Tmic-31. multi-platform genomic landscape of intra-tumoral hypoxia in glioblastoma

Abstract Hypoxia is a prominent characteristic of aggressive tumors, including glioblastoma (GB), and is linked to therapy resistance. Understanding the molecular mechanisms underlying hypoxia-induced changes in neoplastic and non-neoplastic cells is crucial for developing targeted therapies. In this study, we took a comprehensive approach to investigate the genomic, epigenomic, and spatial transcriptomic profiles of intra-tumoral hypoxia in GB at bulk and single-cell levels. Our objectives were to identify a GB-specific hypoxia gene signature and uncover novel mechanisms of GB's response to hypoxia. Pimonidazole (PIMO), a hypoxia marker, was administered to 77 GB patients. We utilized immune-guided laser microdissection to extract DNA and perform methylome profiling of PIMO+ (hypoxic) and PIMO- regions. Visium 10X spatial transcriptomics was employed to identify differentially expressed genes in PIMO+ tumor regions. Through single-nuclear multi-omics profiling, we examined gene expression and chromatin accessibility of the hypoxic neoplastic and non-neoplastic cells at the single-cell level. The hypoxia transcriptome in GB exhibited general commonalities, in addition to unique features, compared to other reported hypoxia signatures. Some of these alterations were accompanied by changes at the DNA methylome level. Importantly, we found a heterogenous molecular landscape across PIMO+ regions within each sample. While a general transition to a MES-like state was observed, we found a mixture of cell states across the different PIMO+ regions. We also found distinct sub-hypoxic microenvironments harboring unique immune cell populations that overlapped with specific cell type states, cell cycle states, and deregulation of specific cellular pathways. These sub-regions were associated with specific anatomic features and displayed prognostic utility in GBM patient cohorts. Our single-cell multi-omics analysis further revealed intra-tumoral copy number aberrations and changes in chromatin accessibility associated with hypoxic cells. Our findings demonstrate that the hypoxic microenvironment in GB is heterogenous with unique targetable features that could lead to novel treatment modalities.