Thrombospondin Structural Domains Thrombospondin: Relationship to Metastatic Potential and Motility of Human Carcinoma Cells in Response to

The interactions of tumorigenic cells with the extracellular matrix play a critical role in the establishment of metastases. Thrombospondin (TSP) is prominent at sites of tissue injury and promotes the attachment, spreading, and motility of several cell types. We have investigated the relationship between human carcinoma cell metastatic potential and TSP-mediated cell motility by comparing highly metastatic l lB carcinoma cells with a nonmetastatic counterpart, 22B carcinoma cells, l lB cells demonstrated motility in response to soluble TSP with a maximal effect observed at 1 laM TSP. Checkerboard analysis indicated that motility was directional with a significant chemokinetic component. Monoclonal antibody C6.7, specific for the distal COOH terminus of TSP, inhibited chemotaxis by 60%. Studies with TSP fragments demonstrated that the Mr 140,000 COOHterminal domain (140K) supported chemotaxis to the same extent as intact TSP. The NH2-terminal heparin-binding domain was ineffective in stimulating chemotaxis. Substrate-bound TSP also elicited l lB cell motility with a maximal response at 100 nM TSP. Directionality of this response was confirmed by checkerboard analysis. Interestingly, as in chemotaxis, haptotaxis was mediated exclusively by 140K as demonstrated by TSP fragment studies and inhibition with monoclonal antibody C6.7. Therefore, 140K appeared to mediate both chemotaxis and haptotaxis. Compared with l lB cells, 22B carcinoma cells are nonmetastatic and synthesize and secrete low levels of TSP. Immunoprecipitation and Northern blot analysis confirmed that l lB cells expressed much higher levels of TSP than 22B cells. Although 22B cells are able to attach to TSP, they did not exhibit either chemotaxis or haptotaxis in response to TSP or TSP fragments. Similarly, an antisense TSP cell line responded poorly in chemotaxis assays to TSP and 140K. These data suggest that the ability of metastatic cells in vivo to establish secondary sites of proliferation may be related to their ability to migrate in response to extracellular matrix proteins such as TSP incorporated into basement membranes or interstitial matrices.