Neutrophil extracellular traps induced by tumour-derived NGAL reprogram CAF subtypes via ITGA5 to promote tumour progression in pancreatic cancer

Abstract

Background Pancreatic ductal adenocarcinoma (PDAC) is largely refractory to immune checkpoint blockade, owing to its immunosuppressive tumour microenvironment. Neutrophil extracellular traps (NETs) accumulate in PDAC and correlate with disease progression, yet whether NETs reprogram cancer-associated fibroblast (CAF) heterogeneity and the upstream tumour-intrinsic signals that sustain pathological NETosis remain undefined. Objective To delineate how NETs instruct CAF subtype specification and immunosuppression in PDAC, identify upstream NETosis drivers and evaluate combinatorial therapeutic strategies targeting this axis. Design Quantitative spatial analysis of human PDAC specimens, NETs–pancreatic stellate cell and patient-derived CAF cocultures, biotinylated DNA pull-down with liquid chromatography-tandem mass spectrometry, live-cell integrin-blocking assays, Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing, orthotopic and hepatic colonisation models, CD8+ T-cell functional assays and single-cell RNA sequencing of an eight-arm therapeutic study were employed. Results NETs-DNA directly engaged integrin α5β1 (ITGA5) on fibroblasts via its N-terminal domain, initiating a FAK (focal adhesion kinase)/SRC (Src family tyrosine kinase)–YAP (Yes-associated protein)–IL-6 (interleukin-6)–JAK (Janus kinase)/STAT3 (signal transducer and activator of transcription 3) autocrine cascade specifying inflammatory CAF (iCAF) differentiation. Tumour-derived neutrophil gelatinase-associated lipocalin (NGAL), induced by IL-17, drove extracellular signal-regulated kinase (ERK)–reactive oxygen species-mediated NETosis. NETs-reprogrammed iCAFs accelerated tumour growth, hepatic colonisation and CD8+ T-cell exhaustion, with exhausted T cells spatially enriched in iCAF-rich regions. A composite NETs/ITGA5 signature stratified overall survival in patients with PDAC. Single-cell transcriptomics demonstrated that triple therapy (anti-IL-17+AV3+anti-PD-1) shifted fibroblasts from iCAF towards myofibroblastic CAF dominance, restored CD8+ T-cell effector programmes and achieved the greatest tumour suppression and survival benefit. Conclusion We define an IL-17/NGAL/NETs/ITGA5 axis linking neutrophil-derived extracellular DNA to iCAF specification and immunosuppression in PDAC. Cotargeting IL-17 and ITGA5 synergises with PD-1 blockade, providing a rationale for combinatorial immunotherapy.