Dysregulation of adaptive immunity in the uninvolved regional lymph nodes of patients undergoing resection for aggressive non-small cell lung cancer
Embargo Date
2028-06-15
OA Version
Citation
Abstract
Lung cancer remains the leading cause of cancer-related death worldwide, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Regional lymph node (LN) involvement is a pivotal prognostic marker and staging component in NSCLC. While the presence of overt LN metastasis is well-recognized as a driver of poor outcome, the immunologic changes that occur within tumor-surrounding LNs prior to detectable metastasis remain poorly understood. We hypothesize that as NSCLC tumors increase in size, regional LNs—particularly the tumor-draining N1 nodes—undergo immunologic dysregulation that precedes and potentially facilitates metastatic progression.To address this, we pursued two specific aims. Aim 1 focused on the development and optimization of lung tissue dissociation and cryopreservation protocols that maximize recovery and viability of innate and adaptive immune cells while preserving cell surface protein integrity for downstream CITE-seq analysis. Using mouse lung tissue as a model, we systematically evaluated dissociation and freezing strategies, ultimately establishing a workflow combining post-thaw collagenase digestion of 1 mm³ chopped tissue with cryopreservation media that reliably preserved cellular viability and phenotype. These protocols were translated to human lung and lymph node tissues for multimodal single-cell profiling.
In Aim 2, we applied single-cell CITE-seq and Imaging Mass Cytometry (IMC) to 36 lymph nodes from 11 treatment-naïve early-stage NSCLC patients, stratified by tumor stage and LN location (N1 vs. N2). We identified significant enrichment of immunosuppressive cell populations—including dysfunctional CD8⁺ T cells, FOXP3⁺ regulatory T cells (Tregs), and mature regulatory dendritic cells (mregDCs)—specifically in N1 LNs of higher-stage patients (stage IB–IIIA). These cell types were largely absent from N2 LNs and from N1 LNs of lower-stage patients (stage IA), suggesting local immune suppression at the primary tumor site that emerges prior to nodal metastasis. Using Imaging Mass Cytometry (IMC), we revealed stage-associated architectural disruption of B cell follicles within N1 LNs. While lower-stage nodes exhibited organized secondary follicles with germinal centers (GCs) encapsulated by mantle zone (MZ) structures, higher-stage N1 LNs displayed frequent loss of MZ integrity and emergence of “aberrant follicles” featuring GC-like cores without surrounding MZ niches. These aberrant follicles were enriched for plasma cells, immunosuppressive macrophages, and Tregs, and were surrounded by altered spatial niches lacking CD4⁺ T helper cells and B cell–FRC interactions, indicative of impaired humoral immunity. In addition, we observed spatial enrichment of dysfunctional CD8+ T cells and regulatory T cells (Tregs) co-localized with mature regulatory dendritic cells (mregDCs) expressing CD1c, TIM-3, and LAMP3, forming immunosuppressive niches unique to higher-stage N1 nodes. These findings indicate a dual impairment of both humoral and cell-mediated immunity, and that immune alterations were absent in N2 LNs and in all LNs from stage IA patients, suggesting that regional immune dysfunction arises locally at the tumor site prior to distant dissemination.
Together, these findings demonstrate that early, spatially localized immune dysregulation within regional N1 LNs—characterized by both suppressive T cell infiltration and follicular disorganization—correlates with increased tumor stage even in the absence of nodal metastasis. Our data support a model in which immune remodeling within tumor-surrounding LNs is an early feature of NSCLC progression, providing new avenues for nodal immune profiling in risk stratification and immunotherapeutic targeting. Future work will validate these immune features in expanded clinical cohorts and preclinical models to define their role in metastasis and therapeutic response.
Description
2025