Osteosarcoma-derived exosomes induces M2 polarization of macrophages and promotes metastasis of MG63 cells through Tim-3 / 中国肿瘤生物治疗杂志

ABSTRACT

@#[Abstract] Objective: To investigate the effect of exosomes derived from osteosarcoma on the differentiation of tumor-related macrophages and its mechanism. Methods: From March 2018 to October 2019, tumor tissues and corresponding normal tissues from 18 patients with primary osteosarcoma who underwent osteosarcoma resection and pathological diagnosis in the Departments of Orthopedics and Pediatric Surgery of the Affiliated Hospital of North Sichuan Medical College were collected. The expression level of Tim-3 was detected by Western blotting; Exosomes of osteosarcoma MG63 cells (MG63-Exo) were isolated and identified by transmission electron microscopy and nanoparticle size analysis, and its phagocytosis by macrophages was verified by Dual fluorescent staining; The effects of MG63-Exo on macrophage differentiation and the expression levels of IL-10, TGF-β and VEGF were detected by qPCR; The effects of MG63-Exo induced macrophages on the migration and invasion of MG63 cells and the expression of EMT related proteins were detected by Transwell invasion and migration assay and Western blotting; CRISPR/cas9 was used to knock out Tim-3 in MG63 cells, and its knockout efficiency was verified by Western blotting, and then qPCR, transwell assay and Western blotting were used to detect the effect of MG63-Exo with Tim-3 knock-out on macrophage differentiation, as well as migration, invasion and expression of EMT related proteins in MG63 cells; Finally, the mouse model of osteosarcoma lung metastasis was used to verify the effect of exosomes from different sources on the lung metastasis of osteosarcoma. Results: Transmission electron microscopy and nanoparticle size assay confirmed that MG63-Exo were successfully isolated, and Confocal fluorescence results confirmed that it could be phagocytized by macrophages; qPCR results showed that MG63-Exo significantly promoted M2 differentiation of macrophages compared with PBS (P<0.05); Compared with PBS control group, M2 macrophages induced by MG63-Exo significantly promoted the migration, invasion and EMT of osteosarcoma cells (all P<0.05); The mRNA and protein expressions of Tim-3 in the MG63 cells knocked out by CRISPR/cas9 (Tim-3-KO) were significantly reduced (all P<0.05), and Tim-3 could be transferred into macrophages in the form of exosomes; Compared with MG63-Exo co-cultured macrophages, the M2 type differentiation of macrophages treated with Tim-3-KO-exo was significantly decreased (P<0.05); Compared with the MG63 cells co-cultured with macrophages induced by MG63-Exo, the migration, invasion and EMT were significantly reduced while the lung metastasis was significantly promoted in MG63 cells co-cultured with macrophages induced by Tim-3-KO-Exo (all P<0.05). Conclusion: Exosomes derived from osteosarcoma can induce M2 polarization of macrophages through Tim-3 and promote the invasion and metastasis of tumor.