Abstract 2686: P2X4 purinergic receptor activation on macrophages inhibits phagocytosis and promotes tumor-promoting cytokine secretion

Abstract Prostate cancer (PCa), the most frequent cancer and the second leading cause of cancer-related deaths among men in the U.S., necessitates improved therapeutic strategies. Our previous studies demonstrated elevated P2X4 purinergic receptor expression in PCa tissues, which correlated with poor prognoses. We found that P2X4 receptor knock down or blockade in PCa cells reduces subcutaneous allograft tumor growth in vivo and inhibits human PCa cell viability, migration, and invasion in vitro. However, the expression profile and functional role of the P2X4 receptor in the PCa tumor microenvironment (TME) remain poorly defined. This study aims to characterize P2X4 receptor expression and functional significance within the prostate TME. Multiplex immunohistochemistry (IHC) analyses revealed P2X4 receptor expression on CD163+ M2 macrophages, but not in CD8+ T cytotoxic cells, CD4+ T helper cells and FAP+ fibroblast cells in human PCa tissues. IHC performed on FVB mouse allograft primary and metastatic tumors also suggested P2X4 expression on F4/80+ macrophages. Treatment of human THP-1-derived M2 macrophages with CTP (400 µM), a P2X4-specific agonist, significantly reduced phagocytosis in vitro (p = 0.0115). A human phospho-kinase array revealed that P2X4 activation decreased phosphorylation of AKT (S473) and p70S6 (Thr389), indicating a potential role in suppressing Fcγ receptor-mediated phagocytosis through the PI3K > AKT > mTORC1 > p70S6 pathway. Additionally, decreased phosphorylation of Src and Src family kinases (Hck, Fgr, and Lyn) suggested a Src-mediated mechanism of impaired phagocytosis. Further, a human cytokine array revealed that P2X4 receptor activation with CTP (400 µM) reduced the secretion of tumor-suppressive factors such as Endoglin, IGFBP3, and Serpin E1, while increasing the secretion of tumor-promoting factors including BAFF, RANTES, FGF-19, IL-8, MCP-1, MIP-3α, PDGF-AB/BB, and Relaxin, contributing to PCa growth, metastasis, and recurrence. We generated a macrophage-specific P2X4 receptor knockout mouse (LyzM-P2X4-/-), confirmed by flow cytometry and IHC. Supporting our findings in human THP-1 derived M2 macrophages, peritoneal TIM4+ F4/80+ macrophages from LyzM-P2X4-/- mice exhibited enhanced phagocytosis compared to wild-type controls (p= <0.0001). However, we observed no apparent difference in TRAMP-C2 tumor growth rates between wild-type and LyzM-P2X4-/- mice. In summary, our findings demonstrate that P2X4 receptors are present on tumor-associated M2 macrophages in PCa tissues. We identified a role for P2X4 purinergic receptors in regulating macrophage cytokine secretion and phagocytic activity in vitro, suggesting that P2X4 receptors in macrophages within the prostate TME may contribute to PCa progression. Our ongoing studies are aimed at determining the efficacy of P2X4 purinergic receptors as a potential therapeutic target. Citation Format: Vahinipriya Manoharan, Kennedy M. Rains, Jillian K. Landers, Sharvari S. Saraf, Tristan R. Ibarra, Angelo M. De Marzo, David E. Sanin, Janielle P. Maynard. P2X4 purinergic receptor activation on macrophages inhibits phagocytosis and promotes tumor-promoting cytokine secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2686.