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

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Alonso-Crisostomo, Luz 
Trendell, Jennifer 
Ferraresso, Marta 
Bailey, Shivani 
Ward, Dawn 

Malignant germ-cell-tumours (GCTs) are characterised by microRNA (miRNA/miR-) dysregulation, 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 within 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 co-culture, TGCT-derived EVs, and a miRNA overexpression system (miR-371a-OE) to assess functional relevance. TGCT cells secreted EVs into culture media. MiR-371~373 and miR-302/367 cluster miRNAs were overexpressed 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. TGCT co-culture resulted in increased levels of miRNAs from the miR-371~373 and miR-302/367 clusters in TME (fibroblast) cells. Next, fluorescent labelling demonstrated TGCT-derived EVs were internalised by all TME (fibroblast/endothelial/macrophage) 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 (eg, miR-205-5p/miR-148-3p) and subtype-specific (seminoma, eg, miR-203a-3p; yolk-sac-tumour, eg, miR-375-3p) miRNAs. MiR-371a-OE in TME cells resulted in increased collagen contraction (fibroblasts) and angiogenesis (endothelial cells), via direct mRNA downregulation and alteration of relevant pathways. TGCT cells communicate with nontumour stromal TME cells through release of EVs enriched in oncogenic miRNAs, potentially contributing to tumour progression.

extracellular vesicles, germ cell tumour, microRNA, testicular, tumour microenvironment, Male, Female, Humans, MicroRNAs, Seminoma, Endothelial Cells, Tumor Microenvironment, Biomarkers, Tumor, Testicular Neoplasms, Neoplasms, Germ Cell and Embryonal, Extracellular Vesicles
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Int J Cancer
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St Baldrick's Foundation (via Dana-Farber Cancer Institute) (2015-0743)
National Institute for Health and Care Research (IS-BRC-1215-20014)
The authors acknowledge grant funding the St. Baldrick’s Foundation (reference 358099), the Gwyneath Pretty Research Fund PhD Studentship (Department of Pathology, University of Cambridge), and the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). We are grateful for support from the Max Williamson Fund and from Christiane and Alan Hodson, in memory of their daughter Olivia. The funders were not involved in study design, data collection or interpretation, or decision to submit for publication. Views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care.