Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness

Paul P. Szotek^*, Rafael Pieretti-Vanmarcke^*, Peter T. Masiakos^*, Daniela M. Dinulescu, Denise Connolly, Rosemary Foster, David Dombkowski^¶, Frederic Preffer^¶, David T. MacLaughlin^*, and Patricia K. Donahoe^*,||

*Pediatric Surgical Research Laboratories, Department of Surgery, and ^¶Flow Cytometry Laboratory, Department of Pathology and Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, 185 Cambridge Street, Boston, MA 02114; Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111; Department of Pathology, Eugene Braunwald Research Center, Brigham and Women’s Hospital, Harvard Medical School, 221 Longwood Avenue, Room 401a, Boston, MA 02115; and Department of Medicine, Division of Hematology/Oncology, Massachusetts General Hospital, Harvard Medical School, 70 Blossom Street, Boston, MA 02114

The recent identification of “side population” (SP) cells in a number of unrelated human cancers and their normal tissue sources has renewed interest in the hypothesis that cancers may arise from somatic stem/progenitor cells. The high incidence of recurrence attributable to multidrug resistance and the multiple histologic phenotypes indicative of multipotency suggests a stem cell-like etiology of ovarian cancer. Here we identify and characterize SP cells from two distinct genetically engineered mouse ovarian cancer cell lines. Differential efflux of the DNA-binding dye Hoechst 33342 from these cell lines defined a human breast cancer-resistance protein 1-expressing, verapamil-sensitive SP of candidate cancer stem cells. In vivo, mouse SP cells formed measurable tumors sooner than non-SP (NSP) cells when equal numbers were injected into the dorsal fat pad of nude mice. The presence of Mullerian Inhibiting Substance (MIS) signaling pathway transduction molecules in both SP and NSP mouse cells led us to investigate the efficacy of MIS against these populations in comparison with traditional chemotherapies. MIS inhibited the proliferation of both SP and NSP cells, whereas the lipophilic chemotherapeutic agent doxorubicin more significantly inhibited the NSP cells. Finally, we identified breast cancer-resistance protein 1-expressing verapamil-sensitive SPs in three of four human ovarian cancer cell lines and four of six patient primary ascites cells. In the future, individualized therapy must incorporate analysis of the stem cell-like subpopulation of ovarian cancer cells when designing therapeutic strategies for ovarian cancer patients.

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