Testicular germ cell tumours (TGCT) are the most common malignancy in young adult men and are characterised by their broad heterogeneity in terms of differentiation stage, histological presentation and capacity for progression. In addition, the tumour microenvironment (TME) of these tumours varies depending on TGCT histological subtype and clinical aggressiveness. Despite this heterogeneity, all malignant TGCTs overexpress the microRNA (miRNA) clusters miR-371~373 and miR-302/367, which robustly segregate malignant samples from non-malignant controls. These miRNAs are found at high levels in serum of malignant TGCT patients and represent highly sensitive and specific biomarkers for this disease. However, very little is known about the potential role of these miRNAs in TGCT progression, the mechanisms through which they are secreted by tumour cells, and whether they exert any biological function in surrounding stromal cells before reaching the bloodstream. The present body of work aimed to gain new insights into the influence of TGCT cells on their surrounding microenvironment via the exchange of miRNAs and extracellular vesicles (EVs) and shed light on the cellular mechanisms underpinning TGCT modulation of the TME.

EVs are key mediators of intercellular communication and have been widely shown to promote tumour progression through the exchange of oncogenic molecules between the tumour and the TME in many cancer types. Here, TGCT-overexpressed miRNAs were found to be highly abundant in EVs derived from TGCT cells. Moreover, TGCT-derived EVs were efficiently internalised by TME cells, including fibroblasts, macrophages and endothelial cells. EV-uptake was followed by increased detection of TGCT-overexpressed miRNAs in recipient cells, suggesting the existence of an EV-dependent active transport of these molecules between tumour cells and other surrounding non-tumour cells. Interestingly, the treatment of fibroblasts with TGCT-derived EVs resulted in changes in myofibroblast-associated gene expression suggesting movement towards a cancer-associated fibroblast phenotype. However, in silico prediction analysis did not find strong correlations between the studied TGCT-overexpressed miRNAs and the markers being modulated after TGCT-derived EV treatment, implying that other gene expression regulators might be contained within TGCT-derived EVs.

In order to elucidate the drivers of this gene expression modulation on EV-recipient cells, as well as discovering new molecules involved in TGCT progression, the transcriptional landscape of TGCT cells and EVs was investigated. Next-generation sequencing (NGS) was employed for this purpose and small RNA species defined in the various TGCT subtypes and controls. Various EV-contained miRNAs were newly identified and were shown to segregate malignant from non-malignant samples, as well as characterise specific TGCT-subtypes. Interestingly, the -5p form of some TGCT-overexpressed miRNAs was found to be significantly more abundant in EVs than cells, despite the -3p form also being predominantly detected in affected patient sera, opening new questions about the role of these -5p miRNA strands on TME modulation and uptake.

Finally, the potential oncogenic function of the most commonly overexpressed miRNAs in malignant TGCT tissues (miR-371a-3p) and TGCT-derived EVs (miR-371a-5p) was investigated in cells typically found within the testis TME. To achieve this, stable overexpression of the double stranded miRNA-371a precursor was induced in fibroblasts, macrophages and endothelial cells and phenotypic alterations observed; changes in transcriptional modulation were interrogated by global mRNA microarray gene expression analysis.

Taken together, the data presented in this PhD thesis provides evidence that TGCTs are able to communicate with their surrounding microenvironment through the release of EVs and that these TGCT-derived EVs contain high levels of miRNAs. Work is ongoing to establish how these miRNAs may alter the post-transcriptional response in EV-recipient cells, a potential mechanism through which they could contribute to tumour development. In parallel, EV-contained miRNAs associated with specific TGCT subtypes were identified, which could potentially represent new candidate biomarkers of disease.