HPV⁺ Carrying OSCC-EVs Transfer Viral DNA Into Recipient Cells

Objectives: To identify HPV DNA as a component of small extra cellular vesicles (EVs) isolated from supernatants of oral squamous cell carcinoma (OSCC) cells. To demonstrate that EVs produced by HPV⁺OSCC cells transfer viral DNA to HPV ^- cells in vitro. To evaluate biological effects of EVs-mediated transfer on HPV ^- cells
Methods: HPV⁺ OSCC47 cells supernatants were centrifuged at 10000 rpm for 30 min, followed by filtration on 0.22µm filters. Supernatants were concentrated on vivaspin concentrators (MWCO 100000) and 1 ml was then loaded on size exclusion chromatography (SEC) columns to separate EVs. Numbers of isolated HPV⁺ OSCC47-EVs were measured by nanoparticles tracking (NTA) and protein concentration assay BCA, were cocultured with HPV ^- PCI-13 cells for 3 and 6 hours. RT-PCR was used to confirm HPV DNA in isolated HPV⁺ EVs and to detect viral DNA in HPV ^- recipient cells after coincubation. HPV⁺ EVs were labeled with PKH26 stain and coincubated with PCI-13 cells for 30 min,3h and 6h. Uptake of stained HPV⁺ EVs by HPV ^- recipient cells was quantified by flow cytometry. Jurkat cells were cocultured with 2.5ug, 5ug and 10ug of HPV⁺ EVs for 6 hours,and annexin apoptosis assay was perform to analyze biological changes on HPV ^- recipient cells.
Results: PCR results showed an average of 30306 copies of HPV DNA in PCI-13 cells after 6h of coincubation. Flow cytometry analysis showed PKH26-HPV⁺ EVs uptake by PCI-13 cells after 30min,3 and 6h. HPV⁺ EVs induced apoptotic changes in Jurkat T cells after 6h of coincubation.
Conclusions: We demonstrated that HPV DNA is a component of EVs produced by HPV⁺ OSCC cells. HPV⁺ OSCC-EVs can transfer viral DNA and induce apoptosis in HPV ^- recipient cells after coincubation