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

SIMPLE SUMMARY: Tissue hypoxia is a hallmark of head and neck squamous cell carcinoma (HNSCC) and is considered to drive tumor progression and resistance to anti-cancer therapies. The aim of our study was to characterize the influence of hypoxic environments on the release and proteomic cargo composition of small extracellular vesicles (sEVs). We demonstrated in three HNSCC cell lines that sEV secretion is enhanced in response to hypoxic conditions and that hypoxic sEVs carry distinct proteomic profiles, which can not only discriminate between normoxic and hypoxic conditions, but also discriminate between various degrees of tissue hypoxia. Therefore, sEVs are a potential resource for monitoring tissue hypoxia in HNSCC or even anti-angiogenic or vessel normalization therapies. ABSTRACT: 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.