| Metastatic spread of cancer remains the major barrier to cancer cure. Mounting evidence indicates that there is a specific tropism between cancer cells and the preferred, metastasized organ. This dissertation was designed to characterize the molecular interaction between DPP IV and its counter-ligand expressed on tumor cell surfaces and to explore the important role endothelial DPP IV plays in the lung metastasis of rat breast cancer cells.;In order to confirm that DPP IV is indeed an endothelial cell adhesion molecule for rat breast cancer cells, in vitro cell-to-protein and cell-to-cell adhesion assays were performed. Metabolic labeling and co-immunoprecipitation techniques were carried out to identify the DPP IV ligand on tumor cell surfaces. Rat breast cancer cells specifically adhered to DPP IV as anti-DPP IV mAb 6A3 and soluble DPP IV were able to abolish the adhesion. Tumor cell surface-associated fibronectin was identified as the DPP IV ligand. Accordingly, anti-FN rabbit serum also blocked the adhesion.;To exert a maximal inhibitory effect, a truncated DPP IV was prepared by acidic extraction of rat lungs and the importance of DPP IV in the lung metastasis of rat breast cancer cells was evaluated by incubating lung-metastatic MTF7 cells with truncated DPP IV (DPP IV(31--767)) before tail vein injection. DPP IV(31--767) was found to colocalize with tumor cell-surface-associated FN globules, thereby acting as a competitive inhibitor of the MTF7/wtDPP IV adhesion and impeding lung colonization by these cancer cells.;To further substantiate the role of DPP IV/FN in metastasis, a rat substrain, Fischer 344/CRJ, that harbors a point-mutation in DPP IV leading to rapid degradation in ER, was used as a "DPP IV protein knock-out" model. The lung colony assay revealed only a moderate reduction in the number of metastases compared to wild-type Fischer 344 rats. Detailed immunohistochemical, biochemical analyses, and lung colony assays demonstrated that, although in greatly reduced amount, mutant DPP IV was still expressed on cell surfaces and sufficient to arrest breast cancer cells in the lung vasculature. Therefore it is an incomplete protein knock-out model for DPP IV-mediated lung metastasis of breast cancer cells.;FN is an extracellular matrix component that has many functions including heparin binding via 3 major heparin binding domains. Several glycosaminoglycans were used to block adhesion. Heparin and pentosan polysulfate were found to completely inhibit DPP IV adhesion and MTF7 lung-colonization. The gel overlay assay, ELISA, and adhesion assay revealed that the FN 12th--15 th type III repeats harboring the second heparin binding domain (FNIII 12--15) was the DPP IV-binding FN fragment and was able to inhibit MTF7 cell adhesion to DPP IV. Further localization is needed to pinpoint the exact DPP IV binding site before an anti-adhesion therapeutic strategy becomes feasible. |
