Evaluation of extracellular vesicle biogenesis in oral squamous cell carcinoma

Oral cancer cells produce increased numbers of extracellular vesicles (EVs) compared to normal controls. However, the underlying mechanism was previously unknown. The endosomal sorting complex required for transport (ESCRT) subunits are implicated in cellular processing of ubiquitinated proteins such as epidermal growth factor receptor (EGFR) and EV biogenesis. We hypothesised that ESCRT members are overexpressed in oral cancer cells, leading to increased production of EVs. In order to test this hypothesis, gene expression and protein abundance of representative ESCRT-0, -I, -II subunits in an immortal normal oral keratinocyte cell line and oral cancer cell lines were compared to primary oral keratinocytes by qPCR and western blotting, respectively. Co-localisation of HGS (ESCRT-0) with early and late endosomal markers (EEA1 and RAB7) was determined by immunofluorescence. CRISPR-Cas9 genome editing was used to knockout HGS in the H357 oral cancer cell line. Endosomal structures were imaged by transmission electron microscopy (TEM). EVs were enriched by ultracentrifugation and analysed by nanoparticle tracking analysis (NTA) and western blotting. Processing of EGFR was tracked by EGF pulse-chase experiments. HGS protein abundance was >4-fold higher in oral cancer cell lines compared to normal controls. Immunofluorescence revealed that HGS co-localised with early endosomal structures (EEA1). Enlarged endosomes were visualised in the HGS knockout cell line by TEM and a 3-fold reduction in particle release was observed by NTA. EGFR processing was stalled in the HGS mutant leading to cellular accumulation and a significant decrease in EV-associated EGFR.