胶质瘤初发与自然复发后肿瘤干细胞克隆及分子遗传学特征比较

Glioma stem cell clones and molecular genetics characteristics of primary and recurrent gliomas

目的比较初发和复发胶质母细胞瘤(GBM)标本、体外培养的原代胶质瘤干细胞及患者原代肿瘤组织来源的肿瘤移植模型(PDX)的分子遗传学特征。方法(1)取同一患者手术切除的初发及复发GBM标本,免疫组化染色检测标本胶质纤维酸性蛋白(GFAP)、巢蛋白、Ki-67的表达;基因检测分析标本O6-甲基鸟嘌呤DNA甲基转移酶(MGMT)基因甲基化、异柠檬酸脱氢酶(IDH)基因突变及表皮生长因子受体(EGFR)基因扩增情况。(2)体外原代培养初发及复发GBM干细胞,分别命名为SU5-1和SU5-2细胞。采用免疫荧光染色检测细胞巢蛋白、CD133的表达;诱导分化后采用免疫荧光染色检测细胞GFAP的表达,CCK-8实验检测细胞的生长曲线;Transwell侵袭实验检测细胞的侵袭能力;CCK-8实验检测细胞对替莫唑胺(TMZ)、卡铂(CBP)、顺铂(DDP)、阿霉素(ADM)的耐药性;Western blotting实验检测细胞程序性死亡受体-配体1(PD-L1)蛋白的表达;流式细胞仪检测细胞PD-L1阳性率的表达;基因检测分析同上。(3)建立裸小鼠原位初发及复发PDX模型,免疫组化染色检测PDX模型巢蛋白、GFAP、Ki-67的表达,基因检测分析同上。结果(1)与初发GBM比较,复发GBM标本Ki-67、巢蛋白阳性细胞数所占比例较高,而GFAP阳性细胞数所占比例较低,差异均有统计学意义(P<0.05);基因检测分析显示初发及复发GBM组织中IDH基因均无突变,初发GBM组织中MGMT基因呈甲基化状态,EGFR基因未扩增,而复发GBM组织中MGMT基因为非甲基化状态,EGFR基因扩增阳性。(2)SU5-1和SU5-2细胞均表达巢蛋白、CD133。诱导分化后均表达GFAP。生长曲线显示SU5-2细胞的增殖启动期早于SU5-1细胞,第3~5天两者持平,第6天起SU5-1细胞的增殖快于SU5-2细胞。SU5-2细胞的侵袭能力高于SU5-1细胞,差异有统计学意义(P<0.05)。5、10、15 mmol/L CBP、0.3125、1.25、5 mmol/L DDP,0.5、2 mmol/L ADM,125、500 mmol/L TMZ作用的SU5-2细胞的抑制率低于同浓度、同药物作用的SU5-1细胞抑制率,差异均有统计学意义(P<0.05)。SU5-2细胞中PD-L1蛋白的表达高于SU5-1细胞。SU5-2细胞PD-L1阳性率高于SU5-1细胞,差异有统计学意义(P<0.05)。基因检测分析结果与初发、复发GBM标本一致。(3)与初发PDX模型比较,复发PDX模型中巢蛋白、Ki-67的表达较高,GFAP的表达较低,差异有统计学意义(P<0.05)。基因检测分析结果与初发、复发GBM标本一致。结论初发及术后未放化疗自然复发的GBM分子遗传特征存在一定差异,后者侵袭性、耐药性、抑制抗瘤免疫能力更强;源于手术标本的原代肿瘤干细胞及相应PDX模型可忠实复制患者脑肿瘤的分子遗传特征,为肿瘤转化医学研究、临床药物靶点筛选提供可靠的实验平台。

Objective To explore the molecular genetic characteristics of primary and recurrent glioblastomas (GBMs) from the same patient in vivo, primary glioma stem cells cultured in vitro, and patient-derived xenograft (PDX). Methods (1) The primary and recurrent GBM specimens from one patient during surgical resection were collected;and the expressions of glial fibrillary acidic protein (GFAP), nestin and Ki-67 were detected by immunohistochemical staining;the methylation of O6-methylguanine DNA methyltransferase (MGMT) gene, mutation of isocitrate dehydrogenase (IDH) gene and amplification of epidermal growth factor receptor (EGFR) gene were analyzed.(2) The primary and recurrent GBM stem cells were cultured in vitro and named as SU5-1 and SU5-2 cells, respectively;the expressions of nestin and CD133 were detected by immunohistochemical staining;GFAP expression was detected by immunohistochemical staining after induced differentiation, and the growth curve was detected by CCK-8 assay;Transwell invasion assay was used to detect the invasion ability;cell resistance to temozolomide (TMZ), carboplatin (CBP), cisplatin (DDP) and adriamycin (ADM) was detected by CCK-8 assay;the protein expression of programmed death receptor-ligand 1(PD-L1) was detected by Western blotting. The rate of PD-L1 positive cells was detected by flow cytometry;genetic testing analysis was as above.(3) The primary and recurrent in situ PDX models in nude mice were established, and the expressions of nestin, GFAP and Ki-67 were detected by immunohistochemical staining. Results (1) As compared with the primary GBM, the recurrent GBM had significantly higher percentages of Ki-67 and nestin positive cells, while statistically lower percentage of GFAP positive cells (P<0.05);genetic analysis showed that there was no mutation in IDH gene in the primary GBM tissues and recurrent GBM tissues;the MGMT gene in the primary GBM tissues was methylated and EGFR gene was not amplified, while the MGMT gene in recurrent GBM tissues was demethylated and EGFR gene amplification was positive.(2) Both SU5-1 and SU5-2 cells expressed nestin and CD133, and GFAP was expressed after induced differentiation;the growth curve showed that the proliferation of SU5-2 cells started earlier than that of SU5-1 cells, the two were equal on the 3rd, 4th, and 5th d, and the proliferation of SU5-1 cells was faster than that of SU5-2 cells from the 6th d;the invasion ability of SU5-2 cells was statistically stronger than that of SU5-1 cells (P<0.05);the inhibition rates of SU5-2 cells treated with 5, 10, and 15 mmol/L CBP, 0.3125, 1.25, and 5 mmol/L DDP, 0.5 and 2 mmol/L ADM, and 125 and 500 mmol/L TMZ were significantly lower than those of SU5-1 cells treated with the same concentrations and same drugs (P<0.05);the protein expression of PD-L1 in SU5-2 cells was higher than that in SU5-1 cells;the positive rate of PD-L1 in SU5-2 cells was statistically higher than that in SU5-1 cells (P<0.05);the results of genetic analysis were consistent with those of the primary and recurrent GBM samples.(3) As compared with those in the primary PDX model, the nestin and Ki-67 expressions were significantly higher and GFAP expression was significantly lower in the recurrent PDX model (P<0.05);the results of genetic analysis were consistent with those of the primary and recurrent GBM samples. Conclusions Genetic differences are detected between primary and recurrent GBMs;recurrent GBM has stronger invasive capacity and multi-drug resistance. The primary stem cells derived from surgical specimens and corresponding PDX models could replicate the molecular genetic characteristics of original tumors, which provide a reliable experimental platform for both tumor translation researches and screening of molecular therapeutic targets.