The role of extracellular vesicles in the oral squamous cell carcinoma tumour microenvironment

Head and neck cancer is the 6th most common cancer worldwide with 90% being classified as oral squamous cell carcinoma (OSCC). Recent findings have implicated the tumour microenvironment (TME), which includes cells such as cancer-associated fibroblasts (CAFs), in cancer progression. Tumour cells are known to communicate with stromal cells via a network of cytokines, growth factors and extracellular vesicles (EVs). There is evidence that some cargo are presented on the surface of EVs, such as transforming growth factor beta 1 (TGF-β1), and can induce CAF differentiation.
We therefore sought to determine the role of EVs in promoting a CAF like phenotype.
A cell panel including an immortalised normal oral keratinocyte cell line, an OSCC cell line and primary patient derived normal oral fibroblasts (NOFs) and CAFs were used in this study. An EV deficient OSCC cell line (H357ΔHGS) and a fluorescent EV reporter OSCC cell line (H357CD63-GFP) were characterised and validated by nanoparticle tracking analysis (NTA), quantitative polymerase chain reaction (qPCR), western blotting and immunofluorescence microscopy (IF). GFP tagged EV uptake in NOFs was visualised by fluorescence imaging. EVs were enriched by size exclusion chromatography (SEC) and characterised by NTA and western blotting. TGF-β1 was quantified by ELISA. CAF markers and phenotype was assessed using molecular techniques and functional assays.
There were significantly higher levels of TGF-β1 associated with EVs derived from oral cancer cells (H357) compared to normal cells (FNB6). TGF-β1 levels were significantly greater in EV preparations compared to conditioned media (CM). Treatment of NOFs with soluble recombinant TGF-β1 or H357 derived EVs led to an increase in α-SMA stress fibre formation, however this was abrogated with the use of a TGF-β neutralising antibody, indicating an EV-TGF-β dependent role in NOF activation. Furthermore, compared to untreated cells, H357 derived EVs enhanced NOF migration and contraction. The cancer derived EVs promoted greater NOF contraction at 24 and 48 hours compared to their normal cell -derived EV counterparts.. These results indicate that inhibition of EV associated TGF-β1 might be a novel strategy to reduce stromal cell activation in the TME.