levels in VEGFR-1-expressing ABCB5⁺ but not VEGFR-1-negative ABCB5⁻ melanoma cells, to 36.2±5.7% vs. 4.8±2.7% of cells (mean±s.e.m., n=3), respectively (P<0.01) (FIG. 1 d). Preincubation with a blocking monoclonal antibody (mAb) to VEGFR-1 abrogated the ability of VEGF to induce CD144 expression in human melanoma cells (0.0±0.0% in VEGFR-1 mAb-treated vs. 64±1% or 57±3% in untreated or isotype control mAb-treated cultures, respectively, mean±s.e.m., n=3, P<0.01) (FIG. 1 e). Moreover, VEGFR-1 mAb strongly inhibited VEGF-induced multicellular capillary-like tube formation by human melanoma cells in established in vitro vasculogenic differentiation assays¹¹, with significantly reduced numbers of tubes formed/microscopy field (6.7±0.9 in VEGFR-1 mAb-treated vs. 99.0±24.0 or 76.7±3.3% in untreated or isotype control mAb-treated cultures, respectively, mean±s.e.m., n=3, P<0.05), and significantly lower average tube length (33.2±4.5 μm in VEGFR-1 mAb-treated vs. 92.1±1.6 μm or 86.5±1.7 μm in untreated or isotype control mAb-treated cultures, respectively, mean±s.e.m., n=3, P<0.001) (FIG. 1 f). In contrast, both ABCB5⁺ MMIC and ABCB5⁻ melanoma bulk population exhibited similar adipogenic and osteogenic differentiation capacity previously detected in human melanoma cells¹²(adipogenesis: 100.0±0.0% vs. 93.2±6.9% of cells Oil Red-positive, respectively; mean±s.e.m., n=3, NS; osteogenesis: 90.8±9.2% vs. 98.3±1.7% of cells Alizarin Red-positive, respectively; mean±s.e.m., n=3, NS) (FIG. 1 g,h), and neither ABCB5⁺ MMIC nor ABCB5 melanoma bulk population exhibited capacity for myogenic differentiation¹⁰(0.0±0.0% vs. 0.0±0.0% of cells myogenin-positive, respectively; mean±s.e.m., n=3, NS) (FIG. 1 h). The selective in vitro vasculogenic differentiation capacity of ABCB5⁺ MMIC in response to VEGF/VEGFR-1 signaling indicated a potential role of this CSC subset in tumour vasculogenesis.

TABLE 5

Differentially expressed genes between ABCB5⁺MMIC and
ABCB5⁻melanoma bulk populations (P < 0.05).

	Molecules	ID	Fold Change

AABHD7	239579_at	0.661
ACBD6	225317_at	0.83
AK3	224655_at	0.845
AKAP9	215483_at	2.168
AKR1CL2	1559982_s_at	1.732
AMZ2	227567_at	1.377
ANAPC5	235926_at	2.631
ANK2	202921_s_at	4.162
ANKH	229176_at	0.776
ANKRD28	241063_at	2.297
ANKRD44	226641_at	1.218
ANKRD52	228257_at	0.762
ANXA4	201302_at	0.83
AOC3	204894_s_at	1.894
APBB2	40148_at	1.139
ARS2	201679_at	1.307
ASCC3L1 (includes EG: 23020)	214982_at	3.009
ASPM	232238_at	1.411
ATAD2	235266_at	1.304
ATP5I	207335_x_at	0.737
ATXN2L	207798_s_at	1.656
BARD1	205345_at	1.559
BAT3	230513_at	0.697
BCL9L	227616_at	1.291
BDP1	224227_s_at	1.632
BLID	239672_at	1.91
BRI3	223376_s_at	0.792
BUB1 (includes EG: 699)	216277_at	1.856
BUB1 (includes EG: 699)	233445_at	3.209
C10ORF18	244165_at	2.046
C12ORF45	226349_at	0.688
C12ORF48	220060_s_at	1.216
C12ORF51	230216_at	2.874
C12ORF51	1557529_at	3.632
C14ORF135	1563259_at	1.353
C14ORF156	221434_s_at	0.867
C16ORF63	225087_at	0.872
C18ORF10	213617_s_at	0.754
C18ORF10	212055_at	0.737
C19ORF42	219097_x_at	0.813
C20ORF4	234654_at	1.731
C22ORF28	200042_at	0.829
C22ORF30	216555_at	1.521
C2ORF30	224630_at	0.851
C5ORF24	229098_s_at	1.531
C9ORF78	218116_at	0.789
C9ORF85	244160_at	1.52
CABIN1	1557581_x_at	3.052
CAMK2D	225019_at	0.823
CAMK2D	228555_at	0.758
CANX	238034_at	0.8
CAPZB	201949_x_at	0.764
CASC5	228323_at	1.144
CBS	240517_at	1.818
CCDC127	226515_at	0.835
CCDC14	240884_at	1.771
CCDC52	234995_at	1.166
CCDC57	214818_at	1.703
CCDC73	239848_at	1.294
CCDC93	219774_at	1.208
CDC14B	234605_at	2.512
CDC16	242359_at	6.261
CENPJ	234023_s_at	1.22
CENPJ	220885_s_at	1.64
CEP27	228744_at	0.651
CEP55	218542_at	1.096
CGGBP1	224600_at	0.913
CHD2	244443_at	1.757
CHD8	212571_at	1.27
CLN8	229958_at	1.344
CNIH3	232758_s_at	1.451
COBRA1	1556434_at	1.985
COIL	203653_s_at	1.259
COL4A2	211966_at	0.729
COQ4	218328_at	1.328
CPEB2	226939_at	1.251
CPNE3	202119_s_at	0.833
CREB1	204313_s_at	0.791
CREB3L2	237952_at	2.013
CRIPAK	228318_s_at	1.486
CROP	242389_at	2.121
CSE1L	201112_s_at	0.911
CSE1L	210766_s_at	0.885
CUL4A	232466_at	2.607
CYB5R3	1554574_a_at	0.793
DARS	201624_at	0.928
DCLRE1C	242927_at	1.187
DCUN1D2	240478_at	1.76
DDX17	213998_s_at	1.528
DDX52	212834_at	0.771
DEGS1	209250_at	0.804
DEPDC1	232278_s_at	1.119
DHX40	218277_s_at	0.812
DNAJC21	230893_at	0.829
DNM1L	236032_at	1.503
DTX3	49051_g_at	1.32
ECHDC1	233124_s_at	0.943
EIF2S1	201142_at	0.717
EIF2S1	201144_s_at	0.824
EIF4G3	201935_s_at	1.174
ELOVL2	213712_at	0.699
EMP2	225079_at	0.781
ENAH	222433_at	0.783
ENDOD1	212573_at	0.775
ENTPD5	231676_s_at	0.867
ERBB3	1563253_s_at	0.691
ERRFI1	224657_at	0.881
ETS1	241435_at	1.797
EWSR1	229966_at	1.686
EXT1	242126_at	2.116
FAM62C	239770_at	1.551
FAM98A	212333_at	0.767
FHL3	218818_at	0.546
FLJ10357	241627_x_at	2.31
FLJ31306	239432_at	1.753
FLT1	232809_s_at	1.861
FOXN3	218031_s_at	0.721
FUBP1	240307_at	2.087
GABARAPL2	209046_s_at	0.863
GABPA	243498_at	2.03
GALNT1	201722_s_at	0.926
GBF1	233114_at	2.03
GGT1	211417_x_at	1.555
GHITM	1554510_s_at	0.764
GMFB	202544_at	0.904
GNPDA1	202382_s_at	0.787
GOLGA8A	213650_at	2.289
GPD2	243598_at	2.13
GPR107	211979_at	0.843
GPR135	241085_at	1.851
HDAC3	240482_at	2.062
HEATR2	241352_at	0.784
HECW1	237295_at	11.843
HELLS	242890_at	1.359
HERC5	219863_at	1.156
HIAT1	225222_at	0.832
HNRNPC	235500_at	1.769
HNRPD	235999_at	1.92
HNRPD	241702_at	1.962
HNRPH1	213472_at	2.332
HOXA2	228642_at	1.44
HOXB9	216417_x_at	0.766
HOXD3	206601_s_at	1.897
HPS1	239382_at	1.749
HSD17B1	228595_at	0.753
HSDL2	209513_s_at	0.803
HSPA4L	205543_at	0.786
HUWE1	214673_s_at	1.858
IDS	1559136_s_at	2.001
IFNGR1	242903_at	2.171
IGHMBP2	215980_s_at	0.893
IL13RA1	201887_at	0.775
INSIG2	209566_at	0.872
IPO7	200993_at	0.875
IPW	213447_at	1.399
IRS2	236338_at	2.162
JARID1A	226367_at	1.192
JARID2	232835_at	2.139
KIAA0841	36888_at	1.389
KIAA0907	230028_at	1.83
KIAA1267	224489_at	1.355
KIAA1618	231956_at	2.27
KIAA1737	225623_at	0.837
KIAA2013	1555933_at	2.18
KIDINS220	1557246_at	2.97
KPNA6	226976_at	0.814
KRTAP19-1	1556410_a_at	2.07
KSR2	230551_at	3.211
LBA1	213261_at	1.225
LIMS1	212687_at	0.822
LOC126917	225615_at	0.819
LOC137886	212934_at	0.886
LOC145757	1558649_at	2.779
LOC145786	229178_at	1.907
LOC146325	1553826_a_at	3.943
LOC203547	225556_at	0.802
LOC219731	1557208_at	0.419
LOC254128	1557059_at	2.164
LOC283888	1559443_s_at	2.56
LOC285147	236166_at	2.377
LOC338799	226369_at	1.137
LOC388135	230475_at	1.979
LOC388969	232145_at	1.555
LOC389203	225014_at	0.79
LOC641298	208118_x_at	1.419
LOC645166	228158_at	0.823
LOC645513	239556_at	2.24
LOC729397	236899_at	2.231
LRCH3	229387_at	1.793
LRRFIP1	239379_at	1.796
MAEA	207922_s_at	0.765
MALAT1	224568_x_at	1.699
MALAT1	223940_x_at	1.659
MAP1LC3B	208785_s_at	0.808
MAP2K4	203266_s_at	0.881
MAP3K15	200979_at	0.741
6-Mar	201737_s_at	1.219
MBNL1	201152_s_at	0.867
MDM4	235589_s_at	1.629
MECR	218664_at	0.832
MED19	226300_at	0.782
MEF2C	236395_at	2.104
MET	213816_s_at	1.283
MIA3	1569057_s_at	0.759
MLL	212079_s_at	1.599
MOBKL1B	214812_s_at	0.762
MRPL42 (includes EG: 28977)	217919_s_at	0.866
MRPL51	224334_s_at	0.846
MTERFD3	225341_at	1.422
MTUS1	239576_at	1.975
MYO10	243159_x_at	2.528
MYO10	244350_at	1.677
N4BP2L1	213375_s_at	2.01
N4BP2L2	235547_at	1.631
N4BP2L2	242576_x_at	2.349
NAALAD2	1554506_x_at	0.464
NANP	228073_at	0.817
NAPA	239362_at	1.624
NAPE-PLD	242635_s_at	1.216
NARG1	1556381_at	2.827
NAT8B	206964_at	2.513
NBPF16	201104_x_at	1.411
NBR1	1568856_at	1.957
NCKAP1L	209734_at	2.071
NDFIP1	217800_s_at	0.815
NDUFAF2	228355_s_at	0.722
NDUFB6	203613_s_at	0.712
NEK1	213328_at	1.381
NFATC2IP	217527_s_at	1.272
NPAS2	1557690_x_at	1.76
NPTN	228723_at	2.086
NRP2	210841_s_at	1.106
NUCB2	203675_at	0.812
NUDT4	212183_at	0.685
NUPL1	241425_at	2.179
OCIAD1	235537_at	1.794
ORMDL1	223187_s_at	1.171
OSBPL5	233734_s_at	1.261
OSGEP	242930_at	1.541
PABPN1	213046_at	2.228
PAK1	226507_at	0.869
PAPD4	222282_at	3.39
PDE4B	215671_at	3.457
PDHB	211023_at	0.827
PDHB	208911_s_at	0.807
PDK1	239798_at	1.654
PDLIM5	212412_at	0.752
PDSS1	236298_at	1.64
PDXDC1	1560014_s_at	2.105
PGRMC2	213227_at	0.686
PHC1	218338_at	1.123
PHF20L1	219606_at	2.3
PIGY (includes EG: 84992)	224660_at	0.793
PIP5K3	1557719_at	2.227
PITPNA	201190_s_at	0.863
PMP22	210139_s_at	0.865
PMS2L3	214473_x_at	1.159
POFUT2	207448_at	1.759
POLR2J2	1552622_s_at	1.828
POLR2J2	1552621_at	1.652
POP4	202868_s_at	0.847
PPP1R3D	204554_at	0.805
PPP1R7	201213_at	0.698
PPP3CA	202457_s_at	0.867
PRO1073	228582_x_at	1.607
PRPF38B	230270_at	1.888
PSEN1	242875_at	1.851
PSMA2	201316_at	0.839
PSMA3	201532_at	0.798
PTK2	234211_at	2.539
PTPMT1	229535_at	0.769
RAB11FIP3	228613_at	2.546
RAB11FIP3	216043_x_at	0.551
RAB14	200927_s_at	0.772
RAB1A	213440_at	0.81
RAD54L	204558_at	1.483
RADIL	223693_s_at	2.126
RBM25	1557081_at	1.57
RBM26	229433_at	1.43
RBM4	213718_at	1.53
RBM5	209936_at	2.249
RFT1	240281_at	1.426
RHOA	240337_at	2.143
RHOBTB2	1556645_s_at	1.538
RLBP1L1	224996_at	0.835
RNF43	228826_at	1.401
RP11-139H14.4	1569124_at	11.472
RPE	221770_at	0.766
RPE	225039_at	0.787
RPL7L1	225515_s_at	0.899
RUNX3	204198_s_at	1.233
SDAD1	242190_at	3.009
SDCCAG8	243963_at	2.67
SEC16B	1552880_at	1.877
SEPHS1	208940_at	0.875
11-Sep	201307_at	0.784
SF1	210172_at	2.452
SF3B1	201070_x_at	1.35
SF3B1	214305_s_at	1.359
SFRS15	222311_s_at	1.818
SFRS15	243759_at	2.028
SGCA	1562729_at	2.395
SGOL2	235425_at	1.591
SH2B3	203320_at	0.806
SKP1	200718_s_at	0.898
SLC16A1	202235_at	0.83
SLC20A1	230494_at	1.884
SLC2A11	232167_at	1.529
SLC2A8	239426_at	2.012
SLC30A9	237051_at	2.063
SMA4	238446_at	2.035
SMC6	218781_at	1.203
SMYD2	212922_s_at	0.867
SNORA28	241843_at	1.628
SNRPA1	242146_at	3.54
SON	201085_s_at	1.144
SPOPL	225659_at	0.828
SQLE	213577_at	1.502
SRP72	208801_at	0.751
SRP72	208803_s_at	0.766
SRPRB	218140_x_at	0.767
STK36	234005_x_at	1.335
STK36	231806_s_at	1.362
STRAP	1558002_at	2.189
STX11	235670_at	0.778
STX8	204690_at	0.819
SUPT7L	201838_s_at	0.865
SVIL	215279_at	2.228
SYNE2	202761_s_at	1.356
TAF15	227891_s_at	1.971
TAF1B	239046_at	1.468
TAOK3	220761_s_at	1.195
TBC1D5	201814_at	0.782
TBC1D8	221592_at	1.246
TBC1D8	204526_s_at	1.373
TBXA2R	207554_x_at	0.877
TBXA2R	336_at	0.73
TCAG7.907	238678_at	1.546
TCOF1 (includes EG: 6949)	202385_s_at	1.169
TFB1M	228075_x_at	0.87
THRAP3	217847_s_at	1.464
TIMM23	218119_at	0.723
TM6SF1	1558102_at	0.704
TMEM126B	221622_s_at	0.843
TMEM165	1560622_at	1.756
TMEM30A	232591_s_at	0.771
TNFAIP1	201207_at	0.88
TNPO1	1556116_s_at	1.739
TNRC6A	234734_s_at	1.268
TOX4	201685_s_at	0.73
TPM4	235094_at	2.079
TRAPPC2	219351_at	0.821
TRAPPC2L	218354_at	0.837
TRIM33	239716_at	2.496
TRIM46	238147_at	1.96
TRIO	240773_at	2.607
TRNT1	243236_at	2.295
TRPV1	1556229_at	2.636
TSPAN31	203227_s_at	0.744
TTC26	233999_s_at	1.184
TTC3	208664_s_at	1.396
TTC9C	1569189_at	1.55
TTLL4	1557611_at	2.092
TXNDC12	223017_at	0.849
TXNL1	243664_at	1.98
UBE2E3	210024_s_at	0.758
UBE3C	1560739_a_at	0.815
UBXD7	212840_at	0.754
UGT1A6	206094_x_at	3.86
UNK	1562434_at	1.637
UQCC	229672_at	1.451
USP36	224979_s_at	1.393
USP8	229501_s_at	0.808
VPS37B	236889_at	2.85
VTI1B	209452_s_at	0.821
WDR41	218055_s_at	0.789
WDR68	233782_at	1.924
WFS1	1555270_a_at	1.315
WIPF2	216006_at	2.916
WTAP	1560274_at	1.747
XRCC5	232633_at	2.106
YY1	224711_at	0.821
ZFHX3	215828_at	1.737
ZFR	238970_at	2.655
ZFX	207920_x_at	1.625
ZMYND8	209049_s_at	1.102
ZNF154	242170_at	2.667
ZNF224	216983_s_at	2.986
ZNF226	219603_s_at	1.332
ZNF251	226754_at	1.313
ZNF292	236435_at	3.201
ZNF326	241720_at	1.418
ZNF337	1565614_at	2.096
ZNF536	233890_at	3.303
ZNF567	242429_at	2.103
ZNF618	226590_at	0.75
ZNF668	219047_s_at	0.691
ZNF800	227101_at	1.484
ZUFSP	228330_at	1.205

TABLE 6

Differentially expressed genes between ABCB5⁺and ABCB5⁻cells as detected by
RT-PCR. PCR Array Catalog #: PAHS-024

A01	Hs.525622	NM_005163	AKT1	V-akt murine thymoma	AKT/PKB	1.2687
				viral oncogene homolog 1
A02	Hs.369675	NM_001146	ANGPT1	Angiopoietin 1	AGP1/AGPT	1.2953
A03	Hs.583870	NM_001147	ANGPT2	Angiopoietin 2	AGPT2/ANG2	2.7007
A04	Hs.209153	NM_014495	ANGPTL3	Angiopoietin-like 3	ANGPT5	3.0596
A05	Hs.9613	NM_001039667	ANGPTL4	Angiopoietin-like 4	ANGPTL2/ARP4	1.6974
A06	Hs.1239	NM_001150	ANPEP	Alanyl (membrane)	APN/CD13	1.3597
				aminopeptidase
				(aminopeptidase N,
				aminopeptidase M,
				microsomal
				aminopeptidase, CD13,
				p150)
A07	Hs.194654	NM_001702	BAI1	Brain-specific	FLJ41988	3.0596
				angiogenesis inhibitor 1
A08	Hs.54460	NM_002986	CCL11	Chemokine (C-C motif)	SCYA11	1.8834
				ligand 11
A09	Hs.303649	NM_002982	CCL2	Chemokine (C-C motif)	GDCF-	2.0326
				ligand 2	2/GDCF-2HC11
A10	Hs.76206	NM_001795	CDH5	Cadherin 5, type 2, VE-	7B4/CD144	3.0596
				cadherin (vascular
				epithelium)
A11	Hs.517356	NM_030582	COL18A1	Collagen, type XVIII,	KNO	1.9634
				alpha 1
A12	Hs.570065	NM_000091	COL4A3	Collagen, type IV, alpha	TUMSTATIN	2.1634
				3 (Goodpasture antigen)
B01	Hs.789	NM_001511	CXCL1	Chemokine (C—X—C	FSP/GRO1	1.2086
				motif) ligand 1
				(melanoma growth
				stimulating activity,
				alpha)
B02	Hs.632586	NM_001565	CXCL10	Chemokine (C—X—C	C7/IFI10	−2.1987
				motif) ligand 10
B03	Hs.89690	NM_002090	CXCL3	Chemokine (C—X—C	CINC-	2.061
				motif) ligand 3	2b/GRO3
B04	Hs.89714	NM_002994	CXCL5	Chemokine (C—X—C	ENA-	1.8834
				motif) ligand 5	78/SCYB5
B05	Hs.164021	NM_002993	CXCL6	Chemokine (C—X—C	CKA-3/GCP-2	2.1936
				motif) ligand 6
				(granulocyte chemotactic
				protein 2)
B06	Hs.77367	NM_002416	CXCL9	Chemokine (C—X—C	CMK/Humig	−1.1225
				motif) ligand 9
B07	Hs.592212	NM_001953	TYMP	Thymidine	ECGF1/MNGIE	1.5837
				phosphorylase
B08	Hs.154210	NM_001400	EDG1	Endothelial	CHEDG1/D1S3362	1.0377
				differentiation,
				sphingolipid G-protein-
				coupled receptor, 1
B09	Hs.516664	NM_182685	EFNA1	Ephrin-A1	B61/ECKLG	1.4573
B10	Hs.516656	NM_004952	EFNA3	Ephrin-A3	EFL2/EPLG3	1.3692
B11	Hs.149239	NM_004093	EFNB2	Ephrin-B2	EPLG5/HTKL	1.1355
B12	Hs.419815	NM_001963	EGF	Epidermal growth factor	HOMG4/URG	187.8365
				(beta-urogastrone)
C01	Hs.76753	NM_000118	ENG	Endoglin (Osler-Rendu-	CD105/END	1.1514
				Weber syndrome 1)
C02	Hs.437008	NM_004444	EPHB4	EPH receptor B4	HTK/MYK1	1.3692
C03	Hs.115263	NM_001432	EREG	Epiregulin	ER	1.8834
C04	Hs.483635	NM_000800	FGF1	Fibroblast growth factor	AFGF/ECGF	1.5511
				1 (acidic)
C05	Hs.284244	NM_002006	FGF2	Fibroblast growth factor	BFGF/FGFB	1.1355
				2 (basic)
C06	Hs.1420	NM_000142	FGFR3	Fibroblast growth factor	ACH/CD333	1.7092
				receptor 3
				(achondroplasia,
				thanatophoric dwarfism)
C07	Hs.11392	NM_004469	FIGF	C-fos induced growth	VEGF-	3.5884
				factor (vascular	D/VEGFD
				endothelial growth factor
				D)
C08	Hs.654360	NM_002019	FLT1	Fms-related tyrosine	FLT/VEGFR1	2.4172
				kinase 1 (vascular
				endothelial growth
				factor/vascular
				permeability factor
				receptor)
C09	Hs.388245	NM_021973	HAND2	Heart and neural crest	DHAND2/Hed	2.0186
				derivatives expressed 2
C10	Hs.396530	NM_000601	HGF	Hepatocyte growth factor	F-	4.542
				(hepapoietin A; scatter	TCF/HGFB
				factor)
C11	Hs.654600	NM_001530	HIF1A	Hypoxia-inducible factor	HIF-	−1.0918
				1, alpha subunit (basic	1alpha/HIF1
				helix-loop-helix
				transcription factor)
C12	Hs.44227	NM_006665	HPSE	Heparanase	HPA/HPR1	286.6871
D01	Hs.504609	NM_002165	ID1	Inhibitor of DNA binding	ID	−1.0329
				1, dominant negative
				helix-loop-helix protein
D02	Hs.76884	NM_002167	ID3	Inhibitor of DNA binding	HEIR-1	−1.3535
				3, dominant negative
				helix-loop-helix protein
D03	Hs.37026	NM_024013	IFNA1	Interferon, alpha 1	IFL/IFN	1.8834
D04	Hs.93177	NM_002176	IFNB1	Interferon, beta 1,	IFB/IFF	1.8834
				fibroblast
D05	Hs.856	NM_000619	IFNG	Interferon, gamma	IFG/IFI	1.8834
D06	Hs.160562	NM_000618	IGF1	Insulin-like growth factor	IGFI	4.7022
				1 (somatomedin C)
D07	Hs.126256	NM_000576	IL1B	Interleukin 1, beta	IL-1/IL1-	2.0898
					BETA
D08	Hs.654458	NM_000600	IL6	Interleukin 6 (interferon,	BSF2/HGF	1.7331
				beta 2)
D09	Hs.624	NM_000584	IL8	Interleukin 8	3-	1.1674
					10C/AMCF-I
D10	Hs.436873	NM_002210	ITGAV	Integrin, alpha V	CD51/DKFZp686A08142	1.217
				(vitronectin receptor,
				alpha polypeptide,
				antigen CD51)
D11	Hs.218040	NM_000212	ITGB3	Integrin, beta 3 (platelet	CD61/GP3A	−1.0619
				glycoprotein IIIa, antigen
				CD61)
D12	Hs.224012	NM_000214	JAG1	Jagged 1 (Alagille	AGS/AHD	1566.5046
				syndrome)
E01	Hs.479756	NM_002253	KDR	Kinase insert domain	CD309/FLK1	1.234
				receptor (a type III
				receptor tyrosine kinase)
E02	Hs.473256	NM_005560	LAMA5	Laminin, alpha 5	KIAA1907	3.8727
E03	Hs.421391	NM_007015	LECT1	Leukocyte cell derived	BRICD3/CHM-I	1.8834
				chemotaxin 1
E04	Hs.194236	NM_000230	LEP	Leptin	OB/OBS	2.1485
E05	Hs.82045	NM_002391	MDK	Midkine (neurite growth-	MK/NEGF2	1.4573
				promoting factor 2)
E06	Hs.513617	NM_004530	MMP2	Matrix metallopeptidase	CLG4/CLG4A	1.674
				2 (gelatinase A, 72 kDa
				gelatinase, 72 kDa type
				IV collagenase)
E07	Hs.297413	NM_004994	MMP9	Matrix metallopeptidase	CLG4B/GELB	1.9097
				9 (gelatinase B, 92 kDa
				gelatinase, 92 kDa type
				IV collagenase)
E08	Hs.436100	NM_004557	NOTCH4	Notch homolog 4	INT3/NOTCH3	1.2002
				(Drosophila)
E09	Hs.131704	NM_003873	NRP1	Neuropilin 1	CD304/DKFZp686A03134	1.1755
E10	Hs.471200	NM_003872	NRP2	Neuropilin 2	NP2/NPN2	1.4373
E11	Hs.707991	NM_002607	PDGFA	Platelet-derived growth	PDGF-	1.2002
				factor alpha polypeptide	A/PDGF1
E12	Hs.514412	NM_000442	PECAM1	Platelet/endothelial cell	CD31/PECAM-1	11.9037
				adhesion molecule
				(CD31 antigen)
F01	Hs.81564	NM_002619	PF4	Platelet factor	4	CXCL4/SCYB4	2.9966
				(chemokine (C—X—C
				motif) ligand 4)
F02	Hs.252820	NM_002632	PGF	Placental growth factor,	D12S1900/PGFL	−1.1865
				vascular endothelial
				growth factor-related
				protein
F03	Hs.77274	NM_002658	PLAU	Plasminogen activator,	ATF/UPA	1.6396
				urokinase
F04	Hs.143436	NM_000301	PLG	Plasminogen	DKFZp779M0222	1.8834
F05	Hs.125036	NM_020405	PLXDC1	Plexin domain containing 1	DKFZp686F0937/TEM3	3.4184
F06	Hs.528665	NM_021935	PROK2	Prokineticin 2	BV8/KAL4	1.8446
F07	Hs.201978	NM_000962	PTGS1	Prostaglandin-	COX1/COX3	1.2086
				endoperoxide synthase 1
				(prostaglandin G/H
				synthase and
				cyclooxygenase)
F08	Hs.532768	NM_002615	SERPINF1	Serpin peptidase	EPC-1/PEDF	1.1121
				inhibitor, clade F (alpha-
				2 antiplasmin, pigment
				epithelium derived
				factor), member 1
F09	Hs.68061	NM_021972	SPHK1	Sphingosine kinase 1	SPHK	1.192
F10	Hs.301989	NM_015136	STAB1	Stabilin 1	CLEVER-	4.357
					1/FEEL-1
F11	Hs.89640	NM_000459	TEK	TEK tyrosine kinase,	CD202B/TIE-2	−1.2805
				endothelial (venous
				malformations, multiple
				cutaneous and mucosal)
F12	Hs.170009	NM_003236	TGFA	Transforming growth	TFGA	3549.3357
				factor, alpha
G01	Hs.645227	NM_000660	TGFB1	Transforming growth	CED/DPD1	1.1837
				factor, beta 1
G02	Hs.133379	NM_003238	TGFB2	Transforming growth	TGF-beta2	1.3787
				factor, beta 2
G03	Hs.494622	NM_004612	TGFBR1	Transforming growth	AAT5/ACVRLK4	1.7695
				factor, beta receptor I
				(activin A receptor type
				II-like kinase, 53 kDa)
G04	Hs.164226	NM_003246	THBS1	Thrombospondin 1	THBS/TSP	−1.0619
G05	Hs.371147	NM_003247	THBS2	Thrombospondin 2	TSP2	−1.203
G06	Hs.522632	NM_003254	TIMP1	TIMP metallopeptidase	CLGI/EPA	−1.1147
				inhibitor 1
G07	Hs.633514	NM_003255	TIMP2	TIMP metallopeptidase	CSC-21K	1.2864
				inhibitor 2
G08	Hs.701968	NM_000362	TIMP3	TIMP metallopeptidase	HSMRK222/	1.8834
				inhibitor 3 (Sorsby	K222
				fundus dystrophy,
				pseudoinflammatory)
G09	Hs.241570	NM_000594	TNF	Tumor necrosis factor	DIF/TNF-	4.0652
				(TNF superfamily,	alpha
				member 2)
G10	Hs.525607	NM_006291	TNFAIP2	Tumor necrosis factor,	B94	1.8834
				alpha-induced protein 2
G11	Hs.73793	NM_003376	VEGFA	Vascular endothelial	VEGF/VEGF-A	2.4509
				growth factor A
G12	Hs.435215	NM_005429	VEGFC	Vascular endothelial	Flt4-L/VRP	446.7529
				growth factor C
H01	Hs.534255	NM_004048	B2M	Beta-2-microglobulin	B2M	−1.2983
H02	Hs.412707	NM_000194	HPRT1	Hypoxanthine	HGPRT/HPRT	−1.2894
				phosphoribosyltransferase
				1 (Lesch-Nyhan
				syndrome)
H03	Hs.523185	NM_012423	RPL13A	Ribosomal protein L13a	RPL13A	1.1837
H04	Hs.544577	NM_002046	GAPDH	Glyceraldehyde-3-	G3PD/GAPD	−1.146
				phosphate dehydrogenase
H05	Hs.520640	NM_001101	ACTB	Actin, beta	PS1TP5BP1	−1.0329
H06	N/A	SA_00105	HGDC	Human Genomic DNA	HIGX1A	1.8834
				Contamination
H07	N/A	SA_00104	RTC	Reverse Transcription	RTC	1.7451
				Control
H08	N/A	SA_00104	RTC	Reverse Transcription	RTC	1.7451
				Control
H09	N/A	SA_00104	RTC	Reverse Transcription	RTC	1.7695
				Control
H10	N/A	SA_00103	PPC	Positive PCR Control	PPC	1.8067
H11	N/A	SA_00103	PPC	Positive PCR Control	PPC	1.7818
H12	N/A	SA_00103	PPC	Positive PCR Control	PPC	36695.9527

Example 3

We therefore tested the hypothesis that MMIC, as defined by the novel marker ABCB5³, specifically relate to the phenomenon of vasculogenic mimicry whereby melanoma cells form channels capable of conducting nutrients from peripheral blood and thus serving as surrogates for mature tumour vessels⁴. Because we posited that this phenomenon may be more robust during early stages of tumour formation before cancer angiogenesis fully develops, we evaluated experimentally-induced human-derived melanomas grown as tumour xenografts in the subcutis of immunodeficient mice and in the dermis of human skin xenografted to immunodeficient mice (FIG. 2), the latter a humanized model whereby human melanoma develops in the context of the human stromal microenvironment¹³. While only peripheral tumour vessels expressed the mature endothelial marker, CD31, more interior regions of the melanomas exhibited formation of CD31-negative anastomosing channels with histologic, histochemical (PAS-D and laminin reactivity), and ultrastructural findings consistent with established features of vasculogenic mimicry⁴(FIG. 2 a-d). By electron microscopy, lumen-like spaces in these regions were lined by basement membrane-like material and viable melanoma cells and contained erythrocytes surrounded by finely granular matrix consistent with plasma, suggesting communication with the systemic circulation⁴. By immunohistochemistry and in situ hybridization, channels expressed ABCB5 protein and mRNA, respectively (FIG. 2 f-h), which also correlated in vitro when assayed across a panel of human melanoma cell lines (FIG. 4), and the architecture of ABCB5⁺ channels in xenografted tumours was identical to that focally detected in patient-derived melanomas (FIG. 2 f, inset). An identical pattern was also observed for CD133 mRNA (not illustrated), an additional marker for tumourigenic melanoma cells¹⁴and melanoma progression¹⁵. Anti-ABCB5 mAb systemically administered in vivo localized to channels, further confirming their systemic perfusion as well as the intimate association of ABCB5⁺ melanoma cells with channel lumens (FIG. 2 i). Double-labeling demonstrated co-localization of the human endothelial markers CD144 and TIE-1 with ABCB5⁺ cells forming channels (FIG. 2 j,k). Tumours initiated by melanoma cell lines expressing the green fluorescence protein (GFP) transgene confirmed the presence of melanoma cells lining channels that co-expressed melanoma-associated GFP and CD144 (FIG. 2 e), as well as human melanoma—but not human xenograft-associated class I major histocompatibility complex (MHC) antigens (not illustrated). These data show that the formation of perfused vessel-like channels in human melanoma is mediated by the ABCB5⁺ MMIC subpopulation found to selectively display gene profiles and differentiation capacity consistent with its participation in tumour vasculogenesis.

Example 4

We next reasoned that if vasculogenic channel formation mediated by ABCB5⁺ MMIC was functionally required for their capacity to efficiently initiate and drive melanoma growth, depletion of MMIC to low levels should inhibit the melanoma-associated vasculogenic response. To evaluate tumourigenesis and vasculogenesis in a bioassay most relevant to human primary melanoma, we again utilized a human skin/murine xenograft model whereby melanomas develop in the relevant dermal microenvironment of human skin and express architectural features and evolutionary growth patterns more akin to naturally occurring lesions¹³. Intradermal orthotopic transplantation of 2×10⁶unsegregated A375 cutaneous melanoma cells (ABCB5 positivity: 5.2±5.1%; mean±s.e.m., n=9) (FIG. 3 a) or heterotopic transplantation of 2×10⁶unsegregated uveal melanoma cells previously assayed for vasculogenic differentiation^4,16(MUM-2B and MUM-2C, ABCB5 positivity: 2.46±0.46% and 3.81±1.04%, respectively; mean±s.e.m., n=3-4) (FIG. 3 a) to human skin resulted in tumour formation three weeks following microinjections in 14 of 14 recipient skin grafts (A375: n=6, MUM-2B: n=4, MUM-2C: n=4 replicates) when assessed histologically in serial sections of each human skin xenograft in its entirety (FIG. 3 b,c). In contrast, intradermal transplantation of equal numbers of ABCB5⁺-depleted melanoma cells resulted in histologically-assessed tumour formation in only 6 of 14 recipient skin grafts (P<0.002) (FIG. 3 b,c) and histologically-determined mean tumour volumes (TV) were significantly reduced in recipients of ABCB5⁺-depleted vs. unsegregated melanoma inocula (TV=2.8±1.8 mm³vs. 10.9±6.9 mm³, respectively; mean±s.e.m., P<0.005) (FIG. 3 d). When vasculogenic channel formation within tumours was evaluated using quantitative image analysis technology to assess the pixilated density of laminin immunoreactivity, significantly fewer channels per cross-sectional area were detected in tumours that formed from MMIC-depleted inocula compared to those that originated from unsegregated tumour cell grafts (A375, P<0.0032; MUM-2B, P<0.0005; MUM-2C, P<0.0059) (FIG. 3 e,f). In aggregate, these findings show in relevant xenograft models of early melanoma development the participation of ABCB5⁺ MMIC in the genesis of vasculogenic channels, and the interdependency of MMIC-derived channel formation and tumour growth.
Discovery of MMIC-driven vasculogenesis identifies selective differentiation plasticity as a novel CSC-specific function through which these tumourigenic cancer subpopulations may provide a specific growth advantage to developing tumours. Our finding of a propensity of MMIC to differentiate selectively into cells capable of serving a defined tissue function required for more efficient tumour growth parallels hallmark characteristics of physiological stem cells, which similarly give rise to cell lineages capable of serving specific roles required for maintenance of tissue homeostasis through defined differentiation programs. Importantly, we find that MMIC-dependent tumourigenesis and vasculogenesis are operative not only in human melanoma to murine skin xenotransplantation models but also upon human melanoma to human skin transplantation. Therefore, our results provide initial evidence that the tumour-sustaining role of human CSC identified in xenotransplantation assays does not merely reflect the limited ability of human tumour cells to adapt to growth in a foreign (mouse) milieu, as has been postulated based on the results of murine tumour transplantation experiments utilizing histocompatible murine hosts¹⁷
The now widely-accepted concept of cancer angiogenesis advanced by Folkman in 1971 states that human cancers are critically dependent upon tumour-related blood-vessel growth and development¹⁸. In addition to classical angiogenesis whereby cancer cells, including CSC¹⁹, induce in-growth of mature, CD31-positive vessels from surrounding stroma²⁰, evidence has been generated that cancer cells may also directly form surrogate vessel-like spaces by the process of vasculogenic mimicry whereby aggressive human melanomas develop patterned networks composed of periodic acid-Schiff (PAS)- and laminin-reactive basement membranes and associated perfusable channels formed by tumour cells that express some but not all endothelium-related genes and proteins⁴. The present study identifies the cells and underlying mechanisms responsible for vasculogenic mimicry, and establishes that in addition to self-renewal, MMIC selectively express vasculogenic genes and form channels consistent with the function of promoting nutrition to rapidly growing tumours. Thus, cancer angiogenesis and MMIC-driven vasculogenesis may represent independent yet potentially interrelated mechanisms whereby aggressive and metabolically-active tumours obtain those nutrients requisite for critical stages of growth and evolution. This may be particularly important during tumour initiation and early phases of tumourigenic growth when hypoxia-dependent, mTOR-driven angiogenesis from surrounding stroma has not fully evolved²¹.
Recently, proof-of-principle has been established for the potential therapeutic utility of the CSC concept^3,22. Therefore, identification of a vasculogenic mechanism whereby MMIC may contribute to tumour growth has potentially important therapeutic implications. Previous studies revealed that normally resistant human melanoma cells are rendered sensitive in vitro to the effects of chemotherapeutic agents by mAb- or siRNA-mediated blockade of ABCB5^5,6, and that mAb binding to ABCB5 is sufficient to induce an effective anti-melanoma immune response via antibody-dependent cell-mediated cytotoxicity (ADCC) in vivo³. Now, recognition of the role of MMIC in tumour vasculogenesis will also permit development of strategies focused on inhibition of the relevant molecular pathways integral to endothelial-directed CSC plasticity. Moreover, the spatial localization of the MMIC component to channels that communicate with the systemic circulation may render this important determinant of cancer virulence particularly vulnerable to therapeutic targeting.

Example 5

Vasculogenic/Angiogenic Pathways in Human Melanoma

We investigated gene relationships based on Ingenuity Pathway Analysis. We prepared a graphical representation of pathway activation across ABCB5⁺ MMIC. Genes that were overexpressed in ABCB5⁺ relative to ABCB5⁻ human melanoma cells were represented by red nodes (circles) and those expressed at lower levels were represented by black nodes. Black lines were drawn between genes to show known interactions. Known gene functions in vasculogenesis and angiogenesis, and genes known as relevant drug targets were annotated (red lines) (FIG. 1 a). We examined expression of vasculogenic/angiogenic pathway members by RT-PCR in ABCB5⁺ MMIC. Results of this analysis are shown in FIG. 1 b. We used dual color flow cytometry using ABCB5 phenotype-specific cell gating to determining FLT1 (VEGFR-1) protein expression of ABCB5+ MMIC (FIG. 1 c, top) and ABCB5-melanoma cells (FIG. 1 c, bottom). We examined CD144 expression in ABCB5⁺ MMIC or ABCB5⁻ melanoma cell subpopulations by immunofluorescence staining prior to (t=0 h) and upon 48 h of culture (t=48 h) in the presence of 100 ng/ml VEGF¹¹. Representative immunofluorescence staining for CD144 expression (Texas red) are shown in FIG. 1 d, with nuclei counterstained in blue (DAPI). Mean percentages (mean±s.e.m., n=3 replicate experiments) of cells staining positively for CD144 in each sample are shown on the right. We examined CD144 expression in melanoma cells in the presence of 100 ng/ml VEGF as in above, but in the presence or absence of anti-FLT1 (VEGFR-1) blocking mAb or isotype control mAb. Representative immunofluorescence staining for CD144 expression (Texas red) by melanoma cells cultured for 48 h (t=48 h) are shown in FIG. 1 e, with nuclei counterstained in blue (DAPI). Mean percentages (mean±s.e.m., n=3 replicate experiments) of cells staining positively for CD144 in each sample are shown in the far right panel. We examined tube formation by phase contrast light microscopy of melanoma cells cultured for 24 h (t=24 h) in the presence of 100 ng/ml VEGF and the presence or absence of anti-FLT1 (VEGFR-1) blocking mAb or isotype control mAb (FIG. 10. Number of tubes/microscopy field (mean±s.e.m., n=3 replicate experiments) and tube length (μm) (mean±s.e.m., n=3 replicate experiments) are illustrated for the different experimental conditions on the far right panels, respectively. We examined the differentiation potential of ABCB5⁺ and ABCB5⁻ human melanoma cells along a adipogenic pathway (FIG. 1 h, Oil Red O staining, nuclei are counterstained with hematoxylin) and osteogenic pathway (FIG. 1 i, Alizarin Red staining). Myogenic differentiation potential of ABCB5⁺ and ABCB5⁻human melanoma cells was also examined (FIG. 1 j). Absence of myogenin staining (FITC, green) was detected in ABCB5⁺ or ABCB5⁻ human melanoma cells (nuclei are counterstained with DAPI).

Example 6

MMIC-Driven In Vivo Vasculogenesis

We investigated MMIC driven vasculogenesis in vivo. Sections of human melanoma growing at melanoma cell injection site within human dermis of skin xenograft to NOD/SCID mouse were conventionally-stained by hematoxylin and eosin (FIG. 2 a). We also examined by immunohistochemistry the expression of human CD31 which indicated angiogenic response at perimeter of melanoma within human xenograft. (FIG. 2 b, broken line represents interface of tumour nodule with dermal connective tissue). We used periodic-acid Schiff (PAS) stain (with diastase), an immunochemical stain of CD31-negative interior regions of melanoma xenografts, to reveal numerous anastomosing channels (FIG. 2 c, inset is laminin immunohistochemistry indicating identical pattern). We conducted transmission electron micrographs of interior regions of melanoma xenografts (FIG. 2 d), and found that lumenal spaces containing blood products (erythrocytes) are lined by melanoma cells and associated basement membrane-like extracellular matrix. We examined the interior zone of melanoma xenograft derived from cells expressing GFP transgene and immunohistochemically stained for endothelial marker CD144 (red chromogen); results are shown in (FIG. 2 e). We found that CD144 expression is confined to cells forming lumen-like spaces lined by cells that co-express GFP and CD144 (indicated as yellow-orange). We also performed immunohistochemistry, at low (FIG. 20 and high (FIG. 2 g) magnification, for ABCB5 protein; our results show that reactivity is restricted to anastomosing channels identical to those seen in FIG. 2 c. The inset in FIG. 2 f depicts similar formation of ABCB5-reactive channels in a patient-derived melanoma biopsy. We performed in situ hybridization for ABCB5 mRNA (FIG. 2 h). Our results reveal a channel pattern corresponding to that of ABCB5 protein expression (compare with FIG. 2 f; inset is sense control). We examined the expression of ABCB5 in melanoma xenografts after intravenous administration in vivo (FIG. 2 h). Detection of anti-ABCB5 mAb was accomplished using anti-mouse Ig immunohistochemistry; note localization to channels (inset represents anti-mouse Ig staining after intravenous administration of irrelevant isotype-matched control mAb). Dual-labeling immunofluorescence microscopy was performed for both ABCB5 (green), CD144 (red), and ABCB5 & CD144 (mix) (FIG. 2 j) and ABCB5 (green), TIE-1 (red), and ABCB5 & TIE-1 (mix) (FIG. 2 j).

Example 7

Interdependency of MMIC-Driven Vasculogenesis and Tumourigenesis

We examined ABCB5 expression by flow cytometry; ABCB5 or control staining (FITC, Fl1) was plotted against forward scatter (FSC) for human A375, MUM-2B, and MUM-2C melanoma cell inocula. Representative data is shown in FIG. 3 a. We examined histologic sections of melanomas that developed from three unsegregated and ABCB5-depleted melanoma cell lines injected intradermally into human skin xenografts. Representative sections are shown in FIG. 3 b. We used histology to determine tumour formation rate (%) 3 weeks following intradermal transplantation of unsegregated vs. ABCB5⁺-depleted human A375, MUM-2B or MUM-2C melanoma cells (2×10⁶/inoculum) into human skin/Rag2^−/− chimeric mice (n=5, respectively). (FIG. 3 c). We determined histological tumour volumes (mean±s.e.m.) 3 weeks following intradermal transplantation of unsegregated vs. ABCB5⁺-depleted human A375, MUM-2B or MUM-2C melanoma cells (2×10⁶/inoculum) into human skin/Rag2^−/− chimeric mice. (FIG. 3 d). We performed immunohistochemistry for laminin. Our results showed the extent of channel formation in melanomas that developed from unsegregated or ABCB5⁺-depleted melanoma cell inocula derived from A375, MUM-2B or MUM-2C lines injected intradermally into human skin xenografts (arrows=necrosis). (FIG. 3 e). We performed image analysis of laminin immunoreactivity for melanomas derived from unsegregated and ABCB5⁺-depleted cell inocula. Data are shown in FIG. 3 f; y-axis is percent of pixelated area with reactivity (mean±s.e.m.); solid bar represents tumours derived from unsegregated melanoma cells, open bars represent tumours derived from ABCB5⁺-depleted cells (A375, P<0.0032; MUM-2B, P<0.0005; MUM-2C, P<0.0059).

Example 8

Correlation of ABCB5 Protein and mRNA Expression Across Human Melanoma Cell Lines

We examined ABCB5 and tubulin expression in a panel of human melanoma cell lines by western blot analysis (FIG. 4 a). We examined relative ABCB5 mRNA expression (log₂) in a panel of human melanoma cell lines plotted against ABCB5 protein expression as determined by ratios of ABCB5 89 kD western blot band intensity and tubulin western blot band intensity for each human melanoma cell line. (FIG. 4 b). Data points are as follows: 1, SK-MEL-2; 2, SK-MEL-5; 3, SK-MEL-28; 4, MDA-MB-435; 5, UACC-62; 6, UACC-257; 7, M14; 8, MALME-3M. Spearman Rank Correlation r (corrected for ties).

Example 9

CSC-Associated Genes Identified at the Protein Level

Using cell surface immunostaining and flow cytometry we identified additional genes to be differentially regulated at the protein level in ABCB5+ CSC versus ABCB5− cancer bulk populations. These are all immunomodulatory molecules and the ones upregulated in ABCB5+ cells may be relevant to the escape from immunosurveillance and be responsible for resistance to immunotherapy in malignant melanoma, i.e. when the genes overexpressed on ABCB5+ cells are targeted, melanoma is predicted to be sensitized to immune attack and therapy.

TABLE 8

Upregulated in ABCB5+ CSC compared to ABCB5−
bulk cancer populations:

	MHC class II
	CD28
	CD86
	PD-1
	CD40-L
	4-1BB-L
	B7-H4
	GITR

TABLE 7

Downregulated in ABCB5+ CSC compared to ABCB5−
cancer bulk populations

	MHC class I
	CD80
	PD-L1
	ICOS-L

REFERENCES FOR DETAILED DESCRIPTION AND EXAMPLES

¹M. Al-Hajj, M. S. Wicha, A. Benito-Hernandez et al., Proc Natl Acad Sci USA 100 (7), 3983 (2003); D. Bonnet and J. E. Dick, Nat Med 3 (7), 730 (1997); C. A. O'Brien, A. Pollett, S. Gallinger et al., Nature 445 (7123), 106 (2007); L. Ricci-Vitiani, D. G. Lombardi, E. Pilozzi et al., Nature 445 (7123), 111 (2007); S. K. Singh, C. Hawkins, I. D. Clarke et al., Nature 432 (7015), 396 (2004).
²T. Schatton and M. H. Frank, Pigment cell & melanoma research 21 (1), 39 (2008).
³T. Schatton, G. F. Murphy, N. Y. Frank et al., Nature 451 (7176), 345 (2008).
⁴A. J. Maniotis, R. Folberg, A. Hess et al., Am J Pathol 155 (3), 739 (1999).
⁵N. Y. Frank, A. Margaryan, Y. Huang et al., Cancer Res 65 (10), 4320 (2005).
⁶Y. Huang, P. Anderle, K. J. Bussey et al., Cancer Res 64 (12), 4294 (2004).
⁷L. Chin, L. A. Garraway, and D. E. Fisher, Genes Dev 20 (16), 2149 (2006).
⁸G. I. Keshet, I. Goldstein, O. Itzhaki et al., Biochem Biophys Res Commun 368 (4), 930 (2008).
⁹J. F. Sousa and E. M. Espreafico, BMC cancer 8, 19 (2008).
¹⁰N. Y. Frank, A. T. Kho, T. Schatton et al., J Cell Biol 175 (I), 99 (2006).
¹¹K. Nishiyama, K. Takaji, Y. Uchijima et al., J Biol Chem 282 (23), 17200 (2007).
¹²D. Fang, T. K. Nguyen, K. Leishear et al., Cancer Res 65 (20), 9328 (2005).
¹³I. Juhasz, S. M. Albelda, D. E. Elder et al., Am J Pathol 143 (2), 528 (1993).
¹⁴E. Monzani, F. Facchetti, E. Galmozzi et al., Eur J Cancer 43 (5), 935 (2007).
¹⁵W. M. Klein, B. P. Wu, S. Zhao et al., Mod Pathol 20 (1), 102 (2007).
¹⁶E. A. Seftor, P. S. Meltzer, D. A. Kirschmann et al., Clin Exp Metastasis 19 (3), 233 (2002).
¹⁷P. N. Kelly, A. Dakic, J. M. Adams et al., Science 317 (5836), 337 (2007).
¹⁸J. Folkman, N Engl J Med 285 (21), 1182 (1971).
¹⁹S. Bao, Q. Wu, S. Sathornsumetee et al., Cancer Res 66 (16), 7843 (2006); R. J. Gilbertson and J. N. Rich, Nat Rev Cancer 7 (10), 733 (2007).
²⁰B. N. Perry and J. L. Arbiser, J Invest Dermatol 126 (10), 2160 (2006).
²¹M. Guba, P. von Breitenbuch, M. Steinbauer et al., Nat Med 8 (2), 128 (2002).
²²Z. F. Yang, D. W. Ho, M. N. Ng et al., Cancer Cell 13 (2), 153 (2008); S. Bao, Q. Wu, Z. Li et al., Cancer Res 68 (15), 6043 (2008).
²³N. Y. Frank, S. S. Pendse, P. H. Lapchak et al., J Biol Chem 278 (47), 47156 (2003).
²⁴D. Donovan, N. J. Brown, E. T. Bishop et al., Angiogenesis 4 (2), 113 (2001).

Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.

Description

1. A method for diagnosing cancer in an individual, comprising:

determining an expression level of a cancer stem cell (CSC)-associated gene in Table 5 in a test sample from the individual; and

comparing the expression level of the CSC-associated gene to a reference value, wherein results of the comparison are diagnostic of cancer.

2. The method of claim 1, wherein the cancer is melanoma, breast cancer, prostate cancer, colon cancer or lung cancer.

3. (canceled)

4. The method of claim 1, wherein the determining comprises detecting in the test sample a mRNA that is encoded by the CSC-associated gene.

5. The method of claim 1, wherein the determining comprises detecting in the test sample a polypeptide that is encoded by the CSC-associated gene.

6-12. (canceled)

13. The method of claim 1, wherein the reference value is the expression level of the CSC-associated gene in a non-cancer reference sample, and wherein, if the expression level of the CSC-associated gene in the test sample is about equal to the expression level of the CSC-associated gene in the non-cancer reference sample, then the comparison does not indicate cancer.

14. The method of claim 1, wherein the reference value is the expression level of the CSC-associated gene in a cancer reference sample, and wherein, if the expression level of the CSC-associated gene is about equal to the expression level of the CSC-associated gene in the cancer reference sample, then the comparison indicates cancer.

15. The method of claim 1, wherein the CSC-associated gene is in Table 1 or 8, wherein the reference value is the expression level of the CSC-associated gene in a non-cancer reference sample, and wherein, if the expression level of the CSC-associated gene in the test sample is significantly higher than the expression level of the CSC-associated gene in the non-cancer reference sample, the comparison indicates cancer.

16-17. (canceled)

18. The method of claim 1, wherein the CSC-associated gene is in Table 2 or 7, wherein the reference value is the expression level of the CSC-associated gene in a non-cancer reference sample, and wherein, if the expression level of the CSC-associated gene in the test sample is significantly lower than the expression level of the CSC-associated gene in the non-cancer reference sample, the comparison indicates cancer.

19-42. (canceled)

43. A method for treating an individual having, or at risk of having, cancer, comprising:

administering a therapeutically effective amount of a composition that targets a product of a CSC-associated gene selected from the group set forth in Table 1 or 8.

44. The method of claim 43, wherein the cancer is melanoma, breast cancer, prostate cancer, colon cancer or lung cancer.

45. The method of claim 43, wherein the CSC-associated gene is selected from the group set forth in Table 4.

46. The method of claim 45, wherein the CSC-associated gene is selected from the group consisting of: ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8.

47. The method of claim 43, wherein the composition comprises a small interfering nucleic acid that inhibits expression of the CSC-associated gene.

48-51. (canceled)

52. The method of claim 45, wherein the composition comprises an isolated molecule that selectively binds to a polypeptide encoded by the CSC-associated gene.

53. The method of claim 52, wherein the isolated molecule is conjugated to a therapeutic agent.

54. The method of claim 53, wherein the isolated molecule is an antibody or antigen-binding fragment.

55-65. (canceled)

66. A method of delivering a therapeutic agent to a cancer stem cell, comprising:

contacting a cancer stem cell with an isolated molecule that selectively binds to a polypeptide encoded by a CSC-associated gene selected from the group set forth in Table 4 conjugated to a therapeutic agent in an effective amount to deliver the therapeutic agent to the cancer stem cell.

67. The method of claim 66, wherein the CSC-associated gene is selected from the 5 group consisting of: ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8.

68. (canceled)

69. The method of claim 66, wherein the therapeutic agent is selected from: a toxin, a small-interfering nucleic acid, and a chemotherapeutic agent.

70-71. (canceled)

72. A nucleic acid array consisting essentially of at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, or more CSC-associated genes set forth in Table 5.

73-84. (canceled)