Targeting Cystatin F Activation Enhances NK Cell Cytotoxicity in Glioblastoma Models

Emanuela Senjor^1,2Anamarija Habič^3,4Urban Švajger⁵Ana Mitrović^1,2Matic Proj²Andrej Porčnik⁶Borut Prestor⁶Miha Jerala⁷Matic Bošnjak⁷Stanislav Gobec²Barbara Breznik^3,8Janko Kos^1,2Milica Perisic Nanut^1*

• ¹Department of Biotechnology, Institut Jožef Stefan (IJS), Ljubljana, Slovenia
• ²Univerza v Ljubljani Fakulteta za farmacijo, Ljubljana, Slovenia
• ³Nacionalni institut za biologijo Oddelek za genetsko toksikologijo in biologija raka, Ljubljana, Slovenia
• ⁴Mednarodna podiplomska sola Jozefa Stefana, Ljubljana, Slovenia
• ⁵Zavod Republike Slovenije za transfuzijsko medicino, Ljubljana, Slovenia
• ⁶Klinični oddelek za nevrokirurgijo, Univerzitetni klinicni center Ljubljana, Ljubljana, Slovenia
• ⁷Univerza v Ljubljani Institut za patologijo, Ljubljana, Slovenia
• ⁸Univerza v Ljubljani Fakulteta za Kemijo in Kemijsko tehnologijo, Ljubljana, Slovenia

Glioblastoma (GBM) is a highly invasive brain tumor with limited therapeutic options and a poor prognosis. Natural killer cells (NK cells) are important effectors of antitumor immunity, as they can eliminate cancer stem-like cells. However, GBM is also characterized as an immunosuppressive tumor. One of the contributing immunosuppressive factors identified in this study is cystatin F, which is involved in the regulation of the granule-mediated cytotoxicity of NK cells. We demonstrate that cystatin F expression in GBM correlates with immune exhaustion and suppression markers. Furthermore, we show reduced function of GBM patients' NK cells compared to healthy donors. Using in vitro co-culture models, we show that cystatin F-expressing microglial cells impair NK cell cytotoxicity against glioblastoma stem-like cells. To counteract this effect, we inhibited the activation of cystatin F from dimeric to active monomeric form by using a small molecular inhibitor of cathepsin V, a protease responsible for cystatin F activation. Enhanced NK cell cytotoxicity was observed using standard cytotoxicity assays, 3D spheroid models, and perfused models in microfluidic devices.