Isolation and Interrogation of Ovarian Cancer Stem Cells
Brendan Ffrench, Michael Gallagher, Aoife Cooke, Britta Stordal, Sharon O'Toole, Cara Martin, Orla Sheils, John O'Leary. Trinity College Dublin, Dublin, Ireland
Background: Mice tumourgenicity studies identify cancer stem cells (CSCs) as the founding cells of tumours. CSCs have also been linked to chemoresistance, metastasis and disease relapse. Therapeutically targeting CSCs could remove the tumours malignant potential and circumvent chemoresistance, relapse and metastasis. Disease progression of Ovarian Cancer correlates with the predictions of the CSC hypothesis. For this reason ovarian malignancy was chosen as a system in which to study CSCs, with the intention of understanding the variation of CSC markers reported in the literature and to further understanding of the various aspects of ovarian malignancy.
Design: Six models of various stages of ovarian malignancy and one model of non-malignant ovarian surface epithelium were screened for the presence of CSCs and somatic stem cells respectively. Three flow cytometry based CSC screens were implemented; ALDEFLUORTM, Hoechst Side-Population and Cell Surface Protein Assays (CD44, CD117, CD133, CXCR4). Cells of interest were isolated via Fluorescence-Assisted Cell Sorting. Putative CSCs (pCSCs) were validated in NOD.SCID Mouse Tumourgenicity Assays and Single Cell Asymmetric Division Assays (AsyD Assay).
Results: Each screening technique identified pCSCs in one or more model systems. There was poor overlap of the cells marked by each of the markers. A pair of cisplatin sensitive and cisplatin adapted cell lines have been sorted into their pCSC and non-pCSC sub-populations, based on ALDEFLUORTM (ALDH) positivity. It was found that both ALDH+ and ALDH- cells were able to efficiently form tumours in NOD.SCID mice. All ALDH+ clones in the AsyD Assay made both ALDH+ and ALDH- cells. Some of the ALDH- (NegA) clones made both ALDH+ and ALDH-. Other ALDH- (NegB) clones made only ALDH- cells. ALDH+ and ALDH_NegA are able to asymmetrically divide, ALDH_NegB cells are not.
Conclusions: The various techniques for isolation of CSCs do not mark the same cells within an ovarian cancer context. This may reflect different stages/histologies of ovarian disease. There may be additional novel markers for Ovarian CSCs, surplus to the markers described above. Mouse Tumourgenicity Assays combined with AsyD Assays and microarray analysis has the potential identify such novel CSC markers in any malignancy. Mice experiments are currently under way to validate the ALDH_NegB clones as non-tumourigenic non-CSCs. Microarray analysis is currently under way to identify the differences between ALDH+ ALDH_NegA and ALDH_NegB cell types.
Category: Gynecologic & Obstetrics
Wednesday, March 6, 2013 1:00 PM
Poster Session VI # 193, Wednesday Afternoon