SEV Released By Glioma Under Hypoxia Induce Transition Of Glioma Stem Cells Into Pericytes And Promote Tumor Angiogenesis

Objective:This study sought to assess whether glioma-derived small extracellular vesicles(sEVs)generated under hypoxic conditions could drive glioma stem cell(GSC)pericyte differentiation and tumor angiogenesis.Methods: An ultracentrifugation strategy was used to isolate glioma-derived sEVs isolated under hypoxic or normoxic conditions.Transmission electron microscopy(TEM),Western blotting,and nanoparticle tracking analysis(NTA)were used to after which these particles were characterized these isolated particles.These sEVs were then used to treat GSCs and HUVEC cells,with cytochalasin D being added to disrupt cellular sEV uptake.A CCK-8 assay was used to evaluate GSC and HUVEC proliferation,while wound healing and Transwell assays were used to assess cell migratory activity.A tube formation assay was used to evaluate angiogenic activity,while ELISAs and Western blotting were used to assess changes in protein levels.The interactions between GSCs and sEVs in vivo were additionally evaluated using a subcutaneous xenograft tumor model system in nude mice.Results: The sEV under hypoxic conditions induced significant higher migration,proliferation and tube formation in HUVEC and GSC,as compared to those under normoxic conditions.Hypoxia-derived sEV treatment was associated with the upregulation of pericyte-related proteins including ?-SMA,Desmin,and CD146 in GSCs and with the activation of TGF-? pathway signaling and p-Smad2/3 expression.Contrastingly,this treatment was also associated with the downregulation of GSC markers including Sox2,Nestin,and CD133.Cytochalasin D treatment partially disrupted these effects.In addition,we found that treatment with Ibrutinib and Bevacizumab reduced tumor growth,angiogenesis,and pericyte quantities in vivo.Conclusion: Our findings suggest that glioma-derived sEVs produced under hypoxic conditions can promote HUVEC and GSC proliferation,migration,and tube formation while inducing differentation of GSCs into pericytes.These effects of the hypoxia-derived sEVs were also involved in triggering tumorigenesis and angiogenesis in vivo.