Glioblastoma-derived Cancer Stem Research Articles

PTEN potentiation of oncolytic HSV therapy for glioblastoma

PTEN potentiation of oncolytic HSV therapy for glioblastoma

Exome sequencing of glioblastoma-derived cancer stem cells reveals rare clinically relevant frameshift deletion in MLLT1 gene

BackgroundGlioblastoma multiforme (GBM) is a heterogeneous CNS neoplasm which causes significant morbidity and mortality. One reason for the poor prognostic outcome of GBM is attributed to the presence of cancer stem cells (CSC) which confer resistance against standard chemo- and radiotherapeutics modalities. Two types of GBM-associated CSC were isolated from the same patient: tumor core- (c-CSC) and peritumor tissue-derived cancer stem cells (p-CSC). Our experiments are focused on glioblastoma–IDH-wild type, and no disease-defining alterations were present in histone, BRAF or other genes.MethodsIn the present study, potential differences in genetic variants between c-CSC versus p-CSC derived from four GBM patients were investigated with the aims of (1) comparing the exome sequences between all the c-CSC or p-CSC to identify the common variants; (2) identifying the variants affecting the function of genes known to be involved in cancer origin and development.ResultsBy comparative analyses, we identified common gene single nucleotide variants (SNV) in all GBM c-CSC and p-CSC, a potentially deleterious variant was a frameshift deletion at Gln461fs in the MLLT1 gene, that was encountered only in p-CSC samples with different allelic frequency.ConclusionsWe discovered a potentially harmful frameshift deletion at Gln461fs in the MLLT1 gene. Further investigation is required to confirm the presence of the identified mutations in patient tissue samples, as well as the significance of the frameshift mutation in the MLLT1 gene on GBM biology and response to therapy based on genomic functional experiments.

Identification of Global DNA Methylation Signatures in Glioblastoma-Derived Cancer Stem Cells

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis. In this study, we performed an in-depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down-regulated in GBMs such as SPINT2, NEFM and PENK. Forced re-expression of SPINT2 reduced glioma cell proliferative capacity, anchorage independent growth, cell motility, and tumor sphere formation in vitro. The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM.

Suggestive evidence on the involvement of polypyrimidine-tract binding protein in regulating alternative splicing of MAP/microtubule affinity-regulating kinase 4 in glioma

MAP/microtubule affinity-regulating kinase 4 (MARK4) is a serine–threonine kinase that phosphorylates microtubule-associated proteins taking part in the regulation of microtubule dynamics. MARK4 is expressed in two spliced isoforms characterized by inclusion (MARK4S) or exclusion (MARK4L) of exon 16. The distinct expression profiles in the central nervous system and their imbalance in gliomas point to roles of MARK4L and MARK4S in cell proliferation and cell differentiation, respectively. Having ruled out mutations and transcription defects, we hypothesized that alterations in the expression of splicing factors may underlie deregulated MARK4 expression in gliomas.Bioinformatic analysis revealed four putative polypyrimidine-tract binding (PTB) protein binding sites in MARK4 introns 15 and 16. Glioma tissues and glioblastoma-derived cancer stem cells showed, compared with normal brain, significant overexpression of PTB, correlated with high MARK4L mRNA expression. Splicing minigene assays revealed a functional intronic splicing silencer in MARK4 intron 15, but mutagenesis of the PTB binding site in this region did not affect minigene splicing, suggesting that PTB may bind to a splicing silencer other than the predicted one and synergistically acting with the other predicted PTB sites. Electrophoretic mobility shift assays coupled with mass spectrometry confirmed binding of PTB to the polypyrimidine tract of intron 15, and thus its involvement in MARK4 alternative splicing. This finding, along with evidence of PTB overexpression in gliomas and glioblastoma-derived cancer stem cells and differentiated progeny, merged in pointing out the involvement of PTB in the switch to MARK4L, consistent with its established role in driving oncogenic splicing in brain tumors.

Abstract 1379: Identification of global DNA methylation signatures in glioblastoma-derived cancer stem cells

Abstract Glioblastoma (GBM) is the most common and most aggressive brain tumor in adults. Its frequent recurrence after resection and dismal prognosis are thought to be due to a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy. In this study, we performed an in depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary GBM tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified two groups hyper- and hypomethylated of genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also be found in primary GBM-derived xenograft tumors suggesting that they were not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down regulated in GBMs such as SPINT2, NEFM, and PENK. The comparison between the NSC line and the normal brain tissue sample leads to the identification of a group of NSC-specific differentially methylated regions (nsDMRs). Cluster analysis using nsDMRs revealed the presence of stem cell-specific DNA methylation signatures in both primary GBM and GSC lines. Higher expression of genes associated with stem cell-specific DNA methylation signatures such as DNMT3A, STAT5A, CSTB, PMEPA1 and G6PD in GBM patients was found to be associated with poor patient survival. The results from this study demonstrate the utility of using cancer stem cell models for advancing understanding of the pathobiology of gliomas. Citation Format: Eun Joon Lee, Prakash Rath, Jimei Liu, Dungsheng Ryu, Alan Free, Lirong Pei, Douglas C Anthony, Suash Sharma, Mark D Kirk, John J. Laterra, Duck Hwan Ryu, Jeong-Hyeon Choi, Huidong Shi, Douglas C. Miller, N. Scott Litofsky, Qi Feng. Identification of global DNA methylation signatures in glioblastoma-derived cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1379. doi:10.1158/1538-7445.AM2014-1379

Enhanced antitumor efficacy of an oncolytic herpes simplex virus expressing an endostatin-angiostatin fusion gene in human glioblastoma stem cell xenografts.

Viruses have demonstrated strong potential for the therapeutic targeting of glioblastoma stem cells (GSCs). In this study, the use of a herpes simplex virus carrying endostatin–angiostatin (VAE) as a novel therapeutic targeting strategy for glioblastoma-derived cancer stem cells was investigated. We isolated six stable GSC-enriched cultures from 36 human glioblastoma specimens and selected one of the stable GSCs lines for establishing GSC-carrying orthotopic nude mouse models. The following results were obtained: (a) VAE rapidly proliferated in GSCs and expressed endo–angio in vitro and in vivo 48 h and 10 d after infection, respectively; (b) compared with the control gliomas treated with rHSV or Endostar, the subcutaneous gliomas derived from the GSCs showed a significant reduction in microvessel density after VAE treatment; (c) compared with the control, a significant improvement was observed in the length of the survival of mice with intracranial and subcutaneous gliomas treated with VAE; (d) MRI analysis showed that the tumor volumes of the intracranial gliomas generated by GSCs remarkably decreased after 10 d of VAE treatment compared with the controls. In conclusion, VAE demonstrated oncolytic therapeutic efficacy in animal models of human GSCs and expressed an endostatin–angiostatin fusion gene, which enhanced antitumor efficacy most likely by restricting tumor microvasculature development.

The Inhibition of KCa3.1 Channels Activity Reduces Cell Motility in Glioblastoma Derived Cancer Stem Cells

In the present study we evaluated the expression of the intermediate conductance calcium-activated potassium (KCa3.1) channel in human glioblastoma stem-like cells (CSCs) and investigated its role in cell motility. While the KCa3.1 channel is not expressed in neuronal- and glial-derived tissues of healthy individuals, both the KCa3.1 mRNA and protein are present in the glioblastoma tumor population, and are significantly enhanced in CSCs derived from both established cell line U87MG and a primary cell line, FCN9. Consistent with these data, voltage-independent and TRAM-34 sensitive potassium currents imputable to the KCa3.1 channel were recorded in the murine GL261 cell line and several primary human glioblastoma cells lines. Moreover, a significantly higher KCa3.1 current was recorded in U87MG-CD133 positive cells as compared to the U87MG-CD133 negative subpopulation. Further, we found that the tumor cell motility is strongly associated with KCa3.1 channel expression. Blockade of the KCa3.1 channel with the specific inhibitor TRAM-34 has in fact a greater impact on the motility of CSCs (reduction of 75%), which express a high level of KCa3.1 channel, than on the FCN9 parental population (reduction of 32%), where the KCa3.1 channel is expressed at lower level. Similar results were also observed with the CSCs derived from U87MG. Because invasion of surrounding tissues is one of the main causes of treatment failure in glioblastoma, these findings can be relevant for future development of novel cancer therapeutic drugs.

Lectins Identify Glycan Biomarkers on Glioblastoma-Derived Cancer Stem Cells

Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. Despite aggressive therapy with surgery, radiotherapy, and chemotherapy, nearly all patients succumb to disease within 2 years. Several studies have supported the presence of stem-like cells in brain tumor cultures that are CD133-positive, are capable of self-renewal, and give rise to all cell types found within the tumor, potentially perpetuating growth. CD133 is a widely accepted marker for glioma-derived cancer stem cells; however, its reliability has been questioned, creating a need for other identifiers of this biologically important subpopulation. We used a panel of 20 lectins to identify differences in glycan expression found in the glycocalyx of undifferentiated glioma-derived stem cells and differentiated cells that arise from them. Fluorescently labeled lectins that specifically recognize α-N-acetylgalactosamine (GalNAc) and α-N-acetylglucosamine (GlcNAc) differentially bound to the cell surface based on the state of cellular differentiation. GalNAc and GlcNAc were highly expressed on the surface of undifferentiated cells and showed markedly reduced expression over a 12-day duration of differentiation. Additionally, the GalNAc-recognizing lectin Dolichos biflorus agglutinin was capable of specifically selecting and sorting glioma-derived stem cell populations from an unsorted tumor stock and this subpopulation had proliferative properties similar to CD133(+) cells in vitro and also had tumor-forming capability in vivo. Our preliminary results on a single cerebellar GBM suggest that GalNAc and GlcNAc are novel biomarkers for identifying glioma-derived stem cells and can be used to isolate cancer stem cells from unsorted cell populations, thereby creating new cell lines for research or clinical testing.

An Off-Target Nucleostemin RNAi Inhibits Growth in Human Glioblastoma-Derived Cancer Stem Cells

Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133+ neurospheres, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Here we report that nucleostemin is expressed in GBM-CSCs isolated from patient samples, and that its expression, conversely to what it has been described for ordinary stem cells, does not disappear when cells are differentiated. The significance of nucleostemin expression in CSCs was addressed by targeting the corresponding mRNA using lentivirally transduced short hairpin RNA (shRNA). In doing so, we found an off-target nucleostemin RNAi (shRNA22) that abolishes proliferation and induces apoptosis in GBM-CSCs. Furthermore, in the presence of shRNA22, GBM-CSCs failed to form neurospheres in vitro or grow on soft agar. When these cells are xenotransplanted into the brains of nude rats, tumor development is significantly delayed. Attempts were made to identify the primary target/s of shRNA22, suggesting a transcription factor involved in one of the MAP-kinases signaling-pathways or multiple targets. The use of this shRNA may contribute to develop new therapeutic approaches for this incurable type of brain tumor.

Abstract 2304: 2-DG inhibits of N-glycosylation in glioblastoma-derived cancer stem cells

Abstract Cancer stem cells (CSCs) are capable of unlimited self-renewal and multi-lineage differentiation. We have shown that 2-deoxy-D-glucose (2-DG), a known inhibitor of glycolysis, that is also a 2-deoxy-D-mannose can inhibits the growth of glioma-derived stem cells (GSC11) under normoxic conditions, and we hypothesize that 2-DG can affect D-glucose and D-mannose metabolism in glioblastoma derived cancer stem. Our studies showed that 2-DG affects formation of N-glycans by replacing D-mannose in glycosylation processes. We have synthesized 2-DG, D-glucose, and D-mannose labeled with deuterium at C-2 and C-6 and treated GSC11 cells with these monosaccharides to measure their effects on global N-glycan formation. Our results demonstrated that deuterium labeled 2-DG was incorporated into the N-glycans leading to the termination of the extension of oligosaccharide chain. Comparative glycomic analysis of control, 2-DG-treated, and D-mannose-rescued GSC11cells revealed a distinct modulation of the N-glycan profile. The levels of all types of N-glycans were decreased (by ∼4-fold) in 2-DG-treated GSC11 cells compared with control cells. In contrast, N-glycan synthesis in GSC11 cells could be rescued to almost “normal control” levels by adding exogenous D-mannose. D-Mannose rescue of 2-DG-treated GSC11 cells drastically reduced the incorporation of 2-DG into the N-glycans. These results indicate that 2-DG can interfere with biochemical transformations of D-mannose and that such interference might contribute to the overall antitumor effects of 2-DG. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2304. doi:10.1158/1538-7445.AM2011-2304

Expression of the Stem Cell Markers Nestin and CD133 on Circulating Melanoma Cells

Different molecular markers have been identified for melanoma-initiating cells including CD133 and nestin. Assuming that metastasis requires a dissemination of tumor-initiating cells, presence of circulating tumor-initiating cells should be associated with worse patient outcome. In this study, 20 ml blood was collected from 32 consecutive patients affected by metastatic melanoma and blood was enriched for circulating melanoma cells (CMCs) by CD45 depletion of the non-melanoma cell fraction. Multiparameter cytometry was carried out to co-stain with combinations of CD133 and nestin (NES). Six tissue samples from metastatic lesions of six different patients were stained with the same antibodies by immunohistochemistry. Percentage of NES-positive CMCs correlated with tumor burden and number of metastatic sites. Cox regression analysis revealed levels of lactate dehydrogenase (LDH; hazard ratio: 12.8 (1.35-121.5); P=0.02), number of metastatic sites (hazard ratio 3.87 (1.66-9.03); P=0.02), tumor burden (hazard ratio 5.72 (1.57-20.9); P=0.01), and percentage of NES-expressing CMCs ≥ 35% (hazard ratio 5.73 (1.66-19.7); P=0.006) to be factors related to shorter overall survival. CD133- and NES-expression profiles on CMCs were similar to matched metastatic tissue. These findings show that CMCs expressed stem cell-associated markers NES and CD133. Higher expression of NES on CMCs might represent an index of poor prognosis.

Differential Signature of the Centrosomal MARK4 Isoforms in Glioma

Background: MAP/microtubule affinity-regulating kinase 4 (MARK4) is a serine-threonine kinase expressed in two spliced isoforms, MARK4L and MARK4S, of which MARK4L is a candidate for a role in neoplastic transformation. Methods: We performed mutation analysis to identify sequence alterations possibly affecting MARK4 expression. We then investigated the MARK4L and MARK4S expression profile in 21 glioma cell lines and 36 tissues of different malignancy grades, glioblastoma-derived cancer stem cells (GBM CSCs) and mouse neural stem cells (NSCs) by real-time PCR, immunoblotting and immunohistochemistry. We also analyzed the sub-cellular localisation of MARK4 isoforms in glioma and normal cell lines by immunofluorescence. Results: Mutation analysis rules out sequence variations as the cause of the altered MARK4 expression in glioma. Expression profiling confirms that MARK4L is the predominant isoform, whereas MARK4S levels are significantly decreased in comparison and show an inverse correlation with tumour grade. A high MARK4L/MARK4S ratio also characterizes undifferentiated cells, such as GBM CSCs and NSCs. Accordingly, only MARK4L is expressed in brain neurogenic regions. Moreover, while both MARK4 isoforms are localised to the centrosome and midbody in glioma and normal cells, the L isoform exhibits an additional nucleolar localisation in tumour cells. Conclusions: The observed switch towards MARK4L suggests that the balance between the MARK4 isoforms is carefully guarded during neural differentiation but may be subverted in gliomagenesis. Moreover, the MARK4L nucleolar localisation in tumour cells features this MARK4 isoform as a nucleolus-associated tumour marker.

Differential signature of the centrosomal MARK4 isoforms in glioma.

Background: MAP/microtubule affinity-regulating kinase 4 (MARK4) is a serine-threonine kinase expressed in two spliced isoforms, MARK4L and MARK4S, of which MARK4L is a candidate for a role in neoplastic transformation. Methods: We performed mutation analysis to identify sequence alterations possibly affecting MARK4 expression. We then investigated the MARK4L and MARK4S expression profile in 21 glioma cell lines and 36 tissues of different malignancy grades, glioblastoma-derived cancer stem cells (GBM CSCs) and mouse neural stem cells (NSCs) by real-time PCR, immunoblotting and immunohistochemistry. We also analyzed the sub-cellular localisation of MARK4 isoforms in glioma and normal cell lines by immunofluorescence. Results: Mutation analysis rules out sequence variations as the cause of the altered MARK4 expression in glioma. Expression profiling confirms that MARK4L is the predominant isoform, whereas MARK4S levels are significantly decreased in comparison and show an inverse correlation with tumour grade. A high MARK4L/MARK4S ratio also characterizes undifferentiated cells, such as GBM CSCs and NSCs. Accordingly, only MARK4L is expressed in brain neurogenic regions. Moreover, while both MARK4 isoforms are localised to the centrosome and midbody in glioma and normal cells, the L isoform exhibits an additional nucleolar localisation in tumour cells. Conclusions: The observed switch towards MARK4L suggests that the balance between the MARK4 isoforms is carefully guarded during neural differentiation but may be subverted in gliomagenesis. Moreover, the MARK4L nucleolar localisation in tumour cells features this MARK4 isoform as a nucleolus-associated tumour marker.

Identification of CD133(-)/telomerase(low) progenitor cells in glioblastoma-derived cancer stem cell lines.

Glioblastoma multiforme (GBM) is paradigmatic for the investigation of cancer stem cells (CSC) in solid tumors. The CSC hypothesis implies that tumors are maintained by a rare subpopulation of CSC that gives rise to rapidly proliferating progenitor cells. Although the presence of progenitor cells is crucial for the CSC hypothesis, progenitor cells derived from GBM CSC are yet uncharacterized. We analyzed human CD133(+) CSC lines that were directly derived from CD133(+) primary astrocytic GBM. In these CSC lines, CD133(+)/telomerase(high) CSC give rise to non-tumorigenic, CD133(-)/telomerase(low) progenitor cells. The proliferation of the progenitor cell population results in significant telomere shortening as compared to the CD133(+) compartment comprising CSC. The average difference in telomere length as determined by a modified multi-color flow fluorescent in situ hybridization was 320 bp corresponding to 4-8 cell divisions. Taken together, we demonstrate that CD133(+) primary astrocytic GBM comprise proliferating, CD133(-)/telomerase(low) progenitor cell population characterized by low telomerase activity and shortened telomeres as compared to CSC.

Abstract 5149: Nucleostemin is a key factor in maintenance of human glioblastoma-derived cancer stem cells

Abstract Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133+ neurospheres, of retaining their multi-lineage potential over time, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. We will report that nucleostemin is expressed in GBM-CSCs isolated from patient samples. The significance of its expression was addressed by targeting the corresponding mRNA using lentivirally transduced short hairpin RNA (shRNA). Knockdown of nucleostemin in GBM-CSC abolishes proliferation and induces apoptosis. Furthermore, GBM-CSCs with decreased nucleostemin levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised rats. These findings suggest a central role of nucleostemin in regulating proliferation, self-renewal and survival of GBM-CSC, and targeting its expression may offer a new therapeutic approach for this incurable type of brain tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5149.

A Hierarchy of Self-Renewing Tumor-Initiating Cell Types in Glioblastoma

The neural stem cell marker CD133 is reported to identify cells within glioblastoma (GBM) that can initiate neurosphere growth and tumor formation; however, instances of CD133(-) cells exhibiting similar properties have also been reported. Here, we show that some PTEN-deficient GBM tumors produce a series of CD133(+) and CD133(-) self-renewing tumor-initiating cell types and provide evidence that these cell types constitute a lineage hierarchy. Our results show that the capacities for self-renewal and tumor initiation in GBM need not be restricted to a uniform population of stemlike cells, but can be shared by a lineage of self-renewing cell types expressing a range of markers of forebrain lineage.

Transcriptional Profiles of CD133+ and CD133− Glioblastoma-Derived Cancer Stem Cell Lines Suggest Different Cells of Origin

Glioblastoma multiforme (GBM) is paradigmatic for the investigation of cancer stem cells (CSC) in solid tumors. Growing evidence suggests that different types of CSC lead to the formation of GBM. This has prompted the present comparison of gene expression profiles between 17 GBM CSC lines and their different putative founder cells. Using a newly derived 24-gene signature, we can now distinguish two subgroups of GBM: Type I CSC lines display "proneural" signature genes and resemble fetal neural stem cell (fNSC) lines, whereas type II CSC lines show "mesenchymal" transcriptional profiles similar to adult NSC (aNSC) lines. Phenotypically, type I CSC lines are CD133 positive and grow as neurospheres. Type II CSC lines, in contrast, display (semi-)adherent growth and lack CD133 expression. Molecular differences between type I and type II CSC lines include the expression of extracellular matrix molecules and the transcriptional activity of the WNT and the transforming growth factor-beta/bone morphogenetic protein signaling pathways. Importantly, these characteristics were not affected by induced adherence on laminin. Comparing CSC lines with their putative cells of origin, we observed greatly increased proliferation and impaired differentiation capacity in both types of CSC lines but no cancer-associated activation of otherwise silent signaling pathways. Thus, our data suggest that the heterogeneous tumor entity GBM may derive from cells that have preserved or acquired properties of either fNSC or aNSC but lost the corresponding differentiation potential. Moreover, we propose a gene signature that enables the subclassification of GBM according to their putative cells of origin.

Human Glioblastoma–Derived Cancer Stem Cells: Establishment of Invasive Glioma Models and Treatment with Oncolytic Herpes Simplex Virus Vectors

Glioblastoma, the most malignant type of primary brain tumor, is one of the solid cancers where cancer stem cells have been isolated, and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here, we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serum-free medium with epidermal growth factor and fibroblast growth factor 2, varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem-like cells (GBM-SC), we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)-deleted mutants kill GBM-SCs as efficiently as wild-type HSV, the deletion of gamma34.5 significantly attenuated the vectors due to poor replication. However, this was significantly reversed by the additional deletion of alpha47. Infection with oHSV G47Delta (ICP6(-), gamma34.5(-), alpha47(-)) not only killed GBM-SCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly, despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs, intratumoral injection of G47Delta significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs, and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover, the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis.

CD133+ and CD133− Glioblastoma-Derived Cancer Stem Cells Show Differential Growth Characteristics and Molecular Profiles

Although glioblastomas show the same histologic phenotype, biological hallmarks such as growth and differentiation properties vary considerably between individual cases. To investigate whether different subtypes of glioblastomas might originate from different cells of origin, we cultured tumor cells from 22 glioblastomas under medium conditions favoring the growth of neural and cancer stem cells (CSC). Secondary glioblastoma (n = 7)-derived cells did not show any growth in the medium used, suggesting the absence of neural stem cell-like tumor cells. In contrast, 11/15 primary glioblastomas contained a significant CD133(+) subpopulation that displayed neurosphere-like, nonadherent growth and asymmetrical cell divisions yielding cells expressing markers characteristic for all three neural lineages. Four of 15 cell lines derived from primary glioblastomas grew adherently in vitro and were driven by CD133(-) tumor cells that fulfilled stem cell criteria. Both subtypes were similarly tumorigenic in nude mice in vivo. Clinically, CD133(-) glioblastomas were characterized by a lower proliferation index, whereas glial fibrillary acidic protein staining was similar. GeneArray analysis revealed 117 genes to be differentially expressed by these two subtypes. Together, our data provide first evidence that CD133(+) CSC maintain only a subset of primary glioblastomas. The remainder stems from previously unknown CD133(-) tumor cells with apparent stem cell-like properties but distinct molecular profiles and growth characteristics in vitro and in vivo.

N-CoR Pathway Targeting Induces Glioblastoma Derived Cancer Stem Cell Differentiation

Nuclear receptor co-repressor (N-CoR) is a critical regulator of neural stem cell differentiation. Nuclear localization of N-CoR is a feature of undifferentiated neural stem cells and cytoplasmic translocation of N-CoR leads to astrocytic differentiation. Comparative proteomic analysis of microdissected glioblastoma multiforme (GBM) specimens and matched normal glial tissue reveals increased expression of N-CoR in GBM. In GBM primary cell cultures, tumor cells with nuclear localization of N-CoR demonstrate an undifferentiated phenotype, but are subject to astroglial differentiation upon exposure to agents promoting phosphorylation of N-CoR and its subsequent translocation to the cytoplasm. Treatment of glioma cell lines with a combination of retinoic acid and low-dose okadaic acid decreases the co-repressor effect of N-CoR and has a striking synergistic effect on growth inhibition. The identification of N-CoR in GBM provides insights into the tumorigenesis process and supports the development of differentiation-based therapeutic strategies.