Histochemical localization of cathepsin B at the invasion front of the rabbit V2 carcinoma

To clarify the role of cathepsin B in tumor invasion, the enzyme was visualized in tissue frozen sections of the subcutaneously growing rabbit V2 carcinoma. Localization of cathepsin B was achieved by immunofluorescent staining and by enzyme histochemistry. For the former approach, a sheep antiserum was raised against purified cathepsin B from rabbit liver. The antibodies, isolated by immunoadsorption, reacted monospecifically with rabbit liver cathepsin B in Ouchterlony double diffusion and in immunoelectrophoresis. In the enzyme histochemical assay, Z-Ala-Arg-Arg-methoxynaphtylamide was used as fluorogenic substrate and nitrosalicylaldehyde as coupling agent. With both methods, cathepsin B was found to be localized within fibroblasts and leukocytes assembled at the tumor invasion front. In addition, immunofluorescent staining demonstrated the occurrence of the enzyme in the extracellular matrix surrounding tumor cell clusters. Carcinoma cells always remained unstained. The conclusion is drawn that cathepsin B is chiefly produced by host cells which are stimulated to increase synthesis and to release the enzyme under the influence of the tumor. A dual function can be ascribed to cathepsin B concentrated in the vicinity of the tumor: it operates intracellularly (in host cells) through degradation of endocytosed protein and extracellularly through activation of collagenase. The resulting lytic action on host structures appears to be a prerequisite for local spread of the V2 carcinoma.