Exosomes Derived from Post-Surgical Drain Fluid of HPV+ Oropharyngeal Cancer Patients Contain HPV DNA and can Induce E6 mRNA Transcription in HPV-Negative Cells

Abstract

<h3>Purpose/Objective(s)</h3> Human papillomavirus (HPV) infection predisposes to oropharyngeal squamous cell carcinoma (OPC). Exosomes are a component of cell-free DNAs that are actively shed by tumor and immune cells. The role of exosomes in cancer metastasis and in HPV-induced carcinogenesis remains unknown. We hypothesize that exosomes derived from surgical drain fluid (SDF) in HPV+ OPC patients contain HPV viral DNA that can be transmitted directly into HPV-negative cells, resulting in transcription of viral oncogenes. <h3>Materials/Methods</h3> Following surgical neck dissection, SDF samples were collected and processed from 21 HPV+ and 5 HPV- OPC patients (discovery cohort). Exosomes were isolated from SDFs using either the Qiagen exoEasy kit or the SBI SEC EV isolation kit. Exosome structure was characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Immunoblotting was performed to verify exosome-specific biomarkers. Exosomes were pretreated with Dnase (NEB), then exosomal DNA was extracted using a cell-free DNA isolation kit (Qiagen). HPV16 copy number was determined by TaqMan quantitative PCR. Validation was then performed using SDF from a separate cohort of 10 HPV+ OPC patients. To examine transcription of exosome-derived HPV DNA, exosomes were isolated from 3 HPV+ SDF and 1 HPV- SDF. After incubating these exosomes with two HPV- cell lines (SCC22A and SCC25), we isolated total cell line RNA, and performed reverse transcriptase PCR (RT-PCR). <h3>Results</h3> TEM and NTA results revealed rounded structures with a size of 70-150 nm, respectively. Immunoblots detected CD63, CD9, and TSG101 in the protein lysates, indicating the vesicles isolated from SDF were indeed exosomes. In the discovery cohort, HPV16 DNA was detected in SDF exosomes isolated from 81% (n = 17) of patients with HPV+ OPC, while SDF-derived exosomes from all (n=5) HPV- patients had undetectable HPV16 DNA. Importantly, paired comparison of HPV16 levels revealed a significant proportion (46.7±34.4%) of HPV16 cell-free DNA in SDF was contained within exosomes. Results from our validation cohort showed that all the exosome samples (100%) isolated from SDF from 10 HPV+ OPC patients contained HPV DNA, regardless of the exosome isolation method; 24.4±14.4% of HPV16 cell-free DNA was localized in exosomes. Furthermore, an <i>in vitro</i> cell culture assay showed that exosomal HPV16 DNA can undergo uptake by SCC22A and SCC25 cells. RT-PCR of RNA from SCC22A cells incubated with HPV DNA-containing exosomes revealed that exosomal HPV16 DNA can be transcribed into HPV16 E6 mRNA in HPV- recipient cells. <h3>Conclusion</h3> SDF, a novel post-surgical biofluid analyte, is enriched for HPV-containing exosomes from HPV+ OPC patients. Exosomes from these HPV+ OPC patients can transmit viral oncogenes to HPV-negative cells, suggesting a novel mode of virus-mediated spread of disease. Furthermore, exosomal HPV DNA can mediate HPV16 E6 mRNA transcription in HPV- recipient cells, indicating its potential role in OPC tumorigenesis.