| 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.
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