Abstract A079: Conditional silencing of neutrophil-restricted Nlrp3 reveals an unrecognized role of MDSC-inflammasomes in driving stromal inflammation and chemoresistance in PDAC

Abstract INTRODUCTION: Pivotal contributors to chemoresistance in PDAC are myeloid cell infiltration/signaling, and interleukin-1 (IL1)-mediated inflammatory polarization of cancer-associated fibroblasts (CAF). We have previously shown that neutrophilic myeloid-derived suppressor cells (MDSC) are the dominant source of NLRP3 inflammasome-derived IL-1β in the tumor microenvironment (TME) and instigate iCAF polarization. Paradoxically, prior data also indicate that DC/Th1-intrinsic NLRP3 inflammasomes are critical for antitumor T-cell immunity. We hypothesized that conditional silencing of neutrophil-restricted NLPR3 inflammasomes would attenuate stromal inflammation while preserving cytotoxic CD8+ T-cell function, thereby enhancing chemosensitivity. METHODS: We generated a neutrophil-restricted B6.MRP8Cre/+ Nlrp3 flox/flox model (Neu-Nlrp3 KO) by crossing Nlrp3 flox/flox mice with mice bearing Cre-recombinase under control of S100a8/MRP8 neutrophilic promoter, and confirmed >98% reduction in ASC speck formation in circulating neutrophils. We performed scRNAseq on orthotopically KPC tumors in Neu-Nlrp3 KO vs littermate mice. In vitro CRISPR/Cas9 silenced Nlrp3 and Il1r1 in MDSC-like J774M cells and CAFs, respectively. RESULTS: In tumor-bearing Neu-Nlrp3 KO mice, compared with littermate controls, Nlrp3 expression, ASC speck formation, and IL-1β levels were significantly reduced in intratumoral Ly6G+ neutrophilic MDSCs. We observed striking growth arrest of orthotopically implanted KPC tumors in Neu-Nlrp3 KO mice vs littermates. scRNAseq of KPC tumors in Neu-Nlrp3 KO mice revealed significant attenuation in inflammatory gene modules (Il6, Cxcl1, Ccl2, Cxcl12), hypoxia signaling, and metabolic dysregulation in CAF transcriptomes. J774-Nlrp3 -/- cells reduced CAF-Il6 expression in vitro. We observed preserved DC/Th1 inflammasome module expression in Neu-Nlrp3 KO vs littermate tumors, which correlated with enrichment in activation/memory and Gzmb + Ifng + cytotoxic programs in single-cell CD8+ T-cell transcriptomes. This was corroborated by flow cytometry in Neu-Nlrp3 KO vs. littermate tumors, revealing an increased abundance of CD44+CD62L-CD8+ effector memory cells. Cell chat analysis indicated reduced IL6-STAT3 signaling in cancer cell clusters—a key chemoresistance signaling node—from Neu-Nlrp3 KO vs littermate mice. Conditioned media from co-cultures of MDSC-like J774M cells and KPC CAFs induced pSTAT3Tyr705 in KPC tumor cells in vitro, which was reduced when either J774-Nlrp3 -/- or CAF-Il1r1 -/-- cells were used in co-cultures. KPC tumor-bearing Neu-Nlrp3 KO mice receiving gemcitabine+nab-paclitaxel showed improved survival compared with respective controls. We validated these preclinical observations in the HTAN human PDAC repository, where neutrophil-specific inflammasome gene module expression was greatly enriched in chemoresistant samples. CONCLUSIONS: Neutrophilic MDSC-specific inhibition of NLPR3 inflammasomes may be a provocative strategy to mitigate stromal inflammation and improve chemosensitivity in PDAC. Citation Format: Karthik Rajkumar, Andrew Adams, Haleh Amirian, Manan Patel, Erin Dickey, Harper M. Marsh, Siddharth Mehra, Nagaraj Nagathihalli, Nipun B. Merchant, Anna Bianchi, Jashodeep Datta. Conditional silencing of neutrophil-restricted Nlrp3 reveals an unrecognized role of MDSC-inflammasomes in driving stromal inflammation and chemoresistance in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A079.