Radiation responses of 2D and 3D glioblastoma cells: a novel, 3D-specific radioprotective role for VEGF activation through NHEJ

Natividad Gomez-Roman1,Katrina Stevenson1,Lesley Gilmour1,Anthony Chalmers1

1University of Glasgow, Glasgow, UK

Presenting date: Monday 2 November
Presenting time: 16.50-17.05

Background

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.

Method

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.

Results

2D and 3D cultures of three different primary GSC lines exhibited similar radiation sensitivities as measured by clonogenic survival. Previous studies have demonstrated radiopotentiating efficacy of the EGF receptor (EGFR) inhibitor erlotinib against GBM cell lines in 2D cultures; however it failed in GBM clinical trials. Thus we evaluated the radiation modifying effects of erlotinib on 2D and 3D GSC cultures. Erlotinib enhanced radiosensitivity of 2D GSC cultures but had no effect on radiation responses of 3D GSC or in neurosphere formation assays, where cells grow in 3D conditions devoid of a scaffold or extrinsic ECM. We next examined VEGF inhibition, since anti-VEGF therapy in combination with standard radio-chemotherapy increases progression-free survival of GBM patients. VEGF deprivation was associated with significant radiosensitisation of 3D GSC cultures but had no effect on 2D GSC. Erlotinib treatment of VEGF-deprived 3D cultures increased the radiation resistance of 3D cells to the same extent as VEGF addition, indicating epistasis. EGFR has been shown to regulate repair of radiation-induced double-strand breaks by activating the non-homologous end-joining (NHEJ) repair protein DNA-PKcs. A correlation between radiosensitivity, increased gammaH2AX foci and phospho-DNA-PK nuclear foci after radiation treatment was observed, In contrast, increased numbers of foci of the homologous recombination (HR) repair protein Rad51 were observed in radioresistant populations.

Conclusion

Our results show that EGFR inhibition and/or VEGF signalling induce a switch from ineffective NHEJ to more accurate HR repair leading to radiation protection. These 3D effects recapitulate data from clinical trials, strongly supporting the clinical relevance of this 3D model and its potential value in preclinical studies.