Gastric Cancer-derived Exosomes Promote Malignant Progression Of Gastric Cancer Through Glycolytic Activation Of Neutrophils

Objective: To investigate the regulation of gastric cancer-derived exosomes on the glycolysis of neutrophils.To explore the role and mechanism of glycolysis in the neutrophil-dependent promotion of gastric cancer progression.To provide an experimental and mechanism basis for revealing that tumor derived-exosomes promote the malignant progression of gastric cancer via activating neutrophil polarization.And provide a new idea for the clinical treatment of gastric cancer targeting tumor-associated neutrophils.Methods: The exosomes were isolated from the conditioned medium of gastric cancer cell lines MGC-803 and HGC-27 by ultracentrifugation,and identified by transmission electron microscope,Western blot,Nanosight nanoparticle analysis and dynamic light scattering;Neutrophils were isolated by Polymorphprep solution and identified by Wright’s staining.The expression of glycolysis related genes in neutrophils after treatment of gastric cancer-derived exosomes was detected by q RT-PCR;The expression of glycolysis related proteins in neutrophils was detected by Western blot;Glucose,lactate,ATP and lactate dehydrogenase analysis kits were used to detect the changes of glycolysis level of neutrophils;The conditioned medium of neutrophils after treatment of gastric cancer-derived exosomes was collected and applied to gastric cancer cells,cell counting assay,colony formation assay and Transwell migration and invasion experiment were utilized to observe the ability of gastric cancer cell proliferation,migration and invasion;Neutrophils were pretreated with glycolysis inhibitor 2-deoxyglucose(2-DG)to observe the role of neutrophil glycolysis in the malignant progression of gastric cancer,including cancer cell proliferation,migration and invasion;q RT-PCR was applied to detect the expression of inflammatory factors;Western blot was employed to detect activation of signal pathways in neutrophils after treatment of gastric cancer-derived exosomes;Use NF-κB pathway inhibitor BAY11-7082 to pretreat neutrophils,the expression of glycolysis related genes and proteins in neutrophils was detected by q RT-PCR and Western blot respectively;the changes of glycolysis level of neutrophils were evaluted by glucose,lactate,ATP and lactate dehydrogenase analysis kits.Results: The exosomes isolated from the conditioned medium of gastric cancer cells MGC-803 and HGC-27 displayed as small round vesicles under transmission electron microscope;The exosomes were about 100 nm in diameter according to Nanosight visual nanoparticle analysis;Western blot showed that exosome markers CD9,CD63 and TSG101 were expressed on the exosomes;The polydispersity index of Exosomes was less than 0.3;Those results indicated the exosomes possess high purity and uniform distribution.After the treatment of gastric cancerderived exosomes,the expression levels of glycolysis related genes(PFKFB3,LDHA,HK-2,PKM2,PFKL,PGK1 and GLUT1)and proteins(PFKFB3,LDHA,HK-2 and PKM2)upregulated,and the levels of glucose uptake,lactate production,ATP production and lactate dehydrogenase activity of neutrophils increased;The neutrophil conditioned medium promoted gastric cancer cell growth,clone formation,cell migration and invasion,which was weakened after pretreatment with glycolysis inhibitor 2-DG;The activation of inflammatory factor(IL-6,IL-8 and TNF-α)expression in neutrophils was repressed after 2-DG treatment.NF-κB signal pathway was activated after treatment of gastric cancer-derived exosomes in neutrophils,pretreatment with NF-κB pathway inhibitor BAY11-7082 can inhibit the promotion of glycolysis related genes and proteins,glucose uptake,lactate production,lactate dehydrogenase activity and ATP level in neutrophils.Conclusions: Gastric cancer-derived exosomes can trigger the activation of glycolysis in neutrophils via activating NF-κB pathway,and thus promote gastric cancer progression.It reveals the new effect of tumor-derived exosomes on neutrophils,and also provides a new idea for the neutrophil-targetd gastric cancer therapy.It also provides a new idea for the clinical treatment of gastric cancer targeting neutrophil glucose metabolism.