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Tissue hypoxia is commonly observed in head and neck squamous cell carcinomas (HNSCCs), resulting in molecular and functional alterations of the tumor cells. The aim of this study was to characterize tumor-derived small extracellular vesicles (sEVs) released under hypoxic vs. normoxic conditions and analyze their proteomic content. HNSCC cells (FaDu, PCI-30, SCC-25) and HaCaT keratinocytes were cultured in 21, 10, 5, and 1% O 2 . sEVs were isolated from supernatants using size exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis, electron microscopy, immunoblotting, and high-resolution mass spectrometry. Isolated sEVs ranged in size from 125-135 nm and contained CD63 and CD9 but not Grp94. sEVs reflected the hypoxic profile of HNSCC parent cells: about 15% of the total detected proteins were unique for hypoxic cells. Hypoxic sEVs expressed a common signature of seven hypoxia-related proteins (KT33B, DYSF, STON2, MLX, LIPA3, NEK5, P12L1) and were enriched in pro-angiogenic proteins. Protein profiles of sEVs reflected the degree of tumor hypoxia and could serve as potential sEV-based biomarkers for hypoxic conditions. Adaptation of HNSCC cells to hypoxia is associated with increased release of sEVs, which are enriched in a unique protein profile. Thus, tumor-derived sEVs can potentially be useful for evaluating levels of hypoxia in HNSCC.

Small Extracellular Vesicles from Head and Neck Squamous Cell Carcinoma Cells Carry a Proteomic Signature for Tumor Hypoxia