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.

中文翻译：

---

胱抑素 F 在胶质母细胞瘤中充当免疫抑制介质

目的

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群体组成。Cystatin F 是一种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哪种类型的细胞在胶质母细胞瘤中表达胱抑素 F，并确定胱抑素 F 在疾病进展过程中的作用。

方法

RT-qPCR 和免疫组织化学用于确定胶质母细胞瘤组织样本中的胱抑素 F mRNA 和蛋白质水平。通过蛋白质印迹分析胱抑素F的内化。酶动力学、实时侵袭和钙黄绿素释放细胞毒性测定用于评估内化胱抑素 F 的作用。

结果

我们发现胱抑素 F 在非癌脑组织中不表达，但其表达随着神经胶质瘤的进展而增加。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) 细胞的细胞毒活性的敏感性。

结论

我们的数据暗示胱抑素 F 是胶质母细胞瘤免疫抑制的介质。免疫细胞、胶质母细胞瘤和胶质母细胞瘤干细胞样细胞或反式内化的胱抑素 F mRNA 和蛋白质水平升高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。