Simon Bousseau

Marion Marchand

Raffaella Soleti

Luisa Vergori

Grégory Hilairet

Sylvain Recoquillon

Morgane Le Mao

Naig Gueguen

Salim Khiati

Ludovic Clarion

Norbert Bakalara

M Carmen Martinez

Stéphane Germain

Guy Lenaers

Ramaroson Andriantsitohaina
email: ramaroson.andriantsitohaina@univ-angers.fr

Phostine 3.1a as a pharmacological compound with anti-angiogenic properties against diseases with excess vascularization

Keywords: Anti-angiogenic properties of PST 3.1a Angiogenesis, Cancer growth, Pharmacotherapy, Endothelial metabolism, Glycosylation .1a PST 3.1a CTL PST 3.1a

Nonstandard Abbreviations

3-Hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5- [START_REF] Dimova | Angiogenesis in cancer -general pathways and their therapeutic implications[END_REF][START_REF] Eelen | Endothelial cell metabolism[END_REF] Vascular endothelial growth factor receptor 2 (VEGFR2)

Introduction

Angiogenesis is a complex process related to the growth of new blood vessels from existing vasculature, triggered by local pro-angiogenic factors such as the vascular endothelial growth factor (VEGF). In healthy adult, angiogenesis contributes to menstruation, pregnancy and wound healing, and can be activated in various pathological conditions such as tumor growth, diabetic retinopathy, rheumatoid arthritis, and ischemic diseases [START_REF] Dimova | Angiogenesis in cancer -general pathways and their therapeutic implications[END_REF][START_REF] Eelen | Endothelial cell metabolism[END_REF]. Endothelial cells (ECs) form the innermost layer of blood vessels and remain quiescent for years in healthy adults in physiological conditions. In contrast, ECs can rapidly increase their metabolism to initiate angiogenesis in pathological conditions. During this process, quiescent ECs quickly switch to an angiogenic phenotype and differentiate to migratory "tip" and proliferating "stalk" cells [START_REF] Treps | Manipulating angiogenesis by targeting endothelial metabolism: hitting the engine rather than the drivers-a new perspective?[END_REF], which display specific metabolic phenotypes [START_REF] Bock | Role of endothelial cell metabolism in vessel sprouting[END_REF][START_REF] Stapor | Angiogenesis revisited -role and therapeutic potential of targeting endothelial metabolism[END_REF].

Current standards of care against excessive pathological angiogenesis aim to inhibit vascular growth signals, such as VEGF. Indeed, the VEGF pathway is widely recognized as critical in angiogenesis, and most current anti-angiogenic therapies targeting VEGF have been clinically approved [START_REF] Croci | Glycosylation-dependent lectin-receptor interactions preserve angiogenesis in anti-VEGF refractory tumors[END_REF]. However, in cancer therapy, anti-VEGF treatments provide less success than expected, due to restricted efficacy, intrinsic refractoriness and resistance acquisition, implying that alternative strategies are needed [START_REF] Bergers | Modes of resistance to anti-angiogenic therapy[END_REF][START_REF] Mcintyre | Metabolic and hypoxic adaptation to anti-angiogenic therapy: a target for induced essentiality[END_REF]. As a matter of fact, EC metabolism is a common underlying mechanism towards which proangiogenic vascular growth signals converge, and consequently represents an innovative antiangiogenic opportunity based on an entirely distinct novel strategy [START_REF] Stapor | Angiogenesis revisited -role and therapeutic potential of targeting endothelial metabolism[END_REF][START_REF] Harjes | Targeting fatty acid metabolism in cancer and endothelial cells[END_REF]. In this respect, new targets involved in EC metabolism, energy production and protein modifications, such as glycosylation, are emerging [START_REF] Potente | The Link Between Angiogenesis and Endothelial Metabolism[END_REF]. Although energy production and its main regulator 6phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) are now well described to regulate neovascularization [START_REF] Schoors | Partial and transient reduction of glycolysis by PFKFB3 blockade reduces pathological angiogenesis[END_REF][START_REF] Cantelmo | Inhibition of the glycolytic activator PFKFB3 in endothelium induces tumor vessel normalization, impairs metastasis, and improves chemotherapy[END_REF], the impact of glycosylation on angiogenesis is less clear. This post-translational modification allows the addition of glycans to glycoconjugates on growth factor receptors and nutrient transporters. In particular, glycosylation of vascular endothelial growth factor receptor 2 (VEGFR2) and Notch contributes to their stability, solubility, localization, trafficking and function [START_REF] Benedito | The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis[END_REF][START_REF] Markowska | Galectin-3 protein modulates cell surface expression and activation of vascular endothelial growth factor receptor 2 in human endothelial cells[END_REF]. Addition of glycans on Nlinked glycoproteins is modulated by the enzymatic activity of N-acetylglucosaminyltransferases (GnTs), which catalyze the addition of N-acetylglucosamine to N-linked proteins. GnT-V is particularly associated with malignancies and correlates with cell migration, invasion, and metastatic properties in numerous cancer cell lines [START_REF] Elola | Assembly, organization and regulation of cell-surface receptors by lectin-glycan complexes[END_REF][START_REF] Liu | β1,6-N-acetylglucosaminyltransferase V predicts recurrence and survival of patients with clear-cell renal cell carcinoma after surgical resection[END_REF][START_REF] Hassani | Phostine PST3.1a targets MGAT5 and inhibits glioblastomainitiating cell invasiveness and proliferation[END_REF]. Furthermore, galectins, a family of endogenous glycan-binding proteins specifically interact with -galactoside partners in the cell membrane to regulate stability and signaling. Galectin-1 (Gal-1) was shown to regulate angiogenesis and its interaction with VEGFR2 could thus be responsible for tumor adaptations to antiangiogenic strategies [START_REF] Croci | Glycosylation-dependent lectin-receptor interactions preserve angiogenesis in anti-VEGF refractory tumors[END_REF][START_REF] Griffioen | Galectins in tumor angiogenesis[END_REF].

Phosphinosugars or Phostines, a class of glycomimetic compounds in which the hemiacetal group of an hexopyranose is replaced by a chemically and configurationally stable phosphinolactone group [START_REF] Clarion | Oxaphosphinanes: new therapeutic perspectives for glioblastoma[END_REF][START_REF] Clarion | C-glycoside mimetics inhibit glioma stem cell proliferation, migration, and invasion[END_REF], have been synthetized to test their capacity to alter tumor growth by interfering with N-glycosylation. In particular, the lead compound, Phostine 3.1a (3-Hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5- [START_REF] Dimova | Angiogenesis in cancer -general pathways and their therapeutic implications[END_REF][START_REF] Eelen | Endothelial cell metabolism[END_REF]oxaphosphinane, PST 3.1a) displays antitumoral activity against glioblastoma (GBM), the most common primary malignant brain tumor. By specifically inhibiting GnT-V enzymatic activity, PST 3.1a has shown promising properties to inhibit proliferation, migration and invasiveness of GBM initiating cells [START_REF] Hassani | Phostine PST3.1a targets MGAT5 and inhibits glioblastomainitiating cell invasiveness and proliferation[END_REF]. It is now described that hyper-vascularization is an important feature of GBM tumors that also regulates invasion, migration, and proliferation of the tumor cells [START_REF] Valdés-Rives | Apoptotic signaling pathways in glioblastoma and therapeutic implications[END_REF][START_REF] Arbab | Major challenges and potential microenvironment-targeted therapies in glioblastoma[END_REF]. However, the effect of PST 3.1a on angiogenesis has never been addressed. Thus, the aim of this study was to investigate the effect of PST 3.1a on angiogenesis under physiological conditions both in vitro and in vivo as well as in a mouse model of glioblastoma. In this study, we show that PST 3.1a is an innovative approach to efficiently target angiogenesis and might constitute a new relevant therapeutic tool against glioblastoma.

Materials and Methods

Cell culture

Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from Lonza (Basel, Switzerland) and grown on plastic flasks in EGM-2 medium supplemented with 0.1% human epidermal growth factor, 0.1% ascorbic acid, 0.1% GA-1000 (Gentamicin, Amphotericin-B), 0.1% heparin, 2% fetal bovine serum, 0.1% VEGF, 0.4% human basic fibroblast growth factor (bFGF), 0.04% hydrocortisone, 0.1% R3-IGF-1, and 1% streptomycin/penicillin (all chemicals were purchased from Lonza). HUVECs were grown at 37 °C in a humidified incubator gassed with 5% CO 2 in air and were used for experiments at the third to seventh passage. PST 3.1a (provided from Phost'in SAS, Montpellier, France) solutions were prepared in 100% dimethylsulfoxyde (DMSO). Cells were grown for 24 h in the absence or presence systematically, or otherwise stated, of 1, 5 and 10 µM PST 3.1a with a final DMSO concentration of 0.1 %.

Animal model

The local ethics committee approved the animal protocol followed in the current study (CEEA.PdL 00509.01). This study was carried out in strict accordance with the guidelines and authorization with French Ministry of Agriculture regulations based on European Community. Six week-old male NMRI-nude mice (Janvier labs, Le Genest-Saint-Isle, France) were maintained at room temperature of 23 ± 2 °C under a 12 h light/12 h dark cycle (lights on, 8:00 AM). The animals were allowed free access to water and standard diet.

Wound healing assay

HUVECs (6 x 10 4 ) were seeded in 12-well plates. When the confluence of cells reached about 90%, cells were scratched with a sterile pipet tip to make a gap. Cells were then rinsed with medium to remove any floating cells and debris, then treated either with PST 3.1a (1, 5 or 10 µM), either with tunicamycin (1µg/mL) for 24 h to compare their efficacy. Photographic images of the plates were taken under a microscope immediately after healing and 24 h after. The area of the scratch was measured using the T-scratch software.

Proliferation assay

Evaluation of PST 3.1a on HUVEC proliferation was performed using Trypan blue exclusion. For this, 1.2 x 10 5 cells per well were seeded into 6-well plates overnight, then treated in absence or presence of PST 3.1a or tunicamycin during 24 h, and counted using Trypan blue solution (Sigma-Aldrich, Saint-Louis MO, USA).

Apoptosis analysis

HUVECs (1.5 x 10 5 ) were exposed or not to PST 3.1a (10 µM) for 24 h and then fixed in 70 % ethanol at 4°C for at least 4 h. After a centrifugation at 15,000 g for 5 min, cells were re-suspended in PBS containing 50 μg/mL RNase (Sigma-Aldrich) and 10 μg/mL propidium iodide (Sigma-Aldrich). Cellular DNA content was analyzed on a Cytomics FC 500 MPL flow cytometer (Beckman Coulter).

ATP/ADP measurements

HUVECs were plated in 96-well microplates treated or not with PST 3.1a (10 µM) for 24 h. The ATP/ADP ratio was performed using bioluminescent detection (ADP/ATP Ratio Assay Kit, Abcam) according to the manufacturer's instructions. The bioluminescent intensities were measured on a multi-mode microplate reader (Clariostar, BMG labtech, NC, USA).

Adhesion assay

Effect of PST 3.1a on HUVEC adhesion was analyzed by crystal violet staining. For this, 10 4 cells per well were seeded into 96-well plates overnight, then PST 3.1a was added. After 24 h of incubation, the plate was shaken for 15 sec. The supernatant with non-adherent cells was removed by three washes with the washing buffer (0.1% bovine serum albumin (BSA) in medium without serum; Sigma-Aldrich). Attached cells were fixed with 4% of paraformaldehyde (Fisher Scientific, Hampton, NJ, USA) for 15 min at room temperature.

Cells were rinsed twice with washing buffer, stained with crystal violet (Sigma-Aldrich) (1 mg/mL in 2% of ethanol) for 10 min at room temperature protected from light, and extensively washed with distilled water.

Then, sodium dodecyl sulfate (SDS) 2% was added and incubated for 30 min at room temperature. Absorbance was evaluated using a microplate reader at 550 nm (Synergy HT Biotek, Winooski, VT, USA).

In vitro capillary network formation on ECM gel

Endothelial capillary network formation was assessed using matrigel (ECM gel ® Sigma-Aldrich), as previously described [START_REF] Soleti | Microparticles harboring Sonic Hedgehog promote angiogenesis through the upregulation of adhesion proteins and proangiogenic factors[END_REF]. Briefly, 10 µL of ECM gel substrate was added into a 15 µ-slide Angiogenesis plate (Ibidi, Planegg, Germany), and allowed to solidify for 1 h at 37 °C. Then, HUVECs were seeded with a density of 10 x 10 4 cells per well and allowed to adhere for 1 h after which the different treatments were added.

Photographic images of the plates were taken under a microscope after 24 h. Tube formation was assessed using ImageJ software.

In vivo matrigel plug assay

For in vivo studies of angiogenesis, HUVECs were treated or not with PST 3.1a (10 µM) for 24 h. After treatment, cells were detached and mixed with 500 µL of matrigel (ECM gel ® Sigma-Aldrich) with recombinant bFGF-2 (300 ng/mL, Peprotech, Rocky Hill, NJ, USA). This mixture was subcutaneously injected on the back of NMRI-nude mice fed with normal chow (Janvier labs, Le Genest-Saint-Isle, France) At day 7, mice were sacrificed and matrigel plugs were removed. Plugs were homogenized in RIPA lysis buffer and incubated for 24 h 4 • C, then disrupted with a Polytron (PRO Scientific, Oxford, CT, USA). Hemoglobin levels were determined using Drabkin's reagent (Sigma-Aldrich) according to the manufacturer's instructions.

Immunohistochemistry of matrigel plugs

After the surgery, matrigel plugs were embedded in Tissue-Tek® O.C.T. compound (Sakura Finetek, Torrance, CA, USA), and serial frozen sections 10-µm thick were obtained using a Leica CM3050S cryostat (Leica, Wetzlar, Germany) and then briefly fixed with 4% paraformaldehyde. Slides were incubated with rat anti-mouse CD31 antibody (BD Biosciences, Franklin Lakes, NJ, USA) followed by incubation with goat antirat IgG-FITC (Southern Biotech, Birmingham, AL, USA). Nuclei were counterstained with 4,6-diamidino-2phenylindole (DAPI) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 5 min.

Confocal microscopy (Zeiss CLMS 700, Oberkochen, Germany) and Digital image recording were used using ZEN software.

Zebrafish experiments

General maintenance of zebrafish was carried out according to common practices at 28 °C. PST 3.1a (50, 75 or 100 µM) was added in one-cell eggs water during 72, 96 or 120 h post fecundation (hpf). Embryos were anesthetized with 0.01% tricaine (Sigma-Aldrich) and mounted in 1% agarose. GFP-expressing ECs and DsRed-expressing red blood cells were visualized in Tg(fli1a:EGFP/gata1a:dsRed) zebrafish with a Spinning disk confocal microscope (Zeiss Axio Observer) as previously shown [START_REF] Bignon | Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane[END_REF]. Stacks of 20-50 focal planes were processed with MetaMorph. The number of embryo heartbeats were counted in Tg(fli1a:EGFP/gata1a:dsRed) zebrafish with a stereomicroscope (Motic, Carlsbad, CA, USA).

Tumor Xenograft

SNB75 glioblastoma cells (Stanford University, Stanford, CA, USA) were dissociated and resuspended in 50% PBS and 50% matrigel. Cells (4.5 x 10 6 ) were subcutaneously injected into the back of 6 weeks-old male NMRI-nude mice under isoflurane anesthesia. Transplanted animals were allocated randomly to two treatment groups by the end of the surgery. Six-week post-surgery, animals were treated daily for 14 consecutive days, with one per os administration of a nano-suspension of PST 3.1a (12 mg/kg in accordance with Hassani et al ( 17)), or with control solution (sham-operated). Mice were then weighted every two days and tumor volume was measured using a caliper. The animals were sacrificed at the end of the treatment and the xenograft removed.

Echography examination

In vivo echography was assessed in mice anesthetized with isoflurane using a VEVO 770 ultrasound machine from FUJIFILM Visualsonics (Toronto, Canada) equipped with a 30-MHz imaging transducer. Threedimensional M-mode data was acquired along the entire length of the xenograft at day 0, day 7 and day 14 in order to evaluate tumor growth.

Immunohistochemistry of tumors

Frozen tumor sections (10 µm thickness) were prepared using a Leica CM3050S cryostat (Leica). Tumor sections were incubated with rat anti-mouse CD31 antibody (BD Biosciences) followed by incubation with goat anti-rat IgG-FITC (Southern Biotech) or with rabbit anti-human VE-Cadherin antibody (Abcam) followed by incubation with goat anti-rabbit IgG-FluoProbes 546 (Interchim, San Diego, CA, USA). Nuclei were counterstained DAPI. Confocal microscopy and digital image recording were performed.

Quantification of angiogenic factors production by RT-qPCR

SNB75 were treated in absence or presence of 10 µM PST 3.1a during 24 h. Total mRNA was isolated from SNB75 according to the manufacturer's protocol (Qiagen, Hilden, Germany). The mRNA copy number was determined by RT-qPCR using genes coding for the VEGF-A, platelet-derived growth factor (PDGF), interleukin-6 (IL-6) and transforming growth factor- (TGF-). Total mRNA was measured on a Light cycler 480 II (Roche, Basel, Switzerland).

Quantification of the mitochondrial DNA content

HUVECs (1.2 x 10 5 per well) were seeded into 6-well plates overnight, then treated in absence or presence of PST 3.1a during 24 h. Total DNA was isolated from HUVECs according to the manufacturer's protocol (Qiagen). The mitochondrial DNA copy number was determined by qPCR using a mitochondrial gene coding for the NADH dehydrogenase subunit 1 (ND1) and a nuclear gene coding for β2 microglobulin (B2M) as respective markers of mitochondrial and genomic DNA. Total DNA was measured on a Light cycler 480 II (Roche).

Citrate synthase activity measurement

HUVECs (2.5 x 10 5) were harvested and resuspended in cell buffer containing 250 mM sucrose, Tris 20 mM, EDTA 2 mM, BSA 1 mg/ml, pH 7.2. Analysis of citrate synthase activity was performed by measuring absorbance of citrate synthase product, the thionitrobenzoic acid (412 nm), on cell lysates, thermostatically maintained at 37 • C, using Safas UVmc2 ® spectrophotometer (Safas, Monaco, Monaco). Protein dosage was finally made to normalize the quantity of cells.

Measurement of HUVEC respiration

Respiration rates were measured in the HUVEC cell line, stimulated with or without PST 3.1a (1, 5 or 10 µM). The cells were collected by trypsinization, washed once in culture medium and centrifuged (500 g × 5 min). The pellet was resuspended in respiratory medium (DMEM, Ham's F-12 1:1, 1% l-Glutamine, 1% aminopterin/thymidin/hypoxanthin, 1% non-essential amino acids). Mitochondrial oxygen consumption was recorded at 37 • C using a high-resolution Oxygraph-2K respirometer (Oroboros, Innsbruck, Austria). The cell's basal respiration rate was determined by measuring the linear rate of oxygen consumption, which reflects the cell's aerobic metabolic activity with physiological substrates in culture medium. Oligomycin (4 mg/mL) was then added to determine the respiration uncoupled to ATP synthesis. The uncoupled respiratory rate was assessed by stepwise additions of FCCP (carbonilcyanide p-triflouromethoxyphenylhydrazone, 200 µM) up to an optimal concentration that represents the capacity of the respiratory chain without the regulation of ATP synthase. Finally, non-mitochondrial oxygen consumption rate was determined by adding antimycin A (1 mg/mL) and rotenone (2.5 mM). Protein concentrations were determined using the Lowry method. Respiration rate was expressed in nanomoles of O 2 consumed per minute and per milligram of protein. Respiratory ratios were determined as described below. The respiratory control ratio (RCR) is the ratio between the FCCP and the oligomycin respiration, and reflects the component of the maximal respiratory chain capacity that is used for the compensation of the proton leak. The phosphorylating respiratory control ratio (RCRp) was obtained by subtraction of oligomycin to basal respiration, and then by dividing with the FCCP respiration. The RCRp ratio reflects the part of the maximal respiratory chain capacity used for ATP synthesis. Finally, the uncoupling control ratio (UCR) is the ratio between the FCCP and the basal respiration and reflects the reserve of capacity of the respiratory chain.

Glucose and lactate measurements

Glucose and lactate concentrations in the culture media were determined by spectrophotometry, using appropriate enzymatic kits (Boerhinger, Ingelheim am Rhein, Germany) on a Hitachi-Roche apparatus (Roche).

Western blot

After treatment, cells were homogenized and lysed. Proteins (20 µg) were separated on 4-12% precast SDS-polyacrylamide gel electrophoresis (Invitrogen, Carlsbad, CA, USA). Blots were probed with anti-P-VEGR2 (Y1175), anti-VEGFR2, anti-phosphorylated focal adhesion kinase (FAK) (Y925), anti-FAK, antiphosphorylated cyclin-dependent kinase 1 (CDK1), anti-CDK1 (Cell Signaling, Danvers, MA, USA), anti-PFKFB3 (Proteintech, Manchester, United Kingdom), anti-Glut1, anti-Galectin-1 and anti-Galectin-3 (Abcam, Cambridge, United Kingdom), anti-GnT-V (R&D systems, Minneapolis, MN, USA), anti-GnT-III (Thermo Fisher, Waltham, MA, USA) antibodies. Monoclonal anti--actin antibody (Sigma-Aldrich) was used to visualize protein gel loading. After washing, bound antibodies were detected with a secondary peroxidaseconjugated anti-rabbit antibody (US Biological, Salem, MA, USA) or anti-mouse antibody (US Biological). The protein-antibody complexes were detected by enhanced chemiluminescence system and quantified by densitometry (Amersham Biosciences, Little Chalfont, United Kingdom).

Cell cycle analysis

HUVECs (1.5 x 10 5 ) were seeded in 6-well plates and treated with or without PST 3.1a for 24 h. Cells were then trypsinized, washed in PBS and fixed with 70% ethanol at 4 °C overnight. The cells were first incubated with RNAse A (50 μg/mL, Sigma-Aldrich) at 37 °C for 10 min and then labelled with propidium iodide (0.1 mg/mL, Sigma-Aldrich) and incubated at room temperature in the dark for 30 min. DNA content was then analyzed on a Cytomics FC 500 MPL flow cytometer (Beckman Coulter, Brea, CA, USA).

Glycosylation spectrum measurement

After treatment with or without PST 3.1a (10 µM), cells were homogenized and lysed. Proteins (20 µg) were separated on 4-12% precast SDS-polyacrylamide gel electrophoresis (Invitrogen) and transferred on nitrocellulose membranes. Measurement of the full glycosylation spectrum was performed on membranes following manufacturer's instructions (Thermo Fisher).

Immunofluorescence on cells

After treatment, HUVECs were fixed with 4 % paraformaldehyde for 15 min at room temperature, and then blocked with 5 % BSA in PBS for 30 min at room temperature. Cells were treated with two types of anti-VEGFR2: (i) One antibody against the extracellular domain of VEGFR2 (ref MAB 3571, R&D system) in nonpermeabilized cells; (ii) In another set of experiments, an antibody against intracellular domain of VEGFR2 (ref 2479, Cell Signaling), and in this case, HUVECs were permeabilized with 0.2 % Triton X-100 and 0.2 % BSA in PBS. Finally, anti-Galectin-1 (Abcam) antibodies was also used. After washing with PBS, cells were respectively treated with goat anti-mouse R-phycoerythrin antibody (Invitrogen) and goat anti-rabbit fragment-FITC antibody (Sigma-Aldrich) in 5 % BSA in PBS for 1 h at room temperature.

Co-immunoprecipitation

Anti-VEGFR2 antibody (R&D systems) or anti-Gal-1 antibody (Abcam) were incubated with protein A-G Sepharose beads (Santa Cruz Biotechnology) at 4 °C for 2 h to form beads-antibody complex. Then, HUVEC protein extracts treated or not with PST 3.1a (10 µM) were added at 4 °C overnight to allow antibody interaction with VEGFR2 or Gal-1 protein. Subsequently, the tubes containing the immunoprecipitates were centrifuged, and the supernatants were eliminated. Pellets were washed two times with 100 µL lysis buffer diluted to 1:5 with PBS. Gel-loading bugger (15 µL) was then added to the pellets. The tubes were heated for 10 min at 70 °C, after which proteins were separated on 4-12% SDS-PAGE gel. The gel was trans-blotted to perform Western analysis for VEGFR2 and Gal-1 proteins as described above.

Data analysis

Data are represented as mean ± SEM; n represents the number of repeated experiments. For in vivo experiments, n represents the number of mice. Statistical analyses were performed either using one-way ANOVA (parametric) for all in vitro angiogenesis experiments, or using Mann-Whitney U-tests (nonparametric) for other experiments, using Prism software package 5.00 (GraphPAD Software, La Jolla, CA, USA). p <0.05 was considered to be statistically significant.

Results

PST 3.1a inhibits angiogenesis steps in vitro

First, the effects of PST 3.1a were assayed in vitro on HUVECs on collective migration, proliferation, adhesion and tube formation. Using a wound healing assay, we showed that PST 3.1a decreased migration of HUVECs in a concentration-dependent manner (Fig. 1A). In addition, PST 3.1a significantly decreased EC proliferation at 10 µM, as assayed by trypan blue exclusion test (Fig. 1B). It should be noted that PST 3.1a (10 µM) did modify neither apoptosis nor ATP/ADP ratio (Supplemental Fig. 1A and1B), demonstrating that PST 3.1a did not display any toxicity under the experimental conditions used. PST 3.1a at 10 µM also reduced HUVEC adhesion as compared to controls (Fig. 1C). In addition, our results highlight that PST 3.1a reduced both migration and proliferation steps in comparable proportions with tunicamycin, a broad spectrum glycosylation inhibitor (Supplemental Fig 1C and1D).

On matrigel, HUVECs reorganized and formed capillary-like structures. PST 3.1a did modify neither the areas nor the number of branching of capillary-like structures at 1 and 5 µM (Fig. 1D). Interestingly, PST 3.1a (10 µM) reduced both areas and number of branching by 50% compared to controls. Overall, these results indicate that PST 3.1a reduces the angiogenic processes in vitro at 10 µM.

PST 3.1a inhibits neovascularization in vivo

We next characterized whether PST 3.1a might also affect angiogenesis in vivo using the matrigel plug assay. bFGF-containing plugs with HUVECs treated or not with PST 3.1a at 10 µM for 24 h were subcutaneously injected into mice. After one week, the plug hemoglobin content was reduced in samples containing PST 3.1a-treated HUVECs as compared to control plugs (Fig. 2A and2B). Additional histological analyses using CD31 immunostaining confirm that PST 3.1a significantly reduced plug vascularization (Fig. 2C). In order to study the in vivo effects of PST 3.1a in more detail, we next investigated the impact of PST 3.1a on the vascular development of zebrafish embryos. We first studied formation of the sub-intestinal venous (SIV) plexus that sprout from the posterior cardinal vein as it is well established that VEGF promotes initial sprouting and proliferation of the vessels of the plexus. Our results showed that 100 µM PST 3.1a inhibited SIV formation at 72 hpf. Lower concentrations such as 75 µM showed a similar inhibitory effect of PST 3.1a on SIV but at later time points (96 hpf and 120 hpf) (Fig. 2D). No effects were observed at a concentration of 50 µM.

The number of SIV compartments was significantly reduced at 120 hpf using 75 µM and 100 µM of PST 3.1a (Fig. 2E). Quantification of SIV area confirmed alteration of SIV development, as early as 72 hpf using 100 µM of PST 3.1a, and as 96 hpf and 120 hpf using PST 3.1a at 75 µM and 100 µM (Fig. 2F).

In addition, whereas no effects were observed on intersegmental vessels (ISV) formation, PST 3.1a treatment reduced perfusion in ISV at 75 µM and 100 µM at 72 hpf, 96 hpf and 120 hpf. Fifty µM of PST 3.1a also reduced ISV perfusion at 120 hpf (Fig. 3A and3B). Lack of ISV circulation is associated with a reduction of vessels diameter at 72 hpf and 120 hpf using both 75 µM and 100 µM PST 3.1a. (Fig. 3C). We further investigated whether ISV defects may be explained by an alteration of heart function. We thus analyzed heartbeats in embryos and revealed no difference whatever the treatment time and the PST 3.1a concentrations that were used (Supplemental Fig. 2A and2B) suggesting that under these conditions PST 3.1a does not display any toxicity in zebrafish.

We next explored whether PST 3.1a also affects angiogenesis in a murine model of subcutaneous glioblastoma tumors generated by the injection of SNB75 cells in NMRI-Nude mice. After tumor development and when tumors reached up to 1 cm 3 , animals were fed for 14 days either with PST 3.1a or with control solution. Animals treated with PST 3.1a displayed a significant decrease of tumor volume in comparison to controls (Fig. 4A, 4B and4D). This effect was not associated with a modification of mouse weight during the treatment (Fig. 4C). Further histological analyses using both CD31 and vascular endothelial-cadherin immunostaining showed that PST 3.1a induced a significantly reduction of tumor vascularization (Fig. 4E,4F and Supplemental Fig 3A). Finally, PST 3.1a did not disrupt the production of tumor-secreted angiogenic factors such as VEGF-A, platelet-derived growth factor, interleukin-6 and transforming growth factor- (Supplemental Fig 3B).

PST 3.1a decreases the expression of master angiogenic regulators

We then explored whether the vascular defects caused by PST 3.1a are associated with changes in the expression and phosphorylation levels of key angiogenic regulators such as VEGFR2 and FAK. PST 3.1a (10 µM) reduced by more than 50% VEGFR2 expression but did not modify its phosphorylation level on Tyr. 1175 that regulates both cell proliferation and migration (Fig. 5A). Moreover, FAK expression, but not its phosphorylation on Tyr. 925, was significantly decreased upon PST 3.1a treatment (Fig. 5B). Finally, PFKFB3 expression, a potent glycolytic and angiogenic regulator, was strongly reduced in the presence of PST 3.1a (Fig. 5C).

PST 3.1a does not affect energy production in ECs

We next wondered whether PST 3.1a could reduce the energy production in ECs. We first measured the effects of PST 3.1a on mitochondrial activities. PST 3.1a treatment did not disrupt mitochondrial DNA content, mitochondrial biogenesis and respiration (Supplemental Fig. 4A to 4C). Moreover, EC glycolysis, analyzed by measuring the ratio between glucose consumption and lactate production, and the abundance of the glucose transporter GLUT1 was not disturbed by PST 3.1a (Supplemental Fig. 4D and4E). Together, these results suggest that PST 3.1a reduces angiogenesis without a significant effect on EC energetic metabolism.

PST 3.1a impairs HUVEC cell cycle progression via inhibition of G2/M phase transition

Besides its direct involvement in glycolysis, PFKFB3 has also been described to regulate the cell cycle progression by a mechanism involving CDK1 [START_REF] Yalcin | Nuclear targeting of 6-phosphofructo-2-kinase (PFKFB3) increases proliferation via cyclin-dependent kinases[END_REF]. To investigate whether PST 3.1a affects progression through the cell cycle, HUVECs treated or not by PST 3.1a (10 µM) were evaluated by flow cytometry. PST 3.1a treatment significantly reduced the number of cells at the G2/M phase transition, in comparison with control cells, while no significant modification was observed on G0/G1 and S phases (Fig. 6A). We next assessed the effect of PST 3.1a on CDK1 expression, a major cell cycle regulator of the G2/M transition, and found that its expression was significantly reduced with 10 µM of PST 3.1a, without modification of its inhibitory phosphorylation site on Tyr 15 (Fig. 6B). Furthermore, the abundances of cyclins A and B, the main CDK1 partners, were not affected (Fig. 6C).

PST 3.1a decreases GnT-V and disrupts VEGFR2-Gal-1 interaction

To study the implication of PST 3.1a on glycosylation, we first measure the full glycosylation spectrum of endothelial cells. PST 3.1a did not reduced the glycosylation profile of HUVECs in comparison with control (Fig. 7A). We next investigate the effect of PST 3.1a on the expression of GnT-III and GnT-V, the enzymes involved in glycosylation linkage. Whereas we did not find any changes on GnT-III expression, we found a significant reduction of GnT-V expression by PST 3.1a, in comparison to control (Fig. 7B). In addition, PST 3.1a did significantly reduce the glycan-binding proteins Gal-1 expression by 30%, but not that of Gal-3 (Fig. 7C). Since VEGFR2 interacts with Gal-1, and both protein abundances are decreased by PST 3.1a, we tested whether PST 3.1a affects the interaction between both proteins. Using confocal microscopy, we found that PST 3.1a treatment reduced VEGFR2/Gal-1 co-localization, compared to control cells using antibody that recognizes either extracellular domain (Fig. 7D and7E) or intracellular domain (Supplemental Fig. 5). Moreover, coimmunoprecipitation revealed that the physical association between Gal-1 and VEGFR2 was reduced in the presence of PST 3.1a treatment (Fig. 7F and7G). These results suggest that PST 3.1a. decreases of interaction between both protein is probably due to a decrease of GnT-V expression that might lead to a loss of VEGFR2 glycosylation.

Discussion

Herein, we evaluated PST 3.1a activity on the angiogenesis process. PST 3.1a, which is a small glycomimetic inhibitor with an efficient activity on GBM initiating cell invasiveness and proliferation, showed a remarkable ability to alter angiogenesis in vitro as well as in vivo. Importantly, we demonstrated that PST 3.1a intervenes in vitro on all the different steps leading to the angiogenesis process, such as migration, proliferation, adhesion and tube formation, and alters angiogenesis in two distinct in vivo models. Of particular interest, we highlight a novel mechanism that modulates the interaction of VEGFR2 with its glycan-binding protein Gal-1 in ECs, leading to a reduction of the metabolic process associated with the inhibition of the master glycolytic regulator PFKFB3, and reducing the cell cycle timing at the G2/M transition.

As a first step, we found that PST 3.1a at a concentration of 10 µM decreases HUVEC migration, proliferation, adhesion and tube formation, all of them being required in angiogenesis. Furthermore, and in accordance with these results, PST 3.1a reduced the expression of key angiogenic factors such as VEGFR2 and FAK that are implicated in proliferation and migration of ECs, respectively. In addition, PST 3.1a reduced the expression of the glycolytic regulator, PFKFB3, which is a potent angiogenic modulator. This observation fits with the fact that other inhibitors of PFKFB3, as the 3PO (3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one), have powerful antiangiogenic capacities [START_REF] Schoors | Partial and transient reduction of glycolysis by PFKFB3 blockade reduces pathological angiogenesis[END_REF]. This type of effect has been reported recently in human hepatocellular carcinoma drug-resistant cell line [START_REF] Li | Effect of GnT-V knockdown on the proliferation, migration and invasion of the SMMC7721/R human hepatocellular carcinoma drugresistant cell line[END_REF]. Indeed, knockdown of GnT-V significantly suppressed the proliferation, migration and invasion of the SMMC-7721/R cells by acting on the reduction of expression of specific proteins involved in these processes. Such type of association could take place in the present study with regard to inhibition of GnT-V expression and decreased FAK and PFKFB3 and subsequent inhibition of all the pathways leading to angiogenesis in endothelial cells.

Angiogenesis is a highly energetic process associated with an increase of mitochondrial respiration capacity and biogenesis [START_REF] Duluc | Modulation of mitochondrial capacity and angiogenesis by red wine polyphenols via estrogen receptor, NADPH oxidase and nitric oxide synthase pathways[END_REF]. However, we observed that the treatment with PST 3.1a does not modify either the mitochondrial biogenesis, or the mitochondrial DNA content in HUVECs. Moreover, PST 3.1a did not modify the mitochondrial respiration, suggesting that the regulation of mitochondrial activities do not contribute to the anti-angiogenic property of PST 3.1a. These results reinforce the critical involvement of the glycolytic pathway, via the modulation of PFKFB3, as an anti-angiogenic target of the compound. Indeed, besides its implication in controlling the energetic process, PFKFB3 also traffics to the nucleus to promote cell cycle progression by regulating CDK1 activity [START_REF] Yalcin | Nuclear targeting of 6-phosphofructo-2-kinase (PFKFB3) increases proliferation via cyclin-dependent kinases[END_REF]. In accordance with these evidences, treatment with PST 3.1a reduced CDK1 expression, but not its phosphorylation nor the abundances of its partner cyclins A and B, and consequently the accumulation of cells at the G2/M transition. However, unlike other reports, the inhibition of PFKFB3 expression by PST 3.1a did not induce apoptosis in HUVECs [START_REF] Yalcin | 6-Phosphofructo-2-kinase (PFKFB3) promotes cell cycle progression and suppresses apoptosis via Cdk1-mediated phosphorylation of p27[END_REF].

Altogether, these results suggest that PST 3.1a has an anti-proliferative action on ECs, which could be mediated by a cell cycle alteration at the G2/M transition, but without promoting apoptosis as summarized on Supplemental Fig. 6. Furthermore, we found that, in HUVECs, PST 3.1a reduces GnT-V expression, although without affecting significantly the global glycosylation pattern. Nevertheless, the influence of specific glycosylation on tumor growth is nowadays well recognized, its implication in angiogenesis remains poorly documented [START_REF] Potente | The Link Between Angiogenesis and Endothelial Metabolism[END_REF][START_REF] Merchan | Antiangiogenic activity of 2-deoxy-D-glucose[END_REF]. Indeed, it was reported that master angiogenic regulators, such as VEGFR2 and Notch can be glycosylated, in order to modulate their functions. In this respect, galectins that are directly connected to GnT have emerged as powerful angiogenic modulators. Notably, Gal-1 and Gal-3 are among the most powerful galectins regulating angiogenesis and pathological vascularization [START_REF] Griffioen | Galectins in tumor angiogenesis[END_REF][START_REF] Stanley | Galectin-1 pulls the strings on VEGFR2[END_REF][START_REF] Méndez-Huergo | Galectins: emerging regulatory checkpoints linking tumor immunity and angiogenesis[END_REF]. Consequently, Gal-1 inhibitors were synthetized to target melanoma, ovarian, breast and colon cancers, but none of them were reported until now to be efficient on GBM [START_REF] Le | Galectin-1: a link between tumor hypoxia and tumor immune privilege[END_REF][START_REF] Ito | Thiodigalactoside inhibits murine cancers by concurrently blocking effects of galectin-1 on immune dysregulation, angiogenesis and protection against oxidative stress[END_REF][START_REF] Ito | Galectin-1 as a potent target for cancer therapy: role in the tumor microenvironment[END_REF]. Interestingly, Gal-1 mediates the resistance to a number of anticancer therapies, therefore the finding that PST 3.1a treatment reduces Gal-1 expression in HUVECs parallels a similar observation in GBM [START_REF] Hassani | Phostine PST3.1a targets MGAT5 and inhibits glioblastomainitiating cell invasiveness and proliferation[END_REF] and is of particular interest for increasing antiangiogenic treatment efficiency. It was further shown that glycosylation controls the interaction between Gal-1 and VEGFR2 to sustained tumor angiogenesis [START_REF] Méndez-Huergo | Galectins: emerging regulatory checkpoints linking tumor immunity and angiogenesis[END_REF]. Indeed, the extracellular portion of VEGFR2 is composed by seven Ig-like domains which are highly Nglycosylated, a post-translational process crucial for its function, VEGF binding and stabilization [START_REF] Chandler | Site-specific Nglycosylation of endothelial cell receptor tyrosine kinase VEGFR-2[END_REF]. VEGFR2 glycosylating on 3, 4 and 7 sites may promote a Gal-1-dependent retention of VEGFR2 on plasma membrane, therefore enhancing VEGF signaling, even in the presence of VEGF inhibitors [START_REF] Stanley | Galectin-1 pulls the strings on VEGFR2[END_REF]. Therefore, the finding that PST 3.1a reduces Gal-1-VEGFR2 interaction in HUVECs suggests that the anti-angiogenic properties of PST 3.1a may be related to this process. This may represent a promising anti-angiogenic approach in combination with the one currently used to treat tumors growth related to excess angiogenesis.

In conclusion, we provide evidence that PST 3.1a inhibits angiogenesis in cell culture and in both mice and zebrafish models, and reduces GBM progression associated with an alteration of tumor vascularization. Of particular interest is that we highlight a novel mechanism by which PST 3.1a modulates the interaction of VEGFR2 with its glycan-binding protein Gal-1. This leads to a reduction of the metabolic process associated with the inhibition of the master glycolytic regulator PFKFB3 and the cell cycle transition at G2/M check-point.

These results provide information on an innovative approach to target angiogenesis-related diseases including cancer, which could eventually be extended to further diseases with excess vascularization. Most importantly, the present study underscores the potential use of PST 3.1a as new perspective in GBM anti-angiogenic treatments.

Figures:

Fig.

  oxaphosphinane (PST 3.1a) 4,6-diamidino-2-phenylindole (DAPI) 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) Basic fibroblast growth factor (bFGF) Bovine serum albumin (BSA) venous (SIV) Vascular endothelial growth factor (VEGF).

1 :

 1 Fig. 1: PST 3.1a inhibits angiogenesis steps in vitro. HUVECs were incubated in the absence or in the presence of different concentrations of PST 3.1a (1, 5 or 10 µM) during 24 h. A. Wound healing was performed to evaluate anti-migratory properties of PST 3.1a. B. HUVECs were counted using Trypan blue exclusion to evaluate proliferation. C. Adhesion was estimated using crystal violet. D. In vitro angiogenesis using matrigel was performed, allowing HUVECs to reorganize and form capillary-like structures. Data represent the mean ± SEM (n=3/8) (* p<0.05; ** p<0.01; *** p<0.001; versus control).

Fig. 2 :

 2 Fig. 2: PST 3.1a decreases angiogenesis in vivo. A. HUVECs were incubated in the absence or in the presence of PST 3.1a (10 µM) during 24 h. Cells were mixed with Matrigel and bFGF (6 µg/mL) and subcutaneously injected into nude mice for 1 week. Pictures show the matrigel plugs of control (left) and PST 3.1a-treated (right) cells. B. Quantitative measurement of hemoglobin (Hb) was reported as absorbance/weight of plugs. C. Representative images of matrigel plug vessel sections. Blue fluorescence represents DAPI (nuclei) and green one represents endothelial marker CD31 labelling. D. Zebrafish Tg (fli1a:EGFP/gata1a:dsRed) eggs were exposed to 50, 75 or 100 µM of PST 3.1a and SIV formation was analyzed at 72, 96 or 120 hpf using spinning disk confocal microscopy. Green fluorescence represents ECs. Scale bars represent 100 μm. E. The mean number of compartments per sub-intestinal plexus was calculated at different times. F. The measure of the SIV area coverage over the yolk was performed at different times. Data represent the mean ± SEM (n=4 for matrigel plug and n=16/21 embryos per condition) (* p<0.05; ** p<0.01; versus control).

Fig. 3 :

 3 Fig. 3: PST 3.1a decreases blood flow in Zebrafish. A. Zebrafish Tg(fli1a:EGFP/gata1a:dsRed) eggs were exposed or not to PST 3.1a at 50 and 75 µM for 72, 96 or 120 hpf. Representative photos showing ISV circulation (see Video Fig. 3 for corresponding videos). Green fluorescence represents ECs and red one represents red blood cells. Scale bars represent 100 μm. B. The number of perfused ISV was quantified in zebrafish embryos. C. Quantification of ISV diameters in zebrafish embryos. Data represent the mean ± SEM (n=16/21 embryos per condition). (* p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 versus control).

Fig. 4 :

 4 Fig. 4: PST 3.1a decreases tumor development. A. After SNB75 injection and tumor growth, mice were fed or not with PST 3.1a (12 mg/kg) during 14 days; representative echography profiles show mouse tumors. B. Histogram shows the tumor volume evolution

Fig. 5 :

 5 Fig. 5: PST 3.1a treatment reduces protein expression of key angiogenic factors in HUVECs. HUVECs were treated with 10 µM of PST 3.1a for 24 h. A. Immunoblots show VEGFR2 phosphorylation (Tyr 1175) and expression. Histograms show densitometric analysis of protein phosphorylation and expression. βactin is used as a loading control. B. Western-Blots show FAK phosphorylation (Tyr 925) and expression. Histograms show densitometric analysis of protein phosphorylation and expression. β-actin is used as a loading control. C. Immunoblots show PFKFB3 expression. Histograms represent densitometric analysis of protein expression. β-actin is used as a loading control. Data represent the mean ± SEM (n=5/7) (* p<0.05; ** p<0.01; versus control).

Fig. 6 :

 6 Fig. 6: PST 3.1a modifies HUVEC cell cycle. Cells were exposed to control medium or to PST 3.1a 10 µM during 24 h. A. Representative histograms showing the percentage of cells in the G0/G1, S and G2/M phases determined by flow cytometric analysis. B. Immunoblot shows CDK1 phosphorylation (Tyr 15) and expression. Histograms represent quantification of CDK1 phosphorylation and expression. β-actin is used as a loading control. C. Western-blot representing cyclin A and cyclin B expression in comparison with control. Histograms show densitometric analysis of protein expression. β-actin is used as a loading control. Data represent the mean ± SEM (n=4/7) (* p<0.05; ** p<0.01; versus control).

Fig. 7 :. 1 :. 4 :

 714 Fig. 7: PST 3.1a decreases GnT-V and Gal-1 expression. HUVECs were treated with PST 3.1a during 24 h at 10 µM. A. Quantitative measurement of the full glycosylation spectrum. B. Western-blots represent GnT-III and GnT-V expression. Histograms show the quantitative measurement of GnT-III and GnT-V expression. β-actin is used as a loading control. C. Immunoblots show Gal-1 and Gal-3 expressions. Histograms represent the quantitative measurement of proteins expression. β-actin is used as a loading control. D. Immunofluorescence detection of VEGFR2 (red) and Gal-1 (green) was performed and co-localization of both proteins was measured using co-immunofluorescence. E. Representative histograms of co-immunofluorescence showing the co-localization of VEGFR2 and Gal-1. F.Western-blot revealing content of Gal-1 and VEGFR2 after Gal-1 immunoprecipitation in HUVECs treated

VE-cadherin / DAPI

Acknowledgments

We thank the SCAHU staff (Université d'Angers) for taking care of mice, Dr. Christine Rampon and Matthieu Boukaissi (CIRB, CNRS UMR 7241, Collège de France, Paris) for the access to the zebrafish facilities, the staff of Plateforme d'Analyse Cellulaire et Moléculaire for the qPCR experiments, Dr. Zahra Hassani (Phost'in SAS) for experiment advices. S.B. is a recipient of a doctoral fellowship from French Ministry of Education and Research (University of Angers). This work was supported by INSERM, Région Pays de la Loire, Angers Loire Métropole, Université d'Angers and the CHU d'Angers. 2 Suppl. Fig. 2: PST 3.1a did not disrupts zebrafish heartbeats Zebrafish Tg(fli1a:EGFP/gata1a:dsRed) eggs were exposed or not to PST 3.1a at 50, 72 and 100 µM for 72, 96 or 120 hpf. A. Representative pictures showing Zebrafish embryos heartbeats (see Video Suppl Fig. 2 for corresponding videos) in the absence or in the presence of PST 3.1a. B.

Quantitative measurement of zebrafish heartbeats was reported as number of heartbeats per min.

Data represent the mean ± SEM (n=16/21 embryos per condition).

Immunofluorescence detection of VEGFR2 (red) and Gal-1 (green) was performed and colocalization of both proteins was measured using co-immunofluorescence. In this case, experiments were performed on permeabilized endothelial cells using antibody of VEGFR2 that recognizes the intracellular domain of the receptor.