脑胶质细胞瘤肿瘤干细胞的分离、鉴定以及生物学特性研究
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摘要
[研究背景]
胶质瘤,尤其是胶质母细胞瘤,由于其恶性程度高,生长快,病程短,且对放化疗敏感性差,术后复发快,5年生存率仅为20%左右,因此一直被认为是神经外科治疗中最棘手的难治性肿瘤之一。由于肿瘤细胞具有的高度异质性,基因遗传学上的多样化,我们在认识胶质瘤的起源,细胞调节机制以及生长特性上遇到了很多困难。
肿瘤干细胞假说认为在肿瘤细胞中仅有一小部分(1%)的细胞具有自我更新、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。Bonnet等最早在急性髓性白血病中分离出白血病干细胞,而脑肿瘤的干细胞研究始于Ignatova,紧接着Singh,Galli等先后通过体内外实验在人的髓母细胞瘤,不同级别的胶质瘤中发现了肿瘤干细胞,他们认为只有这些细胞具有导致肿瘤形成的能力。上述的研究为我们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。
常用的鉴定脑肿瘤干细胞的方法主要有三种,即悬浮克隆球形成法,CD133免疫磁珠分选法和SP流式细胞仪筛选法。由于标本来源不同,方法不同使得脑肿瘤干细胞在比例构成、生物学特性等方面还存在很大的争议。Kondo以及国内的学者通过SP细胞流式筛选法和悬浮克隆球法在大鼠胶质瘤C6细胞系中发现了小部分(0.4%、1%)具有肿瘤干细胞样特性的细胞,国内的黄强等利用CD133免疫磁珠分选法也在体外长期培养的SHG—44胶质瘤细胞系中发现了肿瘤干细胞的存在。但是也有作者发现在体外长期培养的细胞系中不存在肿瘤干细胞,它们的基因与母代细胞发生了根本变化,因此不具有体内致瘤性。我们的初步观察发现大鼠胶质瘤C6细胞系生长极其旺盛与人原代胶质瘤细胞相比在克隆球形成能力以及致瘤能力等很多方面存在着差异,因此对两种细胞从脑肿瘤干细胞的概念和细胞生物学特性出发,作了进一步系统的肿瘤干细胞的研究。
[目的]
明确人胶质母细胞瘤以及体外长期培养的大鼠胶质瘤C6细胞系中的肿瘤干细胞及其特性;利用三种不同的分类方法获得肿瘤干细胞并比较它们在构成以及生物学特性上的差异,从而获得最理想的鉴定、分离方式;明确原代肿瘤干细胞和体外长期培养的细胞系中的肿瘤干细胞的异同,进一步阐明肿瘤干细胞在肿瘤发生,发展中的作用。
[方法]
第一部分:通过无血清培养基条件观察5例人原代胶质母细胞瘤细胞中悬浮克隆球的形成以及在含血清和无血清两种培养基中的生长转化情况;利用流式细胞仪比较原代肿瘤细胞与克隆球细胞中CD133阳性细胞的构成;利用单细胞克隆培养技术获得具有自我更新能力的细胞比例;利用细胞免疫荧光技术检测细胞的多向分化能力以及抗原的共表达情况;通过裸鼠致瘤实验验证人原代胶质母细胞瘤细胞中具有连续克隆形成能力的细胞的体内致瘤能力。
第二部分:通过血清和无血清培养基的相互转化,观察大鼠胶质瘤C6细胞系在两种培养基中的生长特性变化;利用流式细胞仪比较在不同培养条件下CD133阳性细胞以及分化细胞的构成变化;利用无血清条件下连续单细胞克隆培养初步获得具有自我更新能力的细胞比例;利用细胞免疫荧光和免疫组织化学技术检测多向分化能力以及抗原的共表达情况;通过裸鼠致瘤实验验证C6单细胞以及种植瘤细胞的肿瘤发生能力。
第三部分:通过CD133免疫磁珠分选技术将原代人胶质瘤细胞和大鼠C6细胞分为CD133阴性和阳性组;利用western blot技术和免疫荧光技术检测细胞纯度;利用Brdu染色检测细胞的增殖能力,利用克隆形成试验、免疫荧光技术、体内致瘤实验检测不同细胞组间肿瘤干细胞样细胞的构成以及生物学特性;利用western blot和细胞免疫组织化学技术监测分选细胞CD133表达变化情况;利用荧光显微镜观察Hoechst33342对细胞的毒性作用和ABCG2蛋白的表达。
[结果]
一、人脑胶质母细胞瘤肿瘤干细胞的分离、鉴定
1.5例人胶质母细胞瘤中有5.8-13.9%的细胞具有克隆形成能力,悬浮克隆球细胞中有20—34.5%左右的细胞具有再次形成克隆球的能力,同时这些细胞具有多向分化为表达GFAP/NSE阳性细胞的能力和体内致瘤的能力。
2.人胶质母细胞瘤克隆球细胞可以连续在血清和无血清条件下相互转化,同时发现有少量细胞会出现CD133与GFAP/NSE的共表达现象。
3.流式细胞仪和免疫组织化学结果显示克隆球中CD133阳性细胞比例要明显高于原代人胶质母细胞瘤实体细胞中CD133阳性细胞比例。
4.裸鼠致瘤实验发现人胶质母细胞瘤中只有克隆球形成能力的细胞具有致瘤能力;种植瘤中的子代细胞同样有母代肿瘤干细胞样细胞的存在。
二、大鼠C6胶质瘤细胞系肿瘤干细胞的分离、鉴定
5.C6细胞在血清和无血清条件下可以相互转化生长,血清培养条件不影响细胞在无血清培养基中的克隆球形成能力。
6.C6胶质瘤细胞系中有＞60%的细胞及其子代细胞可以在无血清培养基中形成克隆球,有＞90%的细胞及其子代细胞可以在含血清培养基中无限扩增。同时这些细胞具有胶质瘤样细胞,神经元样细胞的多向分化能力以及体内致瘤能力。
7.流式细胞仪和免疫组织化学结果显示长期血清培养基条件下C6细胞中约有30%的细胞为CD133阳性表达。在无血清培养条件下克隆球中的比例＞85%。
8.C6细胞在含血清培养基中有CD133与GFAP/NSE的共表达现象,而在克隆球中该现象明显减少。
9.裸鼠致瘤实验发现所有参与种植的C6单细胞克隆株均可成瘤,且种植瘤子代细胞同样具有肿瘤干细胞样特性,与母代细胞一致。
三、不同方法鉴定胶质瘤肿瘤干细胞的比较分析
10.大鼠胶质瘤C6细胞系中CD133阴性和阳性细胞均有肿瘤干细胞样细胞存在,两者在细胞增殖、克隆形成方式,致瘤速度等方面存在一定差异。
11.人胶质母细胞瘤细胞中CD133阴性细胞不具有肿瘤干细胞样特性,也并不是所有的CD133阳性细胞具有克隆形成能力。
12.在长期血清培养条件下,C6细胞中CD133阴性的细胞可以产生阳性细胞,而人胶质母细胞瘤细胞中未发现该现象。
13.Hoechst33342对大鼠C6细胞和人胶质母细胞瘤细胞均有毒性作用。
14.C6细胞系和人胶质母细胞瘤细胞中均发现有1%左右的细胞ABCG2蛋白表达阳性。而且仅该部分细胞在与Hoechst33342共同孵育后仍保留有肿瘤干细胞样特性,而其它细胞均丧失了自我更新能力。
[结论]
1.人胶质母细胞瘤和体外长期血清培养的大鼠胶质瘤C6细胞系中均有肿瘤干细胞样细胞的存在。
2.人胶质母细胞瘤和C6细胞系中的肿瘤干细胞样细胞在比例构成,自我更新能力、干细胞与分化抗原共表达、在血清与无血清两种条件下相互转化能力以及体内致瘤能力上均存在着较大的差异。
3.C6细胞系中同时存在CD133阴性以及CD133阳性的肿瘤干细胞样细胞,同时CD133阴性和阳性细胞可以相互转化。而在人胶质母细胞瘤中仅有CD133阳性细胞表现有自我更新、多向分化和连续致瘤的能力。
4.Hochest33342对人胶质母细胞瘤细胞和C6细胞均有毒性作用,会将那些缺乏ABC转运蛋白(如ABCG2蛋白)表达而没有能力把Hochest33342崩出体外但具有肿瘤干细胞特性的细胞排除在SP细胞范围之外。
5.利用肿瘤干细胞生物学特性分离、鉴定法获得的悬浮克隆球数与CD133免疫磁珠分选以及SP细胞的比例存在一定差异,因此寻找一种新的更能全面反映脑肿瘤干细胞的分子标记是今后研究的重点。
[BACKGROUND]
Gliomas, especially high grade glioblastomas (WHO grade IV), are characterized with highly malignancy, rapid growth, poor prognosis, resistance to radiotherapy and chemotherapy, and early relapse with a 5-year-survival rate of just 20%. Moreover, the tumor cells are highly heterogeneous and diversified in terms of their genetic backgrounds between individual cases. Therefore, the mechanism underlying glioma initiation, cellular regulation and growth characteristics are still remain poorly understood.
The cancer stem cell hypothesis implies that only a relatively small fraction of cells (1%), termed cancer stem cell (CSC), possess the stem cell ability such as indefinite proliferation, extensive self-renewal, and multi-linkage differentiation, and they are the true sources of tumor initiation, metastasis and relapse. Bonnet et al are the first who have isolated the leukemia CSC from acute myeloid leukaemia and the researches on brain CSC are pioneered by Ignatova et al, Singh et al and Galli et al who have identified brain CSC from human medulloblastoma and gliomas of different grades. These studies have provide insights into our understanding of glioma cancer biology and paved the way for the future CSC-targeted therapy.
The most widely adopted brain CSC identification methods are floating tumor spheres culture, CD133 magnetic activated cell sorting (MACS) and flow cytometry sorting (FCMS) for side population (SP). However, the differences in the specimens analyzed and specific identification methods applied usually lead to large controversy over the percentage and biological characteristics of the brain CSC isolated. Kondo et al and Li et al have identified a rare cell faction(0.4%, 1%) with CSC properties from long-term in vitro cultured rat C6 glioma cell line. Moreover, Huang et al also have proved the existence of cancer stem-like cells in SHG-44 glioma cell line. However, there are still some researches believe that there is no cancer stem cell in long-term in vitro cultured cell lines and they are dramatically different from their matched primary tumor in terms of genetic backgrounds. Our preliminary studies also showed that rat C6 cells are different from human primary glioblastoma cells in many respects, such as tumor sphere formation and tumorigenesis. Therefore, a systematic comparison was made between the identified CSCs from C6 glioma cell line and human glioblastomas.
[OBJECTIVES]
To identify the CSCs from primary human glioblastomas and rat C6 glioma cell line and explore their biological characteristics; Three different identification methods are used to identify the CSCs, comparing their composition and characteristics, and the optimal identification method is suggested; To determine the differences between the CSCs from primary human glioma and in vitro cultured glioma cell line and to further illuminate the role CSC plays in the initiation and development of glioma.
[METHODS]
Part 1: Both serum-containing and serum-free medium were used to culture the 5 human primary glioblastomas and the impact of alteration in culture medium on the growth pattern of glioblastoma cells was observed; FCMS was used to determine the percentage of CD133 positive cells in primary tumor cells and floating tumor spheres; Single-cell clonal culture was used to determine the percentage of cells with self-renewal capacity; Cytoimmunofluorescence and immunohistochemistry staining were used to analyze the multi-linkage differentiation capacity and co-expression of differentiations markers of the human glioblastoma cells. Xenograft tumor formation in nude mice was used to determine the in vivo tumorigenecity of the glioblastomacells.
Part 2: Both serum-containing and serum-free medium were used to culture the rat C6 glioma cell line and the impact of alteration in culture medium on the growth pattern of C6 cells was observed; FCMS was used to determine the percentage of CD133 positive cells and differentiated progenies of the C6 cells in different culture medium; Consecutive single-cell clonal culture in serum-free medium was used to determine the percentage of cells with self-renewal capacity; Cytoimmunofluorescence and immunohistochemistry staining were used to analyze the multi-linkage differentiation capacity and co-expression of differentiations markers of the C6 cells. Xenograft tumor formation in nude mice was used to determine the in vivo tumorigenecity of the C6 cells.
Part 3: CD133 MACS was used to isolate CD133 positive cell fractions from the CD133 negative cell fractions in both human primary glioblastoma and C6 line; Western Blot and immunofluorscence assay were used to determine the purity of the cell separated. Brdu staining was used to clear the proliferative capacity of the cells separated. Clonal analysis, immunofluorescence assay, and in vivo nude mice transplantation were used to determine the composition and biological characteristics of the CSCs in the separated cell fractions. Immunofluorescence assay was used to determine the toxicity of Hoechst 33342 to tumor cells and the expression of ABCG2.
[RESULTS]
一、Isolation and identification of cancer stem cells in human glioblastomas.
1. About 5.8-13.9% human primary glioblastoma cells were capable of forming tumor spheres and 20-34.5% of the tumor sphere cells were endowed with self-renewal capacity, multi-linkage differentiation and in vivo tumorigenecity.
2. Human primary glioblastoma cells could readily transform their growth pattern in serum-containing and serum-free medium and co-expression of CD133 and GFAP/NSE was found in few cells.
3. FCMS and immunohistochemistry assay showed CD133 positive rate in primary glioblastoma cells were much lower than in the tumor spheres they formed in serum-free medium.
4. Nude mice transplantation showed only human glioblastoma cells which could form tumor-spheres were tumorigenic and the xenograft tumor cells also contained CSCs.
二、Isolation and identification of cancer stem cells in C6 glioma cell line
5. C6 cells could transform its growth pattern in different culture medium and serum-containing medium did not alter its tumor-sphere formation capacity in serum-free medium.
6. >60% C6 cells and their progenies were capable of forming tumor spheres in serum-free medium and >90% C6 cells and their progenies were capable of extensive self-renewal in serum-containing medium. All these cells had characteristics of neuronal and glial linkage differentiation and in vivo tumorigenecity.
7. FCMS and immunohistochemistry assay showed a 30% CD133 positive rate in long-term serum-containing medium cultured C6 cells and >85% in serum-free meium.
8. C6 cells co-expressed CD133 and NSE/GFAP in serum-containing medium, which is rarely seen in serum-free medium.
9. Nude mice transplantation showed all C6 single cells were tumorigenic and the xenograft tumor cells had the similar CSC properties as their mother cells.
三、Identification of cancer stem cells with different methods
10. Both C6 CD133 positive and negative cell fractions contamed cancer stem-like cells with different proliferation, clone formation and tumorigenecity capacities.
11. CD133 negative cell fraction in human glioblastomas contained no CSC and not all CD133 positive cells were capable of forming clones.
12. CD133 negative cells in C6 cell line could generate CD133 positive cells in serum-containing medium but not the human glioblastoma cells.
13. Hoechst 33342 was toxic to both C6 and human glioblastoma cells.
14. C6 cell line and human glioblastoma cells contained 1% cells which were ABCG2 positive. Only this rare cell fraction still had CSC properties when they were incubated with Hoechst33342 for 2 hours.
[CONCLUTIONS]
1. Both human glioblastoma cells and C6 cells contained cancer stem-like cells.
2. Human glioblastoma cells differed from C6 cells in terms of their CSC percentage, self-renewal capacity, differentiation marker expression, transformation in serum-containing/free medium and in vivo tumorigenecity.
3. Both C6 CD133 positive and negative fractions contained cancer stem-like cells while only CD133 positive human glioblastoma cells had self-renewal capacity, multi-linkage differentiation and in vivo tumorigenecity.
4. Hochest 33342 was toxic to both human glioblastoma cells and C6 cells and thus exclude those cells that could not pump out Hochest 33342, for example ABCG2 negative cells, from being potential CSCs.
5. Identifying CSCs by their biological characteristics yield a much higher CSC percentage than by CD133 MACS or SP FCMS. Therefore finding a more reliable CSC marker should be the aim of future work.
胶质瘤,尤其是胶质母细胞瘤,由于其恶性程度高,生长快,病程短,且对放化疗敏感性差,术后复发快,5年生存率仅为20%左右,因此一直被认为是神经外科治疗中最棘手的难治性肿瘤之一。由于肿瘤细胞具有的高度异质性,基因遗传学上的多样化,我们在认识胶质瘤的起源,细胞调节机制以及生长特性上遇到了很多困难。
肿瘤干细胞假说认为在肿瘤细胞中仅有一小部分(1%)的细胞具有自我更新、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。Bonnet等最早在急性髓性白血病中分离出白血病干细胞,而脑肿瘤的干细胞研究始于Ignatova,紧接着Singh,Galli等先后通过体内外实验在人的髓母细胞瘤,不同级别的胶质瘤中发现了肿瘤干细胞,他们认为只有这些细胞具有导致肿瘤形成的能力。上述的研究为我们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。
常用的鉴定脑肿瘤干细胞的方法主要有三种,即悬浮克隆球形成法,CD133免疫磁珠分选法和SP流式细胞仪筛选法。由于标本来源不同,方法不同使得脑肿瘤干细胞在比例构成、生物学特性等方面还存在很大的争议。Kondo以及国内的学者通过SP细胞流式筛选法和悬浮克隆球法在大鼠胶质瘤C6细胞系中发现了小部分(0.4%、1%)具有肿瘤干细胞样特性的细胞,国内的黄强等利用CD133免疫磁珠分选法也在体外长期培养的SHG—44胶质瘤细胞系中发现了肿瘤干细胞的存在。但是也有作者发现在体外长期培养的细胞系中不存在肿瘤干细胞,它们的基因与母代细胞发生了根本变化,因此不具有体内致瘤性。我们的初步观察发现大鼠胶质瘤C6细胞系生长极其旺盛与人原代胶质瘤细胞相比在克隆球形成能力以及致瘤能力等很多方面存在着差异,因此对两种细胞从脑肿瘤干细胞的概念和细胞生物学特性出发,作了进一步系统的肿瘤干细胞的研究。
[目的]
明确人胶质母细胞瘤以及体外长期培养的大鼠胶质瘤C6细胞系中的肿瘤干细胞及其特性;利用三种不同的分类方法获得肿瘤干细胞并比较它们在构成以及生物学特性上的差异,从而获得最理想的鉴定、分离方式;明确原代肿瘤干细胞和体外长期培养的细胞系中的肿瘤干细胞的异同,进一步阐明肿瘤干细胞在肿瘤发生,发展中的作用。
[方法]
第一部分:通过无血清培养基条件观察5例人原代胶质母细胞瘤细胞中悬浮克隆球的形成以及在含血清和无血清两种培养基中的生长转化情况;利用流式细胞仪比较原代肿瘤细胞与克隆球细胞中CD133阳性细胞的构成;利用单细胞克隆培养技术获得具有自我更新能力的细胞比例;利用细胞免疫荧光技术检测细胞的多向分化能力以及抗原的共表达情况;通过裸鼠致瘤实验验证人原代胶质母细胞瘤细胞中具有连续克隆形成能力的细胞的体内致瘤能力。
第二部分:通过血清和无血清培养基的相互转化,观察大鼠胶质瘤C6细胞系在两种培养基中的生长特性变化;利用流式细胞仪比较在不同培养条件下CD133阳性细胞以及分化细胞的构成变化;利用无血清条件下连续单细胞克隆培养初步获得具有自我更新能力的细胞比例;利用细胞免疫荧光和免疫组织化学技术检测多向分化能力以及抗原的共表达情况;通过裸鼠致瘤实验验证C6单细胞以及种植瘤细胞的肿瘤发生能力。
第三部分:通过CD133免疫磁珠分选技术将原代人胶质瘤细胞和大鼠C6细胞分为CD133阴性和阳性组;利用western blot技术和免疫荧光技术检测细胞纯度;利用Brdu染色检测细胞的增殖能力,利用克隆形成试验、免疫荧光技术、体内致瘤实验检测不同细胞组间肿瘤干细胞样细胞的构成以及生物学特性;利用western blot和细胞免疫组织化学技术监测分选细胞CD133表达变化情况;利用荧光显微镜观察Hoechst33342对细胞的毒性作用和ABCG2蛋白的表达。
[结果]
一、人脑胶质母细胞瘤肿瘤干细胞的分离、鉴定
1.5例人胶质母细胞瘤中有5.8-13.9%的细胞具有克隆形成能力,悬浮克隆球细胞中有20—34.5%左右的细胞具有再次形成克隆球的能力,同时这些细胞具有多向分化为表达GFAP/NSE阳性细胞的能力和体内致瘤的能力。
2.人胶质母细胞瘤克隆球细胞可以连续在血清和无血清条件下相互转化,同时发现有少量细胞会出现CD133与GFAP/NSE的共表达现象。
3.流式细胞仪和免疫组织化学结果显示克隆球中CD133阳性细胞比例要明显高于原代人胶质母细胞瘤实体细胞中CD133阳性细胞比例。
4.裸鼠致瘤实验发现人胶质母细胞瘤中只有克隆球形成能力的细胞具有致瘤能力;种植瘤中的子代细胞同样有母代肿瘤干细胞样细胞的存在。
二、大鼠C6胶质瘤细胞系肿瘤干细胞的分离、鉴定
5.C6细胞在血清和无血清条件下可以相互转化生长,血清培养条件不影响细胞在无血清培养基中的克隆球形成能力。
6.C6胶质瘤细胞系中有＞60%的细胞及其子代细胞可以在无血清培养基中形成克隆球,有＞90%的细胞及其子代细胞可以在含血清培养基中无限扩增。同时这些细胞具有胶质瘤样细胞,神经元样细胞的多向分化能力以及体内致瘤能力。
7.流式细胞仪和免疫组织化学结果显示长期血清培养基条件下C6细胞中约有30%的细胞为CD133阳性表达。在无血清培养条件下克隆球中的比例＞85%。
8.C6细胞在含血清培养基中有CD133与GFAP/NSE的共表达现象,而在克隆球中该现象明显减少。
9.裸鼠致瘤实验发现所有参与种植的C6单细胞克隆株均可成瘤,且种植瘤子代细胞同样具有肿瘤干细胞样特性,与母代细胞一致。
三、不同方法鉴定胶质瘤肿瘤干细胞的比较分析
10.大鼠胶质瘤C6细胞系中CD133阴性和阳性细胞均有肿瘤干细胞样细胞存在,两者在细胞增殖、克隆形成方式,致瘤速度等方面存在一定差异。
11.人胶质母细胞瘤细胞中CD133阴性细胞不具有肿瘤干细胞样特性,也并不是所有的CD133阳性细胞具有克隆形成能力。
12.在长期血清培养条件下,C6细胞中CD133阴性的细胞可以产生阳性细胞,而人胶质母细胞瘤细胞中未发现该现象。
13.Hoechst33342对大鼠C6细胞和人胶质母细胞瘤细胞均有毒性作用。
14.C6细胞系和人胶质母细胞瘤细胞中均发现有1%左右的细胞ABCG2蛋白表达阳性。而且仅该部分细胞在与Hoechst33342共同孵育后仍保留有肿瘤干细胞样特性,而其它细胞均丧失了自我更新能力。
[结论]
1.人胶质母细胞瘤和体外长期血清培养的大鼠胶质瘤C6细胞系中均有肿瘤干细胞样细胞的存在。
2.人胶质母细胞瘤和C6细胞系中的肿瘤干细胞样细胞在比例构成,自我更新能力、干细胞与分化抗原共表达、在血清与无血清两种条件下相互转化能力以及体内致瘤能力上均存在着较大的差异。
3.C6细胞系中同时存在CD133阴性以及CD133阳性的肿瘤干细胞样细胞,同时CD133阴性和阳性细胞可以相互转化。而在人胶质母细胞瘤中仅有CD133阳性细胞表现有自我更新、多向分化和连续致瘤的能力。
4.Hochest33342对人胶质母细胞瘤细胞和C6细胞均有毒性作用,会将那些缺乏ABC转运蛋白(如ABCG2蛋白)表达而没有能力把Hochest33342崩出体外但具有肿瘤干细胞特性的细胞排除在SP细胞范围之外。
5.利用肿瘤干细胞生物学特性分离、鉴定法获得的悬浮克隆球数与CD133免疫磁珠分选以及SP细胞的比例存在一定差异,因此寻找一种新的更能全面反映脑肿瘤干细胞的分子标记是今后研究的重点。
[BACKGROUND]
Gliomas, especially high grade glioblastomas (WHO grade IV), are characterized with highly malignancy, rapid growth, poor prognosis, resistance to radiotherapy and chemotherapy, and early relapse with a 5-year-survival rate of just 20%. Moreover, the tumor cells are highly heterogeneous and diversified in terms of their genetic backgrounds between individual cases. Therefore, the mechanism underlying glioma initiation, cellular regulation and growth characteristics are still remain poorly understood.
The cancer stem cell hypothesis implies that only a relatively small fraction of cells (1%), termed cancer stem cell (CSC), possess the stem cell ability such as indefinite proliferation, extensive self-renewal, and multi-linkage differentiation, and they are the true sources of tumor initiation, metastasis and relapse. Bonnet et al are the first who have isolated the leukemia CSC from acute myeloid leukaemia and the researches on brain CSC are pioneered by Ignatova et al, Singh et al and Galli et al who have identified brain CSC from human medulloblastoma and gliomas of different grades. These studies have provide insights into our understanding of glioma cancer biology and paved the way for the future CSC-targeted therapy.
The most widely adopted brain CSC identification methods are floating tumor spheres culture, CD133 magnetic activated cell sorting (MACS) and flow cytometry sorting (FCMS) for side population (SP). However, the differences in the specimens analyzed and specific identification methods applied usually lead to large controversy over the percentage and biological characteristics of the brain CSC isolated. Kondo et al and Li et al have identified a rare cell faction(0.4%, 1%) with CSC properties from long-term in vitro cultured rat C6 glioma cell line. Moreover, Huang et al also have proved the existence of cancer stem-like cells in SHG-44 glioma cell line. However, there are still some researches believe that there is no cancer stem cell in long-term in vitro cultured cell lines and they are dramatically different from their matched primary tumor in terms of genetic backgrounds. Our preliminary studies also showed that rat C6 cells are different from human primary glioblastoma cells in many respects, such as tumor sphere formation and tumorigenesis. Therefore, a systematic comparison was made between the identified CSCs from C6 glioma cell line and human glioblastomas.
[OBJECTIVES]
To identify the CSCs from primary human glioblastomas and rat C6 glioma cell line and explore their biological characteristics; Three different identification methods are used to identify the CSCs, comparing their composition and characteristics, and the optimal identification method is suggested; To determine the differences between the CSCs from primary human glioma and in vitro cultured glioma cell line and to further illuminate the role CSC plays in the initiation and development of glioma.
[METHODS]
Part 1: Both serum-containing and serum-free medium were used to culture the 5 human primary glioblastomas and the impact of alteration in culture medium on the growth pattern of glioblastoma cells was observed; FCMS was used to determine the percentage of CD133 positive cells in primary tumor cells and floating tumor spheres; Single-cell clonal culture was used to determine the percentage of cells with self-renewal capacity; Cytoimmunofluorescence and immunohistochemistry staining were used to analyze the multi-linkage differentiation capacity and co-expression of differentiations markers of the human glioblastoma cells. Xenograft tumor formation in nude mice was used to determine the in vivo tumorigenecity of the glioblastomacells.
Part 2: Both serum-containing and serum-free medium were used to culture the rat C6 glioma cell line and the impact of alteration in culture medium on the growth pattern of C6 cells was observed; FCMS was used to determine the percentage of CD133 positive cells and differentiated progenies of the C6 cells in different culture medium; Consecutive single-cell clonal culture in serum-free medium was used to determine the percentage of cells with self-renewal capacity; Cytoimmunofluorescence and immunohistochemistry staining were used to analyze the multi-linkage differentiation capacity and co-expression of differentiations markers of the C6 cells. Xenograft tumor formation in nude mice was used to determine the in vivo tumorigenecity of the C6 cells.
Part 3: CD133 MACS was used to isolate CD133 positive cell fractions from the CD133 negative cell fractions in both human primary glioblastoma and C6 line; Western Blot and immunofluorscence assay were used to determine the purity of the cell separated. Brdu staining was used to clear the proliferative capacity of the cells separated. Clonal analysis, immunofluorescence assay, and in vivo nude mice transplantation were used to determine the composition and biological characteristics of the CSCs in the separated cell fractions. Immunofluorescence assay was used to determine the toxicity of Hoechst 33342 to tumor cells and the expression of ABCG2.
[RESULTS]
一、Isolation and identification of cancer stem cells in human glioblastomas.
1. About 5.8-13.9% human primary glioblastoma cells were capable of forming tumor spheres and 20-34.5% of the tumor sphere cells were endowed with self-renewal capacity, multi-linkage differentiation and in vivo tumorigenecity.
2. Human primary glioblastoma cells could readily transform their growth pattern in serum-containing and serum-free medium and co-expression of CD133 and GFAP/NSE was found in few cells.
3. FCMS and immunohistochemistry assay showed CD133 positive rate in primary glioblastoma cells were much lower than in the tumor spheres they formed in serum-free medium.
4. Nude mice transplantation showed only human glioblastoma cells which could form tumor-spheres were tumorigenic and the xenograft tumor cells also contained CSCs.
二、Isolation and identification of cancer stem cells in C6 glioma cell line
5. C6 cells could transform its growth pattern in different culture medium and serum-containing medium did not alter its tumor-sphere formation capacity in serum-free medium.
6. >60% C6 cells and their progenies were capable of forming tumor spheres in serum-free medium and >90% C6 cells and their progenies were capable of extensive self-renewal in serum-containing medium. All these cells had characteristics of neuronal and glial linkage differentiation and in vivo tumorigenecity.
7. FCMS and immunohistochemistry assay showed a 30% CD133 positive rate in long-term serum-containing medium cultured C6 cells and >85% in serum-free meium.
8. C6 cells co-expressed CD133 and NSE/GFAP in serum-containing medium, which is rarely seen in serum-free medium.
9. Nude mice transplantation showed all C6 single cells were tumorigenic and the xenograft tumor cells had the similar CSC properties as their mother cells.
三、Identification of cancer stem cells with different methods
10. Both C6 CD133 positive and negative cell fractions contamed cancer stem-like cells with different proliferation, clone formation and tumorigenecity capacities.
11. CD133 negative cell fraction in human glioblastomas contained no CSC and not all CD133 positive cells were capable of forming clones.
12. CD133 negative cells in C6 cell line could generate CD133 positive cells in serum-containing medium but not the human glioblastoma cells.
13. Hoechst 33342 was toxic to both C6 and human glioblastoma cells.
14. C6 cell line and human glioblastoma cells contained 1% cells which were ABCG2 positive. Only this rare cell fraction still had CSC properties when they were incubated with Hoechst33342 for 2 hours.
[CONCLUTIONS]
1. Both human glioblastoma cells and C6 cells contained cancer stem-like cells.
2. Human glioblastoma cells differed from C6 cells in terms of their CSC percentage, self-renewal capacity, differentiation marker expression, transformation in serum-containing/free medium and in vivo tumorigenecity.
3. Both C6 CD133 positive and negative fractions contained cancer stem-like cells while only CD133 positive human glioblastoma cells had self-renewal capacity, multi-linkage differentiation and in vivo tumorigenecity.
4. Hochest 33342 was toxic to both human glioblastoma cells and C6 cells and thus exclude those cells that could not pump out Hochest 33342, for example ABCG2 negative cells, from being potential CSCs.
5. Identifying CSCs by their biological characteristics yield a much higher CSC percentage than by CD133 MACS or SP FCMS. Therefore finding a more reliable CSC marker should be the aim of future work.
引文
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