Proton minibeam radiation therapy induces distinct metabolic and synaptic remodelling in rats with orthotopic glioblastoma.

Abstract

Glioblastoma (GB) is an aggressive primary brain tumour with limited therapeutic options. Although radiotherapy (RT) remains a cornerstone of treatment, its efficacy is constrained by collateral damage to healthy brain tissue. Proton minibeam radiotherapy (pMBRT), a spatially fractionated modality, has shown promise in preclinical models and early clinical applications by mitigating normal tissue toxicity while enhancing tumour control. Notably, previous studies in rats have reported long-term neuroprotective effects following brain irradiation, as well as the emergence of long-term survivors accompanied by immune memory in GB-bearing models. To investigate the underlying radiobiological mechanisms of pMBRT, we performed an untargeted proteomics profiling comparing pMBRT with conventional proton therapy (CPT) in an orthotopic rat model of GB. Male Fischer rats were orthotopically injected with 50,000 RG2 cells into the right caudate nucleus. After validation of tumour uptake at day 13 by bioluminescence, rats received pMBRT or CPT treatment at day 14. Seven days after irradiation, brains were harvested to perform proteomics analysis and immunofluorescence staining. Both modalities attenuated proliferative signalling in tumours after seven days; however, pMBRT was associated with distinct metabolic modulation in tumours, notably upregulation of vanin-1 and attenuated glutamine synthetase induction, changes that may reflect increased tumour radiosensitivity. These alterations were further validated via immunofluorescence on brain cryosections. In normal brain, pMBRT was associated with preservation of synaptic-related protein expression and modulation of nitrogen metabolism, including altered glutamate dehydrogenase levels. Collectively, these findings highlight the dual capacity of pMBRT to sensitise tumours to radiation while preserving healthy brain tissue, supporting its translational potential for GB treatment.