Differences in Extracellular Vesicle Protein Cargo Are Dependent on Head and Neck Squamous Cell Carcinoma Cell of Origin and Human Papillomavirus Status

Simple Summary Many individuals with head and neck cancer do not survive, even with intense treatment. Patients with HPV-positive tumors generally have better survival; however, for yet unknown reasons, a subset are unresponsive to therapy. One strategy to monitor cancers for progression and recurrence is evaluation of extracellular vesicles, released by tumor cells into the blood and other body fluids. We can also understand differences in tumors and their behavior by comparing the molecules packaged into vesicles that are released from tumor cells. Our study examined differences in the proteins contained within extracellular vesicles released from head and neck cancer cells. We found that key extracellular vesicle proteins differed based on HPV status of the originating cell line and tumor, as well as how responsive the originating tumor was to treatment. Our findings suggest that these extracellular vesicle proteins may be important markers for continued investigation. Abstract To identify potential extracellular vesicle (EV) biomarkers in head and neck squamous cell carcinoma (HNSCC), we evaluated EV protein cargo and whole cell lysates (WCL) from HPV-positive and -negative HNSCC cell lines, as well as normal oral keratinocytes and HPV16-transformed cells. EVs were isolated from serum-depleted, conditioned cell culture media by polyethylene glycol (PEG) precipitation/ultracentrifugation. EV and WCL preparations were analyzed by LC-MS/MS. Candidate proteins detected at significantly higher levels in EV compared with WCL, or compared with EV from normal oral keratinocytes, were identified and confirmed by Wes Simple Western protein analysis. Our findings suggest that these proteins may be potential HNSCC EV markers as proteins that may be (1) selectively included in EV cargo for export from the cell as a strategy for metastasis, tumor cell survival, or modification of tumor microenvironment, or (2) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. This work demonstrates that our method can be used to reliably detect EV proteins from HNSCC, normal keratinocyte, and transformed cell lines. Furthermore, this work has identified HNSCC EV protein candidates for continued evaluation, specifically tenascin-C, HLA-A, E-cadherin, EGFR, EPHA2, and cytokeratin 19.