The Role And Mechanism Of Macrophages Activated By Gastric Cancer Derived Exosomes In Gastric Cancer Progression

Objective: To investigate biological significance of gastric cancer derived exosomes on macrophages and provide theoretical basis and experimental evidence for the promoting effect of exosomes in gastric cancer progression through the modulation of cancer microenvironment.Methods: Exosomes were isolated from the supernatants of cultured cells(GES-1, MGC-803, SGC-7901) by ultracentrifugation. The morphology of exosomes were characterized by transmission electron microscopy(TEM) assay. The size distribution and concentration of exosomes were analyzed by using Nano Sight analysis. The protein markers of exosomes were identified by Western blot. The internalization of exosomes by macrophages was detected by flow cytometry imaging analysis and Image Xpress Ultra Molecular Devices analysis. The cell scratch wound assay, transwell migration assay, cell colony formation assay, cell invasion and apoptosis ability analysis were used to explore the biological effect of exosomes treated macrophages on gastric cancer cells. The activation of NF-κB pathway in macrophages treated by gastric cancer derived exosomes were analyzed by Western blot. Macrophages were treated by NF-κB pathway inhibitor Bay11-7082. The difference of the expression of proinflammatory factors in macrophages treated by gastric cancer derived exosomes were determined by q RT-PCR and ELISA.Results: The isolated exosomes were verified as small vesicles of approximately 50-100 nm in size. The concentration of gastric cancer exosomes was approximately ranging from 1.0 × 1011 to 2.5 × 1011particles/ml. CD9 and CD81, two exosomal protein markers, were expressed in the purified exosomes. Macrophages could take up gastric cancer derived exosomes with high efficiency. The migrating ability, cell colony and cell invasion ability of cells was obviously promoted after the incubation with supernatant from gastric exosomes treated macrophages,while the apoptosis ability had no significant difference. NF-κB pathway were activated in macrophages treated by MGC-803 and SGC-7901 derived exosomes. The expression of P-P65 and m RNA expression of IL-6, TNF-α and CCL2 were up-regulated compared with control groups.The activation of NF-κB pathway in macrophages after gastric cancer derived exosomes treatment were eliminated with the pretreatment of NF-κB pathway inhibitor Bay11-7082. Moreover, we found that gastric cancer cells derived exosomes could also activate macrophages from human peripheral blood monocytes through the activation of NF-κB. The primary macrophages produced high level of proinflamatory factors such as IL-6, TNF-α and CCL2 after activated by exosomes to promote the gastric cancer progression.Conclusions: Exosomes derived from gastric cancer activate macrophages in microenvironment to promote the gastric cancer progression. Our results provide novel evidence for the role of exosomes in gastric cancer progression and present potential therapeutic ideas for clinical treatment.