Pleural mesothelioma cell-derived sEVs promote epithelial-to-mesenchymal transition and T-cell suppression through the PD-L1 pathway

Background Pleural mesothelioma (PM) is a highly aggressive malignancy with limited durable responses to immune checkpoint blockade, largely due to poorly defined tumour-intrinsic mechanisms of immune evasion. Small extracellular vesicles (sEVs) have emerged as key mediators of tumour progression, yet their role in coordinating phenotypic plasticity and suppressing anti-tumour immunity in PM remains poorly understood. Methods Given the central role of epithelial-to-mesenchymal transition (EMT) in tumour aggressiveness, we investigated whether PM cells of the biphasic histologic subtype exploit sEVs to propagate EMT-associated and immunomodulatory signals. sEVs were isolated from human (MSTO-211H, NCI-H28) and murine (AB1, AB22) PM cell lines using differential ultracentrifugation and comprehensively characterized via nanoparticle analysis, immunoblotting, proteomics, and cytokine profiling. A 3D multicellular organoid model incorporating immune cells was used to assess sEV-mediated effects on the tumour immune microenvironment. Results Biphasic/mesenchymal PM cells and their sEVs were selectively enriched in TGF-β1 and EMT-related transcriptional regulators. Consistently, exposure of epithelial NCI-H28 and AB22 cells to sEVs from biphasic PM cells induced hallmark EMT features, including reduced EpCAM, increased vimentin expression, and enhanced migration and proliferation, as confirmed by immunofluorescence. Notably, PD-L1 was significantly elevated in biphasic cell-derived sEVs, prompting functional evaluation in a physiologically relevant 3D tumour–immune co-culture system. PD-L1–positive sEVs suppressed CD8⁺ T-cell proliferation and decreased CD8 expression, demonstrating a direct vesicle-mediated mechanism of immune suppression. Tumour microenvironment profiling of biphasic PM further revealed a stromal-rich and immune-suppressed niche consistent with these effects. These findings establish that biphasic PM-derived sEVs act as drivers of EMT and PD-L1–mediated T-cell inhibition, implicating sEV-associated signalling pathways as promising targets to overcome immunotherapy resistance in pleural mesothelioma. Conclusions Biphasic/mesenchymal PM-derived sEVs drive phenotypic transition and immune reprogramming in pleural mesothelioma. Targeting sEV-mediated intercellular communication may represent a novel strategy to enhance immunotherapy efficacy in this disease.