Abstract
Osteosarcoma, the most prevalent primary malignancy of the bone, is often presented with high-grade subclinical metastatic disease that metastasizes at very early stages. Exosomes, as molecular information carriers, may play a potent role in the occurrence and development of tumors through oncogenic molecular reprogramming of tumor-associated macrophages (TAMs). In this study, we will investigate the effect of osteosarcoma-derived exosomes on the polarization of TAMs and decipher its underlying molecular mechanism. Osteosarcoma-derived exosomes from MG63cells were isolated and characterized by transmission electron microscopy, and nano-particle size analysis. Double fluorescence staining was performed to confirm the macrophages phagocytosis of exosomes. Western blot, qRT-PCR, and transwell assays were conducted to assess the effect of exosomes on migration, invasion, and macrophage differentiation. The mouse model of osteosarcoma was established to evaluate the effects of exosomes on lung metastasis invivo. MG63 exosomes were successfully isolated and verified to be phagocytized by macrophages through fluorescence confocal microscopy. The results revealed that osteosarcoma cells could induce M2 type differentiation of macrophages largely through Tim-3 mediated by exosomes, which in turn could promote the migration, invasion, epithelial-mesenchymal transition (EMT), and lung metastasis of osteosarcoma cells through the secretion of cytokines including IL-10, TGF-β, and VEGF. Our results demonstrated that osteosarcoma-derived exosomes induced M2 polarization of macrophages and promoted the invasion and metastasis of tumors through Tim-3; besides, the study also suggests a novel therapeutic target for future studies.
Highlights
Osteosarcomas, the second most prevalent primary malignancy of the bone, are often presented with high-grade subclinical metastatic disease that metastasizes at very early stages
Our results demonstrated that osteosarcoma-derived exosomes induced M2 polarization of macrophages and promoted the invasion and metastasis of tumors through T cell immunoglobulin and mucin domain (Tim)-3; besides, the study suggests a novel therapeutic target for future studies
The green fluorescence around the nuclei of macrophages was observed under confocal fluorescence microscopy, indicating that exosomes were phagocytized by macrophages (Figure 1F). Quantitative Real-time PCR (qRT-PCR) data suggested that compared with the PBS control group or macrophages cocultured with hFOB-Exo, MG63-Exo significantly promoted the expression of M2-type macrophage markers CD206, CD163, and Arginase–1; no alterations in the expression of iNOS, the marker of M1 macrophage, was noted
Summary
Osteosarcomas, the second most prevalent primary malignancy of the bone, are often presented with high-grade subclinical metastatic disease that metastasizes at very early stages. The polarization of macrophages have been more recently demonstrated by Low et al.[7,8,9,10], wherein, the coculture of nasopharyngeal carcinoma (NPC) cells and macrophages exerted pro-tumoral functions by inducing various inflammatory cytokines during the early stage; up-regulated the induction of cancer-related genes in macrophages in the later stages, suggesting that recruited macrophages into the NPC tumor microenvironment was tuned to promote tumor progression. The effect of TAMs on tumor progression possibly depends on the tumor type, TME, and the localization of TAMs. Given the increasing evidence supporting active crosstalk between TAMs and EMT in tumor metastasis and progression, investigating the signaling pathways involved in this process might provide an insight into new strategies for the treatment of invasive cancer including osteosarcoma
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