Tumor-derived TNF-α promotes CCL4⁺ tumor-associated neutrophil formation and immunotherapy resistance in non-small cell lung cancer

Immune checkpoint inhibitors (ICIs) have improved outcomes in advanced non-small cell lung cancer (NSCLC), but primary and acquired resistance remain frequent. Because neutrophils are abundant and functionally plastic myeloid cells, we examined whether defined tumor-associated neutrophil (TAN) states contribute to ICI resistance in NSCLC. We analyzed 496 institutional NSCLC patients treated with immunotherapy, integrated public single-cell RNA sequencing, bulk transcriptomic and spatial transcriptomic datasets, and performed functional experiments using A549- and PC-9-conditioned medium, recombinant TNF-α, infliximab (a chimeric anti-TNF-α monoclonal antibody), peripheral blood neutrophils (PBNs), and PBN/CD8⁺ T-cell coculture. High pretreatment peripheral neutrophil levels were independently associated with non-response, shorter progression-free survival, and shorter overall survival. Single-cell analysis identified a CCL4-high Neutrophil_2_CCL4 subset with TAN features that was enriched in non-responding (stable disease) tumors and associated with shorter progression-free survival. Pseudotime, regulon, cell-communication, and spatial analyses linked this subset to tumor cell–neutrophil TNF/TNF receptor-dependent NF-κB activation. A549-CM, PC-9-CM, and recombinant TNF-α increased p-p65 and PD-L1 protein and CCL3, CCL4, CXCL8, and CD274 mRNA in PBNs, whereas infliximab attenuated these effects. TAN-like PBNs suppressed IFNγ production by CD8⁺ T cells. An eight-gene tumor-associated neutrophil-related signature (TANRS) derived from CCL4⁺ TAN features was prognostic in public NSCLC cohorts and was associated with response, progression-free survival, and overall survival in ICI-treated patients in an independent institutional cohort, whereas it stratified neither response nor survival in an institutional chemotherapy-treated comparator cohort. These data identify a tumor-derived TNF-α/NF-κB/CCL4⁺ TAN axis associated with NSCLC immunotherapy resistance and support TANRS as a candidate blood-accessible biomarker that is prognostic and outcome-associated in ICI-treated NSCLC.