Hypoxia enhance interactions of tumor-derived small extracellular vesicles with immune cells through glycosylation modification in head and neck squamous cell carcinoma 9409

Description Introduction Hypoxia, a hallmark of solid tumors, drives aggressive phenotypes. HNSCC exhibits extensive hypoxic regions and high recurrence rates. Our previous research demonstrated that hypoxia induces excessive release of tumor-derived sEVs that reprogram the tumor microenvironment. Materials and Methods HNSCC cell lines (FaDu, PCI-30, SCC-25) and normal keratinocytes (HaCaT) were cultured under hypoxic (1% O2) or normoxic (21% O2) conditions. sEVs were isolated from supernatants and plasma of HNSCC patients (n = 10) and healthy donors (n = 10) using SEC, characterized by WB, TEM, and NTA. Glycosylation modifications were analyzed using mass spectrometry, bulk and single-cell RNA sequencing, and publicly databases (eg.TCGA). Glycan patterns were analyzed by flow cytometry. Results sEVs (85-135nm) expressed CD63, CD9, TSG101, and Alix, but lacked Calnexin and Grp94. TCGA analysis idetified glucosylation-related genes (LG3BP, LEG1, OST48, DAD1, B4GALNT1, PYGL, ALG3, EXT2, KDELC1) significanlty upregulated, correlated with advanced tumor stage, and associated with poorer survival in HNSCC patients, with hypoxia and vesiculation-related gene sets, and preferentially expressed in HPV(-) tumors, enriched in hypoxic areas. Galectin-1 and -3 expression was predominant in immune cells subsets, while Galectin-3 binding protein in hypoxia-derived HNSCC sEVs. Conclusions Tumor cells adapt to hypoxia by glycosylation modifications of sEVs which might interact with immune cells.