The inflammatory micro-environment induced by targeted CNS radiotherapy is underpined by disruption of DNA methylation [Array]

Although targeted radiotherapy (RT) is integral to the increasing survival of cancer patients, it has significant side-effects, the cellular and molecular mechanisms of which are not fully understood. During RT epigenetic changes occur in neoplastic tissue, but few studies have assessed these in non-neoplastic tissue and results are highly variable. Using bulk DNA methylation and RNA sequencing as well as spatial transcriptomics (ST) in a unique cohort of patient tissue samples, we show distinct differences in DNA methylation patterns in irradiated brain tissue, whilst ST characterisation identifies specific micro-environmental niches present after irradiation and highlights neuropeptides that could be propagating neuroinflammation. We also show that in a cerebral organoid (CO) model of early changes in neurons after irradiation there are similar DNA methylation alterations and disruption of the DNA methylation machinery, suggesting that early but persistent epigenetic dysregulation plays a role in neurotoxicity. We provide a link between radiotherapy induced neuroinflammation and disruption of DNA methylation for the first time and suggest possible driving mechanisms for this chronic neuroinflammation. DNA methylation was performed on brain tissue from surplus FFPE surgical materials of patients who received radiotherapy for the treatment of cerebral lesions. The experimental samples comprised cases where neurosurgical removal of a brain lesion occurred following radiotherapy, and the control samples included peri-lesional CNS tissue that had not received radiotherapy. H&E and subsequent unstained sections were obtained from the representative FFPE blocks and non-lesional brain tissue was morphologically identified and marked. Extraction of nucleic acids was performed from the delineated healthy tissue. DNA methylation profiles were compared between the irradiated and non-irradiated cases. For the DNA methylation analysis of COs, organoids were treated with 24 Gy dose at day 48 and collected at 4 weeks post-irradiation and the unirradiated organoids served as controls. Subsequent extraction of nucleic acids were performed from each CO.