Abstract
Background:
Tumor-derived small extracellular vesicles (EVs) play a crucial role in modulating immune responses and shaping the tumor microenvironment; however, their functional impact on airway immunity in NSCLC remains largely unexplored. This study represents the first attempt to investigate the immunomodulatory and tumor-promoting effects of NSCLC-derived EVs in a human 3D bronchial airway model, which closely mimics the human lung microenvironment.
Methods:
EVs were isolated from the plasma and bronchoalveolar lavage fluid (BALF) of NSCLC patients and analyzed via nanoparticle tracking analysis (NTA) and high-resolution imaging flow cytometry. The lymphocyte compositions of the matched blood and BALF samples were profiled. To assess the functional effects of EVs, we employed a pioneering in vitro 3D airway coculture model that combines primary human airway epithelial cells and alveolar macrophages at the air‒liquid interface. Proteomic analysis of EV-treated cells and their secretome was performed to identify key molecular pathways underlying EV-driven immunomodulation.
Results:
Surprisingly, no significant molecular differences were detected between EVs from cancerous (cBALF) and opposite (oBALF) lung compartments, despite a localized increase in regulatory T cells (Tregs) in the cBALF, suggesting regional immunosuppression. Plasma-derived EVs exhibited highly diverse, patient-specific molecular signatures but were not directly correlated with clinical or immune parameters. Functional studies of EVs with high and low surface molecular cargo in a 3D airway model revealed that both EV subgroups promoted monocyte/macrophage recruitment, angiogenesis, and epithelial-to-mesenchymal transition (EMT) via MCP-1 secretion and induced an immunosuppressive airway microenvironment, enhancing IL-10 production and shifting macrophages toward a tumor-promoting M2 phenotype. Proteomic analysis revealed distinct differentially expressed protein (DEP) profiles across epithelial and macrophage populations, ultimately resulting in protumorigenic and immunosuppressive outcomes. Notably, functional enrichment analysis of macrophages revealed that EV-driven M2 polarization occurred through the suppression of EGFR activity, a previously underrecognized mechanism that links EV-mediated immune suppression to lung cancer progression.
Conclusions:
This study provides the first functional evidence that NSCLC-derived EVs drive immune suppression and tumor-supportive changes in a human 3D airway model, closely mimicking in vivo lung conditions. The identification of EGFR suppression as a driver of macrophage polarization underscores the need to consider macrophage-specific EGFR regulation in anti-EGFR therapies to prevent unintended protumorigenic effects. These findings pave the way for future studies exploring EV cargo, such as miRNAs, as potential therapeutic targets in NSCLC.