Tumor-associated macrophages secrete TGFBI to promote malignancy of glioma stem cells in vitro

doi:10.3969/j.issn.1672-6731.2019.12.006

Abstract

Abstract:

Objective To investigate the expression heterogeneity of transforming growth factor-β-induced protein (TGFBI) in glioblastoma (GBM) multiform and the mechanism of its promotion on malignancy of glioma stem cells (GSCs) in vitro. Methods Using The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), REMBRANDT and Oncomine database to analyse the potential associations between grades, subtypes, overall survival (OS) of GBM and the expression level of TGFBI and the correlation between TGFBI and tumor-associated macrophages (TAMs) marker CD11b and CD163. Treating human monocytes cell line U937 with sequential peripheral blood mononuclear cells and IL-4 to get Primed-and M2-TAMs, quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression level of markers for M1 -, M2-TAMs and TGFBI. Performing immunofluorescent and immunohistochemical stain to reveal the distribution of TGFBI, M2-TAMs and GSCs in specimen of GBM patients, adding rhTGFBI to the culture medium to see its impacts on the sphere formation and invasion capacity of NCH-421K. Results According to the RNA-seq and microarray data of GBM patients in each database, expression level of TGFBI positively correlated to GBM grades and negatively to OS, Futhermore, expression level of TGFBI were strongly correlate with either pan TAMs marker CD11b or M2-TAMs marker CD163, and as the induction of U937-derived M2-TAMs, the expression level of M2-TAMs marker and TGFBI increased accompanied by decreasingly M1-TAMs marker. Also, we found an obvious co-location between TGFBI and CD163, SOX2 in frozen and paraffin sections of GBM specimens. Lastly, we demonstrate that rhTGF86BI could enhance the sphere formation and invasion capacity of NCH-421K in vitro. Conclusions M2-TAMs secrete TGFBI to promote the sphere formation and invasion capacity of GSCs in GBM.

Key words: Glioblastoma, Macrophages, Transforming growth factor beta, Neoplastic stem cells, Tumor cells, cultured

摘要：

目的探讨转化生长因子-β诱导蛋白（TGFBI）在胶质母细胞瘤中的异质性表达及其促进胶质瘤干细胞恶性行为的作用机制。方法检索肿瘤基因组学图谱计划（TCGA）、中国脑胶质瘤基因组图谱计划（CGGA）、脑肿瘤分子数据库（REMBRANDT）、Oncomine数据库中TGFBI在不同级别胶质瘤、不同亚型胶质母细胞瘤中的表达水平，以及TGFBI表达变化与患者生存期、胶质瘤相关巨噬细胞（TAMs）标志物CD11b和CD163的相关性。体外诱导人外周血单核细胞系U937分化为Primed-U937细胞和M2型巨噬细胞样细胞，实时定量聚合酶链反应检测诱导过程中TGFBI，M1型TAMs标志物CD80、白细胞介素-6（IL-6），以及M2型TAMs标志物CC趋化因子配体18（CCL18）、血管内皮生长因子A（VEGFA）的表达变化。免疫荧光染色和免疫组织化学染色观察胶质母细胞瘤细胞TGFBI与TAMs标志物CD163和胶质瘤干细胞标志物SOX2的分布情况。体外培养人原代胶质瘤干细胞系NCH-421K，测定重组人TGFBI（rhTGFBI）对细胞成球能力和侵袭能力的影响。结果（1）来自TCGA、CCGA、REMBRANDT、Oncomine数据库的数据分析显示，TGFBI表达变化与胶质瘤分级和恶性程度呈正相关，与患者生存期呈负相关，与TAMs标志物CD11b和CD163表达水平呈正相关。（2）在U937细胞诱导分化为M2型TAMs过程中，M1型TAMs标志物CD80（P=0.000）和IL-6（P=0.001）表达水平降低，M2型TAMs标志物CCL18（P=0.000）和VEGFA（P=0.002）表达水平升高，TGFBI表达水平亦升高（P=0.001）。（3）免疫荧光染色和免疫组织化学染色显示，胶质母细胞瘤细胞中TGFBI与TAMs标志物CD163和胶质瘤干细胞标志物SOX2均存在共定位。（4）体外成球实验和侵袭实验显示，经rhTGFBI处理后，NCH-421K细胞成球数目多于（P=0.000）、细胞球侵袭能力强于（P=0.001）对照组。结论胶质母细胞瘤TGFBI可由M2型TAMs分泌并促进胶质瘤干细胞成球、侵袭等恶性行为。

关键词: 胶质母细胞瘤, 巨噬细胞, 转化生长因子β, 肿瘤干细胞, 肿瘤细胞, 培养的

ZHU Hong-tao, LIU Dan, CHEN Zi-rong, LI Ya-kun, WANG Zhen, CHENG Fang-ling, LIU Jia-min, ZHANG Suo-jun, ZHANG Bin, WAN Feng. Tumor-associated macrophages secrete TGFBI to promote malignancy of glioma stem cells in vitro[J]. Chinese Journal of Contemporary Neurology and Neurosurgery, 2019, 19(12): 936-944.

朱洪涛, 刘丹, 陈籽荣, 厉亚坤, 王震, 程芳玲, 刘佳敏, 张所军, 张斌, 万锋. 肿瘤相关巨噬细胞分泌转化生长因子-β诱导蛋白影响胶质瘤干细胞特性的体外研究[J]. 中国现代神经疾病杂志, 2019, 19(12): 936-944.

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