Anti-metastasis Role Of ATRA In Osteosarcoma By Tumor-associated Macrophages Polarization Intervention

Objective:Osteosarcoma which is the most primary malignant bone tumor, arises primarily in children and adolescents. High metastatic potential is the most important characteristic of osteosarcoma, and it is the key factor to limit patient survival. Though the anti-metastasis strategy by inhibition of tumor-associated macrophages (TAM) M2 type polarization has gained wide attention, its relation with osteosarcoma metastasis remains elusive, and the effect of TAM M2 type polarization on osteosarcoma metastasis has not been reported. Recent studies have reported that ATRA plays an important role in inhibiting the invasion and metastasis of various tumors, but its molecular mechanism remains unclear, and there is no correlative report in osteosarcoma metastasis. In this study, we firstly investigated the effect of M2-polarized macrophages on the osteosarcoma metastasis, and then based on the model of macrophages polarization, we explored the influence of ATRA on macrophages polarization, as well as the anti-metastasis role of ATRA in osteosarcoma, lastly we clarified the underlying molecular mechanism of anti-metastasis role of ATRA in osteosarcoma. The aim of this study is to determine the role of macrophages polarization in regulating osteosarcoma metastasis, and then to clarify the molecular mechanism of ATRA-controlled macrophages polarization and anti-metastasis role of ATRA in osteosarcoma, which would provide novel directions for intervening osteosarcoma metastasis.Methods:Murine macrophages and osteosarcoma cell lines were used in this study. (1) SRB assay was used to detect the proliferation effect of ATRA on macrophages and conditioned-medium of macrophages on osteosarcoma cells; (2) Flow cytometer was performed to analyze the cell surface antigen F4/80、CD206 and CD86, and then determined the influence of ATRA on macrophages polarization; (3) H&E staining was used to detect the metastatic niches of osteosarcoma; (4) Immunohistochemistry staining was used to analyze the expression of macrophages-related proteins and MMP12 in tumor paraffin tissue sections; (5) Immunofluorescence staining was performed to explore the expression levels of macrophages-related proteins in tumor frozen tissue sections; (6) Real time PCR assay was used to analyze the mRNA levels of M1 and M2 type macrophages markers; (7) Transwell assay and wound healing were used to detect the migration and invasion abilities of osteosarcoma cells; (8) Tail-vein injection of K7M2 WT cells and orthotopic mouse osteosarcoma model in balb/c mice were established to simulate the lung metastasis of osteosarcoma, and investigated the effect of macrophages on the metastasis of osteosarcoma and the anti-metastasis role of ATRA in osteosarcoma; (9) Differential genes were compared through microarray approach; (10) ELISA assay was used to detect the secretion of related matrix metalloproteinase.Results:(1) Firstly, we established the osteosarcoma pulmonary metastasis model through the tail-vein inoculation of murine osteosarcoma K7M2 WT cells alone or mixed with macrophages in balb/c mice, and on this model we observed that M2 macrophages could promote the pulmonary metastasis of osteosarcoma. (2) In vitro, we used macrophages cell line RAW264.7 and primary macrophages BMDM cells, on the classical model of IL-13 or IL-4 induced M2-polarized macrophages, Flow cytometry results showed that ATRA significantly inhibited the expression of CD206 (a marker of M2-type macrophages) induced by IL-13 or IL-4 both in RAW264.7 and BMDM cells. Additionly, real time PCR result was also demonstrated that ATRA could efficiently inhibit the mRNA levels of MRC1 and PPAR-y; on the other hands, on the model of IFN-y or LPS induced M1-polarized macrophages, ATRA could partly promote RAW264.7 macrophages to M1-like phenotype while had no influence in BMDM cells. (3) To further investigate the anti-metastasis effect of ATRA on osteosarcoma cells in vitro, wound healing and transwell assay were applied and the results showed that ATRA could obviously suppress the movement and migration of K7M2 WT cells induced by supernatants of M2-type macrophages which were treated with IL-13 or IL-4. (4) Next, we established two mouse osteosarcoma metastasis models, which were tail-vein injection and orthotopic inoculation of K7M2 WT cells. On these two models, we further evaluated the anti-metastasis effcet of ATRA on osteosarcoma in vivo. H&E staining, detecting osteosarcoma metastatic nodes, demonstrated that ATRA had the ability to significantly inhibit pulmonary metastasis of osteosarcoma in vivo. In addition, via introducing macrophage scavenger clodronate lipsome, we found that ATRA exerted anti-metastasis role might through suppressing M2-polarized macrophages in tumor tissues. Immunohistochemistry staining results further verified that ATRA could effectively inhibit the expression of M2-type macrophages in metastatic tumor tissues, but had no effect on the expression of M1-type macrophages. (5) Microarray analyzed the underlying mechanism of ATRA-regulated macrophages polarization and found that MMP12 might be the potential target. ELISA results displayed that ATRA decreased MMP12 secretion stimulated by IL-13 in macrophages. Mmp408, an inhibitor of MMP12, could suppress the migration and invasion of K7M2 WT induced by supernatants of M2-type macrophages treated with IL-13 or IL-4. Immunohistochemistry staining results demonstrated that MMP12 was generally high expressed in osteosarcoma pulmonary metastatic tissues; moreover ATRA could obviously decrease the expression of MMP12 in metastatic nodes.Conclusion:M2 macrophages could promote the pulmonary metastasis of osteosarcoma, and ATRA could specifically regulate M2-type macrophages and thus inhibit metastasis of osteosarcoma. MMP12 might be a potential target for ATRA-controlled macrophages polarization and anti- metastasis role in osteosarcoma.