918: Is single amino acid arginine deprivation a strategy to treat non-auxotrophic glioblastoma?

Abstract

Introduction: Arginine deprivation is a promising strategy in metabolic anticancer therapy. It was shown to inhibit tumor growth of arginine-auxotrophic cancers in vitro and in vivo. We previously reported that acute arginine withdrawal sensitizes cancer cells in a multicellular spheroid (MCTS) assay to radiotherapy even in the absence of complete arginine auxotrophy. Because of its systemic applicability enzymatic arginine deprivation could thus be of interest for the treatment of therapeutically challenging glioblastomas. In the present study, we evaluated the impact of arginine withdrawal on 3-D growth and invasive capacity of glioblastoma cells with wildtype and mutated/knockdown p53 status. Material and Methods: U87-MG, U87-MG-shp53 (p53-knockdown) and U251- MG (p53-mutant) transduced with eGFP were applied. Spheroids were cultured and analysed in liquid overlay using a standardized semi-automated set-up. Arginine deprivation was realized by transfer of MCTS into arginine- free medium containing dialysed serum. Upon treatment, spheroid volume was monitored by phase contrast imaging, viable cell numbers were determined, and invasion distance of spheroid cells was evaluated in a 3-D collagen-I gel by fluorescence microscopy. Results and Discussion: All three glioblastoma cell lines form MCTS but only two of them (U87-MG-wt, U87-MG-shp53) show spheroid volume growth. In contrast, all spheroid types clearly invade into collagen-I gel. Interestingly, the p53-knockdown U87-MG-shp53 cells exhibit a slightly enhanced growth in both monolayer and spheroid culture but appeared to be less invasive in the collagen-I gel as compared to their U87-MG-wt counterpart. Acute arginine deprivation results in a complete loss of cell growth in monolayer and MCTS culture independent of the p53-status. In parallel, acute arginine starvation affects the invasion capacity of U87-MG-wt but neither of the p53- knockdown descendant nor of the p53-mutated U251-MG spheroids. Both invasion capacity and MCTS volume growth are restored after completing an arginine-free diet even after long-term exposure. Conclusions: Arginine deprivation effectively inhibits 3-D volume growth independent of the p53 status and appears to reduce the invasion of at least p53-wt glioblastoma cells. The strategy has a clear potential and shall thus be further tested in other models and in combination with standard treatments for glioblastoma patients in particular radiotherapy. This project is funded by the European Social Fund (ESF) and the Free State of Saxony.