Yabkowitz R, Mansfield P J, Dixit V M, Suchard S J
Department of Pathology, University of Michigan Medical School, Ann Arbor 48109.
Cancer Res. 1993 Jan 15;53(2):378-87.
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 11B carcinoma cells with a nonmetastatic counterpart, 22B carcinoma cells. 11B cells demonstrated motility in response to soluble TSP with a maximal effect observed at 1 microM 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 M(r) 140,000 COOH-terminal 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 11B 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 11B cells, 22B carcinoma cells are nonmetastatic and synthesize and secrete low levels of TSP. Immunoprecipitation and Northern blot analysis confirmed that 11B 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.
致瘤细胞与细胞外基质的相互作用在转移的形成过程中起着关键作用。血小板反应蛋白（TSP）在组织损伤部位很突出，并促进几种细胞类型的黏附、铺展和运动。我们通过比较高转移性的11B癌细胞和非转移性的对应细胞22B癌细胞，研究了人癌细胞转移潜能与TSP介导的细胞运动之间的关系。11B细胞对可溶性TSP有反应，在1 microM TSP时观察到最大效应。棋盘分析表明运动是有方向性的，且有显著的化学动力学成分。对TSP远端COOH末端特异的单克隆抗体C6.7可使趋化作用抑制60%。对TSP片段的研究表明，分子量为140,000的COOH末端结构域（140K）支持趋化作用的程度与完整的TSP相同。NH2末端肝素结合结构域在刺激趋化作用方面无效。底物结合的TSP也能引起11B细胞运动，在100 nM TSP时反应最大。这种反应的方向性通过棋盘分析得到证实。有趣的是，与趋化作用一样，触觉趋化作用也仅由140K介导，这在TSP片段研究和单克隆抗体C6.7的抑制实验中得到了证明。因此，140K似乎介导了趋化作用和触觉趋化作用。与11B细胞相比，22B癌细胞是非转移性的，合成和分泌的TSP水平较低。免疫沉淀和Northern印迹分析证实，11B细胞表达的TSP水平比22B细胞高得多。尽管22B细胞能够黏附到TSP上，但它们对TSP或TSP片段没有表现出趋化作用或触觉趋化作用。同样，一个反义TSP细胞系在趋化作用实验中对TSP和140K反应不佳。这些数据表明，体内转移细胞建立继发性增殖位点的能力可能与其响应细胞外基质蛋白（如整合到基底膜或间质基质中的TSP）而迁移的能力有关。
