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

Abstract 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. Significance This manuscript demonstrates that non-metastatic regional lymph nodes in advanced NSCLC are not immunologically normal but instead show coordinated remodeling of T cell immunity and B-cell follicular architecture. By integrating single-cell and spatial profiling, it identifies dysfunctional CD8 T cells, regulatory T cells, mregDC-associated suppressive niches, and disrupted follicular organization as features of early nodal immune dysregulation before overt metastasis. These findings support regional lymph nodes as clinically relevant immune sites that may improve risk stratification and guide future perioperative or adjuvant immunotherapy strategies in resected NSCLC.