Characterisation of extracellular vesicles released by HPV+ and HPV- oropharyngeal carcinoma cells in vitro

Key differences between Human papilloma positive (HPV+) and negative (HPV-) oropharyngeal squamous cell carcinoma (OPSCCs), such as tumour stability and patient prognosis, have yet to be fully understood resulting in insufficient treatment and detection options for patients. Extracellular vesicles (EV) are involved in complex cellular signalling and can be secreted from tumour cells to manipulate their environment aiding in tumour growth and survivability through the transfer of protein and RNA molecules. As such, EV research is becoming increasingly important to biomarker and drug development. EVs secreted from four cell lines were characterized by tunable resistive pulse sensing (TRPS) analysis showing a higher rate of EV secretion from 2 HPV- OPSCC lines compared to 2 HPV+ OPSCC lines. Utilizing size exclusion chromatography (SEC) and ultracentrifugation, EVs were purified and RNA and protein cargo extracted. Next generation sequencing showed miRNA profiles differ between these two cancers as hierarchical clustering grouped cell lines by HPV status and differential expression identified 14 miRNAs more abundant in the EVs of HPV+ OPSCC and 19 miRNAs more abundant in HPV- OPSCC EVs. Protein cargo of HPV- OPSCC EVs showed a greater variety of proteins than HPV+, approximately doubling the number of HPV+ EV proteins, and showing an association to cell-adhesion and ECM interactions. Exposing fibroblasts to EVs from both OPSCC types induced changes to α-SMA expression, with immune-fluorescent imaging highlighting a myo-fibroblastic phenotype similar to TGF-β treatment. EVs also stimulated tubule formation of endothelial cells on a matrigel layer suggesting pro-angiogenic properties of these EVs in the tumour stroma. This study demonstrates the importance of EVs in OPSCC development and identifies several key differences between HPV+ and HPV- tumours which may aid in future biomarker and drug development studies.