Abstract LB-105: Identifying molecular markers associated with chemo-resistance in ovarian cancer stem cells

Abstract

Abstract Stem-like cancer cells, or cancer stem cells (CSC) have been proposed to sustain the growth of tumour cells and give rise to drug resistance in cancer. Side populations (SP) of cells, characterised by their differential ability to efflux Hoechst 33342 from their cytoplasm via multi-drug transporters, are also thought to be enriched in CSCs. The aim of this study was to identify SPs of cancer cells in epithelial ovarian cancer (EOC), and genes within SP populations that both characterize SP cell in EOC cell lines and play a role in the development of resistance to chemotherapeutic agents. Using flow cytometry, we isolated SP cells from a panel of EOC cell lines. Detectable levels of viable SP cells were observed in the cell lines IGROV1 (1.8–5%), OVCAR3 (0.8%), OVCAR8 (0.2%), and the matched pairs of cisplatin-sensitive cell lines and their resistant variants OV2008 (2.5%)/C13*(1.5%) and A2780 (0.2%)/A2780CP (0.2%). In vitro characterization of SP cells from IGROV1, OVCAR3 and OVCAR8, compared to the non-SP cells showed that SP cells have higher colony forming efficiency when cultured as three-dimensional spheroids in non-adherent culture conditions. A proportion of single SP cells also had the ability to form multi-cellular spheroids in culture (a stem-cell ‘like’ feature) while non-SP cells did not. Under adherent culture conditions, SP cells show a range of morphologies, consisting of holoclones (which are enriched in SP cells) and paraclones (depleted of SP cells). Sub-cloning of holoclones generated both holoclones and paraclones; culturing paraclones led to the formation of only a few paraclones, which senesced. Differential whole-genome gene expression microarray profiling of SP vs non-SP cells identified the P-glycoprotein ABCB1 as the most highly upregulated gene in SP cells, consistent with other studies. Profiling of A2780 and its chemo-resistant derivative A2780CP identified Lin28, an embryonic stem cell marker, as the most differentially over-expressed gene in A2780CP SP cells. SiRNAs to ABCB1 were used to test the hypothesis that knockdown of this marker reduce proportionally the stem cell population in the IGROV1 EOC cell line; siRNAs to Lin28 were used to test whether knockdown of this gene re-sensitized A2780CP cells to cisplatin. Ongoing studies are evaluating the effects on chemosensitivity in vivo, by knocking down Lin28 in A2780CP cells expressing a luciferase reporter. Using Xenogen live animal imaging technologies, tumor burden is being evaluated in real-time, comparing Lin28 knockdown xenografted cell lines with non-silencing siRNA controls. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-105. doi:10.1158/1538-7445.AM2011-LB-105