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Archive > Volume 27 Issue 8 > 2020,27(8):911-919. DOI:10.3872/j.issn.1007-385X.2020.08.012 Prev Next
Clinical Research
Osteosarcoma-derived exosomes induces M2 polarization of macrophages and promotes metastasis of MG63 cells through Tim-3
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Abstract:
Objective: To investigate the effect of exosomes derived from osteosarcoma on the differentiation of tumor-related macrophagesanditsmechanism.Methods:FromMarch2018toOctober2019,tumortissuesandcorrespondingnormaltissuesfrom18patients with primary osteosarcoma who underwent osteosarcoma resection and pathological diagnosis in the Departments of Orthopedics and Pediatric Surgery of theAffiliated HospitalofNorthSichuanMedicalCollegewerecollected. 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.
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Project Supported:
Project supported by theApplication Foundation Project of Sichuan Department of Science and Technology (No. 2018jy0250)
Clc Number:
