Single-cell and spatial transcriptomics identify a JUNB+ neutrophil subset enriched in immune-excluded regions of lung adenocarcinoma.

BACKGROUND

Tumor-associated neutrophils (TANs) display functional plasticity in lung adenocarcinoma (LUAD), but subsets linked to immune exclusion are poorly defined.

METHODS

We integrated single-cell RNA-seq, spatial transcriptomics, and bulk multi-omics data to characterize neutrophil heterogeneity in LUAD. Key findings were validated by immunofluorescence co-localization and qPCR on clinical blood and isolated neutrophils.

RESULTS

Neutrophils exhibited the highest scores for an EMT-associated transcriptional program and immune exclusion among all immune cell types. A distinct JUNB+ neutrophil subpopulation was spatially enriched in immune-excluded tumor regions and correlated with unfavorable prognosis. Spatial transcriptomic analysis revealed a significant positive correlation between JUNB expression and neutrophil marker module scores across tissue spots (Pearson r = 0.216 and 0.205, P = 1.84 × 10^-10 and 9.06 × 10^-11). Immunofluorescence confirmed JUNB protein co-localization with CD66b+ neutrophils in LUAD tissues (54.3% ± 8.2% of CD66b+ cells JUNB +). qPCR revealed specific upregulation of JUNB mRNA in circulating neutrophils from LUAD patients, whereas no difference was observed in whole blood.

CONCLUSION

This study identifies JUNB as a marker of a neutrophil state associated with immune exclusion and poor prognosis in LUAD. The neutrophil-specific upregulation of JUNB highlights its potential as a circulating biomarker for TAN-mediated immunosuppression, which warrants validation in prospective cohorts.