Study On The Effects And Mechanism Of TIM-3 On Invasion And Metastasis Of Osteosarcoma

Background: Osteosarcoma is the most common type of non-hematopoietic primary malignant bone tumor. Despite significant progress in chemotherapy, patients who have metastases at diagnosis continue to have poor prognosis. Therefore, it is essential to identify additional biomarkers for use in diagnosis and novel therapeutic strategies. Signals from the T cell Ig- and mucin-domain-containing molecules (TIMs) have been demonstrated to be actively involved in regulating the progression of carcinomas. Inceasing studies indicates that TIMs plays an essential role in the progression of epithelial-mesenchymal transition (EMT), and it is known that EMT is an important mechanism in the progression of cancer invasion and metastasis. However, the expression and distribution of TIMs in osteosarcoma, the most common primary bone malignancy with poor prognosis, have not been investigated.Purposes: This study aims to investigate the expression of TIMs in osteosarcoma tissue sections, and the association between TIMs and EMT biomarkers were further analyzed by dual IF staining and Western blot. Also, we aims to clarify the role of TIM-3 in osteosarcoma progression, to define the specific oncogenic activities of TIM-3 in osteosarcoma in vitro, and to identify the molecular mechanism of TIM-3 underlying the cancer invasion and metastasis of osteosarcoma.Materials and methods: The expression of TIMs (TIM-1, TIM-3, TIM-4) was examined in 38 of invasive human osteosarcoma tissues using immunohistochemistry (IHC), and the phenotypes were detected by dual immunofluorescence (IF) staining and Western blot. Overall survival (OS) rates of all of osteosarcoma patients were calculated using the Kaplan-Meier method. For the further mechanism study, we established TIM-3 siRNA or TIM-3 shRNA-expressing lentivirus (shTIM-3-Lv) to interfere TIM-3 expression in MG-63 osteosarcoma cell line, then to detect TIM-3, proliferation, macrophage and EMT markers in TIM-3 (+) and TIM-3 (-) MG-63 cells using dual IF staining and Western blot assay.Results: Using IHC, it was observed that only TIM-3, rather than TIM-1 or TIM-4, was expressed in these tumor specimens, where it was localized in the cytoplasm and plasma membrane of tumor cells. The positive rate of TIM-3 protein expression was observed in 84.2% (32/38) of osteosarcoma cases. And high TIM-3 expression predicts shorter survival of patients with osteosarcoma. Additionally, transwell assay showed that the invasive ability of MG-63 was significantly inhibited in MG-63 cells with si-TIM-3 transfection compared with the cells with si-Control. Dual IF staining revealed that the expression of TIM-3 was observed in all cell types investigated, including CD68+ macrophages, CD31+ endothelial cells, CK-18+ epithelial cells and PCNA+ tumor cells. Notably, in MG-63 sarcoma cells, TIM-3 was co-expressed with certain biomarkers of epithelial-mesenchymal transition (EMT), including Vimentin, Slug, Snail and Smad.Conclusions:TIM-3 is frequently up-regulated in osteosarcoma, and its over-expression predicts poor prognosis of patients with osteosarcoma. Moreover, TIM-3 can promotes the proliferation and EMT formation induced by TGF-β. These combined results suggest that TIM-3 triggers tumor cells to acquire features of aggressive EMT and may be involved in the pathogenesis of this malignancy.