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Breast cancer grows, metastasizes and relapses from rare, therapy resistant cells with a stem cell phenotype (cancer stem cells/CSCs). However, there is a lack of studies comparing the functions of CSCs isolated using different phenotypes in order to determine if CSCs are homogeneous or heterogeneous.    Methods  Cells with various stem cell phenotypes were isolated by sorting from Clone 66 murine breast cancer cells that grow orthotopically in immune intact syngeneic mice. These populations were compared by  in vitro  functional assays for proliferation, growth, sphere and colony formation; and  in vivo  limiting dilution analysis of tumorigenesis.    Results  The proportion of cells expressing CD44 high CD24 low/neg , side population (SP) cells, ALDH1 + , CD49f high , CD133 high , and CD34 high  differed, suggesting heterogeneity. Differences in frequency and size of tumor spheres from these populations were observed. Higher rates of proliferation of non-SP, ALDH1 + , CD34 low , and CD49f high  suggested properties of transit amplifying cells. Colony formation was higher from ALDH1 −  and non-SP cells than ALDH1 +  and SP cells suggesting a progenitor phenotype. The frequency of clonal colonies that grew in agar varied and was differentially altered by the presence of Matrigel™.  In vivo,  fewer cells with a stem cell phenotype were needed for tumor formation than “non-stem” cells. Fewer SP cells were needed to form tumors than ALDH1 +  cells suggesting further heterogeneities of cells with stem phenotypes. Different levels of cytokines/chemokines were produced by Clone 66 with RANTES being the highest. Whether the heterogeneity reflects soluble factor production remains to be determined.    Conclusions  These data demonstrate that Clone 66 murine breast cancer cells that express stem cell phenotypes are heterogeneous and exhibit different functional properties, and this may also be the case for human breast cancer stem cells.

Heterogeneity of Functional Properties of Clone 66 Murine Breast Cancer Cells Expressing Various Stem Cell Phenotypes