THROMBOSPONDIN MODULATES HUMAN BREAST ADENOCARCINOMA CELL-ADHESION TOHUMAN VASCULAR ENDOTHELIAL-CELLS

Thrombospondin (TSP), a M(r) 450,000 cytoadhesive glycoprotein, has be en shown to potentiate tumor cell metastasis in mice by a mechanism th at involves the hemostatic system of the host. In this study, the pote ntial involvement of TSP in the interaction of human mammary adenocarc inoma MCF-7 cells with human umbilical vein endothelial cells (HUVECs) in culture was investigated. Using an ELISA, preconfluent HUVECs synt hesized 100-foid more TSP than did MCF-7 cells during 24 h of culture (20 versus 0.2 mu g/10(6) cells). Confocal microscopy localized TSP wi thin intercellular junctions between aggregated MCF-7 cells in suspens ion. On adherent cells, TSP exhibited a patchy distribution both on th e cell surface and in the cytosol, In HUVECs, TSP strongly stained the perinuclear space and was also found in association with cytoskeletal microfibrils. Flow cytometric analysis indicated the presence of a la rge number of unoccupied receptors for TSP on MCF-7 cells. Binding stu dies using [I-125]TSP demonstrated the presence of 1.6 x 10(6) sites/c ell with an apparent K-d of 28 nM. Attachment of radiolabeled MCF-7 ce lls to a TSP-coated substrate and to HUVEC monolayers was inhibited in the presence of a polyclonal antibody to TSP (10 mu g/ml) or increasi ng concentrations (1-10 mu g/ml) of soluble TSP. Neither nonimmune IgG nor the cell adhesion peptide Gly-Arg-Gly-Asp-Ser (100 mu g/ml) inhib ited these interactions. Inhibition was also observed with heparin (10 mu g/ml), suggesting the participation of TSP heparin-binding domain( s) and heparin-like molecules. In the presence of an excess of soluble TSP or anti-TSP antibody, MCF-7 cells did not form aggregates in susp ension and preformed aggregates were readily dissociated by the additi on of soluble TSP. These results indicate that mammary adenocarcinoma cells use TSP to form aggregates and to attach to human endothelial ce lls. These interactions may have physiological implications during the hematogenous spread of tumor cells.