Project Summary/Abstract Glioblastoma, IDH-wildtype (GBM; WHO grade 4), the most malignant primary brain tumor, has well-defined pre-necrotic, necrotic and recurrent growth phases. Transition to the necrotic phase in primary and recurrent states involves a massive restructuring of the tumor microenvironment (TME), in which there is a dramatic increase in tumor- associated macrophages (TAMs) that are polarized to an immunosuppressive state. TAMs emerge from the perivascular niche and aggregate in high density with hypoxic glioma cells within the peri-necrotic niche (PN) with remarkable temporal similarity. We hypothesize that the spatial arrangement of TAMs and glioma cells within the highly hypoxic PN represents a biologic engine that serves as a sustainable source of TAM immunosuppression and is reliant on their synergistic interdependencies. We propose investigations to better understand mechanisms underlying these events using patient- derived, molecularly characterized GBM neurosphere cultures and RCAS-tv-a mouse model that allows study of the evolution of TME events following necrosis in real-time in vivo and with advanced spatial transcriptomics. We focus on specific signaling events within the PN that cause polarization of TAMs and have preliminary data indicating that the hypoxic expression of podoplanin (PDPN) by glioma cells activates CLEC5A on TAMs, causing an immunosuppressive phenotype through Syk/STAT3 signaling. We also suggest that the direct effects of hypoxia and TGFβ1 activate Hippo signaling and PDPN expression within this niche. Pharmacologic inhibition of Syk as a single agent prolongs survival and enhances the presence of CD8+ T cells in the RCAS/tv-a model of GBM. We hypothesize that therapeutic targeting of CLEC5A signaling by using Syk inhibitors in addition to standard of care chemoradiation, with or without checkpoint inhibitors, will diminish immunosuppression, enhance immunity and prolong survival in preclinical models of primary and re