Multimodal single-cell spatial profiling reveals altered T cell immunity and B-cell follicular architecture in non-metastatic lymph nodes of advanced NSCLC patients

Regional lymph nodes (LNs) in the thoracic cavity are essential immunological hubs that coordinate humoral and cell-mediated immunity during non-small cell lung cancer (NSCLC) progression. To investigate immune dysregulation in non-metastatic regional LNs from patients with advanced-stage NSCLC, we performed multimodal profiling of 36 LNs from 11 patients undergoing curative-intent resection using CITE-seq, scRNA-seq, and Imaging Mass Cytometry (IMC). Regional N1 LNs from our advanced-stage patients (stage IB-IIIA) displayed visceral pleural invasion (VPI) or lymphovascular invasion (LVI), and exhibited significant enrichment of dysfunctional CD8+; T cells and regulatory T cells compared with N2 LNs and stage IA LNs. These subsets spatially co-localized with mature regulatory dendritic cells (CD1c+, TIM3+, LAMP3+), forming an immunosuppressive niche enriched in advance-stage N1 LNs. Concurrently, advanced-stage N1 LNs contained more "decorticated" B-cell follicles characterized by decreased encapsulation of the mantle zone layer surrounding the germinal centers. Together, these findings reveal parallel disruption of humoral and cell-mediated immunity in regional LNs of advanced-stage NSCLC patients.