Exosomes Regulate Glycolysis To Promote Gastric Cancer Progression

Objective:Exosomes(Ex)are key cell-to-cell signal trasmitters that regulate numerous cells in the tumor microenvironment(TME).Glycolysis is linked to the development of cancer.The purpose of this work is to investigate the role of Ex from gastric cancer(GC)cells,in the malignant progression of GC,as well as the role of glycolysis in this process,and to propose new ideas for the relationship between Ex and the GC pathogenesis.Methods:1.PKM2 was selected as the molecule for our study according to the mass spectrometry results of GC cells-derived Ex and literature reports.The Ex secreted from GC cells(MGC-803,HGC-27 and MKN-45)and normal gastric mucosa cells(GES-1)was extracted by ultracentrifugation and identified by Western Blot,nanoparticle analyzer(NTA)and transmission electron microscopy(TEM).The expression of PKM2 in cells and Ex was detected by Western Blot,and the GC cells with high expression of PKM2 were knocked down by si RNA transfection.2.After the treatment of Ex,the ability of cell proliferation,colony formation,migration and invasion was observed by the cell growth curve,the cell colony formation experiment and the Transwell migration and invasion assays,the glycolytic ability of GC cells was measured by an ATP production level detection kit,a glucose uptake capacity detection kit and a lactate excretion level detection kit,and the expression of glycolysis-related genes(GLUT1,PKM2,HK-2,LDHA,PGK1,PFKL and PFKFB3)was detected by q RT-PCR.The expression of glycolysis-related proteins(PKM2,HK-2 and LDHA)was detected by Western Blot.2-Deoxyglucose(2-DG),the inhibitor of glycolysis,was used to act on the GC cells together with Ex to observe the ability of cell proliferation,colony formation,migration and invasion.3.The expression of PKM2 in the nucleus of GC cells after Ex treatment was detected by a nuclear/cytosol fraction assay,and the expression of phosphorylated STAT3 was detected by Western Blot.Using Stattic as the inhibitor of STAT3 phosphorylation to treat GC cells together with Ex,the expression level of EMT-related proteins,cell apoptosis-related proteins,cell cycle-related proteins and the glycolytic ability of GC cells were detected.4.PMA-stimulated THP-1 cells were treated with Ex.The glycolytic ability of macrophages was detected by an ATP production level detection kit,a glucose uptake capacity detection kit,a lactate excretion level detection kit,q PT-PCR and Western Blot.The expression of cytokines(ARG-1,IL-10,TGF-β)secreted by M2 macrophages was detected by q RT-PCR,and the expression of the M2 macrophage-associated surface marker CD206 was determined by flow cytometry.The supernatant collected from macrophages treated with Ex was applied to GC cells to observe the changes in cell proliferation,migration and invasion abilities.Results:1.The cup-like vesicles with a particle size of around 140 nm were extracted,showing positive expressions of CD63,TSG101 and CD9 while negative expression of calnexin.Fluorescence microscopy showed that Ex was efficiently taken by GC cells.GC cells-derived Ex expressed high levels of PKM2,and the expression of PKM2 in Ex decreased after knocking down PKM2 with small interfering RNA.2.Compared with the PBS group,the ability of proliferation,colony formation,migration and invasion was promoted after Ex treatment,and inhibited in PKM2 knockdown Ex group.Moreover,ATP production level,the glucose uptake,the lactate excretion level and the expression of glycolysis-related genes and enzymes in GC cells were also enhanced after Ex treatment,and decreased in PKM2 knockdown Ex group.After treatment with 2-DG,a glycolysis inhibition,the proliferation,colony formation,migration and invasion abilities of GC cells were decreased.3.Ex treatment promoted the nuclear translocation of PKM2 in GC cells,and the expression level of P-STAT3 increased in a time-dependent manner.Stattic,a STAT3 inhibitor,decreased the ability of glycolysis and the expression of cancer progression-related proteins in GC cells after Ex treatment.4.Ex stimulated the glycolytic ability and M2-type polarization of macrophages.The supernatants from macrophages treated with Ex could substantially stimulate the proliferation,migration and invasion of GC cells.After pretreatment of macrophages with 2-DG,the expression level of CD206 and M2-related cytokines was inhibited,and the promotion of proliferation,colony formation,migration and invasion abilities of GC by macrophages were inhibited.Conclusions:Ex could directly promote the glycolytic capacity of GC cells through transmitting PKM2,and activate the STAT3 signaling pathway through the nuclear translocation of PKM2 protein to promote the glycolysis of GC cells,enhancing the proliferation and metastasis ability of cells eventually.In addition,Ex could indirectly promote the proliferation and metastasis of GC cells by enhancing the glycolysis ability of macrophages and inducing the M2 polarization of macrophages.These results indicated that Ex plays a dual role in regulating GC cells and macrophages through PKM2-mediate glycolysis,which jointly promotes the malignant progression of GC.