Divergence from the human astrocyte developmental trajectory in glioblastoma [MeDIP-seq]

Glioblastoma (GBM) is defined by heterogenous and resilient cell populations that closely reflect neurodevelopmental cell types. While it is clear that GBM echoes early and immature cell states, identifying the specific developmental programs disrupted in these tumors has been hindered by a lack of high-resolution trajectories of glial and neuronal lineages. Here, we delineate the course of human astrocyte maturation to uncover discrete developmental stages and attributes mirrored by GBM. We generated a transcriptomic and epigenomic map of human astrocyte maturation using cortical organoids maintained in culture for nearly two years. Through this approach, we chronicled a multi-phase developmental process orchestrated by a series of transcription factor and gene regulatory networks. Our time course of human astrocyte maturation includes a novel and molecularly distinct intermediate period that serves as a lineage commitment checkpoint upstream of mature quiescence. This intermediate stage acts as a site of developmental deviation separating IDH-wildtype neoplastic astrocyte lineage cells from quiescent astrocyte populations. Interestingly, IDH1-mutant tumor astrocyte lineage cells are the exception to this developmental perturbation, where immature properties are suppressed as a result of D2HG oncometabolite exposure. We propose that this defiance is likely a consequence of IDH1mt-associated epigenetic dysregulation and we identified biased DNA hydroxymethylation (5hmC) in maturation genes as a possible mechanism. Together, this study illustrates a novel cellular state aberration in GBM astrocyte lineage cells and presents new developmental targets for experimental and therapeutic exploration. Overall design: This study consists of multiple sequencing datasets. (1) We purified astrocytes from fresh human cortical organoids and GBM tumor and margin tissue samples for bulk RNA-seq and ATAC-seq. (2) Nuclei were isolated from frozen GBM tumor and margin tissue samples for single-nucleus multiome (ATAC- and RNA-seq) sequencing. (3) We performed MeDIP and hMe-Seal with flash-frozen tissue from IDH1mt and IDHwt tumors (with corresponding margin samples) for 5mC and 5hmC analyses. (4) Bulk RNA-seq data for ASCL1 overexpression experiments and D2HG functional experiments with human fetal astrocytes.

胶质母细胞瘤（Glioblastoma, GBM）以异质性且强韧的细胞群体为特征，此类群体紧密模拟神经发育细胞类型。尽管学界已明确胶质母细胞瘤重现早期未成熟细胞状态，但由于缺乏胶质细胞与神经元谱系的高分辨率发育轨迹，识别此类肿瘤中受干扰的特异性发育程序始终存在阻碍。本研究通过解析人类星形胶质细胞（astrocyte）成熟过程，揭示胶质母细胞瘤所模拟的离散发育阶段与特征。我们利用体外培养近2年的大脑皮层类器官（cortical organoids），构建了人类星形胶质细胞成熟过程的转录组（transcriptomic）图谱与表观基因组（epigenomic）图谱。借助该模型，我们刻画了由一系列转录因子（transcription factor）与基因调控网络（gene regulatory networks）协同调控的多阶段发育进程。我们的人类星形胶质细胞成熟时序分析中，发现了一个全新的、分子特征独特的中间阶段，该阶段是成熟静止状态（quiescence）上游的谱系定型（lineage commitment）检查点。该中间阶段是发育偏离的节点，可区分异柠檬酸脱氢酶野生型（IDH-wildtype）肿瘤性星形胶质谱系细胞与静止态星形胶质细胞群体。有趣的是，异柠檬酸脱氢酶1突变型（IDH1-mutant）肿瘤星形胶质谱系细胞则是此发育扰动的例外：由于暴露于D2HG致癌代谢物（oncometabolite），其未成熟特性受到抑制。我们推测这种抗性可能源于IDH1突变相关的表观遗传失调（epigenetic dysregulation），并发现成熟基因的DNA羟甲基化（DNA hydroxymethylation, 5hmC）偏倚可能是其潜在机制。综上，本研究揭示了胶质母细胞瘤星形胶质谱系细胞中一种全新的细胞状态异常，并为实验与治疗探索提供了全新的发育靶点。整体实验设计：本研究包含多组测序数据集：(1) 从新鲜人类大脑皮层类器官、胶质母细胞瘤肿瘤及癌旁组织中纯化星形胶质细胞，进行批量RNA测序（bulk RNA-seq）与转座酶可及性测序（ATAC-seq）；(2) 从冻存的胶质母细胞瘤肿瘤及癌旁组织中分离细胞核，进行单细胞核多组学（single-nucleus multiome）测序（涵盖ATAC-seq与RNA-seq）；(3) 针对异柠檬酸脱氢酶1突变型与野生型肿瘤（及对应癌旁组织）的速冻组织，采用甲基化DNA免疫沉淀（MeDIP）与hMe-Seal技术，开展5-甲基胞嘧啶（5mC）与5-羟甲基胞嘧啶（5hmC）分析；(4) 对过表达ASCL1的实验以及人胎儿星形胶质细胞的D2HG功能实验，进行批量RNA测序。