Cistatin F kot mediator imunske supresije v tumorskem mikrookolju

Immunosuppressive microenvironment causes decreased function of cytotoxic immune cells and contributes to the immune escape of cancer cells. Natural killer (NK) cells represent a very promising candidate for cancer therapy. They target tumor cells with granule-mediated cytotoxic pathway when the balance between activatory and inhibitory signals caused by the interaction of suitable ligands with their receptors is disrupted towards the activation of NK cells. Effector molecules, granzymes and perforin are stored in their inactive form and are activated from precursor forms by cathepsins C, H, and L. Cystatin F acts as a modulator of NK cell cytotoxicity as it can inhibit those cathepsins, thereby attenuating NK cell cytotoxicity. Cystatin F is glycosylated and is activated from dimeric to monomeric form by cathepsin V. It is localized in endosomes/lysosomes, but can also be secreted and further internalized to bystander cells. Cystatin F is normally expressed by immune cells, but several studies implicate cystatin F expression to other cell types, in particular under various pathological conditions. We have shown that cystatin F expression is increased in glioblastoma by either glioblastoma tumor or immune cells, both infiltrating immune cells and resident immune cells such as microglia. Moreover, glioblastoma cells were able to internalise cystatin F, which contributed to the decreased susceptibility of glioblastoma cells to NK cell cytotoxicity. We demonstrated that N-glycosylation profile of cystatin F differed between cell types and between NK cells of different cytotoxic potential. High-mannose glycosylation was associated with increased localization in lysosomes, cathepsin C interaction and attenuation of NK cell cytotoxicity. Further, cystatin F internalisation to NK cells by means of endocytosis, enhanced split anergy of NK cells. We characterised super-charged NK cells, potent candidates for cancer therapy, with increased cytotoxicity, cytokine secretion and ability to proliferate. Super-charged NK cells had increased expression and activity of cathepsin C and granzyme B and increased expression of inactive dimeric cystatin F. Finally, we identified a small molecular inhibitor of cathepsin V, which prevented activation of cystatin F from inactive dimeric form and increased NK cell cytotoxicity. We have therefore established cystatin F as an important mediator of immunosuppression in the tumor microenvironment and proposed novel therapeutic approaches to improve cancer immunotherapy by targeting cystatin F.Imunosupresivno mikrookolje povzroča zmanjšano delovanje citotoksičnih imunskih celic in prispeva k imunskemu pobegu rakavih celic. Naravne celice ubijalke (NK) so uporabne kandidatke za zdravljenje raka, ki se še ne uporabljajo v klinični praksi. Celice NK ciljajo tumorske celice s citotoksičnimi granulami, potem ko se ravnovesje signalov, ki jih sproži vezava ustreznih ligandov na aktivacijske in inhibitorne receptorje na njihovih površinah, prevesi v aktivacijo celic NK. Neaktivno shranjene efektorske molekule, grancime in perforin aktivirajo katepsini C, H in L. Cistatin F deluje kot modulator citotoksičnosti celic NK, saj lahko zavira omenjene katepsine in s tem oslabi citotoksično delovanje celic NK. Cistatin F je glikoziliran. Da lahko deluje, se mora pretvoriti iz dimerne v monomerno obliko, kar katalizira katepsin V. Nahaja se v endosomih/lizosomih, lahko pa se tudi izloča in internalizira v sosednje celice. Cistatin F se običajno izraža v imunskih celicah, vendar vedno več študij kaže tudi na njegovo izražanje v patoloških stanjih. Pokazali smo, da je izražanje cistatina F povečano v glioblastomu in da ga izražajo tako glioblastomske kot infiltrirajoče ter stalno prisotne imunske celice, kot npr. mikroglija. Poleg tega, so bile glioblastomske celice sposobne privzeti cistatin F z endocitozo, kar je prispevalo k njihovi zmanjšani občutljivosti na citotoksičnost celic NK. Dokazali smo tudi, da se N-glikozilacija cistatina F razlikuje med različnimi tipi celic in med celicami NK z različnim citotoksičnim potencialom. Visoko-manozna glikozilacija cistatina F je bila povezana z njegovo povečano lokalizacijo v lizosomih, interakcijo s katepsinom C in zmanjšanjem citotoksičnosti celic NK. Internalizacija cistatina F v celice NK je vplivala na njihovo delno anergijo. Analizirali smo tudi supervzbujene celice NK, ki imajo dober potencial za zdravljenje raka, saj so zanje značilni povečana citotoksičnost, izločanje citokinov in zmožnost proliferacije. Supervzbujene celice NK izkazujejo povečano izražanje in aktivnost katepsina C in grancima B, ter povečano izražanje neaktivnega dimernega cistatina F. Nazadnje smo identificirali še nizko-molekularni zaviralec katepsina V, ki je preprečil aktivacijo cistatina F in povečal citotoksičnost celic NK. Tako smo cistatin F opredelili kot pomemben dejavnik imunosupresije v tumorskem mikrookolju in zasnovali nove terapevtske pristope za izboljšanje imunske terapije raka

Cysteine Cathepsins as Therapeutic Targets in Immune Regulation and Immune Disorders

Cysteine cathepsins, as the most abundant proteases found in the lysosomes, play a vital role in several processes—such as protein degradation, changes in cell signaling, cell morphology, migration and proliferation, and energy metabolism. In addition to their lysosomal function, they are also secreted and may remain functional in the extracellular space. Upregulation of cathepsin expression is associated with several pathological conditions including cancer, neurodegeneration, and immune-system dysregulation. In this review, we present an overview of cysteine-cathepsin involvement and possible targeting options for mitigation of aberrant function in immune disorders such as inflammation, autoimmune diseases, and immune response in cancer

Cysteine Cathepsins as Therapeutic Targets in Immune Regulation and Immune Disorders

Cysteine cathepsins, as the most abundant proteases found in the lysosomes, play a vital role in several processes—such as protein degradation, changes in cell signaling, cell morphology, migration and proliferation, and energy metabolism. In addition to their lysosomal function, they are also secreted and may remain functional in the extracellular space. Upregulation of cathepsin expression is associated with several pathological conditions including cancer, neurodegeneration, and immune-system dysregulation. In this review, we present an overview of cysteine-cathepsin involvement and possible targeting options for mitigation of aberrant function in immune disorders such as inflammation, autoimmune diseases, and immune response in cancer

New inhibitors of cathepsin V impair tumor cell proliferation and elastin degradation and increase immune cell cytotoxicity

Cathepsin V is a human lysosomal cysteine peptidase with specific functions during pathological processes and is as such a promising therapeutic target. Peptidase inhibitors represent powerful pharmacological tools for regulating excessive proteolytic activity in various diseases. Cathepsin V is highly related to cathepsin L but differs in tissue distribution, binding site morphology, substrate specificity, and function. To validate its therapeutic potential and extend the number of potent and selective cathepsin V inhibitors, we used virtual high-throughput screening of commercially available compound libraries followed by an evaluation of kinetic properties to identify novel potent and selective cathepsin V inhibitors. We identified the ureido methylpiperidine carboxylate derivative, compound 7, as a reversible, selective, and potent inhibitor of cathepsin V. It also exhibited the most preferable characteristics for further evaluation with in vitro functional assays that simulate the processes in which cathepsin V is known to play an important role. Compound 7 exerted significant effects on cell proliferation, elastin degradation, and immune cell cytotoxicity. The latter was increased because compound 7 impaired conversion of immunosuppressive factor cystatin F to its active monomeric form. Taken together, our results present novel potent inhibitors of cathepsin V and provide new hit compounds for detailed development and optimization. Further, we demonstrate that cathepsin V is a potential target for new approaches to cancer therapy

Sequential therapy with supercharged NK cells with either chemotherapy drug cisplatin or anti-PD-1 antibody decreases the tumor size and significantly enhances the NK function in Hu-BLT mice

Introduction and methodsIn this study we report that sequential treatment of supercharged NK (sNK) cells with either chemotherapeutic drugs or check-point inhibitors eliminate both poorly differentiated and well differentiated tumors in-vivo in humanized-BLT mice.Background and resultssNK cells were found to be a unique population of activated NK cells with genetic, proteomic, and functional attributes that are very different from primary untreated or IL-2 treated NK cells. Furthermore, NK-supernatant differentiated or well-differentiated oral or pancreatic tumor cell lines are not susceptible to IL-2 activated primary NK cell-mediated cytotoxicity; however, they are greatly killed by the CDDP and paclitaxel in in-vitro assays. Injection of one dose of sNK cells at 1 million cells per mouse to aggressive CSC-like/poorly differentiated oral tumor bearing mice, followed by an injection of CDDP, inhibited tumor weight and growth, and increased IFN-γ secretion as well as NK cell-mediated cytotoxicity substantially in bone marrow, spleen and peripheral blood derived immune cells. Similarly, the use of check point inhibitor anti-PD-1 antibody increased IFN-γ secretion and NK cell-mediated cytotoxicity, and decreased the tumor burden in-vivo, and tumor growth of resected minimal residual tumors from hu-BLT mice when used sequentially with sNK cells. The addition of anti-PDL1 antibody to poorly differentiated MP2, NK-differentiated MP2 or well-differentiated PL-12 pancreatic tumors had different effects on tumor cells depending on the differentiation status of the tumor cells, since differentiated tumors expressed PD-L1 and were susceptible to NK cell mediated ADCC, whereas poorly differentiated OSCSCs or MP2 did not express PD-L1 and were killed directly by the NK cells.ConclusionsTherefore, the ability to target combinatorially clones of tumors with NK cells and chemotherapeutic drugs or NK cells with checkpoint inhibitors at different stages of tumor differentiation may be crucial for successful eradication and cure of cancer. Furthermore, the success of check point inhibitor PD-L1 may relate to the levels of expression on tumor cells