INTRODUCTION: We have recently shown that neutrophilic myeloid-derived suppressor cells (MDSC) are the dominant source of TNF in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). Moreover, neutrophil-derived TNF amplifies stromal fibrosis and immune tolerance via transmembraneTNF-TNFR2 interactions to promote therapeutic resistance in PDAC. Here, we develop a novel tumor-bearing murine model with efficient neutrophil lineage-restricted inactivation of Tnf to mechanistically dissect the impact of neutrophil-intrinsic TNF on stromal and T-cell dysfunction in PDAC. METHODS: We generated a neutrophil-lineage restricted MRP8Cre/+Tnfflox/flox model (Neu-TnfKO) by crossing Tnfflox/flox mice with mice bearing Cre-recombinase under control of MRP8 neutrophilic promoter, and confirmed >98% Tnf reduction in circulating neutrophils. Orthotopically implanted KPC tumors in Neu-TnfKO vs. littermates (MRP8Cre+/−Tnf+/+ or MRP8Cre-/−Tnffl/fl) were subjected to single-cell RNAseq (scRNAseq), flow cytometric immunophenotyping, and tissue histology. RESULTS: Compared with littermate controls, primary tumor weights and frequency of liver metastasis were significantly reduced in Neu-TnfKO mice. ScRNAseq in CD45 fraction of d21 tumors in Neu-TnfKO vs. control mice revealed downregulation of myofibroblastic features (i.e., Ccn2, Tgfb1), hypoxia signaling, and metabolic dysregulation in CAF single-cell transcriptomes. Tissue histology in Neu-TnfKO tumors revealed striking attenuation in fibrotic collagen deposition, and myofibril organization. Moreover, Lrrc15—implicated as a central regulator of CAF-driven immunotherapy resistance—was downregulated in Neu-TnfKO CAFs. This remodeling of stromal fibrosis manifested in increased trafficking of T-cells into Neu-TnfKO vs. littermate tumors via immunostaining and flow cytometry. ScRNAseq in CD45+ fractions of Neu-TnfKO mice corroborated enrichment in effector/helper T-cell and attenuation of MDSC and macrophage populations. In investigating if neutrophil-restricted Tnf silencing invokes reprogramming of T-cell single-cell transcriptomes, we observed global skewness towards effector vs exhausted programming in Neu-TnfKO tumors; enrichment of pathways related to ribosomal activation, interferon signaling, and negative regulation of T-cell apoptosis; and overexpression of multiple effector function genes—e.g., Adgre5, Cam1kd, Dgka—together indicating functional T-cell activation. Flow cytometry validated this activated CD8+ T-cell phenotype in Neu-TnfKO tumor-infiltrating by highlighting increased antigen-experienced (PD1+CD39neg) and short-lived effector populations (P<0.0001), and also revealed reduction in terminally exhausted (CD69+Ly108 in PD1hi) CD8+ T-cell subsets (P=0.028). CONCLUSIONS: Conditional silencing of neutrophilic-restricted TNF reprograms the immunotherapy-restrictive CAF and T-cell landscapes in the PDAC TME. Compartment-specific inhibition of TNF may be a provocative strategy to overcome immunotherapy resistance in PDAC.

Citation Format: Anna Bianchi, Haleh Amirian, Iago De Castro Silva, Ifeanyichukwu Ogobuiro, Karthik Rajkumar, Andrew Mark Adams, Vanessa Garrido, Samara Singh, Siddharth Mehra, Christine Rafie, Nagarj Nagathihalli, Erietta Stelekati, Nipun Merchant, Jashodeep Datta. Neutrophil-intrinsic tumor necrosis factor (TNF) is a novel driver of T-cell and cancer-associated fibroblast (CAF) dysfunction 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 PR16.