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

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. RNA sequencing 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 healthy brain tissue was morphologically identified and marked. Extraction of nucleic acids was performed from the delineated healthy tissue. Expression profiles were compared between the irradiated and non-irradiated cases.

尽管靶向放射治疗（radiotherapy, RT）在提升癌症患者生存率方面不可或缺，但其引发的严重副作用的细胞及分子机制尚未完全明晰。放射治疗过程中，肿瘤组织会发生表观遗传改变，但目前鲜有研究针对非肿瘤组织开展此类评估，且相关研究结果差异显著。本研究依托独特的患者组织样本队列，结合批量DNA甲基化测序、RNA测序以及空间转录组学（spatial transcriptomics, ST）分析，证实受照射脑组织的DNA甲基化模式存在显著差异；同时空间转录组学分析揭示了照射后特有的微环境生态位，并筛选出可能介导神经炎症传播的神经肽。本研究还发现，在照射后神经元早期变化的脑类器官（cerebral organoid, CO）模型中，同样存在DNA甲基化改变与DNA甲基化调控机制的紊乱，提示早期且持续的表观遗传失调在神经毒性中发挥了关键作用。本研究首次建立了放射治疗诱导的神经炎症与DNA甲基化紊乱之间的关联，并提出了这种慢性神经炎症的潜在驱动机制。本研究针对因脑部病变接受放射治疗的患者，利用其剩余的福尔马林固定石蜡包埋（Formalin-Fixed Paraffin-Embedded, FFPE）手术样本脑组织开展了RNA测序。实验样本为放射治疗后接受神经外科手术切除脑部病变的患者组织，对照样本则为未接受放射治疗的病变周围中枢神经系统（central nervous system, CNS）组织。从代表性FFPE包埋块中获取苏木精-伊红（hematoxylin-eosin, H&E）染色切片及后续未染色切片，通过形态学鉴定并标记出健康脑组织区域。从标记出的健康脑组织中提取核酸，并对比受照射与未受照射样本的基因表达谱。