Treatment of SKOV3ip1, HeyA8, and CaOV3 ovarian cancer cells with the u-PAR antibody inhibited cell invasion, migration and adhesion. levels in xenograft ovarian cancer tumors (images) are taken at Z-plane (depth) of contact (Z=0 m) between ovarian cancer cell and plastic/omental ECM, and the Z image (and (Supplementary Fig. S3). Open in a separate window Fig. 5 Anti-u-PAR treatment decreases integrin signalingin the SKOV3ip1 and CaOV3 xenograft models and in SKOV3ip1 and CaOV3 cells grown on plastic or the 3D omental culture. Anti-u-PAR treatment significantly reduced 3-integrin and FGFR1 mRNA (qRT-PCR) and protein (immunoblot) levels in xenograft ovarian cancer tumors and in ovarian cancer cells cultured on plastic or the 3D culture. Bar graph shows results from three independent experiments (n=5, Colocalization of u-PAR and 5-integrin on omental ECM in ovarian cancer cells. SKOV3ip1 cells Hydroxyfasudil cultured on omental ECM were stained with FITC-labeled uPA and cofocal immunofluorescence for 5-integrin was performed. Bar graph shows mean percent of uPA-FITC colocalization with 5-integrin as calculated using the Imaris colocalization application (details in in two cell lines (SKOV3ip1 and CaOV3). Treatment of cells on plastic, as well as on the 3D culture, resulted in an inhibition of 3-integrin and FGFR1 mRNA and protein expression (Fig. 5B). Previous studies have Hydroxyfasudil shown that the association of u-PAR with the fibronectin receptor (51-integrin) affects the expression and activation state of u-PAR, and that Hydroxyfasudil u-PAR is important for tumor cell invasion [26, 27]. Therefore, we tested to determine if the antibody would affect the expression of 5-integrin and the interaction of u-PAR and 5-integrin. Indeed, treatment with the u-PAR antibody in the CaOV3 xenograft model inhibited 5-integrin mRNA and protein expression (Fig. 5C). Hydroxyfasudil (Fig. 6B). Treatment with the u-PAR antibody increased expression of active caspase 3 in SKOV3ip1 and CaOV3 cells, DNA fragmentation of SKOV3ip1 cells, and the SPTAN1 percent of apoptotic cells in SKOV3ip1 cells when compared to control antibody treated cells (Fig. 6B). Open in a separate window Fig. 6 Treatment with an u-PAR antibody increases apoptosis of ovarian cancer cellsand and in a 3D model, as well as to inhibit metastasis in 3 different ovarian xenograft models. These results are in agreement with those of other investigators who have identified u-PAR as a therapeutic target in preclinical models of cancer. Two studies by the same group specifically investigated the targeting of u-PAR in ovarian cancer. First, Sato et al., using the OVMZ-6 ovarian cancer xenograft mouse model, identified two cyclic peptides which act as competitive antagonists of the uPA/u-PAR-interaction and were able to reduce tumor weight [33]. Second, Knor et al. successfully targeted u-PAR in OVMZ-6 ovarian cancer cells in culture, which inhibited colony formation [34]. The urokinase receptor has also been successfully inhibited using various techniques in other cancers, including DNAzymes (osteosarcoma) [35], siRNA (glioma) [9], monoclonal antibodies (pancreatic, colorectal and prostate) [10, 28, 36], and u-PAR antagonists (melanoma and colorectal cancer) [37, 38]. While most of these studies successfully targeted u-PAR in preclinical models of cancer and resulted in various degrees of disruption of known u-PAR functions, most of the reported methods used to inhibit u-PAR are not ready for further clinical development because of short half life of the agents, and concerns involving their purity, stable delivery, and safety. Monoclonal antibodies, however, have finally come of age as therapeutics, and several molecules have recently been approved as cancer therapies. Therefore, given the encouraging results in this Hydroxyfasudil and other pre-clinical studies [10, 36], we believe that an antibody against u-PAR has the potential to be advanced into clinical testing. In view of previous observations that u-PAR directly binds to adhesion molecules, we investigated whether anti-u-PAR treatment affects u-PAR interaction with integrins. We.