Cell Of Origin For Glioma Research Articles

Abstract 5801: Inhibition of muscarinic acetylcholine receptors in glioma stem cells blocks tumor progression

Abstract Primary gliomas arising within the brain are the deadliest form of brain cancer and account for 78% of all malignant brain tumors. Glioma patients have a 5 percent five-year survival rate and drug-resistant tumors often recur following surgical resection and treatment with radiation and/or chemotherapy. The cancer stem cell hypothesis suggests the presence of a subset of undifferentiated cells, namely glioma stem cells (GSCs), in the heterogenous tumor mass responsible for disease progression. Understanding mechanisms responsible for maintaining GSCs and developing strategies to deplete the GSC population could improve glioma treatment and prognosis. Some GSCs are similar to oligodendrocyte precursor cells (OPCs), which are observed in neural development and the adult brain. OPCs are prone to malignant transformation and are believed to be a cell of origin for glioma. Recent findings indicate that the well-characterized neurotransmitter acetylcholine (ACh) maintains the primitive state of normal OPCs via muscarinic ACh receptors (mAChRs) preventing maturation and cell cycle exit. We hypothesized that cholinergic signaling may also maintain the primitive state of OPC-like GSCs. We analyzed publicly available single nuclei RNASeq data of patient glioblastoma samples and observed high expression of CHRM3, encoding the M3 mAChR, in OPC-like cells of the proneural subtype of glioma. Studies in mouse OPC-like GSCs confirmed high levels of CHRM3 expression and demonstrated that ACh generated voltage changes and rapid (< 1 second) increases in cytosolic calcium from internal calcium stores. Exposure to a brain permeant FDA-approved anti-muscarinic drug targeting M3mAChR suppressed proliferation, evoked calcium release, and the activation of intracellular second messengers PKC and ERK. Pharmacologic inhibition of mAChRs in established patient derived glioma grafts prevented re-initiation of tumors in subsequent host animals compared with vehicle treated tumors. These studies suggest that the cholinergic microenvironment maintains GSCs in a manner similar to normal OPCs and provides a platform for repositioning available small molecule mAChR antagonists for treatment of glioma. Citation Format: Sumyuktha V. Anand, Min K. Lee, Alexander G. Skorput, Isabella B. Fox, Alison L. Young, Brock C. Christensen, Allan Gulledge, Matthew C. Havrda. Inhibition of muscarinic acetylcholine receptors in glioma stem cells blocks tumor progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5801.

Premalignant Oligodendrocyte Precursor Cells Stall in a Heterogeneous State of Replication Stress Prior to Gliomagenesis.

Cancer evolves from premalignant clones that adopt unusual cell states to achieve transformation. We previously pinpointed the oligodendrocyte precursor cell (OPC) as a cell of origin for glioma, but the early changes of mutant OPCs during premalignancy remained unknown. Using mice engineered for inducible Nf1-Trp53 loss in OPCs, we acutely isolated labeled mutant OPCs by laser-capture microdissection, determined global gene-expression changes by bulk RNA sequencing, and compared with cell-state fluctuations at the single-cell level by stochastic profiling, which uses RNA-sequencing measurements from random pools of 10 mutant cells. At 12 days after Nf1-Trp53 deletion, bulk differences were mostly limited to mitotic hallmarks and genes for ribosome biosynthesis, and stochastic profiling revealed a spectrum of stem-progenitor (Axl, Aldh1a1), proneural, and mesenchymal states as potential starting points for gliomagenesis. At 90 days, bulk sequencing detected few differentially expressed transcripts, whereas stochastic profiling revealed cell states for neurons and mural cells that do not give rise to glial tumors, suggesting cellular dead-ends for gliomagenesis. Importantly, mutant OPCs that strongly expressed key effectors of nonsense-mediated decay (Upf3b) and homology-dependent DNA repair (Rad51c, Slx1b, Ercc4) were identified along with DNA-damage markers, suggesting transcription-associated replication stress. Analysis of 10-cell transcriptomes at 90 days identified a locus of elevated gene expression containing an additional repair endonuclease (Mus81) and Rin1, a Ras-Raf antagonist and possible counterbalance to Nf1 loss, which was microdeleted or downregulated in gliomas at 150 days. These hidden cell-state variations uncover replication stress as a potential bottleneck that must be resolved for glioma initiation. SIGNIFICANCE: Profiling premalignant cell states in a mouse model of glioma uncovers regulatory heterogeneity in glioma cells-of-origin and defines a state of replication stress that precedes tumor initiation.See related articles by Singh and colleagues, p. 1840 and Schaff and colleagues, p. 1853.

OS12.1 Editing of IDH1 R132H mutation in human induced pluripotent stem cells to investigate tumor genesis in glioma

Abstract BACKGROUND Low grade gliomas and secondary glioblastomas are characterised by a hot-spot mutation in the Isocitrate dehydrogenase 1 (IDH1). The mutation causes a new catalytic function of the enzyme resulting in the production of 2-Hydroxyglutarat (2-HG), which is known as an oncometabolite. To this day, the pathomechanism by which IDH1 mutation promotes tumorigenesis is not completely understood and the tumour cell of origin for glioma is not known. Moreover, reliable cell models reflecting the patient ́s situation are not available. Thus, we aim at creating human induced pluripotent stem cells (hiPSC) carrying the IDH1 R132H mutation. We will utilize this cell model to investigate how the mutation influences stem cell properties and cell differentiation in neuronal progenitor cells. MATERIAL AND METHODS For designing the cell model, we applied the novel CRISPR/Cas9 based genome editing tool Base Editor 3 and 4. We transiently transfected the cells with two plasmids containing the Cas9 enzyme and sgRNA respectively. To determine editing efficiency, cells were investigated using the T7 Endonuclease 1 assay and Next Generation Sequencing. Single cell clones were picked to analyse allele status using allele-specific PCR with subsequent Sanger Sequencing. RESULTS Using fluorescence-activated cell sorting we measured a transfection efficiency in iPSCs for one plasmid of 33.8% ± 5.92%. Out of 96 picked clones one clone showed the IDH1 mutation. Thus, the overall editing efficiency was about 1%. We confirmed expression of mutated IDH1 R132H by Western Blot. Measurement of the tricarboxylic acid cycle metabolites using liquid chromatography tandem mass spectrometry (LC-MS/MS) showed a forty times increased concentration of 2-HG in IDH-mutated compared to the wildtype iPSCs, proving that the mutated enzyme is functional. To investigate effects of the IDH1 R132H on cell differentiation, we generated cerebral organoids from our iPSC-models. The IDH1 R132H mutation did not inhibit the cell differentiation in maturation of cerebral organoids. However, we observed differences in the growth between mutated and wildtype brain organoids. We additionally characterized our cell models using genome wide methylation arrays and RNA-Seq; these results will also be presented. CONCLUSION We successfully established a heterozygous IDH1 R132H mutated hiPSC cell line, which represents a promising cell model for future studies of early glioma genesis.

P53 and NF 1 loss plays distinct but complementary roles in glioma initiation and progression.

Malignant glioma is one of the deadliest types of cancer. Understanding how the cell of origin progressively evolves toward malignancy in greater detail could provide mechanistic insights and lead to novel concepts for tumor prevention and therapy. Previously we have identified oligodendrocyte precursor cell (OPC) as the cell of origin for glioma following the concurrent deletion of p53 and NF1 using a mouse genetic mosaic system that can reveal mutant cells prior to malignancy. In the current study, we set out to deconstruct the gliomagenic process in two aspects. First, we determined how the individual loss of p53 or NF1 contributes to aberrant behaviors of OPCs. Second, we determined how signaling aberrations in OPCs progressively change from pre-malignant to transformed stages. We found that while the deletion of NF1 leads to mutant OPC expansion through increased proliferation and decreased differentiation, the deletion of p53 impairs OPC senescence. Signaling analysis showed that, while PI3K and MEK pathways go through stepwise over-activation, mTOR signaling remains at the basal level in pre-transforming mutant OPCs but is abruptly up-regulated in tumor OPCs. Finally, inhibiting mTOR via pharmacological or genetic methods, led to a significant blockade of gliomagenesis but had little impact on pre-transforming mutant OPCs, suggesting that mTOR is necessary for final transformation but not early progression. In summary, our findings show that deconstructing the tumorigenic process reveals specific aberrations caused by individual gene mutations and altered signaling events at precise timing during tumor progression, which may shed light on tumor-prevention strategies.

Abstract B16: Deciphering individual roles of p53 and NF1 in the cell of origin for malignant glioma and the implications of targeted therapy

Abstract Glioma is currently an incurable disease. Identifying the cell of origin for glioma could provide critical insights for developing effective therapies. We previously used a mouse genetic system termed Mosaic Analysis using Double Markers (MADM) to introduce p53 and NF1 mutations into neural stem cells to determine which cell type is responsible for gliomagenesis. The unequivocal GFP-labeling of sparse mutant cells generated by MADM enabled us to study the entire course of tumor development, from pre-transforming stages to full malignancy. Based on thorough histological, transcriptomic, and genetic analyses we identified oligodendrocyte precursor cells (OPCs) as the cell of origin in this glioma model. Here, we sought to understand the distinct role of p53 and NF1 in the transformation of OPCs since understanding these roles can give us a more thorough insight into how future therapies may be designed. The MADM system generates sparse GFP+ null and RFP+ sibling WT OPCs in an otherwise colorless, heterozygous mouse, allowing us to assess the impact of TSG loss on the capacity of cells to proliferate, survive, and differentiate. The loss of NF1 alone significantly increased mutant OPC proliferation with a proportional decrease in differentiation. However, massively over-expanded NF1-null OPCs did not transform into malignant gliomas, implicating the critical role of p53 in tumor suppression. Surprisingly, the loss of p53 alone had no effect on mutant OPC number, proliferation, or differentiation, suggesting that p53 function as a braking system that won't manifest its activity in the absence of driver mutation, such as NF1 loss. Next, we set out to test whether restoring either TSG function in tumor OPC cell lines would have any therapeutic efficacy. The restoration of wild type p53 led to a significant increase in both cell-cycle arrest and cell death. Since p53 function is commonly altered from mutations in patients, rather than complete loss of the protein, we tested whether restoring mutant p53 function with PRIMA-1 would have the same biological response as WT p53. We found that the restoration of mutant p53 function resulted in similar levels of cell cycle arrest and cell death as WT p53. This finding suggests that rescuing p53 function using small molecules may be of high value for future therapies. It also suggests that the loss of p53 activity is not a permanent fixture of tumor cells but that p53 can still exert it's tumor suppressor activity after transformation. Next we tested the therapeutic effects of the restoration of the GAP domain of NF1, which should reduce Ras activity in tumor OPCs. We found that the expression of WT but not the “GAP-dead” NF1-GAP led to a decrease in proliferation and an increase in differentiation of glioma cells. To test pharmacological compounds, we explore the possibility of targeting downstream effectors of Ras signaling, specifically mTOR inhibition because phospho-Akt level is elevated in tumor OPCs compared to WT OPCs. When we inhibited mTOR activity in MADM tumor mice at an age that is critical for mutant OPC expansion prior to transformation, we found that it blocked the ability of mutant OPCs to over-expand, suggesting that mTOR inhibition could potentially prevent gliomagenesis. In summary our data demonstrates that while NF1 loss increases OPC proliferation and decreases differentiation, p53 loss is critical for transformation and that restoring the function of either of these pathways has profound effects on tumor OPC biology that could be translated into future therapeutic strategies. Citation Format: Phillippe P. Gonzalez, Hui Zong. Deciphering individual roles of p53 and NF1 in the cell of origin for malignant glioma and the implications of targeted therapy. [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 B16.

PM-03 * ROLE OF Olig2 PHOSPHORYLATION IN PEDIATRIC GLIOMA FORMATION

Malignant gliomas are a leading cause of tumor-related mortality in children. Current therapies often have significant adverse effects on brain development and are largely ineffective against aggressive gliomas. The development of rational therapies has been limited by poor information regarding the cell of origin for pediatric glioma and the relevant underlying genetic alterations. Recent findings now reveal that mutations existing in pediatric glioma are different than those seen in adult gliomas. For example, BRAFV600E activating mutations are associated with deletion of the tumor suppressor gene, p16 (Ink4a-Arf), in about 15% of pediatric gliomas. We have generated a faithful genetic mouse model in which there is concomitant activation of BRAFV600E and deletion of Ink4a-Arf in oligodendrocyte progenitor cells (OPCs) leading to the generation of pediatric high-grade glioma. OPCs have been shown to be a potential cell of origin for glioma and expression of Olig2 is found in 100 percent of human pediatric and adult high-grade gliomas. Given that Olig2 is also required for normal myelin development, targeting all Olig2-expressing cells with small molecule inhibitors would likely have detrimental effects. We have recently found that Olig2 protein is present in a phosphorylated form that promotes proliferation of adult glioma cells. In this study, we demonstrate that phospho-Olig2 is expressed in tumorigenic oligodendrocyte progenitor cells and is required for pediatric glioma generation. In addition, we found that phospho-Olig2 and Olig2 interact with different proteins, some of which are feasible drug targets. This study gives insights into new therapies by understanding key protein interactions in normal oligodendrocyte development as well as in tumorigenic oligodendrocyte progenitor cells.

Abstract LB-206: Role of Olig2 phosphorylation in pediatric glioma formation

Abstract Malignant gliomas are a leading cause of tumor-related mortality in children. Current therapies often have significant adverse effects on brain development and are largely ineffective against aggressive gliomas. The development of rational therapies has been limited by poor information regarding the cell of origin for pediatric glioma and the relevant underlying genetic alterations. Recent findings now reveal that mutations existing in pediatric glioma are different than those seen in adult gliomas. For example, BRAFV600E activating mutations are associated with deletion of the tumor suppressor gene, p16 (Ink4a-Arf), in about 15% of pediatric gliomas. We have generated a faithful genetic mouse model in which there is concomitant activation of BRAFV600E and deletion of Ink4a-Arf in oligodendrocyte progenitor cells (OPCs) leading to the generation of pediatric high-grade glioma. OPCs have been shown to be a potential cell of origin for glioma and expression of Olig2 is found in 100 percent of human pediatric and adult high-grade gliomas. Given that Olig2 is also required for normal myelin development, targeting all Olig2-expressing cells with small molecule inhibitors would likely have detrimental effects. We have recently found that Olig2 protein is present in a phosphorylated form that promotes proliferation of adult glioma cells. In this study, we demonstrate that phospho-Olig2 is expressed in tumorigenic oligodendrocyte progenitor cells and is required for pediatric glioma generation. In addition, we found that phospho-Olig2 and Olig2 interact with different proteins, some of which are feasible drug targets. This study gives insights into new therapies by understanding key protein interactions in normal oligodendrocyte development as well as in tumorigenic oligodendrocyte progenitor cells. Citation Format: Amelie GRIVEAU, An-Chi TIEN, David H. Rowitch. Role of Olig2 phosphorylation in pediatric glioma formation. [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 LB-206. doi:10.1158/1538-7445.AM2014-LB-206

Abstract 5007: Delineating human fetal CNS stem cell hierarchy reveals a progenitor cell of origin for human Gliobastoma Multiforme.

Abstract Glioblastoma Multiforme (GBM) is the most common and highly aggressive human brain tumor. Even with great advancement of surgical procedures and treatment regimens with radiation and chemotherapy, malignant gliomas remain incurable. This is due to their resistance to all conventional therapies and the diffuse infiltrative nature of the tumor cells, which makes complete tumor resection impossible. One of the major difficulties for the identification of the cell of origin for gliomas is the complex cellular composition of this disease. It is now hypothesized that the different subtypes of high-grade glioma may have different cell of origin and in mouse models it has been shown that the cell of origin resides within the progenitor populations. In order to identify the cell of origin we decided to first dissect out the various stages of CNS stem cell differentiation. After testing a panel of 34 antibodies to prospectively isolate neurospheres forming cells from human fetal brain tissue we identified a panel of four antibodies (i.e CD15, Notch1, EGFR, and CD90). Subpopulations of cells derived after FACS were analyzed for single cell lineage potency and the ability to generate neurospheres. Using an in-vitro limiting dilution assay, we ascertained that double-positive (DP; CD15+Notch1+EGFR-CD90-)) populations have the highest frequency of self-renewing cells, followed by quadruple-positive (QP) populations (CD15+Notch1+EGFR-CD90-). In-vitro differentiation assays reveal both DP and QP to be multipotent in nature however serial passaging showed that only the DP cells have a consistent self-renewing capacity, whereas QP derived cells were unable to survive after 5-6 passages. In glioma samples however we observed by limiting dilution and serial passaging assays that the tumorsphere forming frequency was predominant in the QP population and not in the DP populations. Furthermore the GBM derived DP cells serially passaged if grown under normal neural stem cell growth condition suggesting that this population is still at a pre-malignant state. In-vivo tumorigenic assay via orthotopic implation of as few as a 1000 cells into mouse brain showed significantly higher tumor formation from the QP cells and not in the DP cells. Taken together our data suggests the presence of “normal” or pre-malignant stem cells in glioblastoma, which suggests a possible mechanism of relapse. These pre-malignant cells could comprise a cellular reservoir that may need to be targeted for future therapies. Citation Format: Siddhartha S. Mitra, Sharareh Gholamin, Abdullah Feroze, Samuel H. Cheshier, Irving L. Weissman. Delineating human fetal CNS stem cell hierarchy reveals a progenitor cell of origin for human Gliobastoma Multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5007. doi:10.1158/1538-7445.AM2013-5007

The cellular origin for malignant glioma and prospects for clinical advancements

Glioma remains incurable despite great advancements in medicine. Targeting the cell of origin for gliomas could bring great hope for patients. However, as a collection of diverse diseases, each subtype of glioma could derive from a distinct cell of origin. To resolve such a complex problem, one must use multiple research approaches to gain deep insights. Here we review current evidence regarding the cell of origin from clinical observations, whole-genome molecular pathology and glioma animal models. We conclude that neural stem cells, glial progenitors (including oligodendrocyte progenitor cells) and astrocytes could all serve as cells of origin for gliomas, and that cells incurring initial mutations (cells of mutation) might not transform, while their progeny cells could instead transform and act as cells of origin. Further studies with multidisciplinary approaches are needed to link each subtype to a particular cell of origin, and to develop effective therapies that target the signaling network within these cells.

On the origin of glioma

Glioma is the most frequent primary brain tumor of adults that has a presumably glial origin. Although our knowledge regarding molecular mechanisms and signaling pathways involved in gliomagenesis has increased immensely during the past decade, high-grade glioma remains a lethal disease with dismal prognosis. The failure of current therapies has to a large extent been ascribed the functional heterogeneity of glioma cells. One reason for this heterogeneity is most certainly the large number of variations in genetic alterations that can be found in high-grade gliomas. Another factor that may influence glioma heterogeneity could be the cell type from which the glioma is initiated. The cell of origin for glioma is still undefined, and additional knowledge about this issue may prove critical for a more complete understanding of glioma biology. Based on information from patients, developmental biology, and experimental glioma models, the most putative target cells include astrocytes, neural stem cells, and oligodendrocyte precursor cells, which are all discussed in more detail in this article. Animal modeling of glioma suggests that these three cell types have the capability to be the origin of glioma, and we have reason to believe that, depending on the initiating cell type, prognosis and response to therapy may be significantly different. Thus, it is essential to explore further the role of cellular origin in glioma.

The origins of glioma

Liu and colleagues identify oligodendrocyte precursor cells as the cell of origin for glioma that is caused by inactivating mutations in neurofibromatosis 1 (Nf1) and Trp53.

Oligodendrocyte progenitor cells can act as cell of origin for experimental glioma

Gliomas are primary brain tumors mainly affecting adults. The cellular origin is unknown. The recent identification of tumor-initiating cells in glioma, which share many similarities with normal neural stem cells, has suggested the cell of origin to be a transformed neural stem cell. In previous studies, using the RCAS/tv-a mouse model, platelet-derived growth factor B (PDGF-B)-induced gliomas have been generated from nestin or glial fibrillary acidic protein-expressing cells, markers of neural stem cells. To investigate if committed glial progenitor cells could be the cell of origin for glioma, we generated the Ctv-a mouse where tumor induction would be restricted to myelinating oligodendrocyte progenitor cells (OPCs) expressing 2',3'-cyclic nucleotide 3'-phosphodiesterase. We showed that PDGF-B transfer to OPCs could induce gliomas with an incidence of 33%. The majority of tumors resembled human WHO grade II oligodendroglioma based on close similarities in histopathology and expression of cellular markers. Thus, with the Ctv-a mouse we have showed that the cell of origin for glioma may be a committed glial progenitor cell.

Anaplastic oligodendrogliomas with 1p19q codeletion have a proneural gene expression profile

BackgroundIn high grade gliomas, 1p19q codeletion and EGFR amplification are mutually exclusive and predictive of dramatically different outcomes. We performed a microarray gene expression study of four high grade gliomas with 1p19q codeletion and nine with EGFR amplification, identified by CGH-array.ResultsThe two groups of gliomas exhibited very different gene expression profiles and were consistently distinguished by unsupervised clustering analysis. One of the most striking differences was the expression of normal brain genes by oligodendrogliomas with 1p19q codeletion. These gliomas harbored a gene expression profile that partially resembled the gene expression of normal brain samples, whereas gliomas with EGFR amplification expressed many genes in common with glioblastoma cancer stem cells. The differences between the two types of gliomas and the expression of neuronal genes in gliomas with 1p19q codeletion were both validated in an independent series of 16 gliomas using real-time RT-PCR with a set of 22 genes differentiating the two groups of gliomas (AKR1C3, ATOH8, BMP2, C20orf42, CCNB1, CDK2, CHI3L1, CTTNBP2, DCX, EGFR, GALNT13, GBP1, IGFBP2, IQGAP1, L1CAM, NCAM1, NOG, OLIG2, PDPN, PLAT, POSTN, RNF135). Immunohistochemical study of the most differentially expressed neuronal gene, alpha-internexin, clearly differentiated the two groups of gliomas, with 1p19q codeletion gliomas showing specific staining in tumor cells.ConclusionThese findings provide evidence for neuronal differentiation in oligodendrogliomas with 1p19q codeletion and support the hypothesis that the cell of origin for gliomas with 1p19q codeletion could be a bi-potential progenitor cell, able to give rise to both neurons and oligodendrocytes.

Bmi1 Controls Tumor Development in an Ink4a/Arf-Independent Manner in a Mouse Model for Glioma

The Polycomb group and oncogene Bmi1 is required for the proliferation of various differentiated cells and for the self-renewal of stem cells and leukemic cancer stem cells. Repression of the Ink4a/Arf locus is a well described mechanism through which Bmi1 can exert its proliferative effects. However, we now demonstrate in an orthotopic transplantation model for glioma, a type of cancer harboring cancer stem cells, that Bmi1 is also required for tumor development in an Ink4a/Arf-independent manner. Tumors derived from Bmi1;Ink4a/Arf doubly deficient astrocytes or neural stem cells have a later time of onset and different histological grading. Moreover, in the absence of Ink4a/Arf, Bmi1-deficient cells and tumors display changes in differentiation capacity.

Spontaneous Transformation of Human Adult Nontumorigenic Stem Cells to Cancer Stem Cells Is Driven by Genomic Instability in a Human Model of Glioblastoma

The presence of a CD133+/nestin+ population in brain tumors suggests that a normal neural stem cell may be the cell of origin for gliomas. We have identified human CD133-positive NSCs from adult glioma tissue and established them as long-term in vitro cultures human neuroglial culture (HNGC)-1. Replicative senescence in HNGC-1 led to a high level of genomic instability and emergence of a spontaneously immortalized clone that developed into cell line HNGC-2 with features of cancer stem cells (CSCs), which include the ability for self-renewal and the capacity to form CD133-positive neurospheres and develop intracranial tumors. The data from our study specify an important role of genomic instability in initiation of transformed state as well as its progression into highly tumorigenic CSCs. The activated forms of Notch and Hes isoforms were expressed in both non-neoplastic neural stem cells and brain tumor stem cells derived from it. Importantly, a significant overexpression of these molecules was found in the brain tumor stem cells. These findings suggest that this model comprised of HNGC-1 and HNGC-2 cells would be a useful system for studying pathways involved in self-renewal of stem cells and their transformation to cancer stem cells. Disclosure of potential conflicts of interest is found at the end of this article.