TMIC-105. P2RY12 as a potential therapeutic target for disrupting tumor microenvironment– microglia cross talk

Abstract Gliomas are highly heterogeneous tumors composed of dense cores and diffusely infiltrating margins, where glioma cells interact with non-neoplastic cells, including microglia—the brain’s resident immune cells. To investigate spatial microglial states, we isolated live microglia from paired patient glioma core and margin samples, and performed single-cell RNA sequencing. We found that margin-associated microglia displayed distinct transcriptional profiles from those in the core. Specifically, purinergic signaling pathways were upregulated at the margin, while chemokine signaling, interferon response, and antigen presentation pathways were enriched in the core. Using Monocle pseudotime analysis, we constructed microglial activation trajectories and identified P2RY12 as the gene most negatively correlated with inflammatory progression, suggesting it may play a role in early-stage activation at the tumor margin. To model this in vitro, we used human iPSC-derived microglia, which recapitulate microglial activation states seen in vivo. ATP stimulation of iPSC-microglia, mimicking increased levels of extracellular ATP (eATP) in the glioma microenvironment, significantly upregulated inflammatory genes seen in the late-stage pseudotime signatures, and downregulated genes in the early-stage pseudotime signature. This indicates that ATP stimulation induces a shift along the inflammatory trajectory towards later stage activation phenotypes. To probe therapeutic relevance, we treated microglia with the purinergic receptor inhibitor PPADS, which attenuated ATP-induced transcriptional programs, including interferon signaling. These results suggest that P2RY12 activation by eATP modulates early microglial activation. We are currently testing the effects of P2RY12 knockdown in the iPSC-microglia model to assess responses to ATP and glioma co-culture. Future directions include acute brain slices and in vivo mouse glioma models to evaluate how targeting P2RY12 affects microglial behavior and tumor progression, potentially informing novel immunomodulatory strategies for glioma.