Glioblastoma (GB) is the most common and malignant primary adult brain cancer with a median survival of 15 months despite treatment with surgical resection followed by chemo-radiotherapy. The clonal diversity and evolutionary dynamics inherent to GBs is considered a major obstacle to effective treatment response. While studies have focused on temozolomide, a role for radiotherapy as an independent driver of GB evolution has not been investigated. We addressed the impact of radiation on glioblastoma evolution and potential treatment implications by examining the influence of intratumoral heterogeneity (ITH) on intrinsic radiosensitivity, by determining the effects of radiation on glioma stem-like cell (GSC) initiated orthotopic xenografts, and by assessing radioresistance with a reirradiation protocol.

To determine the impact of ITH on intrinsic radiosensitivity, we performed whole-exome sequencing (WES) of multiple tumour fragments and corresponding patient-derived cell lines that underwent γH2AX foci analysis and limiting dilution assay analysis. Cell lines from the same tumour seem to display similar levels of intrinsic radiosensitivity despite genomic differences, suggesting that radiotherapy regimens may be effective for the whole of the tumour. To test the ability of radiation to drive GB evolution, we utilised GSC-initiated orthotopic xenograft models treated with or without fractionated radiation (3x5Gy) to examine differences in survival, morphology/histology, Viral integration site analysis (VISA), and WES. Irradiated mice experienced a survival advantage and harboured less invasive tumours compared to control mice. VISA revealed that control tumours harbour fewer clones than in vitro lines and that irradiated tumours harbour the fewest clones of all suggesting that radiation, particularly in the context of the brain microenvironment, drives GBM evolution. WES results demonstrated that variants from irradiated tumours mapped to different COSMIC mutational signatures and displayed a considerable amount of subpopulation shifting compared to control tumours, consistent with radiation-induced evolution and subpopulation selection. By adding a reirradiation protocol to this GSC-initiated orthotopic xenograft model, we sought to better understand the functional impact of radiotherapy on recurrent GB evolution and to establish an in vivo model for studying reirradiation. After initial treatment, mice were rerandomised into control (3x5Gy-Control) and radiation therapy groups (3x5Gy-3x5Gy) and retreated once the average BLI ratio began to increase. A further survival advantage was found for mice undergoing reirradiation compared to mice receiving only one course of radiation. This survival advantage was supported by clonogenic survival and reimplantation studies of cell lines derived from control and irradiated NSC11 tumours that did not demonstrate a difference in survival after radiation regardless of the previous tumour’s treatment regimen. Whereas radiation-induced evolution may not influence radioresponse, it may lead to the identification of novel targets for sensitisation which may ultimately yield more effective treatment strategies.

Our results demonstrate that radiation, a treatment component for almost all glioblastoma patients, can have wide-ranging effects on the evolution of this dynamic tumour. In particular, the pressures imposed by radiation treatment seem to lead to the selection of a reduced number of clones. This selection may have future implications for tumour evolution and the treatment of recurrent GB. In addition, we have demonstrated for the first time the utility of a GSC-initiated orthotopic xenograft model for studying retreatment protocols and recurrent GB biology. This reirradiation model may provide the opportunity to design and test more effective recurrent GB treatment strategies centered around recurrent biology.