Thrombospondin 1 Triggers Osteosarcoma Cell Metastasis and Tumor Angiogenesis

Abstract

Osteosarcomas, especially those with metastatic or unresectable disease, have limited treatment options. The antitumor effects of pharmacologic inhibitors of angiogenesis in osteosarcomas are hampered in patients by the rapid development of tumor resistance, notably through increased invasiveness and accelerated metastasis. Here we demonstrated that thrombospondin 1 (TSP-1) is a potent inhibitor of the growth and metastasis of the osteosarcoma cell line MG-63. Moreover, we demonstrate that upregulation of TSP-1 facilitated expression of vasculostatin in MG-63 cells. In angiogenesis assays, overexpression of TSP-1 inhibited MG-63 cells and induced tube formation of human umbilical vein endothelial cells (HUVECs) in a CD36-dependent fashion. Finally, in xenografted tumors, we observed that TSP-1 overexpression inhibited angiogenesis and tumor growth. These results provided strong evidence for an important role of the TSP-1/CD36/vasculostatin signaling axis in mediating the antiangiogenic activity of osteosarcoma.

Figure 1

Thrombospondin 1 (TSP-1) overexpression inhibits the malignancy of osteosarcoma MG-63 cells. (A) Relative expression of TSP-1 in a panel of osteosarcoma cell lines. (B) Quantitative real-time PCR (qRT-PCR) analysis of TSP-1 expression after infection of TSP-1 lentivirus vectors. The data are presented as means ± SD. For indicated comparisons, **p < 0.01. (C) Western blotting analysis of TSP-1 protein expression. GAPDH was used as internal control. (D) 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. (E) TSP-1-overexpressing MG-63 cells showed decreased efficiency of cell colony formation compared to control cells. Cell colonies were counted in five random fields of vision. Scale bar: 200 μm. The data are presented as means ± SD. For indicated comparisons, **p < 0.01. (F) Effects of TSP-1 overexpression on MG-63 cell migration were determined by wound healing assay. The percentage of wound healing was calculated as the width at indicated time. Scale bar: 200 μm. The data are presented as means ± SD. For indicated comparisons, **p < 0.01. (G) Invasion assay. MG-63 cells were seeded on the upper chamber precoated with Matrigel for 24 h. Cells on the underside of the membrane were fixed and stained with crystal violet. Scale bar: 200 μm. The data are presented as means ± SD. For indicated comparisons, **p < 0.01.

Figure 2

TSP-1-overexpressing MG-63 cells inhibit human umbilical vein endothelial cell (HUVEC) tube formation and induce apoptosis. (A) HUVECs were seeded on Matrigel. Cultures were treated with either PBS, or culture medium (CM) from control MG-63 cells, or CM from MG-63 cells overexpressing TSP-1. HUVECs were incubated with both CM from MG-63 cells overexpressing TSP-1 and blocking CD36 antibody. Incubation of HUVECs with a blocking CD36 antibody abolished the inhibition of HUVEC tube formation induced by TSP-1 overexpression. Scale bar: 50 μm. The data are presented as means ± SD. **p < 0.01 compared to control, ##p < 0.01 compared to cells transfected with TSP-1. (B) Flow cytometric apoptosis assay. Cell apoptosis was assessed by flow cytometry. The data are presented as means ± SD. **p < 0.01 compared to control, ##p < 0.01 compared to cells transfected with TSP-1.

Figure 3

MG-63 expressing TSP-1 inhibits tumorigenicity in mice models. (A) Representative picture of tumor mass. (B) Tumor growth kinetics (mean ± SD) of vector cells versus TSP-1-overexpressing MG-63 cells in nude mice. (C) Mean weight of tumor spheres. The data are presented as means ± SD. **p < 0.01. (D). Percentages of CD31 staining in tumor mass from vector cells versus TSP-1-overexpressing MG-63 cells. Scale bar: 200 μm.

Figure 4

Effects of TSP-1/CD36 on regulation of vasculostatin gene expression. (A) Western blotting analysis of the expression of vasculostatin in TSP-1-overexpressing cells or CD36-overexpressing MG-63 cells. (B) The expression of vasculostatin in TSP-1-overexpressing or CD36-overexpressing MG-63 cells was determined by qRT-PCR. GAPDH was used as an internal control. The data are presented as means ± SD. **p < 0.01 compared to vector. (C) Representative immunohistochemistry staining for expression of vasculostatin in MG-63 osteosarcoma cell grafts grown in nude mice. Scale bar: 200 μm.

Figure 5

Overexpression of TSP-1 inhibits metastases of MG-63 cells in vivo. (A) Photographs of lungs from each group. (B) Representative lungs after hematoxylin and eosin (H&E) staining from each group. Visible lung metastasis number of each group was counted. Scale bar: 200 μm.