Isolation and use of solid tumor stem cells

Abstract

A small percentage of cells within an established solid tumor have the properties of stem cells. These solid tumor stem cells give rise to both more tumor stem cells and to the majority of cells in the tumor that have lost the capacity for extensive proliferation and the ability to give rise to new tumors. Thus, solid tumor heterogeneity reflects the presence of tumor cell progeny arising from a solid tumor stem cell. We have developed a xenograft model in which we have been able to establish tumors from primary tumors via injection of tumors in the mammary gland of severely immunodeficient mice. These xenograft assay have allowed us to do biological and molecular assays to characterize clonogenic solid tumor stem cells. We have also developed evidence that strongly implicates the Notch pathway, especially Notch 4, as playing a central pathway in carcinogenesis.

1 - 44 . (canceled) 
     
     
         45 . An enriched population of human solid tumor stem cells obtained from a solid tumor of epithelial origin, wherein the solid tumor stem cells:
 (a) express CD44;   (b) are enriched at least two-fold compared to unfractionated tumor cells; and   (c) are tumorigenic;   
       wherein the solid tumor is a primary tumor or a xenograft tumor established from a primary tumor. 
     
     
         46 . The enriched population of  claim 45 , wherein the population is enriched at least 5-fold. 
     
     
         47 . The enriched population of  claim 45 , wherein the population is enriched at least 10-fold. 
     
     
         48 . The enriched population of  claim 45 , wherein the solid tumor stem cells further express epithelial specific antigen (ESA). 
     
     
         49 . The enriched population of  claim 45 , wherein the solid tumor stem cells are from a solid tumor selected from the group consisting of colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, cervical cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, and melanoma. 
     
     
         50 . A cellular composition comprising the enriched population of human solid tumor stem cells of  claim 45 . 
     
     
         51 . A method of enriching for a population of solid tumor stem cells, the method comprising:
 (a) obtaining an initial mixture of solid tumor stem cells and solid tumor cells from a human solid tumor of epithelial origin, wherein the solid tumor is a primary tumor or a xenograft tumor established from a primary tumor; and   (b) separating a fraction of solid tumor stem cells from the mixture of solid tumor stem cells and solid tumor cells, wherein the solid tumor stem cells:
 (i) express CD44; 
 (ii) are enriched at least two-fold compared to the initial unfractionated mixture of solid tumor stem cells and solid tumor cells; and 
 (iii) are tumorigenic. 
   
     
     
         52 . A method of enriching for a population of CD44+solid tumor stem cells, the method comprising:
 (a) dissociating a human solid tumor into an initial mixture of solid tumor stem cells and solid tumor cells, wherein the solid tumor is a primary tumor or a xenograft tumor established from a primary tumor;   (b) contacting the dissociated cells with a first reagent that binds CD44; and   (c) selecting cells that bind to the first reagent, wherein the selected cells are:
 (i) CD44+; 
 (ii) enriched at least two-fold compared to the initial unfractionated mixture of tumor cells; and 
 (iii) tumorigenic. 
   
     
     
         53 . The method of  claim 52 , wherein the first reagent is an antibody. 
     
     
         54 . The method of  claim 52 , wherein the first reagent is conjugated to a fluorochrome or magnetic particles. 
     
     
         55 . The method of  claim 52 , wherein the selecting is performed by flow cytometry, fluorescence activated cell sorting (FACS) sorting, panning, affinity chromatography, or magnetic selection. 
     
     
         56 . The method of  claim 52 , wherein the dissociated cells are contacted with at least 1 additional reagent, at least 2 additional reagents, at least 3 additional reagents, or at least 5 additional reagents. 
     
     
         57 . The method of  claim 52 , wherein the additional reagent(s) binds human ESA, CD3, CD2, CD10, CD14, CD16, CD31, CD45, CD140b, CD64, CD24, and/or a mouse cell marker. 
     
     
         58 . The method of  claim 52 , wherein the solid tumor is selected from the group consisting of colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, cervical cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, and melanoma. 
     
     
         59 . A method of obtaining a cellular composition comprising an enriched population of human CD44+CD24−/low solid tumor stem cells, the method comprising:
 (a) obtaining an initial mixture of solid tumor stem cells and solid tumor cells from a human breast, ovarian, or prostate solid tumor, wherein the solid tumor is a primary tumor or a xenograft tumor established from a primary tumor; 
 (b) separating a fraction of solid tumor stem cells from the mixture of solid tumor stem cells and solid tumor cells, wherein the solid tumor stem cells are:
 (i) CD44+; 
 (ii) CD24−/low; 
 (iii) enriched at least two-fold compared to the initial unfractionated mixture of solid tumor stem cells and solid tumor cells; and 
 (iv) tumorigenic. 
 
 
     
     
         60 . The method of  claim 59 , wherein the separating is performed by flow cytometry, fluorescence activated cell sorting (FACS), panning, affinity chromatography, or magnetic selection. 
     
     
         61 . The method of  claim 59 , further comprising: (c) isolating the separated solid tumor stem cells. 
     
     
         62 . The method of  claim 59 , wherein the solid tumor stem cells are enriched at least five to six-fold compared to the initial unfractionated mixture.