Effect Of Extracellular Vesicles Derived From Esophageal Squamous Cell Carcinoma On Angiogenesis

ObjectiveTo investigate the effect of extracellular vesicles(EVs)derived from esophageal squamous cell carcinoma(ESCC)on angiogenesis,thus laying a foundation for studying the mechanism of EVs on angiogenesis in ESCC and providing a new therapeutic target for ESCC.Methods(1)EVs in supernatant of ESCC cell line Eca-109 was extracted by ultra-high speed centrifugation,and its morphological characteristics were observed by transmission electron microscope.Western blot was used to detect the relevant surface markers;(2)After incubation with green fluorescent dye DIO labeled EVs and red fluorescent dye CM-Dil labeled human venous endothelial cell(HUVEC),the uptake of EVs by HUVEC was observed by laser confocal microscope;(3)CCK-8 and Brd U assay were used to detect the effect of EVs on the activity and proliferation of HUVEC;(4)The effect of EVs on HUVEC migration ability was tested through scratch assay and Transwell assay;(5)The effect of EVs on HUVEC angiogenesis was detected by tubule formation assay;(6)After subcutaneous tumor-bearing of ESCC cell line Eca-109 into nude mice,EVs was injected into the tumor body in situ to evaluate the tumorigenicity of EVs;Immunohistochemistry was used to detect the effect of EVs on CD31 expression of vascular endothelial cell marker in tumor body.Results(1)EVs extracted by ultra-high-speed centrifugation shows round or oval vesicle structure under electron microscope;Western blot results showed that ESCC cell line Eca-109 and its EVs expressed CD9,CD63.(2)Confocal microscopy showed that EVs could be internalized by HUVEC;(3)The rsesults of CCK8 assay showed that the activity of HUVEC increased significantly after EVs treatment with different concentrations,which showed a does-dependent manner;Brd U results also confirmed that HUVEC proliferation increased significantly after EVs treatment with different concentrations.(4)Scratch and Transwell assay results showed that HUVEC migration ability increased significantly after EVs treatment at different concentrations,which showed a does-dependent manner;(5)The experimental results of tubule formation showed that EVs of different concentrations can significantly promote the formation of blood vessels,which showed a does-dependent manner;(6)The subcutaneous tumor-bearing test results of nude mice showed that the size and weight of EVs tumor after injection were significantly higher than those in control group.Immunohistochemical results showed that CD31 expression in the experimental group was significantly higher than that in control group.Conclusions(1)EVs secreted by ESCC can significantly promote the proliferation,migration and tubule formation of HUVEC,the mechanism of which may be related to the endocytosis of EVs by HUVEC;(2)EVs can promote angiogenesis in tumor body,thus promoting tumor growth.