Francesca Pistollato

Sara Abbadi

Elena Rampazzo

Giampietro Viola
email: giampietro.viola.1@unipd.it

Alessandro Della Puppa

Lucia Cavallini

Chiara Frasson

Luca Persano

David M Panchision

Giuseppe Basso

Alessandro Della Puppa

Hypoxia and succinate antagonize 2-deoxyglucose effects on glioblastoma

Keywords: 2-deoxyglucose, Hypoxia, Hypoxia Inducible Factor-1α, proline hydroxylase 2, GBM differentiation GBM, glioblastoma multiforme, BMP2, bone morphogenetic protein2, HIF-1α., hypoxia inducible factor 1α, PHD2, Proline hydroxylase2, SDH, succinate dehydrogenase

A c c e p t e d M a n u s c r i p t 3

1.Introduction

Brain tumors and particularly glioblastoma multiforme (GBM) are highly proliferative tumors characterized by a hypoxic microenvironment. Recent literature shows that this hypoxic microenvironment correlates with tumor aggressiveness [START_REF] Azuma | Hypoxia and differentiation in squamous cell carcinomas of the uterine cervix: pimonidazole and involucrin[END_REF][START_REF] Helczynska | Hypoxia promotes a dedifferentiated phenotype in ductal breast carcinoma in situ[END_REF][START_REF] Jogi | Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crestlike phenotype[END_REF] and over-activity of hypoxia inducible factor-1α (HIF-1α) [START_REF] Smith | Silencing of epidermal growth factor receptor suppresses hypoxia-inducible factor-2-driven VHL-/-renal cancer[END_REF] is implicated in tumor progression and in the maintenance of a de-differentiated (i.e. stem) tumor cell phenotype [START_REF] Jogi | Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crestlike phenotype[END_REF]. Particularly, HIF-1α has been found to contribute to a differentiation defect in malignant gliomas [START_REF] Lu | Hypoxia-inducible factor-1alpha blocks differentiation of malignant gliomas[END_REF], even following exogenous bone morphogenetic protein2 (BMP2) treatment, known to induce glial commitment in GBM [START_REF] Piccirillo | Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumourinitiating cells[END_REF][START_REF] Pistollato | Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma[END_REF][START_REF] Pistollato | Molecular mechanisms of HIF-1alpha modulation induced by oxygen tension and BMP2 in glioblastoma derived cells[END_REF]. These evidences shed new light on the differentiation therapy of solid tumors by HIF-1α targeting. Hypoxia has a role in normal physiological responses, such as carotid body growth and generation of new neural crest derived glomus cells [START_REF] Pardal | Glia-like stem cells sustain physiologic neurogenesis in the adult mammalian carotid body[END_REF], and angiogenesis [START_REF] Wong | Antiangiogenesis treatment for glioblastoma multiforme challenges and opportunities[END_REF]. It is implicated in the regulation of several developmentally important signaling pathways, such as Notch [START_REF] Gustafsson | Hypoxia requires notch signaling to maintain the undifferentiated cell state[END_REF] and BMP pathways [START_REF] Pistollato | Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma[END_REF], as well as glucose metabolism [START_REF] Dang | Oncogenic alterations of metabolism[END_REF]. Moreover, hypoxia alters cancer cells metabolism by increasing tumor anaerobic glycolysis and reducing mitochondrial activity [START_REF] Hsu | Cancer cell metabolism: Warburg and beyond[END_REF]. Particularly SDH, the enzyme complex II bound to the inner mitochondrial membrane that converts succinate to fumarate via FAD reduction to FADH2, has been found inactive in some solid tumors [START_REF] King | Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer[END_REF]. This has been described as a key event leading to the accumulation of intra-cytoplasmic succinate and reactive oxygen species (ROS) causative of the "pseudo-hypoxic" effect, a major tumorrelated hallmark, characterized by the activation of HIF even under normoxia [START_REF] King | Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer[END_REF][START_REF] Mackenzie | Cell-permeating alpha-ketoglutarate derivatives alleviate pseudohypoxia in succinate dehydrogenase-deficient cells[END_REF]. As intra-tumoral hypoxia increases glycolytic activity in many types of cancer, including GBM, limiting glucose uptake or glucose metabolism is a promising therapeutic approach for cancer treatment [START_REF] Jin | Metabolic catastrophe as a means to cancer cell death[END_REF][START_REF] Zhu | 2-Deoxyglucose as an energy restriction mimetic agent: effects on mammary carcinogenesis and on mammary tumor cell growth in vitro[END_REF] in order to reduce tumor growth as shown in animal models [START_REF] Michels | Caloric restriction and incidence of breast cancer[END_REF]. 2deoxyglucose (2-DG) is a glucose analogue which is converted by hexokinase to phosphorylated 2-DG, inhibiting glycolysis. Inhibition of glycolysis by phosphorylated 2-A c c e p t e d M a n u s c r i p t 4 DG, 2-DG-6-phosphate, is thought to occur via inhibition of hexokinase and phosphohexoisomerase [START_REF] Liu | Dual mechanisms for glucose 6-phosphate inhibition of human brain hexokinase[END_REF][START_REF] Nelson | Crane PD The interaction among glucose transport, hexokinase, and glucose-6-phosphatase with respect to 3H-2-deoxyglucose retention in murine tumor models[END_REF] 2-DG is effective in several mouse tumor models, particularly in combination therapy [START_REF] Zhu | 2-Deoxyglucose as an energy restriction mimetic agent: effects on mammary carcinogenesis and on mammary tumor cell growth in vitro[END_REF]. Indeed, metformin, a widely used antidiabetic agent, has been seen to exert antitumoral and antiproliferative action in prostate cancer cells, especially when used in combination with 2-DG [START_REF] Sahra | Targeting cancer cell metabolism: the combination of metformin and 2-deoxyglucose induces p53-dependent apoptosis in prostate cancer cells[END_REF]. Moreover, earlier studies have shown that 2-DG significantly enhances the cytotoxic effects of anticancer agents like topoisomerase inhibitors and a radiomimetic drug (bleomycin) in established human tumor cell lines. Also, it has been shown that 2-DG can be used as a modulator of radiation response [START_REF] Singh | Optimizing cancer radiotherapy with 2-deoxy-d-glucose dose escalation studies in patients with glioblastoma multiforme[END_REF][START_REF] Gupta | Enhancement of radiation and chemotherapeutic drug responses by 2-deoxy-D-glucose in animal tumors[END_REF]. These studies demonstrate that targeting carbohydrate metabolism may be an effective therapeutic approach. Here we investigated how 2-DG affects primary GBM-derived cells growth, by promoting differentiation and an increase in SDH activity, leading to HIF-1α degradation.

Addition of succinate and preserving cells in a hypoxic microenvironment limit 2-DG effects by maintaining GBM in an undifferentiated status. Finally, our results unravel a novel mechanistic control on GBM cell metabolism and phenotype by 2-DG, succinate and oxygen tension. S1 (see Supplementary Information).

2.Materials and Methods

Isolation and gas-controlled expansion of cells

We dissociated and cultured cells as previously described [START_REF] Pistollato | Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma[END_REF]. Briefly, cells were cultured in control medium consisted of HAM'S-F12/DME with 25 mM glucose, (Irvine Scientific, Santa Ana, CA), BIT9500, 10%, as serum substitute (Stem Cell Technologies, Milano, Italy).

For continuous expansion, one-half of the medium was replaced every day and cells were passaged every 7-10 days using TrypLE (Invitrogen, Carlsbad, CA) to dissociate them. Cells were not cultured for more than 8 passages in vitro in order to avoid long term culture related effects. Cell viability was evaluated by trypan blue exclusion. For low glucose experiments, cells were cultured in DMEM/F12, glucose-free, (Gibco BRL, Milano, Italy) with BIT9500 (10%), for a final glucose concentration of 2.5 mM. Two different glucose concentrations were established by adding D-glucose (12.5 and 25 mM final concentration). In experimental groups 2-deoxyglucose (2-DG) (Sigma, Milano, Italy) was added to a final concentration of 12.5 or 25 mM as reported in literature [START_REF] Hunter | Blekkenhorst GH The effects of 2-deoxyglucose and amino-oxyacetic acid on the radiation response of mammalian cells in vitro[END_REF][START_REF] Jelluma | Glucose withdrawal induces oxidative stress followed by apoptosis in glioblastoma cells but not in normal human astrocytes[END_REF] . Cells were maintained in an atmosphere of 5% carbon dioxide and 2% (Ruskinn C300, Ruskinn Technology Ltd, Bridgend, UK) or 20% oxygen balanced with nitrogen [START_REF] Pistollato | Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma[END_REF]. In some experiments, esterificated diethyl-succinate (5 mM, Sigma-Aldrich, Milano, Italy) or pyruvate (1 mM, Sigma-Aldrich, Milano, Italy) were added for 18 hr. Pyruvate, the physiological substrate of pyruvate dehydrogenase (PDH), was used to confirm specificity of antibody specific for PDH E1α.

Immunocytochemistry

Cells were fixed in cold 4% paraformaldehyde for 15 min, rinsed and stored prior to analysis.

Primary antibody staining was performed for nestin (mouse, 1:200, Millipore, Billerica, MA), glial fibrillary acidic protein (GFAP, mouse, 1:1000, Sigma-Aldrich, Milano, Italy), β-IIItubulin (mouse, monoclonal Tuj-1, 1:1000, Covance, Milano, Italy). After incubation, cells

CD133, CD90, cell cycle and Annexin-V/Propidium Iodide analyses

Cells (2x10 5 cells/ml) were incubated with anti-human CD133 (clone AC133/2, PE conjugated, (Miltenyi Biotec, Bologna, Italy) as described in [START_REF] Panchision | Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24[END_REF] and anti-human CD90 clone 5E10, FITC conjugated, ( BioLegend, Milano, Italy). Viability was assessed by adding 7-amino-actinomycin-D (7-AAD, 50 ng/ml, BD Biosciences, Milano, Italy) prior to analysis.

Cells were analyzed on a Cytomics FC 500 flow cytometer (Beckman Coulter, Milano, Italy).

Relative percentages of different subpopulations were calculated based on live gated cells.

Unlabeled cells and cells incubated with appropriate isotypic control antibodies were first acquired to ensure labeling specificity. For cell cycle phase analysis, cells were incubated with BrdU (1 mM, BD, Milano, Italy) for 45 min then washed and prepared for analysis as directed (BrdU Flow Kit Staining Protocol, BD, Milano, Italy). Analysis of apoptosis was performed as recommended by using the Annexin Fluo kit (Roche Biochemicals, Milano, Italy).

Mitochondrial membrane potential, reactive oxygen species (ROS) and GSH analyses

The mitochondrial membrane potential was measured with the lipophilic cation 5,5′,6,6′tetrachlo-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine (JC-1, Invitrogen, Milano, Italy), as described in [START_REF] Viola | Induction of apoptosis by photoexcited tetracyclic compounds derivatives of benzo[b]thiophenes and pyridines[END_REF], a fluorescent probe known to selectively enter into the mitochondria and used to measure changes in mitochondrial membrane potential (Δψ mt ). JC-1 forms aggregates (red color) at higher potential and monomers (green color) at low membrane potential. To (not shown). The production of ROS was measured by flow cytometry using hydrohethidine (HE) (Invitrogen, Milano, Italy) [START_REF] Viola | Induction of apoptosis by photoexcited tetracyclic compounds derivatives of benzo[b]thiophenes and pyridines[END_REF]. Analysis of total GSH level was measured by using monobromobimane (mBrB), (Invitrogen, Milano, Italy) (50 μM) [START_REF] Chow | Flow cytometric determination of glutathione in clinical samples[END_REF][START_REF] Rice | Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo[END_REF] and chloromethyldichlorofluorescein diacetate (CM-DCFDA) (5 μM, Invitrogen, Milano Italy), preincubating cells for 15 and 30 min at 37 °C respectively prior to analysis.

Western blot analyses

Total protein extracts were isolated in lysis buffer prepared as follows: TPER Reagent (Pierce, Milano, Italy), NaCl 300 mM, orthovanadate 1 mM (Sigma, Milano, Italy), AEBSF 2 mM (PEFABLOC, Roche Biochemicals, Milano, Italy), Aprotinin 1 μg/mL (Sigma-Aldrich, Milano, Italy), Pepstatin-A 5 μg/ml (Sigma-Aldrich, Milano, Italy) and Leupeptin 1 μg/ml (Sigma-Aldrich, Milano, Italy). Equal amounts of protein (10 µg) were resolved using SDS-PAGE gels and transferred to PVDF Hybond-p membrane (GE Healthcare, Catania, Italy).

Membranes were blocked with I-blockTM Blocking (Tropix, Applied Biosystem, Monza, Italy) for at least 2 hr under rotation at room temperature. Membranes were then incubated with primary antibodies against HIF-1α (mouse, 1:250, BD Biosciences, Milano, Italy), HIF-2α (mouse, 1:250, Novus Biologicals, Milano, Italy), PHD2 (goat, 1:300, Santa Cruz, Santa Cruz, CA, USA), p21 cip1 (mouse, 1:1000, Sigma-Aldrich, Milano, Italy), Bcl-2, Bcl-XL, PARP, (all rabbit, 1:1000, Cell Signaling, Milano, Italy), phosphorylated PDH E1α serine 293 (rabbit, 1:5000, Novus Biologicals, Milano, Italy), total PDH E1α subunit (1:1000, Invitrogen, Carlsbad, CA, USA) or β-actin (mouse, 1:10000, Sigma-Aldrich, Milano, Italy) for 2 hr. Membranes were next incubated with peroxidase-labeled goat anti-rabbit IgG (1:100000, Santa Cruz, Santa Cruz, CA, USA) for 60 min. All membranes were visualized using ECL Advance (GE Healthcare, Catania, Italy) and exposed to Hyperfilm MP (GE Healthcare, Catania, Italy).

Lactate Assay

Extracellular lactate measurement was performed using the Lactate Assay Kit, (BioVision, Torino, Italy) kindly provided by Dr. Alberto Burlina (University of Padova), following the manufacturer instructions.

Succinate dehydrogenase activity

To evaluate aerobic metabolism succinate dehydrogenase (SDH) activity has been measured, since enzyme activity is considered an indicator of tricarboxylic acid (TCA) cycle activity.

To measure SDH activity, we used the methods described by Levine et al. with little modifications [START_REF] Levine | Bioenergetic adaptation of individual human diaphragmatic myofibers to severe COPD[END_REF]. Briefly, cell monolayers (not fixed) were snap-frozen at -80°C for at least 1 hr and were incubated for 20 min in the dark at 37°C in a reaction buffer containing 12.3 mM diethyl-succinate (Sigma, Milano, Italy), 0.2 mM 1-methoxy-5-methylphenazine methyl sulphate (Sigma-Aldrich, Milano, Italy), 1.2 mM nitro blue tetrazolium (NBT, Sigma-Aldrich, Milano, Italy) in 50 mM Tris-HCl, pH 7.6. We used 12.3 mM malonate (Sigma, Milano, Italy) to test aspecific reactions, and 3-nitropropionic acid (3-NPA, 500 μM, Sigma-Aldrich, Milano, Italy), to irreversibly inactivate SDH, as negative control. The enzyme activity is determined by measuring the formation of nitro blue diformazan (NBT-dfz) due to NBT reduction and the end product (i.e. blue cells) is visible by bright field microscopy or measured at 570 nm, by absorption spectroscopy.

A c c e p t e d M a n u s c r i p t 9

ATP assay

Cells were cultured with 2-DG for 18 or 24 hr at either 2% or 20% oxygen, collected and counted. The ATP content per 100000 cells was determined using the CellTiter-Glo luminescent assay (Promega, Milano, Italy) according to manufacturer instruction, using a Victor 3TM luminometer (Perkin Elmer, Milano, Italy). Data were normalized to ATP content in cells cultured with glucose at 2% O 2 .

MTT assay

Individual wells of a 96-well tissue culture microtiter plate (Falcon, BD, Milano, Italy) were inoculated with 100 l of medium containing 5x10 3 of GBM cells. The plates were incubated overnight prior to the analyses. After medium removal, 2-DG was added and cells were incubated for 18 or 24 hr at either 2% or 20% oxygen. Cell viability was assayed by the MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide)] test as previously described [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF].

Real-Time PCR analysis

RNA was isolated from cells using Trizol (Invitrogen, Milano, Italy) and 1 μg of total RNA reverse-transcribed using SuperScript RNAse H-Reverse Transcriptase (Invitrogen, Milano, Italy). Quantitative RT-PCR reactions were run in triplicate using Brilliant® SYBR® Green QPCR Core Reagent Kit (Stratagene, La Jolla, CA, USA). Fluorescent emission was recorded in real-time (Sequence Detection System 7900HT, Applied Biosystems, Foster City, CA, USA). Gene expression profiling was completed using the comparative Ct method of relative quantification. PCR amplification conditions consisted of 50 cycles with primers annealing at 60°C. These set of primers were used: PHD2, F: 5'-GGGACATTCATTGCCTCACT-3', R: (http://frodo.wi.mit.edu/primer3/input.htm) and the specificity of the primers for GUSB, HIF-1α and PHD2 sequences has been evaluated by using the software Human BLAT Search (http://genome.ucsc.edu/cgi-bin/hgBlat?command=start). PCR amplicons had been previously evaluated on agarose gel, and during SYBR green analyses primers dissociation curves have been checked in each run to ensure primers specificity. Relative RNA quantities were normalized to GUSB as a housekeeping gene and 2% oxygen T0 was used as the calibrating condition (ΔΔCt Method). GBM cells were transfected either with a wHRE or with a mHRE. Along with these vectors, also a Renilla luciferase vector has been transfected in order to normalize luciferase detection (Promega, Madison, WI, USA). 12 hr after transfection, total medium change was done and Finally, cells were processed and analysis of HRE-luciferase activity was performed as described (Dual-Luciferase Reporter Assay System, Promega, Madison, WI, USA) by using a plate-reading luminometer (Victor 3TM , Perkin Elmer, Milano, Italy).

Hypoxia Responsive Element (HRE)-luciferase reporter assay

Statistical analysis

Graphs and statistical analyses were prepared using Prism 3.03 (Graph Pad, La Jolla, CA).

All values were presented as mean ± standard error of the mean (S.E.M.). Statistical significance was measured by one-way ANOVA with post-hoc Newman-Keul's test, *p < 0.05, **p < 0.01, ***p < 0.001. For all graphs, an asterisk over a bracket indicates a significant difference with a variable as indicated, an asterisk over a bar indicates a significant difference between the 2% and 20% oxygen at either a particular time point or culture condition.

3.Results

2-DG induces loss of GBM cell adhesive properties and decreases cell proliferation.

It has been suggested that hypoxia is one of the major regulatory factors in the brain tumor niche, since it positively correlates with brain tumor aggressiveness, supporting the idea that hypoxia may confer a growth advantage to brain tumor stem cells [START_REF] Helczynska | Hypoxia promotes a dedifferentiated phenotype in ductal breast carcinoma in situ[END_REF][START_REF] Jogi | Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crestlike phenotype[END_REF][START_REF] Lu | Hypoxia-inducible factor-1alpha blocks differentiation of malignant gliomas[END_REF][START_REF] Pistollato | Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma[END_REF]. Under hypoxia cancer cell metabolism is pushed toward increased anaerobic glycolysis, which has been extensively described as a hallmark of tumor cell biology [START_REF] Hsu | Cancer cell metabolism: Warburg and beyond[END_REF]. Here, we used primary cultures of adult GBM tumors to study the interaction between oxygen levels and the effects elicited by 2-DG, thereby inducing a block of glycolysis. We evaluated cells grown in 2% oxygen, which represents the condition typically found in hypoxic solid tumors [START_REF] Ljungkvist | Dynamics of tumor hypoxia measured with bioreductive hypoxic cell markers[END_REF], versus cells that were acutely exposed to higher oxygen tension (20% oxygen). We found that (Fig. 1C), consistent with GBM cell metabolic dependence on anaerobic glycolysis. Addition of 2-DG under both oxygen tensions led to a strong extracellular lactate decrease in both GBM and SVZ-derived cells (Fig. 1B,C). Moreover, 2-DG lowered GBM cells growth (Fig. 1D), especially in cells acutely exposed to high oxygen, while SVZ cells did not undergo morphological changes (Suppl. Fig. 2A) and underwent proliferation reduction only when acutely exposed to high oxygen tension (Fig. 1E).

Analysis of GBM cell viability by trypan blue revealed no significant differences among conditions (not shown), while methyltetrazolium reduction (MTT) (Fig. 1F), indicative of extra-mitochondrial NADH and NADPH [START_REF] Berridge | Characterization of the cellular reduction of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction[END_REF] occurred. Importantly, re-addition of glucose after 2-DG exposure did not recover GBM cells growth (data not shown). Also, intracellular ATP content in GBM cells, which was found higher at 20% oxygen tension consequentially to stimulated mitochondrial activity, was decreased following addition of 2-DG (Fig. 1G).

2-DG increases succinate dehydrogenase and pyruvate dehydrogenase activity.

We sought to investigate if 2-DG, besides determining a glycolytic block, was able to perturb mitochondrial metabolism. Thus, we analyzed succinate dehydrogenase (SDH) activity. It is Additionally, we analyzed the phosphorylation state of pyruvate dehydrogenase E1α (pPDH E1α) in order to test if addition of 2-DG induced an increase of mitochondrial metabolism.

The PDH complex contributes to transforming pyruvate into acetyl-CoA, which is used in the citric acid cycle to carry out cellular respiration. The activation state of PDH is controlled by the enzyme pyruvate dehydrogenase kinase (PDK), which inhibits PDH activity by phosphorylating it using ATP [START_REF] Mcfate | Pyruvate dehydrogenase complex activity controls metabolic and malignant phenotype in cancer cells[END_REF]. We found that GBM cells maintained at 2% oxygen had a higher level of pPDH compared to cells acutely exposed to 20% oxygen (see T0 at 2% and T0 at 20% oxygen in Fig. 2C), indicating inactivation of the complex and increased lactate production level under hypoxia, as confirmed in Fig. 1B. After the addition of 2-DG, a transient increase of pPDH level occurred, which decreased after 18 hr (Fig. 2C) and 24 hr (data not shown). Importantly, total PDH level did not significantly change following addition of 2-DG (Fig. 2C). These results, together with the observation of an increase in SDH activation after the addition of 2-DG (Fig. 2A,B) indicate a transient recovery of mitochondrial activity following addition of 2-DG.

As intracellular succinate level and ROS production are associated to SDH activity and defects of electron transfer in the respiratory chain are bound to enhanced ROS production [START_REF] Lenaz | The mitochondrial production of reactive oxygen species: mechanisms and implications in human pathology[END_REF], we evaluated the possible effects of 2-DG on ROS production. We found that hypoxic ) reduction after the addition of 2-DG for 18 hr (Fig. 2D). Importantly, by adding succinate to GBM cells ROS levels were higher under both oxygen tensions and this effect was repressed by the addition of 2-DG (Fig. 2E).

Conversely, significant variations of ROS content under the same conditions were not observed in SVZ-derived cells (Suppl. Fig. 2B).

The decrease in ROS may also relate to higher reduced glutathione (GSH) level. GSH is maintained in cells by reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is primarily generated when glucose is metabolized by pentose phosphate pathway. By measuring total GSH level we could not find any significant change following addition of 2-DG (Suppl. Fig. 3). This suggests that ROS level reduction is primarily connected to higher SDH activity (i.e. lower succinate level) rather than improved cytoplasmic radical scavenging.

2-DG promotes HIF-1α degradation in GBM cells.

It has been previously reported in a fibrosarcoma cell line that the hypoxic accumulation of HIF-1α, an endogenous hypoxia marker, strongly depends on glucose availability, as shown by modulation with 2-DG [START_REF] Staab | Modulation of glucose metabolism inhibits hypoxic accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha)[END_REF]. Nevertheless, the molecular mechanism causing this phenomenon has not been elucidated. HIF-1α is known to be chemically stabilized by hypoxia independent factors, such as mitochondrial ROS [START_REF]Taylor CT Mitochondria and cellular oxygen sensing in the HIF pathway[END_REF], CoCl 2 , Fe 2+ and cytoplasmic succinate [START_REF] King | Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer[END_REF], all known to be cofactors and inhibitors of proline hydroxylases (PHDs) [START_REF] Kang | Costa M Effect of metal ions on HIF-1alpha and Fe homeostasis in human A549 cells[END_REF],

which target HIF-1α degradation through proteasome. Importantly, we found that HIF-1α protein level was reduced by 2-DG under hypoxia, in some cases below detection level (Fig. 3A). Higher-molecular-weight isoform of HIF-1α, indicative of poly-ubiquitylated isoform [START_REF] Mackenzie | Cell-permeating alpha-ketoglutarate derivatives alleviate pseudohypoxia in succinate dehydrogenase-deficient cells[END_REF], eventually occurred (Fig. 3A). In SVZ-derived cells HIF-1α was rapidly reduced A c c e p t e d M a n u s c r i p t 15 following addition of 2-DG but its recovery was visible after 18 hr (Suppl. Fig. 2C). HIF-2α has been described as a promising target for anti-GBM therapies as it has been found highly expressed in glioma stem cells [START_REF] Li | Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells[END_REF]. We found that, oppositely to HIF-1α, HIF-2α level was not significantly decreased by addition of 2-DG (Fig. 3A).

To explain HIF-1α degradation, we tested expression of proline hydroxylase-2 (PHD2), the best described proline hydroxylase involved in HIF-1α protein hydroxylation. We found that after addition of 2-DG, PHD2 protein increased under hypoxia (Fig. 3A) reaching the level observed in cells exposed to 20% oxygen. We hypothesized that 2-DG-mediated PHD2 protein increase was dependent on de novo protein synthesis. Analyses of QRT-PCR revealed that HIF-1α transcript did not change after addition of 2-DG (Fig. 3B), while expression of PHD2 transcript was decreased by 2-DG (Fig. 3C), leading to exclude de novo PHD2 synthesis. Importantly, by silencing HIF-1α with lentiviral vectors GBM-derived cells acquired a more differentiated phenotype, with increased endogenous BMP signalling, and eventually underwent cell death after 5-7 days, as already reported [START_REF] Pistollato | Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma[END_REF][START_REF] Pistollato | Molecular mechanisms of HIF-1alpha modulation induced by oxygen tension and BMP2 in glioblastoma derived cells[END_REF]. Importantly, further addition of 2-DG did not promote any difference in HIF-1α silenced cells (data not shown).

Addition of succinate prevents 2-DG-mediated HIF-1α degradation.

As HIF-1α stability is related to accumulation of intra-cytoplasmic succinate consequential to impaired SDH activity [START_REF] King | Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer[END_REF], we hypothesized that 2-DG dependent HIF-1α degradation may be caused by the observed increase of SDH activity (Fig. 2A,B). To prove this hypothesis we evaluated if addition of succinate in combination with 2-DG, promoted a recovery of HIF-1α protein level in GBM-derived cells. We found that HIF-1α stability was recovered in 2-DG/succinate cultured cells (Fig. 3D). Analysis of HIF-2α did not reveal any significant change among conditions (Fig. 3D).

A c c e p t e d M a n u s c r i p t 16

We investigated if 2-DG, besides modulating HIF-1α protein stability, promoted also inhibition of HIF-1α transcriptional activity, by using a hypoxia responsive element (HRE)luciferase reporter construct. We found a modest reduction of HIF-1α transcriptional activity under hypoxia following 8 hr of 2-DG treatment compared to untreated GBM cells (Fig. 3E).

Notably, by adding succinate together with 2-DG this reduction did not occur, and addition of succinate alone doubled HIF-1α transcriptional activity (Fig. 3E).

Hypoxia inhibits the pro-apoptotic effect caused by 2-DG in GBM-derived cells.

We sought to investigate whether these changes of GBM metabolic profile and the observed reduction of HIF-1α protein level correlated to block of cell cycle and induction of apoptosis.

It has already been reported that downregulation of HIF-1α suppresses tumorigenicity of renal cell carcinoma through induction of apoptosis [START_REF] Xu | Silencing of HIF-1alpha suppresses tumorigenicity of renal cell carcinoma through induction of apoptosis[END_REF] and attenuates glioma growth in vivo [START_REF] Gillespie | Silencing of hypoxia inducible factor-1alpha by RNA interference attenuates human glioma cell growth in vivo[END_REF]. Analysis of cell cycle revealed that addition of 2-DG in GBM cells acutely exposed to high oxygen tension induced a modest but not significant decrease of S phase and a slight increase of apoptotic cells (sub G1 fraction), but these effects were not observed in hypoxia (Fig. 4A). Analysis of apoptosis by using annexin-V/propidium iodide staining (annexin-V/PI) indicated that addition of 2-DG significantly induced early apoptosis (annexin-V + /PI -cells) especially when GBM cells were exposed for 24 and 48 hr to high oxygen tension (Fig. 4B). Oppositely, addition of 2-DG on SVZ-derived cells did not promote significant cell cycle changes and/or induction of apoptosis (not shown).

Apoptosis often implicates disruption of mitochondrial membrane potential (Δψ mt ). We found that addition of 2-DG for 24 and 48 hr caused reduction of Δψ mt especially when cells were exposed to 20% oxygen (Fig. 4C). Moreover, we found that anti-apoptotic proteins (Bcl-2

and Bcl-XL), involved in mitochondria controlled apoptosis, were down-regulated more rapidly by addition of 2DG under 20% oxygen, while PARP cleavage, which is a marker of A c c e p t e d M a n u s c r i p t cells undergoing apoptosis [START_REF] Oliver | Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant[END_REF], occurred earlier under the same conditions (Fig. 4D).

Importantly, even though decrease of Bcl-2 and Bcl-XL proteins occurred even after 18 hr after addition of 2-DG, percentage of early apoptotic cells (annexin-V + /PI -cells) increased significantly only after 48 hr (Fig. 4B). In normal SVZ-derived cells these apoptosis-related proteins were not affected at early time points, but only after prolonged 2-DG stimulus (Suppl. Fig. 2C).

Analysis of p21 cip1 , which is related to cell cycle arrest and induction of differentiation or to cell death [START_REF] Gartel | WAF1/CIP1) and cancer: a shifting paradigm?[END_REF], revealed that GBM cells exposed to 20% oxygen, independently from addition of 2-DG, had a higher p21 cip1 level compared to cells maintained under hypoxia, confirming our previous report [START_REF] Pistollato | Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma[END_REF] (Fig. 4D). Under hypoxia addition of 2-DG slowly upregulated p21 cip1 (Fig. 4D) to level comparable to the one observed at 20% oxygen. In SVZderived cells p21 cip1 did not change following addition of 2-DG (Suppl. Fig. 2C).

2-DG induces neuronal commitment and addition of succinate antagonizes this effect by reducing GBM differentiation.

HIF-1α expression has been related to the maintenance of a more immature phenotype in ductal breast carcinoma and neuroblastoma [START_REF] Helczynska | Hypoxia promotes a dedifferentiated phenotype in ductal breast carcinoma in situ[END_REF][START_REF] Jogi | Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crestlike phenotype[END_REF], a phenomenon that has been recently described also in malignant gliomas [START_REF] Lu | Hypoxia-inducible factor-1alpha blocks differentiation of malignant gliomas[END_REF][START_REF] Pistollato | Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma[END_REF]. As we found that addition of 2-DG within the first 18 hr induced increase of p21 cip1 , with apoptosis significantly occurring after 48 hr (Fig. 4B-D), together with HIF-1α poly-ubiquitylation and degradation even under hypoxia (Fig. 3A), we evaluated if these events correlated to the acquisition of a more differentiated phenotype in live remaining GBM cells. Flow cytometric analysis of cell surface marker CD133, revealed that addition of 2-DG promoted a decrease of CD133 + cells percentage, especially at 20% oxygen (Fig. 5A). Normal SVZ-derived cells contained a lower percentage of CD133 + cells compared to GBM-derived cells, and addition of 2-DG analogously induced a slight A c c e p t e d M a n u s c r i p t 18 decrease of CD133 + cells (Suppl. Fig. 2D). Importantly, we found that addition of succinate under hypoxia increased CD133 + cells in GBM (Fig. 5A), and this did not occur in SVZderived cells (Suppl. Fig. 2D). Addition of both 2-DG and succinate slightly prevented 2-DGmediated decrease of tumor CD133 + cells exposed to 20% oxygen (Fig. 5A). Another described GBM stem cell marker is CD90 [START_REF] Kang | Tumorigenesis of chemotherapeutic drug-resistant cancer stemlike cells in brain glioma[END_REF], which we found to be highly expressed in all our GBM cell cultures. Nevertheless, only a slight decrease of CD90 + cells was observed after addition of 2-DG and succinate did not reverse this effect (Suppl. Fig. 4).

Immunocytochemical analysis on adherent remaining cells of nestin (Fig. 5B,E), indicative of undifferentiated precursors, glial fibrillary acidic protein (GFAP) (Fig. 5C,E), a marker of astroglia and their progenitors, and βIII-tubulin (Fig. 5D,F), expressed in neurons and their committed progenitors, revealed that addition of 2-DG under hypoxia promoted specifically neuronal differentiation (i.e. higher βIII-tubulin + cells percentage), rather than glial commitment, with decrease in nestin + cells. In GBM cells acutely exposed to 20% oxygen a higher basal neuronal and glial commitment was visible and under this condition the addition of 2-DG did not further increase differentiation. Opposite to 2-DG effects, addition of succinate raised fraction of nestin + cells (Fig. 5B,E), lowering the percentage of GFAP + and βIII-tubulin + cells (Fig. 5C-F). Finally, addition of succinate together with 2-DG prevented 2-DG mediated differentiation of GBM-derived cells (Fig. 5B-D). Altogether these data suggest that addition of 2-DG promotes induction of GBM cell differentiation and lately cell death under 20% oxygen, while maintaining cells at 2% oxygen partially inhibits these effects, preserving tumor stem cells survival, despite HIF-1α degradation occurs.

Discussion

There have been increasing efforts to characterize the solid tumor microenvironment and to define which signalling molecules and metabolic responses may be disrupted in tumor cells.

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As the most primitive tumor cells share characteristics with non-tumor stem cells [START_REF] Singh | Identification of a cancer stem cell in human brain tumors[END_REF], the microenvironment that regulates stem cell self-renewal and differentiation may also play a role in the proliferation and fate of tumor cells. In particular, there is evidence that hypoxia plays a key role in normal homeostasis of stem cells [START_REF] Pardal | Glia-like stem cells sustain physiologic neurogenesis in the adult mammalian carotid body[END_REF] and in the initiation, development and aggressiveness of gliomas [START_REF] Diabira | Gliomagenesis and neural stem cells: Key role of hypoxia and concept of tumor "neo-niche[END_REF]. Accordingly, our recent report confirmed that intratumoral hypoxia drives GBM stem cells distribution [START_REF] Pistollato | Intratumoral Hypoxic Gradient Drives Stem Cells Distribution and MGMT Expression in Glioblastoma[END_REF]. Hypoxia could be crucial to recruit cancer stem-like cells, deregulating their differentiation [START_REF] Helczynska | Hypoxia promotes a dedifferentiated phenotype in ductal breast carcinoma in situ[END_REF][START_REF] Jogi | Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crestlike phenotype[END_REF][START_REF] Pistollato | Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma[END_REF] and altering their metabolism, by increasing tumor glycolytic response and reducing mitochondrial metabolism. Particularly, among mitochondrial dehydrogenases, SDH has been found inactive in some solid tumors, and this has been described as a key event leading to the accumulation of intra-cytoplasmic succinate and ROS causative of the "pseudo-hypoxic" effect. This is a major tumor-related hallmark, characterized by the activation of HIF even in the absence of hypoxia [START_REF] King | Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer[END_REF][START_REF] Mackenzie | Cell-permeating alpha-ketoglutarate derivatives alleviate pseudohypoxia in succinate dehydrogenase-deficient cells[END_REF]. Moreover, recent reports indicate that the gene encoding isocitrate dehydrogenase-1 (IDH1), one of the enzyme of citric acid cycle which is related to HIF-1 pathway, is mutated in 80% of certain types of GBM [START_REF] Parsons | An integrated genomic analysis of human glioblastoma multiforme[END_REF][START_REF] Pollard | Puzzling patterns of predisposition[END_REF][START_REF] Zhao | Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha[END_REF], thus linking mitochondrial dysfunction to tumorigenesis.

2-DG, a glucose analogue that reduces intracellular utilization of glucose, inhibits glycolysis, through competitive inhibition of some glycolytic enzymes. 2-DG can affect the growth of some tumors in vivo and it has been shown to be cytotoxic in vitro [START_REF] Aft | Welch MJ Enhancing targeted radiotherapy by copper(II)diacetyl-bis(N4-methylthiosemicarbazone) using 2-deoxy-D-glucose[END_REF]. In a recent work HIF-1α has been shown to mediate resistance specifically to glycolytic inhibitors [START_REF] Maher | Lampidis TJ Hypoxia-inducible factor-1 confers resistance to the glycolytic inhibitor 2-deoxy-D-glucose[END_REF],

however, the molecular mechanisms by which hypoxia may counteract 2-DG effects in brain tumor has not been so far fully elucidated. A decrease in provision of glucose (from 25 mM to 2,5 mM) and also of oxygen (2%) can be found in the internal perinecrotic/hypoxic region of the solid tumor mass compared to peripheral, relatively well-nourished regions of the tumor [START_REF] Dearling | Combining radioimmunotherapy with antihypoxia therapy 2-deoxy-D-glucose results in reduction of therapeutic efficacy[END_REF]. Thus, in this study we set our control maintaining GBM-derived cells at 2% Degradation of HIF-1α by 2-DG has been described in a previous report [START_REF] Staab | Modulation of glucose metabolism inhibits hypoxic accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha)[END_REF], nevertheless a molecular explanation for this effect has not been so far fully provided. Here we show that 2-DG increases SDH activity, reducing ROS levels and these are reliably causative of the observed HIF-1α degradation, consequentially to increased PHD2 activity. Indeed, HIF-1α is known to be chemically stabilized also by hypoxia independent factors, such as ROS [START_REF]Taylor CT Mitochondria and cellular oxygen sensing in the HIF pathway[END_REF],

CoCl 2 , Fe 2+ [START_REF] Kang | Costa M Effect of metal ions on HIF-1alpha and Fe homeostasis in human A549 cells[END_REF] and cytoplasmic succinate [START_REF] King | Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer[END_REF]. Earlier studies have suggested that ROS produced by mitochondria are required for proper induction of HIF-1α under hypoxic conditions [START_REF] Bell | The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production[END_REF][START_REF] Chandel | Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O 2 sensing[END_REF]. It has been well established, that Fe 2+ is an important cofactor for PHDs [START_REF] Kang | Costa M Effect of metal ions on HIF-1alpha and Fe homeostasis in human A549 cells[END_REF], and importantly H 2 O 2 , produced by mitochondria, is known to destabilize PHDs activity, either by converting Fe 2+ in Fe 3+ or by induction of post-transcriptional modifications of PHD2 [START_REF] Bell | The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production[END_REF]. Besides ROS acting as second messengers, impaired SDH activity and consequential intra-cytoplasmic succinate accumulation are associated to HIF-1α stabilization in several cancer types [START_REF] King | Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer[END_REF]. We found that addition of succinate reverts 2-DG effects, promoting recovery of HIF-1α protein level and transcriptional activity (i.e. HREluciferase), as well as increase of ROS levels. Besides succinate, PHDs have been shown to be regulated also by other glycolytic and citric acid cycle metabolites, such as pyruvate and oxaloacetate, that could independently modify the oxygen-sensing abilities of cells [START_REF] Dalgard | Verma A Endogenous 2-oxoacids differentially regulate expression of oxygen sensors[END_REF].

While we found that PHD2 protein level is raised by addition of 2-DG, PHD2 transcription is decreased, and this is related to the observed degradation of HIF-1α, known to control PHD2 transcription [START_REF] Metzen | Jelkmann W Regulation of the prolyl hydroxylase domain protein 2 (phd2/egln-1) gene: identification of a functional hypoxia-responsive element[END_REF]. Additionally, HIF-2α has been found highly expressed in glioma stem cells Importantly, HIF-1α is related to maintenance of an undifferentiated cell state and inhibition of cell differentiation [START_REF] Pistollato | Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma[END_REF][START_REF] Gustafsson | Hypoxia requires notch signaling to maintain the undifferentiated cell state[END_REF]. Particularly, HIF-1α contributes to a differentiation defect in malignant gliomas [START_REF] Lu | Hypoxia-inducible factor-1alpha blocks differentiation of malignant gliomas[END_REF], even following exogenous BMP2 treatment [START_REF] Pistollato | Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma[END_REF][START_REF] Pistollato | Molecular mechanisms of HIF-1alpha modulation induced by oxygen tension and BMP2 in glioblastoma derived cells[END_REF], shedding new light on the differentiation therapy of solid tumors by HIF-1α targeting. Therefore, HIF-1α has become in recent years an attractive, although challenging, therapeutic target [START_REF] Poon | Ashcroft M Targeting the hypoxia-inducible factor (HIF) pathway in cancer[END_REF]. Here, we found that after addition of 2-DG, a decrease of CD133 + and nestin + cells and induction of neuronal differentiation occur under hypoxia, condition in which GBM cells are maintained in a less committed state [START_REF] Pistollato | Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma[END_REF][START_REF] Pistollato | Intratumoral Hypoxic Gradient Drives Stem Cells Distribution and MGMT Expression in Glioblastoma[END_REF]. Oppositely, addition of succinate increases GBM cell undifferentiated state, preventing the pro-differentiating effects evoked by 2-DG.

Previous reports describe that highly de-differentiated and fast-growing tumors compared to slow-growing tumors or non-tumor cells are characterized by faster glycolysis and slower oxidative phosphorylation [START_REF] Krieg | Mitochondrial proteome: cancer-altered metabolism associated with cytochrome c oxidase subunit level variation[END_REF][START_REF] Moreno-Sanchez | Energy metabolism in tumor cells[END_REF][START_REF] Pedersen | Tumor mitochondria and the bioenergetics of cancer cells[END_REF][START_REF] Ristow | Oxidative metabolism in cancer growth[END_REF]. Here we found that diverse mechanisms may control GBM cell phenotype: on one side addition of 2-DG and raised oxygen tension induce GBM cell commitment and eventually cell death; oppositely to these, succinate and hypoxia promote GBM immature cells maintenance (Fig. 6).

In conclusion, we show that reducing glycolysis in GBM tumor cells destabilizes cell

  Under institutional-approved protocols, tissue samples were collected from patients and neonates after written informed consent. Neonatal brain subventricular zone (SVZ) tissue was obtained at the Children's Hospital of Orange County, USA, under the auspices of the protocol for the National Human Neural Stem Cell Resource (NHNSCR). Adult GBM samples were obtained at the Padova Academic Hospital, under the auspices of the protocol for the acquisition of human brain tissues obtained from the Ethical Committee board of the A c c e p t e d M a n u s c r i p t 5 University of Padova. Tumor and non-tumor patients characteristics are described in Table

  incubated with species-specific secondary antibodies conjugated to Alexa dyes (1:2000, Invitrogen, Milano, Italy). Cells were counterstained with DAPI (1:10000, Sigma-Aldrich, Milano, Italy) to measure total cell number. Staining was visualized by epifluorescence (Nikon Eclipse 80i, Melville, NY, USA) and images compiled for figures using Adobe Illustrator (Adobe, San Jose, CA).

  1 effectiveness GBM-derived cells have been pretreated with valinomycin (1μM, Sigma-Aldrich, Milano, Italy), facilitating mitochondrial membrane potential depolarization

( 1 :

 1 100000, Sigma-Aldrich, Milano, Italy), peroxidase-labeled goat anti-mouse IgG

A c c e p t e d M a n u s c r i p t 8 ( 1 :

 81 100000, Sigma-Aldrich, Milano, Italy) or peroxidase-labeled donkey anti-goat IgG

  5'-ACACATGTGGTGCTTGCTGT-3'; HIF-1α, F: 5'-CGTTCCTTCGATCAGTTGTC-3', A c c e p t e d M a n u s c r i p t 10 R: 5'-TCAGTGGTGGCAGTGGTAGT-3'; beta-glucuronidase (GUSB), F: 5'-GAAAATACGTGGTTGGAGAGCTCATT-3', R: 5'-CCGAGTGAAGATCCCCTTTTTA-3'. Primers have been designed by using the software Primer 3

  GBM cells were transfected using a Promega Corporation (Madison WI, USA) protocol for transient transfection of adherent cells using Effectene reagent (Qiagen, Milano, Italy). HREluciferase reporter construct used (wHRE) was kindly provided by Dr. Stefano Indraccolo (Istituto Oncologico Veneto -IRCCS, Padova, Italy). It consists of a trimerized 24-mer containing 18 bp of sequence from the PGK promoter including the HRE (5'-TGTCACGTCCTGCACGACTCTAGT, HRE) and an 8-bp linker sequence followed by a 50-bp minimal tyrosine kinase promoter in a pGL2-firefly luciferase basic Vector backbone (Promega, Madison, WI, USA)[START_REF] Arsham | Simon MC Phosphatidylinositol 3-kinase/Akt signaling is neither required for hypoxic stabilization of HIF-1 alpha nor sufficient for HIF-1-dependent target gene transcription[END_REF]. The mutant HRE (mHRE) construct used to evaluate aspecific effects, has the ACG of the HIF-1 binding site mutated to CAT, abolishing binding, as well as a point mutation that abolished a BsgI restriction site for diagnostic purposes.

  A c c e p t e d M a n u s c r i p t 11 cells were treated with 25 mM 2-DG, succinate 5 mM or a combination of both for 8 hr.

  A c c e p t e d M a n u s c r i p t 12 addition of 25 mM 2-DG for 18 hr, under the two oxygen tensions, induced loss of cell adhesive properties compared to GBM cells grown without 2-DG (Fig.1Aand Suppl. Fig.1A,B). Simply decreasing the glucose medium concentration from 25 mM to 2.5 mM, without adding 2-DG (Suppl. Fig.1C) did not cause change of cell morphology. By adding both 2-DG and glucose in the medium (i.e. 25 mM 2-DG + 25 mM glucose, Suppl. Fig.1C, or 12.5 mM 2-DG + 12.5 mM glucose, not shown), we found that loss of cell adherence was prevented and cells showed a flat phenotype with more extensions compared to cells grown in the absence of 2-DG. Measurement of extracellular lactate was generally high in tumor cell culture medium (Fig.1B) compared to non-tumor subventricular zone (SVZ)-derived cells

A c c e p t e d M a n u s c r i p t 13 known

 13 that SDH activation is impaired in several solid tumors, inducing the accumulation of succinate and ROS into the cytoplasm and causing strong HIF-1α stabilization even in the absence of hypoxia[START_REF] King | Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer[END_REF]. Surprisingly, we found that addition of 2-DG increased SDH activity in GBM-derived cells, especially at 20% oxygen (Fig.2A, B). Addition of cell permeable succinate either in presence or absence of 2-DG did not modify SDH activation state (data not shown). Notably, in SVZ-derived cells SDH activity did not change after addition of 2-DG (data not shown), confirming differences in metabolic response between non-tumor and tumor cells.

A c c e p t e d M a n u s c r i p t 14 GBM

 14 -derived cells, which resulted to contain a variable but generally high percentage of ROS, underwent transient superoxide anion (O 2 .-

A c c e p t e d M a n u s c r i p t 20 oxygen

 20 and in presence of high glucose concentration. We show that inhibiting glycolisis by addition of 2-DG, induces a decrease in cell proliferation and late apoptosis in primary GBMderived cells acutely exposed to 20% oxygen, while these effects are partially inhibited by hypoxia, condition in which 2-DG addition causes GBM cell differentiation and HIF-1α level reduction. Importantly, normal SVZ-derived cells are not affected by addition of 2-DG.

A c c e p t e d M a n u s c r i p t 21 [ 39 ]

 2139 , but we did not find change of HIF-2α protein level following neither stimulus with 2-DG, nor addition of succinate.

  functional and metabolic status, forcing cancer cells into a mitochondrial metabolism, as demonstrated by the activation of PDH and SDH, and promoting GBM cell differentiation.These findings improve the knowledge of GBM tumor intra-and extra-cellular microenvironment and the mechanisms by which GBM cells respond to an alteration of their metabolic state.

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Acknowledgments

We thank Dr. Ahmed Mohyeldin for kindly providing the antibody for pPDH E1α, Dr. Hugo Guerrero-Cazares and Dr. Martina Pigazzi for their suggestions on the manuscript; Dr.

Alberto Burlina for kindly providing the Lactate Assay Kit. This work was supported by funds from the Italian Association for the Fight against Neuroblastoma (Pensiero Project) and the Italian Association AIRC (Interregional pediatric project grant).

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Competing interests

The authors declare that they have no competing interests.