Furthermore, and by exploiting the function blocking properties of a cell-impermeable monoclonal antibody named mAb 4C5, specifically targeted against HSP90 we have shown that extracellular HSP90 interacts with HER-2 on the cell surface as well as metalloproteinases MMP-2 and MMP-9. Although a Torin 1 growing number of HSP90’s co-chaperones such as HSP70, Hop and p23 were also found on the cell surface, their action and underlying mechanisms have not been elucidated yet. Taking the above into consideration, in the present work we explore the cell surface localization of Cdc37 and we examine its possible involvement in cancer cell invasion processes as well as its potential interacting partners during this process, using the MDA-MB-453 and MDA-MB-231 human breast cancer cell lines and a commercially available polyclonal antibody against Cdc37. Furthermore and taking into account previously reported data showing that mAb 4C5 inhibits cancer cell invasion by disrupting association of extracellular HSP90 with HER2 and metalloproteinases MMP2 and MMP9 in this work we investigate the possible effect of this cell impermeable anti-HSP90 antibody on the interactions of surface Cdc37 with HSP90 and the ErbB receptors. In the present work we demonstrate that the co-chaperone Cdc37 is localized on the surface of MDA-MB-453 and MDAMB-231 breast cancer cells, where it is necessary for the motility of these cells and similarly to its intracellular counterpart it specifically interacts with the molecular chaperone HSP90. Moreover our findings show that this surface pool of Cdc37 directly interacts with members of the ErbB family of growth factor receptors possibly acting as a co-factor in HSP90 extracellular chaperoning activities implicated in cancer cell invasion processes and that the anti-HSP90 antibody mAb 4C5 disrupts these interactions. Cell surface localization of Cdc37 was demonstrated by both immunocytochemistry on live MDA-MB-453 and MDA-MB-231 breast cancer cells and western blot using membrane fraction lysates derived from these cell lines. The inhibition of MDA-MB-453 and MDA-MB-231 breast cancer cell motility, by the anti-Cdc37 antibody was shown using the wound healing assay. Presence of this antibody in the culture medium significantly reduced the motility rate of both cell lines studied. At this point it is interesting to note that when the antiHSP90 antibody mAb 4C5 and the anti-Cdc37 antibody were included either separately or combined in the culture medium of the above cells no statistically significant differences were observed in the wound closures. This is not surprising since mAb 4C5 and anti-Cdc37 are targeted against two different molecules involved in the cell motility process. To assess the participation of cell surface and not intracellular Cdc37 in the motility process of the cancer cells studied, internalization of the anti-Cdc37 antibody was examined. Interestingly, the antibody remained bound to the cell surface of both MDA-MB-453 and MDA-MB-231 cells, even after 16 h in culture and was not internalized indicating that the previously mentioned inhibitory effect of the anti-Cdc37 antibody is due to binding of the antibody to the cell surface pool of Cdc37. It is well established that intracellular Cdc37 acts as cochaperone of HSP90 by targeting protein kinases to the chaperone machinery and thus contributing to their activation. Taking this into account together with the above mentioned results we next examined the possible association of cell surface Cdc37 with the surface pools of HSP90 and the ErbB receptors.