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.