C6细胞系内脑肿瘤干细胞的分离、培养及移植瘤内分布特点研究
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摘要
研究背景
恶性胶质瘤是最常见的成人脑肿瘤,约占所有成人脑肿瘤的40%。高分级的胶质瘤患者五年生存率低于3%,中位生存时间小于一年。虽然外科手术和其他治疗措施进展很快,但仍很难治愈。经手术、放疗和化疗后,病人几乎无一例外地出现肿瘤复发。近来,许多研究者先后在肿瘤中发现了具有干细胞特性的细胞,提出了肿瘤干细胞(cancer stem cell,CSCs)的概念,认为在肿瘤细胞中有一小部分细胞具有自我更新、无限增殖、多向分化潜能的干细胞样特性,它们是肿瘤发生,转移和复发的根源。而肿瘤中的绝大部分细胞不具有上述特性,无法独立在体内形成种植性肿瘤。肿瘤干细胞最早是在白血病中发现,后来相继在乳腺癌、多发性骨髓瘤,前列腺癌以及胰腺癌中分离并证实,为研究胶质瘤提供了一个新的思路,为今后的靶向治疗奠定了基础。
神经系统的肿瘤干细胞,最早报道的是Ignatova和Singh等,他们将脑胶质瘤细胞在神经干细胞(Neural stem cells,NSC)培养条件下进行培养,分离了一类具有自我更新能力及多向分化潜能的细胞,称之为脑肿瘤干细胞(brain tumor stem cell,BTSC)。这类细胞的特点是可表达神经干细胞的特异性细胞标志—CD133和神经巢蛋白(Nestin)。Singh等还发现,仅10~2个CD133+细胞就可以在免疫缺陷小鼠的额叶形成肿瘤,而接种10~5个CD133-细胞仅在注射部位形成了一条胶质疤痕。某些脑肿瘤干细胞能特征性的表达ABC转运体(ATP-binding cassette),该转运体能将多种药物转运出细胞外,从而对化疗产生耐药;而某些脑肿瘤干细胞具有很强的修复DNA损伤的能力,从而对放疗产生抵抗。
基于肿瘤干细胞与非干细胞肿瘤细胞在致瘤性以及放化疗抵抗能力上的差别,Singh等提出了源于CSCs的肿瘤生成模式:少量正常细胞由于基因突变等原因转化为CSCs,CSCs通过对称和不对称分裂,分别产生少量的子代CSCs和大量的肿瘤细胞,这种分裂方式持续进行直至形成肿块。肿块中的CSCs所占比例较小,多数为CD133-的肿瘤细胞,这种模式说明肿瘤中始终存在一定数量的CSCs,尽管数量可能较少,但它们在肿瘤的发生、发展、侵袭、转移、复发和治疗敏感性中起关键性的作用。
作为经典的胶质瘤模型,多年来C6大鼠胶质瘤细胞系被广泛应用于胶质瘤的实验研究。但是C6胶质瘤细胞系内肿瘤干细胞的分离、培养方法以及肿瘤干细胞所占的比例一直存在争议。Toru Kondo等应用一种复杂的无血清DMEM培养基对C6细胞系内的CSCs进行分离培养,并指出其中CSCs的比例约为0.4%。但Xuesheng Zheng等应用单克隆研究,提出了C6细胞系中大部分细胞都是CSCs的观点。同时,虽然越来越多的研究指出CSCs参与肿瘤的血管生成,肿瘤形成、侵袭以及复发,从而可以作为靶向治疗的新的靶点,但尚未有对于瘤体内肿瘤干细胞分布位置及分布特点的研究报道。
在本实验中,我们应用简化的无血清DMEM培养基对C6细胞系进行培养,对其中的CSCs进行了分离与鉴定,并测定了其内CSCs所占的比例;建立了裸鼠皮下C6胶质瘤致瘤模型,并研究了移植瘤内CSCs的分布特点;提出了肿瘤内CSCs的“爆炸式”分布假说,从形态学上,为CSCs参与肿瘤形成、侵袭以及复发提供了新的证据,为以CSCs为靶点的靶向治疗提供了定位依据。
目的
1.分离、培养C6细胞系内的脑肿瘤干细胞,并对其进行鉴定,测定C6细胞系内脑肿瘤干细胞所占的比例。
2.建立裸鼠皮下C6胶质瘤载瘤模型,查找其中的肿瘤干细胞。
3.研究C6移植瘤内肿瘤干细胞的分布特点。
方法
1.C6细胞系中的脑肿瘤干细胞的分离培养与传代
先将C6细胞系接种于传统含血清培养基中,在37℃、5%CO_2、饱和湿度培养箱中培养。待细胞处于对数生长期,用PBS清洗,0.25%胰酶消化,自制巴斯德吸管机械吹打成单细胞悬液,重悬于添加了表皮生长因子和碱性成纤维生长因子的无血清培养基,待细胞增殖形成肿瘤细胞球4—5天后,在超净工作台内将细胞团连同培养基一起转移至试管内,以800r/min离心10min,弃上清。无血清培养基重悬细胞,自制巴斯德尖嘴吸管反复吹打,制成单细胞悬液,按1:2或者1:3的比例接种于25cm~2底面积培养瓶,添加无血清培养基至6ml;或以相同浓度接种于标准6孔板,每孔加无血清培养基2.5ml,继续在37℃、5%CO_2、饱和湿度培养箱中培养。
2.C6细胞系中的脑肿瘤干细胞的鉴定
以CD133和Nestin为标记,应用免疫荧光技术对普通含血清培养基中的C6细胞和无血清培养基中形成的肿瘤细胞球进行鉴定。取含血清培养基中处于对数生长期的C6细胞,胰酶消化后,调整细胞密度为5×10~5/ml,接种于放置强酸过夜浸泡的载波片的六孔板内,爬片过夜后,将细胞爬片用4.0%多聚甲醛固定。取无血清培养基培养的肿瘤细胞球悬液,离心后滴于0.01%多聚赖氨酸溶液包被的载玻片上,静置待干,4.0%多聚甲醛固定。10%山羊血清封闭。抗CD133或抗Nestin抗体孵育4℃过夜,添加荧光素标记二抗孵育37℃20min,每步骤间均用0.01MPBS冲洗3遍。封片后置荧光显微镜下观察。
3.C6细胞系中的脑肿瘤干细胞所占比例的测定
应用Nestin标记的流式细胞术检测C6细胞系中的脑肿瘤干细胞所占的比例。取处于对数生长期的C6细胞,胰酶消化后,调整细胞密度为1×10~7/ml,取10个样本,每个样本100μl,分别加入TRITC标记的抗鼠Nestin抗体后,于暗处孵育30分钟。设不加抗鼠Nestin抗体的阴性对照10个,分别上机检测每个样本中脑肿瘤干细胞的比例。
4.裸鼠皮下C6胶质瘤载瘤模型的建立
取处于对数生长期的C6细胞,胰酶消化后,调整细胞密度为1×10~6/ml。抽取细胞悬液100μl,接种于裸鼠双侧腋下或股部皮下。定期观察裸鼠皮下肿瘤生长情况,以及体重、精神、饮食、排便等情况。每隔5天测量移植瘤的最长径(a)和最短径(b),按公式V=ab~2×π/6计算肿瘤体积,绘制生长曲线。待移植40天后,脱颈处死裸鼠,切开移植部位皮肤,手术显微镜下观察移植瘤生长情况并将其完整分离。
5.移植瘤内肿瘤干细胞的分布特点研究
(1)将肿瘤标本做连续石蜡切片,应用CD133或Nestin标记的免疫荧光及免疫组化技术查找肿瘤组织内的肿瘤干细胞,直观观察其内肿瘤干细胞的分布位置及分布方式。
(2)取新鲜肿瘤瘤体最大剖面处做厚度0.5cm的切片,由瘤体中心至外缘均分成5部分,显微手术器械分割,所得的5个样本,采用机械研磨法处理,研磨后200目滤网过滤除去细胞团快,所得的单细胞悬液调整细胞密度为1×10~7/ml,CD133标记后,上机检测自瘤体中心至瘤体边缘不同部位脑肿瘤干细胞的比例。
结果
1.无血清培养基中脑肿瘤干细胞球的形成
使用传统含血清培养基进行培养的C6细胞,呈贴壁生长,接种到无血清培养基后1小时,呈单个细胞悬浮生长;接种后24小时,小的肿瘤干细胞球形成;接种48小时后形成典型的肿瘤干细胞球,细胞球呈圆形或卵圆形,大小不一,折光性较强,悬浮生长。
2.C6细胞系中脑肿瘤干细球的鉴定
含血清培养基中贴壁生长的C6细胞爬片进行的免疫荧光,可以查见散在分布的CD133与Nestin表达阳性的细胞。无血清培养基中形成的肿瘤干细胞球进行的免疫荧光显示呈CD133与Nestin的强阳性表达。
3.流式细胞术检测C6细胞系内肿瘤干细胞比例的结果
Nestin标记流式细胞术可检出C6细胞系内的脑肿瘤干细胞,10例样本内测得的所含脑肿瘤干细胞的比例在4%左右。统计学分析表明,所测10个样本中脑肿瘤干细胞的比例为4.02±0.04%,95%可信区间为3.93—4.10%。
4.移植致瘤实验
本实验所用20只裸鼠,均在注射部位形成了肿瘤,致瘤潜伏期为4—6天,肿瘤体积呈指数生长。移植致瘤后40天被处死时,瘤体最长径为1.7—2.0cm。
5.移植瘤内肿瘤干细胞的分布特点
(1)肿瘤组织的免疫组化与免疫荧光结果
以肿瘤干细胞的表面标志CD133与Nestin为指标,对肿瘤组织中的肿瘤干细胞进行鉴定和定位,可见典型的阳性分布,其在瘤体内的分布,主要有以下三种方式:散在的单个阳性细胞,巢状分布和条带状分布。这三种分布方式在瘤体内的分布也有一定的特点,即在肿瘤的中心,阳性细胞很少,即使有,也多是散在的单个细胞,并且多围绕在小血管的周围;在靠外侧的肿瘤基质内,可以看到巢状和条带状分布;而在肿瘤的周边或者边缘区域,多是带状分布,并且越接近肿瘤的边缘,阳性细胞的密度越大。在比较典型的视野中,可以找到呈分层排列的肿瘤干细胞带,在肿瘤边缘可见包裹有一层密集的肿瘤干细胞。
(2)流式细胞术检测自瘤体中心至瘤体边缘不同部位脑肿瘤干细胞比例的结果
肿瘤中心部位脑肿瘤干细胞比例为0.90±0.01%,而边缘部位比例为9.81±0.04%。经oneway-anova分析,结果显示,在由肿瘤中心至肿瘤边缘的组织中,肿瘤干细胞比例呈现线性增高趋势。
结论
1.C6细胞系内存在比例约为4.02%的CD133和Nestin表达阳性的CSCs。简化的无血清培养基可以作为肿瘤干细胞分离、培养的一种有效途径。
2.裸鼠皮下C6胶质瘤载瘤模型成瘤稳定,肿瘤生长良好。移植瘤内可查见脑肿瘤干细胞。
3.C6胶质瘤瘤体内,脑肿瘤干细胞的分布有三种方式,并呈现由中心向四周放射状逐渐增多的趋势,呈“爆炸式”分布特点。
Background:
Malignant glioma represents about 40%of all adult primary brain tumors.The prognosis of human malignant glioma remains poor with an overall 5-year survival rate of less than 3%and a median survival time of about 1 year for higher grade tumors such as glioblastoma.Gliomas present as diffuse tumors with invasion into normal brain tissue,but frequently recur or progress after surgery,radiation or chemotherapy as focal masses,suggesting that only a fraction of tumor cells is responsible for regrowth.Recently,there is accumulating evidence that malignant solid tumors may contain their own stem cells,termed cancer stem cells(CSCs).Despite their small quantity,this subpopulation within tumor possesses the ability of infinite proliferation and multipotency thus may play a crucial role in the initiation,progression and recurrence of cancer.The concept of cancer stem cells was intuitively suggested by clinical experience with leukemia,and also evident in teratocarcinomas and other solid tumors,such as breast cancer,prostate cancer and pancreas cancer. These studies have provided insights into the understanding of glioma cancer biology and paved the way for thefuture CSCs-targeted theropy.
CSCs in nervous system were first reported by Ignatova and Singh.They cultured glioma cells in serum-free neural stem cell medium and obtained a group of cells that were dramatically different from common glioma cells in growth conditions,cell markers,proliferating and differentiating capacity and tumor-initiating ability in vivo.Singh separated CD133- and CD133+ cells with immunomagnetic beads and planted them separately to the front lobe of 6-week-old nude mice.12-24weeks later,he found only 10~2 CD133+ cells could generate a tumor,but 10~5 CD133- cells only made a glial scar. Importantly,elevated expression of transporters that pump out chemotherapeutic agents and an increased capacity to repair DNA damage may also contribute to CSCs' ability to survive conventional modes of therapy.
Based on the ability of tumorgenesis and to survive conventional modes of therapy,Singh proposed a tumorgenic pattern based on CSCs.In the cancer stem cell model of tumors,there is a small subset of cancer cells,the cancer stem cells,which constitute a reservoir of self-sustaining cells with the exclusive ability to self-renew and maintain the tumor.These cancer stem cells have the capacity to both divide and expand the cancer stem cell pool and to differentiate into the heterogeneous nontumorigenic cancer cell types that in most cases appear to constitute the bulk of the tumor.If cancer stem cells are relatively refractory to therapies that have been developed to eradicate the rapidly dividing cells,they are unlikely to be curative and relapses would be expected.
The rat glioma cell line C6 is one of the widely used cell lines in the studies of glioma.However,it remained controversial about the culturing methods and fraction of CSCs in this cell line.For instance,Toru Kondo et al. found only 0.4%of C6 cells acted as cancer stem-like cells in a complicated serum-free DMEM medium.However,Xuesheng Zheng et al.pointed out that most C6 cells were cancer stem cells from clonal and population analyses. Given the difference of tumorigenic activity and resistance to treatment between CSCs and normal tumor cells,CSCs could act as a new target for cancer therapy.However there is a problem:where do they locate? Until now, there is little research on the location of CSCs in tumor bulk.
In our study,we cultured C6 cell line in a simplified serum-free neural stem cell medium and built up C6 subcutaneous xenografts model in nude mice.CD133 and Nestin marked immunohistochemistry and immunofluorescence confirmed the existence of CSCs both in C6 cell line and xenografts.Nestin marked flow cytometry demonstrated that 4.02%cells in C6 cell line presented as CSCs.Immunohistochemistry, immunofluorescence and flow cytometry were applied to detect the location and distribution characteristics of CSCs in subcutaneous xenografts.We proposed the hypothesis of "explosive distribution" of CSCs in tumor bulk and believed it helpful in explaining how CSCs participate in angiogenesis, tumor initiation,invasion and recurrence in a histological view.
Objective
1.To find a simple and effective method to culture,isolate and characterize cancer stem cells and confirm the fraction of cancer stem cells in C6 cell line.
2.To build up C6 subcutaneous xenografts model in nude mice and detect CSCs in C6 xenografts.
3.To study the distribution characteristics of brain cancer stem cells in C6 glioma.
Methods:
1.Tumor sphere culture
C6 glioma cells were firstly seeded in a 100ml culture flask containing 5ml 1640 culture solution,supplemented with 10%fetal bovine serum,100 U/ml penicillin G and 100μg/ml streptomycin.Cells which were in exponential phase of growth were collected and transplanted to a new culture flask with an equal volume of serum-free neural stem cell medium containing DMEM/F12,B27 supplement,recombinant human epidermal growth factor(rhEGF,20 ng/ml), and basic fibroblast growth factor(bFGF,20 ng/ml).Cells were incubated at 37℃with 95%air,5%CO_2,and 100%humidity.When the tumor spheres reached the size of approximately 100-200 cells per sphere,culture propagation was performed at the ratio of 1:2.
2.Characterization of cancer stem cells from C6 glioma cell line
CD133 or Nestin marked immunohistochemistry and immunofluorescence were used for characterization of cancer stem cells.Cells were plated onto poly-L-lysine coated glass coverslips in 1640 culture solution with 10%FBS or serum-free neural DMEM medium for 12 h.Cells were washed and then fixed in 4%paraformaldehyde and processed for immunofluorescence of CD133 or Nestin.The staining was detected under laser confocal scanning microscope
3.The detection of cancer stem cells in C6 glioma cell line
Nestin marked flow cytometry was used to detect the fraction of cancer stem cells in C6 cell line.Cells which were in exponential phase of growth were collected and cell density was adjusted to 1×10~7/ml.Each sample contained 100μl single-cell suspension.Ten C6 cell line samples were taken and incubated with anti-nestin antibody.Flow cytometry was then performed to test the fractions of CSCs in C6 cell line.
4.Xenografts assays
C6 cells(1×10~5 cells ) were implanted subcutaneously to both right and left thighs of 4-week-old female immunocompromised mice(n = 20).Xenograft tumors were harvested and examined 6 weeks after implantation.
5.The distribution characteristics of brain cancer stem cells in C6 glioma.
(1) Xenografts specimens were fixed in 4%paraformaldehyde,and sected into serial sections(5μm in thickness).For each specimen,20 slides were selected with uniform distribution along the tumor macroaxis. Immunohistochemistry and immunofluorescence were performed on the tissue slides for CD133 and Nestin.
(2) For any fresh tumor bulk,a section(0.5cm in thickness) was taken along its macroaxis.The section was equally divided into 5 parts of ring-like tissue from the center to the periphery and these 5 parts were incubated with anti-CD133 antibody.Flow cytometry was then performed to test the percentage of CSCs in each part.
Results:
1.Formation of tumor spheres in serum-free medium
When cultured in traditional serum-supplemented medium,C6 cells grew as single cells attached to the culture fask,while presented as single cells suspending in medium 1 hour after reseeded in serum-free neural stem cell medium,as small tumor spheres 24 hours later and as typical tumor spheres after 48 hours.The tumor spheres were globular,different in size,and floating in medium.
2.Expression of cancer stem cell markers
CD133 and Nestin marked immunofluorescence showed that:sparsly distributed CD133+ and Nestin+ cells could be seen in C6 cells cultured in serum-supplemented medium;in tumor soheres formed in serum-free medium, CD133 and Nestin were strongly abundantly expressed.
3.The fractions of CSCs in C6 cell line
Nestin marked flow cytometry demonstrated that about 4%cells in C6 cell line presented as CSCs.t tests demonstrated the fraction of CSCs in C6 cell line was 4.02±0.04%and 95%CI was 3.93-4.10%.
4.Formation of subcutaneous xenografts in nude mice
All the 20 experimental nude mice formed subcutaneous xenografts 4-6 days after injection.When nude mice were harvested 6 weeks after implantation,the maximum diameter of the tumor bulks was 1.7-2.0cm.
5.The distribution characteristics of brain cancer stem cells in C6 glioma.
(1) Immunohistochemistry and immunofluorescence results
CD133+ or Nestin+ cells could be found in xenografts of C6 glioma with three patterns of distribution:spot-like,nest-like,and zone-like.Interestingly, these three patterns of distribution were found in different parts of tumor bulks. At the center of a tumor bulk,very few CSCs could be found,which usually emerged in a spot-like pattern around capillaries.At the margin of a tumor bulk, CSCs displayed a zone-like pattern of distribution.In the area between the center and the margin,both nest-like and zone-like pattern could be seen,and the density of positivity showed a stepwise increase from the center to the margin.In some typical visual fields,CSCs displayed as cocenric citcles at the margin of the tumor bulk,forming a package of high density of CSCs for the tumor.
(2) Flow cytometry results
At the tumor centre,the fraction of CSCs was 0.90±0.01%.In contrast,at the margin of the tumor,the fraction was as high as 9.81±0.04%.Oneway-ANOVA analysis of the fractions showed that the fraction of CD133+ cells presented a stepwise increase from the center of tumor bulk to the margin.
Conclusions:
1.The fraction of CSCs in C6 cell line is 4.02%and the simplified serum-free neural stem cell medium can act as a simple and effective method for isolation and detection of CSCs to study the initiation and progression of human glioma.
2.C6 subcutaneous glioma in nude mice is a reliable animal model for studying glioma.
3.There are three distributing patterns of CSCs in C6 glioma:spot-like, nest-like and zone-like and their distribution shows a tendency of radioactive enhancement from the center to the margin of tumor bulk as "explosive distribution" pattern.
恶性胶质瘤是最常见的成人脑肿瘤,约占所有成人脑肿瘤的40%。高分级的胶质瘤患者五年生存率低于3%,中位生存时间小于一年。虽然外科手术和其他治疗措施进展很快,但仍很难治愈。经手术、放疗和化疗后,病人几乎无一例外地出现肿瘤复发。近来,许多研究者先后在肿瘤中发现了具有干细胞特性的细胞,提出了肿瘤干细胞(cancer stem cell,CSCs)的概念,认为在肿瘤细胞中有一小部分细胞具有自我更新、无限增殖、多向分化潜能的干细胞样特性,它们是肿瘤发生,转移和复发的根源。而肿瘤中的绝大部分细胞不具有上述特性,无法独立在体内形成种植性肿瘤。肿瘤干细胞最早是在白血病中发现,后来相继在乳腺癌、多发性骨髓瘤,前列腺癌以及胰腺癌中分离并证实,为研究胶质瘤提供了一个新的思路,为今后的靶向治疗奠定了基础。
神经系统的肿瘤干细胞,最早报道的是Ignatova和Singh等,他们将脑胶质瘤细胞在神经干细胞(Neural stem cells,NSC)培养条件下进行培养,分离了一类具有自我更新能力及多向分化潜能的细胞,称之为脑肿瘤干细胞(brain tumor stem cell,BTSC)。这类细胞的特点是可表达神经干细胞的特异性细胞标志—CD133和神经巢蛋白(Nestin)。Singh等还发现,仅10~2个CD133+细胞就可以在免疫缺陷小鼠的额叶形成肿瘤,而接种10~5个CD133-细胞仅在注射部位形成了一条胶质疤痕。某些脑肿瘤干细胞能特征性的表达ABC转运体(ATP-binding cassette),该转运体能将多种药物转运出细胞外,从而对化疗产生耐药;而某些脑肿瘤干细胞具有很强的修复DNA损伤的能力,从而对放疗产生抵抗。
基于肿瘤干细胞与非干细胞肿瘤细胞在致瘤性以及放化疗抵抗能力上的差别,Singh等提出了源于CSCs的肿瘤生成模式:少量正常细胞由于基因突变等原因转化为CSCs,CSCs通过对称和不对称分裂,分别产生少量的子代CSCs和大量的肿瘤细胞,这种分裂方式持续进行直至形成肿块。肿块中的CSCs所占比例较小,多数为CD133-的肿瘤细胞,这种模式说明肿瘤中始终存在一定数量的CSCs,尽管数量可能较少,但它们在肿瘤的发生、发展、侵袭、转移、复发和治疗敏感性中起关键性的作用。
作为经典的胶质瘤模型,多年来C6大鼠胶质瘤细胞系被广泛应用于胶质瘤的实验研究。但是C6胶质瘤细胞系内肿瘤干细胞的分离、培养方法以及肿瘤干细胞所占的比例一直存在争议。Toru Kondo等应用一种复杂的无血清DMEM培养基对C6细胞系内的CSCs进行分离培养,并指出其中CSCs的比例约为0.4%。但Xuesheng Zheng等应用单克隆研究,提出了C6细胞系中大部分细胞都是CSCs的观点。同时,虽然越来越多的研究指出CSCs参与肿瘤的血管生成,肿瘤形成、侵袭以及复发,从而可以作为靶向治疗的新的靶点,但尚未有对于瘤体内肿瘤干细胞分布位置及分布特点的研究报道。
在本实验中,我们应用简化的无血清DMEM培养基对C6细胞系进行培养,对其中的CSCs进行了分离与鉴定,并测定了其内CSCs所占的比例;建立了裸鼠皮下C6胶质瘤致瘤模型,并研究了移植瘤内CSCs的分布特点;提出了肿瘤内CSCs的“爆炸式”分布假说,从形态学上,为CSCs参与肿瘤形成、侵袭以及复发提供了新的证据,为以CSCs为靶点的靶向治疗提供了定位依据。
目的
1.分离、培养C6细胞系内的脑肿瘤干细胞,并对其进行鉴定,测定C6细胞系内脑肿瘤干细胞所占的比例。
2.建立裸鼠皮下C6胶质瘤载瘤模型,查找其中的肿瘤干细胞。
3.研究C6移植瘤内肿瘤干细胞的分布特点。
方法
1.C6细胞系中的脑肿瘤干细胞的分离培养与传代
先将C6细胞系接种于传统含血清培养基中,在37℃、5%CO_2、饱和湿度培养箱中培养。待细胞处于对数生长期,用PBS清洗,0.25%胰酶消化,自制巴斯德吸管机械吹打成单细胞悬液,重悬于添加了表皮生长因子和碱性成纤维生长因子的无血清培养基,待细胞增殖形成肿瘤细胞球4—5天后,在超净工作台内将细胞团连同培养基一起转移至试管内,以800r/min离心10min,弃上清。无血清培养基重悬细胞,自制巴斯德尖嘴吸管反复吹打,制成单细胞悬液,按1:2或者1:3的比例接种于25cm~2底面积培养瓶,添加无血清培养基至6ml;或以相同浓度接种于标准6孔板,每孔加无血清培养基2.5ml,继续在37℃、5%CO_2、饱和湿度培养箱中培养。
2.C6细胞系中的脑肿瘤干细胞的鉴定
以CD133和Nestin为标记,应用免疫荧光技术对普通含血清培养基中的C6细胞和无血清培养基中形成的肿瘤细胞球进行鉴定。取含血清培养基中处于对数生长期的C6细胞,胰酶消化后,调整细胞密度为5×10~5/ml,接种于放置强酸过夜浸泡的载波片的六孔板内,爬片过夜后,将细胞爬片用4.0%多聚甲醛固定。取无血清培养基培养的肿瘤细胞球悬液,离心后滴于0.01%多聚赖氨酸溶液包被的载玻片上,静置待干,4.0%多聚甲醛固定。10%山羊血清封闭。抗CD133或抗Nestin抗体孵育4℃过夜,添加荧光素标记二抗孵育37℃20min,每步骤间均用0.01MPBS冲洗3遍。封片后置荧光显微镜下观察。
3.C6细胞系中的脑肿瘤干细胞所占比例的测定
应用Nestin标记的流式细胞术检测C6细胞系中的脑肿瘤干细胞所占的比例。取处于对数生长期的C6细胞,胰酶消化后,调整细胞密度为1×10~7/ml,取10个样本,每个样本100μl,分别加入TRITC标记的抗鼠Nestin抗体后,于暗处孵育30分钟。设不加抗鼠Nestin抗体的阴性对照10个,分别上机检测每个样本中脑肿瘤干细胞的比例。
4.裸鼠皮下C6胶质瘤载瘤模型的建立
取处于对数生长期的C6细胞,胰酶消化后,调整细胞密度为1×10~6/ml。抽取细胞悬液100μl,接种于裸鼠双侧腋下或股部皮下。定期观察裸鼠皮下肿瘤生长情况,以及体重、精神、饮食、排便等情况。每隔5天测量移植瘤的最长径(a)和最短径(b),按公式V=ab~2×π/6计算肿瘤体积,绘制生长曲线。待移植40天后,脱颈处死裸鼠,切开移植部位皮肤,手术显微镜下观察移植瘤生长情况并将其完整分离。
5.移植瘤内肿瘤干细胞的分布特点研究
(1)将肿瘤标本做连续石蜡切片,应用CD133或Nestin标记的免疫荧光及免疫组化技术查找肿瘤组织内的肿瘤干细胞,直观观察其内肿瘤干细胞的分布位置及分布方式。
(2)取新鲜肿瘤瘤体最大剖面处做厚度0.5cm的切片,由瘤体中心至外缘均分成5部分,显微手术器械分割,所得的5个样本,采用机械研磨法处理,研磨后200目滤网过滤除去细胞团快,所得的单细胞悬液调整细胞密度为1×10~7/ml,CD133标记后,上机检测自瘤体中心至瘤体边缘不同部位脑肿瘤干细胞的比例。
结果
1.无血清培养基中脑肿瘤干细胞球的形成
使用传统含血清培养基进行培养的C6细胞,呈贴壁生长,接种到无血清培养基后1小时,呈单个细胞悬浮生长;接种后24小时,小的肿瘤干细胞球形成;接种48小时后形成典型的肿瘤干细胞球,细胞球呈圆形或卵圆形,大小不一,折光性较强,悬浮生长。
2.C6细胞系中脑肿瘤干细球的鉴定
含血清培养基中贴壁生长的C6细胞爬片进行的免疫荧光,可以查见散在分布的CD133与Nestin表达阳性的细胞。无血清培养基中形成的肿瘤干细胞球进行的免疫荧光显示呈CD133与Nestin的强阳性表达。
3.流式细胞术检测C6细胞系内肿瘤干细胞比例的结果
Nestin标记流式细胞术可检出C6细胞系内的脑肿瘤干细胞,10例样本内测得的所含脑肿瘤干细胞的比例在4%左右。统计学分析表明,所测10个样本中脑肿瘤干细胞的比例为4.02±0.04%,95%可信区间为3.93—4.10%。
4.移植致瘤实验
本实验所用20只裸鼠,均在注射部位形成了肿瘤,致瘤潜伏期为4—6天,肿瘤体积呈指数生长。移植致瘤后40天被处死时,瘤体最长径为1.7—2.0cm。
5.移植瘤内肿瘤干细胞的分布特点
(1)肿瘤组织的免疫组化与免疫荧光结果
以肿瘤干细胞的表面标志CD133与Nestin为指标,对肿瘤组织中的肿瘤干细胞进行鉴定和定位,可见典型的阳性分布,其在瘤体内的分布,主要有以下三种方式:散在的单个阳性细胞,巢状分布和条带状分布。这三种分布方式在瘤体内的分布也有一定的特点,即在肿瘤的中心,阳性细胞很少,即使有,也多是散在的单个细胞,并且多围绕在小血管的周围;在靠外侧的肿瘤基质内,可以看到巢状和条带状分布;而在肿瘤的周边或者边缘区域,多是带状分布,并且越接近肿瘤的边缘,阳性细胞的密度越大。在比较典型的视野中,可以找到呈分层排列的肿瘤干细胞带,在肿瘤边缘可见包裹有一层密集的肿瘤干细胞。
(2)流式细胞术检测自瘤体中心至瘤体边缘不同部位脑肿瘤干细胞比例的结果
肿瘤中心部位脑肿瘤干细胞比例为0.90±0.01%,而边缘部位比例为9.81±0.04%。经oneway-anova分析,结果显示,在由肿瘤中心至肿瘤边缘的组织中,肿瘤干细胞比例呈现线性增高趋势。
结论
1.C6细胞系内存在比例约为4.02%的CD133和Nestin表达阳性的CSCs。简化的无血清培养基可以作为肿瘤干细胞分离、培养的一种有效途径。
2.裸鼠皮下C6胶质瘤载瘤模型成瘤稳定,肿瘤生长良好。移植瘤内可查见脑肿瘤干细胞。
3.C6胶质瘤瘤体内,脑肿瘤干细胞的分布有三种方式,并呈现由中心向四周放射状逐渐增多的趋势,呈“爆炸式”分布特点。
Background:
Malignant glioma represents about 40%of all adult primary brain tumors.The prognosis of human malignant glioma remains poor with an overall 5-year survival rate of less than 3%and a median survival time of about 1 year for higher grade tumors such as glioblastoma.Gliomas present as diffuse tumors with invasion into normal brain tissue,but frequently recur or progress after surgery,radiation or chemotherapy as focal masses,suggesting that only a fraction of tumor cells is responsible for regrowth.Recently,there is accumulating evidence that malignant solid tumors may contain their own stem cells,termed cancer stem cells(CSCs).Despite their small quantity,this subpopulation within tumor possesses the ability of infinite proliferation and multipotency thus may play a crucial role in the initiation,progression and recurrence of cancer.The concept of cancer stem cells was intuitively suggested by clinical experience with leukemia,and also evident in teratocarcinomas and other solid tumors,such as breast cancer,prostate cancer and pancreas cancer. These studies have provided insights into the understanding of glioma cancer biology and paved the way for thefuture CSCs-targeted theropy.
CSCs in nervous system were first reported by Ignatova and Singh.They cultured glioma cells in serum-free neural stem cell medium and obtained a group of cells that were dramatically different from common glioma cells in growth conditions,cell markers,proliferating and differentiating capacity and tumor-initiating ability in vivo.Singh separated CD133- and CD133+ cells with immunomagnetic beads and planted them separately to the front lobe of 6-week-old nude mice.12-24weeks later,he found only 10~2 CD133+ cells could generate a tumor,but 10~5 CD133- cells only made a glial scar. Importantly,elevated expression of transporters that pump out chemotherapeutic agents and an increased capacity to repair DNA damage may also contribute to CSCs' ability to survive conventional modes of therapy.
Based on the ability of tumorgenesis and to survive conventional modes of therapy,Singh proposed a tumorgenic pattern based on CSCs.In the cancer stem cell model of tumors,there is a small subset of cancer cells,the cancer stem cells,which constitute a reservoir of self-sustaining cells with the exclusive ability to self-renew and maintain the tumor.These cancer stem cells have the capacity to both divide and expand the cancer stem cell pool and to differentiate into the heterogeneous nontumorigenic cancer cell types that in most cases appear to constitute the bulk of the tumor.If cancer stem cells are relatively refractory to therapies that have been developed to eradicate the rapidly dividing cells,they are unlikely to be curative and relapses would be expected.
The rat glioma cell line C6 is one of the widely used cell lines in the studies of glioma.However,it remained controversial about the culturing methods and fraction of CSCs in this cell line.For instance,Toru Kondo et al. found only 0.4%of C6 cells acted as cancer stem-like cells in a complicated serum-free DMEM medium.However,Xuesheng Zheng et al.pointed out that most C6 cells were cancer stem cells from clonal and population analyses. Given the difference of tumorigenic activity and resistance to treatment between CSCs and normal tumor cells,CSCs could act as a new target for cancer therapy.However there is a problem:where do they locate? Until now, there is little research on the location of CSCs in tumor bulk.
In our study,we cultured C6 cell line in a simplified serum-free neural stem cell medium and built up C6 subcutaneous xenografts model in nude mice.CD133 and Nestin marked immunohistochemistry and immunofluorescence confirmed the existence of CSCs both in C6 cell line and xenografts.Nestin marked flow cytometry demonstrated that 4.02%cells in C6 cell line presented as CSCs.Immunohistochemistry, immunofluorescence and flow cytometry were applied to detect the location and distribution characteristics of CSCs in subcutaneous xenografts.We proposed the hypothesis of "explosive distribution" of CSCs in tumor bulk and believed it helpful in explaining how CSCs participate in angiogenesis, tumor initiation,invasion and recurrence in a histological view.
Objective
1.To find a simple and effective method to culture,isolate and characterize cancer stem cells and confirm the fraction of cancer stem cells in C6 cell line.
2.To build up C6 subcutaneous xenografts model in nude mice and detect CSCs in C6 xenografts.
3.To study the distribution characteristics of brain cancer stem cells in C6 glioma.
Methods:
1.Tumor sphere culture
C6 glioma cells were firstly seeded in a 100ml culture flask containing 5ml 1640 culture solution,supplemented with 10%fetal bovine serum,100 U/ml penicillin G and 100μg/ml streptomycin.Cells which were in exponential phase of growth were collected and transplanted to a new culture flask with an equal volume of serum-free neural stem cell medium containing DMEM/F12,B27 supplement,recombinant human epidermal growth factor(rhEGF,20 ng/ml), and basic fibroblast growth factor(bFGF,20 ng/ml).Cells were incubated at 37℃with 95%air,5%CO_2,and 100%humidity.When the tumor spheres reached the size of approximately 100-200 cells per sphere,culture propagation was performed at the ratio of 1:2.
2.Characterization of cancer stem cells from C6 glioma cell line
CD133 or Nestin marked immunohistochemistry and immunofluorescence were used for characterization of cancer stem cells.Cells were plated onto poly-L-lysine coated glass coverslips in 1640 culture solution with 10%FBS or serum-free neural DMEM medium for 12 h.Cells were washed and then fixed in 4%paraformaldehyde and processed for immunofluorescence of CD133 or Nestin.The staining was detected under laser confocal scanning microscope
3.The detection of cancer stem cells in C6 glioma cell line
Nestin marked flow cytometry was used to detect the fraction of cancer stem cells in C6 cell line.Cells which were in exponential phase of growth were collected and cell density was adjusted to 1×10~7/ml.Each sample contained 100μl single-cell suspension.Ten C6 cell line samples were taken and incubated with anti-nestin antibody.Flow cytometry was then performed to test the fractions of CSCs in C6 cell line.
4.Xenografts assays
C6 cells(1×10~5 cells ) were implanted subcutaneously to both right and left thighs of 4-week-old female immunocompromised mice(n = 20).Xenograft tumors were harvested and examined 6 weeks after implantation.
5.The distribution characteristics of brain cancer stem cells in C6 glioma.
(1) Xenografts specimens were fixed in 4%paraformaldehyde,and sected into serial sections(5μm in thickness).For each specimen,20 slides were selected with uniform distribution along the tumor macroaxis. Immunohistochemistry and immunofluorescence were performed on the tissue slides for CD133 and Nestin.
(2) For any fresh tumor bulk,a section(0.5cm in thickness) was taken along its macroaxis.The section was equally divided into 5 parts of ring-like tissue from the center to the periphery and these 5 parts were incubated with anti-CD133 antibody.Flow cytometry was then performed to test the percentage of CSCs in each part.
Results:
1.Formation of tumor spheres in serum-free medium
When cultured in traditional serum-supplemented medium,C6 cells grew as single cells attached to the culture fask,while presented as single cells suspending in medium 1 hour after reseeded in serum-free neural stem cell medium,as small tumor spheres 24 hours later and as typical tumor spheres after 48 hours.The tumor spheres were globular,different in size,and floating in medium.
2.Expression of cancer stem cell markers
CD133 and Nestin marked immunofluorescence showed that:sparsly distributed CD133+ and Nestin+ cells could be seen in C6 cells cultured in serum-supplemented medium;in tumor soheres formed in serum-free medium, CD133 and Nestin were strongly abundantly expressed.
3.The fractions of CSCs in C6 cell line
Nestin marked flow cytometry demonstrated that about 4%cells in C6 cell line presented as CSCs.t tests demonstrated the fraction of CSCs in C6 cell line was 4.02±0.04%and 95%CI was 3.93-4.10%.
4.Formation of subcutaneous xenografts in nude mice
All the 20 experimental nude mice formed subcutaneous xenografts 4-6 days after injection.When nude mice were harvested 6 weeks after implantation,the maximum diameter of the tumor bulks was 1.7-2.0cm.
5.The distribution characteristics of brain cancer stem cells in C6 glioma.
(1) Immunohistochemistry and immunofluorescence results
CD133+ or Nestin+ cells could be found in xenografts of C6 glioma with three patterns of distribution:spot-like,nest-like,and zone-like.Interestingly, these three patterns of distribution were found in different parts of tumor bulks. At the center of a tumor bulk,very few CSCs could be found,which usually emerged in a spot-like pattern around capillaries.At the margin of a tumor bulk, CSCs displayed a zone-like pattern of distribution.In the area between the center and the margin,both nest-like and zone-like pattern could be seen,and the density of positivity showed a stepwise increase from the center to the margin.In some typical visual fields,CSCs displayed as cocenric citcles at the margin of the tumor bulk,forming a package of high density of CSCs for the tumor.
(2) Flow cytometry results
At the tumor centre,the fraction of CSCs was 0.90±0.01%.In contrast,at the margin of the tumor,the fraction was as high as 9.81±0.04%.Oneway-ANOVA analysis of the fractions showed that the fraction of CD133+ cells presented a stepwise increase from the center of tumor bulk to the margin.
Conclusions:
1.The fraction of CSCs in C6 cell line is 4.02%and the simplified serum-free neural stem cell medium can act as a simple and effective method for isolation and detection of CSCs to study the initiation and progression of human glioma.
2.C6 subcutaneous glioma in nude mice is a reliable animal model for studying glioma.
3.There are three distributing patterns of CSCs in C6 glioma:spot-like, nest-like and zone-like and their distribution shows a tendency of radioactive enhancement from the center to the margin of tumor bulk as "explosive distribution" pattern.
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