Proteomes of exosomes from HPV(+) or HPV(-) head and neck cancer cells: differential enrichment in immunoregulatory proteins

Human papillomavirus (HPV) is an etiologic factor in head and neck squamous cell carcinoma (HNSCC). HPV(+) cancers respond favorably to therapy potentially due to more robust anti-tumor immune responses. We hypothesized that tumor-derived exosomes (TEX) produced by HPV(+) or HPV(-) HNSCCs differentially modulate anti-tumor immune responses. Proteomes of exosomes from HPV(+) and HPV(-) HNSCC cell lines were compared in search for proteins putatively involved in the communication with immune system. TEX were isolated from supernatants of HPV(+) (SCC-2, SCC-47, and SCC-90) or HPV(-) (PCI-13 and PCI-30) cells by size exclusion chromatography. A comparison of proteome profiles was performed by high-resolution mass spectrometry. The presence and biological activity of selected immunoregulatory proteins were validated by flow cytometry and co-incubation assays. Exosomes produced by SCC-90 and PCI-30 cells contained 711 proteins, including 80 proteins specific for HPV(+) exosomes and 77 specific for HPV(-) exosomes, associated with similar GO terms such as regulation of cell growth, metabolism, communication, and cellular signaling. Search for proteins localized in the membrane and involved in immune regulation identified a few proteins detected specifically in HPV(+) or HPV(-) exosomes. Only HPV(+) exosomes were enriched in immune effector cell-related CD47 and CD276 antigens; only HPV(-) exosomes contained tumor-protective/growth-promoting antigens, MUC-1 and HLA-DA. Flow cytometry and Western blots confirmed the reciprocal presence/paucity of these proteins in a whole panel of tumor cells and corresponding exosomes. The differential content of protein cargos in HPV(+) and HPV(-) exosomes might contribute to the disparity in immune responses that characterize HPV(+) and HPV(-) HNSCC.