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Cellosaurus publication CLPUB00667

Publication number CLPUB00667
Authors Lisio M.-A.
Title Evaluation of platinum-sensitivity in a cell line model of high grade serous ovarian cancer and the induction of resistance from a chemo- sensitive cell line through the repopulation of cells following short-term cisplatin treatment.
Citation Thesis PhD (2019), McGill University Montreal, Canada
Web pages https://escholarship.mcgill.ca/concern/theses/d791sj55t
Abstract Introduction: Resistance to platinum-based therapy develops in most patients treated for High Grade Serous Ovarian Cancer (HGSOC), a fact that largely accounts for this disease's elevated mortality. It was previously shown by Cooke and colleagues (Oncogene, 2010) that platinum (Pt) resistance results from the expansion of sub-clonal populations of resistant cells present prior to treatment. Current in vitro models of Pt- resistance may be confounded by a lack of fidelity to the genetic signature of HGSOC, or by the methods used to elicit the onset of resistance which may lack clinical relevance. This project aims to address whether Pt-resistance can be evolved from a population of Pt-sensitive cells exposed to cisplatin (CDDP) in a clinically relevant manner using patient-derived cell lines such as PEO1. The central aim was to uncover what heterogeneity might exist within this cell line and to determine if in vitro derived Pt-resistance can result from the selection of pre- existent subsets of cells as occurs in vivo. Methods: The Pt-sensitivity of the cell lines used, was established by exposing cells in culture to CDDP for 1 hr. Live cell number and percent viability were assessed 72 hours after drug removal using Guava microcytometry. Long-term assessment of Pt toxicity was provided by use of the clonogenic survival assay. To achieve Pt-resistance in vitro, a culture of PEO1 was exposed to 10 muM CDDP for 1 hour and allowed to repopulate. These cells were passaged and used to establish a novel cell line (PEO1X) with 20-fold diminished Pt-sensitivity confirmed via the clonogenic survival assay. PEO1X cells were assayed for histopathological and cell-fate markers by immunohistochemistry and compared to PEO1. Migratory capacity was assessed via the Boyden chamber method, while cell cycle status 72 hours after CDDP exposure was interrogated using propidium iodide staining. Doubling time was also determined, and a partial genetic signature was established by sequencing a panel of 33 cancer-related genes. Results: PEO1 exhibits morphological asymmetry, with a dichotomy between epithelial and mesenchymal phenotypes. This is further evidenced by the heterogeneous expression of markers such as E-cadherin, vimentin and CA125. Although similar to PEO1 in proliferation rate, PEO1X cells possess obvious differences in morphology, being smaller and more homogenously rounded in appearance with altered expression of markers such as E-cadherin, vimentin, CA125 and CD133. Unlike PEO1, the cell cycle status of PEO1X is barely altered 72 hrs following CDDP exposure even at 10 muM. PEO1X also possesses a more migratory phenotype than PEO1 with significantly more cells transiting through the pores of the Boyden chamber membrane in 30 hrs. Sequencing revealed heterogeneity in the status of P53 in PEO1 with PEO1X being slightly enriched in P53 WT expressing cells. Interestingly, the gene NF1, encoding the tumor- suppressor neurofibromin was found to contain an indel mutation in PEO1 and PEO1X while containing a different loss-of-function point mutation in PEO4 and being WT in PEO6 despite all being established from the same patient. Conclusions: The cell line PEO1 contains a sub-population of Pt- resistant cells that can be selected for, in vitro, by clinically relevant CDDP treatment. These cells are distinct from those having emerged clinically in the original patient.
Cell lines CVCL_2686; PEO1
CVCL_2690; PEO4