Tumor-initiating cells and methods of use

Abstract

Provided herein are an isolated or enriched population of tumor initiating cells derived from normal cells, cells susceptible to neoplasia, or neoplastic cells. Methods of use of the cells for screening for anti-hyperproliferative agents, and use of the cells for animal models of hyperproliferative disorders including metastatic cancer, diagnostic methods, and therapeutic methods are provided.

What is claimed is: 
     
         1 . An isolated self-renewing cell selected as the product of a spontaneous cell-cell fusion or a descendant thereof. 
     
     
         2 . A tumor-initiating cell selected as the product of a spontaneous cell-cell fusion or a descendant thereof, wherein said cell gives rise to a neoplasia in vitro or in vivo under suitable growth conditions. 
     
     
         3 . The cell of  claim 1  or  2 , wherein said cell exhibits a characteristic selected from the group consisting of:
 aneuploidy, 
 increased cellular proliferation rate relative to a corresponding reference cell, 
 periodically increased expression of VEGF, k-ras, myc, MMP-9, CD44 or Bcl-2 relative to a corresponding reference cell, 
 periodically reduced expression of CDKn2A and p53 relative to a corresponding reference cell, 
 formation of a colony in soft agar in vitro, and 
 formation of a tumor in vivo. 
 
     
     
         4 . The cell of any of  claims 1 - 3 , wherein said cell is aneuploid, has periodically increased expression of an oncogene selected from the group consisting of k-ras, myc and Bcl-2 relative to a reference cell, and has periodically reduced expression of CDKn2A, p53, and CD44 relative to a reference cell. 
     
     
         5 . The cell of any of  claims 1 - 4 , wherein the spontaneous fusion occurred between a neoplastic cell and a neoplastic cell, a non-neoplastic cell and a neoplastic cell, or a non-neoplastic cell and a non-neoplastic cell. 
     
     
         6 . The cell of any of  claims 1 - 5 , wherein the cell is selected using two distinct detectable labels. 
     
     
         7 . An isolated cell population comprising cells selected as the product of a spontaneous cell-cell fusion or descendants thereof, and cultured under conditions sufficient to promote further fusion, wherein at least one cell of said population is a self-renewing tumor-initiating cell. 
     
     
         8 . The isolated cell population of  claim 7 , wherein at least about 50-75% of said population are tumor-initiating cells or highly tumorogenic cells. 
     
     
         9 . The isolated cell population of  claim 7  or  8 , wherein said cell population exhibits a characteristic selected from the group consisting of:
 aneuploidy, 
 increased cellular proliferation rate relative to a corresponding reference cell, 
 periodically increased expression of a VEGF, k-ras, myc, MMP-9, CD44 or Bcl-2 relative to a corresponding reference cell, 
 periodically reduced expression of CDKn2A and p53 relative to a corresponding reference cell, 
 able to form a colony in soft agar in vitro, and 
 able to form a tumor in vivo. 
 
     
     
         10 . The isolated cell population of any of  claims 7 - 9 , wherein at least about 50% of said cells are aneuploid and show increased expression of an oncogene selected from the group consisting of k-ras, myc and Bcl-2, and show decreased expression of CDKn2A and p53 relative to a corresponding reference cell. 
     
     
         11 . The isolated cell population of any of  claims 7 - 10 , wherein the population has a tumor producing efficiency that is at least about 10-fold greater than a reference cell. 
     
     
         12 . The isolated cell population of any of  claims 7 - 11 , wherein injection of at least about 10-500 cells of said population forms a tumor of about 1 cm diameter in at least about 50% of nude mice within about 2-6 weeks. 
     
     
         13 . The isolated cell population of any of  claims 7 - 12 , wherein at least about 50% of the cells in the population exhibit synchronous differential expression of at least two markers selected from the group consisting of Myc, K-ras, Bcl-2, p53, CDKn2a, CD44, SPP1, and VEGF relative to a corresponding reference population. 
     
     
         14 . The isolated cell population of any of  claims 7 - 13 , wherein the spontaneous fusion occurred between a neoplastic cell and a neoplastic cell, a non-neoplastic cell and a neoplastic cell, or a non-neoplastic cell and a non-neoplastic cell. 
     
     
         15 . The isolated cell population of any of  claims 7 - 14 , wherein one of the non-neoplastic cells is a fibroblast or epithelial cell. 
     
     
         16 . The isolated cell population of any of  claims 7 - 15 , wherein the cell-cell fusion does not give rise to a macrophage, muscle cell, hybridoma cell, or virus infected cell. 
     
     
         17 . The isolated cell population of any of  claims 7 - 16 , wherein at least about 10-100% of the population is descended from the fusion of a neoplastic cell and a neoplastic cell, a non-neoplastic cell and a neoplastic cell, or a non-neoplastic cell and a non-neoplastic cell. 
     
     
         18 . The isolated cell population of any of  claims 7 - 17 , wherein the cell is derived from a subject identified as having or having a propensity to develop a neoplasia. 
     
     
         19 . The isolated cell population of  claim 18 , wherein the neoplasia is a solid tumor or blood tumor. 
     
     
         20 . The isolated cell population of any of  claims 7 - 19 , wherein conditions sufficient to promote further fusion include culturing the cells at a density of about 80% confluence. 
     
     
         21 . A method of generating a tumor-initiating cell, comprising selecting a cell that is the product of a spontaneous cell-cell fusion or a descendant thereof and culturing said cells, thereby generating a tumor-initiating cell population. 
     
     
         22 . The method of  claim 21 , further comprising culturing said cells under conditions sufficient to promote further fusion. 
     
     
         23 . The method of  claim 21  or  22 , wherein said cells comprise at least two nuclei. 
     
     
         24 . The method of any of  claims 21 - 23 , wherein the method further comprises selecting a cell comprising at least 3 or 4 nuclei, and allowing said cell to proliferate. 
     
     
         25 . The method of any of  claim 21 - 24 , wherein said fused cell is identified using detectable cellular markers. 
     
     
         26 . The method of any of  claims 21 - 25 , where said detectable markers are cytoplasmic vital dyes. 
     
     
         27 . The method of any of  claims 21 - 26 , wherein at least one of the cells that undergoes spontaneous cell-cell fusion is derived from a subject identified as having or having a propensity to develop a neoplasia. 
     
     
         28 . An isolated tumor-initiating cell made by the process of any of  claims 21 - 27 . 
     
     
         29 . The isolated cell of  claim 28 , wherein the cell or cell population is self-renewing, capable of forming a tumor when injected into a SCID mouse, a nude mouse, an immune competent mouse, or is resistant to exposure to radiation or chemotherapy. 
     
     
         30 . The isolated cell of  claim 28  or  29 , wherein said cell exhibits a characteristic selected from the group consisting of:
 aneuploidy, 
 genomic instability, 
 increased cellular proliferation rate relative to a corresponding reference cell, 
 periodically increased expression of a VEGF, k-ras, myc, MMP-9, CD44 or Bcl-2 relative to a corresponding reference cell, 
 periodically reduced expression of CDKn2A and p53 relative to a corresponding reference cell, 
 able to form a colony in soft agar in vitro, and 
 able to form a tumor in vivo. 
 
     
     
         31 . The isolated cell of any of  claims 28 - 30 , wherein said cell is aneuploid, has increased expression of k-ras, myc and Bcl-2, and has reduced expression of CDKn2A and p53 relative to a control cell. 
     
     
         32 . The isolated cell of any of  claims 28 - 31 , wherein the cell is tumorogenic. 
     
     
         33 . The isolated cell of any of  claims 28 - 32 , wherein said cell has a tumor producing efficiency that is at least 10-fold greater than a reference cell not the product of a cell-cell fusion event. 
     
     
         34 . A composition comprising a population of tumor-initiating cells made by the process of any of  claims 21 - 27 . 
     
     
         35 . The composition of  claim 34 , wherein injection of at least about 10-500 cells of said population forms a tumor of about 1 cm diameter in at least about 50% of nude mice within about 2-6 weeks. 
     
     
         36 . The composition of  claim 34  or  35 , wherein at least about 50% of the cells in the population exhibit periodic differential expression of at least two markers selected from the group consisting of Myc, K-ras, Bcl-2, p53, CDKn2a, CD44, SPP1, and VEGF relative to a corresponding reference population. 
     
     
         37 . A method for identifying an agent for the treatment or prevention of a neoplasia, hyperplasia or a hyperproliferative disorder comprising:
 contacting a tumor-initiating cell or cell population of any one of  claim 1 - 20  or  28 - 36  with an agent; and   detecting a decrease in cell survival or proliferation relative to an untreated control cell or cell population.   
     
     
         38 . A method for identifying an agent that inhibits cell-cell fusion comprising:
 contacting a population of cells cultured under conditions sufficient to induce spontaneous cell-cell fusion with an agent; and   detecting a reduction in cell-cell fusion relative to an untreated control population.   
     
     
         39 . The method of  claim 38 , wherein a reduction in multinucleate cell number in the treated population identifies an agent that inhibits cell-cell fusion. 
     
     
         40 . The method of  claim 37  or  38 , wherein the agent increases cell death. 
     
     
         41 . The method of  claim 37  or  38 , further comprising maintaining the cell in culture under conditions sufficient to generate mononucleate cells from a multinucleate cell, and identifying a reduction in the generation of said cells relative to a corresponding control culture. 
     
     
         42 . A method of selecting a therapeutic regimen for a subject, the method comprising
 contacting a tumor-initiating cell or cell population derived from a subject according to the method of any of  claims 21 - 27  with an agent;   detecting a decrease in cell survival, proliferation, or cell-cell fusion relative to an untreated control cell or cell population; and   identifying the agent as effective in a therapeutic regimen for said subject.   
     
     
         43 . The method of  claim 38  or  42 , wherein the cell is a fibroblast or epithelial cell. 
     
     
         44 . A method of identifying an agent that prevents neoplasia relapse following chemotherapy, the method comprising:
 contacting a tumor-initiating cell or cell population derived from a subject according to the method of any of  claims 21 - 27  with an agent;   detecting a decrease in cell survival, proliferation, or fusion relative to an untreated control cell or cell population, wherein said decrease identifies that agent as preventing neoplasia relapse following chemotherapy.   
     
     
         45 . An immunogenic composition for the treatment or prevention of a neoplasia in a subject, the composition comprising an effective amount of a non-viable cell population of any one of  claim 1 - 20  or  28 - 36  and an excipient. 
     
     
         46 . The immunogenic composition of  claim 45 , further comprising an adjuvant. 
     
     
         47 . The immunogenic composition of  claim 45  or  46 , wherein said cell population is derived from a subject identified as having or having a propensity to develop a neoplasia. 
     
     
         48 . A method for inducing an immune response against a tumor-initiating cell or neoplasia, the method comprising administering the immunogenic composition of  claim 45  to a subject. 
     
     
         49 . The method of  claim 48 , wherein the immunogenic composition comprises a cell derived from the subject to be treated. 
     
     
         50 . The method of  claim 48  or  49 , wherein the method treats or prevents a neoplasia. 
     
     
         51 . The method of any of  claims 48 - 50 , wherein the method treats or prevents the relapse of a neoplasia. 
     
     
         52 . The method of any of  claims 48 - 51 , wherein the method is administered prior to, concurrent with, or following chemotherapy or radiation therapy. 
     
     
         53 . A method of immortalizing a primary cell in culture comprising:
 selecting a cell as having undergone spontaneous cell-cell fusion in a primary cell culture; and   culturing the selected cell under conditions that promote further cell-cell fusions, thereby generating an immortalized cell.   
     
     
         54 . The method of  claim 53 , wherein the selected cell is bi or multinucleate. 
     
     
         55 . The method of  claim 53  or  54 , further comprising the step of maintaining the bi or multinucleate cells in culture for a time sufficient to generate a population comprising at least 50% mononucleate aneuploid cells. 
     
     
         56 . A non-human mammal comprising an injected cell or cell population of any of  claim 1 - 20  or  28 - 36 . 
     
     
         57 . The non-human mammal of  claim 56 , wherein said cell or cell population is sufficient to form a tumor in the mammal. 
     
     
         58 . A method for producing a differentiated cell from a tumor-initiating cell, the method comprising providing a cell according to any of  claim 1 - 20  or  28 - 36 , and contacting the cell with a differentiation agent, thereby producing a differentiated cell. 
     
     
         59 . A cellular composition comprising the differentiated cell of  claim 58 . 
     
     
         60 . A method for treating a subject having a disease or disorder characterized by a deficiency in cell number, the method comprising administering to the subject a cellular composition of  claim 59 .