antibodies to recognize pro-uPA, uPA, and in vitro-formed complexes of uPA with either soluble uPAR or with plasminogen activator Inhibitor

Current evidence regarding the regulation of urokinase-dependent ex tracellular proteolysis indicates that plasminogen activation is a surface associated process. We have compared the histological localization of urokinase plasminogen activator (uPA) and urokinase plasminogen acti vator receptor (uPAR) in breast cancer sections using a panel of mono clonal antibodies. First, the ability of six different anti-uPA monoclonal antibodies to recognize pro-uPA, uPA, and in vitro-formed complexes of uPA with either soluble uPAR or with plasminogen activator Inhibitor type 1 was compared. Then the reactivity ofthe anti-uPAR antibodies was tested, and the occurrence of an uPA receptor of about Mr 55,000 in samples from breast carcinoma was assessed by immunopredpitating the uPA receptor from an in vitro †̃@I-laboled tumor extract. Immunocyto chemical data from adjacent sections of 10 tumor specimens showed that antibodies recognizing free and bound uPA mostly stain the cytoplasm and the membrane of epithelial tumor ceHsin confined areas of the tumor and some fibroblast-like stromal cells. Acid pretreatment of tumor see dons, which removes receptor.bound uPA, causes a strong reduction of the immunocytochemical reactivity of epithelial tumor cells, whereas staining of fibroblast-like cells is not considerably affected. Consistent with these results, epithelial tumor cells were mostly unreactive to anti uPAR antibodies unless pretreated with acidic buffer, whereas fibroblast like stromal cells showed a faint but acid-resistant staining with all anti-uPARs. In conclusion, these results show that occupied uPA receptors are definitely present on the membrane of epitheial tumor cells and suggest the occurrence of uPA-uPAR-dependent proteolytic activity on the surface of breast cancer cells.