Presence of pluripotent CD133+ cells correlates with malignancy of gliomas

BackgroundPresence of CD133⁺ cancer stem cells has been demonstrated within glioblastoma multiforme (GBM), the most malignant phenotype of gliomas (WHO grade IV). Since GBM frequently develops from low grade gliomas (WHO grade II) we assessed a possible qualitative or quantitative correlation of CD133⁺ cells and glioma grade to get new insights in gliomagenesis.ResultsThe amount of CD133⁺ cells within the bulk tumor mass, analyzed by immunostaining and Western blotting, showed a clear quantitative correlation with glioma grade (WHO° II, III and IV). Most of CD133⁺ cells were arranged in clusters frequently associated to tumor vessels. Protein analysis revealed high cellular coexpression of CD133 with Musashi-I but not CD34 indicating a neural, i.e. local origin of these cells. In vitro, no differences in stem cell properties concerning self-renewal and multi-lineage differentiation have been found for CD133⁺ cells isolated from gliomas of different grades.ConclusionsThese findings indicate a solely quantitative correlation of glioma grade with the presence of neural CD133⁺ cells within tumors supporting the concept of a CD133⁺ stem cell dependent gliomagenesis.     

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.     

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.     

The effects of tibolone on the human primary glioblastoma multiforme cell culture and the rat C6 glioma model

Abstract

Objectives: In the light of recent advances in tumor biology and genetics, we hypothesized that tibolone, an estrogen receptor agonist, may have antiproliferative effects on primary human glioblastoma cells and rat C6 malignant glioma cell lines. We thought that tibolone should exert its antiproliferative effects by augmenting glial cell differentiation through the naive, nonhypermethylated estrogen receptors in the glioma cells.

Methods: Human primary glioblastoma multiforme (GBM) cells were acquired perioperatively from ten patients aged between 45 and 69 years, diagnosed clinically and radiologically with GBM. The diagnosis was confirmed using immunohistochemical assays. Human GBM and rat C6 malignant glioma cells were cultivated in vitro to obtain monolayer cell cultures. Tibolone was then applied to these cultures in wells, each containing 500,000 tumor cells.

Results: Tibolone significantly decreased the number of human GBM cells at the concentrations of 10 and 100 mg/ml. For tibolone, a strong dose-dependent correlation in tumor inhibition was found (p=0.001). This antiproliferative effect of tibolone in human GBM cells was not observed in rat C6 malignant glioma cells. Tibolone demonstrated differential effects on human GBM and rat C6 glioma cells.

Discussion: In vitro antiproliferative effects of tibolone on human GBM need to be investigated further in in vivo works.     

Celecoxib enhances radiosensitivity in medulloblastoma-derived CD133-positive cells

Objects

Cyclooxygenase-2 (COX-2), the enzyme that converts arachidonic acid to prostaglandins, is overexpressed in a variety of tumors, including medulloblastoma (MB). CD133, a transmembrane glycoprotein, has been suggested as a marker for cancer stem cells in brain tumors. The aim of the present study was to investigate the role of celecoxib, a selective COX-2 inhibitor, in enhancing the effects of ionizing radiotherapy (IR) on medulloblastoma-derived CD133-positive cells (MB-CD133⁺).

Materials and methods

MB-CD133⁺ were isolated from two medulloblastoma cell lines (Daoy and UW228). Then, they were treated with celecoxib in different concentrations, and cell viability was assessed. The assays of cell survival, soft agar, radiosensitivity, colony formation, and apoptotic activity in MB-CD133⁺ treated with celecoxib alone, radiation alone, or celecoxib combined with radiation were further evaluated.

Results

MB-CD133⁺ showed the self-renew ability to form sphere bodies in vitro and regenerate tumors in vivo. The levels of COX-2 mRNA and protein in MB-CD133⁺ were significantly higher than those in MB-CD133^?. The treatment of 30 μM celecoxib could effectively inhibit the abilities of cell proliferation and colony formation and increase IR-induced apoptosis in treated MB-CD133⁺. Furthermore, in vivo study demonstrated that celecoxib significantly enhanced radiosensitivity in MB-CD133⁺-transplanted grafts. Notably, xenotransplantation analysis demonstrated that the treatment of celecoxib could further suppress the expressions of angiogenic and stemnness-related genes in treated MB-CD133⁺ grafts of SCID mice.

Conclusions

Celecoxib presents the potential of radiosensitizing effect in MB-derived cancer stem cells. Therefore, it should be warranted in future trials to enhance the radiotherapeutic effects in MB patients.     

High expression of RFX4 is associated with tumor progression and poor prognosis in patients with glioblastoma

Abstract

Aim: Glioma stem cells (GSCs) have been shown to contribute to tumor development and recurrence, therapeutic resistance, and cellular heterogeneity of glioblastoma multiforme (GBM). Recently, it has been reported that GSCs lose their self-renewal ability and tumorigenic potential upon differentiation. In this study, we identified Regulatory Factor X4 (RFX4) gene to regulate GSCs’ survival and self-renewal activity in the GBM patients samples.

Materials and methods: We utilized public datasets from the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Ivy Glioblastoma Atlas Project, and The Human Protein Atlas to screen candidate genes which are associated with the development of GBM and poor patients survival. Small hairpin RNA (shRNA) lentivirus was applied to knockdown RFX4 gene in GSCs.

Results: We found that RFX4 mRNA expression among the RFX family was particularly reduced during GSC differentiation. RT-qPCR analysis revealed significant downregulation of RFX4 and stem cell markers (CD15 and CD133) mRNA expressions in primary human GBM-derived GSCs cultured under serum condition. Consistently, GSCs showed significantly elevated RFX4 mRNA expression levels compared to normal astrocytes, NHA, whereas glioma cells did not. Furthermore, analysis of the TCGA data set revealed that RFX4 is highly expressed in GBM, and contributes to the lowering of patient survival. Depletion of RFX4 using shRNA lentivirus in patient GBM-derived GSCs decreased neurosphere formation and cell viability.

Conclusion: These results suggest that RFX4 is a potential risk factor for maintaining the stemness of GSCs and making glioma more malignant, and thus, could be a promising target of GBM treatment.     

Inhibition of phosphorylated STAT3 by cucurbitacin I enhances chemoradiosensitivity in medulloblastoma-derived cancer stem cells

Introduction

CD133 (PROM1) is a potential marker for cancer stem cells (CSCs), including those found in brain tumors. Recently, medulloblastoma (MB)-derived CD133-positive cells were found to have CSC-like properties and were proposed to be important contributors to tumorigenicity, cancer progression, and chemoradioresistance. However, the biomolecular pathways and therapeutic targets specific to MB-derived CSCs remain unresolved.

Materials and methods

In the present study, we isolated CD133⁺ cells from MB cell lines and determined that they showed increased tumorigenicity, radioresistance, and higher expression of both embryonic stem cell-related and drug resistance-related genes compared to CD133^? cells. Bioinformatics analysis suggested that the STAT3 pathway might be important in MB and CD133⁺ cells. To evaluate the effects of inhibiting the STAT3 pathway, MB-derived CD133^+/? cells were treated with the potent STAT3 inhibitor, cucurbitacin I. Treatment with cucurbitacin I significantly suppressed the CSC-like properties and stemness gene signature of MB-derived CD133⁺ cells. Furthermore, cucurbitacin I treatment increased the apoptotic sensitivity of MB-derived CD133⁺ cells to radiation and chemotherapeutic drugs. Notably, cucurbitacin I demonstrated synergistic effects with ionizing radiation to inhibit tumorigenicity in MB-CD133⁺-inoculated mice.

Results

These results indicate that the STAT3 pathway plays a key role in mediating CSC properties in MB-derived CD133⁺ cells. Targeting STAT3 with cucurbitacin I may therefore represent a novel therapeutic approach for treating malignant brain tumors.     

Vascular endothelial growth factor enhances migration of astroglial cells in subventricular zone neurosphere cultures

Vascular endothelial growth factor (VEGF) is an endothelial and neuronal survival factor and a mitogen for endothelial cells and astrocytes in both explant and in vivo injury models. In the CNS, interplay between the vasculature and neural stem progenitor (NSP) cells is required for the maintenance of angiogenic/neurogenic coordination in the germinal niche in the subventricular zone (SVZ) of the lateral ventricle. Using an in vitro SVZ neurosphere (NS) model, this study aimed to understand the direct effects of VEGF and its receptor signaling on neonatal NSP cell growth and migration. Our data indicate that VEGF administration, compared with untreated or brain‐derived neurotrophic factor‐treated NS, significantly increased growth and migratory capacity of glial fibrillary acidic protein (GFAP)⁺ and nestin⁺ NSP cells and in secondary cultures induced a stellate astrocyte morphology. Blockade of both VEGF, which is normally expressed in some NS cells, and its flt‐1 receptor signaling by neutralizing antibodies caused morphological changes specifically in GFAP⁺ cells and disrupted sphere formation and outward migration. These cells did not appear as conventional polygonal astrocytes; their process growth was severely restricted, and overall migration was reduced by up to 76% of control cultures. Blockade of VEGF's flk‐1 receptor reduced VEGF expression and caused a lesser, though significant, decrease (29%) in NSP (GFAP⁺) cell migration. The results show that both VEGF and, in particular, flt‐1 receptor signaling are critical to the proper configuration of the NS and its subsequent development. VEGF is also an important growth and migratory factor particularly for GFAP⁺ cells developing in SVZ‐derived NS in culture. © 2009 Wiley‐Liss, Inc.     

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.     

Isolation, cultivation and identification of brain glioma stem cells by magnetic bead sorting

This study describes a detailed process for obtaining brain glioma stem cells from freshly dissected human brain glioma samples using an immunomagnetic bead technique combined with serum-free media pressure screening.Furthermore,the proliferation,differentiation and self-renewal biological features of brain glioma stem cells were identified.Results showed that a small number of CD133 positive tumor cells isolated from brain glioma samples survived as a cell suspension in serum-free media and proliferated.Subcultured CD133 positive cells maintained a potent self-renewal and proliferative ability,and expressed the stem cell-specific markers CD133 and nestin.After incubation with fetal bovine serum,the number of glial fibrillary acidic protein and microtubule associated protein 2 positive cells increased significantly,indicating that the cultured brain glioma stem cells can differentiate into astrocytes and neurons.Western blot analysis showed that tumor suppressor phosphatase and tensin homolog was highly expressed in tumor spheres compared with the differentiated tumor cells.These experimental findings indicate that the immunomagnetic beads technique is a useful method to obtain brain glioma stem cells from human brain tumors.     

Mobilization of CD133+CD34− cells in healthy individuals following whole‐body acupuncture for spinal cord injuries

Acupuncture can alleviate symptoms of spinal cord injuries (SCI). The underlying mechanism, however, is unknown. We hypothesized that stem cells could be mobilized by acupuncture. Therefore, we enrolled 14 healthy study participants using acupuncture points for the treatment of SCI. The frequency of CD133 and CD34 cells in peripheral blood and the serum concentrations of matrix metalloproteinase (MMP)‐9, brain‐derived neurotrophic factor (BDNF), nerve growth factor (NGF), and interleukin‐6 were determined before and after acupuncture (<1 hr, 24 hr, and 48 hr). CD133⁺34^? cells were doubled 48 hr after acupuncture, with concomitant decreases in BDNF and MMP‐9 levels. Interleukin‐6 remained below detectable levels, eliminating a stress‐induced cell release. Individuals acupunctured on control counterpoints showed no changes in CD133⁺ cells. Our results indicate that acupuncture for SCI can mobilize human CD133⁺34^? cells. © 2009 Wiley‐Liss, Inc.     

人脑胶质瘤干细胞增殖和分化过程中NOTCH-1信号通路的调控

背景：研究发现NOTCH-1信号通路在神经干细胞或神经前体细胞的自我更新、增殖及分化中起重要作用。 目的：探讨NOTCH-1信号通路在人脑胶质瘤干细胞增殖和分化过程中的调控作用。 设计、时间及地点：开放性实验,于2005-01/2007-03在解放军第三军医大学新桥医院完成。 材料：人脑胶质瘤组织、正常成人脑组织由解放军第三军医大学新桥医院神经外科提供。U251胶质瘤细胞株由解放军第三军医大学新桥医院神经外科吕胜青副教授惠赠;CHG-5胶质瘤细胞株由解放军第三军医大学西南医院病理研究所卞修武教授、姚晓红博士惠赠。 方法：取人脑胶质瘤组织、正常成人脑组织、U251及CHG-5胶质瘤细胞株,采用免疫磁珠法分选获得CD133+脑胶质瘤干细胞,加入DMEM/F12无血清培养基进行增殖培养,形成细胞克隆后,加入含体积分数为10%胎牛血清的培养液,2 h后行抗CD133和抗巢蛋白免疫荧光双标染色。 主要观察指标：人脑胶质瘤干细胞的生长和鉴定,采用WST-8法、免疫组化实验、流式细胞仪、免疫荧光双标实验检测NOTCH-1信号通路蛋白的表达。 结果：在无血清培养基中,细胞呈悬浮生长,培养24~48 h可见单个细胞开始分裂生长,形成肿瘤球,将肿瘤球转入含胎牛血清的培养基后,4 h周边细胞伸出突起并逐渐分化,24 h后肿瘤球迁移出的细胞增多,形成单细胞层。肿瘤球能同时表达干细胞标志物CD133和巢蛋白,CD133+脑胶质瘤干细胞核浆比例达2/3~3/4,突起少,胞浆中线粒体等细胞器较少,核糖体丰富,未见胶质丝等分化结构,符合干细胞超微结构特点。NOTCH-1蛋白在人脑胶质瘤组织中的表达明显强于正常成人脑组织（P < 0.01）,在CD133+ U251及CHG-5胶质瘤细胞中有很强的表达,在GFAP+和MAP2+ U251及CHG-5胶质瘤细胞中的表达强弱不等,在MBP+ U251及CHG-5胶质瘤细胞中呈弱表达或不表达。在脑胶质瘤干细胞增殖过程中能检测到较强的NOTCH-1信号通路关键基因NOTCH-1,CBF-1,HES-1 mRNA及NOTCH-1,HES-1蛋白表达;而在细胞分化时NOTCH-1信号通路关键基因NOTCH-1,CBF-1,HES-1 mRNA和HES-1蛋白表达逐渐减弱。 结论：NOTCH-1信号通路的关键蛋白分子NOTCH-1在人脑胶质瘤组织和胶质瘤细胞株中均有表达,而在正常成人脑组织中仅微弱表达。NOTCH-1信号通路关键基因NOTCH-1和HES-1的表达强弱可能参与了胶质瘤干细胞增殖和分化的调控。     

Identification and functional validation of CDH11, PCSK6 and SH3GL3 as novel glioma invasion-associated candidate genes

S. Delic, N. Lottmann, K. Jetschke, G. Reifenberger and M. J. Riemenschneider (2012) Neuropathology and Applied Neurobiology 38, 201–212 Identification and functional validation of CDH11, PCSK6 and SH3GL3 as novel glioma invasion‐associated candidate genes Aims: The molecular mechanisms underlying the infiltrative growth of glioblastomas, the most common primary tumours of the central nervous system in adults, are still poorly understood. We aimed to identify and functionally validate novel glioma invasion‐associated candidate genes. Methods: Microarray‐based expression analysis was applied to identify differentially expressed genes in microdissected infiltrating glioma cells in vivo. Promising candidate genes were selected by the invasion‐associated gene ontology terms cell adhesion, endocytosis, extracellular matrix and cell migration and validated in vitro by invasion assays and in situ by immunohistochemistry. Results: We identified 180 up‐regulated and 61 down‐regulated genes (fold change: ≥2; P < 0.01) in the infiltration zone relative to more central cell‐rich tumour areas of malignant astrocytic gliomas (n = 11). Twenty‐seven of these genes matched to invasion‐related gene ontology terms. From these, we confirmed the genes encoding cadherin‐11 (CDH11), proprotein convertase subtilisin/kexin type 6 (PCSK6) and SH3‐domain GRB2‐like 3 (SH3GL3) as novel glioma invasion‐associated candidate genes, with knockdown of PCSK6 and SH3GL3 inhibiting glioma cell invasion, while inhibition of CDH11 promoted glioma cell invasion in vitro. Immunohistochemistry on glioblastoma tissue sections revealed expression of CDH11 and PCSK6 protein in glioma cells of more central, cell‐rich tumour areas, with only weak or absent CDH11 immunoreactivity but consistent PCSK6 staining in infiltrating glioma cells. Conclusion: Using molecular profiling of microdissected primary tumour tissue specimens followed by functional in vitro analysis, we identified and validated CDH11, PCSK6 and SH3GL3 as novel glioma invasion‐associated candidate genes that likely contribute to the invasive phenotype of malignant gliomas.     

Are gadolinium contrast agents suitable for gadolinium neutron capture therapy?

Abstract

Objective: Gadolinium neutron capture therapy (GdNCT) is a potential treatment for malignant tumors based on two steps: (1) injection of a tumor-specific ¹⁵⁷Gd compound; (2) tumor irradiation with thermal neutrons. The GdNC reaction can induce cell death provided that Gd is proximate to DNA. Here, we studied the nuclear uptake of Gd by glioblastoma (GBM) tumor cells after treatment with two Gd compounds commonly used for magnetic resonance imaging, to evaluate their potential as GdNCT agents.

Methods: Using synchrotron X-ray spectromicroscopy, we analyzed the Gd distribution at the subcellular level in: (1) human cultured GBM cells exposed to Gd-DTPA or Gd-DOTA for 0–72hours; (2) intracerebrally implanted C6 glioma tumors in rats injected with one or two doses of Gd-DOTA, and (3) tumor samples from GBM patients injected with Gd-DTPA.

Results: In cell cultures, Gd-DTPA and Gd-DOTA were found in 84% and 56% of the cell nuclei, respectively. In rat tumors, Gd penetrated the nuclei of 47% and 85% of the tumor cells, after single and double injection of Gd-DOTA, respectively. In contrast, in human GBM tumors 6.1% of the cell nuclei contained Gd-DTPA.

Discussion: Efficacy of Gd-DTPA and Gd-DOTA as GdNCT agents is predicted to be low, due to the insufficient number of tumor cell nuclei incorporating Gd. Although multiple administration schedules in vivo might induce Gd penetration into more tumor cell nuclei, a search for new Gd compounds with higher nuclear affinity is warranted before planning GdNCT in animal models or clinical trials.