Chronic hypoxia remodels the tumor microenvironment to support glioma stem cell growth [scRNAseq+Multiome]. Chronic hypoxia remodels the tumor microenvironment to support glioma stem cell growth [scRNAseq+Multiome]

Cerebral organoids co-cultured with patient derived glioma stem cells (GLICOs) are an experimentally tractable research tool useful for investigating the role of the human brain tumor microenvironment in glioblastoma. Here we develop long-term GLICOs, a novel model in which COs are minimally seeded with GSCs and tumor development is monitored over extended durations (ltGLICOs). Single-cell profiling of ltGLICOs revealed an unexpectedly long latency period prior to GSC expansion, and that normal organoid development was unimpaired by the presence of low numbers of GSCs. However, as organoids age they experience chronic hypoxia and oxidative stress which remodels the tumor microenvironment to promote GSC expansion. Receptor-ligand modelling identified astrocytes, which secreted various pro-tumorigenic ligands including FGF1, as the primary cell type for GSC crosstalk and single-cell multi-omic analysis revealed these astrocytes were under the control of ischemic regulatory networks. Functional validation confirmed hypoxia as a driver of pro-tumorigenic astrocytic ligand secretion and that GSC expansion was accelerated by pharmacological induction of oxidative stress. When controlled for genotype, the close association between glioma aggressiveness with patient age has very few proposed biological explanations. Our findings indicate that age-associated increases in cerebral vascular insufficiency and associated regional chronic cerebral hypoxia may contribute to this phenomenon. Overall design: We performed scRNAseq on a cohort of 19 cerebral organoids (COs) seeded with low levels of glioma stem cells (ltGLICOs) spanning 1-24 months in culture, as well as scMultiome (RNA + ATAC) on 9 further ltGLICOs aged 5, 9 & 12months. Next we performed scRNAseq on 2x 7month control COs & 2x 7month COs maintained under hypoxic culture conditions.

与患者来源胶质瘤干细胞共培养的大脑类器官（cerebral organoids, GLICOs）是一种实验上易于操作的研究工具，可用于探究人脑肿瘤微环境在胶质母细胞瘤中的作用。本研究构建了长期GLICOs模型：该新型模型仅向大脑类器官（cerebral organoids, COs）中少量接种胶质瘤干细胞（glioma stem cells, GSCs），并可在长期培养过程中监测肿瘤发生发展，即ltGLICOs。对ltGLICOs的单细胞谱分析揭示了GSC扩增前一段出乎意料的长潜伏期，且少量GSCs的存在并未干扰正常类器官的发育。然而，随着类器官培养时长增加，其会出现慢性缺氧与氧化应激，重塑肿瘤微环境以促进GSC扩增。受体-配体相互作用建模显示，星形胶质细胞是与GSC发生信号串扰的主要细胞类型，其可分泌包括成纤维细胞生长因子1（fibroblast growth factor 1, FGF1）在内的多种促肿瘤配体；单细胞多组学分析进一步表明，这些星形胶质细胞受缺血调控网络的调控。功能验证实验证实，缺氧可驱动星形胶质细胞分泌促肿瘤配体，且通过药理学手段诱导氧化应激可加速GSC扩增。在控制基因型变量的前提下，目前针对胶质瘤侵袭性与患者年龄之间的紧密关联，仅有极少的合理解释生物学机制。本研究结果表明，与年龄相关的脑血管功能不全及伴随的区域性慢性脑缺氧可能是这一现象的诱因之一。实验整体设计：我们对19株培养周期覆盖1~24个月的低接种量GSCs大脑类器官（ltGLICOs）进行了单细胞RNA测序（single-cell RNA sequencing, scRNAseq），并对另外9株培养时长分别为5、9、12个月的ltGLICOs开展了单细胞多组学测序（single-cell multiome sequencing, scMultiome，包含RNA测序与转座酶可及性染色质测序（assay for transposase-accessible chromatin, ATAC））。此外，我们还对2株7月龄的对照大脑类器官，以及2株在缺氧培养条件下维持的7月龄大脑类器官进行了scRNAseq分析。