Deciphering hypoxia related glioblastoma-associated macrophages and predicting prognostic markers by single-cell, spatially-resolved transcriptomics and experimental analysis

Glioblastomas (GBM), the most common and lethal type of primary brain tumor in adult, currently lack effective treatment and prognostic indicators. Hypoxia, a pathology promoting tumor progression in most solid tumors, has been reported to exhibit a negative correlation with GBM prognosis. Hypoxia related tumor-associated macrophages (hTAMs), the major cellular component of hypoxic regions, have not being systematically studied. We characterized the transcriptomic identity of hTAMs at the single-cell level, simultaneously discovering signatures indicative of poor outcomes. Immunofluorescent staining and spatially resolved transcriptomics revealed pronounced disparities regarding the morphology and distribution of TAMs between hypoxic and non-hypoxic regions of GBMs. We identified ARL4C and HSPA5 as prognostic indicators, exhibiting spatial patterns congruent with hypoxic regions and predicting worse outcomes, verified by qPCR and cell culture. Intercellular communication analysis using single-cell-omics revealed that hypoxia related GBM tumor cells (hGBM cells) exploit the TIMP1/LRP1 ligand-receptor axis to modulate hTAMs, resulting in a worse prognosis. Our study has offered clinically prognostic indicators, along with potentially therapeutic targets for GBMs.