Tumor-derived exosomes induced M2 macrophage polarization and promoted the metastasis of osteosarcoma cells through Tim-3

Background: 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. Exosomes, as molecular information carriers, may play a potent role in the occurrence and development of tumors through oncogenic molecular reprogramming of tumor-associated macrophage (TAM). In this study, we will investigate the effect of osteosarcoma-derived exosomes on the polarization of TAM and decipher its underlying molecular mechanism. Material and Methods: Osteosarcoma-derived exosomes from MG63 cells 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 in vivo . Results: 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, EMT, and lung metastasis of osteosarcoma cells through the secretion of cytokines including IL-10, TGFβ, and VEGF. Conclusions: 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. and hFOB as detected by qRT-PCR; D: the expression of Tim-3 protein in MG63 and hFOB as detected by Western blot assay; E: the expression of Tim-3 protein in hFOB-Exo or MG63-Exo was detected by Western blot assay; F: the expression of Tim-3 protein in macrophages following treatment with PBS, or MG63-Exo as detected by Western blot; G: RT-PCR was used to detect the expression of Tim-3 mRNA following the Tim-3 gene is knocked out in MG63 cells; H: Western blot assay was used to detect the expression of Tim-3 protein after knocking out the Tim-3 gene in MG63 cells; I: the size and density distribution of Tim-3 KO-Exo particles as detected by NanoSight size analyzer; J: the expression of exosome marker including, CD9, CD81, and CD63 in Tim-3 KO-Exo as detected by Western blot assay; K: the expression of Tim-3 protein in NC-Exo, MG63-Exo, and Tim-3 KO-Exo as detected by Western blot; L: the expression of Tim-3 protein in macrophages treated with NC-Exo, MG63-Exo and Tim-3 KO-Exo as detected by Western blot assay. <0.05, <0.01.


Background
Osteosarcoma represents the fourth most frequent malignant primary neoplasm of the bone, constituting approximately 35% of all the primary malignant bone tumors. Predominantly, osteosarcomas originate in the metaphyseal region of tubular long bone during adolescence and early adulthood and are histologically characterized by the presence of malignant mesenchymal cells producing osteoid or immature bone. Approximately 15%~20% of osteosarcoma patients are presented with high-grade subclinical metastatic disease that metastasizes at very early stages [1,2].
Although chemotherapy combined with surgery is the standard of care for osteosarcoma, the prognosis in patients with metastatic osteosarcoma remains poor; besides, 20-40% of patients still experience tumor recurrence or distant metastasis following this treatment [2]. Increasing evidence also suggested that exosomes may participate in the formation and progression of different cancer processes, including remodeling of the tumor microenvironment (TME), angiogenesis, immune escape, dissemination and metastasis [18].  [28,29]. In osteosarcoma, it has been documented that the expression of Tim-3 is significantly increased in tumor tissue [30], and the overexpression of Tim-3 could promote the occurrence and development of osteosarcoma. However, whether the overexpression of Tim-3 in osteosarcoma could also regulate the differentiation of tumor-associated macrophages and the underlying molecular mechanism of this regulation remains elusive.
Therefore, this study was initiated to investigate the effect of osteosarcoma-derived exosomes on M2 macrophage polarization and to elucidate the underlying molecular mechanism in the occurrence and development of osteosarcoma. Furthermore, this study also provides a theoretical basis and experimental evidence for refining the therapeutical strategy and improving the prognosis of patients with osteosarcoma. and intact cells and cell debris were removed and exosomes were isolated by differential centrifugation at 300×g for 10 min, and 2,000×g for 10 min, 1,000×g for 30 min, respectively. The Western blot assay.

Labeling and tracking of exosomes
The extracted and purified exosomes were collected and labeled KPH67 reagent according to the manufacturer's instructions. The nuclei of macrophages were counter-stained with DAPI dye following the manufacturer's instructions. KPH67 labeled exosomes were added to DAPI stained macrophages and co-cultured at 37 °C in a humidified atmosphere of 5% CO 2 for 12 h. After incubation, the exosomes were analyzed by a fluorescence confocal microscope.

Cell migration and invasion
The cell invasion assay was carried out using 24-well transwell chambers with 6.5 mm diameter polycarbonate filters ( were performed in triplicates.

Pulmonary metastases of osteosarcoma
The animals were cared for in accordance with the Guide for the  Figure 1A). The size of exosomes was ranged from about 30 to 150 nm based on size analysis ( Figure 1B). Western blot assay results indicated that exosome-specific molecular marker including CD9, D81, and CD63 was significantly highly expressed in hFOB-Exo and MG63-Exo ( Figure 1C). Macrophages were the most abundantly expressed immune cells in the TME. Furthermore, the effect of osteosarcoma-derived exosomes on macrophage differentiation was also investigated. Human monocyte line THP-1 was differentiated into macrophages under PMA stimulation. As illustrated in Figure 1D, the suspension of THP-1 monocytes could be differentiated into adherent-growing macrophages and extended pseudopodia from the cell body under an optical microscope.
Further, qRT-PCR results revealed that the expression of macrophage marker CD68 in THP-1 was significantly over-expressed following stimulation with PMA in comparison with the control group ( Figure 1E). In order to confirm that the isolated exosomes could be phagocytized by macrophages, we labeled the exosomes with the green fluorescent dye PKH67 beforehand, which was co-cultured with macrophages for 12 h. Notably, the green fluorescence around the nuclei of macrophages was observed under confocal fluorescence microscopy, indicating that exosomes were phagocytized by macrophages ( Figure 1F). qRT-PCR data suggested that compared with the PBS control group or macrophages co-cultured with hFOB-Exo, MG63-Exo significantly promoted the expression of M2-type macrophage markers CD206, CD163, and Arginase-1; however, no alterations in the expression of iNOS, the marker of M1 macrophage, was noted. Conceivably, hFOB-Exo exhibited no apparent effect on the M1 or M2 phenotype differentiation of macrophages ( Figure 1G). We further applied qRT-PCR to detect the effect of macrophages on the expression levels of M2 type cytokines, TGF-β, IL-10 and VEGF under different treatment conditions. The results indicated that compared with the control group, MG63-Exo significantly promoted the expression of TGF-β, IL-10 and VEGF; however, hFOB-Exo did not affect macrophages for the expression of the above cytokines ( Figure 1H).

M2 macrophages induced by MG63-Exo promoted the migration, invasion, and EMT of osteosarcoma cells
To clarify the effects of M2 macrophages induced by MG63-Exo on the migration, invasion, and EMT of osteosarcoma cell in vitro, we used a co-culture system for the detection (Figure 2A)      by binding to its ligand Galectin-9 (Gal-9) could induce apoptosis and immune tolerance of T cells. In this study, Tim-3 was also found to be expressed in macrophages, NK cells, and other natural immune cells, which may play an important role in maintaining immune homeostasis [30]. Using Western blot assay, we detected high expression of Tim-3 protein in osteosarcoma tissues and cells.,

Conclusions
This study identified that osteosarcoma-derived exosomes induced M2 type polarization of macrophages through regulation of the expression of Tim-3. Furthermore, the differentiated macrophages promoted the migration, invasion, EMT, and distant metastasis ability of osteosarcoma cells through the secretion of IL-10, TGF β, VEGF. However, the role of exosomes in tumor microenvironment remains complex and variable and therefore the above-mentioned mechanism may represent a probable mechanism and warrant further investigations.

Declarations Ethics approval and consent to participate
This study was approved by the Institutional Ethics Review Board of Huanggang Central Hospital. Written informed consent was obtained from all the patients. Tissue samples were collected at surgery, immediately frozen in liquid nitrogen and stored until analysis in the present study.

Availability of data and materials
All data are available in the included figures and tables.