Isolation of glioma cancer stem cells in relation to histological grades in glioma specimens

Purpose

The existence of cancer stem cells (CSCs) in glioblastoma has been proposed. However, the unknown knowledge that is yet to be revealed is the presence of glioma CSCs (gCSCs) in correlation to each WHO grades of glioma. We approached this study with a hypothesis that specimens from high-grade gliomas would have higher isolation rate of gCSCs in comparison to those of lower-grade gliomas.

Methods

The glioma specimens were obtained from patients and underwent gliomasphere assay. The gliomaspheres were chosen to be analyzed with immunocytochemisty for surface markers. Then the selected gliomaspheres were exposed to neural differentiation conditions. Lastly, we made mouse orthotopic glioma models to examine the capacity of gliomagenesis.

Results

The gliomaspheres were formed in WHO grade IV (13 of 21) and III (two of nine) gliomas. Among them, WHO grade IV (11 of 13) and III (two of two) gliomaspheres showed similar surface markers to gCSCs and were capable of neural differentiation. Lastly, among the chosen cells, 10 of 11 WHO grade IV and two of two WHO grade III gliomaspheres were capable of gliomagenesis. Thus, overall, the rates of existence of gCSCs were more prominent in high-grade gliomas: 47.6 % (10 of 21) in WHO grade IV gliomas and 22.2 % (two of nine) in WHO grade III gliomas, whereas WHO grade II and I gliomas showed virtually no gCSCs.

Conclusions

This trend of stage-by-stage increase of gCSCs in gliomas showed statistical significance by chi-square test linear-by-linear association. We prove that the rates of existence of gCSCs increase proportionally as the WHO grades of gliomas rise.     

The pathological characteristics of glioma stem cell niches

Brain tumor stem cells (BTSC) are predicted to be critical drivers of tumor progression due to their self-renewal capacity and limitless proliferative potential. Recent studies suggest that stem cells are controlled by a particular microenvironment known as a “niche”. We therefore analysed human glioma tissues and found that the CD133⁺ and nestin⁺ niches are perivascularly localized in all glioma tissues. Furthermore, there is a positive correlation between the CD133⁺ niches and CD133⁺ blood vessels, which is similar to the correlation between the nestin⁺ niches and nestin⁺ blood vessels. We demonstrate that both CD133⁺ blood vessels and nestin⁺ blood vessels have an important role in maintaining the structure of the glioma stem cell niche. Moreover, the abundance of CD133⁺ niches and nestin⁺ niches increases significantly as tumor grade increases. These findings provide a new insight into the biology of BTSC and open a new perspective for targeted therapy against the brain tumors.     

Enhancer of Zeste 2 (EZH2) is up-regulated in malignant gliomas and in glioma stem-like cells

F. Orzan, S. Pellegatta, P. L. Poliani, F. Pisati, V. Caldera, F. Menghi, D. Kapetis, C. Marras, D. Schiffer and G. Finocchiaro (2011) Neuropathology and Applied Neurobiology 37, 381–394
Enhancer of Zeste 2 (EZH2) is up‐regulated in malignant gliomas and in glioma stem‐like cells Aims: Proteins of the Polycomb repressive complex 2 (PRC2) are epigenetic gene silencers and are involved in tumour development. Their oncogenic function might be associated with their role in stem cell maintenance. The histone methyltransferase Enhancer of Zeste 2 (EZH2) is a key member of PRC2 function: we have investigated its expression and function in gliomas. Methods: EZH2 expression was studied in grade II–IV gliomas and in glioma stem‐like cells (GSC) by quantitative PCR and immunohistochemistry. Effects of EZH2 down‐regulation were analysed by treating GSC with the histone deacetylase (HDAC) inhibitor suberoylanide hydroxamic acid (SAHA) and by shRNA. Results: DNA microarray analysis showed that EZH2 is highly expressed in murine and human GSC. Real‐time PCR on gliomas of different grade (n = 66) indicated that EZH2 is more expressed in glioblastoma multiforme (GBM) than in low‐grade gliomas (P = 0.0013). This was confirmed by immunohistochemistry on an independent set of 106 gliomas. Treatment with SAHA caused significant up‐regulation of PRC2 predicted target genes, GSC disruption and decreased expression of EZH2 and of the stem cell marker CD133. Inhibition of EZH2 expression by shRNA was associated with a significant decrease of glioma proliferation. Conclusion: The data suggest that EZH2 plays a role in glioma progression and encourage the therapeutic targeting of these malignancies by HDAC inhibitors.     

CD133鉴定胶质瘤干细胞血管周围壁龛的实验研究

目的 检测脑肿瘤干细胞(BTSC)标记物CD133、巢蛋白(Nestin)和增殖细胞核抗原(PCNA)在74例脑胶质瘤标本中的表达,探讨肿瘤干细胞生存的微环境一壁龛的组成、形态及其在脑肿瘤组织中的分布. 方法 选取安徽医科大学附属省立医院神经外科自2007年1月至2008年10月间手术切除的74例胶质瘤标本,按照WH02000年的神经系统肿瘤分类分级标准分为Ⅱ级22例(低级别组)、Ⅲ级27例和Ⅳ级25例(高级别组),采用免疫组织化学染色和免疫荧光双标法分别检测标本中CD133的表达及其与Nestin、PCNA的共表达情况.计算并比较不同级别胶质瘤组织CD133+细胞、CD133+血管和CD133+壁龛所占的百分比.并对CD133+血管和CD133+壁龛的表达进行相关性分析. 结果 CD133+细胞聚集于壁龛内生长,低级别组胶质瘤中CD133+壁龛阳性率较低.壁龛内增殖细胞较少,与相邻肇龛之间界限清晰,周围CD133+血管分布较少.高级别组胶质瘤中CD133+壁龛阳性率高,壁龛之间无明显界限,壁龛内细胞增殖活跃,周围可见丰富的CD133+血管分布:壁龛中除CD133+/Nestin+BTSC外,可见CD133+/Nestin-细胞、CD133/PCNA+细胞等亚群细胞;不同级别胶质瘤CD133+细胞、CD133+血管、CD133+壁龛百分比不同,且肿瘤级别越高,三者表达越高,差异有统计学意义(P<0.05).CD133+壁龛与CD133+血管的表达呈正相关(r=0.425,P=0.000). 结论 在脑胶质瘤组织中存在着由CD133+/Nestin+BTSC和一些亚群细胞组成的壁龛结构,CD133+血管对于壁龛结构的维持起着非常重要的作用.     

脑肿瘤干细胞的增殖活性与病理级别的相关性研究

目的检测增殖细胞核标记物Ki-67与脑肿瘤干细胞（BTSCs）标记物CD133、Nestin在60例脑胶质瘤组织中的表达,探讨BTSCs的增殖活性与病理级别的相关性。方法采用免疫组织化学SP法检测CD133、Nestin和Ki-67在60例胶质瘤组织中的表达;采用免疫荧光双染法检测Ki-67、CD133和Nestin之间的共表达情况。计算两种方法中CD133＋细胞、Nestin＋细胞和Ki-67＋细胞所占的百分率,并将Ki-67＋细胞与CD133＋细胞、Nestin＋细胞和肿瘤病理分级分别进行相关性分析。结果按照WHO 2000的神经系统肿瘤分类分级标准所有标本分为Ⅱ级18例,Ⅲ级23例,Ⅳ级19例,在不同的病理级别组中,CD133＋、Nestin＋和Ki-67＋细胞的表达有明显差异,并且免疫荧光双染相比免疫组化单染更能代表BTSCs的增殖活性研究。在免疫荧光共表达中,随着病理级别的升高,CD133＋/Ki-67＋细胞（F=30.668,P=0.000）或Nestin＋/Ki-67＋细胞（F=15.316,P=0.000）的百分比差异都有显著性,并且同时均与CD133＋/Nestin＋BTSCs的表达成正相关。结论Ki-67＋指标与肿瘤级别成正相关,可以作为预后判断的指标,同时与BTSCs标记物CD133、Nestin的表达之间也有明显的相关性,并且,免疫荧光双染得出的BTSCs的增殖活性与病理级别的相关性研究更有统计学意义。     

Cytotoxic CD4+ T cells are correlated with better prognosis in Han Chinese grade II and grade III glioma subjects and are suppressed by PD-1 signaling

Purpose: Malignant gliomas are the most common tumors in the central nervous system with a poor prognosis. Recently, CD4⁺ cytotoxic T cells (CTLs) are being increasingly recognized as possessing antitumor capacity. However, their presence, activity and regulation in glioma have not been investigated in detail. Methods: To examine this, 72 grade II and grade III Han Chinese glioma patients and 30 Han Chinese healthy controls were investigated. Results: We found that compared to healthy controls, glioma patients had significantly upregulated frequencies of circulating CD4⁺ CTLs, identified by the expression of granzyme A (GzmA), granzyme B (GzmB) and/or perforin. The stimulated CD4⁺ CTLs in grade II and grade III glioma patients also had less proliferative ability than those in healthy controls, a feature of suppression that progressed with tumor grade. The frequencies of GzmB-expressing circulating CD4⁺ CTLs were directly associated with prognosis. We hypothesized that the programed death 1 (PD-1)/PD-ligand 1 (L1) interaction possibly contributed to the suppression of CD4⁺ CTLs in grade II and grade III glioma, since an upregulation of PD-1 was observed on CD4⁺ CTLs in glioma compared to those in the healthy individuals. Blockade of the PD-1/PD-L1 interaction with neutralizing antibodies significantly increased the proliferation and granzyme or perforin production by CD4⁺ CTLs in grade II and grade III glioma patients. Conclusions: These data suggest that the CD4⁺ CTLs in grade II and grade III glioma patients contribute to antitumor immunity and could be suppressed by PD-1 signal transduction.     

Formation of new lymphatic vessels in glioma: An immunohistochemical analysis

We investigated the distribution and formation of new lymphatic vessels in gliomas. Specimens from seven glioma cases were analyzed by immunohistochemical staining for CD34, lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1), prospero-related homeobox 1 (Prox1), nestin, and hypoxia-inducible factor 1α (HIF-1α). Three types of vessels were observed in glioma specimens: LYVE-1⁺ lymphatic vessels, CD34⁺ blood vessels, and LYVE-1⁺/CD34⁺ blood vessels. Prox1⁺/LYVE-1⁺ cells were distributed in some lymphatic vessels as well as among vascular endothelial cells and glioma cells. Nestin⁺ cells were scattered throughout the gliomas, and some lymphatic cells also expressed nestin. HIF-1α⁺ Prox1⁺ cells were widely distributed within the glioma specimens. The present immunohistochemical analysis revealed upregulation of Prox1 and HIF-1α in some glioma tissues as well as the differentiation of nestin⁺ tumor stem cells into LYVE-1⁺ lymphatic vessels.     

人脑胶质瘤中CD133、SSEA-1、Nestin的表达和意义

目的：研究胶质瘤干细胞标志物CD133、SSEA-1和Nestin在胶质瘤组织中表达及其与病理分级的相关性;探讨三者在人脑胶质瘤的诊断及恶性程度判断中的临床意义。方法应用免疫组织化学方法检测54例脑胶质瘤组织和6例正常脑组织标本中CD133、SSEA-1及Nestin的表达。结果 CD133、SSEA-1和Nestin在胶质瘤组织中的阳性细胞平均表达率分别为25.38%、26.62%和22.39%,而在正常脑组织中均无表达。CD133、SSEA-1和Nestin阳性细胞率在胶质瘤各病理级别间比较,差异均有统计学意义,且三者的表达与胶质瘤病理级别呈正相关（P＜0.05）。SSEA-1与CD133、CD133与Nestin及SSEA-1与Nestin阳性细胞表达均呈正相关（P＜0.05）。结论检测CD133、SSEA-1、Nestin表达,有利于胶质瘤的诊断及恶性程度判断,并在胶质瘤的个性化综合治疗和预后评估发挥作用。     

CD133基因表达与人脑胶质瘤恶性程度相关性分析

目的探讨人脑胶质瘤组织中胶质瘤干细胞标志物CD133的表达与肿瘤恶性程度的关系。方法应用实时荧光定量PCR方法检测75例不同病理级别胶质瘤组织及4例正常脑组织中CD133基因的表达情况，并与肿瘤病理级别进行相关性分析；同时采用免疫组化法检测35例肿瘤组织和2例正常脑组织中CD133的表达情况，在蛋白表达水平予以验证。结果CD133在基因转录水平和蛋白表达水平具有良好的相关性。CD133在正常脑组织中未见表达，在各级别胶质瘤中均有表达，且差异有显著性（P〈0．01）；CD133表达量与肿瘤的恶性程度呈正相关（P〈0．01）。结论检测胶质瘤CD133表达水平有助于评价肿瘤的生物学行为，并为针对肿瘤干细胞的靶向治疗提供参考依据。     

脑胶质瘤干细胞鉴定与增殖及耐药特性的实验研究

目的 从胶质瘤组织中分离和培养出肿瘤干细胞,并初步探讨其生长特性. 方法 收集脑胶质瘤手术标本并获取细胞,用含有表皮生长因子(EGF)、白血病细胞抑制因子(LIE)和碱性成纤维生长因子(bFGF)的无血清培养液原代培养,再经免疫磁珠分离得到CD133+细胞.并用免疫细胞化学技术检测CD133、NSE和GFAP在细胞中的表达以鉴定CD133+细胞,比较不同恶性级别胶质瘤组织的CD133+细胞生长情况,并用CCK8法比较CD133+和CD133-细胞对替尼泊苷(VM-26)的耐药性. 结果 从胶质瘤组织中成功分选获得CD133+细胞,这些细胞能自我更新,增殖,并分化成NSE+和GFAP+的细胞.恶性度高的胶质瘤组织中CD133+细胞生长速度明显比低级别中的CD133+细胞快,且CD133+细胞在含有VM-26培养基中的存活细胞数显著多于CD133-细胞(P＜0.05). 结论 胶质瘤组织中存在肿瘤干细胞,这类细胞具有很强的耐药性,高恶性度胶质瘤组织中的CD133+细胞具有更强的增殖能力.     

IDH1/2 mutations in gliomas

The NADP+-dependent isocitrate dehydrogenases 1 and 2 (IDH1/2) catalyze the oxidative decarboxylation of isocitrate into α-ketoglutarate (α-KG). IDH1 and IDH2 mutations have been frequently found in some types of gliomas (low-grade diffuse gliomas WHO grade II, anaplastic gliomas WHO grade III, and secondary glioblastomas WHO grade IV), and have received significant attention because of their specificity to single codons. Since the unveiling of IDH1/2 mutations, many studies have investigated their clinical impact on gliomas. While the favorable influence of these mutations in high-grade gliomas has been well established, their prognostic impact on low-grade diffuse gliomas is much less clear. While the mechanism of IDH1/2 mutations in gliomagenesis remains to be clarified, its elucidation might lead to novel therapeutic strategies against gliomas.     

Circulating progenitor cells: a comparison of patients with glioblastoma or meningioma

Blood circulating endothelial cells and circulating hematopoietic progenitor cells (CPCs) are two cell populations that are thought to play important role in angiogenesis. In the present study, we investigated the role of CPCs in patients with brain tumors. We prospectively studied 19 brain tumor patients. Ten healthy individuals were used as controls. Variables that were analyzed included age, sex, Ki-67 index, symptom duration, tumor location, tumor size and preoperative Karnofsky performance status score (KPS). CPCs were determined as CD45^dim/CD34+/CD133+ in the peripheral blood. Twelve patients had glioblastoma (GBM), 1 patient had a grade II glioma and 6 patients had meningioma. Brain tumor patients had significantly higher CPC levels compared to healthy volunteers. Patients with gliomas had significantly higher CPC levels than patients with meningiomas. In GBM patients no correlation was found between CPC levels and sex, age, Ki-67 index, tumor location, size and KPS. Patients with CPC levels lower than 1,743 cells/ml had a higher progression-free survival but the difference was not statistically significant. Glioma patients had higher CPC levels compared to patients with meningiomas. Larger studies are obviously needed to verify the role of CPC levels in patients with brain tumors.     

Macrophage migration inhibitory factor (MIF) expression in human malignant gliomas contributes to immune escape and tumour progression

Macrophage migration inhibitory factor (MIF), which inhibits apoptosis and promotes angiogenesis, is expressed in cancers suppressing immune surveillance. Its biological role in human glioblastoma is, however, only poorly understood. We examined in-vivo expression of MIF in 166 gliomas and 23 normal control brains by immunohistochemistry. MIF immunoreactivity was enhanced in neoplastic astrocytes in WHO grade II glioma and increased significantly in higher tumour grades (III–IV). MIF expression was further assessed in 12 glioma cell lines in vitro. Quantitative RT-PCR showed that MIF mRNA expression was elevated up to 800-fold in malignant glioma cells compared with normal brain. This translated into high protein levels as assessed by immunoblotting of total cell lysates and by ELISA-based measurement of secreted MIF. Wild-type p53-retaining glioma cell lines expressed higher levels of MIF, which may be connected with the previously described role of MIF as a negative regulator of wild-type p53 signalling in tumour cells. Stable knockdown of MIF by shRNA in glioma cells significantly increased tumour cell susceptibility towards NK cell-mediated cytotoxicity. Furthermore, supernatant from mock-transfected cells, but not from MIF knockdown cells, induced downregulation of the activating immune receptor NKG2D on NK and CD8⁺ T cells. We thus propose that human glioma cell-derived MIF contributes to the immune escape of malignant gliomas by counteracting NK and cytotoxic T-cell-mediated tumour immune surveillance. Considering its further cell-intrinsic and extrinsic tumour-promoting effects and the availability of small molecule inhibitors, MIF seems to be a promising candidate for future glioma therapy.     

Presence of glioma stroma mesenchymal stem cells in a murine orthotopic glioma model

Purpose

High-grade gliomas are closely related to the mesenchymal phenotype which might be explained by unorthodox differentiation of glioma cancer stem cells (gCSCs). We reasoned that other non-neural stem cells, especially mesenchymal stem cells (MSCs), might play a role in expresssing mesenchymal phenotype of high-grade gliomas. Thus we hypothesized that cells resembling MSCs exist in glioma specimens.

Methods

We created a mouse (m) orthotopic glioma model using human gCSCs. Single-cell suspensions were isolated from glioma specimens and cultured according to the methods for mMSCs or gliomaspheres. These cells were analyzed by fluorescence-activated cell sorting (FACS) for surface markers associated with mMSCs or gCSCs. Glioma stroma (GS)-MSCs were exposed to mesenchymal differentiation conditions. To decide the location of GS-MSCs, sections of orthotopic glioma models were analyzed by immunofluorescent labeling.

Results

GS-MSCs were isolated which were morphologically similar to mMSCs. FACS analysis showed that the GS-MSCs had similar surface markers to mMSCs (stem cell antigen-1 [Sca-1]⁺, CD9⁺, CD45^?, CD11b^?, CD31^?, and nerve/glial antigen 2 [NG2]^?). GS-MSCs were capable of mesenchymal differentiation. Immunofluorescent labeling indicated that GS-MSCs are located around blood vessels, are distinct from endothelial cells, and have features that partially overlap with vascular pericytes.

Conclusions

Our results indicate that cells similar to mMSCs exist in glioma specimens. The GS-MSCs might be located around vessels, which suggests that GS-MSCs may provide the mesenchymal elements of the vascular niche. GS-MSCs may represent non-neural stem cells that act as an important source of mesenchymal elements, particularly during the growth of gliomas.     

Enhanced cell growth and tumorigenicity of rat glioma cells by stable expression of human CD133 through multiple molecular actions

CD133 (Prominin‐1/AC133) is generally treated as a cell surface marker found on multipotent stem cells and tumor stem‐like cells, and its biological function remains debated. Genetically modified rat glioma cell lines were generated by lentiviral gene delivery of human CD133 into rat C6 glioma cells (hCD133⁺‐C6) or by infection of C6 cells with control lentivirus (mock‐C6). Stable hCD133 expression promoted the self‐renewal ability of C6‐formed spheres with an increase in the expression of the stemness markers, Bmi‐1 and SOX2. Akt phosphorylation, Notch‐1 activation, and Notch‐1 target gene expression (Hes‐1, Hey1 and Hey2) were increased in hCD133⁺‐C6 when compared to mock‐C6. The inhibition of Akt phosphorylation, Notch‐1 activation, and Hes‐1 in hCD133⁺‐C6 cells effectively suppressed their clonogenic ability, indicating that these factors are involved in expanding the growth of hCD133⁺‐C6. An elevated expression of GTPase‐activating protein 27 (Arhgap27) was detected in hCD133⁺‐C6. A decline in the invasion of hCD133⁺‐C6 by knockdown of Arhgap27 expression indicated the critical role of Arhgap27 in promoting cell migration of hCD133⁺‐C6. In vivo study further showed that hCD133⁺‐C6 formed aggressive tumors in vivo compared to mock‐C6. Exposure of hCD133⁺‐C6 to arsenic trioxide not only reduced Akt phosphorylation, Notch‐1 activation and Hes‐1 expression in vitro, but also inhibited their tumorigenicity in vivo. The results show that C6 glioma cells with stable hCD133 expression enhanced their stemness properties with increased Notch‐1/Hes‐1 signaling, Akt activation, and Arhgap27 action, which contribute to increased cell proliferation and migration of hCD133⁺‐C6 in vitro, as well as progressive tumor formation in vivo.     

MiR-29a-Mediated CD133 Expression Contributes to Cisplatin Resistance in CD133<Superscript>+</Superscript> Glioblastoma Stem Cells

CD133 positive (CD133⁺) cells are cancer stem cells in glioblastoma that are associated with poor prognosis and resistance to radiotherapy. However, the role of CD133 in chemoresistance is inconclusive, although recent studies suggest that increased CD133 expression may lead to increased cisplatin resistance under certain circumstances. In this study, we further explored the mechanism underlying CD133-mediated cisplatin resistance in glioblastoma stem cells. We sorted human glioblastoma T98G and U87MG cells into CD133⁺ and CD133^? pools and measured apoptosis and CD133 expression levels in response to cisplatin treatment. We predicted candidate microRNAs that might target CD133 and assessed their levels in cisplatin-treated CD133⁺ cells. Finally, we overexpressed miR-29a in CD133⁺ cells and tested its effects in cisplatin-mediated apoptosis and survival of CD133⁺ tumor bearing mice receiving cisplatin treatment. We found that CD133⁺ glioblastoma stem cells showed more resistance to cisplatin treatment. Cisplatin increased CD133 expression by suppressing miR-29a levels. MiR-29a overexpression improved sensitivity of cisplatin in CD133⁺ cells and significantly suppressed tumor growth in CD133⁺ tumor bearing mice in response to cisplatin treatment. Our data show that miR-29a ameliorates CD133-mediated chemoresistance in glioblastoma stem cells, suggesting it as a potential therapeutic target for treating glioblastoma.