Emanuela Senjor1,2, Milica Perišić Nanut1, Barbara Breznik3, Ana Mitrović1, Jernej Mlakar4, Ana Rotter3, Andrej Porčnik5, Tamara Lah Turnšek3, Janko Kos6,7. 1. Department of Biotechnology, Jožef Stefan Institute, Jamova cesta 39, SI-1000, Ljubljana, Slovenia. 2. Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, SI-1000, Ljubljana, Slovenia. 3. Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111, SI-1000, Ljubljana, Slovenia. 4. Institute of Pathology, Medical Faculty, University of Ljubljana, Korytkova 2, SI-1000, Ljubljana, Slovenia. 5. Department of Neurosurgery, University Clinical Centre Ljubljana, SI-1000, Ljubljana, Slovenia. 6. Department of Biotechnology, Jožef Stefan Institute, Jamova cesta 39, SI-1000, Ljubljana, Slovenia. janko.kos@ffa.uni-lj.si. 7. Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, SI-1000, Ljubljana, Slovenia. janko.kos@ffa.uni-lj.si.
Abstract
PURPOSE: Glioblastoma, the most aggressive type of brain cancer, is composed of heterogeneous populations of differentiated cells, cancer stem cells and immune cells. Cystatin F, an endogenous inhibitor of lysosomal cysteine peptidases, regulates the function of cytotoxic immune cells. The aim of this study was to determine which type of cells expresses cystatin F in glioblastoma and to determine the role of cystatin F during disease progression. METHODS: RT-qPCR and immunohistochemistry were used to determine cystatin F mRNA and protein levels in glioblastoma tissue samples. The internalization of cystatin F was analyzed by Western blotting. Enzyme kinetics, real time invasion and calcein release cytotoxicity assays were used to assess the role of internalized cystatin F. RESULTS: We found that cystatin F was not expressed in non-cancer brain tissues, but that its expression increased with glioma progression. In tumor tissues, extensive staining was observed in cancer stem-like cells and microglia/monocytes, which secrete cystatin F into their microenvironment. In trans activity of cystatin F was confirmed using an in vitro glioblastoma cell model. Internalized cystatin F affected cathepsin L activity in glioblastoma cells and decreased their invasiveness. In addition, we found that cystatin F decreased the susceptibility of glioblastoma cells to the cytotoxic activity of natural killer (NK) cells. CONCLUSIONS: Our data implicate cystatin F as a mediator of immune suppression in glioblastoma. Increased cystatin F mRNA and protein levels in immune, glioblastoma and glioblastoma stem-like cells or trans internalized cystatin F may have an impact on decreased susceptibility of glioblastoma cells to NK cytotoxicity.
PURPOSE: Glioblastoma, the most aggressive type of brain cancer, is composed of heterogeneous populations of differentiated cells, cancer stem cells and immune cells. Cystatin F, an endogenous inhibitor of lysosomal cysteine peptidases, regulates the function of cytotoxic immune cells. The aim of this study was to determine which type of cells expresses cystatin F in glioblastoma and to determine the role of cystatin F during disease progression. METHODS: RT-qPCR and immunohistochemistry were used to determine cystatin F mRNA and protein levels in glioblastoma tissue samples. The internalization of cystatin F was analyzed by Western blotting. Enzyme kinetics, real time invasion and calcein release cytotoxicity assays were used to assess the role of internalized cystatin F. RESULTS: We found that cystatin F was not expressed in non-cancer brain tissues, but that its expression increased with glioma progression. In tumor tissues, extensive staining was observed in cancer stem-like cells and microglia/monocytes, which secrete cystatin F into their microenvironment. In trans activity of cystatin F was confirmed using an in vitro glioblastoma cell model. Internalized cystatin F affected cathepsin L activity in glioblastoma cells and decreased their invasiveness. In addition, we found that cystatin F decreased the susceptibility of glioblastoma cells to the cytotoxic activity of natural killer (NK) cells. CONCLUSIONS: Our data implicate cystatin F as a mediator of immune suppression in glioblastoma. Increased cystatin F mRNA and protein levels in immune, glioblastoma and glioblastoma stem-like cells or trans internalized cystatin F may have an impact on decreased susceptibility of glioblastoma cells to NK cytotoxicity.
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