Tmic-82. spatial and single-cell proteomic landscaping of the hypoxic microenvironment in glioblastoma

Abstract Glioblastoma (GBM) is a fatal adult solid tumour with median overall survival of 18-20 months post-diagnosis; contributing factors to therapeutic inefficacy include acquisition of genomic alterations post-therapy, immune evasion, deregulated hypervascularization, and tumor microenvironmental factors such as hypoxia. Combined with extensive inter- and intra-tumoral heterogeneity at bulk and single-cell level, hypoxia contributes to a gradient of molecular alterations that are specific to different cell populations that make up tumour bulk and reside in specific niches. Hitherto, high-dimensional histopathologic analyses of hypoxic regions within GBM tissue have not been performed. We took a combined spatial and single-cell proteomic profiling approach to investigate the histopathologic features of hypoxia by leveraging a unique clinical study wherein the exogenous hypoxia marker, pimonidazole (PIMO), was administered to GBM-patients preoperatively. Tissue specimens were subjected to imaging mass cytometry, high-resolution imaging, and serial immunohistochemistry using a panel of markers associated with cellular hallmarks of hypoxia, metabolism, proliferation, stemness, angiogenesis, and immune cell types. Our findings showed that PIMO staining is associated with histopathologic features of hypoxia and correlates with specific metabolic, immune, and stemness markers in GBM; specific lymphocyte populations were depleted from hypoxic regions alongside alterations in macrophagic and microglial landscape in a niche-specific manner; hypoxia reduced the proportion of proliferating glioma initiating cells and altered the proliferative, transcriptional, and translational capacity of different cell populations; microvessel density was reduced in hypoxic microenvironment. Cytometry by time-of-flight further validated the altered proportions of specific immune cell types enriched in hypoxic populations of GBM microenvironment. Our study is the first to report use of PIMO to interrogate spatial, single-cell, and phenotypic architecture associated with tissue hypoxia and altered expression of biomarkers associated with hypoxia/glycolysis, immune infiltration, proliferation, and stemness. Identification of targetable biomarkers and mediators of hypoxia-driven habitats in GBM may provide direction for future immunotherapeutic research.