Effect Of Extracellular Vesicles Derived From Glioma On Tumor Microenvironment

Objectives:The microenvironment of glioma is a key factor in the progression of glioma.Glioma cells communicate with and manipulate various cells in the glioma microenvironment through extracellular vesicles(EVs),thereby reprograming their survival microenvironment to support tumor progression and resistance to treatment.This topic is to study the effects of Cavin1 on the production and function of glioma-derived-EVs,as well as the role of EVs played in communacation of glioma cells to neighborhood glioma cells,microglia and endothelial cells,thereby providing new strategies for the treatment of glioma.Methods:1.Through computational structural biology methods,including sequence alignment,homology modeling and molecular docking simulation,we design a variant Cavin1(vCavin1)that does not interact with Caveolin1.Through scanning electron microscopy analysis,BCA assay,Western blot experiments,NTA analysis,etc.,the effect of Cavin1 on the production of glioma EVs was detected.2.Flow cytometry and confocal fluorescence microscopy analysis were performed to detect the effect of Cavin1 on the uptake of glioma EVs by recipient glioma cells.The CCK-8 cell proliferation assay was used to detect the effect of glioma EVs on the proliferation of recipient glioma cells.Through fluorescent labeling and live animal imaging,the ability of glioma EVs homing to glioma is detected.3.Through in vitro microglial cell migration experiments and orthotopic glioma mouse models,the effects of glioma cells on microglia through EVs were explored.4.The iTRAQ/TMT mass spectrometry method was used to study the proteomic changes of endothelial cells after treatment with glioma EVs,and the mass spectrometry results were verified through Western blot.The relevant signal pathway was found out through GO analysis and protein network analysis and verified in endothelial cells using STAT3 phosphorylation inhibitor WP1066.5.The influence of glioma-EVs and NGAL protein on endothelial cell membrane fluidity was detected through FRAP assay,and transmission electron microscope was used to observe and analyze the endocytosis of endothelial cells.The influence of glioma EVs and NGAL protein on the permeability of BBB to nanocapsules was detedcted.6.Effect of MK2206,an inhibitor of glioma EVs,on the entry of nanocapsules through the BBB into glioma tissue was studied using the orthotopic glioma model.The effects of different application sequences of tAb nanocapsules and MK2206 on the treatment of gliomas were explored through the orthotopic glioma model.Results:1.The high expression of Cavin1 significantly increased the EV production,while the expression of vCavin1 had no significant effect.2.Cavin1 promoted the uptake of glioma-EVs by recipient glioma cells LN229 and EVs expressing Cavin1 promoted the proliferation of recipient cells LN229 in a dosedependent manner,but vCavin1 could not.3.Cavin1 promoted the recruitment and activation of microglial BV2 caused by glioma EVs,but vCavin1 cannot.4.Mass spectrometry results showed that glioma EVs caused a significant increase in endothelial cell NGAL levels,and it was probably caused by the JAKs-STAT3signaling pathway.5.Through improving NGAL level in endothelial cells,glioma EVs significantly increased the membrane fluidity and the number of endocytic vesicles in endothelial cells,and improved the permeability of BBB to nanocapsules.6.The small molecule inhibitor MK2206 leads to a significant decrease in the number of nanocapsules passing through the BBB,which is related to its blocking of AKT phosphorylation,resulting in a decrease in the release of extracellular vesicles in gliomas.Compared with the sequential administration of tAb nanocapsules-MK2206,the number of nanoparticles entering the glioma area was significantly less in the sequential administration of MK2206-tAb nanocapsules or the tAb nanocapsules alone.Conclusions:1.Cavin1 increased the production of EVs secreted by glioma cells U87,promoted the uptake of EVs by recipient cells,enhance the promoting effect of EVs on the proliferation of recipient glioma cells,improved the homing ability of glioma EVs to glioma,and enhanced microglia recruitment and activation mediated by glioma EVs.The above effects of Cavin1 were achieved through the interaction with Caveolin1.Therefore,the Cavin1-Caveolin1 interaction is expected to become a new therapeutic target for gliomas with high Cavin1 expression levels.2.EVs secreted by gliomas acted on cerebral vascular endothelial cells b End.3,through the JAKs-STAT3 signaling pathway,causing increased NGAL levels,resulting in enhanced membrane fluidity and endocytosis of endothelial cells,so that more applied tAb-nanomicrocapsules can enter through the BBB into glioma tissue.Therefore,the application of tAb nanocapsules before the application of MK2206 can achieve the greatest benefit in the treatment of glioma.