Abstract 3347: Enrichment of a CD44+/CD24- putative cancer stem cell population drives tumor progression and growth factor independence in a mouse model of postmenopausal breast cancer

Abstract

Abstract MMTV-Wnt-1 transgenic mice develop heterogeneous mammary tumors that accurately recapitulate human basal-like breast cancer. Additionally, targeted alterations in the Wnt signaling pathway in mammary epithelial cells preferentially induce mammary cancers from progenitor cells. Therefore, we hypothesized that MMTV-Wnt-1 mammary tumors harbor cells with cancer stem cell characteristics. To test this hypothesis, we characterized (in vitro and in vivo) two newly generated, clonal mammary epithelial cell lines (WA4 and WG4) from MMTV-Wnt-1 transgenic mouse tumors. The cell lines were analyzed using flow cytometry for CD44 and CD24 cell surface markers (a CD44+/CD24- profile is indicative of putative human and murine mammary cancer stem cells), mammosphere formation (low density seeding on non-adherent plates in stem-cell media), in vitro scratch assay (to measure wound healing), and by an in vitro Matrigel invasion chamber assay (to quantitate cell invasion). Finally, unsorted WA4 and WG4 cell lines were transplanted into syngeneic mammary fat pads of female ovariectomized (to model the postmenopausal state) C57BL/6 mice (1×10^7 cells/mouse; n=5/cell line), and tumor burden was assessed after 3 weeks. Tumors were analyzed by qRT-PCR and immunohistochemistry (IHC) for pathological and genetic markers of progression. Flow cytometric analysis demonstrated that the percentage of CD44+/CD24- cells was 62.2% in the WA4 line and 2.4% in the WG4 line. Serum starvation enriched the WA4 line for CD44+/CD24- cells (from 62.2% to 82.0%), without altering the composition of the WG4 line. Approximately 30% of unsorted WA4 cells, but <1% of unsorted WG4 cells, formed mammospheres. Additionally, the WA4 line showed increases in wound healing by scratch assay and significant increases in invasion (226-fold increase versus WG4 cells after 30 hours). In vivo, the WA4 cell line was associated with larger, more aggressive tumors on average (+/− SD) than the WG4 line (2.09 +/− 0.68 g and 0.03 +/− 0.02 g, respectively, p<0.0001). WA4 tumors were characterized by increased cellular proliferation, microvascular proliferation, and a basal appearance lacking ductal structures. WA4 tumors also demonstrated a loss of ERα/β expression (qRT-PCR), lacked IGF1-R activation (by IHC), and showed an increase in Wnt-1 progenitor signaling (qRT-PCR). In conclusion, MMTV-Wnt-1 transgenic mouse mammary tumors harbor a CD44+/CD24- putative cancer stem cell population, and an enrichment of this population corresponds with a more aggressive tumor phenotype in a mouse model of postmenopausal breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3347.