Selective Targeting uPAR-driven Neutrophil Extracellular Traps Functional Clusters to Attenuate Tumor Progression and Enhance the Response to Immunotherapy in Intrahepatic Cholangiocarcinoma

Cholangiocarcinoma (CCA) is an aggressive malignancy with dismal prognosis; PD-1/PD-L1 blockade benefits few patients, as the tumor microenvironment (TME) is immunologically “cold”. A key CCA-TME feature is massive tumor-associated neutrophil (TAN) infiltration releasing neutrophil extracellular traps (NETs). Conventionally deemed pro-tumorigenic, NETs’ anti-tumor potential is overlooked, leaving their dual roles and regulatory circuits in CCA undefined. Our study finds prominent NETs enrichment in CCA clinical specimens and preclinical models, correlating with poor outcomes. Functional studies show NETs exert dual effects: a dominant pro-tumor arm accelerating growth/metastasis, and a latent immunostimulatory arm rendering the TME “hot”. Integrated multi-omics and bioinformatics analyses dissect these functions into distinct molecular clusters with divergent prognostic value. Pro-tumor clusters are selectively activated by MAPK signaling, present in ~25% of CCA with KRAS mutations. Mechanistically, uPA-loaded NETs engage uPAR on CCA cells; TLR co-reception licenses downstream MAPK activation, tipping toward tumor promotion. We devised a cluster-directed combination: DNase I dismantling NETs scaffolds plus uPAR blockade neutralizing residual pro-tumor fragments. This strategy abolishes oncogenic signaling while sparing—even boosting—STING-dependent anti-tumor immunity, sensitizing KRAS-mutant and wild-type CCA to anti-PD-L1 therapy. Human transcriptomic datasets link low pro-tumor/high immunostimulatory NETs signatures with durable immunotherapy responses. We establish a functional-cluster framework for CCA NETs biology and provide a precision co-targeting regimen turning pro-tumor function into immunotherapeutic opportunity.