Tumorigenicity Of Glioma Stem Cells Research Articles

E3 ubiquitin-ligase RNF138 may regulate p53 protein expression to regulate the self-renewal and tumorigenicity of glioma stem cells.

Glioblastoma multiforme (GBM), the most malignant tumor of the central nervous system, is characterized by poor survival and high recurrence. Glioma stem cells (GSCs) are key to treating GBM and are regulated by various signaling pathways. Ubiquitination, a post-translational modification, plays an important regulatory role in many biological processes. Ring finger protein 138 (RNF138) is an E3 ubiquitin-protein ligase that is highly expressed in several tumors; however, its role in GBM is unclear. This study investigated whether RNF138 regulates the self-renewal ability of glioma stem GSCs to treat GBM. The expression of RNF138 in glioma tissues and its correlation with GSCs were analyzed using bioinformatics. Short hairpin ribonucleic acid (RNA) was designed to downregulate the expression of RNF138 in GSCs, and immunofluorescence, secondary pellet formation, and western blotting were used to detect changes in GSC markers and self-renewal ability. The effects of RNF138 on p53 protein expression were determined by immunofluorescence and western blotting. The effects of RNF138 on the self-renewal and tumorigenic abilities of GSCs were evaluated in vivo. RNF138 expression was higher in glioma tissues than in normal brain tissues, and was highly expressed in GSCs. RNF138 downregulation significantly decreased the expression of the GSC markers cluster of differentiation 133 (CD133) and nestin. Mechanistically, RNF138 may interfere with the self-renewal ability of GSCs by regulating the expression of p53. RNF138 downregulation in vivo prolonged survival time and regulated the expression of p53 protein in tumor-bearing mice. RNF138 may regulate the expression of p53 protein through ubiquitination, thereby affecting the self-renewal and tumorigenic ability of GSCs. This study provides a scientific basis for the treatment of glioblastoma by targeting RNF138 to inhibit GSCs.

SPOPL induces tumorigenicity and stemness in glioma stem cells by activating Notch signaling.

Recent studies have increasingly shown that glioma stem cells (GSCs) are extremely important for developing and treating glioblastoma multiforme (GBM). The Broad-complex, Tram-track, and Bric-a-brac protein family is functionally related to a variety of tumor stem cells, and the role of SPOPL as a member of this family in GSCs deserves to be investigated. To investigate the expression of SPOPL in GSCs and its impact on the prognosis of GBM patients by using clinical specimens, patient-derived primary GSCs and public databases. In vivo and in vitro, the effect of SPOPL on the proliferation, self-renewal, and differentiation ability of GSCs was explored. Probing the mechanism by which SPOPL affects the biological function of GSCs using RNA sequencing (RNA-seq) and rescue experiments. The expression of SPOPL was significantly upregulated in GSCs and GBM, and patients with high SPOPL expression had a poorer prognosis. SPOPL enhanced the proliferation and self-renewal ability of GSCs and enhanced the tumorigenicity of GSCs. The Notch signaling pathway was significantly inhibited in SPOPL knockdown GSCs. Activation or inhibition of the Notch signaling pathway rescued changes in the biological function of GSCs caused by altered SPOPL expression. SPOPL can be used as a potential prognostic biomarker for GBM in clinical work and promotes the proliferation and stemness of GSCs by activating the Notch signaling pathway, which may be a potential molecule for targeting GSCs to treat GBM.

CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma.

MAD2 is a spindle assembly checkpoint protein that participates in the formation of mitotic checkpoint complex, which blocks mitotic progression. RNF8, an established DNA damage response protein, has been implicated in mitotic checkpoint regulation but its exact role remains poorly understood. Here, RNF8 proximity proteomics uncovered a role of RNF8-MAD2 in generating the mitotic checkpoint signal. Specifically, RNF8 competes with a small pool of p31comet for binding to the closed conformer of MAD2 via its RING domain, while CAMK2D serves as a molecular scaffold to concentrate the RNF8-MAD2 complex via transient/weak interactions between its p-Thr287 and RNF8's FHA domain. Accordingly, RNF8 overexpression impairs glioma stem cell (GSC) mitotic progression in a FHA- and RING-dependent manner. Importantly, low RNF8 expression correlates with inferior glioma outcome and RNF8 overexpression impedes GSC tumorigenicity. Last, we identify PLK1 inhibitor that mimics RNF8 overexpression using a chemical biology approach, and demonstrate a PLK1/HSP90 inhibitor combination that synergistically reduces GSC proliferation and stemness. Thus, our study has unveiled a previously unrecognized CAMK2D-RNF8-MAD2 complex in regulating mitotic checkpoint with relevance to gliomas, which is therapeutically targetable.

MEK5-ERK5 Axis Promotes Self-renewal and Tumorigenicity of Glioma Stem Cells.

In this study, we demonstrated that MEK5-ERK5-STAT3 axis plays a critical role in maintaining stemness and tumorigenicity in GSCs by using genetic, pharmacologic, and bioinformatics tools, identifying the MEK5-ERK5-STAT3 axis as a potential target for GSC-directed therapy.

Induction of cancer cell stemness in glioma through glycolysis and the long noncoding RNA HULC-activated FOXM1/AGR2/HIF-1α axis

Gliomas are the most common primary intracranial tumor, accounting for more than 70% of brain malignancies. Studies indicate that highly upregulated in liver cancer (HULC), a long noncoding RNA (lncRNA), functions as an oncogene in gliomas. However, the underlying mechanism of HULC in gliomas remains under-studied and was subsequently investigated in the current study. Brain tissues were clinically collected from 50 patients with glioblastoma (GBM) and 35 patients with acute craniocerebral injury, followed by immunohistochemical detection of the expression patterns of Forkhead box M1 (FOXM1), anterior gradient 2 (AGR2), and hypoxia-inducible factor-1α (HIF-1α). After flow cytometry-based sorting of the CD133+ glioma stem cells (GSCs) from the U251 cell line, the obtained cells were subjected to lentivirus infection. Afterwards, the proliferation, stemness, and apoptosis of GSCs were evaluated using sphere formation, immunofluorescence, and flow cytometry assays, respectively. In addition, the interactions among HULC, FOXM1, AGR2, and HIF-1α were identified using RNA immunoprecipitation (RIP), RNA pull-down, Chromatin immunoprecipitation (ChIP), IP, and dual luciferase reporter assays. Last, the specific effects were validated in vivo. HULC was upregulated in GBM tissues and GSCs, which may promote the progression of glioma. On the other hand, silencing of HULC reduced the stemness, inhibited the proliferation, and promoted the apoptosis and differentiation of GSCs. In addition, HULC further stabilized FOXM1 expression in GSCs through ubiquitination, while FOXM1 activated AGR2 transcription to promote HIF-1α expression. Moreover, HULC promoted the glycolysis and stemness of GSCs through its regulation of the FOXM1/AGR2/HIF-1α axis, consequently exacerbating the occurrence and development of glioma. The findings obtained in our study indicate that HULC stabilizes the FOXM1 protein by ubiquitination to upregulate the expression of AGR2 and HIF-1α, which further promote the glycolysis of and maintain the stemness of GSCs, to enhance the tumorigenicity of GSCs, highlighting a novel therapeutic target for glioma.

Oct4A palmitoylation modulates tumorigenicity and stemness in human glioblastoma cells.

Glioblastoma multiforme and other solid malignancies are heterogeneous, containing subpopulations of tumor cells that exhibit stem characteristics. Oct4, also known as POU5F1, is a key transcription factor in the self-renewal, proliferation, and differentiation of stem cells. Although it has been detected in advanced gliomas, the biological function of Oct4, and transcriptional machinery maintained by the stemness of Oct4 protein-mediated glioma stem cells (GSC), has not been fully determined. The expression of Oct4 variants was evaluated in brain cancer cell lines, and in brain tumor tissues, by quantitative real-time PCR, western blotting, and immunohistochemical analysis. The palmitoylation level of Oct4A was determined by the acyl-biotin exchange method, and the effects of palmitoylation Oct4A on GSCs were investigated by a series of in vitro (neuro-sphere formation assay, double immunofluorescence, pharmacological treatment, luciferase assay, and coimmunoprecipitation) and in vivo (xenograft model) experiments. Here, we report that all three variants of Oct4 are expressed in different types of cerebral cancer, while Oct4A is important for maintaining tumorigenicity in GSCs. Palmitoylation mediated by ZDHHC17 was indispensable for preserving Oct4A from lysosome degradation to maintain its protein stability. Oct4A palmitoylation also helped to integrate Sox4 and Oct4A in the SOX2 enhancement subregion to maintain the stem performance of GSCs. We also designed Oct4A palmitoylation competitive inhibitors, inhibiting the self-renewal ability and tumorigenicity of GSCs. These findings indicate that Oct4A acts on the tumorigenic activity of glioblastoma, and Oct4A palmitoylation is a candidate therapeutic target.

SMURF2 phosphorylation at Thr249 modifies glioma stemness and tumorigenicity by regulating TGF-\u03b2 receptor stability

Glioma stem cells (GSCs) contribute to the pathogenesis of glioblastoma, the most malignant form of glioma. The implication and underlying mechanisms of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) on the GSC phenotypes remain unknown. We previously demonstrated that SMURF2 phosphorylation at Thr249 (SMURF2Thr249) activates its E3 ubiquitin ligase activity. Here, we demonstrate that SMURF2Thr249 phosphorylation plays an essential role in maintaining GSC stemness and tumorigenicity. SMURF2 silencing augmented the self-renewal potential and tumorigenicity of patient-derived GSCs. The SMURF2Thr249 phosphorylation level was low in human glioblastoma pathology specimens. Introduction of the SMURF2T249A mutant resulted in increased stemness and tumorigenicity of GSCs, recapitulating the SMURF2 silencing. Moreover, the inactivation of SMURF2Thr249 phosphorylation increases TGF-β receptor (TGFBR) protein stability. Indeed, TGFBR1 knockdown markedly counteracted the GSC phenotypes by SMURF2T249A mutant. These findings highlight the importance of SMURF2Thr249 phosphorylation in maintaining GSC phenotypes, thereby demonstrating a potential target for GSC-directed therapy.

ERK5 regulates stemness and tumorigenicity of glioma stem cells by controlling the STAT3 signaling

ERK5 regulates stemness and tumorigenicity of glioma stem cells by controlling the STAT3 signaling

MCM8 is regulated by EGFR signaling and promotes the growth of glioma stem cells through its interaction with DNA-replication-initiating factors.

Mini-chromosome maintenance (MCM) proteins are critical components of DNA-replication-licensing factors. MCM8 is an MCM protein that exhibits oncogenic functions in several human malignancies. However, the role of MCM8 in glioblastomas (GBMs) has remained unclear. In the present study, we investigated the biological functions and mechanisms of MCM8 in glioma stem cells (GSCs). The clinical relevance of MCM8 mRNA expression was explored via TCGA and REMBRANDT datasets. The effects of MCM8 on the self-renewal and tumorigenicity of GSCs were examined both in vitro and in vivo. The regulation of MCM8 expression and its interacting proteins were also evaluated. We found that the expression of MCM8 was elevated in high-grade gliomas and classical molecular subtypes and was inversely correlated with patient prognosis. GSCs had a significantly higher expression of MCM8 compared with that in normal glioma cells. Silencing of MCM8 induced G0/G1 arrest and apoptosis, as well as inhibited the proliferation and self-renewal of GSCs. Forced expression of MCM8 enhanced clonogenicity of GSCs both in vitro and in vivo. MCM8 expression was regulated by EGFR signaling, which was mediated by NF-κB (p65). MCM8 interacted with DNA-replication-initiating factors-including EZH2, CDC6, and CDCA2-and influenced these factors to associate with chromatin. In addition, MCM8 knockdown increased the sensitivity of GSCs to radiation and TMZ treatments. Our findings suggest that MCM8, regulated by the EGFR pathway, maintains the clonogenic and tumorigenic potential of GSCs through interaction with DNA-replication-initiating factors; hence, MCM8 may represent a novel therapeutic target in GBMs.

CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity.

Glioma stem cells (GSCs) drive propagation and therapeutic resistance of glioblastomas, the most aggressive diffuse brain tumors. However, the molecular mechanisms that maintain the stemness and promote therapy resistance remain poorly understood. Here we report CD109/STAT3 axis as crucial for the maintenance of stemness and tumorigenicity of GSCs and as a mediator of chemoresistance. Mechanistically, CD109 physically interacts with glycoprotein 130 to promote activation of the IL-6/STAT3 pathway in GSCs. Genetic depletion of CD109 abolished the stemness and self-renewal of GSCs and impaired tumorigenicity. Loss of stemness was accompanied with a phenotypic shift of GSCs to more differentiated astrocytic-like cells. Importantly, genetic or pharmacologic targeting of CD109/STAT3 axis sensitized the GSCs to chemotherapy, suggesting that targeting CD109/STAT3 axis has potential to overcome therapy resistance in glioblastoma.

CDK8 maintains stemness and tumorigenicity of glioma stem cells by regulating the c-MYC pathway.

Glioblastoma (GBM) is the most malignant form of glioma. Glioma stem cells (GSCs) contribute to the initiation, progression, and recurrence of GBM as a result of their self-renewal potential and tumorigenicity. Cyclin-dependent kinase 8 (CDK8) belongs to the transcription-related CDK family. Although CDK8 has been shown to be implicated in the malignancy of several types of cancer, its functional role and mechanism in gliomagenesis remain largely unknown. Here, we demonstrate how CDK8 plays an essential role in maintaining stemness and tumorigenicity in GSCs. The genetic inhibition of CDK8 by shRNA or CRISPR interference resulted in an abrogation of the self-renewal potential and tumorigenicity of patient-derived GSCs, which could be significantly rescued by the ectopic expression of c-MYC, a stem cell transcription factor. Moreover, we demonstrated that the pharmacological inhibition of CDK8 significantly attenuated the self-renewal potential and tumorigenicity of GSCs. CDK8 expression was significantly higher in human GBM tissues than in normal brain tissues, and its expression was positively correlated with stem cell markers including c-MYC and SOX2 in human GBM specimens. Additionally, CDK8 expression is associated with poor survival in GBM patients. Collectively, these findings highlight the importance of the CDK8-c-MYC axis in maintaining stemness and tumorigenicity in GSCs; these findings also identify the CDK8-c-MYC axis as a potential target for GSC-directed therapy.

CDK8 maintains stemness and tumorigenicity of glioma stem cells by controlling the c-MYC pathway

CDK8 maintains stemness and tumorigenicity of glioma stem cells by controlling the c-MYC pathway

Phosphorylation of Smurf2, a novel therapeutic target, regulates stemness and tumorigenicity of glioma stem cells

Phosphorylation of Smurf2, a novel therapeutic target, regulates stemness and tumorigenicity of glioma stem cells

MicroRNA-30a suppresses self-renewal and tumorigenicity of glioma stem cells by blocking the NT5E-dependent Akt signaling pathway.

Over the past decade, increasing researches have demonstrated the implication of microRNAs (miRNAs or miRs) in tumorigenicity of glioma stem cells (GSCs). The regulatory functions of miRNAs in GSCs have emerged as potential therapeutic candidates for glioma treatment. Herein, we aim to investigate the role of miR-30a in the proliferation and self-renewal of GSCs and the possible mechanism in relation to ecto-5'-nucleotidase (NT5E)-dependent Akt signaling pathway. RT-qPCR and Western blot analysis were performed to determine the expression of miR-30a and NT5E in glioma tissues and cell lines. GSCs were isolated from glioma cells and identified using flow cytometry. The relationship between miR-30a and NT5E was determined by dual-luciferase reporter gene assay. Gain- and loss-of-function experiments were performed to examine the effects of miR-30a and NT5E on sphere formation, colony formation, and proliferation of GSCs in vitro, as well as orthotopic tumor growth of GSCs in nude mice. Additionally, the Akt signaling pathway was blocked with an Akt inhibitor, LY294002, to investigate its involvement in the regulatory effect of miR30a. miR-30a was poorly expressed in glioma tissues and cell lines as well as GSCs. NT5E, highly expressed in GSCs, was identified as a target of miR-30a. In addition, miR-30a upregulation or NT5E silencing could reduce GSC sphere formation, clone formation, proliferation, and orthotopic tumor growth in nude mice. Moreover, miR-30a inhibited the activation of the Akt signaling pathway by targeting NT5E, and ultimately suppressing the self-renewal and orthotopic tumor growth of GSCs. Our results demonstrate that miR-30a targets NT5E to inhibit the Akt signaling pathway, by which could suppress the self-renewal and orthotopic tumor growth of GSCs. Those findings may provide theoretical basis of miR-30a as a therapeutic target to suppress the glioma progression.

Long Noncoding RNA HOTAIRM1 Maintains Tumorigenicity of Glioblastoma Stem-Like Cells Through Regulation of HOX Gene Expression.

Noncoding RNAs regulate transcription of gene expression and play an important role in the pathogenesis of glioblastomas. These tumors are heterogeneous with some glioma stem cells (GSCs) that are highly tumorigenic subpopulations of cells contributing to recurrence and treatment resistance. In this study, GSCs were established by neurosphere cultures of primary glioblastoma cells and validated by the expression of GSC marker CD133. The expression of the long noncoding RNA HOTAIRM1 was detected using real-time quantitative reverse transcription PCR (qRT-PCR). The role of HOTAIRM1 in the proliferation, apoptosis, stemness, and tumorigenicity of GSCs was investigated by soft agar colony formation, flow cytometry, TUNEL analysis, sphere formation, and in vivo xenograft models through silencing of HOTAIRM1. The expression of HOTAIRM1 and the neighboring HOX genes were analyzed by qRT-PCR in different grades of gliomas and nontumor tissues. We found that HOTAIRM1 is significantly elevated in GSCs. The silencing of HOTAIRM1 significantly impairs the proliferation, apoptosis, self-renewal, tumorigenesis of GSCs. In addition, HOTAIRM1 is significantly upregulated in gliomas and associated with tumor grade and patient survival. HOTAIRM1 neighboring genes, HOXA1, HOXA2, and HOXA3, are also significantly upregulated in gliomas and correlate with the expression of HOTAIRM1. Among them, HOXA2 and HOXA3 were identified as being upregulated in GSCs and contributed to the self-renewal of these stem cells. Taken together, our results demonstrate that HOTAIRM1 plays a critical role in the self-renewal of GSCs. These data also suggest that overexpression of HOTAIRM1 can be a negative prognostic factor for patient survival in malignant glioma and may be a promising potential therapeutic target.

SHP2 regulates proliferation and tumorigenicity of glioma stem cells.

SHP2 is a cytoplasmic protein tyrosine phosphatase (PTPase) involved in multiple signaling pathways and was the first identified proto-oncogene PTPase. Previous work in glioblastoma (GBM) has demonstrated the role of SHP2 PTPase activity in modulating the oncogenic phenotype of adherent GBM cell lines. Mutations in PTPN11, the gene encoding SHP2, have been identified with increasing frequency in GBM. Given the importance of SHP2 in developing neural stem cells, and the importance of glioma stem cells (GSCs) in GBM oncogenesis, we explored the functional role of SHP2 in GSCs. Using paired differentiated and stem cell primary cultures, we investigated the association of SHP2 expression with the tumor stem cell compartment. Proliferation and soft agar assays were used to demonstrate the functional contribution of SHP2 to cell growth and transformation. SHP2 expression correlated with SOX2 expression in GSC lines and was decreased in differentiated cells. Forced differentiation of GSCs by removal of growth factors, as confirmed by loss of SOX2 expression, also resulted in decreased SHP2 expression. Lentiviral-mediated knockdown of SHP2 inhibited proliferation. Finally, growth in soft-agar was similarly inhibited by loss of SHP2 expression. Our results show that SHP2 function is required for cell growth and transformation of the GSC compartment in GBM.

Abstract A45: Semaphorin 3C mediates glioma stem cell tumorigenicity and radioresistance

Abstract Purpose: Different cancer cell compartments often communicate through soluble factors to facilitate tumor growth. Glioma stem cells (GSCs) are a subset of tumor cells that resist standard therapy to contribute to disease progression. How GSCs employ a distinct secretory program to communicate with and nurture each other over the non-stem tumor cell (NSTC) population is not well defined. Methods/Materials: GSCs were enriched from patient specimens and functionally validated. We used a candidate approach to screen for secreted factors that were preferentially used by GSCs but not NSTCs. Expression was assessed by Western blot, and confirmed by immunohistochemistry and immunofluorescence in vitro and in human and mouse GBM tissues. Ligand-receptor interactions were confirmed by co-immunoprecipitation. Rac activity was assessed by Rac-GTP pulldown. Co-culture epistasis experiments were performed using knockdown and recombinant protein approaches. Glioma stem cell marker analysis (CD133, Sox2, Olig2), cell viability (ATP-dependent assays), apoptosis (TUNEL, PI/AnnexinV, cleaved caspase 3/7), self-renewal (tumorsphere formation and limiting dilution assay) and invasion (transwell migration assay) were performed. GSCs were separated in PlexinD1 hi and lo populations by FACS. Radiation (3 Gy) was delivered by Cs-137 irradiator. GSCs were used to establish orthotopic xenograft models of glioblastoma and tumor growth and animal survival were assessed. Results: We have found that GSCs preferentially secrete Sema3C and coordinately express PlexinA2/D1 receptors to activate Rac1/NFkB signaling in an autocrine and paracrine loop to promote their own survival. Importantly, Sema3C is not expressed in neural progenitor cells (NPCs) or NSTCs. Disruption of Sema3C induced apoptosis of GSCs, but not NPCs or NSTCs, and suppressed tumor growth in orthotopic models of glioblastoma. Introduction of activated Rac1 rescued the Sema3C knockdown phenotype in vivo. Furthermore, GSCs that expressed high levels of PlexinD1 receptors were more resistant to radiation, and Sema3C and PlexinD1 were upregulated after irradiation. Together, these data support that Sema3C signaling contributes to radiation resistance. Conclusions: Our study supports targeting Sema3C to break this GSC-specific autocrine/paracrine loop to improve glioblastoma treatment, potentially with a high therapeutic index. Citation Format: Jennifer Yu, Jianghong Man, Shoemake Jocelyn, Wu Qiulian, Jeremy Rich, Shideng Bao. Semaphorin 3C mediates glioma stem cell tumorigenicity and radioresistance. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A45.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Sema3C Promotes the Survival and Tumorigenicity of Glioma Stem Cells through Rac1 Activation

Different cancer cell compartments often communicate through soluble factors to facilitate tumor growth. Glioma stem cells (GSCs) are a subset of tumor cells that resist standard therapy to contribute to disease progression. How GSCs employ a distinct secretory program to communicate with and nurture each other over the nonstem tumor cell (NSTC) population is not well defined. Here, we show that GSCs preferentially secrete Sema3C and coordinately express PlexinA2/D1 receptors to activate Rac1/nuclear factor (NF)-κB signaling in an autocrine/paracrine loop to promote their own survival. Importantly, Sema3C is not expressed in neural progenitor cells (NPCs) or NSTCs. Disruption of Sema3C induced apoptosis of GSCs, but not NPCs or NSTCs, and suppressed tumor growth in orthotopic models of glioblastoma. Introduction of activated Rac1 rescued the Sema3C knockdown phenotype invivo. Our study supports the targeting of Sema3C to break this GSC-specific autocrine/paracrine loop in order to improve glioblastoma treatment, potentially with a high therapeutic index.

FoxM1 and Wnt/β-Catenin Signaling in Glioma Stem Cells

Cancer stem cells may be responsible for tumor initiation and maintenance. The molecular mechanisms that control cancer stem cells are related to alterations in various signaling pathways, including the Wnt/β-catenin signaling pathway. The canonical Wnt/β-catenin signaling pathway is one of the major signaling systems in stem and progenitor cells, and aberrant activation of the Wnt/β-catenin signaling pathway is common in human cancers. As with β-catenin, FoxM1 has been found to play important roles in a number of cancers. In this review, we discuss the evidence that FoxM1 affects the expression and function of a variety of genes that are critical to the survival, proliferation, invasion, angiogenesis, and self-renewal of cancer stem cells. We highlight the pivotal roles of the Wnt/β-catenin and FoxM1 signaling pathways in neural stem and progenitor cells and glioma stem cells. We also discuss the evidence for cross-talk between the β-catenin and FoxM1 signaling pathways in the regulation of the stemness and tumorigenicity of glioma stem cells.