A systematic approach to the study of cancer stem cells applied to ovarian cancer

Background: Tumorgenicity studies show that Cancer Stem Cells (CSCs) but not their differentiated counterparts efficiently generate tumors. Therapeutically targeting CSCs could remove the malignant potential from tumours, while circumventing chemoresistance, relapse and metastasis. CSCs are difficult to study as they usually represent a small fraction of tumors. We will present a systematic approach we developed to investigate CSCs within any malignancy. We will present the data obtained thus far in the application of this system to the study of Ovarian CSCs. Methods: We developed a four phase model for investigating CSCs: Screening - Validation - Characterisation - Identification of novel therapeutic targets. Six cell lines modelling various stages of ovarian cancer (A2780, A2780cis, IGROV-1, IGROV(CDDP), SK-OV-3 and 59M) and one cell line (HIO-80) modelling non malignant ovarian surface epithelium were screened for the presence of CSCs and somatic stem cells respectively. Three flow cytometry based screens were implemented; ALDEFLUOR, Hoechst Side Population and Cell Surface Protein Screens. Cells of interest were isolated via Fluorescence Activated Cell Sorting. Mouse Tumorgenicity and Asymmetric Division Assays have been developed to validate putative CSCs (pCSCs) as true CSCs. Hypoxic growth, chemotherapeutic and matrigel invasion assays have all been developed to investigate the role of Ovarian CSCs in these commonly observed phenotypes of Ovarian cancer. Results: Six pCSC populations have been identified. No overlap has been observed between the three screening approaches. ALDEFLUOR identified pCSCs within A2780 and A2780cis. Hoechst Side Population identified pCSCs within IGROV-1 and IGROV(CDDP). CD44/CD117 identified pCSCs within SK-OV-3 and 59M. No somatic stem cells were identified within HIO-80. To date pCSC and non-pCSCs have been sorted from two cell lines. In both models the isolated pCSCs regenerated the non-pCSC phenotype. Both the isolated pCSCs and non-pCScs from each model are resistance to cisplatin and paclitaxel. In both cases the parent lines were chemoresistant, so this finding is not unexpected. All pCSCs are currently being validated via Mouse Tumourgenicity and Single Cell Asymmetric Division Assays. All validated CSCs will be described. Conclusion: A systematic approach has been established for the isolation and study of CSCs. Each ‘type’ of ovarian CSC appears to be mutually exclusive. This may reflect different stages/histologies of ovarian disease, or perhaps the selective conditions of tissue culture. pCSCs have been identified within chemosensitive cell lines. Investigation of the differences between chemoresistant and chemosensitive CSCs could elucidate the mechanisms behind chemoresistant relapse.