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Cancer stem–like cells (CSC) could be a novel target for cancer therapy, including dendritic cell (DC) immunotherapy. To address this, we developed experiments aimed at DC targeting of neurospheres (NS) from GL261 glioma cells because neurospheres can be enriched in CSC. We obtained murine neurospheres by growing GL261 cells in epidermal growth factor/basic fibroblast growth factor without serum. GL261-NS recapitulated important features of glioblastoma CSC and expressed higher levels of radial glia stem cell markers than GL261 cells growing under standard conditions (GL261 adherent cells, GL261-AC), as assessed by DNA microarray and real-time PCR. GL261-NS brain gliomas were highly infiltrating and more rapidly lethal than GL261-AC, as evidenced by survival analysis (P < 0.0001), magnetic resonance imaging and histology. DC from the bone marrow of syngeneic mice were then used for immunotherapy of GL261-NS and GL261-AC tumors. Strikingly, DC loaded with GL261-NS (DC-NS) cured 80% and 60% of GL261-AC and GL261-NS tumors, respectively (P < 0.0001), whereas DC-AC cured only 50% of GL261-AC tumors (P = 0.0022) and none of the GL261-NS tumors. GL261-NS expressed higher levels of MHC and costimulatory molecules (CD80 and CD86) than GL261-AC; the JAM assay indicated that DC-NS splenocytes had higher lytic activity than DC-AC splenocytes on both GL261-NS and GL261-AC, and immunohistochemistry showed that DC-NS vaccination was associated with robust tumor infiltration by CD8+ and CD4+ T lymphocytes. These findings suggest that DC targeting of CSC provides a higher level of protection against GL261 gliomas, a finding with potential implications for the design of clinical trials based on DC vaccination. (Cancer Res 2006; 66(21): 10247-52)

Supplementary Methods and Figures 1-8 from Neurospheres Enriched in Cancer Stem–Like Cells Are Highly Effective in Eliciting a Dendritic Cell–Mediated Immune Response against Malignant Gliomas

Supplementary Table S2 from Neurospheres Enriched in Cancer Stem–Like Cells Are Highly Effective in Eliciting a Dendritic Cell–Mediated Immune Response against Malignant Gliomas

Aims The repressor element-1 silencing transcription factor/neurone-restrictive silencer factor (REST/NRSF) is a master regulator of neuronal gene expression. REST/NRSF functions by recruiting other cofactors to genomic loci that contain the repressor element 1/neurone restrictive silencer element (RE1/NRSE) binding motif. In brain, demonstration of REST protein presence in neurones has remained controversial. However, RE1/NRSE containing neuronal genes are actively modulated and REST dysregulation is implicated in Huntington's disease (HD). We aimed to investigate REST distribution in autopsy brain from control and HD patients. Methods Brain tissues from six controls and six HD cases (Vonsattel grade 3 and 4) were investigated using immunohistochemical analysis. Results REST was present in neurones and glial cells of the cortex, caudate nucleus, hippocampus and cerebellum. REST labelling was mainly cytoplasmic in neurones while preferential nuclear staining of REST was found in glial cells. We also found that REST and huntingtin (HTT) colocalize in human neurones. Low levels of cytoplasmic REST were detected in neurones of the HD cortex and caudate but no direct relationship between decreased neuronal REST expression and disease grade was observed. Conclusions These data support the notion of REST presence in human brain neurones and glial cells and indicate the importance of developing compounds able to restore REST-regulated transcription of neuronal genes in HD.

Glioblastoma (GBM) stem cells (GSCs), responsible for tumor growth, recurrence, and resistance to therapies, are considered the real therapeutic target, if they had no molecular mechanisms of resistance, in comparison with the mass of more differentiated cells which are insensitive to therapies just because of being differentiated and nonproliferating. GSCs occur in tumor niches where both stemness status and angiogenesis are conditioned by the microenvironment. In both perivascular and perinecrotic niches, hypoxia plays a fundamental role. Fifteen glioblastomas have been studied by immunohistochemistry and immunofluorescence for stemness and differentiation antigens. It has been found that circumscribed necroses develop inside hyperproliferating areas that are characterized by high expression of stemness antigens. Necrosis developed inside them because of the imbalance between the proliferation of tumor cells and endothelial cells; it reduces the number of GSCs to a thin ring around the former hyperproliferating area. The perinecrotic GSCs are nothing else that the survivors remnants of those populating hyperproliferating areas. In the tumor, GSCs coincide with malignant areas so that the need to detect where they are located is not so urgent.

Glioblastoma multiforme (GBM) is the most malignant and frequent intra-axial tumor of the central nervous system. Despite improvements in neurosurgery, radiotherapy, and chemotherapy, no local control of the disease is today possible.1,2 The failure of chemotherapy is due to the tumor's polyclonality, to its modalities of invasion, and to resistance to therapies. The presence of so-called cancer stem cells (CSCs) within GBM has been suggested as another possible reason for chemoresistance.3–5 GBM CSCs are believed to originate from the transformation of neural stem cells of the embryonic matrix and the neurogenic zones of the adult brain6 or from the dedifferentiation of hemispheric astrocytes or tumor cells. The latter reacquire stemness through embryonic regression upon the progressive accumulation of mutations.7–10 GBM CSCs show self-renewal, clonogenicity, and tumorigenicity when transplanted into mice.11 In culture systems without a coated matrix, the occurrence of CSCs in a tumor is denoted by the generation of neurospheres (NSs) beside adherent cells (ACs) after the addition of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).12 GBM CSCs share with neural stem cells stemness antigens, such as CD133, Musashi1, nestin, and Sox2,12,13 along with genetic alterations with primary tumors.10,14 They often show high activity of transforming growth factor/bone morphogenetic protein, Notch, Wnt, and sonic hedgehog homolog pathways, which are involved in self-renewal, proliferation, and invasion.15 The molecular basis of chemoresistance in GBM CSCs is under intensive investigation.3,16–18 The main drugs used in the therapy of GBM are DNA-damaging agents, such as the alkylating agents temozolomide (TMZ) and carmustine (BCNU), the topoisomerase II inhibitor etoposide, the topoisomerase I inhibitor irinotecan, and the DNA-intercalating drug doxorubicin.2 TMZ methylates the O6 position on guanine that mismatches with thymine in genomic DNA. The subsequent DNA aberration blocks replication and induces cell death. It is generally recognized that the overexpression of O6-methylguanine-DNA methyltransferase (MGMT), which removes the alkyl group from guanine, mediates the resistance to TMZ in GBM.3,19 However, MGMT alone is not always correlated with resistance to TMZ in GBM CSCs. A plethora of additional intrinsic factors (eg, the activity of DNA-repairing systems or the activity of specific signaling pathways involved in cell proliferation and apoptosis protection) or extrinsic factors (eg, the hypoxic environment or the presence of CSC niches rich in protective growth factors) may contribute to the chemoresistant phenotype of GBM CSCs.3,20 The role of membrane ATP-binding cassette (ABC) transporters—such as P-glycoprotein (Pgp/ABCB1), multidrug-resistance related protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2)—as inducers of the chemoresistant phenotype in GBM CSCs is still a matter of debate.3 Pgp,21,22 MRP1/3/5,21 and BCRP23,24 have been found highly expressed in GBM CSCs, either derived from primary tumors21,23 or obtained in vitro by growing ACs in the NS-generating medium.22 The overexpression of Pgp in NS U87MG cells is predictive of resistance to doxorubicin, etoposide, carboplatin, BCNU,22 and vincristine.25 TMZ has been reported to be a substrate of Pgp,26 although this observation is still controversial. To our knowledge, the regulation of ABC transporters in GBM CSCs has not yet been investigated. In this work we found that Pgp levels in GBM CSCs are controlled by Wnt3a oncogene and that clinically achievable doses of TMZ interfere with Wnt3a-dependent signaling and decrease the expression of Pgp in this tumor population.

GBM Cancer stem cells (CSCs) are responsible for growth, recurrence and resistance to chemo- and radio-therapy. They are supposed to originate from the transformation of Neural stem cells (NSC) of the Sub-ventricular zone (SVZ) or Sub-granular zone (SGZ) of hippocampus. Alternatively, they can be the expression of a functional status of competence or dedifferentiated cells of the tumor re-acquiring stemness properties. The origin of gliomas has been put in relation with the primitive neuroepithelial cells of the SVZ or NSC or progenitors, as showed by the development of experimental tumors in rats by transplacental ethylnitrosourea administration. The demonstration of CSCs in GBM is based on Neurosphere (NS) and Adherent cell (AC) development in culture. NS share the same genetic alterations with primary tumors and express stemness antigens, whereas AC show differentiation antigens. NS are generated by the most malignant areas of GBM. CSCs are considered at the top of a hierarchy of tumor cells of which the most immature are Nestin+/CD133– cells or established on the basis of EGFR amplification or delta-EGFR. NS in serum conditions differentiate and give origin to AC, the real nature of which is still a matter of discussion. Cells in culture could be simply in vitro entities depending on culture methodology. CSCs in GBM could be tumor cells at the end of a dedifferentiation process re-acquiring stemness properties, in an opposite way to what is realized in normal cytogenesis, where stemness is lost progressively with cell differentiation. This interpretation could fit with the origin of the two GBM types, primary and secondary. In primary GBM the tumor originates directly from stem cells or progenitors from SVZ with an accelerated transformation, whereas secondary GBM originates by transformation from astrocytomas arisen through a slow transformation from migrating stem cells and progenitors.

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 Neurobiology37, 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.

Signal transducer and activator of transcription-3 (Stat3) was studied along with several steps of the PI3/Akt pathway in a series of 64 gliomas that included both malignant and low-grade tumors, using quantitative immunohistochemistry, Western blotting, and molecular biology techniques. The goal of the study was to investigate whether activated Stat3 (phospho-Stat3) levels correlated with cell proliferation, apoptosis, and autophagy. Stat3 and activated Akt (phospho-Akt) expression increased with malignancy grade, but did not correlate with proliferation and survival within the category of glioblastomas. A correlation of Stat3 with Akt was found, indicating a regulation of the former by the PI3/Akt pathway, which, in turn, was in relation with EGFR amplification. Stat3 and Akt did not show any correlation with apoptosis, whereas they showed an inverse correlation with Beclin 1, a stimulator of autophagy, which was rarely positive in glioblastomas. Autophagy seems then to be inactivated in malignant gliomas.

Therapeutic resistance in glioblastoma multiforme (GBM) has been linked to a subpopulation of cells with stem cell-like properties, the glioma stem cells (GSCs), responsible for cancer progression and recurrence. This study investigated the in vitro cytotoxicity of three chemotherapeutics, temozolomide (TMZ), doxorubicin (Dox) and paclitaxel (PTX) on glioma cell lines, by analyzing the molecular mechanisms leading to DNA repair and cell resistance, or to cell death. The drugs were tested on 16 GBM cell lines, grown as neurospheres (NS) or adherent cells (AC), by studying DNA damage occurrence by Comet assay, the expression by immunofluorescence and western blotting of checkpoint/repair molecules and apoptosis. The three drugs were able to provoke a genotoxic injury and to inhibit dose- and time-dependently cell proliferation, more evidently in AC than in NS. The first cell response to DNA damage was the activation of the damage sensors (p-ATM, p-53BP1, γ-H2AX), followed by repair effectors; the expression of checkpoint/repair molecules appeared higher in NS than in AC. The non-homologous repair pathway (NHEJ) seemed more involved than the homologous one (HR). Apoptosis occurred after long treatment times, but only a small percentage of cells in NS underwent death, even at high drug concentration, whereas most cells survived in a quiescent state and resumed proliferation after drug removal. In tumor specimens, checkpoint/repair proteins were constitutively expressed in GBMs, but not in low-grade gliomas.

Paclitaxel loaded solid lipid nanoparticles (SLN) of behenic acid were prepared with the coacervation technique. Generally, spherical shaped SLN with mean diameters in the range 300–600 nm were obtained. The introduction of charged molecules, such as stearylamine and glycol chitosan into the formulation allowed to obtain positive SLN with Zeta potential in the 8-20 mV range and encapsulation efficiency in the 25–90% range.Blood–brain barrier (BBB) permeability, tested in vitro through hCMEC/D3 cells monolayer, showed a significantly increase in the permeation of Coumarin-6, used as model drug, when vehicled in SLN. Positive-charged SLN do not seem to enhance permeation although stearylamine-positive SLN resulted the best permeable formulation after 24 h.Cytotoxicity studies on NO3 glioblastoma cell line demonstrated the maintenance of cytotoxic activity of all paclitaxel-loaded SLN that was always unmodified or greater compared with free drug. No difference in cytotoxicity was noted between neutral and charged SLN.Co-culture experiments with hCMEC/D3 and different glioblastoma cells evidenced that, when delivered in SLN, paclitaxel increased its cytotoxicity towards glioblastoma cells.

The major obstacle to glioblastoma pharmacological therapy is the overcoming of the blood–brain barrier (BBB). In literature, several strategies have been proposed to overcome the BBB: in this experimental work, solid lipid nanoparticles (SLN), prepared according to fatty acid coacervation technique, are proposed as the vehicle for doxorubicin (Dox), to enhance its permeation through an artificial model of BBB. The in vitro cytotoxicity of Dox-loaded SLN has been measured on three different commercial and patient-derived glioma cell lines. Dox was entrapped within SLN thanks to hydrophobic ion pairing with negatively charged surfactants, used as counterions. Results indicate that Dox entrapped in SLN maintains its cytotoxic activity toward glioma cell lines; moreover, its permeation through hCMEC/D3 cell monolayer, assumed as a model of the BBB, was increased when the drug was entrapped in SLN. In conclusion, SLN proved to be a promising vehicle for the delivery of Dox to the brain in glioblastoma treatment.

Low delivery of many anticancer drugs across the blood-brain barrier (BBB) is a limitation to the success of chemotherapy in glioblastoma. This is because of the high levels of ATP-binding cassette transporters like P-glycoprotein (Pgp/ABCB1), which effluxes drugs back to the bloodstream. Temozolomide is one of the few agents able to cross the BBB; its effects on BBB cells permeability and Pgp activity are not known. We found that temozolomide, at therapeutic concentration, increased the transport of Pgp substrates across human brain microvascular endothelial cells and decreased the expression of Pgp. By methylating the promoter of Wnt3 gene, temozolomide lowers the endogenous synthesis of Wnt3 in BBB cells, disrupts the Wnt3/glycogen synthase kinase 3/?-catenin signaling, and reduces the binding of ?-catenin on the promoter of mdr1 gene, which encodes for Pgp. In co-culture models of BBB cells and human glioblastoma cells, pre-treatment with temozolomide increases the delivery, cytotoxicity, and antiproliferative effects of doxorubicin, vinblastine, and topotecan, three substrates of Pgp that are usually poorly delivered across BBB. Our work suggests that temozolomide increases the BBB permeability of drugs that are normally effluxed by Pgp back to the bloodstream. These findings may pave the way to new combinatorial chemotherapy schemes in glioblastoma. © 2013 Springer Basel.

Valproic acid (VPA), an histone deacetylase inhibitor, is emerging as a promising therapeutic agent for the treatments of gliomas by virtue of its ability to reactivate the expression of epigenetically silenced genes. VPA induces the unfolded protein response (UPR), an adaptive pathway displaying a dichotomic yin yang characteristic; it initially contributes in safeguarding the malignant cell survival, whereas long-lasting activation favors a proapoptotic response. By triggering UPR, VPA might tip the balance between cellular adaptation and programmed cell death via the deregulation of protein homeostasis and induction of proteotoxicity. Here we aimed to investigate the impact of proteostasis on glioma stem cells (GSC) using VPA treatment combined with subversion of SEL1L, a crucial protein involved in homeostatic pathways, cancer aggressiveness, and stem cell state maintenance. We investigated the global expression of GSC lines untreated and treated with VPA, SEL1L interference, and GSC line response to VPA treatment by analyzing cell viability via MTT assay, neurosphere formation, and endoplasmic reticulum stress/UPR-responsive proteins. Moreover, SEL1L immunohistochemistry was performed on primary glial tumors. The results show that (i) VPA affects GSC lines viability and anchorage-dependent growth by inducing differentiative programs and cell cycle progression, (ii) SEL1L down-modulation synergy enhances VPA cytotoxic effects by influencing GSCs proliferation and self-renewal properties, and (iii) SEL1L expression is indicative of glioma proliferation rate, malignancy, and endoplasmic reticulum stress statuses. Targeting the proteostasis network in association to VPA treatment may provide an alternative approach to deplete GSC and improve glioma treatments.

The repressor element-1 silencing transcription factor/neurone-restrictive silencer factor (REST/NRSF) is a master regulator of neuronal gene expression. REST/NRSF functions by recruiting other cofactors to genomic loci that contain the repressor element 1/neurone restrictive silencer element (RE1/NRSE) binding motif. In brain, demonstration of REST protein presence in neurones has remained controversial. However, RE1/NRSE containing neuronal genes are actively modulated and REST dysregulation is implicated in Huntington's disease (HD). We aimed to investigate REST distribution in autopsy brain from control and HD patients.Brain tissues from six controls and six HD cases (Vonsattel grade 3 and 4) were investigated using immunohistochemical analysis.REST was present in neurones and glial cells of the cortex, caudate nucleus, hippocampus and cerebellum. REST labelling was mainly cytoplasmic in neurones while preferential nuclear staining of REST was found in glial cells. We also found that REST and huntingtin (HTT) colocalize in human neurones. Low levels of cytoplasmic REST were detected in neurones of the HD cortex and caudate but no direct relationship between decreased neuronal REST expression and disease grade was observed.These data support the notion of REST presence in human brain neurones and glial cells and indicate the importance of developing compounds able to restore REST-regulated transcription of neuronal genes in HD.

The discovery of somatic isocitrate dehydrogenase (IDH) mutations in gliomas is an example of the powerful impact of the next-generation sequencing on the comprehension of both tumor biology and human diseases. IDHs catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate with production of NADH/NADPH. Thus, they are key enzymes in the Krebs cycle. For this family of metabolic genes, no previous role in human cancer has been described. However, in a recent genomewide study, recurrent somatic mutations in the IDH1 gene have been identified in patients affected by Glioblastoma Multiforme (GBM) [1]. In successive studies, IDH mutations have also been found in low-grade gliomas, as well as in acute myeloid leukemia. The aim of this chapter is to review the findings on the epidemiology and significance of IDH mutations in human gliomas, from the discovery to the current knowledge about their molecular pathogenesis. Special attention will be paid to the powerful diagnostic and prognostic relevance of the IDH mutations in the clinical practice of neuro-oncology.

The epithelial membrane protein 3 (EMP3) is a candidate tumor suppressor gene in the critical region 19q13.3 for several solid tumors, including tumors of the nervous systems. The aim of this study was to investigate theEMP3promoter hypermethylation status in a series of 229 astrocytic and oligodendroglial tumors and in 16 GBM cell lines. The analysis was performed by methylation-specific PCR and capillary electrophoresis. Furthermore, the EMP3 expression at protein level was evaluated by immunohistochemistry and Western blotting analysis. Associations ofEMP3hypermethylation with total 1p/19q codeletion,MGMTpromoter hypermethylation,IDH1/IDH2andTP53mutations, andEGFRamplification were studied, as well as its prognostic significance. TheEMP3promoter hypermethylation has been found in 39.5% of gliomas. It prevailed in low-grade tumors, especially in gliomas with an oligodendroglial component, and in sGBMs upon pGBMs. In oligodendroglial tumors, it was strongly associated with bothIDH1/IDH2mutations and total 1p/19q codeletion and inversely withEGFRgene amplification. No association was found withMGMThypermethylation andTP53mutations. In the whole series, theEMP3hypermethylation status correlated with 19q13.3 loss and lack of EMP3 expression at protein level. A favorable prognostic significance on overall survival of theEMP3promoter hypermethylation was found in patients with oligodendroglial tumors.

Cancer stem cells (CSCs) in gliomas are associated with resistance to radio- and chemotherapy, based on O(6)-methylguanine-DNA methyltransferase (MGMT) hypermethylation and the Multidrug resistance (MDR) system activation.Samples from 21 glioblastomas (GBMs) were put in culture with growth factors or serum in order to obtain neurospheres or adherent cells. Both were genetically and immunohistochemically characterized for ATP-binding cassette, sub-family B (MDR/TAP), member 1 (ABCB1), ATP-binding cassette, sub-family C (CFTR/MRP), member 1 (ABCC1) and MGMT expression together with primary tumors.ABCB1 expression was positive in endothelial cells of primary tumors. ABCC1 expression was variably positive in tumor cells and positive in neurospheres, and less expressed in adherent cells. MGMT was methylated and unmethylated in primary tumors and in neurospheres, respectively, and unmethylated in adherent cells.Methylation is an epigenetic event affecting progenitors before the separation of the two glia lineages and maximally the future initiating cells. ABCB1 expression is limited to endothelial cells, whereas ABCC1 expression could mark a minority of tumor cells approaching a stem-like status.

Gliomas represent the classical intra-axial tumors of the brain and glioblastoma multiforme (GBM) is the most frequent and malignant glioma. It is an extremely aggressive tumor with a high invasive potential. After treatments, it invariably resumes proliferation and its recurrences occur most often within 2 cm from the resection margins (Hochberg & Pruitt, 1980; Wallner et al., 1989; Oppitz et al., 1999). The dispersion of glioma cells in the surrounding normal brain puts them out of reach of surgery, radioand chemotherapy, because outside of the limits of surgical resection and of the irradiated volume, established in order to avoid damages to the normal brain. The BAT, therefore, is the place where tumor cells migrate and invade and where a series of biological, pathological and molecular events occur as far as the interaction between host and tumor is concerned. Migrating cells from the tumor, reacting astroglia and microglial cells, elements of the immunological response or belonging to the monocytic phagocyting system reaching the tumor from the blood flow, and cells from gliogenetic zones of the brain make the BAT a melting pot of interactions among cells and factors. It has special importance also from the neuroimaging point of view for the recognition of the tumor limits and of peritumoral edema, which may have, at the same time, a prognostic significance (Ramakrishna et al., 2010). With MRI, beside the peripheral increase of T2 signal, uptake of gadolinium, hypodensities corresponding to vasogenic edema, necrotic cyst formation, other features can be shown by special technical procedures. The correlation, therefore, between neuroimaging and histopathology and molecular biology in the BAT is of the greatest interest.

MGMT (O6-methylguanine-DNA methyltransferase) promoter hypermethylation is a helpful prognostic marker for chemotherapy of gliomas, although with some controversy for low-grade tumors. The objective of this study was to retrospectively investigate MGMT promoter hypermethylation status for a series of 350 human brain tumors, including 275 gliomas of different malignancy grade, 21 glioblastoma multiforme (GBM) cell lines, and 75 non-glial tumors. The analysis was performed by methylation-specific PCR and capillary electrophoresis. MGMT expression at the protein level was also evaluated by both immunohistochemistry (IHC) and western blotting analysis. Associations of MGMT hypermethylation with IDH1/IDH2 mutations, EGFR amplification, TP53 mutations, and 1p/19q co-deletion, and the prognostic significance of these, were investigated for the gliomas. MGMT promoter hypermethylation was identified in 37.8% of gliomas, but was not present in non-glial tumors, with the exception of one primitive neuroectodermal tumor (PNET). The frequency was similar for all the astrocytic gliomas, with no correlation with histological grade. Significantly higher values were obtained for oligodendrogliomas. MGMT promoter hypermethylation was significantly associated with IDH1/IDH2 mutations (P = 0.0207) in grade II–III tumors, whereas it had a borderline association with 1p deletion (P = 0.0538) in oligodendrogliomas. No other association was found. Significant correlation of MGMT hypermethylation with MGMT protein expression was identified by IHC in GBMs and oligodendrogliomas (P = 0.0001), but not by western blotting. A positive correlation between MGMT protein expression, as detected by either IHC or western blotting, was also observed. The latter was consistent with MGMT promoter hypermethylation status in GBM cell lines. In low-grade gliomas, MGMT hypermethylation, but not MGMT protein expression, was associated with a trend, only, toward better survival, in contrast with GBMs, for which it had favorable prognostic significance.

The transcription factor SOX2 controls gene expression during development and has roles in both neurogenesis and gliogenesis.The current study utilized immunohistochemistry, immunofluorescence, molecular genetics (gene copy number analysis) and Western blotting to examine SOX2 expression in surgical samples from 133 brain gliomas of different grades of malignancy, and in cell lines from 16 glioblastomas.Our results reveal a positive correlation between SOX2 expression and malignancy grade in gliomas and identify the hypercellular and hyperproliferative areas of glioblastomas as the areas with the highest SOX2 expression. SOX2 gene amplification was found in neurospheres and 14.4% and 11.1% of glioblastomas and anaplastic oligodendrogliomas, respectively. In contrast, SOX2 expression was not observed in neuronal tumors and varied in medulloblastomas depending upon their neuronal differentiation status.It is concluded that SOX2 is a marker for undifferentiated and proliferating cells and that its expression is up-regulated in the most anaplastic areas of glioblastomas and oligodendrogliomas. The hypothesis is that antigenic and genetic analogy exist between these areas and neurospheres and that glioblastoma stem cells may actually be dedifferentiated tumor cells that re-acquire a stem cell-like status.

Formation of neurospheres (NS) in cultures of glioblastomas (GBMs), with self-renewal, clonogenic capacities, and tumorigenicity following transplantation into immunodeficient mice, may denounce the existence of brain tumor stem cells (BTSCs) in vivo. In sixteen cell lines from resected primary glioblastomas, NS showed the same genetic alterations as primary tumors and the expression of stemness antigens. Adherent cells (AC), after adding 10% of fetal bovine serum (FBS) to the culture, were genetically different from NS and prevailingly expressed differentiation antigens. NS developed from a highly malignant tumor phenotype with proliferation, circumscribed necrosis, and high vessel density. Beside originating from transformed neural stem cells (NSCs), BTSCs may be contained within or correspond to dedifferentiated cells after mutation accumulation, which reacquire the expression of stemness antigens.

This review begins with the description of the nervous cytogenesis, proliferation of primitive cells, migration and differentiation with antigen expression and regulation through factors. Emphasis is given to neural stem cells, progenitors and to radial glia as belonging to early stages of gliogenesis. Experimental production of brain tumors in the rat by transplacental administration of ethylnitrosourea and systemic administration of methylnitrosourea effective through neural stem cells and progenitors is briefly described as a preamble to the recent conception of gliomas as originating from stem cells or brain tumor stem cells (BTSCs). The nature and origin of BTSCs, their molecular regulation and their recognition in vivo are discussed. In the growth of tumors, the role of nestin, migration of neural stem cells toward the tumor from the subventricular zone or from the tumor to the brain and the migration from bone marrow of mesenchymal stem cells for angiogenesis of the tumors are considered. Special mention is made of the relationship between glio-vascular niches in the subventricular zone and the neo-niches around the tumors with their importance in the tumor growth as points of joint between cytogenesis and angiogenesis. Finally, besides the importance of hypoxia, cell traffic in the brain adjacent to tumor is emphasized in relation to the growth of gliomas.

The mammalian target of rapamycin (mTOR) controls cell growth through protein synthesis regulation. It can be activated by protein kinase B (AKT) or through ribosomal S6 kinase (S6K1). In malignant glioma, mTOR inhibitors have antiproliferative and proapoptotic effects and mTOR has been suggested as a target of therapies, thus it is worthwhile to verify its relations with the phosphatidylinositol-3-kinase (PI3)/AKT cascade, proliferation and apoptosis in human gliomas.In a series of 64 gliomas, including high- and low-grade tumors, AKT, mTOR, S6, caspase-3, poly(ADP-ribose) polymerase 1 (PARP1) and cleaved PARP1, Ki-67/MIB.1 and beclin 1 were studied by molecular biology techniques, quantitative immunohistochemistry and Western blotting.mTOR (phospho-mTOR), S6 (phospho-S6), AKT (phospho-AKT) levels and Ki-67/MIB.1 labelling index (LI) increased with increasing grade of malignancy. Epithelial growth factor receptor (EGFR) amplification correlated with EGFRwt and EGFRvIII immunohistochemistry, and with AKT expression; the latter correlated with mTOR expression, whereas S6 expression correlated with Ki-67/MIB.1 LI. Within the category of glioblastoma, S6 but not mTOR correlated with proliferation. mTOR did not show correlation with apoptosis, whereas it was inversely correlated with beclin 1, in line with the observation that autophagy is not activated in many malignancies.The relationship of S6 with the proliferation markers emphasizes the importance of the position of S6K1 downstream AKT in the PI3/AKT pathway.

Survivin is expressed in proliferating tissues and in tumors. It is a member of the inhibitory apoptosis protein (IAP) family known to regulate mitosis and to inhibit apoptosis. It has therefore been regarded as a target for therapies. In malignant gliomas it increases with malignancy, even though in glioblastomas it does not seem to correlate with outcome.Survivin was immunohistochemically studied in 39 selected viable glioblastoma areas belonging to 20 cases which were assayed for apoptosis, using a TUNEL assay, caspase-3, poly(ADP-ribose)polymerase 1 (PARP-1), Bid (BH3-interacting domain death agonist) and with the proliferation index Ki-67/MIB-1 and mitotic index (MI).A positive linear correlation was found between the survivin labelling index (LI) and the Ki-67/MIB-1 LI and MI. No inverse correlation was found with apoptosis.This double behavior can be attributed to mechanisms mediating survivin activity, either as a mitosis regulator and apoptosis inhibitor, and should be taken into account in therapeutic strategies using survivin.

Cancer stem–like cells (CSC) could be a novel target for cancer therapy, including dendritic cell (DC) immunotherapy. To address this, we developed experiments aimed at DC targeting of neurospheres (NS) from GL261 glioma cells because neurospheres can be enriched in CSC. We obtained murine neurospheres by growing GL261 cells in epidermal growth factor/basic fibroblast growth factor without serum. GL261-NS recapitulated important features of glioblastoma CSC and expressed higher levels of radial glia stem cell markers than GL261 cells growing under standard conditions (GL261 adherent cells, GL261-AC), as assessed by DNA microarray and real-time PCR. GL261-NS brain gliomas were highly infiltrating and more rapidly lethal than GL261-AC, as evidenced by survival analysis (P < 0.0001), magnetic resonance imaging and histology. DC from the bone marrow of syngeneic mice were then used for immunotherapy of GL261-NS and GL261-AC tumors. Strikingly, DC loaded with GL261-NS (DC-NS) cured 80% and 60% of GL261-AC and GL261-NS tumors, respectively (P < 0.0001), whereas DC-AC cured only 50% of GL261-AC tumors (P = 0.0022) and none of the GL261-NS tumors. GL261-NS expressed higher levels of MHC and costimulatory molecules (CD80 and CD86) than GL261-AC; the JAM assay indicated that DC-NS splenocytes had higher lytic activity than DC-AC splenocytes on both GL261-NS and GL261-AC, and immunohistochemistry showed that DC-NS vaccination was associated with robust tumor infiltration by CD8+ and CD4+ T lymphocytes. These findings suggest that DC targeting of CSC provides a higher level of protection against GL261 gliomas, a finding with potential implications for the design of clinical trials based on DC vaccination. (Cancer Res 2006; 66(21): 10247-52)

The Repressor Element 1 Silencing Transcription factor (REST/NRSF) is a master repressor of neuronal programs in non-neuronal lineages shown to function as a central regulator of developmental programs and stem cell physiology. Aberrant REST function has been associated with a number of pathological conditions. In cancer biology, REST has been shown to play a tumor suppressor activity in epithelial cancers but an oncogenic role in brain childhood malignancies such as neuroblastoma and medulloblastoma. Here we examined REST expression in human glioblastoma multiforme (GBM) specimens and its role in GBM cells carrying self-renewal and tumorigenic competence. We found REST to be expressed in GBM specimens, its presence being particularly enriched in tumor cells in the perivascular compartment. Significantly, REST is highly expressed in self-renewing tumorigenic-competent GBM cells and its knock down strongly reduces their self-renewal in vitro and tumor-initiating capacity in vivo and affects levels of miR-124 and its downstream targets. These results indicate that REST contributes to GBM maintenance by affecting its self-renewing and tumorigenic cellular component and that, hence, a better understanding of these circuitries in these cells might lead to new exploitable therapeutic targets.