Effect Of Extracellular Vesicles Derived From Head And Neck Squamous Cell Carcinoma On Macrophages After Hypoxia And Radiotherapy

Head and neck cancer is the sixth cancer in the world,with a 5-year overall survival rate of only 50%.More than 90% of head and neck cancer are head and neck squamous cell carcinoma.According to previous research of our group,extracellular vesicles play a role in information communication in the tumor microenvironment,and cancer cells are keen to use this communication tool to interact with immune cells and assist tumor immune escape.Therefore,this study explored the immune effects of extracellular vesicles(EV)secreted by head and neck squamous cell carcinoma under hypoxia and radiation stimulation on macrophages,and we used metabolomics to explore some different metabolic pathways.Methods:(1)Extraction and identification of extracellular vesicles: Collect the cell supernatants of head and neck squamous cell carcinoma cells after different treatments of normoxia,normoxia radiotherapy,hypoxia,and hypoxia radiotherapy,and extract sufficient extracellular vesicles by ultracentrifugation.The extracellular vesicles were identified by Transmission Electron Microscope,Nanosight,and Western Blot.(2)Co-culture model of extracellular vesicles and macrophages: The supernatants and extracellular vesicles derived from head and neck squamous cell carcinoma after different treatments and the obtained,which were co-cultured with human acute monocytic leukemia cells(THP-1),leukemia cells in mouse macrophage(RAW264.7)and mouse bone marrow-derived macrophages(BMDM)to investigate the effect on macrophages.(3)Metabolomics analysis: The extracellular vesicles extracted from the supernatant of head and neck squamous cell carcinoma cells after hypoxia or radiotherapy treatments were subjected to high-throughput non-targeted metabolomics sequencing by HPLC-MS/MS,and then using statistical methods such as PCA,heat map for difference analysis.Research result:(1)Extracellular vesicles,which were derived from head and neck squamous cell carcinoma cells after different treatments,have a complete lipid bilayer structure with a size between 30 and 200 nm,and the membrane surface expresses specific biomarkers CD63,CD81,HSP-70,etc.And they can be effectively taken up and utilized by macrophages.(2)Compared with normal oxygen,the supernatant and extracellular vesicles from cancer cells can inhibit the growth of macrophages under hypoxia.Under normal oxygen concentration,the supernatant and extracellular vesicles from cancer cells after radiotherapy inhibit the proliferation of macrophages,but under hypoxic conditions,the opposite results are shown.In addition,the supernatant of cancer cells treated with hypoxia and their secreted vesicles,compared with the normoxia group,can promote the migration of macrophages.The supernatants and vesicles of cancer cells after radiotherapy can promote the migration of macrophages.Both under normoxia and hypoxia conditions inhibit the migration of macrophages.(3)THP-1,RAW264.7 and BMDM cells will promote the polarization of M2 after hypoxic EV treatment,compared with normoxia EV treatment group.Non-targeted metabolomics analysis shows that different cells and different treatment groups involve multiple metabolic pathways.Conclusions: The cell supernatant and extracellular vesicles are derived from head and neck squamous cell carcinoma after hypoxia or radiotherapy have different effects on the proliferation,migration and polarization of macrophages.In addition,through metabolomics analysis,we found that the extracellular vesicles of SCC-Ⅶ and Cal-27 have many different metabolites between different treatment groups,and multiple metabolic pathways are involved.These pathways may be potential mechanisms affecting the proliferation,migration and polarization of macrophages.