胶质瘤相关小胶质细胞/巨噬细胞中的 CCL5 通过钙依赖性基质金属蛋白酶 2 调控胶质瘤迁移和侵袭。
CCL5 of glioma-associated microglia/macrophages regulates glioma migration and invasion via calcium-dependent matrix metalloproteinase 2.
机构信息
Graduate Institute of Biomedical Sciences, Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Taoyuan, Taiwan.
Department of Neurosurgery, Keelung Chang Gung Memorial Hospital, Keelung, Taiwan.
出版信息
Neuro Oncol. 2020 Feb 20;22(2):253-266. doi: 10.1093/neuonc/noz189.
BACKGROUND
Glioma-associated microglia/macrophages (GAMs) comprise macrophages of peripheral origin and brain-intrinsic microglia, which support tumor progression. Chemokine C-C ligand 5 (CCL5) is an inflammatory mediator produced by immune cells and is involved in tumor growth and migration in several cancers, including glioma. However, the mechanisms detailing how CCL5 facilitates glioma invasion remain largely unresolved.
METHODS
Glioma migration and invasion were determined by wound healing, transwell assay, and 3D µ-slide chemotaxis assay. The expression levels of CCL5, CD68, matrix metalloproteinase 2 (MMP2), phosphorylated Ca2+/calmodulin-dependent protein kinase II (p-CaMKII), p-Akt, and phosphorylated proline-rich tyrosine kinase 2 were determined by cytokine array, quantitative PCR, western blot, or immunohistochemistry. Zymography and intracellular calcium assays were used to analyze MMP2 activity and intracellular calcium levels, respectively.
RESULTS
CCL5 modulated the migratory and invasive activities of human glioma cells in association with MMP2 expression. In response to CCL5, glioma cells underwent a synchronized increase in intracellular calcium levels and p-CaMKII and p-Akt expression levels. CCL5-directed glioma invasion and increases in MMP2 were suppressed after inhibition of p-CaMKII. Glioma cells tended to migrate toward GAM-conditioned media activated by granulocyte-macrophage colony-stimulating factor (GM-CSF) in which CCL5 was abundant. This homing effect was associated with MMP2 upregulation, and could be ameliorated either by controlling intracellular and extracellular calcium levels or by CCL5 antagonism. Clinical results also revealed the associations between CCL5 and GAM activation.
CONCLUSION
Our results suggest that modulation of glioma CaMKII may restrict the effect of CCL5 on glioma invasion and could be a potential therapeutic target for alleviating glioma growth.
背景
神经胶质瘤相关的小胶质细胞/巨噬细胞（GAMs）包括外周来源的巨噬细胞和脑固有小胶质细胞，它们支持肿瘤的进展。趋化因子 C-C 配体 5（CCL5）是一种由免疫细胞产生的炎症介质，参与包括神经胶质瘤在内的几种癌症的肿瘤生长和迁移。然而，详细阐明 CCL5 如何促进神经胶质瘤侵袭的机制在很大程度上仍未解决。
方法
通过划痕愈合试验、Transwell 检测和 3D µ-slide 趋化性试验测定神经胶质瘤的迁移和侵袭。通过细胞因子阵列、定量 PCR、Western blot 或免疫组织化学测定 CCL5、CD68、基质金属蛋白酶 2（MMP2）、磷酸化钙/钙调蛋白依赖性蛋白激酶 II（p-CaMKII）、磷酸化 Akt 和磷酸化富含脯氨酸的酪氨酸激酶 2 的表达水平。分别使用明胶酶谱法和细胞内钙测定法分析 MMP2 活性和细胞内钙水平。
结果
CCL5 调节人神经胶质瘤细胞的迁移和侵袭活性，与 MMP2 的表达相关。对 CCL5 作出反应后，神经胶质瘤细胞经历了细胞内钙水平和 p-CaMKII 和 p-Akt 表达水平的同步增加。抑制 p-CaMKII 后，CCL5 介导的神经胶质瘤侵袭和 MMP2 的增加受到抑制。神经胶质瘤细胞倾向于向粒细胞-巨噬细胞集落刺激因子（GM-CSF）激活的 GAM 条件培养基中迁移，其中 CCL5 丰富。这种归巢效应与 MMP2 的上调有关，通过控制细胞内和细胞外钙水平或 CCL5 拮抗作用可以改善这种效应。临床结果也揭示了 CCL5 与 GAM 激活之间的关联。
结论
我们的结果表明，调节神经胶质瘤 CaMKII 可能会限制 CCL5 对神经胶质瘤侵袭的影响，并且可能是缓解神经胶质瘤生长的潜在治疗靶点。
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