TLR9在胶质瘤中的表达及其与胶质瘤生物学行为和预后的关系
摘要
前言
神经胶质瘤是最常见的脑肿瘤,尽管包括手术切除,放射治疗和化学治疗在内的综合治疗策略的联合应用,高级别神经胶质瘤的预后仍不尽人意,特别是多形性胶质母细胞瘤。多形胶质母细胞瘤是颅内恶性程度最高的恶性肿瘤,占原发性颅内肿瘤的15-20%。多形胶质母细胞瘤具有细胞异质性、分裂极度活跃、肿瘤微血管增生和坏死的组织病理特点。多形胶质母细胞瘤病人的平均中位生存期只有大约一年,在过去的十年中没有显著改善。因此,迫切需要开发新的治疗策略或新的辅助治疗方法。
免疫治疗被认为是一种极有前途的治疗方法,因为它能激活淋巴细胞,可以特异性的识别并摧毁肿瘤细胞。近年来对胶质瘤发病机制的研究取得了较大的进展,同时研究者发现中枢神经系统内存在免疫反应,这使得免疫疗法可以应用于脑胶质瘤的治疗。胶质瘤免疫治疗的策略包括被动性免疫治疗,T细胞过继免疫治疗和主动免疫。
未甲基化的CpG二核苷酸在细菌和病毒DNA中相当常见,但是在脊椎动物中却减弱并被甲基化。包含非甲基化的CpG双核苷酸序列在内的特定核苷酸序列具有强有力的免疫调节功能,可以作为癌症疫苗的免疫佐剂。CpG-ODN是人工合成的含有未甲基化CpG基序的单链寡聚脱氧核苷酸,可以模仿细菌或病毒DNA对脊椎动物免疫系统产生刺激作用。根据作用特点的差别,CpG-ODN可被分成三类,其中B类CpG-ODN能刺激B细胞增生和活化,目前已作为癌症疫苗的免疫佐剂在临床试验中使用。B型CpG-ODN对哺乳动物免疫细胞增生活化的刺激作用主要是通过与Toll样受体9结合,进而激活免疫系统,诱导细胞因子的分泌和释放,并诱发强有力的T细胞抗瘤免疫反应。
目前为止,已经鉴定出人类11种Toll样受体和老鼠13种Toll样受体。TLR9就是Toll样受体家族家族的一员,进化上是高度保守的跨膜蛋白,可以识别微生物衍生的分子模式结构。TLR9对于浆细胞样树突状细胞和B细胞的免疫刺激作用得到了广泛的研究。CpG作用于TLR9可以引起受体的结构性变化,然后通过衔接蛋白MyD88的募集反应,促进激酶类有丝分裂原激活蛋白的磷酸化激活相应信号通道,激活核因子-κB并调控相应基因的表达。在细胞水平上,TLR9的激动作用引起先天性和适应性免疫反应的级联效应。TLR9激动药激活浆细胞样树突状细胞分泌Ⅰ型干扰素并上调CD80和CD86等共刺激分子的表达水平。人们认为这会引发一系列免疫效应,如MCP-1,IP-10等细胞因子以及炎症趋化因子的分泌,NK细胞的激活,T细胞群的扩张,尤其是对于TH-1细胞和细胞毒性T细胞的作用。TLR9激动剂作用的结果之一是启动有效的细胞介导的TH-1应答。另外TLR9激动剂可以加快B细胞分化成抗体分泌型浆细胞,还可以促进抗体依赖的细胞毒性反应,这说明TLR9激动剂的另一个作用结果是可以启动体液性免疫应。
对由TLR9激动作用引起的免疫应答的了解,推动了在传染病,癌症,哮喘和过敏领域中TLR9激动药在临床上的发展。然而研究TLR9激动药作为抗癌药的理论基础是一种假说,这一假说认为先天性免疫应答可能有直接的抗肿瘤作用,而TH-1类细胞因子和趋化因子可以增强抗原提呈并进而促进抗肿瘤免疫应答。
最近的一些研究表明,除浆细胞样树突状细胞和B细胞中有表达外,TLR9的也表达在某些肿瘤细胞,研究者证实乳腺、胃、肺和淋巴性的上皮性肿瘤中存在TLR9的表达。然而大量数据表示,TLR9的激动剂CpG寡核苷酸可促进肿瘤的发展和转移。胶质瘤属于神经上皮性肿瘤,我们推测TLR9在胶质瘤中可能存在表达。我们的预试验验证了我们的想法,在细胞系和胶质瘤标本中都检测出了其表达。我们假设,TLR9的信号通路可能在脑胶质瘤生长和进展中非常重要。TLR9信号通路在人脑胶质瘤的确切作用目前还不清楚,其对胶质瘤生长是促进还是抑制,目前还没有相关报道。有实验报道局部使用CpG寡核苷酸免疫治疗可以延长胶质瘤小鼠的生存期。相反,Ginzkeyetal等人发现在大鼠脑胶质瘤模型中瘤内注射免疫刺激剂CpG寡核苷酸,肿瘤大小反而增加,这与我们先前所做的实验结果一致,在以前的实验中我们发现的CpG寡核苷酸瘤内注射并不能增加GL261胶质瘤荷瘤鼠的生存时间,这结果提示,CpG直接局部注射,可能不会在胶质瘤患者产生有益的结果。
已有研究者初步检测了TLR9在胶质母细胞瘤中的表达,但没有发现其对预后的意义。我们认为此研究存在一定的局限性,其研究仅进行了TLR9 mRNA水平的评价,而没有进行蛋白质水平的研究,由于蛋白质是基因功能的体现,因此检测TLR9蛋白在胶质瘤中表达是十分必要的,这对于提高对CpG-ODN恶性胶质瘤的免疫辅助治疗的认识是至关重要的。目前还没有系统的TLR9蛋白在胶质瘤中的表达情况和作用机制的研究。本研究将应用组织芯片技术,探讨TLR9蛋白在大样本的脑胶质细胞瘤样品中的表达情况,分析TLR9的表达与临床病理特点的关系以及TLR9表达与胶质母细胞瘤患者生存期之间的关联。为了进一步阐述TLR9的表达对肿瘤细胞可能发挥的作用,我们还将探讨CpG寡核苷酸在体外对的胶质瘤细胞增殖和侵袭的直接影响。
研究TLR9在神经胶质瘤中的表达和功能,及其信号传导通路,对于理解神经胶质瘤的形成和恶性进展有重要帮助,同时也提供了胶质瘤新的有效治疗靶点。
方法一、细胞和细胞培养
恶性人脑胶质瘤细胞系U87-MG, U251-MG和大鼠脑胶质瘤C6细胞系从ATCC(美国组织培养细胞库)购得,并在中国医科大学附属第一医院中心实验室培养。胶质瘤细胞系U87-MG、U251-MG和C6细胞以含10%胎牛血清的DMEM高糖培养基培养,于37℃,100U／ml青霉素,100μg／ml链霉素和5%CO2条件下恒温培养箱内培养。
二、RNA提取及RT-PCR检测TLR9的表达采用Trizol试剂一步法提取人脑胶质瘤U87-MG、人脑胶质瘤U251-MG细胞系、大鼠脑胶质瘤C6和手术切除的人脑胶质瘤组织总RNA,紫外分光光度计检测OD260/OD280及核酸浓度。提取细胞系和肿瘤标本的总RNA进行cDNA第一链合成。按照RT-PCR试剂盒的说明,应用特异性引物人类TLR9 (F: 5'-TGTAATAACAGTTGCCGTCCAT-3',R:5'-CAGCCTTTCCTTGTCCTCC-3'),大鼠TLR9 (F:5'-GGACAGTTCTCTCCACTCGC-3', R:5'-TTCTTTGAGACGGGAGTGCT-3');人和大鼠管家基因GAPDH (F:5'-TCCACCACCCTGTTGCTGTA-3', R:5'-ACCACAGTCCATGCCATCAC-3')进行PCR扩增,产物进行1.5%琼脂糖凝胶电泳,并在紫外线下的观察。
三、免疫荧光检测TLR9蛋白在细胞系中的表达
U87-MG和U251细胞在盖玻片上爬片生长,盖玻片用无血清培养液冲洗后,4%多聚甲醛固定,0.5%Trixton X-100的PBS透膜,并在含5%胎牛血清的PBS中封闭1小时。细胞与TLR9的单克隆抗体在4℃孵育过夜,然后由Cy3标记的抗鼠的二抗孵育2小时,细胞核用Hoechest复染。在对照样品中,去掉一抗,胶质瘤细胞用二抗和Hoechest染色。然后应用荧光激发Cy3和Hoechest,共聚焦显微镜下观察TLR9在细胞系中的表达并拍照。
四、脑胶质瘤组织芯片的构建
提取脑胶质瘤组织标本,进行石蜡包埋并进行HE染色,根据世界卫生组织2000年的分类系统的诊断标准重新诊断并且与病理结果比较,由神经病理学家选取代表性的肿瘤区域,构建组织微阵列芯片。手术切除的正常肾脏组织作为阳性对照。
五、脑胶质瘤组织芯片免疫组织化学染色
4um组织芯片切片在65℃烤30分钟,二甲苯脱蜡和水化后,切片浸入乙二胺四乙酸的缓冲液中进行高压抗原修复,然后经过3%过氧化氢灭活内源性过氧化物酶,1%胎牛血清封闭。抗人TLR9抗体,4℃孵育过夜。阴性对照,一抗为正常小鼠血清。人正常肾样品作为阳性对照。根据免疫组化试剂盒说明,辣根过氧化物酶标记的二抗聚合物在室温下孵育30分钟,DAB显色,苏木素复染后封片。结果在光学显微镜下观察并拍照分析。
六、MTT法评估CpG-ODN对脑胶质瘤细胞增殖的影响
对数增长期的人脑胶质瘤U87-MG、U251-MG和大鼠胶质瘤C6细胞系接种到96孔板中。然后孵育让细胞粘附,加入TLR9的激动剂ODN 2006,对照组加λODN 2006 Control或相同体积的培养基。5mg/ml的MTT溶液过滤灭菌,不同时间点加入20ul MTT。然后孵育4小时,吸净培养液,每孔加入二甲基亚砜150μl,振荡10分钟充分溶解结晶后,选择,在酶联免疫监测仪上测定各孔在490nm波长处的光吸收值。
七、Transwell法测定CpG-ODN对脑胶质瘤细胞侵袭力的影响
在具有直径为8um微孔基底膜的小室内预先铺好基质胶。1x104的U87胶质瘤细胞连同200u1的培养基(含1%FBS)置于上室,下室置500μl的培养基(含20%FBS)。上下室内分别加入TLR9的激动剂ODN 2006,对照组加入ODN 2006Control或者相同体积的培养基,氯喹(10uM)作为TLR9的特异性阻断剂。37℃孵育24小时后,用棉签擦去基质胶和上室内的细胞。在过滤器的下表面细胞用甲醇和冰醋酸混合物(3：1)固定30分钟,风干后吉姆萨染色。侵入的细胞数在任意5个200高倍镜下视野观察计数,实验重复3次,进行统计学分析。
八、统计学分析
使用Windows SPSS 13.0统计软件进行所有的统计学分析。肿瘤细胞增殖和侵袭实验的组间比较使用t检验。TLR9的表达水平与胶质瘤恶性分级的关联使用Jonckheere-Terpstra检验,使用卡方检验进行组间比较。构建Cox回归模型,多因素分析TLR9的表达水平与胶质母细胞瘤病人无进展生存期的关系。用Kaplan-Meier方法构建生存曲线,对数秩检验log-rank进行组间比较。P≤0.05认为有显著性差异。
结果
一、TLR9在脑胶质瘤细胞株和肿瘤样本中的表达
RT-PCR方法检测到在胶质瘤细胞株和肿瘤组织中有TLR9 mRNA的表达。
免疫荧光染色说明TLR9蛋白在人脑胶质瘤细胞系中表达。TLR9蛋白定位于胞浆。
二、TLR9的表达与胶质瘤的恶性程度相关
脑胶质瘤组织芯片免疫组化染色结果分析表明,TLR9在非肿瘤脑组织未发现强染色。然而TLR9在所有高级别胶质瘤中均有表达,而且高表达占到43.78%。TLR9的表达水平在低级别胶质瘤(Ⅱ级)与高级别胶质瘤(Ⅲ级和Ⅳ级)组间有显著性差异(P<0.01),说明TLR9的表达与脑胶质瘤恶性进展有关。
三、TLR9的表达及GBM患者的预后的关系
我们评估了69例多形性胶质母细胞瘤患者TLR9的表达,单因素Cox回归分析结果表明,TLR9的蛋白表达水平与患者的无进展生存期呈负相关性(P=0.013)。多变量分析结果表明显示,TLR9蛋白质的表达水平是胶质母细胞瘤患者无进展生存期的独立危险因素(p=0.026)。独立于传统的临床独立变量(风险比[HR],3.205；95%置信区间[CI],1.152-8.920,p=0.026)。在这项研究中,年龄、性别、术前KPS评分及切除范围并没有与胶质母细胞瘤病人的无进展生存期相关。
我们使用Kaplan-Meier方法,评估了TLR9的表达水平(高表达和低表达)对胶质母细胞瘤病人无进展生存期的影响。我们发现,TLR9的表达水平可显着区分胶质母细胞瘤为两个分组,TLR9低表达组无进展生存期为9个月(中位数无进展生存期,11.108个月；95%CI为8.843-13.372),TLR9高表达组无进展生存期为6个月(中位数无进展生存期,6.958个月；95%CI为5.634-8.283)(P=0.001)。
四、CpG-ODN对脑胶质瘤的体外增殖无影响
MTT结果表明,CpG-ODN 2006组与对照组相比,在72小时内对胶质瘤细胞的增殖没有显著性差异。说明CpG-ODN 2006在体外对胶质瘤细胞的增殖没有显著影响。
五、CpG-ODN可以增强胶质瘤侵袭力
TLR9的激动剂CpG-ODN 2006在体外可以明显增加U87胶质瘤细胞侵入的数量。TLR9的抑制剂氯喹,能明显抑制CpG-ODN诱导的U87细胞侵袭能力的增强,这表明U87细胞侵袭力的增强是由CpG-ODN激活TLR9的信号转导引起的。
结论
我们的数据显示,TLR9在胶质瘤细胞系和脑胶质瘤组织有表达,而且TLR9在胶质瘤细胞中是有功能的。TLR9识别含有CpG基序的寡核苷酸,在与配体结合后,TLR9的信号通路可能诱导肿瘤发生炎症或微环境的变化。CpG-ODN可显著提高胶质瘤细胞的侵袭力,我们证实是由CpG-ODN激活的TLR9信号传导促进了胶质瘤细胞侵袭性的增加,这是因为TLR9的抑制剂氯喹能明显抑制CpG-ODN诱导的U87细胞侵袭能力的增强。应用胶质瘤组织芯片技术,通过对胶质瘤大样本的免疫组织化学分析,我们发现TLR9的表达与胶质瘤的恶性程度有关。TLR9的表达是胶质母细胞瘤患者一个独立的预后危险因素,可以预测多形性胶质母细胞瘤患者的无进展生存期。此外我们的研究结果提示,在进行胶质瘤免疫治疗时,不应直接瘤内注射TLR9的激动剂,至少应该持谨慎态度。另外我们认为TLR9也许可以作为胶质瘤治疗的新靶点。
Introduction
Glioma is the most common brain tumors, including surgery, radiotherapy, chemotherapy and combined treatment strategies, high-grade gliomas the prognosis is still unsatisfactory. Glioblastoma multiforme, in particular, is the highest degree of intracranial malignant tumor, accounting for 15-20% of primary intracranial tumors. Its a cell heterogeneity, splitting the extremely active and microvascular hyperplasia and necrosis of tumor histopathological features. GBM patients, the average median survival is only about a year, during the past decade have changed little. Therefore, an urgently need for new treatment strategies and the development of new treatments.
Immunotherapy is a promising treatment because it can activate lymphocytes, inducing target and destroy tumor cells. Glioma in recent years, the progress made in study of biological activity, making it can be used in the treatment of glioma. Treatment strategies include passive immunotherapy, T cell adoptive immunotherapy and active immunization. Glioma vaccine strategies include whole-cell vaccines, glioma-related peptide vaccine and DC vaccines.
Unmethylated CpG dinucleotide in bacterial and viral DNA, very common, but has diminished in vertebrates and is methylated. Containing non-methylated CpG pairs of nucleotide sequences, including the specific nucleotide sequences have a strong immune regulatory function. CpG ODN, a synthetic containing unmethylated CG motif of the single-stranded oligodeoxynucleotides that can mimic bacterial or viral DNA of the vertebrate immune system to produce a stimulating effect. According to the role of differences in the characteristics, CpG-ODN can be divided into three categories, including B class CpG-ODN can stimulate B cell proliferation and activation, now as a cancer vaccine adjuvant used in clinical trials. B-type CpG-ODN stimulate mammalian immune cells, this stimulating effect mainly through the combination with the TLR9 activates the immune system, inducing cytokine secretion, and induce a strong T-cell anti-tumor immune response. So far, has identified 11 species of human Toll-like receptors and Toll-like receptor 13 species of rodents. Toll-like receptor family, TLR9 is a member of the family, evolution is highly conserved transmembrane protein that can recognize molecular patterns derived from microorganisms. The role of TLR9 in immune plasma cells like DC and B cells is most widely used. Containing unmethylated CpG TLR9 and act on the structural changes caused by receptor, which is considered to converge to raise response protein MyD88, kinases of mitogen-activated protein phosphorylation of the activation of the same signaling pathway, and NF-kB activation of results. At the cellular level, TLR9 excitement caused by congenital and reactive role in the immune response cascade. TLR9 agonists activate the plasma cell-like typeⅠIFN secretion in DC and expressed as CD80 and CD86 costimulatory molecules like enhanced level. People think that this will begin a series of side effects, including, for example MCP-1, IP-10 cytokines, inflammatory chemokines secretion, NK cell activation, T cell population expansion, particularly TH-1 cells and CTLs. Result, an effective cell-mediated TH-1 response began. When TLR9 agonists accelerate the B cells to differentiate into antibody secreting plasma cells, may also promote antibody-dependent cellular cytotoxicity, a humoral immune response has begun.
Caused by the TLR9 agonist on the immune response of understanding and promote in infectious diseases, cancer, asthma and allergies in the field some of TLR9 agonists in clinical development. Of TLR9 agonists as anticancer drugs is based on the theoretical hypothesis, this hypothesis is the innate immune response may have a direct effect of anti-cancer antibiotics, and the TH-1 type cytokines and chemokines sector to strengthen the antigen-presenting will promote anti-cancer immune response to antibiotics.
Recent studies have shown that, in addition to plasma cell-like DC and B cells, TLR9 also expressed in some tumor cells, such as breast, stomach, lung and lymphatic found expression in epithelial tumors. Large amounts of data showed that TLR9 agonist CpG-ODN can promote tumor progression and metastasis. Gliomas are neuroepithelial tumors, we speculated that there may be TLR9 expression in gliomas. Our pilot study confirms our idea, in cell lines and glioma specimens were detected in its expression. We assume that, TLR9 signal pathway may in glioma growth and progress is very important. But its exact role of human gliomas is still unclear, it is to promote or inhibit the growth of glioma, there is no relevant reports. It has been reported partial use of CpG-ODN immunotherapy can prolong the survival of glioma in mice. On the contrary, Ginzkeyetal, who found that in rat brain glioma model, intratumoral injection of immune-stimulating agent CpG oligonucleotides, tumor sizes increased, which we have previously found that intratumoral injection of CpG oligonucleotide did not increase in GL261 glioma animal survival time model consistent with this result shows that, CpG direct local injection, may not produce useful results in patients with glioma.
TLR9 in glioblastoma expression has initially been detected, but did not find prognostic significance, however, their study was conducted in mRNA levels, protein levels needed research. There is no system of TLR9 expression in gliomas and anti-tumor mechanism study, which increase the CpG-ODN adjuvant therapy for malignant glioma knowledge is essential. A further step could be identified from, for the greatest benefit of this treatment in this subgroup of patients. This study aims to explore a large sample of the tissue microarray, glioblastoma samples TLR9 expression of TLR9 expression and clinical pathological factors and the association between patient survival. In order to further elaborate the expression of TLR9 on tumor cells, we will examine the direct impact of CpG oligonucleotides on invasion and proliferation of glioma cell in vitro.
Therefore, the study of TLR9 expression in gliomas, its functions, and its signal transduction pathway is important for understanding the formation of malignant glioma, and also provides new and effective therapeutic targets for treatment of glioma.
Methods
1. Cells and Cell Culture
The human glioma cell lines U87-MG, U251-MG and rat glioma C6 cell lines were first obtained from ATCC and maintained in the Central Lab of First Hospital of China Medical University. Glioma cells were grown in Dulbecco's modified essential medium (DMEM, Gibcol, Gaithersburg, MD, USA) supplemented with 10% FBS (Hyclone Laboratories, Logan, USA), 100U/ml penicillin, and 100μg/ml streptomycin at 37℃with 5% CO2 in humidified air.
2. RNA extraction and RT-PCR assay for detection of TLR 9
Total RNA was extracted from 3 glioma cell lines and 34 clinical glioma samples using Trizol reagent, lug RNA was used as template for cDNA Synthesis. RT-PCR analyses were performed using the following primer sets, human TLR9 (sense 5'-TGTAATAACAGTTGCCGTCCAT-3'), antisense:5'-CAGCCTTTCCTTGTC CTC C-3'), rat TLR9 (sense:5'-GGACAGTTCTCTCCACTCGC-3'), antisense:5'-TTC TTTGAGACGGGAGTGCT-3'), GAPDH (sense:5'-TCCACCACCCTGTTGC TGTA-3', antisense:5'-ACCACAGTCCATGCCATCAC-3'). GAPDH was used as housekeeping gene control, GADPH consensus primers were shared for Homo sapiens and Rattus norvegicus. Reactions were carried out and products were analyzed by 1.5% agarose gel electrophoresis and visualized by Gene finder staining under ultraviolet.
3. Immunofluorescence
U87-MG and U251-MG cells were grown on coverslips, the cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 in PBS and blocked 1h in PBS supplemented with 5% FBS. Cells were incubated overnight at 4℃with TLR9 mAb, followed by Cy3-labelled anti-mouse secondary antibody for 2h.. The nuclei were counterstained with Hoechst. In the control samples, glioma cells were stained with secondary antibody and Hoechst, but without anti-TLR9 primary antibody. Results were observed and photographed under confocal microscope.
4. Construction of Glioma Tissue Microarray
The paraffin embedded archival tissue blocks were retrieved, and the matching HE-stained slides were screened for representative tumor regions by a neuropathologist, according to the diagnostic criteria of the WHO 2000 classification system. Construction of the tissue microarray cores, surgical resection of normal kidney tissue as positive control.
5. Glioma Tissue Microarray Immunochemistry
Tissue Microarrays were cutted into 4um sections and baked at 65℃for 30 min. The sections were deparaffinized with xylenes and rehydrated. Sections treated with 3% hydrogen peroxide, followed by incubation with 1% FBS. Anti-TLR9 was added and incubated overnight at 4℃. For negative controls, the primary antibody was replaced by normal mouse serum. Human normal kidney samples were used as a positive control. Horseradish peroxidase (HRP) labeled secondary antibody was applied and incubated for 30 mins, followed by DAB for color development. The sections were finally counterstained with haematoxylin and mounted. Results were visualized and photographed under a light microscope.
6. MTT assay
U87-MG, U251-MG and C6 cells were seeded into 96-well plates, then incubated at 37℃for 24h for cells adherence. CpG ODN (ODN 2006), ODN 2006 control and same volumn of medium were added, cell growth was measured at 24,48,72 hours after the treatment. MTT was dissolved in PBS at 5mg/ml and filtered to be sterilized, 20ul MTT solution was added at different time points. Plates were then incubated at 37℃for 4h, 100ul DMSO was added to each well and mixed thoroughly to dissolve the blue-violet crystals. Cell viability data were measured with an ELISA reader at 490nm.
7. Transwell assay
The invasion assay was done in 24 well cell culture chambers using transwell inserts with 8um Pore membrane precotated with matrigel. U87 cells were plated at the density of 1×104 per upper well in 200ul culture medium (1%FBS), the lower chamber was filled with 500μl medium (20% FBS). ODN 2006, ODN 2006 control, same volume of the control medium, or Chloroquine was added to both upper and lower well. The cells were allowed to invade for 24 hours, after which, the noninvading cells with Matrigel matrix were removed from the upper surface of the membrane by scrubbing with a cotton-tipped swab. Cells on the lower surface of the filter were fixed for 30 min in methanol and glacial acetic acid mixture (3:1), air-dried briefly, and stained with Giemsa. The number of invaded cells was counted from five preselected microscopic fields at 200X magnification, all experiments were performed in triplicate.
8. Statistical analysis
SPSS statistical software for Windows 13.0 (SPSS, Inc., Chicago, Illinois, USA) was used for all statistical analysis. Differences of tumor cell proliferation and invasion between groups were analyzed by t-test. The Jonckheere-Terpstra test was used to correlate cumulative TLR9 expression with glioma grading, and the chi-square test was used for comparisons between groups. Cox regression was used to correlate TLR9 expression with PFS while adjusting for clinicopathological variables. Survival curves were constructed using the Kaplan-Meier method. The log-rank test was used for comparison between groups. P<0.05 was defined as significance.
Results
1. TLR9 is expressed in glioma cell lines and tumor samples
RT-PCR results showed that TLR9 mRNAs is expressed in both human and rat glioma cell lines glioma cell lines and tumor tissues.
Immunofluorescence results were also showed TLR9 protein expressed in human glioma cell lines. A cytoplasmic localization of TLR9 in U87 and U251 was confirmed.
2. Correlation between expression of TLR9 and malignancy of gliomas.
TLR9 mainly as cytoplasmic staining was found to be expressed in gliomas, no strong immunoreactivity was detected in non-neoplastic brain tissues. TLR9 expression was detected in all high grade glioma cases and was highly expressed in 43.78% (88/ 201) cases. Expression was significantly higher in highgrade gliomas (gradeⅢand gradeⅣ) compared to low-grade gliomas (gradeⅡ)(P<0.01), which supported that the TLR9 expression is associated with the progression of glioma.
3. Association between TLR9 expression and GBM patient prognosis
We assessed 69 glioblastoma multiforme (GBM) cases using univariate Cox regression analysis, we found that the expression levels of TLR9 protein were correlated with PFS of GBM patients (p=0.013). The multivariate analysis revealed that the expression levels of TLR9 protein level was a significant prognostic factor in GBM patients.
We assessed the discriminative value of expression levels of TLR9 (low expression and high expression) for survival of patients with GBM using Kaplan-Meier analysis. We found that the expression levels of TLR9 could significantly discriminate the survival of the two GBM subgroups, with a mean PFS of 6 months for high TLR9 expression group versus mean 9 months for low TLR9 expression group(p=0.001).
4. CpG-ODN has no effect on glioma proliferation in vitro
MTT assay results showed that CpG-ODN (ODN 2006) did not influence the proliferation rate of the three glioma cell line tested within 72 hours, when compared with non CpG-ODN or medium control.
5. CpG-ODN enhance glioma invasion in vitro
TLR9 agonist, ODN2006, significantly elevated the invasion of glioma U87 cells compared with that of glioma cells treated with medium. In order to ensure that TLR9 is responsible for the invasion effects induced by CpG ODN, glioma cells were treated with CpG ODN in the presence of chloroquine, which is an inhibitor of endosomal acidification resulting in inhibition of TLR9 signaling. Chloroquine significantly inhibited CpG ODN induced invasion in glioma cells, which suggest that TLR9 signaling is responsible for the enhanced invasion of U87 cells induced by CpG-ODN.
Conclusion
Our data indicated that TLR9 is expressed in glioma cell lines and glioma tissues, and the expression of TLR9 in glioma cells is functional. TLR9 recognizes the ODN with CpG motif. After binding with the ligand, TLR9 signal pathway may induce the proinflammatory or progrowth microenvironment of tumor. CpG-ODN can significantly enhance the invasion of GBM cells, we confirmed that TLR9 signaling was responsible for the enhanced invasion of glioma cells induced by CpG-ODN. Screening a large collection of human glioma samples in tissue array with immunohistochemistry, we found that TLR9 expression correlated to the malignancy of gliomas, and TLR9 expression is an independent prognostic factor to predict patient PFS in GBM. Our data indicated that TLR9 may relate to glioma progression and it may be a biological marker for the prognosis of GBM patients. In addition, our findings warrant caution in the directly injection of TLR9 into glioma tissues for the glioma immunotherapy. TLR9 can be used as a new target for glioma therapy.
神经胶质瘤是最常见的脑肿瘤,尽管包括手术切除,放射治疗和化学治疗在内的综合治疗策略的联合应用,高级别神经胶质瘤的预后仍不尽人意,特别是多形性胶质母细胞瘤。多形胶质母细胞瘤是颅内恶性程度最高的恶性肿瘤,占原发性颅内肿瘤的15-20%。多形胶质母细胞瘤具有细胞异质性、分裂极度活跃、肿瘤微血管增生和坏死的组织病理特点。多形胶质母细胞瘤病人的平均中位生存期只有大约一年,在过去的十年中没有显著改善。因此,迫切需要开发新的治疗策略或新的辅助治疗方法。
免疫治疗被认为是一种极有前途的治疗方法,因为它能激活淋巴细胞,可以特异性的识别并摧毁肿瘤细胞。近年来对胶质瘤发病机制的研究取得了较大的进展,同时研究者发现中枢神经系统内存在免疫反应,这使得免疫疗法可以应用于脑胶质瘤的治疗。胶质瘤免疫治疗的策略包括被动性免疫治疗,T细胞过继免疫治疗和主动免疫。
未甲基化的CpG二核苷酸在细菌和病毒DNA中相当常见,但是在脊椎动物中却减弱并被甲基化。包含非甲基化的CpG双核苷酸序列在内的特定核苷酸序列具有强有力的免疫调节功能,可以作为癌症疫苗的免疫佐剂。CpG-ODN是人工合成的含有未甲基化CpG基序的单链寡聚脱氧核苷酸,可以模仿细菌或病毒DNA对脊椎动物免疫系统产生刺激作用。根据作用特点的差别,CpG-ODN可被分成三类,其中B类CpG-ODN能刺激B细胞增生和活化,目前已作为癌症疫苗的免疫佐剂在临床试验中使用。B型CpG-ODN对哺乳动物免疫细胞增生活化的刺激作用主要是通过与Toll样受体9结合,进而激活免疫系统,诱导细胞因子的分泌和释放,并诱发强有力的T细胞抗瘤免疫反应。
目前为止,已经鉴定出人类11种Toll样受体和老鼠13种Toll样受体。TLR9就是Toll样受体家族家族的一员,进化上是高度保守的跨膜蛋白,可以识别微生物衍生的分子模式结构。TLR9对于浆细胞样树突状细胞和B细胞的免疫刺激作用得到了广泛的研究。CpG作用于TLR9可以引起受体的结构性变化,然后通过衔接蛋白MyD88的募集反应,促进激酶类有丝分裂原激活蛋白的磷酸化激活相应信号通道,激活核因子-κB并调控相应基因的表达。在细胞水平上,TLR9的激动作用引起先天性和适应性免疫反应的级联效应。TLR9激动药激活浆细胞样树突状细胞分泌Ⅰ型干扰素并上调CD80和CD86等共刺激分子的表达水平。人们认为这会引发一系列免疫效应,如MCP-1,IP-10等细胞因子以及炎症趋化因子的分泌,NK细胞的激活,T细胞群的扩张,尤其是对于TH-1细胞和细胞毒性T细胞的作用。TLR9激动剂作用的结果之一是启动有效的细胞介导的TH-1应答。另外TLR9激动剂可以加快B细胞分化成抗体分泌型浆细胞,还可以促进抗体依赖的细胞毒性反应,这说明TLR9激动剂的另一个作用结果是可以启动体液性免疫应。
对由TLR9激动作用引起的免疫应答的了解,推动了在传染病,癌症,哮喘和过敏领域中TLR9激动药在临床上的发展。然而研究TLR9激动药作为抗癌药的理论基础是一种假说,这一假说认为先天性免疫应答可能有直接的抗肿瘤作用,而TH-1类细胞因子和趋化因子可以增强抗原提呈并进而促进抗肿瘤免疫应答。
最近的一些研究表明,除浆细胞样树突状细胞和B细胞中有表达外,TLR9的也表达在某些肿瘤细胞,研究者证实乳腺、胃、肺和淋巴性的上皮性肿瘤中存在TLR9的表达。然而大量数据表示,TLR9的激动剂CpG寡核苷酸可促进肿瘤的发展和转移。胶质瘤属于神经上皮性肿瘤,我们推测TLR9在胶质瘤中可能存在表达。我们的预试验验证了我们的想法,在细胞系和胶质瘤标本中都检测出了其表达。我们假设,TLR9的信号通路可能在脑胶质瘤生长和进展中非常重要。TLR9信号通路在人脑胶质瘤的确切作用目前还不清楚,其对胶质瘤生长是促进还是抑制,目前还没有相关报道。有实验报道局部使用CpG寡核苷酸免疫治疗可以延长胶质瘤小鼠的生存期。相反,Ginzkeyetal等人发现在大鼠脑胶质瘤模型中瘤内注射免疫刺激剂CpG寡核苷酸,肿瘤大小反而增加,这与我们先前所做的实验结果一致,在以前的实验中我们发现的CpG寡核苷酸瘤内注射并不能增加GL261胶质瘤荷瘤鼠的生存时间,这结果提示,CpG直接局部注射,可能不会在胶质瘤患者产生有益的结果。
已有研究者初步检测了TLR9在胶质母细胞瘤中的表达,但没有发现其对预后的意义。我们认为此研究存在一定的局限性,其研究仅进行了TLR9 mRNA水平的评价,而没有进行蛋白质水平的研究,由于蛋白质是基因功能的体现,因此检测TLR9蛋白在胶质瘤中表达是十分必要的,这对于提高对CpG-ODN恶性胶质瘤的免疫辅助治疗的认识是至关重要的。目前还没有系统的TLR9蛋白在胶质瘤中的表达情况和作用机制的研究。本研究将应用组织芯片技术,探讨TLR9蛋白在大样本的脑胶质细胞瘤样品中的表达情况,分析TLR9的表达与临床病理特点的关系以及TLR9表达与胶质母细胞瘤患者生存期之间的关联。为了进一步阐述TLR9的表达对肿瘤细胞可能发挥的作用,我们还将探讨CpG寡核苷酸在体外对的胶质瘤细胞增殖和侵袭的直接影响。
研究TLR9在神经胶质瘤中的表达和功能,及其信号传导通路,对于理解神经胶质瘤的形成和恶性进展有重要帮助,同时也提供了胶质瘤新的有效治疗靶点。
方法一、细胞和细胞培养
恶性人脑胶质瘤细胞系U87-MG, U251-MG和大鼠脑胶质瘤C6细胞系从ATCC(美国组织培养细胞库)购得,并在中国医科大学附属第一医院中心实验室培养。胶质瘤细胞系U87-MG、U251-MG和C6细胞以含10%胎牛血清的DMEM高糖培养基培养,于37℃,100U／ml青霉素,100μg／ml链霉素和5%CO2条件下恒温培养箱内培养。
二、RNA提取及RT-PCR检测TLR9的表达采用Trizol试剂一步法提取人脑胶质瘤U87-MG、人脑胶质瘤U251-MG细胞系、大鼠脑胶质瘤C6和手术切除的人脑胶质瘤组织总RNA,紫外分光光度计检测OD260/OD280及核酸浓度。提取细胞系和肿瘤标本的总RNA进行cDNA第一链合成。按照RT-PCR试剂盒的说明,应用特异性引物人类TLR9 (F: 5'-TGTAATAACAGTTGCCGTCCAT-3',R:5'-CAGCCTTTCCTTGTCCTCC-3'),大鼠TLR9 (F:5'-GGACAGTTCTCTCCACTCGC-3', R:5'-TTCTTTGAGACGGGAGTGCT-3');人和大鼠管家基因GAPDH (F:5'-TCCACCACCCTGTTGCTGTA-3', R:5'-ACCACAGTCCATGCCATCAC-3')进行PCR扩增,产物进行1.5%琼脂糖凝胶电泳,并在紫外线下的观察。
三、免疫荧光检测TLR9蛋白在细胞系中的表达
U87-MG和U251细胞在盖玻片上爬片生长,盖玻片用无血清培养液冲洗后,4%多聚甲醛固定,0.5%Trixton X-100的PBS透膜,并在含5%胎牛血清的PBS中封闭1小时。细胞与TLR9的单克隆抗体在4℃孵育过夜,然后由Cy3标记的抗鼠的二抗孵育2小时,细胞核用Hoechest复染。在对照样品中,去掉一抗,胶质瘤细胞用二抗和Hoechest染色。然后应用荧光激发Cy3和Hoechest,共聚焦显微镜下观察TLR9在细胞系中的表达并拍照。
四、脑胶质瘤组织芯片的构建
提取脑胶质瘤组织标本,进行石蜡包埋并进行HE染色,根据世界卫生组织2000年的分类系统的诊断标准重新诊断并且与病理结果比较,由神经病理学家选取代表性的肿瘤区域,构建组织微阵列芯片。手术切除的正常肾脏组织作为阳性对照。
五、脑胶质瘤组织芯片免疫组织化学染色
4um组织芯片切片在65℃烤30分钟,二甲苯脱蜡和水化后,切片浸入乙二胺四乙酸的缓冲液中进行高压抗原修复,然后经过3%过氧化氢灭活内源性过氧化物酶,1%胎牛血清封闭。抗人TLR9抗体,4℃孵育过夜。阴性对照,一抗为正常小鼠血清。人正常肾样品作为阳性对照。根据免疫组化试剂盒说明,辣根过氧化物酶标记的二抗聚合物在室温下孵育30分钟,DAB显色,苏木素复染后封片。结果在光学显微镜下观察并拍照分析。
六、MTT法评估CpG-ODN对脑胶质瘤细胞增殖的影响
对数增长期的人脑胶质瘤U87-MG、U251-MG和大鼠胶质瘤C6细胞系接种到96孔板中。然后孵育让细胞粘附,加入TLR9的激动剂ODN 2006,对照组加λODN 2006 Control或相同体积的培养基。5mg/ml的MTT溶液过滤灭菌,不同时间点加入20ul MTT。然后孵育4小时,吸净培养液,每孔加入二甲基亚砜150μl,振荡10分钟充分溶解结晶后,选择,在酶联免疫监测仪上测定各孔在490nm波长处的光吸收值。
七、Transwell法测定CpG-ODN对脑胶质瘤细胞侵袭力的影响
在具有直径为8um微孔基底膜的小室内预先铺好基质胶。1x104的U87胶质瘤细胞连同200u1的培养基(含1%FBS)置于上室,下室置500μl的培养基(含20%FBS)。上下室内分别加入TLR9的激动剂ODN 2006,对照组加入ODN 2006Control或者相同体积的培养基,氯喹(10uM)作为TLR9的特异性阻断剂。37℃孵育24小时后,用棉签擦去基质胶和上室内的细胞。在过滤器的下表面细胞用甲醇和冰醋酸混合物(3：1)固定30分钟,风干后吉姆萨染色。侵入的细胞数在任意5个200高倍镜下视野观察计数,实验重复3次,进行统计学分析。
八、统计学分析
使用Windows SPSS 13.0统计软件进行所有的统计学分析。肿瘤细胞增殖和侵袭实验的组间比较使用t检验。TLR9的表达水平与胶质瘤恶性分级的关联使用Jonckheere-Terpstra检验,使用卡方检验进行组间比较。构建Cox回归模型,多因素分析TLR9的表达水平与胶质母细胞瘤病人无进展生存期的关系。用Kaplan-Meier方法构建生存曲线,对数秩检验log-rank进行组间比较。P≤0.05认为有显著性差异。
结果
一、TLR9在脑胶质瘤细胞株和肿瘤样本中的表达
RT-PCR方法检测到在胶质瘤细胞株和肿瘤组织中有TLR9 mRNA的表达。
免疫荧光染色说明TLR9蛋白在人脑胶质瘤细胞系中表达。TLR9蛋白定位于胞浆。
二、TLR9的表达与胶质瘤的恶性程度相关
脑胶质瘤组织芯片免疫组化染色结果分析表明,TLR9在非肿瘤脑组织未发现强染色。然而TLR9在所有高级别胶质瘤中均有表达,而且高表达占到43.78%。TLR9的表达水平在低级别胶质瘤(Ⅱ级)与高级别胶质瘤(Ⅲ级和Ⅳ级)组间有显著性差异(P<0.01),说明TLR9的表达与脑胶质瘤恶性进展有关。
三、TLR9的表达及GBM患者的预后的关系
我们评估了69例多形性胶质母细胞瘤患者TLR9的表达,单因素Cox回归分析结果表明,TLR9的蛋白表达水平与患者的无进展生存期呈负相关性(P=0.013)。多变量分析结果表明显示,TLR9蛋白质的表达水平是胶质母细胞瘤患者无进展生存期的独立危险因素(p=0.026)。独立于传统的临床独立变量(风险比[HR],3.205；95%置信区间[CI],1.152-8.920,p=0.026)。在这项研究中,年龄、性别、术前KPS评分及切除范围并没有与胶质母细胞瘤病人的无进展生存期相关。
我们使用Kaplan-Meier方法,评估了TLR9的表达水平(高表达和低表达)对胶质母细胞瘤病人无进展生存期的影响。我们发现,TLR9的表达水平可显着区分胶质母细胞瘤为两个分组,TLR9低表达组无进展生存期为9个月(中位数无进展生存期,11.108个月；95%CI为8.843-13.372),TLR9高表达组无进展生存期为6个月(中位数无进展生存期,6.958个月；95%CI为5.634-8.283)(P=0.001)。
四、CpG-ODN对脑胶质瘤的体外增殖无影响
MTT结果表明,CpG-ODN 2006组与对照组相比,在72小时内对胶质瘤细胞的增殖没有显著性差异。说明CpG-ODN 2006在体外对胶质瘤细胞的增殖没有显著影响。
五、CpG-ODN可以增强胶质瘤侵袭力
TLR9的激动剂CpG-ODN 2006在体外可以明显增加U87胶质瘤细胞侵入的数量。TLR9的抑制剂氯喹,能明显抑制CpG-ODN诱导的U87细胞侵袭能力的增强,这表明U87细胞侵袭力的增强是由CpG-ODN激活TLR9的信号转导引起的。
结论
我们的数据显示,TLR9在胶质瘤细胞系和脑胶质瘤组织有表达,而且TLR9在胶质瘤细胞中是有功能的。TLR9识别含有CpG基序的寡核苷酸,在与配体结合后,TLR9的信号通路可能诱导肿瘤发生炎症或微环境的变化。CpG-ODN可显著提高胶质瘤细胞的侵袭力,我们证实是由CpG-ODN激活的TLR9信号传导促进了胶质瘤细胞侵袭性的增加,这是因为TLR9的抑制剂氯喹能明显抑制CpG-ODN诱导的U87细胞侵袭能力的增强。应用胶质瘤组织芯片技术,通过对胶质瘤大样本的免疫组织化学分析,我们发现TLR9的表达与胶质瘤的恶性程度有关。TLR9的表达是胶质母细胞瘤患者一个独立的预后危险因素,可以预测多形性胶质母细胞瘤患者的无进展生存期。此外我们的研究结果提示,在进行胶质瘤免疫治疗时,不应直接瘤内注射TLR9的激动剂,至少应该持谨慎态度。另外我们认为TLR9也许可以作为胶质瘤治疗的新靶点。
Introduction
Glioma is the most common brain tumors, including surgery, radiotherapy, chemotherapy and combined treatment strategies, high-grade gliomas the prognosis is still unsatisfactory. Glioblastoma multiforme, in particular, is the highest degree of intracranial malignant tumor, accounting for 15-20% of primary intracranial tumors. Its a cell heterogeneity, splitting the extremely active and microvascular hyperplasia and necrosis of tumor histopathological features. GBM patients, the average median survival is only about a year, during the past decade have changed little. Therefore, an urgently need for new treatment strategies and the development of new treatments.
Immunotherapy is a promising treatment because it can activate lymphocytes, inducing target and destroy tumor cells. Glioma in recent years, the progress made in study of biological activity, making it can be used in the treatment of glioma. Treatment strategies include passive immunotherapy, T cell adoptive immunotherapy and active immunization. Glioma vaccine strategies include whole-cell vaccines, glioma-related peptide vaccine and DC vaccines.
Unmethylated CpG dinucleotide in bacterial and viral DNA, very common, but has diminished in vertebrates and is methylated. Containing non-methylated CpG pairs of nucleotide sequences, including the specific nucleotide sequences have a strong immune regulatory function. CpG ODN, a synthetic containing unmethylated CG motif of the single-stranded oligodeoxynucleotides that can mimic bacterial or viral DNA of the vertebrate immune system to produce a stimulating effect. According to the role of differences in the characteristics, CpG-ODN can be divided into three categories, including B class CpG-ODN can stimulate B cell proliferation and activation, now as a cancer vaccine adjuvant used in clinical trials. B-type CpG-ODN stimulate mammalian immune cells, this stimulating effect mainly through the combination with the TLR9 activates the immune system, inducing cytokine secretion, and induce a strong T-cell anti-tumor immune response. So far, has identified 11 species of human Toll-like receptors and Toll-like receptor 13 species of rodents. Toll-like receptor family, TLR9 is a member of the family, evolution is highly conserved transmembrane protein that can recognize molecular patterns derived from microorganisms. The role of TLR9 in immune plasma cells like DC and B cells is most widely used. Containing unmethylated CpG TLR9 and act on the structural changes caused by receptor, which is considered to converge to raise response protein MyD88, kinases of mitogen-activated protein phosphorylation of the activation of the same signaling pathway, and NF-kB activation of results. At the cellular level, TLR9 excitement caused by congenital and reactive role in the immune response cascade. TLR9 agonists activate the plasma cell-like typeⅠIFN secretion in DC and expressed as CD80 and CD86 costimulatory molecules like enhanced level. People think that this will begin a series of side effects, including, for example MCP-1, IP-10 cytokines, inflammatory chemokines secretion, NK cell activation, T cell population expansion, particularly TH-1 cells and CTLs. Result, an effective cell-mediated TH-1 response began. When TLR9 agonists accelerate the B cells to differentiate into antibody secreting plasma cells, may also promote antibody-dependent cellular cytotoxicity, a humoral immune response has begun.
Caused by the TLR9 agonist on the immune response of understanding and promote in infectious diseases, cancer, asthma and allergies in the field some of TLR9 agonists in clinical development. Of TLR9 agonists as anticancer drugs is based on the theoretical hypothesis, this hypothesis is the innate immune response may have a direct effect of anti-cancer antibiotics, and the TH-1 type cytokines and chemokines sector to strengthen the antigen-presenting will promote anti-cancer immune response to antibiotics.
Recent studies have shown that, in addition to plasma cell-like DC and B cells, TLR9 also expressed in some tumor cells, such as breast, stomach, lung and lymphatic found expression in epithelial tumors. Large amounts of data showed that TLR9 agonist CpG-ODN can promote tumor progression and metastasis. Gliomas are neuroepithelial tumors, we speculated that there may be TLR9 expression in gliomas. Our pilot study confirms our idea, in cell lines and glioma specimens were detected in its expression. We assume that, TLR9 signal pathway may in glioma growth and progress is very important. But its exact role of human gliomas is still unclear, it is to promote or inhibit the growth of glioma, there is no relevant reports. It has been reported partial use of CpG-ODN immunotherapy can prolong the survival of glioma in mice. On the contrary, Ginzkeyetal, who found that in rat brain glioma model, intratumoral injection of immune-stimulating agent CpG oligonucleotides, tumor sizes increased, which we have previously found that intratumoral injection of CpG oligonucleotide did not increase in GL261 glioma animal survival time model consistent with this result shows that, CpG direct local injection, may not produce useful results in patients with glioma.
TLR9 in glioblastoma expression has initially been detected, but did not find prognostic significance, however, their study was conducted in mRNA levels, protein levels needed research. There is no system of TLR9 expression in gliomas and anti-tumor mechanism study, which increase the CpG-ODN adjuvant therapy for malignant glioma knowledge is essential. A further step could be identified from, for the greatest benefit of this treatment in this subgroup of patients. This study aims to explore a large sample of the tissue microarray, glioblastoma samples TLR9 expression of TLR9 expression and clinical pathological factors and the association between patient survival. In order to further elaborate the expression of TLR9 on tumor cells, we will examine the direct impact of CpG oligonucleotides on invasion and proliferation of glioma cell in vitro.
Therefore, the study of TLR9 expression in gliomas, its functions, and its signal transduction pathway is important for understanding the formation of malignant glioma, and also provides new and effective therapeutic targets for treatment of glioma.
Methods
1. Cells and Cell Culture
The human glioma cell lines U87-MG, U251-MG and rat glioma C6 cell lines were first obtained from ATCC and maintained in the Central Lab of First Hospital of China Medical University. Glioma cells were grown in Dulbecco's modified essential medium (DMEM, Gibcol, Gaithersburg, MD, USA) supplemented with 10% FBS (Hyclone Laboratories, Logan, USA), 100U/ml penicillin, and 100μg/ml streptomycin at 37℃with 5% CO2 in humidified air.
2. RNA extraction and RT-PCR assay for detection of TLR 9
Total RNA was extracted from 3 glioma cell lines and 34 clinical glioma samples using Trizol reagent, lug RNA was used as template for cDNA Synthesis. RT-PCR analyses were performed using the following primer sets, human TLR9 (sense 5'-TGTAATAACAGTTGCCGTCCAT-3'), antisense:5'-CAGCCTTTCCTTGTC CTC C-3'), rat TLR9 (sense:5'-GGACAGTTCTCTCCACTCGC-3'), antisense:5'-TTC TTTGAGACGGGAGTGCT-3'), GAPDH (sense:5'-TCCACCACCCTGTTGC TGTA-3', antisense:5'-ACCACAGTCCATGCCATCAC-3'). GAPDH was used as housekeeping gene control, GADPH consensus primers were shared for Homo sapiens and Rattus norvegicus. Reactions were carried out and products were analyzed by 1.5% agarose gel electrophoresis and visualized by Gene finder staining under ultraviolet.
3. Immunofluorescence
U87-MG and U251-MG cells were grown on coverslips, the cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 in PBS and blocked 1h in PBS supplemented with 5% FBS. Cells were incubated overnight at 4℃with TLR9 mAb, followed by Cy3-labelled anti-mouse secondary antibody for 2h.. The nuclei were counterstained with Hoechst. In the control samples, glioma cells were stained with secondary antibody and Hoechst, but without anti-TLR9 primary antibody. Results were observed and photographed under confocal microscope.
4. Construction of Glioma Tissue Microarray
The paraffin embedded archival tissue blocks were retrieved, and the matching HE-stained slides were screened for representative tumor regions by a neuropathologist, according to the diagnostic criteria of the WHO 2000 classification system. Construction of the tissue microarray cores, surgical resection of normal kidney tissue as positive control.
5. Glioma Tissue Microarray Immunochemistry
Tissue Microarrays were cutted into 4um sections and baked at 65℃for 30 min. The sections were deparaffinized with xylenes and rehydrated. Sections treated with 3% hydrogen peroxide, followed by incubation with 1% FBS. Anti-TLR9 was added and incubated overnight at 4℃. For negative controls, the primary antibody was replaced by normal mouse serum. Human normal kidney samples were used as a positive control. Horseradish peroxidase (HRP) labeled secondary antibody was applied and incubated for 30 mins, followed by DAB for color development. The sections were finally counterstained with haematoxylin and mounted. Results were visualized and photographed under a light microscope.
6. MTT assay
U87-MG, U251-MG and C6 cells were seeded into 96-well plates, then incubated at 37℃for 24h for cells adherence. CpG ODN (ODN 2006), ODN 2006 control and same volumn of medium were added, cell growth was measured at 24,48,72 hours after the treatment. MTT was dissolved in PBS at 5mg/ml and filtered to be sterilized, 20ul MTT solution was added at different time points. Plates were then incubated at 37℃for 4h, 100ul DMSO was added to each well and mixed thoroughly to dissolve the blue-violet crystals. Cell viability data were measured with an ELISA reader at 490nm.
7. Transwell assay
The invasion assay was done in 24 well cell culture chambers using transwell inserts with 8um Pore membrane precotated with matrigel. U87 cells were plated at the density of 1×104 per upper well in 200ul culture medium (1%FBS), the lower chamber was filled with 500μl medium (20% FBS). ODN 2006, ODN 2006 control, same volume of the control medium, or Chloroquine was added to both upper and lower well. The cells were allowed to invade for 24 hours, after which, the noninvading cells with Matrigel matrix were removed from the upper surface of the membrane by scrubbing with a cotton-tipped swab. Cells on the lower surface of the filter were fixed for 30 min in methanol and glacial acetic acid mixture (3:1), air-dried briefly, and stained with Giemsa. The number of invaded cells was counted from five preselected microscopic fields at 200X magnification, all experiments were performed in triplicate.
8. Statistical analysis
SPSS statistical software for Windows 13.0 (SPSS, Inc., Chicago, Illinois, USA) was used for all statistical analysis. Differences of tumor cell proliferation and invasion between groups were analyzed by t-test. The Jonckheere-Terpstra test was used to correlate cumulative TLR9 expression with glioma grading, and the chi-square test was used for comparisons between groups. Cox regression was used to correlate TLR9 expression with PFS while adjusting for clinicopathological variables. Survival curves were constructed using the Kaplan-Meier method. The log-rank test was used for comparison between groups. P<0.05 was defined as significance.
Results
1. TLR9 is expressed in glioma cell lines and tumor samples
RT-PCR results showed that TLR9 mRNAs is expressed in both human and rat glioma cell lines glioma cell lines and tumor tissues.
Immunofluorescence results were also showed TLR9 protein expressed in human glioma cell lines. A cytoplasmic localization of TLR9 in U87 and U251 was confirmed.
2. Correlation between expression of TLR9 and malignancy of gliomas.
TLR9 mainly as cytoplasmic staining was found to be expressed in gliomas, no strong immunoreactivity was detected in non-neoplastic brain tissues. TLR9 expression was detected in all high grade glioma cases and was highly expressed in 43.78% (88/ 201) cases. Expression was significantly higher in highgrade gliomas (gradeⅢand gradeⅣ) compared to low-grade gliomas (gradeⅡ)(P<0.01), which supported that the TLR9 expression is associated with the progression of glioma.
3. Association between TLR9 expression and GBM patient prognosis
We assessed 69 glioblastoma multiforme (GBM) cases using univariate Cox regression analysis, we found that the expression levels of TLR9 protein were correlated with PFS of GBM patients (p=0.013). The multivariate analysis revealed that the expression levels of TLR9 protein level was a significant prognostic factor in GBM patients.
We assessed the discriminative value of expression levels of TLR9 (low expression and high expression) for survival of patients with GBM using Kaplan-Meier analysis. We found that the expression levels of TLR9 could significantly discriminate the survival of the two GBM subgroups, with a mean PFS of 6 months for high TLR9 expression group versus mean 9 months for low TLR9 expression group(p=0.001).
4. CpG-ODN has no effect on glioma proliferation in vitro
MTT assay results showed that CpG-ODN (ODN 2006) did not influence the proliferation rate of the three glioma cell line tested within 72 hours, when compared with non CpG-ODN or medium control.
5. CpG-ODN enhance glioma invasion in vitro
TLR9 agonist, ODN2006, significantly elevated the invasion of glioma U87 cells compared with that of glioma cells treated with medium. In order to ensure that TLR9 is responsible for the invasion effects induced by CpG ODN, glioma cells were treated with CpG ODN in the presence of chloroquine, which is an inhibitor of endosomal acidification resulting in inhibition of TLR9 signaling. Chloroquine significantly inhibited CpG ODN induced invasion in glioma cells, which suggest that TLR9 signaling is responsible for the enhanced invasion of U87 cells induced by CpG-ODN.
Conclusion
Our data indicated that TLR9 is expressed in glioma cell lines and glioma tissues, and the expression of TLR9 in glioma cells is functional. TLR9 recognizes the ODN with CpG motif. After binding with the ligand, TLR9 signal pathway may induce the proinflammatory or progrowth microenvironment of tumor. CpG-ODN can significantly enhance the invasion of GBM cells, we confirmed that TLR9 signaling was responsible for the enhanced invasion of glioma cells induced by CpG-ODN. Screening a large collection of human glioma samples in tissue array with immunohistochemistry, we found that TLR9 expression correlated to the malignancy of gliomas, and TLR9 expression is an independent prognostic factor to predict patient PFS in GBM. Our data indicated that TLR9 may relate to glioma progression and it may be a biological marker for the prognosis of GBM patients. In addition, our findings warrant caution in the directly injection of TLR9 into glioma tissues for the glioma immunotherapy. TLR9 can be used as a new target for glioma therapy.
引文
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