Methods and Means Related to Cancer Stem Cells

Abstract

This invention relates to the methods for the identification and isolation of cancer stem cells from cultured cancer cell lines. Cell line-derived cancer stem cells isolated using the present methods may be useful, for example, in assays to screen compounds for anti-cancer stem cell activity and in target discovery methods for identifying novel expressed genes and druggable targets. The invention also relates to the screening of compounds for activity against cell line-derived cancer stem cells.

1 . A method of identifying and/or obtaining a cell line-derived cancer stem cell comprising;
 providing a population of cancer cells from a cultured cancer cell line, and;   determining the presence of a cancer stem cell marker in one or more cells in the population,   to thereby identify and/or obtain a cell line-derived cancer stem cell.   
     
     
         2 .- 27 . (canceled) 
     
     
         28 . A method of identifying a cancer stem cell marker comprising;
 comparing a level or amount of one or more polypeptides in a cancer stem cell obtained from a population of cultured cancer cells by the method of  claim 1  with a level or amount of the one or more polypeptides in a non-cancer stem cell, and;   identifying a polypeptide from the one or more polypeptides whose level or amount is increased in the cancer stem cell relative to the non-cancer stem cell as a cancer stem cell marker polypeptide.   
     
     
         29 .- 62 . (canceled) 
     
     
         63 . A method for the identification of a compound that inhibits the growth, viability, and/or proliferation of cell line-derived cancer stem cells in a cancer cell line comprising:
 (i) contacting the cancer cell line with one or more test compounds, wherein the cancer cell line comprises cell line-derived cancer stem cells and tumor bulk;   (ii) determining the effect of the one or more test compounds on the growth, viability, and/or proliferation of cell-line derived cancer stem cells in the cancer cell line and determining the effect of the one or more test compounds on the growth, viability and/or proliferation of cells from the tumor bulk in the cancer cell line; and   (iii) comparing the effect of the one or more test compounds on the growth, viability, and/or proliferation of cell line-derived cancer stem cells in the cancer cell line with the effect of the one or more test compounds on the growth, viability, and/or proliferation of cells from the tumor bulk in the cancer cell line,   wherein a test compound is identified as a compound that inhibits the growth, viability, and/or proliferation of cell line-derived cancer stem cells if it: (a) inhibits the growth, viability, and/or proliferation of the cell line-derived cancer stem cells in the cancer cell line but does not inhibit the growth, viability, and/or proliferation of cells from the tumor bulk in the cancer cell line; or (b) inhibits the growth, viability, and/or proliferation of the cell line-derived cancer stem cells in the cancer cell line and also inhibits the growth, viability, and/or proliferation of cells from the tumor bulk in the cancer cell line.   
     
     
         64 .- 71 . (canceled) 
     
     
         72 . A method for treating cancer in a subject comprising administering the compound of  claim 63  to the subject. 
     
     
         73 . The method of  claim 63 , wherein the growth, viability, and/or proliferation of the cell line-derived cancer stem cells is monitored using a detectable marker for cell death, proliferation, and/or differentiation. 
     
     
         74 . The method of  claim 73 , wherein the detectable marker is Annexin or propidium iodide. 
     
     
         75 . The method of  claim 63 , wherein the effect of the one or more test compounds on the growth, viability, and/or proliferation of the cell line-derived cancer stem cells and tumor bulk is determined by monitoring the binding of the compound to the cell line-derived cancer stem cells. 
     
     
         76 . The method of  claim 63 , wherein the effect of the one or more test compounds on the growth, viability, and/or proliferation of the cell line-derived cancer stem cells and tumor bulk is determined by monitoring the expression or activity of one or more cell line-derived cancer stem cell markers. 
     
     
         77 . The method of  claim 63 , wherein the expression or activity of one or more cell line-derived cancer stem cell markers is monitored by a detectable reporter that is specifically expressed by the cell line-derived cancer stem cells and not by the cells from the tumor bulk in the cancer cell line. 
     
     
         78 . The method of  claim 77 , wherein a gene for the detectable reporter is linked to a promoter, and wherein the promoter belongs to a cell line-derived cancer stem cell marker. 
     
     
         79 . The method of  claim 78 , wherein the one or more cell line-derived cancer stem cell markers comprise stem cell factor, SCF ligand, c-Kit ligand, telomerase, TRA-1-60, TRA-1-81, vimentin, genesis, germ cell nuclear factor, hepatocyte nuclear factor-HNF-4, nestin, breast cancer resistance protein (BCRP), verapamil, NG2, A2B5, polysialylated form of neuronal cell-adhesion molecule (PSA-NCAM), nucleostemin, sox-2, musashi-1 and -2, hairy and enhancer-of-splits (Hes-1, -3, -5), melk, PSP, Inhibitor of differentiation (Id-1, -2, -3, -4), Bmi-1, brca-1, Oct-4, Nanog, FGF-4, Pax6, Stage-specific embryonic antigens (SSEA-1, -3, -4), Cluster designation 30 (CD30), CD34, CD44, Notch, CD123, CD133, CD24, Cripto (TDGF-1) ATA-4 gene, GCTM-2, Alkaline phosphatase, Alpha-fetoprotein (AFP), Bone morphogenetic protein-4, mdr-1, hiwi, prominin-1, Brachyury, Wnt1, Wnt10, Wnt11, Notch 1, Notch 2, Notch 3, Notch 4, integrin alpha-6, mucin-1 (EMA), estrogen receptor-alpha, estrogen receptor-beta, cytokeratin-14, cytokeratin-18, and cytokeratin 19. 
     
     
         80 . The method of  claim 79 , wherein the cell line-derived cancer stem cell marker is Oct-4. 
     
     
         81 . The method of  claim 77 , wherein the detectable reporter is a fluorescent reporter. 
     
     
         82 . The method of  claim 81 , wherein the detectable reporter is green fluorescent protein (GFP) or luciferase. 
     
     
         83 . The method of  claim 77 , wherein the effect of the one or more test compounds on the growth, viability, and/or proliferation of the cell line-derived cancer stem cells and cells from the tumor bulk in the cancer cell line is determined by monitoring the expression or activity of the detectable reporter. 
     
     
         84 . The method of  claim 63 , wherein the effect of the one or more test compounds on the growth, viability and/or proliferation of the cell line-derived cancer stem cells and tumor bulk is determined using at least one of the following techniques: flow cytometry, fluorimetry, microscopy, immunofluorescence, ELISA, radioimmunoas say, immunoenzymatic assay, fluorescence activated cell sorting (FACS), PCR, RT-PCR, differential display, representational difference analysis, microarray, suppressive subtractive hybridization, direct sequencing, Western blotting, immunohistochemical staining, and immunocytochemical staining. 
     
     
         85 . The method of  claim 63 , wherein the cell line is a human cell line. 
     
     
         86 . The method of any one of  claim 63 , wherein the cell line is one of the following: HeLa cells, MCF-7 cells, C6 cells, or B104 cells. 
     
     
         87 . The method of  claim 63 , wherein the cell line is a prostate cancer cell line, an adenocarcinoma cell line, a lung cancer cell line, a gastrointestinal cancer cell line, a colon cancer cell line, a breast carcinoma cell line, an ovarian carcinoma cell line, a testicular cancer cell line, a glioma cell line, a liver cancer cell line, a kidney cancer cell line, a bladder cancer cell line, a pancreatic cancer cell line, a brain cancer cell line, a neuroblastoma cell line, a sarcoma cell line, an osteosarcoma cell line, a melanoma cell line, a lymphoma cell line, a retinoblastoma cell line, a skin cancer cell line, or a leukemia cell line.