The goal of the master’s thesis was to investigate the expression and activity of cysteine cathepsins in indirect-contact co-cultures established between macrophages and breast cancer cells. Cysteine cathepsins are important targets of anticancer therapies, and their expression is influenced by various factors within the tumor microenvironment (TME). In this thesis, we studied the interactions between the tumor cell lines MCF7 and MDA-MB-231 and different types of macrophages differentiated from the monocytic cell lines THP-1 and U937. Indirect-contact co-cultures were established by exchanging culture media between tumor cells and macrophages, while the success of macrophage differentiation was confirmed by flow cytometry. Successful macrophage differentiation was confirmed by flow cytometry. The expression of cysteine cathepsins was analyzed in cell lysates prepared from different co-cultures using primary and secondary antibodies following protein separation using SDS-PAGE and western blot transfer. Cathepsin activity was determined using enzyme kinetics with specific fluorogenic substrates. The results showed that the added conditioned medium diefferently affected the expression and activity of cathepsins across individual co-cultures. In macrophages treated with tumor cell-conditioned medium, we generally detected higher relative amounts of cathepsins compared to the inversely established co-cultures, suggesting that tumor cells secrete cathepsins into the medium, which are subsequently taken up by macrophages. The effect of macrophage-conditioned medium on tumor cells was less pronounced. Analysis of enzymatic activity further revealed differences in cathepsin activity depending on the conditioned media used and the type of co-culture. The results confirm that intercellular interactions significantly contribute to the proteolytic potential of the tumor microenvironment and represent an important basis for the development of targeted antitumor therapies.
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