脑肿瘤干细胞分化抑制机制的研究
摘要
【目的】从新鲜的人脑胶质瘤手术标本中分离、培养脑肿瘤干细胞(brain tumor stem cells,BTSCs),观察研究它们的细胞生物学特征,尤其是分化特征。在此基础上,利用最新的分子遗传学检测方法CGH-array(array-based comparative genomic hybridization, CGH-array )检测脑肿瘤干细胞中基因组发生的异常改变,从中寻找脑肿瘤发生及恶性进展相关的基因。对检测出的感兴趣基因进一步通过组织芯片技术,检测其在胶质瘤组织中的表达情况,并分析其意义。在脑肿瘤的发生和恶性进展过程中,细胞融合很可能是其中的机制之一,因此,本研究,我们利用致瘤能力低下的胶质瘤细胞系T98G和饲养细胞共培养,观察有无细胞融合现象发生,并就细胞融合对肿瘤细胞生物学特征产生的影响进行初步探讨。
【方法】①从人胎脑组织中培养神经球;从一例混合性胶质瘤的原发和复发手术标本中利用神经干细胞培养方法培养脑肿瘤球;利用免疫磁珠细胞分离系统从脑肿瘤球中分离出脑肿瘤干细胞,从神经球中分离出神经干细胞(neural stem cells, NSCs),并通过流式细胞仪评价分离系统的效率;利用纯化的脑肿瘤干细胞和神经干细胞进行诱导分化实验,观察BTSCs和NSCs的形态变化,同期利用免疫细胞组化和流式细胞仪检测脑肿瘤干细胞和神经干细胞分化过程中CD133、Nestin、GFAP、β-TubulinIII等分化相关标志物表达的变化。②分别从脑肿瘤干细胞和神经干细胞中抽取出DNA,利用美国Spectral Genomics公司提供的CGH-array芯片检测脑肿瘤干细胞中发生的基因组改变,根据异常BAC克隆(bacterial artificial clone, BAC)在基因组序列中对应的位置,寻找感兴趣的基因。进一步利用免疫细胞化学或组织芯片,检测感兴趣基因在胶质瘤组织中的表达情况。③从转基因绿色荧光小鼠腹腔中收集饲养细胞,和难致瘤的胶质瘤细胞系TG98共培养,利用相差显微镜、荧光显微镜观察共培养过程中细胞形态特征的改变;由于饲养细胞本身发绿色荧光,故对胶质瘤细胞特异性标志物nestin的单抗进行红色荧光标记,在两种细胞共培养过程中定期爬片,在激光扫描共聚焦显微镜下观察有无融合细胞出现;通过流式细胞仪检测共培养细胞中绿色荧光细胞所占比例的动态变化及染色体倍体有无异常变化;收集共培养前后的瘤细胞,按照5×106 /只行裸小鼠皮下及颅内接种,比较致瘤能力有无差异。
【结果】①从原发和复发肿瘤标本中均成功地培养出了悬浮生长的肿瘤球;通过免疫磁珠分离系统成功地分离出了纯度较高的CD133+瘤细胞,即脑肿瘤干细胞,而且悬浮肿瘤球中脑肿瘤干细胞所占的比例明显高于贴壁生长的细胞;克隆形成实验表明:只有CD133+瘤细胞能形成肿瘤球。体外分化实验显示:BTSCs起初贴壁生长,细胞形态也转变为梭形,然而从第十天开始,瘤细胞形态逐步向圆形回复,并相互聚集,再次形成悬浮肿瘤球;NSCs按固有规律分化,从圆形到多角形和梭形,再到星形胶质细胞和长纤维状的神经元,未能再形成神经球。与此同时,分化相关标志物的检测表明:刚开始分化时,两者都以CD133和nestin表达为主,而GFAP、β-TubulinIII的表达很低;然而分化开始后,神经干细胞中CD133, Nestin的表达逐渐、持续降低,直至消失,而分化标记物GFAP和β-tubulinⅢ则逐渐、持续升高;在脑肿瘤干细胞,CD133和nestin的表达刚开始也下降,但自第10天起出现反弹而呈上升趋势;与此相反, GFAP和β-TubulinIII表达则呈先升后降的趋势,同样也在第10天出现反弹。增殖周期及倍体检测表明,脑肿瘤干细胞比神经干细胞的增殖更活跃,染色体倍体的变化也更复杂。原发肿瘤干细胞和复发肿瘤干细胞相比,后者在细胞增殖及倍体变化方面均显示了恶性进展的特征。②神经干细胞没有发现基因组拷贝的扩增或丢失,而在脑肿瘤干细胞中则发现了大量的异常克隆,其中,在原发BTSCs中,异常克隆数为160个,而在复发BTSCs中,异常克隆数更多,达182个,原发和复发BTSCs相比较,变化相同的克隆,即共变化克隆占大多数。从共变化克隆中,发现了众多与肿瘤发生及恶性进展密切相关的基因,包括CDK7,p18INK,Rb,4ING3,ING5,CHES1,MYCL1,ERBB4,RAB40C等。值得关注的是,INK4-cyclin D/CDK4/6-Rb-E2F细胞周期信号传导通路中的上游负性调控子,INK4的家族成员,新发现的抑瘤基因,p18INK4存在明显的拷贝丢失,而其下游的著名抑瘤基因Rb也存在拷贝丢失。另外,在原发BTSCs和复发BTSCs中还发现了一些各自异常的克隆,值得关注的是,在复发BTSCs,寻找到的肿瘤相关基因大多与RAS癌基因家族相关,其中RAB13和RAB27属于RAS癌基因家族成员,而RASAL2、RRAS2和RASA3虽不属于RAS癌基因家族,但均可激活RAS基因,上述基因在复发BTSCs均表现为拷贝扩增。KSR2可抑制RAS基因活性,但在复发BTSCs中表现为丢失。进一步检测了抑瘤基因p18INK4在脑肿瘤干细胞及胶质瘤组织中的表达情况,结果在脑肿瘤干细胞中为阴性,而在大多数的胶质瘤组织中为阳性,且随着恶性程度的增加而表达增强。③胶质瘤细胞和饲养细胞共培养过程中,在相差显微镜下观察到巨大型细胞,荧光显微镜下观察到巨大、多突起的绿色荧光细胞;流式细胞检测表明,共培养过程中,绿色荧光细胞的比例一开始就呈快速下降趋势,但降至0.6%左右时保持相对稳定;通过流式细胞仪从共培养细胞中分离出荧光细胞后行染色体倍体检测,结果发现约8%的细胞为异倍体细胞。上述结果提示饲养细胞与胶质瘤细胞共培养过程中,少数细胞有可能发生了融合。共培养细胞爬片后在激光扫描共聚焦显微镜下观察,发现个别发绿色荧光的大细胞同时也发红色荧光,并清晰地显示出了双核征,直观地显示细胞融合的发生;动物致瘤实验表明,共培养前,胶质瘤细胞移植裸小鼠致瘤率为25%,而共培养后胶质瘤细胞致瘤能力提高至100%。
【结论】①成功地从原发和复发胶质瘤标本中培养出了脑肿瘤干细胞,和神经干细胞相比,脑肿瘤干细胞突出地展现出了内在的分化抑制潜能;②p18INK4和RB基因拷贝丢失导致的INK4-cyclin D/CDK4/6-Rb-E2F信号传导通路异常可能是胶质瘤来源细胞分裂周期加快而无法正常分化,继而发生恶性转变的原因。③细胞生物学和分子遗传学结果均显示了复发BTSCs比原发BTSCs更高的恶性特征,这一差异表明脑肿瘤干细胞是一功能性概念,是处于动态变化中的,同时也反映了胶质瘤复发往往意味着恶性程度增高的特点。④p18INK4功能的丧失可能是部分胶质瘤发生的原因。作为抑瘤基因在大多数胶质瘤标本中表达,且随着恶性度增高而表达增加的事实并不否认p18INK4的抑瘤功能,相反,p18INK4的高表达很可能是作为负反馈调节机制而被动高表达,目的是减缓细胞分裂,但在其它因子驱动下,变得“无能为力”了。⑤胶质瘤细胞和饲养细胞共培养后出现了细胞融合,尽管融合的比例很低,但对肿瘤细胞生物学特征的改变不可低估,饲养细胞的主要成分是巨噬细胞,推测胶质瘤细胞与巨噬细胞融合后,不但可以逃避免疫系统的清除,而且还借助巨噬细胞高度的运动性、适应性和可塑性在机体中存活下来,继续增殖,从而形成生物学特征更恶性的肿瘤。
PartⅠ. Cellular Biological Behaviors of Brain Tumor Stem Cells: A Comparative Study with Neural Stem Cells
Background and Objective Abnormal differentiation is central to the formation and progression of gliomas, the most common neoplasm of central nervous system in human. Understanding of the differentiation profile of brain tumor stem cells (BTSCs), the key ones among tumor cell population, through comparison with neural stem cells (NSCs) would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Singh et al. observed that, under differentiation condition, all tumor spheres grew attached as monolayer, lacking expression of undifferentiated cell marker CD133 and nestin. However, the fact that only a small proportion of cells within tumor spheres are true stem cells makes it necessary to isolate and focus on tumor stem cells if the exact biological behaviors of this special cell fraction are to be fully understood. So, in this study, we try to culture and purify BTSCs from newly surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property.
Methods Brain tumor spheres were cultured, with the technique used in neural stem cell culturing, from both primary and recurrent mixed gliomas harbored in the same patient. Brain tissue from a 1-month-old embryo was subjected to the same procedure as that mentioned above to get neurospheres. Both BTSCs and NSCs were purified with magnetic cell sorting system and evaluated with flow cytometry. Induction of differentiation was carried with culture medium added with 10% fetal bovine serum. The morphological changes taking place in both BTSCs and NSCs were observed under phase-contrast microscopy, and the expressing tendency of differentiation-related surface markers, like CD133, nestin, GFAP,β-TubulinIII, were detected with immunofluorescent staining and flow cytometry.
Results 1)Tumor spheres were produced from both primary and recurrent glioma samples. 2)BTSCs (CD133+glioma cell), as well as NSCs, were purified effectively with magnetic cell sorting system. 3)In differentiation assay, NSCs changed morphologically along it’s routine way, whereas, BTSCs underwent rather complicated course: changing from round at the beginning to short fusiform, polygon and long fusiform by day 7, after then, cells gradually pulled back their process, huddling together and reforming suspending tumor sphere 21 days after subject to differentiation condition. 4) The differentiation assay with immunoflurescent staining and flow cell cytometry highly coincided with the morphological changes mentioned above. In BTSCs, the expressing rate of CD133 dropped sharply from 80.76% at the beginning to 16.33% on day 3 and to the lowest point of 3.65% by day 7, but bounded up from day 10. During the same period, the expression of nestin was also examined, with the similar results. Inversely, the expressing rates of GFAP andβ-tubulinⅢincreased gradually till day 7, but decreased after then. Same experiments were also performed on NSCs, however, the expressing rates of CD133 and nestin kept decreasing, till to undetectable level on day 10; at the same time the expression of GFAP andβ-tubulinⅢwere remarkably increased without turn point. 6) Detection of cell cycling with flow cytometry showed that all BTSCs from both primary and recurrent glioma were hypodiploid, while NSCs were all normal diploid. After differentiation, NSCs were still normal diploid, however, the population of BTSCs were more complicated: in BTSCs from primary tumor mass, cells were mainly hypodiploid with minority diploid; in BTSCs from recurrent lesion, cell were comprised of the following three types: hypodiploid(54.81%), hyperploid (44.2%) and diploid. It was also revealed that cells of S and/or G2-M phase in BTSCs were much richer than those in NSCs regardless of differentiation status. When BTSCs from primary and recurrent lesions were compared, more cells of S and/or G2-M phase were detected in recurrent BTSCs.
Conclusions BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma. Brain tumor stem cell is a functional concept, the malignant progression of BTSCs actually represent the evolving of gliomas
PartⅡThe Molecular Cytogenetic Profile of Brain Tumor Stem Cells: A Comparative Study with Neural Stem Cells
Background and Objective This difference in differentiation exhibited by BTSCs and NSCs indicates internal signaling pathway regulating differentiation has been severely shifted by accumulated gene mutation(s) in BTSCs; in other words, genes giving rise to differentiation have been suppressed by genes keeping stem cell undifferentiated. To determine the genes responsible for the dedifferentiation through cytogenetic methods and other methods is of great significance in probing the molecular mechanism of brain tumor initiation. The recently developed array based comparative genomic hybridization (array CGH) has resulted in a significant increase of sensitivity of detecting DNA copy number gains and losses from a resolution of 10-20 Mb, which is routinely achieved with traditional CGH, to an average of 1 Mb. The additional advantage of array-based CGH is that the mapping information of BAC clones printed on the arrays are already known, therefore a direct link between the affected regions and candidate genes can be quickly established. In this part, array-based CGH technique was employed to the end of searching for genes that are candidate partners in the malignant transformation of neural stem cells or precursors.
Methods DNA was extracted from brain tumor stem cells purified with magnetic cell sorting system with DNA isolation kit (Qiagen, Valencia, CA), DNA from neural stem cells was also collected as control. The array used in this study consists of 2632 human BACs (Spectral Genomics, Houston, TX). The experiments were performed according to the manufacturer’s protocols. Data analysis was carried out using the SpectralWare 2.2.23 (Spectral Genomics, Houston, TX). The very interesting gene discovered by array-CGH was further examined with tissue microarray technique to determine it’s protein expression in 68 glioma specimens of different malignant grades.
Results 1)Among the 2621 clones spotted on the array, 2457 were mapped to non-sex chromosomes. No DNA copy loss or gain was detected in neural stem cells, while large amount of BAC clones were amplified or deleted in both primary and recurrent brain tumor stem cells, of which, 160 clones were found in primary BTSCs, and 182 clones in recurrent BTSCs. Moreover, most of the altered clones were detected simultaneously in both primary and recurrent BTSCs, from which, dozens of candidate genes associated with tumorigenesis were mapped, including CDK7, p18INK, Rb, 4ING3, ING5, CHES1, MYCL1, ERBB4, RAB40C, and so on. INK4-cyclin D/CDK4/6-Rb-E2F is one of the most essential signal pathways regulating cell progression. In this study, p18INK4, a core member of the upper negative regulator INK4, and the famous tumor suppressor Rb was found to be synchronously lost in both primary and recurrent BTSCs. Besides the common changes detected by array CGH, some candidate genes related to tumor formation or malignant progression were found to be exclusively amplified or lost in BTSC-1 and BTSC-2 respectively. Intriguingly, in recurrent BTSC, genes activating oncogene RAS were found to gain DNA copy, while genes inhibiting RAS were lost. 2) We also examined the expression of p18INK in BTSCs, normal brain tissue, and glioma specimens of various grades with tissue microarray. As a result, no p18INK staining was revealed in BTSCs, but p18 nuclear immunoexpression was found in 46(67.6%) of the glioma tissues we studied. p18 immunoreactivity also exhibited a clear tendency to elevate with increasing tumor grade.
Conclusions Dozens of candidate genes related to malignant transformation and progression of glioma were selected out through array-based CGH technique. Disruption of INK4-cyclin D/CDK4/6-Rb-E2F signal pathway resulting from DNA copy loss of both p18INK and Rb may account for the abnormal differentiation and malignant transformation of neural stem cells or precursors. Up-regulation of oncogene RAS maybe associated with the malignant progression of brain tumor stem cells we studied. The ambivalent result that p18, as a tumor suppressor, was positively immunostained in most of the glioma specimens does not deny the tumor suppressing role of p18, on the contrary, the up-regulation of p18 expression is possibly due to a negative feedback mechanism. Like other tumor suppressor, the loss of p18 function is responsible for the development of some but not all glioma cases.
PartⅢCell-Cell Fusion and the Malignant Progression of Glioma: A Preliminary Study
Background and Objective Cell-cell fusion is a highly regulated and dramatic cellular event that is required for development and homeostasis. More and more evidences indicate cell fusion may also play a critical role in the development and malignant progression of cancer. Not long ago, GBM, a glioma cell line unable to produce tumor mass when transplanted, reduced dramatically in vitro culture. To save this glioma cell line, we co-cultured it with feeding cells harvested from Balb/c mice. As a result, cells were successfully expanded and saved. Unexpectedly, cells undergoing co-culturing with feeding cells formed tumor mass when injected into immunodeficient mice. At that time, we didn’t know why. Here, we conducted a preliminary study to investigate whether cell-cell fusion take place in co-culture of glioma cells and feeding cells consisting mainly of macrophages and lymphocytes.
Methods T98G, a human glioma cell line with very poor potential to produce tumor mass in immunodeficient mice, were raised with feeding cells harvested from GFP(green fluorescent protein) transgenic mouse. During the course of co-culturing, mixed culture was checked under phase-contrast microscope and fluorescent microscope. Laser scanning confocal microscope was employed to search for cells that co-express GFP( green) from feeding cells and nestin (red) from T98G glioma cells. The ploidy and the proportional change of cells giving off green fluorescence were analyzed with flow cytometry. 5×106Glioma cells before and after co-culture were injected into immunodeficient mice to compare their tumorigenesis.
Results 1)During the co-culturing of feeding cells and glioma cells, round giant cells emerged, and some of the giant round cells present green under fluorescent microscope. 2)After plated to glass coverslips and immunofluorescently stained, cells presented green and red simultaneously were discovered among the mixed culture, moreover, binucleate was observed in some double-color-stained cells. 3)The percentage of“green”cells among co-culture decreased continuously from the beginning of co-culturing, but kept at a very low but relatively stable level of 0.6% 10 days later. 4)Compared with feeding cells, the“green”cells purified from mixed culture were found to contain cells of aneuploidy which accounting for about 8% of total. 5)The incidence of tumor mass formation in immunodeficient nude mice was dramatically improved from 25% before co-culture to 100% after co-culture.
Conclusions The phenomenon of hybridization between glioma cells and feeding cells (macrophage or lymphocytes) do exist, the chance of fusion is very small, but the significance is great. Due to fusion, the features possessed by macrophage, like motility, plasticity, and adaptability were added to glioma cells, which resulted in the progression of glioma cells.
【方法】①从人胎脑组织中培养神经球;从一例混合性胶质瘤的原发和复发手术标本中利用神经干细胞培养方法培养脑肿瘤球;利用免疫磁珠细胞分离系统从脑肿瘤球中分离出脑肿瘤干细胞,从神经球中分离出神经干细胞(neural stem cells, NSCs),并通过流式细胞仪评价分离系统的效率;利用纯化的脑肿瘤干细胞和神经干细胞进行诱导分化实验,观察BTSCs和NSCs的形态变化,同期利用免疫细胞组化和流式细胞仪检测脑肿瘤干细胞和神经干细胞分化过程中CD133、Nestin、GFAP、β-TubulinIII等分化相关标志物表达的变化。②分别从脑肿瘤干细胞和神经干细胞中抽取出DNA,利用美国Spectral Genomics公司提供的CGH-array芯片检测脑肿瘤干细胞中发生的基因组改变,根据异常BAC克隆(bacterial artificial clone, BAC)在基因组序列中对应的位置,寻找感兴趣的基因。进一步利用免疫细胞化学或组织芯片,检测感兴趣基因在胶质瘤组织中的表达情况。③从转基因绿色荧光小鼠腹腔中收集饲养细胞,和难致瘤的胶质瘤细胞系TG98共培养,利用相差显微镜、荧光显微镜观察共培养过程中细胞形态特征的改变;由于饲养细胞本身发绿色荧光,故对胶质瘤细胞特异性标志物nestin的单抗进行红色荧光标记,在两种细胞共培养过程中定期爬片,在激光扫描共聚焦显微镜下观察有无融合细胞出现;通过流式细胞仪检测共培养细胞中绿色荧光细胞所占比例的动态变化及染色体倍体有无异常变化;收集共培养前后的瘤细胞,按照5×106 /只行裸小鼠皮下及颅内接种,比较致瘤能力有无差异。
【结果】①从原发和复发肿瘤标本中均成功地培养出了悬浮生长的肿瘤球;通过免疫磁珠分离系统成功地分离出了纯度较高的CD133+瘤细胞,即脑肿瘤干细胞,而且悬浮肿瘤球中脑肿瘤干细胞所占的比例明显高于贴壁生长的细胞;克隆形成实验表明:只有CD133+瘤细胞能形成肿瘤球。体外分化实验显示:BTSCs起初贴壁生长,细胞形态也转变为梭形,然而从第十天开始,瘤细胞形态逐步向圆形回复,并相互聚集,再次形成悬浮肿瘤球;NSCs按固有规律分化,从圆形到多角形和梭形,再到星形胶质细胞和长纤维状的神经元,未能再形成神经球。与此同时,分化相关标志物的检测表明:刚开始分化时,两者都以CD133和nestin表达为主,而GFAP、β-TubulinIII的表达很低;然而分化开始后,神经干细胞中CD133, Nestin的表达逐渐、持续降低,直至消失,而分化标记物GFAP和β-tubulinⅢ则逐渐、持续升高;在脑肿瘤干细胞,CD133和nestin的表达刚开始也下降,但自第10天起出现反弹而呈上升趋势;与此相反, GFAP和β-TubulinIII表达则呈先升后降的趋势,同样也在第10天出现反弹。增殖周期及倍体检测表明,脑肿瘤干细胞比神经干细胞的增殖更活跃,染色体倍体的变化也更复杂。原发肿瘤干细胞和复发肿瘤干细胞相比,后者在细胞增殖及倍体变化方面均显示了恶性进展的特征。②神经干细胞没有发现基因组拷贝的扩增或丢失,而在脑肿瘤干细胞中则发现了大量的异常克隆,其中,在原发BTSCs中,异常克隆数为160个,而在复发BTSCs中,异常克隆数更多,达182个,原发和复发BTSCs相比较,变化相同的克隆,即共变化克隆占大多数。从共变化克隆中,发现了众多与肿瘤发生及恶性进展密切相关的基因,包括CDK7,p18INK,Rb,4ING3,ING5,CHES1,MYCL1,ERBB4,RAB40C等。值得关注的是,INK4-cyclin D/CDK4/6-Rb-E2F细胞周期信号传导通路中的上游负性调控子,INK4的家族成员,新发现的抑瘤基因,p18INK4存在明显的拷贝丢失,而其下游的著名抑瘤基因Rb也存在拷贝丢失。另外,在原发BTSCs和复发BTSCs中还发现了一些各自异常的克隆,值得关注的是,在复发BTSCs,寻找到的肿瘤相关基因大多与RAS癌基因家族相关,其中RAB13和RAB27属于RAS癌基因家族成员,而RASAL2、RRAS2和RASA3虽不属于RAS癌基因家族,但均可激活RAS基因,上述基因在复发BTSCs均表现为拷贝扩增。KSR2可抑制RAS基因活性,但在复发BTSCs中表现为丢失。进一步检测了抑瘤基因p18INK4在脑肿瘤干细胞及胶质瘤组织中的表达情况,结果在脑肿瘤干细胞中为阴性,而在大多数的胶质瘤组织中为阳性,且随着恶性程度的增加而表达增强。③胶质瘤细胞和饲养细胞共培养过程中,在相差显微镜下观察到巨大型细胞,荧光显微镜下观察到巨大、多突起的绿色荧光细胞;流式细胞检测表明,共培养过程中,绿色荧光细胞的比例一开始就呈快速下降趋势,但降至0.6%左右时保持相对稳定;通过流式细胞仪从共培养细胞中分离出荧光细胞后行染色体倍体检测,结果发现约8%的细胞为异倍体细胞。上述结果提示饲养细胞与胶质瘤细胞共培养过程中,少数细胞有可能发生了融合。共培养细胞爬片后在激光扫描共聚焦显微镜下观察,发现个别发绿色荧光的大细胞同时也发红色荧光,并清晰地显示出了双核征,直观地显示细胞融合的发生;动物致瘤实验表明,共培养前,胶质瘤细胞移植裸小鼠致瘤率为25%,而共培养后胶质瘤细胞致瘤能力提高至100%。
【结论】①成功地从原发和复发胶质瘤标本中培养出了脑肿瘤干细胞,和神经干细胞相比,脑肿瘤干细胞突出地展现出了内在的分化抑制潜能;②p18INK4和RB基因拷贝丢失导致的INK4-cyclin D/CDK4/6-Rb-E2F信号传导通路异常可能是胶质瘤来源细胞分裂周期加快而无法正常分化,继而发生恶性转变的原因。③细胞生物学和分子遗传学结果均显示了复发BTSCs比原发BTSCs更高的恶性特征,这一差异表明脑肿瘤干细胞是一功能性概念,是处于动态变化中的,同时也反映了胶质瘤复发往往意味着恶性程度增高的特点。④p18INK4功能的丧失可能是部分胶质瘤发生的原因。作为抑瘤基因在大多数胶质瘤标本中表达,且随着恶性度增高而表达增加的事实并不否认p18INK4的抑瘤功能,相反,p18INK4的高表达很可能是作为负反馈调节机制而被动高表达,目的是减缓细胞分裂,但在其它因子驱动下,变得“无能为力”了。⑤胶质瘤细胞和饲养细胞共培养后出现了细胞融合,尽管融合的比例很低,但对肿瘤细胞生物学特征的改变不可低估,饲养细胞的主要成分是巨噬细胞,推测胶质瘤细胞与巨噬细胞融合后,不但可以逃避免疫系统的清除,而且还借助巨噬细胞高度的运动性、适应性和可塑性在机体中存活下来,继续增殖,从而形成生物学特征更恶性的肿瘤。
PartⅠ. Cellular Biological Behaviors of Brain Tumor Stem Cells: A Comparative Study with Neural Stem Cells
Background and Objective Abnormal differentiation is central to the formation and progression of gliomas, the most common neoplasm of central nervous system in human. Understanding of the differentiation profile of brain tumor stem cells (BTSCs), the key ones among tumor cell population, through comparison with neural stem cells (NSCs) would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Singh et al. observed that, under differentiation condition, all tumor spheres grew attached as monolayer, lacking expression of undifferentiated cell marker CD133 and nestin. However, the fact that only a small proportion of cells within tumor spheres are true stem cells makes it necessary to isolate and focus on tumor stem cells if the exact biological behaviors of this special cell fraction are to be fully understood. So, in this study, we try to culture and purify BTSCs from newly surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property.
Methods Brain tumor spheres were cultured, with the technique used in neural stem cell culturing, from both primary and recurrent mixed gliomas harbored in the same patient. Brain tissue from a 1-month-old embryo was subjected to the same procedure as that mentioned above to get neurospheres. Both BTSCs and NSCs were purified with magnetic cell sorting system and evaluated with flow cytometry. Induction of differentiation was carried with culture medium added with 10% fetal bovine serum. The morphological changes taking place in both BTSCs and NSCs were observed under phase-contrast microscopy, and the expressing tendency of differentiation-related surface markers, like CD133, nestin, GFAP,β-TubulinIII, were detected with immunofluorescent staining and flow cytometry.
Results 1)Tumor spheres were produced from both primary and recurrent glioma samples. 2)BTSCs (CD133+glioma cell), as well as NSCs, were purified effectively with magnetic cell sorting system. 3)In differentiation assay, NSCs changed morphologically along it’s routine way, whereas, BTSCs underwent rather complicated course: changing from round at the beginning to short fusiform, polygon and long fusiform by day 7, after then, cells gradually pulled back their process, huddling together and reforming suspending tumor sphere 21 days after subject to differentiation condition. 4) The differentiation assay with immunoflurescent staining and flow cell cytometry highly coincided with the morphological changes mentioned above. In BTSCs, the expressing rate of CD133 dropped sharply from 80.76% at the beginning to 16.33% on day 3 and to the lowest point of 3.65% by day 7, but bounded up from day 10. During the same period, the expression of nestin was also examined, with the similar results. Inversely, the expressing rates of GFAP andβ-tubulinⅢincreased gradually till day 7, but decreased after then. Same experiments were also performed on NSCs, however, the expressing rates of CD133 and nestin kept decreasing, till to undetectable level on day 10; at the same time the expression of GFAP andβ-tubulinⅢwere remarkably increased without turn point. 6) Detection of cell cycling with flow cytometry showed that all BTSCs from both primary and recurrent glioma were hypodiploid, while NSCs were all normal diploid. After differentiation, NSCs were still normal diploid, however, the population of BTSCs were more complicated: in BTSCs from primary tumor mass, cells were mainly hypodiploid with minority diploid; in BTSCs from recurrent lesion, cell were comprised of the following three types: hypodiploid(54.81%), hyperploid (44.2%) and diploid. It was also revealed that cells of S and/or G2-M phase in BTSCs were much richer than those in NSCs regardless of differentiation status. When BTSCs from primary and recurrent lesions were compared, more cells of S and/or G2-M phase were detected in recurrent BTSCs.
Conclusions BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma. Brain tumor stem cell is a functional concept, the malignant progression of BTSCs actually represent the evolving of gliomas
PartⅡThe Molecular Cytogenetic Profile of Brain Tumor Stem Cells: A Comparative Study with Neural Stem Cells
Background and Objective This difference in differentiation exhibited by BTSCs and NSCs indicates internal signaling pathway regulating differentiation has been severely shifted by accumulated gene mutation(s) in BTSCs; in other words, genes giving rise to differentiation have been suppressed by genes keeping stem cell undifferentiated. To determine the genes responsible for the dedifferentiation through cytogenetic methods and other methods is of great significance in probing the molecular mechanism of brain tumor initiation. The recently developed array based comparative genomic hybridization (array CGH) has resulted in a significant increase of sensitivity of detecting DNA copy number gains and losses from a resolution of 10-20 Mb, which is routinely achieved with traditional CGH, to an average of 1 Mb. The additional advantage of array-based CGH is that the mapping information of BAC clones printed on the arrays are already known, therefore a direct link between the affected regions and candidate genes can be quickly established. In this part, array-based CGH technique was employed to the end of searching for genes that are candidate partners in the malignant transformation of neural stem cells or precursors.
Methods DNA was extracted from brain tumor stem cells purified with magnetic cell sorting system with DNA isolation kit (Qiagen, Valencia, CA), DNA from neural stem cells was also collected as control. The array used in this study consists of 2632 human BACs (Spectral Genomics, Houston, TX). The experiments were performed according to the manufacturer’s protocols. Data analysis was carried out using the SpectralWare 2.2.23 (Spectral Genomics, Houston, TX). The very interesting gene discovered by array-CGH was further examined with tissue microarray technique to determine it’s protein expression in 68 glioma specimens of different malignant grades.
Results 1)Among the 2621 clones spotted on the array, 2457 were mapped to non-sex chromosomes. No DNA copy loss or gain was detected in neural stem cells, while large amount of BAC clones were amplified or deleted in both primary and recurrent brain tumor stem cells, of which, 160 clones were found in primary BTSCs, and 182 clones in recurrent BTSCs. Moreover, most of the altered clones were detected simultaneously in both primary and recurrent BTSCs, from which, dozens of candidate genes associated with tumorigenesis were mapped, including CDK7, p18INK, Rb, 4ING3, ING5, CHES1, MYCL1, ERBB4, RAB40C, and so on. INK4-cyclin D/CDK4/6-Rb-E2F is one of the most essential signal pathways regulating cell progression. In this study, p18INK4, a core member of the upper negative regulator INK4, and the famous tumor suppressor Rb was found to be synchronously lost in both primary and recurrent BTSCs. Besides the common changes detected by array CGH, some candidate genes related to tumor formation or malignant progression were found to be exclusively amplified or lost in BTSC-1 and BTSC-2 respectively. Intriguingly, in recurrent BTSC, genes activating oncogene RAS were found to gain DNA copy, while genes inhibiting RAS were lost. 2) We also examined the expression of p18INK in BTSCs, normal brain tissue, and glioma specimens of various grades with tissue microarray. As a result, no p18INK staining was revealed in BTSCs, but p18 nuclear immunoexpression was found in 46(67.6%) of the glioma tissues we studied. p18 immunoreactivity also exhibited a clear tendency to elevate with increasing tumor grade.
Conclusions Dozens of candidate genes related to malignant transformation and progression of glioma were selected out through array-based CGH technique. Disruption of INK4-cyclin D/CDK4/6-Rb-E2F signal pathway resulting from DNA copy loss of both p18INK and Rb may account for the abnormal differentiation and malignant transformation of neural stem cells or precursors. Up-regulation of oncogene RAS maybe associated with the malignant progression of brain tumor stem cells we studied. The ambivalent result that p18, as a tumor suppressor, was positively immunostained in most of the glioma specimens does not deny the tumor suppressing role of p18, on the contrary, the up-regulation of p18 expression is possibly due to a negative feedback mechanism. Like other tumor suppressor, the loss of p18 function is responsible for the development of some but not all glioma cases.
PartⅢCell-Cell Fusion and the Malignant Progression of Glioma: A Preliminary Study
Background and Objective Cell-cell fusion is a highly regulated and dramatic cellular event that is required for development and homeostasis. More and more evidences indicate cell fusion may also play a critical role in the development and malignant progression of cancer. Not long ago, GBM, a glioma cell line unable to produce tumor mass when transplanted, reduced dramatically in vitro culture. To save this glioma cell line, we co-cultured it with feeding cells harvested from Balb/c mice. As a result, cells were successfully expanded and saved. Unexpectedly, cells undergoing co-culturing with feeding cells formed tumor mass when injected into immunodeficient mice. At that time, we didn’t know why. Here, we conducted a preliminary study to investigate whether cell-cell fusion take place in co-culture of glioma cells and feeding cells consisting mainly of macrophages and lymphocytes.
Methods T98G, a human glioma cell line with very poor potential to produce tumor mass in immunodeficient mice, were raised with feeding cells harvested from GFP(green fluorescent protein) transgenic mouse. During the course of co-culturing, mixed culture was checked under phase-contrast microscope and fluorescent microscope. Laser scanning confocal microscope was employed to search for cells that co-express GFP( green) from feeding cells and nestin (red) from T98G glioma cells. The ploidy and the proportional change of cells giving off green fluorescence were analyzed with flow cytometry. 5×106Glioma cells before and after co-culture were injected into immunodeficient mice to compare their tumorigenesis.
Results 1)During the co-culturing of feeding cells and glioma cells, round giant cells emerged, and some of the giant round cells present green under fluorescent microscope. 2)After plated to glass coverslips and immunofluorescently stained, cells presented green and red simultaneously were discovered among the mixed culture, moreover, binucleate was observed in some double-color-stained cells. 3)The percentage of“green”cells among co-culture decreased continuously from the beginning of co-culturing, but kept at a very low but relatively stable level of 0.6% 10 days later. 4)Compared with feeding cells, the“green”cells purified from mixed culture were found to contain cells of aneuploidy which accounting for about 8% of total. 5)The incidence of tumor mass formation in immunodeficient nude mice was dramatically improved from 25% before co-culture to 100% after co-culture.
Conclusions The phenomenon of hybridization between glioma cells and feeding cells (macrophage or lymphocytes) do exist, the chance of fusion is very small, but the significance is great. Due to fusion, the features possessed by macrophage, like motility, plasticity, and adaptability were added to glioma cells, which resulted in the progression of glioma cells.
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