PO-478 Chemosensitization of cisplatin-resistant testicular germ cell tumours cells

Abstract

IntroductionMajority of testicular germ cell tumours (TGCTs) respond well to cisplatin (CDDP)-based chemotherapy. However, patients with refractory disease have limited treatment modalities associated with poor prognosis. CDDP resistance is a complex phenomenon, and there is need for modalities to overcome the chemoresistance in TGCTs.Material and methodsCDDP-resistant variants of human TGCTs cell lines JEG-3, NTERA-2, NOY-1 were derived by cultivation in the gradually increasing drug concentrations for 6 months. Chemoresistance to CDPP, oxaliplatin and carboplatin was determined by luminescent viability assay. Cell migration was quantitated in wound healing assay, morphology visualised by α-F-actin immunofluorescent staining. Differences in gene expression and protein levels were evaluated by quantitative RT-PCR, proteome profilers for stem cell markers and apoptosis-related proteins. Tumorigenicity was determined on immunodeficient mouse model. Upregulated biomarkers were targeted by pharmacological inhibition to determine the effect on CDDP resistance in 3D tumorosphere assay and in vivo.Results and discussionsCDDP-resistant TGCT cell lines exhibited significantly higher IC50 values for cisplatin associated with major morphology alterations, increased migratory capacity and tumorigenicity. Resistance was retained also in the prolonged absence of CDDP and cells were cross-resistant to other platinum-based compounds. Multiple alterations in pro- and anti-apoptotic proteins, which were cell line-specific, were observed. More importantly, increased or high expression of genes associated with stemness such as aldehyde dehydrogenase (ALDH), prominin-1 (CD133) and ATP binding cassette subfamily G member 2 (ABCG2) was detected in all chemoresistant cell line variants. CDDP-resistance could be reverted by ALDH inhibitor disulfiram, hypomethylating agent decitabine and inhibitor of poly (ADP-ribose) polymerase veliparib, when used in combination with CDDP in 3D culture conditions and in vivo.ConclusionWe derived chemoresistant variants of TGCT cell lines as novel clinically relevant model suitable for the evaluation of therapeutic strategies in vitro or in vivo. More importantly, our results suggest novel treatment option for refractory TGCTs with acquired CDDP-resistance.This work was supported by Slovak Research and Development Agency (APVV-15–0086, APVV-15–0697, APVV-16–0178) and VEGA 2/0124/17. We thank Cancer Research Foundation and League against Cancer for financial support.