In the light of growing understanding of tumor-immune interactions, immune modulating agents are becoming integral part of improved therapeutic strategies for cancer patients. While the primary goal is to engage cytotoxic cells in tumor elimination, targeting immunosuppressive environment is another topmost priority. Immunosuppression is mediated by innate myeloid cells, in particular myeloid-derived suppressor cells (MDSC), which are known to over-express cysteine cathepsins. Increased activities of several cathepsins, including cathepsins L and X, were associated with cancer progression for many cancer types. Therefore, cancer patients could benefit from selective small-molecule inhibitors that would balance cathepsins' excessive activity. However, little is known on their concurrent impact on the immune system, where cathepsins are known to regulate several important functions such as antigen presentation, regulation of cytotoxicity, adhesion etc. Recently, we identified novel selective cathepsin X inhibitor, Z9, with potential for clinical applications due to its reversible mechanism of action. Z9, along with established irreversible inhibitor AMS36, was used to study the impact of cathepsin X inhibition on antitumor immune response. For this purpose, we established and validated protocol for in vitro generation of MDSC. We showed that MDA-MB-231 breast cancer cell line promoted transition of human monocytic cells towards MDSC. Such MDSC potently reduced T cell proliferation in in vitro assay. Moreover, we demonstrated increased expression of several cathepsins in MDSC, most notably cathepsin L and cathepsin X. Inhibition of cathepsin X resulted in increased invasion of MDA-MB-231 cells in co-culture with immune cells. On the other hand, cathepsin L inhibition by CLIK-148 improved cytotoxicity of CD8+ cells during tumor-immune cell interactions. Inhibition of both cathepsins, however, potentiated MDSC immunosuppressive activity. Additionally, we hypothesized that proteolytic processing of [beta]2 integrin LFA-1 by cathepsin X could impact the immunological synapse formation and cytotoxicity against target cells. Surprisingly, we discovered that cathepsin X was regulated similarly to cathepsin W, and it was found to be secreted after cytotoxic cell degranulation. Based on our experiments we conclude that cathepsin X does not play essential role in regulation of LFA-1 in immunological synapse. In this regard cathepsin X inhibitors would not exert negative impact on cytotoxicity. We have demonstrated advantage of co-culture systems over tumor cell mono-cultures for in vitro testing of cysteine cathepsin inhibitors, since they provide additional insights into reciprocal interactions between tumor and immune cells. By using human cells, we succeeded in bridging the inter-species differences and in supplementing the lack of knowledge with novel information about the role of cysteine cathepsins in MDSC. Also, by revealing the functional impact on MDSC and cytotoxic cells, we provided consequential data that should be taken into consideration when evaluating cathepsin X inhibitors in vivo.
|
