Exosome Uptake from Lung Adenocarcinoma and Squamous Cell Carcinoma Alters T Cell Cytokine Expression and Modulates Protein Profiles in Exosome Biogenesis

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Abstract
Despite advances in immunotherapy, non-small cell lung carcinoma (NSCLC) clinical success is limited, possibly due to substantial immunological alterations in advanced cancer pa-tients. This study examines the immunomodulatory effects of exosomes derived from lung adeno-carcinoma (ADC) and squamous cell carcinoma (SCC) on T cells. Methods: Exosomes were isolated from A549 (lung adenocarcinoma), SKMES1 (lung squamous cell carcinoma), and Jurkat-E6.1 (T cells). Exosome size and morphology were analysed by NTA and TEM, respectively, while western blotting confirmed exosome markers. Exosome quantity was measured by the BCA assay. Exosome uptake was assessed, followed by resazurin assay, RNA isolation, quantification, cDNA prepara-tion, RT-PCR, nano LC-MS, and bioinformatic analysis, before and after treating Jurkat-E6.1 cells with exosomes from A549 and SKMES1. Results: Cancer-derived exosomes were efficiently in-ternalized by immune cells, reducing T-cell viability. Real-time PCR showed increased expression of TGFB, GZMB, and BAX, alongside decreased IL2, indicating an immunosuppressive effect. IL10 was increased more in ADC-treated cells than in SCC-treated cells. Higher levels of KI67 and BCL2 suggested a compensatory T-cell response. Proteomic analysis revealed 39 differentially abundant proteins (DAPs) in ADC-treated T cells and 276 in SCC-treated T cells, with 19 shared DAPs. Gene Ontology (GO) analysis of these DAPs highlighted processes such as exosome biogenesis, metabolic pathways, and regulatory functions, with ADC exosomes influencing NAD metabolism, ECM binding, and oxidoreductase activity, while SCC exosomes affected mRNA stability, amino acid metabolism, and cadherin binding. Shared DAPs (19) were mostly linked to nucleic acid metabo-lism. The cytoplasmic colocalization suggested the presence of these proteins in the cellular and extracellular lumen, indicating further release of these proteins in the vesicles by T cells. Conclusion: Lung cancer-derived exosomes regulate T-cell activities through immunoregulatory signaling without cell-cell contacts. The molecular interactions between exosomes and immune cells can re-veal novel tumor immune regulatory mechanisms and therapeutic targets.
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