Senjor Emanuela, Perišić Nanut Milica, Breznik Barbara, Mitrović Ana, Mlakar Jernej, Rotter Ana, Porčnik Andrej, Lah Turnšek Tamara, Kos Janko
Department of Biotechnology, Jožef Stefan Institute, Jamova cesta 39, SI-1000, Ljubljana, Slovenia.
Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, SI-1000, Ljubljana, Slovenia.
Cell Oncol (Dordr). 2021 Oct;44(5):1051-1063. doi: 10.1007/s13402-021-00618-9. Epub 2021 Jun 29.
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.
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.
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.
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.
胶质母细胞瘤是最具侵袭性的脑癌类型，由分化细胞、癌症干细胞和免疫细胞的异质性群体组成。胱抑素F是溶酶体半胱氨酸肽酶的内源性抑制剂，可调节细胞毒性免疫细胞的功能。本研究的目的是确定胶质母细胞瘤中哪种类型的细胞表达胱抑素F，并确定胱抑素F在疾病进展过程中的作用。
采用逆转录定量聚合酶链反应（RT-qPCR）和免疫组织化学方法测定胶质母细胞瘤组织样本中胱抑素F的mRNA和蛋白水平。通过蛋白质印迹法分析胱抑素F的内化情况。采用酶动力学、实时侵袭和钙黄绿素释放细胞毒性试验评估内化胱抑素F的作用。
我们发现胱抑素F在非癌脑组织中不表达，但其表达随胶质瘤进展而增加。在肿瘤组织中，在癌症干细胞样细胞和小胶质细胞/单核细胞中观察到广泛染色，这些细胞将胱抑素F分泌到其微环境中。使用体外胶质母细胞瘤细胞模型证实了胱抑素F的反式活性。内化的胱抑素F影响胶质母细胞瘤细胞中的组织蛋白酶L活性并降低其侵袭性。此外，我们发现胱抑素F降低了胶质母细胞瘤细胞对自然杀伤（NK）细胞细胞毒性活性的敏感性。
我们的数据表明胱抑素F是胶质母细胞瘤免疫抑制的介质。免疫细胞、胶质母细胞瘤细胞和胶质母细胞瘤干细胞样细胞中胱抑素F mRNA和蛋白水平的增加或反式内化的胱抑素F可能会影响胶质母细胞瘤细胞对NK细胞毒性的敏感性降低。
