miRNA cargo from pleural mesothelioma-derived extracellular vesicles reprograms fibroblasts toward an inflammatory CAF-like phenotype

Abstract Pleural mesothelioma (PM) is an aggressive cancer with a low survival rate. A better understanding of disease pathogenesis can aid the development of therapies. This study profiles the miRNA cargo of PM-derived extracellular vesicles (EVs) and examines their effects on fibroblasts, the predominant cell type in the PM tumour microenvironment (TME). High-throughput profiling revealed that EVs from epithelioid and non-epithelioid PM have distinct miRNA cargoes differing from each other and from non-malignant controls. Our data suggest that oncogenic miRNAs were upregulated and tumour-suppressive miRNAs were downregulated in PM-derived EVs. Further, we identified miRs that are exclusively present in either epithelioid or non-epithelioid PM subtypes. These miRs could serve as biomarkers to distinguish PM subtypes and aid in treatment decisions. To analyse the EV-mediated fibroblast response, fibroblasts were treated with isolated EVs, followed by RNA-seq and molecular analyses. Our miRNA–mRNA integrative analysis predicts that differential miRNA cargo from PM-derived EVs modulates multiple pathways in treated fibroblasts. GSEA analysis on KEGG_2021_Human and oncogenic signatures predicts upregulation of pathways including Cell Cycle, Neutrophils and Extracellular Trap Formation, and EZH2-Regulated pathways in treated fibroblasts. Through molecular analysis, we report that a predominant inflammatory CAF-like phenotype was observed post-EV treatment, as reflected by increased levels of markers like IL-6/8, MMP-1/3, ICAM1, ENG, and RRAGD. This was also accompanied by enhanced oncogenic signalling reflected by elevated Osteopontin, thrombospondin, Cystatin C, autotaxin, AXL, and ERBB2 levels. Additionally, our data show that PM-EVs modulate epigenetic regulation by altering EZH2 phosphorylation at Thr345: epithelioid EVs increased p-EZH2 levels, and non-epithelioid EVs decreased p-EZH2 levels. Further, our data show that PM-EV-mediated fibroblast reprogramming can increase PM cell migration and proliferation, thus promoting a tumour-supportive microenvironment. Collectively, this study highlights the regulatory roles of PM-EVs. The predicted EV-driven signalling pathways in PM-TME may represent potential therapeutic avenues for future therapeutic research.