Glycosylation patterns of tumor-derived small extracellular vesicles and hypoxia signatures in head and neck squamous cell carcinoma.

Abstract

e18067 Background: Hypoxia characterizes solid tumors and results in increased tumor resistance. Head and neck squamous cell carcinoma (HNSCC) characterizes hypoxic areas and despite new treatment modalities high recurrence level. Hypoxia interferes in glycosylation modifications. In previous study we demonstrated that hypoxia induces release of small extracellular vesicles (sEVs) by tumor cells. Specific sEV glycosylation profiles were found to be distinct in cancer, but not have been studied in HNSCC yet. Methods: HNSCC cell lines FaDu, PCI-30, SCC-25 and normal keratinocytes HaCaT were cultured under hypoxic (1% O 2 ) or normoxic (21% O 2 ) through 72h. sEVs were isolated from supernatants and from HNSCC patients plasma (n=10), and heathy donors (n=10) using size exclusion chromatography; characterized by western blot, electron microscopy and nanoparticle tracking analysis. Proteomic and transcriptomic analysis of glycosylation modifications were performed with the use of high resolution mass spectrometry and bulk and single cells RNAseq analysis with the use of TCGA and HNSC_GSE103322, HNSC_GSE139324, LSCC_GSE150321, NPC_GSE150430, NPC_GSE162025, OSCC_GSE172577, HNSC_GSE180268 databases. Flow cytometry was used to assess glycan patterns. Results: sEVs ranges in size from 85-135 nm, and carry CD63, CD9, TSG101 and Alix, but not Calnexin nor Grp94. TCGA analysis revealed glycosylation-associated genes which were upregulated, significantly correlated with poorer survival of HNSCC patients and with tumor stage including LG3BP, LEG1, OST48, DAD1, B4GALNT1, PYGL, ALG3, EXT2, KDELC1. Negative correlation with survival showed FUT2 and Siglec receptor 2. For all of these genes correlation with hypoxia hallmark and vesiculation-related geneset was significantly higher in HNSCC patients compared to normal controls. Significantly higher expression of LG3BP, LEG1, B4GALNT1, PYGL, EXT2, KDELC1 was observed in HPV(-) tumors, which are more abundant in hypoxic areas. Among genes of significantly negative impact on survival single cells sequencing revealed upregulation of Galectin-1 (LEG1) in immune cells subsets. MS-based comparative analysis between tumor and normal cells revealed that hypoxia significantly upregulated mannose receptor (MPRD) and downregulated oligosaccharyl transferase (OST48) and dolichyl-diphosphooligosaccharide-protein glycosyltransferase (RPN1)in tumor cells, while in sEVs uniquely induce packaging galectin-3 binding protein (LG3BP). HNSCC patients sEV glycan profiling revealed significant downregulation of ConA lectin binding compared to normal controls, similarly in hypoxia-derived tumor sEVs. Conclusions: Tumor cells adapt to hypoxia by alterations in glycosylation pattern of sEVs and parent cells which might serve as a hypoxia signature and pharmacological intervention target.