Abstract B035: NLRP3 activation drives gemcitabine-mediated metabolic changes in pancreatic cancer tumor-associated macrophages (TAMs)

Abstract Background: Pancreatic cancer is the third leading cause of cancer deaths in the US and pancreatic ductal adenocarcinoma (PDAC) accounts for approximately 90% of all pancreatic cancer cases and for the majority of deaths.1,2 For patients with metastatic disease (80-85% patients at diagnosis), chemotherapy is the main treatment option but has shown to be highly debilitating, only limited effective and patients are at high risk for progression. While primarily targeting cancer cells, cytotoxic agents significantly impact the tumor microenvironment (TME), influencing its composition and response to treatment.3-5 Specifically to PDAC, it has been demonstrated that gemcitabine (GEM) induces distinct phenotypic changes in tumor-associated macrophages (TAMs), including: 1) immunosuppressive and “alternatively activated” polarization and 2) increased oxidative phosphorylation (OXPHOS).6 Notably, immunosuppressive and “alternative activated” macrophages have been found to worsen prognosis in patients with advance PDAC and to reduce response to chemotherapy. However, the molecular mechanism(s) that drive these GEM-mediated changes in TAMs are unclear. Our study investigated the effect of GEM on TAMs and discovered novel inflammatory vulnerabilities that could be exploited to increase/maintain response to chemotherapy. Methods: C57BL/6 and NLRP3−/− mice underwent intrapancreatic injection of PDAC cells (KPC). GEM was administrated intraperitoneally at 50 mg/kg every 3 days starting 10 days after surgery. At sacrifice (28 days post-tumor implantation), primary tumors were isolated and scRNA-seq, metabolomic and functional investigations were conducted to determine the role of NLRP3 in TAMs polarization and metabolism following chemotherapy. Results: Wild-type (WT) PDAC-bearing mice treated with GEM showed increased NLRP3 expression in the primary tumors when compared to the controls. NLRP3-/- mice treated with GEM had further reduction in tumor growth compared to the GEM-treated WT. Sc-RNA-seq analysis revealed that GEM increased the expression of mitochondrial genes (i.e. mt-ND1,3,4,5; mt-Cytb; mt-CO1-3; mt-ATP6,8) associated with OXPHOS in TAMs when compared to vehicle group. This increase was prevented in NLRP3-/- treated with GEM (p<3.1E-16). Phenotypically, TAMs isolated from NLRP3-/- mice showed increased membrane expression of CD68 and reduction in CD163 when compared to the WT group. Metabolomic analysis revealed increased production of metabolites associated with the fatty acid oxidation pathway and increased oxygen consumption in the TAMs isolated from GEM-treated WT mice when compared to the NLRP3 deficient mice. Further, imaging analysis revealed co-localization of NLRP3 on the mitochondria membrane following GEM. Conclusions: Our findings suggest that NLRP3 is a key driver for the GEM-induced changes in TAMs that correlate with the immunosuppressive and pro-tumor phenotype. By limiting chemotherapy-induced TAM modifications,6 NLRP3 blockade could represent a new opportunity for combinational therapies. Citation Format: Migachelle Romano, Makenna May, Laura Pike, Christopher H. Lieu, Wells A. Messersmith, Todd M. Pitts, Carlo Marchetti. NLRP3 activation drives gemcitabine-mediated metabolic changes in pancreatic cancer tumor-associated macrophages (TAMs) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B035.