Abstract
OBJECTIVE: The major drivers of therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) are myeloid cell-derived signaling that sustains immune tolerance and interleukin-1 (IL1)-mediated inflammatory polarization of cancer-associated fibroblasts (iCAF) which promotes stromal inflammation by elaborating soluble factors (i.e., CXCL1, IL6) that further accelerate myeloid chemotaxis. We have recently shown that granulocytic myeloid-derived suppressor cell (gMDSC)-derived IL1β promotes chemoresistance in PDAC via a CAF:tumor cell IL6:STAT3 axis. Since NLRP3 inflammasome activation is the dominant source of IL1β generation in innate immune cells, we hypothesized that gMDSC-derived NLRP3 inflammasomes are a novel driver of iCAF polarization in PDAC. METHODS: We examine single-cell RNA sequencing (scRNAseq) data in human and PKT/KPC PDAC models to identify cellular source of inflammasome-derived IL1β. Gene set enrichment analysis in bulk RNAseq data and signal transduction studies examined novel pathways associated with inflammasome activation in gMDSCs. gMDSCs treated with/without novel NLRP3 inhibitor SB-NL02 were co-cultured with Il6/Acta2 dual-reporter CAF system in vitro to examine iCAF polarization. ASC oligomerization in intratumoral gMDSCs via confocal microscopy and CAF polarization via flow cytometry were evaluated in mice treated with NLRP3i SB-NL02. RESULTS: scRNAseq in human and PKT/KPC PDAC models revealed gMDSCs as the dominant source of inflammasome activation-derived IL1β. Lineage trajectory reconstruction revealed strong co-expression of Cxcr2, Nlrp3, and Il1β in gMDSC single-cell transcriptomes. Mechanistically, in vitro signal transduction and RNAseq studies revealed activation of gMDSC-intrinsic NLRP3 inflammasomes not only via canonical TLR4-MyD88 signaling but also cooperative regulation via CXCR2-p38 MAPK induction. As such, intratumoral gMDSCs retrieved from mice treated with CXCR2 inhibitor AZD5069 or those from CRISPR-edited KPC-Cxcl1KO (cognate ligand of CXCR2) tumor-bearing mice showed reduced Nlrp3 expression, inflammasome activation, and IL1β generation. To investigate the impact of gMDSC-NLRP3 on iCAF polarization, co-culture of J774M gMDSCs with Il6/Acta2 dual reporter CAFs revealed strong induction of CAF-intrinsic Il6 expression, which was mitigated by treatment of gMDSCs with NLRP3i SB-NL02 or CAFs with IL-1 receptor antagonist Anakinra. Treatment of PKT mice with NLRP3i SB-NL02 reduced primary tumor volume, as well as ASC speck oligomerization and IL1β production in intratumoral Ly6G+ gMDSCs. Moreover, we observed significant attenuation in iCAF (CD31−PDPN+Ly6C+MHCII−) skewness in PKT mice treated with NLRP3i compared with vehicle. CONCLUSIONS: gMDSC-derived NLRP3 inflammasome activation—via cooperative TLR4 and p38 signaling induction—and ensuing IL1β production is a novel regulator of iCAF polarization. Therapeutic approaches—such as gMDSC compartment-specific NLRP3 inhibition—may mitigate stromal inflammation and overcome therapeutic resistance in PDAC.
Citation Format: Karthik Rajkumar, Nilesh U. Deshpande, Iago de Castro Silva, Anna Bianchi, Ifeanyichukwu Ogobuiro, Haleh Amirian, Andrews Adams, Vanessa Garrido, Siddharth Mehra, Samara Singh, Nagaraj Nagathihalli, Nipun B. Merchant, Jashodeep Datta. Granulocytic MDSC-derived NLRP3 inflammasome activation is a novel regulator of inflammatory CAF skewness in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C021.
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