Abstract A04: DNA double-strand break repair assay in primary cultures of patient-derived ovarian carcinoma xenografts.
Authors: Guffanti Federica , Kaufmann Julia , Bizzaro Francesca , Ricci Francesca , Ganzinelli Monica , Lupi Monica , Giavazzi Raffaella , Wiesmüller Lisa , Damia Giovanna 
Published: May 2016

Abstract
Introduction: Epithelial ovarian cancer (EOC) represents the most fatal gynecological malignancy. The DNA repair capacity is a key determinant for the cellular response to DNA damaging agents and is inversely correlated with their cytotoxicity. It has been reported that half of high grade EOC display defects in DNA repair, accounting for the sensitivity of this tumor to the first line chemotherapy. The aim of the present work was to evaluate the functional DNA repair activity, focusing on DNA double strand-break (DSB) repair, in EOC cell lines with different sensitivity to cisplatin (DDP) and in EOC primary cultures obtained from patient-derived ovarian carcinoma xenografts (PDX), recently established in our laboratory.
Material and Method: Primary cultures derived from intraperitoneal ascites or from subcutaneous EOC xenograft solid masses were mechanically disaggregated and cultured in RPMI. The experiments were performed within the third in vitro passage. To functionally evaluate DSB repair pathways, cells were transfected with various reporter substrates for EGFP-based quantification of specific DSB repair activities: homologous recombination (HR), total non-homologous end-joining (tNHEJ), and micro-homology mediated NHEJ (mmEJ) pathway. Briefly, 10^6 cells were transfected with different plasmid mixtures, containing the meganuclease expression plasmid (pCMV-I-SceI) together with one of the recombination substrates, and wtEGFP plasmid in split samples for determination of transfection efficiencies. Cellular fluorescence was quantified by flow cytometry and recombination frequencies were calculated from the fraction of wtEGFP positive cells normalized for transfection efficiency.
Results: Among cell lines, OVCAR5 showed very low DSB repair frequencies in all the three sub-pathways investigated. Interestingly, OVCAR433 showed the highest DSB repair frequencies and also the lowest sensitivity to DDP. MmEJ repair frequencies were lower than HR and tNHEJ both in cell lines and in primary EOC cultures derived from PDX. In EOC primary cultures tNHEJ frequencies were 2 to 10 times higher than HR.
Conclusion: We were able to analyze distinct DSB repair activities in EOC primary cultures obtained from PDX. The application of such DNA repair assays to a panel of PDX xenografts with known sensitivity to DDP treatment will allow to assess the power of these tests to predict the response to chemotherapy.
Citation Format: Federica Guffanti, Julia Kaufmann, Francesca Bizzaro, Francesca Ricci, Monica Ganzinelli, Monica Lupi, Raffaella Giavazzi, Lisa Wiesmüller, Giovanna Damia. DNA double-strand break repair assay in primary cultures of patient-derived ovarian carcinoma xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A04.