EH1.2 Radiation responses of 2D and 3D glioblastoma cells: a novel 3D-specific radioprotective role of VEGF/Akt signaling through functional activation of NHEJ

摘要

Glioblastoma (GBM) is the most common primary brain tumour with dismal prognosis. Tumours exhibit inherent resistance to radiation and chemotherapy which has been attributed to a subpopulation of cancer cells termed ‘GBM stem-like cells’ (GSC) characterised by multipotentiality and potent tumorigenic capacity. The use of established cancer cell lines in simplified two-dimensional (2D) in vitro cultures might explain the observed discrepancy between pre-clinical and clinical responses to cytotoxic treatments. We developed a customised, 3D GSC culture system using a polystyrene scaffold (Alvetex®) that recapitulates key histological features of GBM including high cellularity and sparse extracellular matrix (ECM) and compared it to conventional 2D GSC cultures. 2D and 3D cultures of three different primary GSC lines exhibited similar radiation sensitivities as measured by clonogenic survival. Previous studies have demonstrated increases in progression-free survival of GBM patients following anti-VEGF therapy in combination with standard radio-chemotherapy. Bevacizumab enhanced the radiosensitivity of 3D GSC cultures in clonogenic and neurosphere formation assays, but had no effect on radiation responses of 2D GSC. Similar radiosensitising properties were observed by VEGF deprivation, with significant radiosensitisation of 3D GSC cultures but no effect on 2D GSC. Analysis of the DNA damage response after radiation treatment showed a correlation between radiosensitivity, increased double-strand breaks (γH2AX foci), increased non-homologous end-joining (NHEJ) repair protein phospho-DNA-PK nuclear foci and increased number of cells undergoing mitotic catastrophe or with micronuclei following radiation treatment. In contrast, increased numbers of foci of the homologous recombination (HR) repair protein Rad51 were observed in radioresistant populations after radiation. Our results show that in the 3D model, VEGF signalling is required for optimal NHEJ activation with fast kinetics. This effect allows access to HR repair proteins at the remaining unrepaired DSBs at later time points, facilitating their repair and conferring radiation protection. Detailed analysis of the signalling pathways involved in the radiation resistance conferred by VEGF signalling in the 3D model demonstrated a radioprotective role of the downstream signaling molecule Akt. Specific inhibition of Akt using the small molecule inhibitor MK-2206 increased radiation sensitivity to the same extent as VEGF deprivation in 3D cells, and no additivity was observed when these agents were combined. To our knowledge this is the first report demonstrating a role for VEGF in the regulation of the DNA damage response. Our results for anti-VEGF therapy in the 3D model recapitulate data from clinical trials and strongly support its clinical relevance and its potential value in preclinical GBM studies.