Molecular Classification of Normal and Cancer Mammospheres.

Introduction: For decades, scientists have attempted to develop models for growing and studying primary tumors outside of the human body. These techniques have ranged from growth assays in immuno-compromised mice to simple monolayer cultures. Unfortunately, these techniques are either too complex and expensive (animal models), or too simple (monolayer cultures) to effectively recapitulate the growth characteristics of human tumors. The recent discovery of cancer stem cells (CSCs) has provided new insight into the reasons that ex-vivo cultures have been unable to provide an accurate model for in-vivo tumors.Hypothesis: Our hypothesis is that three-dimensional mammosphere culture represents a practical means of enriching cancer stem cells and assessing, ex-vivo, the growth characteristics of human breast cancer. We believe this technique will provide a more accurate model system for testing novel drug targets specific for cancer stem cells and tailor specific therapies for patients.Method: By combining ideas from a number of previously described methods, we have recently developed a protocol to grow and maintain breast cancer specimens in a simple three-dimensional culture system using serum-free media, MEGM supplemented with BT-20, insulin, EGF and bFGF. We have successfully cultured both normal and tumor cells from clinical samples as small as a fine needle aspiration. In each case, small subset of epithelial cells from each sample grow into mammospheres.Results: Our preliminary data in characterization of the mammospheres shows that different morphologies and histotypes can be achieved in culture. We are currently using protein expression and transcriptional profiling methods to characterize the cell types within mammospheres and the histotypes of cancer that can generate mammospheres. Using immunofluorescence of a few key protein markers we have found that mammospheres, derived both from normal breast tissue and from tumor tissue samples, express putative stem cells markers as determined by expression of CD44 in the absence of CD24. Only a subset (30-50%) of CD44 positive cells in mammospheres are also positive for ALDH1 in both normal and cancer derived cultures suggesting an undifferentiating population of cells, potentially indicative of the presence of stem cells. Cells in the mammospheres are also positive for both epithelial and myoepithelial markers (e.g. broad-range cytokeratin, CK8, CK5, CK17, vimentin, P63) suggesting variable differentiation potential of progenitor cells. Preliminary transcriptional profiling data suggests that gene sets useful in molecular subtyping of primary tumors may also be informative in mammospheres, and that segregation of profiles of stromal and tumor cells can be elucidated through studies of mammospheres vs. core biopsies.Conclusion: This model system has the potential to be an inexpensive, accurate and robust model for breast cancer. It could help in the understanding of mechanisms of drug resistance, recurrence and metastasis, as well as in predicting treatment success or failure as we move towards personalized medicine. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 501.