Testicular germ cell tumour cells release microRNA-containing extracellular vesicles that induce phenotypic and genotypic changes in cells of the tumour microenvironment

MicroRNAs (miRNAs/miR-) are short, non-protein coding RNAs that are dysregulated in malignant germ cell tumours (GCTs), with universal over-expression of miR-371~373 and miR-302/367 clusters regardless of patient age, tumour site or subtype (seminoma/yolk-sac-tumour/embryonal carcinoma). These miRNAs are released into the bloodstream, presumed via extracellular vesicles (EVs), and represent promising biomarkers. Here, we comprehensively examined the role of EVs, and their miRNA cargo, on (fibroblast/endothelial/macrophage) cells representative of the testicular GCT (TGCT) tumour microenvironment (TME). Small RNA next generation sequencing was performed on 34 samples, comprising representative malignant GCT cell lines/EVs and controls [testis fibroblast (Hs1.Tes) cell-line/EVs and testis/ovary samples]. TME cells received TGCT-derived EV treatment, miRNA quantification by qRT-PCR, and a miRNA overexpression system (miR-371a-OE) to perform functional assays and assess mRNA changes. TGCT cells secreted EVs into culture media. MiR-371~373 and miR-302/367 cluster miRNAs were over-expressed in all TGCT cells/subtypes compared with control cells and were highly abundant in TGCT-derived EVs, with miR-371a-3p/miR-371a-5p the most abundant compared with normal EVs. Fluorescent labelling demonstrated TGCT-derived EVs were internalised by all TME cells. TME (fibroblast/endothelial) cell treatment with EVs derived from different TGCT subtypes resulted in increased miR-371~373 and miR-302/367 miRNA levels, and other generic (e.g., miR-205-5p/miR-148-3p), and subtype-specific (seminoma, e.g., miR-203a-3p; yolk-sac-tumour, e.g., miR-375-3p) miRNAs. MiR-371a-OE in TME cells resulted in increased collagen contraction (fibroblasts) and angiogenesis (endothelial cells), associated with dysregulation of mRNAs and relevant cellular pathways. TGCT cells communicate with non-tumour stromal TME cells through release of EVs enriched in oncogenic miRNAs, likely contributing to tumour progression.