Temozolomide preferentially depletes cancer stem cells in glioblastoma

The prognosis of patients suffering from glioblastoma (GBM) is dismal despite multimodal therapy. Although chemotherapy with temozolomide may contain tumor growth for some months, invariable tumor recurrence suggests that cancer stem cells (CSC) maintaining these tumors persist. We have therefore investigated the effect of temozolomide on CD133(+) and CD133(-) GBM CSC lines. Although differentiated tumor cells constituting the bulk of all tumor cells were resistant to the cytotoxic effects of the substance, temozolomide induced a dose- and time-dependent decline of the stem cell subpopulation. Incubation with sublethal concentrations of temozolomide for 2 days completely depleted clonogenic tumor cells in vitro and substantially reduced tumorigenicity in vivo. In O(6)-methylguanine-DNA-methyltransferase (MGMT)-expressing CSC lines, this effect occurred at 10-fold higher doses compared with MGMT-negative CSC lines. Thus, temozolomide concentrations that are reached in patients were only sufficient to completely eliminate CSC in vitro from MGMT-negative but not from MGMT-positive tumors. Accordingly, our data strongly suggest that optimized temozolomide-based chemotherapeutic protocols might substantially improve the elimination of GBM stem cells and consequently prolong the survival of patients.     

Patient-derived glioblastoma stem cells are killed by CD133-specific CAR T cells but induce the T cell aging marker CD57

The AC133 epitope of CD133 is a cancer stem cell (CSC) marker for many tumor entities, including the highly malignant glioblastoma multiforme (GBM). We have developed an AC133-specific chimeric antigen receptor (CAR) and show that AC133-CAR T cells kill AC133+ GBM stem cells (GBM-SCs) both in vitro and in an orthotopic tumor model in vivo. Direct contact with patient-derived GBM-SCs caused rapid upregulation of CD57 on the CAR T cells, a molecule known to mark terminally or near-terminally differentiated T cells. However, other changes associated with terminal T cell differentiation could not be readily detected. CD57 is also expressed on tumor cells of neural crest origin and has been preferentially found on highly aggressive, undifferentiated, multipotent CSC-like cells. We found that CD57 was upregulated on activated T cells only upon contact with CD57+ patient-derived GBM-SCs, but not with conventional CD57-negative glioma lines. However, CD57 was not downregulated on the GBM-SCs upon their differentiation, indicating that this molecule is not a bona fide CSC marker for GBM. Differentiated GBM cells still induced CD57 on CAR T cells and other activated T cells. Therefore, CD57 can apparently be upregulated on activated human T cells by mere contact with CD57+ target cells.     

Adult mouse subventricular zones stimulate glioblastoma stem cells specific invasion through CXCL12/CXCR4 signaling

Background

Patients with glioblastoma multiforme (GBM) have an overall median survival of 15 months. This catastrophic survival rate is the consequence of systematic relapses that could arise from remaining glioblastoma stem cells (GSCs) left behind after surgery. We previously demonstrated that GSCs are able to escape the tumor mass and specifically colonize the adult subventricular zones (SVZs) after transplantation. This specific localization, away from the initial injection site, therefore represents a high-quality model of a clinical obstacle to therapy and relapses because GSCs notably retain the ability to form secondary tumors.

Method

In this work, we questioned the role of the CXCL12/CXCR4 signaling in the GSC-specific invasion of the SVZs.

Results

We demonstrated that both receptor and ligand are respectively expressed by different GBM cell populations and by the SVZ itself. In vitro migration bio-assays highlighted that human U87MG GSCs isolated from the SVZs (U87MG-SVZ) display stronger migratory abilities in response to recombinant CXCL12 and/or SVZ-conditioned medium (SVZ-CM) compared with cancer cells isolated from the tumor mass (U87MG-TM). Moreover, in vitro inhibition of the CXCR4 signaling significantly decreased the U87MG-SVZ cell migration in response to the SVZ-CM. Very interestingly, treating U87MG-xenografted mice with daily doses of AMD3100, a specific CXCR4 antagonist, prevented the specific invasion of the SVZ. Another in vivo experiment, using CXCR4-invalidated GBM cells, displayed similar results.

Conclusion

Taken together, these data demonstrate the significant role of the CXCL12/CXCR4 signaling in this original model of brain cancer invasion.     

Inhibition of CXCR7 extends survival following irradiation of brain tumours in mice and rats

Background:

In experimental models of glioblastoma multiforme (GBM), irradiation (IR) induces local expression of the chemokine CXCL12/SDF-1, which promotes tumour recurrence. The role of CXCR7, the high-affinity receptor for CXCL12, in the tumour''s response to IR has not been addressed.

Methods:

We tested CXCR7 inhibitors for their effects on tumour growth and/or animal survival post IR in three rodent GBM models. We used immunohistochemistry to determine where CXCR7 protein is expressed in the tumours and in human GBM samples. We used neurosphere formation assays with human GBM xenografts to determine whether CXCR7 is required for cancer stem cell (CSC) activity in vitro.

Results:

CXCR7 was detected on tumour cells and/or tumour-associated vasculature in the rodent models and in human GBM. In human GBM, CXCR7 expression increased with glioma grade and was spatially associated with CXCL12 and CXCL11/I-TAC. In the rodent GBM models, pharmacological inhibition of CXCR7 post IR caused tumour regression, blocked tumour recurrence, and/or substantially prolonged survival. CXCR7 expression levels on human GBM xenograft cells correlated with neurosphere-forming activity, and a CXCR7 inhibitor blocked sphere formation by sorted CSCs.

Conclusions:

These results indicate that CXCR7 inhibitors could block GBM tumour recurrence after IR, perhaps by interfering with CSCs.     

Chemokine receptor CXCR3 promotes growth of glioma

Human glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The poor prognosis and minimally successful treatments of GBM indicates a need to identify new therapeutic targets. In this study, we examined the role of CXCR3 in glioma progression using the GL261 murine model of malignant glioma. Intracranial GL261 tumors express CXCL9 and CXCL10 in vivo. Glioma-bearing CXCR3-deficient mice had significantly shorter median survival time and reduced numbers of tumor-infiltrated natural killer and natural killer T cells as compared with tumor-bearing wild-type (WT) mice. In contrast, pharmacological antagonism of CXCR3 with NBI-74330 prolonged median survival times of both tumor-bearing WT and CXCR3-deficient mice when compared with vehicle-treated groups. NBI-74330 treatment did not impact tumor infiltration of lymphocytes and microglia. A small percentage of GL261 cells were identified as CXCR3(+), which was similar to the expression of CXCR3 in several grade IV human glioma cell lines (A172, T98G, U87, U118 and U138). When cultured as gliomaspheres (GS), the human and murine lines increased CXCR3 expression; CXCR3 expression was also found in a primary human GBM-derived GS. Additionally, CXCR3 isoform A was expressed by all lines, whereas CXCR3-B was detected in T98G-, U118- and U138-GS cells. CXCL9 or CXCL10 induced in vitro glioma cell growth in GL261- and U87-GS as well as inhibited cell loss in U138-GS cells and this effect was antagonized by NBI-74330. The results suggest that CXCR3 antagonism exerts a direct anti-glioma effect and this receptor may be a potential therapeutic target for treating human GBM.     

Targeting cancer stem‐like cells in glioblastoma and colorectal cancer through metabolic pathways

Cancer stem‐like cells (CSCs) are thought to be the main cause of tumor occurrence, progression and therapeutic resistance. Strong research efforts in the last decade have led to the development of several tailored approaches to target CSCs with some very promising clinical trials underway; however, until now no anti‐CSC therapy has been approved for clinical use. Given the recent improvement in our understanding of how onco‐proteins can manipulate cellular metabolic networks to promote tumorigenesis, cancer metabolism research may well lead to innovative strategies to identify novel regulators and downstream mediators of CSC maintenance. Interfering with distinct stages of CSC‐associated metabolics may elucidate novel, more efficient strategies to target this highly malignant cell population. Here recent discoveries regarding the metabolic properties attributed to CSCs in glioblastoma (GBM) and malignant colorectal cancer (CRC) were summarized. The association between stem cell markers, the response to hypoxia and other environmental stresses including therapeutic insults as well as developmentally conserved signaling pathways with alterations in cellular bioenergetic networks were also discussed. The recent developments in metabolic imaging to identify CSCs were also summarized. This summary should comprehensively update basic and clinical scientists on the metabolic traits of CSCs in GBM and malignant CRC.     

Activation of Multiple ERBB Family Receptors Mediates Glioblastoma Cancer Stem-like Cell Resistance to EGFR-Targeted Inhibition

Epidermal growth factor receptor (EGFR) signaling is strongly implicated in glioblastoma (GBM) tumorigenesis. However, molecular agents targeting EGFR have demonstrated minimal efficacy in clinical trials, suggesting the existence of GBM resistance mechanisms. GBM cells with stem-like properties (CSCs) are highly efficient at tumor initiation and exhibit therapeutic resistance. In this study, GBMCSC lines showed sphere-forming and tumor initiation capacity after EGF withdrawal from cell culture media, compared with normal neural stem cells that rapidly perished after EGF withdrawal. Compensatory activation of related ERBB family receptors (ERBB2 and ERBB3) was observed in GBM CSCs deprived of EGFR signal (EGF deprivation or cetuximab inhibition), suggesting an intrinsic GBM resistance mechanism for EGFR-targeted therapy. Dual inhibition of EGFR and ERBB2 with lapatinib significantly reduced GBM proliferation in colony formation assays compared to cetuximab-mediated EGFR-specific inhibition. Phosphorylation of downstream ERBB signaling components (AKT, ERK1/2) and GBM CSC proliferation were inhibited by lapatinib. Collectively, these findings show that GBM therapeutic resistance to EGFR inhibitors may be explained by compensatory activation of EGFR-related family members (ERBB2, ERBB3) enabling GBM CSC proliferation, and therefore simultaneous blockade of multiple ERBB family members may be required for more efficacious GBM therapy.     

The emerging role of tumor-suppressive microRNA-218 in targeting glioblastoma stemness

Glioblastoma multiforme (GBM) is by far the most common and most aggressive malignant primary tumor in humans and has poor outcomes despite many advances in treatment using combinations of surgery, radiotherapy and chemotherapy. Recent studies demonstrate that GBM contains a subpopulation of cancer cells with stem cell characteristics, including self-renewal and multipotentiality, and that these cancer stem cells contribute to disease progression. MicroRNAs (miRNAs) are small non-coding regulatory RNA molecules that regulate a variety of cellular processes, including stem cell maintenance. An accumulating body of evidence shows that miR-218 may act as a tumor suppressor by inhibiting glioblastoma invasion, migration, proliferation and stemness through its different targets, indicating the great potential and relevance of miR-218 as a novel class of therapeutic target in glioblastoma.     

MicroRNA-137 is downregulated in glioblastoma and inhibits the stemness of glioma stem cells by targeting RTVP-1

Glioblastomas (GBM), the most common and aggressive malignant astrocytic tumors, contain a small subpopulation of cancer stem cells (GSCs) that are implicated in therapeutic resistance and tumor recurrence. Here, we study the expression and function of miR-137, a putative suppressor miRNA, in GBM and GSCs. We found that the expression of miR-137 was significantly lower in GBM and GSCs compared to normal brains and neural stem cells (NSCs) and that the miR-137 promoter was hypermethylated in the GBM specimens. The expression of miR-137 was increased in differentiated NSCs and GSCs and overexpression of miR-137 promoted the neural differentiation of both cell types. Moreover, pre-miR-137 significantly decreased the self-renewal of GSCs and the stem cell markers Oct4, Nanog, Sox2 and Shh. We identified RTVP-1 as a novel target of miR-137 in GSCs; transfection of the cells with miR-137 decreased the expression of RTVP-1 and the luciferase activity of RTVP-1 3''-UTR reporter plasmid. Furthermore, overexpression of RTVP-1 plasmid lacking its 3''-UTR abrogated the inhibitory effect of miR-137 on the self-renewal of GSCs. Silencing of RTVP-1 decreased the self-renewal of GSCs and the expression of CXCR4 and overexpression of CXCR4 abrogated the inhibitory effect of RTVP-1 silencing on GSC self-renewal. These results demonstrate that miR-137 is downregulated in GBM probably due to promoter hypermethylation. miR-137 inhibits GSC self-renewal and promotes their differentiation by targeting RTVP-1 which downregulates CXCR4. Thus, miR-137 and RTVP-1 are attractive therapeutic targets for the eradication of GSCs and for the treatment of GBM.     

Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor-related apoptosis-inducing ligand and induces an alternative cell death pathway

Human glioblastoma (GBM) cells are notorious for their resistance to apoptosis-inducing therapeutics. We have identified lanatoside C as a sensitizer of GBM cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death partly by upregulation of the death receptor 5. We show that lanatoside C sensitizes GBM cells to TRAIL-induced apoptosis in a GBM xenograft model in vivo. Lanatoside C on its own serves as a therapeutic agent against GBM by activating a caspase-independent cell death pathway. Cells treated with lanatoside C showed necrotic cell morphology with absence of caspase activation, low mitochondrial membrane potential, and early intracellular ATP depletion. In conclusion, lanatoside C sensitizes GBM cells to TRAIL-induced cell death and mitigates apoptosis resistance of glioblastoma cells by inducing an alternative cell death pathway. To our knowledge, this is one of the first examples of use of caspase-independent cell death inducers to trigger tumor regression in vivo. Activation of such mechanism may be a useful strategy to counter resistance of cancer cells to apoptosis.     

CXCR4 expression mediates glioma cell invasiveness

Glioblastoma multiforme is a highly invasive tumor bearing a dismal prognosis. Experimental strategies that focus on the specific biological cues governing the invasive capacity of these tumors may hold significant therapeutic promise. In this context, we describe the in vitro and in vivo association of the cell surface chemokine receptor, CXCR4, with the development of an invasive phenotype in malignant glioblastoma. We demonstrate that invasive populations of glioma cells overexpress CXCR4 at the message and protein levels, and that this expression ranges from 25- to 89-fold higher than that found in noninvasive tumor cells. Furthermore, neutralization of CXCR4 significantly impairs the in vitro invasive capacity of malignant glial cells. In addition, glioma cells secrete CXCL12 and demonstrate robust invasive capacity toward a CXCL12 gradient in vitro. These findings underscore the importance of CXCR4 as a potential therapeutic target for the treatment of invasive glioblastoma.     

Abrogation of radioresistance in glioblastoma stem‐like cells by inhibition of ATM kinase

Resistance to radiotherapy in glioblastoma (GBM) is an important clinical problem and several authors have attributed this to a subpopulation of GBM cancer stem cells (CSCs) which may be responsible for tumour recurrence following treatment. It is hypothesised that GBM CSCs exhibit upregulated DNA damage responses and are resistant to radiation but the current literature is conflicting. We investigated radioresistance of primary GBM cells grown in stem cell conditions (CSC) compared to paired differentiated tumour cell populations and explored the radiosensitising effects of the ATM inhibitor KU‐55933.We report that GBM CSCs are radioresistant compared to paired differentiated tumour cells as measured by clonogenic assay. GBM CSC''s display upregulated phosphorylated DNA damage response proteins and enhanced activation of the G2/M checkpoint following irradiation and repair DNA double strand breaks (DSBs) more efficiently than their differentiated tumour cell counterparts following radiation.Inhibition of ATM kinase by KU‐55933 produced potent radiosensitisation of GBM CSCs (sensitiser enhancement ratios 2.6–3.5) and effectively abrogated the enhanced DSB repair proficiency observed in GBM CSCs at 24 h post irradiation. G2/M checkpoint activation was reduced but not abolished by KU‐55933 in GBM CSCs.ATM kinase inhibition overcomes radioresistance of GBM CSCs and, in combination with conventional therapy, has potential to improve outcomes for patients with GBM.     

Na+/K+-ATPase β2-subunit (AMOG) expression abrogates invasion of glioblastoma-derived brain tumor-initiating cells

Background

Mechanisms of glioma invasion remain to be fully elucidated. Glioma cells within glioblastoma multiforme (GBM) range from well-differentiated tumor cells to less-differentiated brain tumor-initiating cells (BTICs). The β2-subunit of Na⁺/K⁺-ATPase, called the adhesion molecule on glia (AMOG), is highly expressed in normal glia but is thought to be universally downregulated in GBM. To test our hypothesis that expression of AMOG is heterogeneous in GBM and confers a less invasive phenotype, we compared it between BTICs and differentiated cells from patient-matched GBM and then tested GBM invasion in vitro after AMOG overexpression.

Methods

Immunohistochemistry, immunoblotting, and real-time PCR were used to characterize AMOG protein and mRNA expression in tumor samples, BTICs, and differentiated cells. Matrigel invasion assay, scratch assay, and direct cell counting were used for testing in vitro invasion, migration, and proliferation, respectively.

Results

While AMOG expression is heterogeneous in astrocytomas of grades II–IV, it is lost in most GBM. BTICs express higher levels of AMOG mRNA and protein compared with patient-matched differentiated tumor cells. Overexpression of AMOG decreased GBM cell and BTIC invasion without affecting migration or proliferation. Knockdown of AMOG expression in normal human astrocytes increased invasion.

Conclusions

AMOG expression inhibits GBM invasion. Its downregulation increases invasion in glial cells and may also represent an important step in BTIC differentiation. These data provide compelling evidence implicating the role of AMOG in glioma invasion and provide impetus for further investigation.     

Proscillaridin A is cytotoxic for glioblastoma cell lines and controls tumor xenograft growth in vivo

Glioblastoma is the most frequent primary brain tumor in adults. Because of molecular and cellular heterogeneity, high proliferation rate and significant invasive ability, prognosis of patients is poor. Recent therapeutic advances increased median overall survival but tumor recurrence remains inevitable. In this context, we used a high throughput screening approach to bring out novel compounds with anti-proliferative and anti-migratory properties for glioblastoma treatment. Screening of the Prestwick chemical library® of 1120 molecules identified proscillaridin A, a cardiac glycoside inhibitor of the Na⁺/K⁺ ATPase pump, with most significant effects on glioblastoma cell lines. In vitro effects of proscillaridin A were evaluated on GBM6 and GBM9 stem-like cell lines and on U87-MG and U251-MG cell lines. We showed that proscillaridin A displayed cytotoxic properties, triggered cell death, induced G₂/M phase blockade in all the glioblastoma cell lines and impaired GBM stem self-renewal capacity even at low concentrations. Heterotopic and orthotopic xenotransplantations were used to confirm in vivo anticancer effects of proscillaridin A that both controls xenograft growth and improves mice survival. Altogether, results suggest that proscillaridin A is a promising candidate as cancer therapies in glioblastoma. This sustains previous reports showing that cardiac glycosides act as anticancer drugs in other cancers.     

Crosstalk between M2 macrophages and glioma stem cells

Purpose

Given its extremely poor prognosis, there is a pressing need for an improved understanding of the biology of glioblastoma multiforme (GBM), including the roles of tumor subpopulations that may contribute to their growth rate and therapy resistance. The most malignant phenotypes of GBM have been ascribed to the presence of subpopulations of cancer stem cells (CSCs), which are resistant to chemotherapeutic drugs and ionizing radiation and which promote invasiveness and metastasis. The mechanisms by which the CSC state is obtained and by which it promotes tumor maintenance are only beginning to emerge. We hypothesize that M2 polarized macrophages may affect CSC phenotypes via cell-cell communication.

Methods

We investigated the interplay between glioma CSCs and macrophages via co-culture. The invasiveness of CSCs in the absence and presence of macrophages was assessed using collagen degradation and Transwell migration assays. The role of STAT3 as a CSC phenotypic mediator was assessed using siRNA-mediated gene silencing.

Results

We found that the levels of a M2 macrophage-specific secreted cytokine, TGF-β1, were elevated in the presence of CSCs, regardless of whether the cells were plated as contacting or non-contacting co-cultures. In addition, we found that the co-culture resulted in enhanced expression of M2 markers in macrophages that were previously polarized to the M1 phenotype. siRNA-mediated STAT3 silencing was found to reduce the chemo-responsiveness and migratory abilities of the CSCs. Combination treatment of STAT3 siRNA and DNA alkylating agents was found to further abrogate CSC functions.

Conclusions

Our data indicate that the co-culture of CSCs and macrophages results in bi-directional signaling that alters the phenotypes of both cell types. These results provide an explanation for recently observed effects of macrophages on GBM tumor cell growth, motility and therapeutic resistance, and suggest potential therapeutic strategies to disrupt the CSC phenotype by impairing its communication with macrophages.

     

Cancer cell heterogeneity and plasticity: A paradigm shift in glioblastoma

Phenotypic plasticity has emerged as a major contributor to intra-tumoral heterogeneity and treatment resistance in cancer. Increasing evidence shows that glioblastoma (GBM) cells display prominent intrinsic plasticity and reversibly adapt to dynamic microenvironmental conditions. Limited genetic evolution at recurrence further suggests that resistance mechanisms also largely operate at the phenotypic level. Here we review recent literature underpinning the role of GBM plasticity in creating gradients of heterogeneous cells including those that carry cancer stem cell (CSC) properties. A historical perspective from the hierarchical to the nonhierarchical concept of CSCs towards the recent appreciation of GBM plasticity is provided. Cellular states interact dynamically with each other and with the surrounding brain to shape a flexible tumor ecosystem, which enables swift adaptation to external pressure including treatment. We present the key components regulating intra-tumoral phenotypic heterogeneity and the equilibrium of phenotypic states, including genetic, epigenetic, and microenvironmental factors. We further discuss plasticity in the context of intrinsic tumor resistance, where a variable balance between preexisting resistant cells and adaptive persisters leads to reversible adaptation upon treatment. Innovative efforts targeting regulators of plasticity and mechanisms of state transitions towards treatment-resistant states are needed to restrict the adaptive capacities of GBM.     

CXCR4 knockdown by small interfering RNA abrogates breast tumor growth in vivo

Breast cancer cells express the chemokine receptor CXCR4 and frequently metastasize to organs with an abundant source of the CXCR4 ligand, stromal cell-derived factor 1 (SDF-1). The chemokine receptor CXCR4 plays an active role in the metastasis of breast cancer. Here, we show that a small interfering RNA (siRNA) against CXCR4 effectively downregulates CXCR4 expression in human MDA-MB-231 breast cancer cells, leading to significant decrease in breast cancer cell invasion and adhesion. It was further found that tumor cells lacking CXCR4 expression proliferated at a much slower rate than control cells in vitro. Surprisingly, tumor cells lacking CXCR4 expression failed to grow in SCID mice in repeated experiments, providing the first direct evidence for an essential role of CXCR4 in breast cancer growth in vivo. This finding suggests an expanded role for the CXCR4 molecule in tumor cell growth in vivo, in addition to its role in breast cancer metastasis. This study implies the CXCR4 molecule as a potential target to control breast tumor growth as well as metastasis.     

MET signaling regulates glioblastoma stem cells

Glioblastomas multiforme (GBM) contain highly tumorigenic, self-renewing populations of stem/initiating cells [glioblastoma stem cells (GSC)] that contribute to tumor propagation and treatment resistance. However, our knowledge of the specific signaling pathways that regulate GSCs is limited. The MET tyrosine kinase is known to stimulate the survival, proliferation, and invasion of various cancers including GBM. Here, we identified a distinct fraction of cells expressing a high level of MET in human primary GBM specimens that were preferentially localized in perivascular regions of human GBM biopsy tissues and were found to be highly clonogenic, tumorigenic, and resistant to radiation. Inhibition of MET signaling in GSCs disrupted tumor growth and invasiveness both in vitro and in vivo, suggesting that MET activation is required for GSCs. Together, our findings indicate that MET activation in GBM is a functional requisite for the cancer stem cell phenotype and a promising therapeutic target.     

Glycosylation-related gene expression is linked to differentiation status in glioblastomas undifferentiated cells

Glioblastoma Multiforme (GBM) is the most frequent malignant brain tumor with still poor prognosis. Tumor initiation, growth and recurrences might depend on Brain Tumor Stem Cells (BTSCs) which can promote tumor aggressiveness and potentially affords new therapeutic target. Recent works emphasized aberrant cell-surface glyco-conjugate expression in brain tumors suggesting that altered glycosylation is closely linked to cancer tumor metastasis and invasive process. Post-translational changes might play a key role in determining the fates of most aggressive and undifferentiated cells such as self-renewal, proliferation and differentiation. In order to characterize the glycosylation-related genes involved in differentiation status of the BTSCs, two glioblastoma cell lines, U87-MG and U251 have been cultured according to two conditions leading to undifferentiated floating cells or differentiated adherent cells. The expression level of 559 glycosylation related genes has been analyzed by Taqman Low Density Array (TLDA) analysis and allowed to isolate eight up-regulated genes specific of a subpopulation of undifferentiated cells. Protein expression has been confirmed. Among main selected genes, five are also over-expressed in the undifferentiated condition in primary cultures provided by three GBM freshly isolated from patient. This work suggests that new Glycosylation-related gene signature might improve the characterization of the most aggressive and undifferentiated cells and supports that in future, N-linked glycosylation might provide new target to develop therapeutic strategy for inhibiting tumor growth.