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Thrombospondin Binds to the Surface of Human Osteosarcoma Cells and Mediates Platelet-Osteosarcoma Cell Interaction¹

INSERM U. 234, Pavillon F, Hôpital Edouard Herriot, Place d'Arsonval, 69437 Lyon, France [P. C., C-M. S., P. D. D.], INSERM U. 331, Faculté de Médecine Alexis Carrel, rue G. Paradin, 69372 Lyon, France [P. C., M-C. T.], and Division of Hematology, Ottawa General Hospital, Ottawa, Ontario, Canada [J. D.]

We have previously shown that thrombospondin (TSP) is synthesized and secreted by human MG-63 osteosarcoma cells. In this study, the secretion and cell surface expression of TSP by two different human osteosarcoma cell lines (MG-63 and TE-85) as well as the involvement of TSP in the platelet-aggregating activity of these tumor cells were studied. Using a sandwich enzyme-linked immunosorbent assay, MG-63 cells secreted 3-fold as much TSP as TE-85 cells at 48 h (0.17 ± 0.01 (SD) versus 0.06 ± 0.006 µg/10⁶ cells, P = 0.007). Binding of exogenous ¹²⁵I-TSP to MG-63 and TE-85 cells in monolayer indicated that binding was time and concentration dependent, saturable, and inhibited by excess cold TSP. However, despite a similar affinity, MG-63 cells had 10-fold more TSP-binding sites than TE-85 cells (402,394 ± 130,346 versus 36,748 ± 7,708 TSP-binding sites/cell; P = 0.002). Similar binding differences of ¹²⁵I-TSP were observed with both osteosarcoma cell lines in suspension. A fluorescence-activated cell-sorting analysis was used in conjunction with an anti-TSP polyclonal antibody, and binding of endogenous TSP to MG-63 and TE-85 cells in suspension was investigated. Addition of an anti-TSP antibody to MG-63 and TE-85 cells in suspension increased the mean fluorescence intensity 50-fold when compared to an irrelevant antibody. Moreover, the fluorescence intensity of MG-63 cells with an anti-TSP polyclonal antibody was increased by 40% when compared to TE-85 cells. Since TSP was expressed on the surface of osteosarcoma cells, the involvement of this glycoprotein in the platelet-aggregating activity of MG-63 and TE-85 cells was therefore investigated using an anti-TSP polyclonal antibody and two monoclonal antibodies (P10 and MA-II), the epitopes of which lie within the M_r 140,000 nonheparin-binding fragment and the M_r 25,000 heparin-binding fragment of TSP, respectively. Preincubation of MG-63 cells (1 x 10⁶ cells/ml) with either an anti-TSP polyclonal antibody (100 µg/ml) or monoclonal antibody P10 (15 µg/ml) inhibited by 80% the platelet-aggregating activity of these tumor cells, while anti-TSP monoclonal antibody MA-II (15 µg/ml) had no effect. In sharp contrast, the anti-TSP polyclonal antibody (100 µg/ml) only exhibited a slight inhibitory effect on platelet aggregation induced by TE-85 cells when using a low concentration of tumor cells (0.6 x 10⁶ cells/ml). Electron microscopy studies showed that platelet aggregation induced by both osteosarcoma cell lines resulted from platelet-osteosarcoma and platelet-platelet interactions. Preincubation of MG-63 cells with the anti-TSP polyclonal antibody induced a drastic inhibition of platelet-osteosarcoma cell interactions, whereas platelet-platelet interactions still occurred. On the other hand, interactions between TE-85 cells and platelets were not affected by the anti-TSP antibody. In conclusion, these results demonstrate that the ability of TSP to mediate platelet-osteosarcoma cell interaction depends at least in part upon the amount of TSP secreted and bound on the surface of osteosarcoma cells.

¹ This study was supported in part by grants from the Fondation de France, the Fédération Nationale des Centres de Lutte Contre le Cancer, and the University of Ottawa Medical Research Fund.

² To whom requests for reprints should be addressed, at INSERM U. 234, Pavillon F, Hôpital Edouard Herriot, Place d'Arsonval, 69437 Lyon Cédex 03, France.

Received 10/15/90. Accepted 3/ 5/91.

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