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May 9th, 2025
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Brown University
cancer biology
biorxiv

Multiomic profiling of hypoxic glioblastoma stem cells reveals expansion of subpopulations with distinct epigenetic and CNV profiles

Corseri, A.Open in Google Scholar•Moore, T. W.Open in Google Scholar•Szczepanski, N.Open in Google Scholar•Hwang, H.Open in Google Scholar•Zdon, A.Open in Google Scholar•Yardimci, G.Open in Google Scholar•Tapinos, N.Open in Google Scholar

Glioblastoma is characterized by extensive intratumoral heterogeneity driven by dynamic microenvironmental cues such as hypoxia. While transcriptional and epigenetic variability have been separately linked to hypoxia responses, the integrated impact of hypoxia on gene regulation and clonal architecture in glioblastoma stem cells (GSCs) remains poorly defined. We applied single-nucleus multiomics ntegrating RNA-seq and ATAC-seq to patient-derived GSCs cultured under normoxic or hypoxic conditions. This enabled simultaneous profiling of gene expression and chromatin accessibility within the same cells. Transcription factor (TF) regulatory networks were inferred using Dictys, while RNA-chromatin dynamics were modeled with MultiVelo. Clonal structure and copy number variations (CNVs) were resolved at single-cell resolution using RID- DLER on snATAC-seq data. Hypoxia induced the emergence of four distinct GSC subpopulations with unique transcriptomic and epigenetic profiles enriched for mesenchymal, angiogenic, and proliferative signatures. Regulatory network modeling revealed novel hypoxia-associated TFs like SP2, CREM, and ETV3 that modulate downstream oncogenic pathways. Trajectory analysis uncovered hypoxia-driven reversals in RNA-chromatin coupling, revealing dysregulated future transcriptional states of key genes such as MMP16 and SVIL. CNV profiling identified 13 clonal substructures, with specific clones (e.g., 5, 6, 9) selectively enriched under hypoxia and harboring distinct chromosomal alterations. These results demonstrate coordinated remodeling of GSC gene regulation and clonal fitness in response to hypoxic stress. Our findings reveal that hypoxia drives concurrent epigenetic, transcriptomic, and clonal selection in glioblastoma stem cells. This integrated model of hypoxia-induced plasticity provides mechanistic insights into tumor adaptation and identifies novel regulators that may serve as targets for therapeutic intervention in the hypoxic niche of glioblastoma.

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