Beta-blockers disrupt mitochondrial bioenergetics and increase radiotherapy efficacy independently of beta-adrenergic receptors in medulloblastoma.

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Research in context Evidence before this study In young patients with medulloblastoma, the current multimodal treatment allows 70% of children to survive up to 5 years but is often accompanied by serious and long-lasting side effects. Drug repositioning represents an attractive strategy to fast-track the development of new low-toxicity therapeutic options. Preclinical and clinical studies have shown that β-blockers can increase the efficacy of chemotherapy in drug-refractory cancers, including paediatric tumours such as neuroblastoma. However, far less is known about the ability of β-blockers to potentiate radiotherapy, which has never been studied in paediatric cancers. Moreover, the literature is divided regarding the mechanisms responsible for the anti-tumour properties of β-blockers. Here, we propose to analyse the combination of β-blockers with radiotherapy in models of high-risk medulloblastoma. Added value of this study We provide the evidence that low concentrations of propranolol, carvedilol and nebivolol improve the efficacy of ionising radiation in medulloblastoma cell lines, patient-derived tumour cells and spheroid micromasses, including those poorly responsive to radiation. In response to the ever-increasing need to find alternatives to animal experimentation, we have developed an innovative organotypic cerebellum model that confirmed the benefits of the combinatorial treatment. Although we showed that patient medulloblastoma biopsies exhibit detectable expression of β-adrenergic receptors, the efficacy of β-blockers in medulloblastoma cells does not result from the inhibition of the canonical targets but is instead driven by a rapid disruption of the mitochondrial bioenergetics. This leads to a sustained accumulation of superoxide radicals that potentiate the DNA damages caused by ionising radiation. Implications of all the available evidence Given the few druggable molecular targets identified in high-risk medulloblastoma and the fact that young age of patients limits treatment options, our work proposes an alternative approach in which drug repurposing could be quickly translated to the clinic to improve the efficacy of radiotherapy. In addition, as the dose of ionising radiations can be significantly reduced by adding β-blockers, this may help limit treatment long-term side effects and improve the quality of life of children with medulloblastoma. Lastly, our work highlights the interest of exploiting the ability of selected repositioned drugs to inhibit mitochondrial bioenergetics to design new therapeutic combinations with radiotherapy. Alt-text: Unlabelled box

Introduction

Medulloblastomas (MB) are embryonal tumours of the cerebellum and the most common malignant brain tumours of childhood. They have been classified into four main subtypes. WNT MB, has the most favorable clinical prognosis but accounts for only 10% of cases. SHH MB, and the other Non-WNT/Non-SHH subgroups (Group 3 and Group 4) are somewhat more aggressive and more frequently metastatic, with a poorer prognosis. 1 , 2 , 3 The current multimodal treatment combines surgery, radiotherapy and chemotherapy. 4 , 5 Overall, long-term survival is now achieved in 60-75% of patients but it comes at the expense of serious and often long-lasting side effects that can reduce independence and significantly alter the quality of life of survivors. 6 , 7 High-risk MB are treated with radiation therapy with a cumulative dose of 54 Gy for irradiation of the posterior cerebellar fossa and additional 36 Gy for craniospinal irradiation. Although these doses are not always sufficient to control tumour progression, they cannot be increased as both acute toxicities and the cognitive and endocrinological sequelae would be too important in the long-term. 7 , 8 Since these sequelae are even greater in young patients, radiotherapy is contraindicated in children under 3-5 years of age depending on countries. 9 , 10 Therefore, new treatment options for MB patients are needed to improve the response to radiotherapy, with the aim of increasing the therapeutic benefits of ionising radiation (IR) and/or reducing its doses and associated deleterious side effects while maintaining its efficacy.

Drug repurposing consists in using already-approved drugs for indications that differ from those for which the drugs were originally developed. Toxicity and pharmacokinetic profiles are well documented, so that repurposed drugs can directly enter Phase II clinical trials. By reducing the time, expenses and risks associated with the development process, drug repurposing is an attractive strategy in anticancer therapeutics. 11 , 12 , 13

One of the promising pharmacological classes to be repurposed is the β-adrenergic antagonists, or β-blockers. They are widely known for their regulatory properties in cardiovascular dysfunctions. 14 To date, the use of propranolol for the treatment of severe hemangiomas of infancy represents one of the most successful examples of drug repurposing, with higher efficacy and fewer toxic side effects than the previous standard of care. 15 Since then, our preclinical and clinical studies have shown that β-blockers can increase the efficacy of chemotherapy in drug-refractory cancers, 16 , 17 including paediatric tumours such as neuroblastoma. 18 β-blockers can impair fundamental biologic processes underlying tumour progression, such as cell proliferation, migration, tumour angiogenesis and metastasis. β-blockers have also been shown to sustain the response of irradiated gastric adenocarcinoma, colon adenocarcinoma or non-small cell lung cancer (NSCLC) in vivo , 19 , 20 , 21 , 22 , 23 and improve survival outcomes in adult patients with intracranial meningiomas and NSCLC. 24 , 25 These recent examples provide a strong rationale to combine β-blockers with radiotherapy in paediatric solid tumours, where this type of combination has never been evaluated. Here, we provide evidence that β-blockers can improve the efficacy of IR in MB cell lines and PDX-derived cells, by disrupting mitochondrial bioenergetics, independently of the β-adrenergic receptors.

Material and methods

Cell culture

The human MB cell lines DAOY (RRID: CVCL_1167), D341 Med (RRID: CVCL_0018) and D283 Med (RRID: CVCL_1155) were obtained from the ATCC biobank. HDMB-03 cells (RRID: CVCL_S506), were kindly provided by Sonia Martial and Gilles Pagès from the Institute for Research on Cancer and Aging (Nice, France). The human ONS-76 (RRID: CVCL_1624) and UW228-2 (RRID: CVCL_0572) cell lines were kindly provided by Janet Lindsey and Steven Clifford from the Wolfson Childhood Cancer Research Center (New Castle, UK). DAOY and UW228-2 cells are representatives of the WNT group; HD-MB03, D341 Med, D283 Med and ONS-76 cells are representatives of non-WNT/non-SHH MB.

All the cells were maintained at 37°C and 5% CO 2 . DAOY and D283 Med cells were grown in MEMα medium (Gibco, ref. 12561056) supplemented with 10% fetal calf serum (FCS; Gibco, ref. 26140079) and of 1% penicillin-streptomycin (PS; Gibco, ref. 15140122). HDMB-03 cells were maintained in RPMI 1640 medium (Gibco, ref. 21875034) supplemented with 10% FCS, 1% Non-Essential Amino Acids (Gibco, ref. 11140035) and of 1% PS. ONS-76 cells were grown in RPMI 1640 medium supplemented with 10% FCS and 1% PS. UW228-2 cells were grown in DMEM/F12 medium (Gibco, ref. 11320033) supplemented with 10% FCS and 1% PS. D341 Med cells were maintained in MEMα medium supplemented with 20% FCS and 1% PS.

A mtDsRed plasmid has been transfected in each cell line using Lipofectamine 2000 (Invitrogen, ref. 11668019) following the manufacturer's protocol. Stable transfectants were obtained after geneticin selection (0·8 mg/mL, Gibco, ref. 10131035) and two cycles of fluorescence-activated cell sorting (FACS). To establish β-blocker resistant cell lines, ONS-76 cells were exposed to increasing doses of propranolol (from 10 to 200 µM), carvedilol or nebivolol (from 2·5 to 20 µM), over 3 to 4 months. The resistant cell lines were named: ONS-76 RP, ONS-76 RC and ONS-76 RN, respectively, and were maintained in the same culture conditions as the parental ONS-76 cells ( i.e ., ONS-76 WT).

The murine SHH MB cell lines were obtained from spontaneous medulloblastoma arising from Patched1 +/- C57BL/6 mouse model (RRID: MGI:2159769), as previously described. 26

All the cells were tested for the absence of mycoplasma contamination (MycoAlert™, ref. #LT07-418, Lonza) at least once a month.

Patient-derived xenografts culture

Patient-derived xenografts (PDXs) were generated from primary human MB samples and were maintained into the subscapular fat pad of Nude mice (RRID:MGI:5649750) as previously described. 27 G3-PDX3, G3-PDX7 and SHH-PDX12 correspond to group 3 ICN-MB-PDX-3, group 3 ICN-MB-PDX-7 and SHH ICN-MB-PDX-12, respectively. For in vitro cultures, tumour cells were purified from the PDX using an enzymatic dissociation followed by a Percoll density gradient separation and cultured as previously described. 26

Drugs and reagents

The β-blockers were resuspended in dimethyl sulfoxide (DMSO). Stock solutions were stored at -20°C for Nebivolol (50 mM, Sigma-Aldrich, ref. #N1915) and Carvedilol (50 mM, Sigma-Aldrich, ref. #C3993) and at -80°C for Propranolol (150 mM, Selleckchem, ref. S4076). The antioxidants Mito-TEMPO (MT) and Troxerutin (TROX) were resuspended in DMSO and stock at -20°C. The solutions are diluted in culture medium ex-temporaneously for the experiments.

Irradiation of MB cells, spheroids and organotypic cultures

Exposure to IR of the different culture models was performed in the Radiotherapy Department of Pr. Cowen (Timone Hospital, AP-HM, France). Water-equivalent RW3 phantom with a chamber adaptation plate was used for therapy dosimetry. Cells, spheroids and organotypic cultures were exposed to doses ranging from 1·8 Gy to 10 Gy, using the Synergy MLCi Elekta® linear accelerator with a beam of 6 MV and a flow rate of 400 UM/min. The PDX cells were irradiated in the RadExp platform of the Curie Institute on the X-Rad 320 equipment (Precision X-ray irradiation).

Cell growth and survival assays

Cell viability assays were performed as previously described. 18 Briefly, the human MB cells were seeded in flat bottom 96-well microplates (2,000 cells/well for DAOY, ONS-76 and UW228-2; 9,000 cells/well for HD-MB03 and D283 Med; 12,000 cells/well for D341 Med) for 24 h. Cells were then exposed to β-blockers alone or in combination with IR for 72 h. Metabolic activity was detected by addition of Alamar Blue and spectrofluorimetric analysis using a PHERAstar® FS multi-Plate Reader (BMG LABTECH; λ ex 540 nm / λ em 590 nm). IC 50 values were determined as previously described. 28

For IncuCyte experiments (RRID:SCR_019874), SHH MB tumour cells were plated in 96-well plates (5,000 cells/well for murine SHH-MB and 7,500 cells/well for ICN-MB-PDX-12), pre-coated with poly-D-lysine (EMD Millipore, ref. A-003-E) and Matrigel (BD Biosciences, ref. 354234). The next day, tumour cells were treated with a range of concentrations of β-blockers or the control, as indicated in figures. Propidium iodide (PI, Sigma Aldrich; 0·3 μg/ml) was also added to the medium to evaluate cell death. Then, the plates were scanned for phase contrast and PI staining during 72-96 h, using the IncuCyte® live cell analysis system with a 4X objective. Proliferation was measured using quantitative kinetic processing metrics from time-lapse image acquisition and showed as percentage of culture confluence over time. For the PI staining, the percentage of PI positive cells was divided by the percentage of cell confluence for each well, thus indicating the level of dead cells in each well.

For the CellTiter-Glo® Luminescent Cell Viability Assay, Group 3 MB tumour cells were cultured in neurospheres in round bottom 96-well plates (5,000 cells/well). Tumour cells were then treated either 1) once with a range of concentrations of β-blockers or 2) daily with β-blockers and/or IR for five consecutive days. Then, the cell viability was evaluated 72 h later using the CellTiter-Glo® Luminescent Cell Viability Assay according to the manufacturer's instructions (Promega Corporation, ref. G7570).

Spheroid growth assay

DsRed-expressing MB cells were plated in round bottom 96-well microplates (1,200 cells/well for HD-MB03 – 1,500cells/well for UW228-2 and DAOY – 2,000 cells/well for ONS-76, D283 Med and D341 Med) in a culture medium containing 10% FBS and 20% methyl cellulose (Sigma-Aldrich, ref. M7027) for 72 h. Spheroids were then daily treated with β-blockers and/or IR for five days. Spheroid growth was quantified over time by acquisition of DsRed fluorescence signal using the PHERAstar® FS multi-plate reader (λ ex 580 nm/λ em 620 nm – “well scanning” 10 × 10). Images were captured with the JuLI™Stage live imaging system (NanoEntek).

Cerebellar organotypic model development and analysis

To establish organotypic cultures of cerebellar tissues, mouse cerebellums were surgically harvested and sectioned into 250 µm thick slices using a vibrating blade microtome (RRID:SCR_016495). A spheroid formed from DsRed-expressing MB cells was then grafted onto each cerebellum slice. These organotypic co-culture models were then placed on inserts with 0,4 µm pore size membranes (Falcon®, ref. 353090) and maintained in medium containing 50% MEMα, 25% horse serum (Gibco, ref. 16050122), 25% Hanks' Balanced Salt Solution (HBSS; Gibco, ref. 14065056), 10 mM HEPES buffer (Gibco, ref. 15630106), 28 mM Glucose (Gibco, ref. 15023021), 1% L-Glutamine (Gibco, ref. 25030081) and 1% PS. After daily exposure to IR and/or β-blockers for 5 consecutive days, tumour growth and invasion within the cerebellum slices were analysed over time, using the JuLI™ Stage imaging system and the PHERAstar® FS multi-plate reader (λ ex 580 nm/λ em 620 nm - fluorescence signal acquisition with a 15 × 15 matrix scanning mode).

Sample preparation and immunohistochemistry

Samples were fixed overnight at 4°C with 4% formaldehyde and prepared for paraffin inclusion using automated tissue processor ASP 300 (RRID:SCR_018916). Dehydration, clarification, and infiltration steps were performed by successive absolute ethanol, histolemon and paraffin baths. After FFPE-embedding, samples were cut at 3µm-thickness with HM340E microtome (Thermo Scientific). Hematoxylin Eosin Safran staining was performed using automated H&E staining Dako CoverStainer.

Ki-67- and γH2AX-immunohistochemistry was carried out with rabbit anti-Ki67 antibody (RRID:AB_443209) and with mouse anti-γH2AX antibody (Merck Milliopore, ref. JBW301) on a Ventana Discovery XT (RRID:SCR_018643). After deparaffinisation, antigen retrieval was performed with Citrate-based buffer pH 6·5 (RiboCC Solution, CC2, ref. 760-107). The primary antibodies were incubated for 20 min at 37°C then an OmniMap anti-Rabbit HRP Detection Kit (ref. 760-149) was used with DAB. Finally, the counterstaining was done with hematoxylin and slides were cleaned, dehydrated and coversliped with permanent mounting media. The microscopic analysis of the tissues was carried out by the pathologists of the Neuropathology Department (Timone Hospital, AP-HM, France).

Western-blot

Cells were lysed in RIPA buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% Triton X-100, 0·1% SDS) to which was extemporaneously added a cocktail of phosphatase and protease inhibitors (Sigma-Aldrich, ref. PPC1010). Protein concentration was determined using a Protein Assay Dye Reagent Concentrate (Bio-Rad, ref. #5000006EDU) according to the Bradford method. Proteins (30 µg) were separated by polyacrylamide-SDS gel (10% Mini-PROTEAN® TGX™ Precast Gels, Bio-Rad, ref. 4561034) and were transferred on nitrocellulose membrane (GE Healthcare Life Sciences). Primary antibodies used were directed against COX2, β-actin or γH2AX (RRID: AB_2571729, RRID: AB_2242334 and RRID: AB_2799949, respectively). Secondary antibody coupled to peroxidase (RRID: AB_2099233 for anti-rabbit, or RRID: AB_330924 for anti-mouse IgG antibody) were used for revelation, performed using the Luminata chemiluminescence detection kit (Millipore, ref. WBLUC0100). Images were acquired using the G-BOX phosphoimager (Ozyme) and signal quantification was realised by Image J® software (RRID:SCR_003070).

Measurement of superoxide production

MB cells were seeded on 96-well microplates (2,000 cells/well for ONS-76 and 9000 cells/well for HD-MB03) for 24 h and exposed to IR and/or β-blockers for 6 h. 3D spheroids of MB cells were formed 3 days before treatment, and exposed to IR and/or propranolol for 6 h. Superoxide anion production was assessed by adding 10% V/V of WST-1 reagent (Roche, ref. 11644807001) in the wells for 30 min at 37°C. Absorbance was measured at 450 nm with a PHERAstar® FS multi-plate reader. To normalise superoxide production to the cell number in each condition, cells were fixed with 1% glutaraldehyde and stained with a solution of 1% (W/V) crystal-violet in 20% methanol (Sigma-Aldrich). The dye has finally been solubilised in DMSO to measure absorbance at 600 nm.

Colony formation assay

Ninety-six-well microplates were coated with 1% agarose for 24 h. Two hundred and fifty ONS-76 cells and 500 HD-MB03 cells per well were then plated in a 10% Matrigel®- containing medium (Corning, ref. 354234) for 24 h and exposed to β-blockers and/or IR. Photos of the colonies were captured with the JuLI™ Stage imaging system and quantified using the Image J® software, 7 and 10 days after treatment initiation for ONS-76 and HD-MB03 cells, respectively.

Analysis of energy metabolism

Energy metabolism analysis was performed using the Seahorse XFe24® extracellular flux analyser (RRID:SCR_019539). Adherent MB cells were seeded in XF24 V7-PS plates (10,000 cells/well for DAOY; 12,000 cells/well for ONS-76 and UW228-2; 30,000 cells/well for HD-MB03; Agilent, ref. 102340-100) for 24 h. Cells were then exposed to β-blockers and/or IR for 6 or 24 h. Oxygen consumption rate (OCR) was measured using XF cell Mito Stress test (Agilent, ref. 103015-100). One hour before measurement, culture medium was changed with unbuffered DMEM, 143 mM NaCl, 2 mM glutamine, 1 mM sodium pyruvate and 10 mM glucose. OCR was measured after injection of 1 µM Oligomycin, 0·5-1 µM FCCP and 0·5 µM antimycin A/rotenone mixture. The acidification rate of the extracellular environment (ECAR) was measured using XF Glycolysis stress test (Agilent, ref. 103020-100). Cell culture medium was replaced with unbuffered DMEM, 143 mM NaCl, 2 mM glutamine and 1 mM sodium pyruvate. ECAR was measured after the addition of glycolysis modulators: 10 mM glucose, 1 µM oligomycin and 100 mM 2-deoxy-glucose.

Maximal mitochondrial respiration was measured after injection of FFCP. OCR-linked ATP production was calculated with difference between basal and maximal respiration values, while glycolytic reserve was calculated as the difference between oligomycin-enhanced and glucose-mediated ECAR values.

To normalise the data to cell number, cells were fixed with glutaraldehyde 1%, stained with violet crystal in 20% methanol (Sigma-Aldrich) and solubilised with DMSO to measure absorbance at 600 nm with PHERAstar®FS multi-plate reader. A calibration range established for each type of cell was finally used to convert the absorbance values into cell numbers.

Quantitative RT-PCR analysis

The expression of β1-, β2- and β3-adrenergic receptor genes ( ADRB1, ADRB2 and ADRB3 ) was examined using real-time quantitative RT-PCR. Total cellular RNA was extracted from MB cells using the RNeasy Mini Kit according to the protocol supplied by the manufacturer (Qiagen, ref. 74104). RNA concentration was measured with the NanoVue™ Plus spectrophotometer (GE Healthcare Life Sciences). Reverse transcription of RNAs was done using QuantiTect® Reverse Transcription kit (Qiagen, ref. 205311) and real-time PCR was ran using the QuantiTect® SYBR® Green PCR kit (Qiagen, ref. 204143) on a LightCycler®480 Instrument (RRID:SCR_020502). The primers were synthetised by Qiagen (QuantiTect® Primer Assay, ref. 249900) according to the sequences described in Cao DX et al . 29 Gene expression level of ADRB1, ADRB2 and ADRB3 was determined using 2^-∆Ct or 2^-∆∆Ct method, normalised to GAPDH as housekeeping gene.

siRNA transfection

Small interfering RNAs directed against β1- or β2- adrenergic receptors (Silencer Select siRNA, ref. #4392420 - S1118, S1119 and S1120 for ADRB1 and S1121, S1122 and S1123 for ADRB2 , ThermoFischer), and non-targeted SignalSilence® Control siRNA (Cell signaling, ref. #6568S) were transfected in MB cells using Lipofectamine™ 3000 (Invitrogen, ref. L3000015) according to the protocol supplied by the manufacturer. Verification of the successful transfection was performed by quantitative RT-PCR, as described above.

RNA sequencing

The MB cells ONS-76 WT, ONS-76 RP, ONS-76 RC and ONS-76 RN were homogenised using a Buffer RLT (Qiagen, ref. 79216) and DNA-free cell lysates were obtained using genomic DNA purification columns (Qiagen). Extraction of total RNA was performed using the RNeasy Plus Mini kit (Qiagen, ref: 74134), according to the protocol supplied by the manufacturer. RNA was quantified using a NanoVue™ Plus spectrophotometer (GE Healthcare Life Sciences). RNA-Seq libraries were generated from 600 ng of total RNA using TruSeq Stranded mRNA Library Prep Kit and TruSeq RNA Single Indexes kits A and B (Illumina), as previously described. 30 The final cDNA libraries were checked for quality and quantified using capillary electrophoresis. Libraries were then sequenced on an Illumina HiSeq4000 sequencer (RRID:SCR_016386) as single end 1 × 50 base reads. Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14. Reads were preprocessed using Cutadapt v1.10 31 in order to remove adapter, polyA and low-quality sequences (Phred quality score below 20), reads shorter than 40 bases were discarded for further analysis. Reads mapping to rRNA were also discarded (this mapping was performed using Bowtie v2.2.8. 32 Reads were then mapped onto the hg38 assembly of human genome using STAR v2.5.3a. 33 Gene expression was quantified using htseq-count v0.6.1p1 34 and gene annotations from Ensembl release 99. Statistical analysis was performed using R 3.3.2 and DESeq2 1.16.1 Bioconductor library. 35

Read counts for ADRB1, ADRB2 and ADRB3 expression in primary MB samples from patients were produced by aligning paired end RNA-seq (∼90 M read/sample Illumina HiSeq2500; RRID:SCR_020123) reads to HG19 genome using STAR-align. 33 Read counts were produced using HT-SEQ-count. DESeq 2 (R/Bioconductor) was used to normalise reads to library size and variance stabilised data (VSD) was generated using the vsd function. Statistical testing for differential expression across groups was performed using an ANOVA test.

Ethics

Tumour samples from individuals with confirmed medulloblastoma diagnosis were used for RNA-seq analysis. These were provided as part of UK CCLG-approved biological study BS-2007-04 and/or with approval from Newcastle North Tyneside Research Ethics Committee (study reference 07/Q0905/71); informed, written consent was obtained from parents of all patients younger than 16 years.

All animals for PDX were housed in the animal facility of the Institut Curie, in accordance with the recommendations of the European Community (2010/63/UE) for the care and use of laboratory animals. Experimental procedures were specifically approved by the ethics committee of the Curie Institute CEEA-IC #118 (approval number: 03130.02, C91471108 and Authorisation APAFiS# 26879-2020081315161665-v1 given by National Authority) in compliance with the international guidelines. Cerebellar explants were obtained from the animal facility of the Faculty of Pharmacy, in accordance with the recommendations of the European Community (approval number: E 13 055 20).

Statistics

All experiments were performed in independent replicates and statistical significance was determined by ANOVA or student's t test using the GraphPad Prism 6 software (RRID:SCR_002798). A significant difference between two conditions is defined as: * p < 0·05, ** p < 0·005, *** p < 0·001.

Role of funders

The study sponsors did not have any role in study design, in the collection, analysis, interpretation of data, in the writing of the manuscript or in the decision to submit it for publication.

Results

β-blockers inhibit the proliferation and survival of MB cell lines and patient-derived tumour cells

To determine the anti-proliferative properties of three different β-blockers with different selectivity profiles for adrenergic receptors – i.e., non-selective β-blocker propranolol, mixed α/β-blocker carvedilol and β1-selective antagonist nebivolol –, we first used a panel of six human MB cell lines characteristic of group 2 SHH (UW228-2 and DAOY) and non-WNT/non-SHH group (HD-MB03, ONS-76, D283 Med and D341 Med) tumours. All tested β-blockers inhibited the proliferation of MB cells, irrespective of their group ( Figures 1 a-c), with IC 50 values ranging from 60-120 µM for propranolol, 12-15 µM for carvedilol and 13-15 µM for nebivolol ( Table 1 ). We further showed that the activity of β-blockers results from both the inhibition of cell growth and the induction of cell death in murine SHH-MB cells (Figure S1a-f). To evaluate the three β-blockers in more clinically relevant cellular models, we cultured primary cells from group 3 and SHH patient-derived xenografts MB tumours (G3-PDX7 and SHH-PDX12, respectively). We confirmed the dose-dependent efficacy of propranolol, carvedilol and nebivolol in inhibiting cell survival of these PDX-derived cells ( Figures 1 d-f), as well as their ability to inhibit cell proliferation and induce cell death (Figure S1g-l). Figure 1 Inhibition of MB cell survival by β-blockers . Cell survival analysis in six human MB cell lines by Alamar Blue assay after 72 h of treatment with increase concentrations of propranolol (a) , carvedilol (b) and nebivolol (c) . Values are the average of ten independent experiments ± standard deviation (SD), with a biological triplicate in each experiment. Evaluation of cell survival using CellTiter-Glo® assay in neurosphere cultures of human MB cells from G3-PDX7 and IncuCyte® live cell analysis system for SHH-PDX12 cells, after 96 h of treatment with increase concentrations of propranolol (d) , carvedilol (e) and nebivolol (f) . Values are the average of three independent experiments ± SD, with a biological triplicate in each experiment. * p < 0·05; ** p < 0·005; *** p < 0·001. Figure 1 Table 1 IC 50 values of propranolol, carvedilol and nebivolol after 72 h of treatment of non-Wnt human MB cells (DAOY, UW228-2, HD-MB03, ONS-76, D283 Med, D341 Med), mice MB cells (murine SHH-MB) and PDX-isolated cells (G3-PDX7 and SHH-PDX12), determined by GraphPad Prism software. Values are the average of at least three independent experiments ± SD. Table 1 Propranolol Carvedilol Nebivolol DAOY 124.9 ± 3.2 14.9 ± 0.1 15.0 ± 0.1 UW288-2 101.5 ± 3.8 14.6 ± 0.1 14.5 ± 0.1 HD-MB03 91.1 ± 3.1 12.9 ± 0.4 12.9 ± 0.4 ONS-76 111.8 ± 2.3 13.2 ± 0.2 13.2 ± 0.2 D283 Med 105.3 ± 2.7 13.9 ± 0.4 13.9 ± 0.4 D341 Med 62.0 ± 1.4 13.0 ± 0.3 13.0 ± 0.3 murine SHH-MB 31.2 ± 2.7 6.3 ± 0.4 1.7 ± 0.3 G3-PDX7 93.2 ± 6.0 5.3 ± 0.2 10.9 ± 0.4 SHH-PDX12 133.7 ± 8.2 17.2 ± 0.6 13.0 ± 1.1

β-blockers enhance IR-mediated inhibition of MB cell proliferation and clonogenicity

To study the combination between β-blockers and radiotherapy in MB cells, we first tested a single co-treatment of IR at 2, 5 or 10 Gy and low concentrations – IC 20 – of propranolol, carvedilol or nebivolol. Results showed that the addition of the β-blockers led to a two-fold reduction in the dose of IR while maintaining the same activity in HD-MB03 cells ( Figure 2 a). For example, irradiation at 2 Gy combined with IC 20 of propranolol is as effective in reducing cell survival as irradiation alone at 5 Gy. IR potentiation by propranolol was also found in ONS-76 cells ( Figure 2 b) and in the three other tested MB cell lines (Figure S2a-c). Similar effects were observed with low concentrations of carvedilol or nebivolol combined to IR in the different MB cell lines ( Figures 2 a-b and Figure S2a-c). To better explore the potential of these combinations in MB cell radiosensitivity, we conducted clonogenic assays. HD-MB03 and ONS-76 cells were exposed to propranolol and/or IR at 1·8 Gy, which is the daily radiation dose the most widely used in the clinic. As expected, the number of colonies was reduced by IR by 64 ± 5 % and 60 ± 5 % in HD-MB03 and ONS-76 cultures, respectively ( Figures 2 c-d). Our results also demonstrated that propranolol decreased the clonogenicity of MB cells, in a dose-dependent manner and significantly enhanced the efficacy of IR ( Figures 2 c-d and Figure S2d). For instance, the clonogenic capacity of HD-MB03 and ONS-76 cells exposed to the combination of IR with propranolol IC 20 was reduced by 86 ± 3 % and 82 ± 3 %, respectively ( Figures 2 c-d, p < 0·001 vs control). Figure 2 Increase of IR efficacy by β-blockers in MB cells . HD-MB03 (a) and ONS-76 (b) cell survival analysis by the Alamar Blue assay after 72 h of treatment with IC 20 of propranolol (propra), carvedilol (carve) or nebivolol (nebi) alone and combined to radiotherapy (IR) 2, 5 and 10 Gy. Values are the average of three independent experiments ± standard error of mean (SEM). Quantification of HD-MB03 (c) and ONS-76 (d) cell colonies, using the ImageJ™ software, respectively after 10 or 7 days of treatment with increase concentrations of propranolol (propra) or radiotherapy (IR) 1·8 Gy alone and their combination. Values are the average of three independent experiments ± SEM. * p < 0·05; ** p < 0·005; *** p < 0·001. Figure 2

To confirm the interest of such a combination in 3D tumour micromasses, we developed tumour spheroid models from MB cells stably expressing DsRed. For five consecutive days, spheroids were exposed to daily low doses of β-blockers alone or in combination with IR at carvedilol and nebivolol sustainably potentiate IR in HD-MB03 spheroids, as compared with IR alone ( Figures 3 a-d). While IR did no longer significantly impact the spheroid growth at day 21 (2317 ± 60 % growth in irradiated versus 2206 ± 79 % growth in control spheroids; p > 0·05), the co-treatment with propranolol, carvedilol and nebivolol decreased the spheroid growth to 724 ± 5 %, 335 ± 2 % and 292 ± 5 %, respectively (p < 0·001, Figures 3 a-d). Similar results were obtained in ONS-76 spheroids (Figure S3a). In addition, β-blockers were able to restore IR efficacy in UW228-2 and D283 Med spheroids that were unresponsive or minimally responsive (Figure S3b-c) and they could further increase IR efficacy against the highly radio-sensitive D341 Med spheroids (Figure S3d). Finally, to evaluate the efficacy of daily co-treatment on primary MB cells, we established 3D neurospheres from the G3-PDX7 cells. Our results confirmed that low concentrations of propranolol, carvedilol, or nebivolol highly potentiated the effects of IR ( Figure 3 e). The use of a second group 3 PDX model (G3-PDX3) further validated the relevance of combining β-blockers in co-treatment with daily radiotherapy in MB (Figure S3e). Altogether, our results demonstrated that β-blockers can improve the efficacy of IR in in vitro MB models. Figure 3 β-blockers enhance IR activity in 3D spheroids of MB cell lines and MB PDX cells. For five consecutive days, the 3D tumour micromasses were exposed to daily low doses (IC 10 ) of β-blockers or radiotherapy (IR) 1·8 Gy alone and their combination. (a) Representative images of DsRed-expressing HD-MB03 spheroids, acquired over time with the JuLi™ Stage system. Treatment efficacy was expressed as a percentage of spheroid growth inhibition vs control spheroids (Ctl), determined by quantifying the DsRed signal with the PHERAstar microplate reader. Scale bars: 500 µm. HD-MB03 spheroid growth measured by acquisition of the DsRed signal over 21 days after treatment with IC 10 of propranolol (propra) (b) , carvedilol (carve) (c) and nebivolol (nebi) (d) alone or combined with radiotherapy (IR) 1·8 Gy. Values are the average of n = 15 samples per condition, from at least four independent experiments ± standard deviation (SD). (e) Spheroid viability assessment by using the CellTiter-Glo® assay in G3-PDX7 cells after 72 h of treatment with IC 10 of propranolol (propra), carvedilol (carve) and nebivolol (nebi) alone or combined with radiotherapy (IR) 1·8 Gy. Values are the average of n = 10 samples per condition, from three independent experiments ± SD. * p < 0·05; ** p < 0·005; *** p < 0·001. Figure 3

Fractionated IR is potentiated by daily low doses of β-blockers in cerebellar organotypic models

To evaluate the potential of the β-blockers and IR combination in more clinically relevant conditions, we developed an organotypic cerebellar model in which MB spheroids stably expressing DsRed were grafted into slices of healthy mouse cerebellum. These innovative cultures were daily exposed to IR (1·8 Gy) and/or very low concentrations of propranolol (IC 10 i.e. , 25 µM) for five consecutive days. After seven days, our data showed that monotherapies reduced the growth of HD-MB03 tumour masses by 23 ± 5% and 27 ± 5% in organotypic models subjected to IR and propranolol, respectively ( p < 0·001 and p < 0·05, respectively; Figures 4 a-b). The combinatorial treatment resulted in a reduction of 38 ± 6% ( p < 0·001 compared with control), which significantly increased the efficacy of IR ( p < 0·001, Figures 4 a-b). The potentiating effect persisted over time, with the combination reducing tumour growth by 57 ± 6% after 14 days ( p < 0·001 vs control and p < 0·05 vs IR; Figures 4 a-b). The benefits of combining propranolol with IR were also confirmed in organotypic cerebellar models transplanted with ONS-76 tumour masses (Figure S4a-b). After 14 days, the organotypic cultures were fixed, sectioned, and subjected to HES and Ki67 staining ( Figure 4 c). Microscopic analysis of these labelling patterns showed that the combinatorial treatment did not induce histological lesions in the non-tumour cerebellar tissue, including in the MB periphery. Furthermore, γH2AX staining of the organotypic models showed that the co-treatment with IR and propranolol did not induce DNA damage in the non-tumour tissue either ( Figure 4 c, Table 2 , and positive control in FigureS4c). This suggests that the combination is effective in significantly reducing MB tumour mass without inducing additional damage to the cerebellum. Figure 4 Benefits of daily low concentrations of β-blockers combined to IR in ex-vivo MB organotypic model. For five consecutive days, the organotypic cerebellar co-cultures were exposed to daily low doses (IC 10 ) of propranolol alone or combined to radiotherapy (IR) 1·8 Gy. (a) Representative pictures, acquired with the JuLi™ Stage live imaging system, of DsRed-expressing HD-MB03 tumour micromasses grafted in slices of healthy cerebellum. Scale bars: 1 mm. Results were expressed as percentage of growth inhibition in treated vs control organotypic models (Ctl). (b) HD-MB03 tumour growth was measured over 14 days by acquisition of the DsRed signal with the PHERAstar microplate reader (well-scanning mode). Values are the average of n = 15 samples per condition, from five independent experiments ± standard deviation (SD). (c) Representative pictures of HES, KI67 and γH2AX immunostaining in organotypic models, control (Ctl) and treated with IC 10 of propranolol (propra), radiotherapy (IR) 1·8 Gy or their combination. Scale bars: 100 µm. * p < 0·05; ** p < 0·005; *** p < 0·001. Figure 4 Table 2 yH2AX positive cells (% +/- SD) in the cerebellum of the organotypic models. Quantification was made by microscopic analysis in the Neuropathology Department. p values > 0·05 indicate no significant variations. Table 2 % SD p Control 1.6 1.34 / Propra IC10 1.6 1.30 0.49 vs Control IR 1,8 Gy 2.0 1.00 0.30 vs Control IR + Propra 1.2 0.63 0.28 vs Control 0.08 vs IR

β-blocker efficacy and potentiation of IR are independent of β-adrenergic receptors in MB cells

The strong synergism between IR and β-blockers in MB stresses the need for a better understanding of the underlying mechanism(s). Since β-blockers antagonise the β-adrenergic receptors (β-ARs) in the cardiovascular system, we first evaluated the expression pattern of β-AR genes ADRB1, ADRB2 and ADRB3 in MB tumours from a cohort of 240 patients ( Figures 5 a-c). While there are significant differences in expression of β-AR isoforms across MB groups (each p < 0·001), the median expression of ADRB2 is the highest, followed by ADRB1 , and WNT MB are the only samples that express high levels of ADRB3 . Kaplan Meier and Cox regression analyses revealed that high expression (>median) of ADRB1 and ADRB2 were associated with a good prognosis in a cohort of 222 patients ( Figures 5 d-f). We then quantified β-AR mRNA levels in the six human SHH and non-WNT/non-SHH group MB cell lines studied. Consistently with the results obtained with patient samples, ADRB2 and ADRB3 are the major and the minor isoforms across the panel of cell lines, respectively ( Figure 5 g). Interestingly, despite being as sensitive as the other cell lines to the β1-selective antagonist nebivolol ( Figure 1 c), neither the DAOY nor the D341 Med cell lines express ADRB1 ( Figure 5 g). This suggests that the efficacy of β-blockers in MB cells may not rely on the canonical β-adrenergic pathway. To confirm this hypothesis, we silenced ADRB1 and ADRB2 in HD-MB03 and ONS-76 cells using RNAi technology (Figure S5a-b), β3-AR not being a target of any of the three β-blockers tested here. Our data showed that the efficacy of propranolol, carvedilol and nebivolol alone or in combination with IR was not impacted by β-AR silencing (dotted vs. solid lines, Figure 5 h and Figure S5c-g). Moreover, β-AR siRNA did not improve the effects of IR alone in HD-MB03 cells, regardless of the dose used ( Figure 5 h and Figure S5c-d). In ONS-76 cells, β-AR silencing even significantly reduced the effects of IR ( p < 0·05; Figure S5e-g). These results indicate that β-ARs are neither involved in β-blocker-induced cytotoxicity nor in radio-sensitisation of MB cells and support the idea that β-blockers trigger an alternative signaling pathway to potentiate radiotherapy in MB cells. Figure 5 β-Adrenergic receptors (β-AR) are not involved in the response of MB cells to β-blockers. Boxplot showing expression of ADRB1 (a) , ADRB2 (b) and ADRB3 (c) by subgroup in MB patient cohort (n = 240). Kaplan-Meier curves reporting patient overall survival stratifying patients by > median and < media expression for ADRB1 (d) , ADRB2 (e) and ADRB3 (f) expression levels. (g) Relative gene expression of ADRB1, ADRB2 and ADRB3 quantified by qRT-PCR using GAPDH as housekeeping gene (n = 8 per condition, from four independent experiments). Calculation has been done by the 2^-∆Ct method. Values are the average of independent experiments ± standard error of mean (SEM). (h) Cell viability analysis by using the Alamar Blue assay after 72h of treatment with radiotherapy (IR) 1·8, 5 and 10 Gy and increase concentrations of propranolol in HD-MB03 cells transfected with siRNA control (Ctl) or siRNA ADRB1&2 . Values are the average of three independent experiments ± SEM, with a biological triplicate in each experiment. * p < 0·05; ** p < 0·005; *** p < 0·001. Figure 5

Response to β-blockers is associated with inhibition of energy metabolism in MB cells

Our previous work in triple-negative breast cancer has highlighted the ability of propranolol to affect energy metabolism pathways in tumour cells. 36 To determine whether β-blockers disrupt the energy metabolism in MB cells, we characterised their bioenergetic profiles by measuring the mitochondrial respiration via the oxygen consumption rate (OCR) and the glycolytic activity via the extracellular acidification rate (ECAR). β-blocker treatment induced a significant drop in mitochondrial respiratory functions in ONS-76, HD-MB03, UW228-2 and DAOY cells, regardless of their initial bioenergetic status ( Figure 6 a and Figure S6a). Indeed, our data showed a decrease in both basal and maximal respiration after 24 h treatment with propranolol, carvedilol and nebivolol, for concentrations ranging from IC 20 to IC 80 ( Figures 6 b-c). As a result, ATP production was strongly reduced in all four MB cell lines exposed to the three β-blockers, even at the lowest concentrations ( Figure 6 d and Figure S6b, d, f). In addition, we demonstrated that incubation with propranolol, carvedilol and nebivolol led to a decrease in the glycolytic reserve in ONS-76, HD-MB03, DAOY and UW228-2 cells ( Figure 6 e and Figure S6c, e, g). Thus, treatment with β-blockers results in a metabolic catastrophe that deprives MB cells of their adaptive bioenergetics capacities. Figure 6 β-blockers inhibit MB cell energy metabolism. (a) Metabolic profile of MB cells before (Ctl) and after a 24 h treatment with IC 50 of propranolol (propra) or carvedilol (carve). Mitochondrial respiration (OCR, oxygen consumption rate) and glycolytic activity (ECAR, extracellular acidification rate) were measured with the Seahorse XFe24® analyser. Values are the average of at least six independent experiments ± standard error of mean (SEM). Basal respiration (b) , maximal respiration (c) , ATP production (d) and glycolytic reserve (e) of ONS-76 cells exposed for 24 h to increasing concentrations of propranolol (propra), carvedilol (carve) or nebivolol (nebi). Values are the average of three independent experiments ± SEM, with a biological duplicate in each experiment. ECAR and OCR were normalised to cell number. * p < 0·05; ** p < 0·005; *** p < 0·001. Figure 6

To better understand the importance of bioenergetics in response to treatment, we generated β-blocker-resistant ONS-76 cells by exposing them to increasing concentrations of propranolol, carvedilol or nebivolol for 16 weeks (Figure S7a). The resulting cell lines i.e. , ONS-76 RP, ONS-76 RC and ONS-76 RN, were cross-resistant to all β-blockers (Figure S7b). By qRT-PCR, we showed that the expression of β-AR genes was not altered in these resistant cells (Figure S7c). RNA sequencing further indicated that cell resistance could not be explained by the downregulation of key genes of the β-AR downstream signaling and its transcriptional targets (Figure S7d). Although four of the ten isoforms of adenylate cyclase are overexpressed in ONS-76 RP cells (ADCY1, 2, 5 and 8; p < 0·001), this pattern of overexpression was not found in ONS-76 RC and ONS-76 RN and therefore may not be the common factor behind the cross-resistance of the cell lines to beta-blockers. Analysis of the metabolic energetic activities in the three β-blocker resistant cell lines showed that they had higher mitochondrial OCR and ATP production than the sensitive parental ONS-76 WT cells ( p < 0·001, Figures 7 a-b and Figure S7e), but no significant changes in glycolytic capacity ( Figures 7 c-d). However, as illustrated with ONS-76 RC cells exposed to IC 20 of propranolol, carvedilol or nebivolol, resistant cells were able to counteract both the β-blocker-mediated suppression of ATP production by mitochondria and glycolytic reserve ( Figures 7 e-f). Figure 7 MB cells resistant to β-blockers are not sensitive to the treatment-mediated alteration of energy metabolism. Mitochondrial oxygen consumption rate (OCR) (a) and ATP production (b) in β-blocker sensitive cells (ONS-76 WT) and in three β-blocker resistant cells (ONS-76 RP, ONS-76 RC, ONS-76 RN), measured using the Seahorse XFe24® analyser. Glycolytic extracellular acidification rate (ECAR) (c) and glycolytic reserve (d) in β-blocker sensitive cells (ONS-76 WT) and in β-blocker resistant cells (ONS-76 RP, ONS-76 RC, ONS-76 RN), measured using the Seahorse XFe24® analyser. ATP production (e) and glycolytic reserve (f) measured with the Seahorse XFe24® analyser in sensitive and carvedilol resistant cells (ONS-76 RC) exposed to IC 20 of propranolol (propra), carvedilol (carve) or nebivolol (nebi) for 24 h. ATP production (g) and glycolytic reserve (h) measured by using the Seahorse XFe24® analyser in HD-MB03 cells exposed for six hours to radiotherapy (IR) 1·8 Gy or IC 10 -IC 20 of propranolol (propra) alone and their combination. All the values are the average of four independent experiments ± standard error of mean (SEM), with a biological triplicate in each experiment. Data were normalised to cell number. * p < 0·05; ** p < 0·005; *** p < 0·001. Figure 7

Lastly, we measured the effects of the combination of β-blockers and IR on the bioenergetics of MB cells. This analysis was performed at short time point (6 h) and with low doses of propranolol (IC 10 and IC 20 ) to better examine the effects of the combinatorial treatment. Propranolol alone and IR alone (1·8 Gy) did not significantly affect ATP production nor glycolytic reserve of HD-MB03 cells in these conditions ( Figures 7 g-h). Nevertheless, IR combined with IC 20 propranolol inhibited the two processes by 39 ± 5 % and 63 ± 11 % respectively, as compared with IR monotherapy ( p < 0·05, Figures 7 g-h). This potentiation was also confirmed in ONS-76 cells (Figure S7f-g). Our results thus support the importance of bioenergetic disturbances in the response of MB cells to β-blockers and their combination with radiotherapy.

β-blockers enhance IR-induced oxidative stress and consequently increase DNA damage in MB cells

Reactive oxygen species (ROS) are the major effectors of IR, contributing substantially to radiation-induced DNA damage and cancer cell death. 37 Given the effects of co-treatment on mitochondrial energy metabolism, we first determined whether the combination therapy could disrupt redox balance, by assessing the superoxide ion levels. Six hours post-irradiation, an expected increase in superoxide relative level of 34 ± 4 % was observed in IR HD-MB03 cells compared to control cells ( p < 0·001, Figure 8 a). Propranolol, at 10 µM (IC 5 ) and 25 µM (IC 10 ), also increased the production of ROS by 36 ± 12 % and 31 ± 9 %, respectively ( p < 0·01, Figure 8 a). The combination of IR with these low doses of propranolol led to an additional upregulation of ROS levels, up to 64 ± 4 % and 59 ± 5 %, respectively ( Figure 8 a, p < 0·001). These results were confirmed with carvedilol at 5 (IC 10 ) and 7·5 µM (IC 20 ) in HD-MB03 cells (Figure S8a), as well as in MD 3D spheroids (Figure S8b, c). A significant overproduction of superoxide ions of 96 ± 6 % ( p < 0·001) and 78 ± 7 % ( p < 0·01) was also found in ONS-76 cells exposed to IR and combined with IC 10 of propranolol or IC 10 of carvedilol ( Figure 8 b). However, in the β-blocker-resistant cells ONS-76 RC, both propranolol and carvedilol were unable to amplify the effects of IR on superoxide production ( Figure 8 b). Potentiation of IR efficacy by the two β-blockers was also significantly reduced in these cells (Figure S8b), supporting a tight link between ROS production and response of MB cells to the combinatorial treatment. Figure 8 Low concentrations of β-blockers enhanced IR-mediated MB cell oxidative stress and DNA damage. (a) Superoxide ions production measured with WST-1 in HD-MB03 cells, six hours after treatment with radiotherapy (IR) 1·8 Gy or IC 5 -IC 10 of propranolol (propra) alone and their combination, and with anti-oxydants (mito-TEMPO (MT) 2·5 µM or Troxerutin (Trox) 1 mM). Data were normalised to cell number. Values are the average of five independent experiments ± standard error of mean (SEM), with a biological quadruplicate in each experiment. (b) Superoxide ions production measured with WST-1 in β-blocker sensitive (ONS-76 WT) and carvedilol resistant ONS-76 cell lines (ONS-76 RC), six hours after treatment with radiotherapy (IR) 1·8 Gy or IC 20 of propranolol (propra) or carvedilol (carve) alone and their combination. Data were normalised to cell number. Values are the average of four independent experiments ± SEM, with a biological quadruplicate in each experiment. Protein expression level of COX-2 (c) and γ-H2AX (d) in HD-MB03 cells exposed to radiotherapy (IR) 1·8 Gy or IC 5 -IC 10 of propranolol (propra) alone and their combination, and with anti-oxydants (mito-TEMPO (MT) 2·5 µM or Troxerutin (Trox) 1 mM). Western blots were quantified using ImageJ™ software; data were normalised to β-Actin. Values are the average of at least five independent experiments ± SEM. * p < 0·05; ** p < 0·005; *** p < 0·001. Figure 8

As the overproduction of ROS may contribute to an increase in cyclooxygenase 2 (COX-2) expression, which has been associated with the acquisition of a secondary radioresistance by tumour cells, 38 we ensured that such a feedback loop was not triggered in MB cells. By analysing COX-2 relative expression level in HD-MB03 cells 24 h after treatment, we showed that it was reduced from 1·7 ± 0·3 in 1·8 Gy irradiated cells to 0·8 ± 0·1 and 0·7 ± 0·1 in cells subjected to IR combined with IC 5 and IC 10 propranolol respectively ( p < 0·05, Figure 8 c). The inhibition of IR-mediated increase in COX-2 expression by the combinatorial treatment was confirmed in ONS cells (Figure S8c).

Lastly, we evaluated the phosphorylation level of H2AX, being an early response to DNA double-strand breaks that here may be caused following ROS exposure. In HD-MB03 cells, 4 h after treatment, IR triggered the expected accumulation of yH2AX, as did low concentrations of propranolol ( Figure 8 d). Our results also showed a significant increase in γH2AX relative level from 2·7 ± 0·4 in irradiated cells to 8·6 ± 0·9 and 6·3 ± 0·7 in cells exposed to the co-treatment with IC 5 and IC 10 propranolol, respectively ( p < 0·001, Figure 8 d). By scavenging the superoxide ions ( Figure 8 a), Mito-TEMPO (MT) counteracted the increase in γH2AX level by the combinatorial treatments – which dropped to 2·0 ± 0·4 and 2·6 ± 0·2, respectively – ( p < 0·01 and 0·05 vs co-treatments, respectively, Figure 8 d). Likewise, scavenging of free radicals by Troxerutin (TROX; Figure 8 a) led to a significant reduction in IR-mediated γH2AX accumulation ( p < 0·05 vs co-treatments, Figure 8 d). Taken together, our results suggest that β-blockers can specifically modulate mitochondrial bioenergetics and ROS production in MB cells, thus priming them for IR-induced oxidative stress and DNA damage. Our results therefore show that the efficacy of the combination of IR with β-blockers is, at least in part, based on a strong inhibition of MB cell bioenergetics, linked to the triggering of an endogenous oxidative stress.

Discussion

In recent years, many advances have been made in the management of children with MB. Nevertheless, a real concern remains the long-term sequelae due to the early exposure to toxic treatments. 7 , 10 Drug repurposing appears to be a major tool to rapidly find effective and well-tolerated therapeutic approaches in oncology. 11 , 13 It might especially be an alternative strategy to manage rare cancers such as paediatric tumours. Cardiovascular regulators, anti-helminthic drugs and non-steroidal anti-inflammatory drugs have recently shown to reduce MB tumour cell progression in vitro and in vivo . 39 Here, we evaluated in MB models propranolol, carvedilol and nebivolol, which are lipophilic β-blockers that can cross the blood-brain barrier, and enter the cerebrospinal fluid and intracranial tissue. 40

Our results showed that the three β-blockers potentiate the efficacy of IR in a panel of MB cells, PDX cells and spheroid micromasses, including those poorly responsive to radiation. These results are consistent with the recent study from Chaudhary et al ., that described a propranolol-mediated sensitisation to IR in non-small cell lung cancer cells in vitro . 20 Enhanced effectiveness of IR at reducing the growth of gastric adenocarcinoma in vivo when combined with propranolol was also shown recently. 23 Retrospective clinical studies have also shown that the combination of ß-blockers and radiotherapy did not result in increased toxicities in patients with lung cancer 20 , 41 , 42 and brain tumors such as meningioma. 24 The interest of combining radiotherapy with β-blockers is further supported by the fact that ß-blockers are largely known to be good brain protectors that can be used for instance after head trauma including in children. 43 , 44

In response to the ever-increasing need to find alternatives to animal experimentation, we have developed an innovative organotypic cerebellum model in which MB tumour progression has been analysed over time. These ex vivo tissue cultures are described as highly relevant models to study the evolution of pathologies and to test their response to different therapeutic strategies, including in MB. 45 We further showed that the dose of IR can be significantly reduced while maintaining treatment efficacy in MB cells by adding β-blockers. As the severity of cognitive damages in patients correlates with radiation doses, 10 , 46 this suggests that combining β-blockers with IR may help limit treatment side effects.

One of the advantages of repurposing β-blockers as anti-cancer agents is that they can be translated to the clinic without the need for extensive preclinical studies, including in vivo experiments. For instance, propranolol was first used in a clinical setting in combination with metronomic chemotherapy in patients before it was later confirmed to be active in vivo in mouse models. 17 , 47 , 48 In addition, an ongoing clinical trial ( NCT04682158 ) exploring the combination of propranolol with chemo-radiation is based on in vitro experiments and retrospective clinico-epidemiological experience in patients who received β-blockers for non-cancer purposes in combination. 49 Another potential example is based on multiple myeloma for which clinical trials have been completed ( NCT02420223 ) or recently initiated ( NCT02420223 ) without myeloma-specific in vivo data but based but again on in vitro and clinic-epidemiological experiments. 50 The results of the present article can thus provide a strong basis for initiating an early phase clinical trial.

The literature is divided regarding the mechanisms responsible for the anti-tumour properties of β-blockers. Inhibition of the β-adrenergic signaling pathway has been suggested to be involved in propranolol activity in pancreatic cancer cells. 51 Studies in angiosarcoma cells provide a good illustration of the conflicting hypotheses. Amaya et al. proposed the involvement of the β-adrenergic pathway in the mechanism of action of propranolol, 52 , 53 whereas a recent study by Overman et al. argued the opposite and showed a key role for the SOX18 protein in the response to the β-blocker. 52 , 53 In the paediatric tumours neuroblastoma and hemangioma, the results agree that β-ARs are not responsible for the anti-tumour efficacy of β-blockers, showing that the R-enantiomer of propranolol – which has very low affinity for β-ARs – has the same efficacy as the S-enantiomer that is highly affine for the receptors. 53 , 54 , 55 Although patient MB biopsies showed detectable β-AR expression, we demonstrated herein that their silencing did not alter the efficacy of propranolol, carvedilol and nebivolol in MB cells, suggesting that the efficacy of β-blockers in MB cells may not result from the inhibition of the canonical targets.

We and others have reported that propranolol-exposed cancer cells were sensitised to the metabolic stress induced by metformin, rapamycin, 2-deoxy-D-glucose or dichloroacetate. 36 , 56 , 57 , 58 Here, we showed that the activity of the β-blockers in MB cells was driven by a rapid disruption of the mitochondrial bioenergetics, which led to a sustained accumulation of ROS. This is consistent with the alteration of the mitochondrial fusion/fission balance that we previously observed in neuroblastoma cells treated with propranolol. 18 The significance of cancer cell energy metabolism in response to β-blockers is further strengthened by the fact that the lack of impact on mitochondrial and glycolytic pathways results in resistance of MB cells to these repurposed drugs.

Efficacy of radiation therapy relies on its ability to cause DNA breaks and to subsequently trigger cell death. The DNA damages mainly result from the generation of ROS, such as superoxide and hydroxyl radicals, during H 2 O radiolysis. 37 In the present study, we showed that β-blockers potentiate IR-mediated DNA damages in MB cells by increasing superoxide accumulation. Our results are consistent with the fact that pharmacologic depletion of glutathione, which belongs to the cell antioxidant system, significantly results in radiosensitisation of cancer stem cells. 59 Recently, Gd-doped titania nanoparticules that target mitochondria to enhance ROS accumulation were also shown to sensitise breast cancer cells to radiotherapy-induced apoptosis in vitro and in vivo . 60 Increasing ROS levels in MB tumour cells during radiotherapy may thus significantly enhance the efficiency and decrease the dosage of radiation.

COX-2 overexpression has been associated with resistance to IR in prostate, lung and oral squamous cancer cells. 61 , 62 , 63 Conversely, COX-2 inhibitors can synergise with IR in inducing apoptosis, 63 , 64 including in MB stem-like cells. 65 , 66 COX-2 inhibition has been suggested as a potential strategy in MB to decrease the production of prostaglandin E2 (PGE2) and ultimately promote tumour cell death. 67 Here, we showed that propranolol prevented the increase in COX-2 expression mediated by IR, but the involvement of the PGE2 pathway in improving response of MB cells to combinatorial therapy remains to be better characterised.

To conclude, our work highlights the interest of channeling the ability of β-blockers to inhibit mitochondrial bioenergetics to design new therapeutic combinations with radiotherapy that lower the dose while maintaining anti-tumour activity. Given the few druggable molecular targets identified in non-WNT MB and the fact that young age of patients limits treatment options, our work proposes an alternative approach in which drug repurposing could be quickly translated to the clinic to improve the efficacy of radiotherapy and/or decrease its toxicity.

Contributors

Conceptualisation: E.P., M.C. and N.A. Formal analysis: M.R., J.T., M.M., P.P., M.-P.M., E.A.-C., M., R.A. and D.W. Funding acquisition: N.A., M.R. and M.C. Methodology: J.T., C.K., D.B., L.P., P.P., M.-P.M., E.A.-C., R.A. and V.B. Project administration: M.C., S.C., O.A. and N.A. Supervision: M.C. and N.A. Validation: M.R., O.A.,J.T., M.L.G., P.P., E.P., D.B., M.-P.M. and M.M. Writing, review & editing: M.R., M.C., E.P., M.L.G., N.A., D.W. and J.T. All authors have read and approved the final manuscript.

Data sharing statement

Our RNA-seq information and raw data from ONS-76 cells are publicly available in GEO database (GEO accession numbers: GSE191165 ). The other data and materials are available from the corresponding authors upon reasonable request.

Declaration of interests

The authors have declared no conflict of interest.

References

1 Schwalbe EC Lindsey JC Nakjang S Novel molecular subgroups for clinical classification and outcome prediction in childhood medulloblastoma: a cohort study Lancet Oncol 18 7 2017 958 971 28545823 10.1016/S1470-2045(17)30243-7 PMC5489698

2 Northcott PA Buchhalter I Morrissy AS The whole-genome landscape of medulloblastoma subtypes Nature 547 7663 2017 311 317 28726821 10.1038/nature22973 PMC5905700

3 Sharma T Schwalbe EC Williamson D Second-generation molecular subgrouping of medulloblastoma: an international meta-analysis of Group 3 and Group 4 subtypes Acta Neuropathol (Berl) 138 2 2019 309 326 31076851 10.1007/s00401-019-02020-0 PMC6660496

4 Massimino M Biassoni V Gandola L Childhood medulloblastoma Crit Rev Oncol Hematol 105 2016 35 51 27375228 10.1016/j.critrevonc.2016.05.012

5 Juraschka K Taylor MD. Medulloblastoma in the age of molecular subgroups: a review J Neurosurg Pediatr 24 4 2019 353 363 31574483 10.3171/2019.5.PEDS18381

6 Ward E DeSantis C Robbins A Kohler B Jemal A. Childhood and adolescent cancer statistics, 2014 CA Cancer J Clin 64 2 2014 83 103 24488779 10.3322/caac.21219

7 Mulhern RK Palmer SL Merchant TE Neurocognitive consequences of risk-adapted therapy for childhood medulloblastoma J Clin Oncol Off J Am Soc Clin Oncol 23 24 2005 5511 5519 10.1200/JCO.2005.00.703 16110011

8 Lafay–Cousin L Bouffet E Hawkins C Amid A Huang A Mabbott DJ Impact of radiation avoidance on survival and neurocognitive outcome in infant medulloblastoma Curr Oncol 16 6 2009 21 28 10.3747/co.v16i6.435 PMC2794676 20016743

9 Rutkowski S Gerber NU von Hoff K Treatment of early childhood medulloblastoma by postoperative chemotherapy and deferred radiotherapy Neuro-Oncol 11 2 2009 201 210 18818397 10.1215/15228517-2008-084 PMC2718992

10 Padovani L Horan G Ajithkumar T. Radiotherapy advances in paediatric medulloblastoma treatment Clin Oncol R Coll Radiol G B 31 3 2019 171 181 10.1016/j.clon.2019.01.001 30655168

11 Bahmad HF Elajami MK El Zarif T Bou-Gharios J Abou-Antoun T Abou-Kheir W. Drug repurposing towards targeting cancer stem cells in pediatric brain tumors Cancer Metastasis Rev 39 1 2020 127 148 31919619 10.1007/s10555-019-09840-2

12 Bertolini F Sukhatme VP Bouche G. Drug repurposing in oncology–patient and health systems opportunities Nat Rev Clin Oncol 12 12 2015 732 742 26483297 10.1038/nrclinonc.2015.169

13 Pantziarka P Verbaanderd C Huys I Bouche G Meheus L. Repurposing drugs in oncology: from candidate selection to clinical adoption Semin Cancer Biol 2020 10.1016/j.semcancer.2020.01.008 31982510

14 Alabed S Sabouni A Al Dakhoul S Bdaiwi Y Beta-blockers for congestive heart failure in children Cochrane Database Syst Rev 7 2020 CD007037 10.1002/14651858.CD007037.pub4 PMC7389334 32700759

15 Léauté-Labrèze C Dumas de la Roque E Hubiche T Boralevi F Thambo JB Taïeb A Propranolol for severe hemangiomas of infancy N Engl J Med 358 24 2008 2649 2651 18550886 10.1056/NEJMc0708819

16 Pasquier E Ciccolini J Carre M Propranolol potentiates the anti-angiogenic effects and anti-tumor efficacy of chemotherapy agents: implication in breast cancer treatment Oncotarget 2 10 2011 797 809 22006582 10.18632/oncotarget.343 PMC3248157

17 Pasquier E André N Street J Effective management of advanced angiosarcoma by the synergistic combination of propranolol and vinblastine-based metronomic chemotherapy: a bench to bedside study EBioMedicine 6 2016 87 95 27211551 10.1016/j.ebiom.2016.02.026 PMC4856748

18 Pasquier E Street J Pouchy C β-blockers increase response to chemotherapy via direct antitumour and anti-angiogenic mechanisms in neuroblastoma Br J Cancer 108 12 2013 2485 2494 23695022 10.1038/bjc.2013.205 PMC3694229

19 MacDonald CR Bucsek MJ Qiao G Adrenergic receptor signaling regulates the response of tumors to ionizing radiation Radiat Res 191 6 2019 585 589 31021732 10.1667/RR15193.1 PMC6774253

20 Chaudhary KR Yan SX Heilbroner SP Effects of β-adrenergic antagonists on chemoradiation therapy for locally advanced non-small cell lung cancer J Clin Med 8 5 2019 [cited 2020 Apr 15] 10.3390/jcm8050575 PMC6572477 31035526

21 Chen M Qiao G Hylander BL Adrenergic stress constrains the development of anti-tumor immunity and abscopal responses following local radiation Nat Commun 11 1 2020 1821 1832 10.1038/s41467-020-15676-0 32286326 PMC7156731

22 Liao X Che X Zhao W Effects of propranolol in combination with radiation on apoptosis and survival of gastric cancer cells in vitro Radiat Oncol Lond Engl 5 2010 98 10.1186/1748-717X-5-98 PMC2974742 20977754

23 Liao X Chaudhary P Qiu G Che X Fan L The role of propranolol as a radiosensitizer in gastric cancer treatment Drug Des Devel Ther 12 2018 639 645 10.2147/DDDT.S160865 PMC5880513 29636598

24 Pintea B Kinfe TM Baumert BG Boström J. Earlier and sustained response with incidental use of cardiovascular drugs among patients with low- to medium-grade meningiomas treated with radiosurgery (SRS) or stereotactic radiotherapy (SRT) Radiother Oncol 111 3 2014 446 450 24998705 10.1016/j.radonc.2014.05.014

25 Wang HM Liao ZX Komaki R Improved survival outcomes with the incidental use of beta-blockers among patients with non-small-cell lung cancer treated with definitive radiation therapy Ann Oncol 24 5 2013 1312 1319 23300016 10.1093/annonc/mds616 PMC3629895

26 Zindy F Uziel T Ayrault O Genetic alterations in mouse medulloblastomas and generation of tumors de novo from primary cerebellar granule neuron precursors Cancer Res 67 6 2007 2676 2684 17363588 10.1158/0008-5472.CAN-06-3418

27 Garancher A Lin CY Morabito M NRL and CRX define photoreceptor identity and reveal subgroup-specific dependencies in medulloblastoma Cancer Cell 33 3 2018 435 449.e6 29533784 10.1016/j.ccell.2018.02.006 PMC6368680

28 Ariey-Bonnet J Carrasco K Le Grand M In silico molecular target prediction unveils mebendazole as a potent MAPK14 inhibitor Mol Oncol 14 12 2020 3083 3099 33021050 10.1002/1878-0261.12810 PMC7718943

29 xing Cao D hao Wu G ang Yang Z Role of beta1-adrenoceptor in increased lipolysis in cancer cachexia Cancer Sci 101 7 2010 1639 1645 20426789 10.1111/j.1349-7006.2010.01582.x PMC11158273

30 Henriques A Huebecker M Blasco H Inhibition of β-glucocerebrosidase activity preserves motor unit integrity in a mouse model of amyotrophic lateral sclerosis Sci Rep 7 2017 5235 28701774 10.1038/s41598-017-05313-0 PMC5507914

31 Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads EMBnet.journal 17 1 2011 10 12

32 Langmead B Salzberg SL. Fast gapped-read alignment with Bowtie 2 Nat Methods 9 4 2012 357 359 22388286 10.1038/nmeth.1923 PMC3322381

33 Dobin A Davis CA Schlesinger F STAR: ultrafast universal RNA-seq aligner Bioinformatics 29 1 2013 15 21 23104886 10.1093/bioinformatics/bts635 PMC3530905

34 Anders S Pyl PT Huber W. HTSeq—a Python framework to work with high-throughput sequencing data Bioinformatics 31 2 2015 166 169 25260700 10.1093/bioinformatics/btu638 PMC4287950

35 Love MI Huber W Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 Genome Biol 15 12 2014 550 25516281 10.1186/s13059-014-0550-8 PMC4302049

36 Rico M Baglioni M Bondarenko M Metformin and propranolol combination prevents cancer progression and metastasis in different breast cancer models Oncotarget 8 2 2017 2874 2889 27926515 10.18632/oncotarget.13760 PMC5356849

37 Srinivas US Tan BWQ Vellayappan BA Jeyasekharan AD. ROS and the DNA damage response in cancer Redox Biol 25 2018 101084 10.1016/j.redox.2018.101084 PMC6859528 30612957

38 Cheki M Yahyapour R Farhood B COX-2 in radiotherapy: a potential target for radioprotection and radiosensitization Curr Mol Pharmacol 11 3 2018 173 183 29468988 10.2174/1874467211666180219102520

39 Hammoud H Saker Z Harati H Fares Y Bahmad HF Nabha S. Drug repurposing in medulloblastoma: challenges and recommendations Curr Treat Options Oncol 22 1 2020 6 33245404 10.1007/s11864-020-00805-0

40 Hjalmarson A. Cardioprotection with beta-adrenoceptor blockers. Does lipophilicity matter? Basic Res Cardiol 95 suppl 1 2000 I41 I45 11192352 10.1007/s003950070008

41 Wang HM Liao ZX Komaki R Improved survival outcomes with the incidental use of beta-blockers among patients with non-small-cell lung cancer treated with definitive radiation therapy Ann Oncol 24 5 2013 1312 1319 23300016 10.1093/annonc/mds616 PMC3629895

42 Wang HM Liao ZX Komaki R Improved survival outcomes with the incidental use of beta-blockers among patients with non-small-cell lung cancer treated with definitive radiation therapy Ann Oncol Off J Eur Soc Med Oncol 24 5 2013 1312 1319 10.1093/annonc/mds616 PMC3629895 23300016

43 Zeeshan M Hamidi M OʼKeeffe T Propranolol attenuates cognitive, learning, and memory deficits in a murine model of traumatic brain injury J Trauma Acute Care Surg 87 5 2019 1140 1147 31425494 10.1097/TA.0000000000002484

44 Fumagalli C Maurizi N Marchionni N Fornasari D. β-blockers: their new life from hypertension to cancer and migraine Pharmacol Res 151 2020 104587 10.1016/j.phrs.2019.104587 31809852

45 Meijer TG Naipal KA Jager A van Gent DC. Ex vivo tumor culture systems for functional drug testing and therapy response prediction Future Sci OA 3 2 2017 FSO190 28670477 10.4155/fsoa-2017-0003 PMC5481868

46 Bindra RS Wolden SL. Advances in Radiation Therapy in Pediatric Neuro-oncology J Child Neurol 31 4 2016 506 516 26271789 10.1177/0883073815597758

47 Banavali S Pasquier E Andre N. Targeted therapy with propranolol and metronomic chemotherapy combination: sustained complete response of a relapsing metastatic angiosarcoma Ecancermedicalscience 9 2015 499 502 10.3332/ecancer.2015.499 25624880 PMC4303616

48 Stiles JM Amaya C Rains S Targeting of beta adrenergic receptors results in therapeutic efficacy against models of hemangioendothelioma and angiosarcoma PLoS One 8 3 2013 e60021 23555867 10.1371/journal.pone.0060021 PMC3610939

49 Farrugia MK Ma SJ Mattson DM Flaherty L Repasky EA Singh AK. Concurrent β-blocker use is associated with improved outcome in esophageal cancer patients who undergo chemoradiation: a retrospective matched pair analysis Am J Clin Oncol 43 12 2020 889 894 33044232 10.1097/COC.0000000000000768 PMC7855691

50 Hwa YL Shi Q Kumar SK Beta-blockers improve survival outcomes in patients with multiple myeloma: a retrospective evaluation Am J Hematol 92 1 2017 50 55 27733010 10.1002/ajh.24582 PMC5217079

51 Zhang D Ma QY Hu HT Zhang M. β2-adrenergic antagonists suppress pancreatic cancer cell invasion by inhibiting CREB, NFκB and AP-1 Cancer Biol Ther 10 1 2010 19 29 20424515 10.4161/cbt.10.1.11944

52 Amaya CN Perkins M Belmont A Non-selective beta blockers inhibit angiosarcoma cell viability and increase progression free- and overall-survival in patients diagnosed with metastatic angiosarcoma Oncoscience 5 3–4 2018 109 119 29854879 10.18632/oncoscience.413 PMC5978448

53 Overman J Fontaine F Wylie-Sears J R-propranolol is a small molecule inhibitor of the SOX18 transcription factor in a rare vascular syndrome and hemangioma eLife 8 2019 e43026 43040 10.7554/eLife.43026 31358114 PMC6667216

54 Sasaki M North PE Elsey J Propranolol exhibits activity against hemangiomas independent of beta blockade NPJ Precis Oncol 3 2019 27 35 10.1038/s41698-019-0099-9 31701018 PMC6825155

55 Seebauer CT Graus MS Huang L McCann AJ Wylie-Sears J Fontaine FR Non-β-blocker enantiomers of propranolol and atenolol inhibit vasculogenesis in infantile hemangioma J Clin Invest 132 3 2021 e151109 151123 10.1172/JCI151109 PMC8803322 34874911

56 Brohée L Demine S Willems J Arnould T Colige AC Deroanne CF. Lipin-1 regulates cancer cell phenotype and is a potential target to potentiate rapamycin treatment Oncotarget 6 13 2015 11264 11280 25834103 10.18632/oncotarget.3595 PMC4484455

57 Brohée L Peulen O Nusgens B Propranolol sensitizes prostate cancer cells to glucose metabolism inhibition and prevents cancer progression Sci Rep 8 2018 7050 29728578 10.1038/s41598-018-25340-9 PMC5935740

58 Lucido CT Miskimins WK Vermeer PD. Propranolol promotes glucose dependence and synergizes with dichloroacetate for anti-cancer activity in HNSCC Cancers 10 12 2018 476 10.3390/cancers10120476 PMC6316475 30513596

59 Diehn M Cho RW Lobo NA Association of reactive oxygen species levels and radioresistance in cancer stem cells Nature 458 7239 2009 780 783 19194462 10.1038/nature07733 PMC2778612

60 Chen Y Li N Wang J Enhancement of mitochondrial ROS accumulation and radiotherapeutic efficacy using a Gd-doped titania nanosensitizer Theranostics 9 1 2019 167 178 30662560 10.7150/thno.28033 PMC6332802

61 Anai S Tanaka M Shiverick KT Increased expression of cyclooxygenase-2 correlates with resistance to radiation in human prostate adenocarcinoma cells J Urol 177 5 2007 1913 1917 17437847 10.1016/j.juro.2007.01.019

62 Terakado N Shintani S Yano J Overexpression of cyclooxygenase-2 is associated with radioresistance in oral squamous cell carcinoma Oral Oncol 40 4 2004 383 389 14969817 10.1016/j.oraloncology.2003.09.005

63 Han ZQ Liao H Shi F Inhibition of cyclooxygenase-2 sensitizes lung cancer cells to radiation-induced apoptosis Oncol Lett 14 5 2017 5959 5965 29113232 10.3892/ol.2017.6940 PMC5661612

64 Salehifar E Hosseinimehr SJ. The use of cyclooxygenase-2 inhibitors for improvement of efficacy of radiotherapy in cancers Drug Discov Today 21 4 2016 654 662 26955911 10.1016/j.drudis.2016.02.019

65 Chen KH Hsu CC Song WS Celecoxib enhances radiosensitivity in medulloblastoma-derived CD133-positive cells Childs Nerv Syst ChNS Off J Int Soc Pediatr Neurosurg 26 11 2010 1605 1612 10.1007/s00381-010-1190-2 20526717

66 Yang MY Lee HT Chen CM Shen CC Ma HI. Celecoxib suppresses the phosphorylation of STAT3 protein and can enhance the radiosensitivity of medulloblastoma-derived cancer stem-like cells Int J Mol Sci 15 6 2014 11013 11029 24945311 10.3390/ijms150611013 PMC4100195

67 Baryawno N Sveinbjörnsson B Eksborg S Tumor-growth–promoting cyclooxygenase-2 prostaglandin E2 pathway provides medulloblastoma therapeutic targets Neuro-Oncol 10 5 2008 661 674 18715952 10.1215/15228517-2008-035 PMC2666243

Supplementary materials

Image, application 1 Image, application 2 Image, application 3 Image, application 4

Acknowledgements

We would like to thank Julie Desrousseaux, Sophie Capdeville and Stephanie Gempp for their availability and help in irradiating cells, Samy Vigier and Anne Rodallec for their help with animal experimentation, and Camille Cordero for defining the conditions of cell irradiation. We also thank Claire Lovo and Marie-Noëlle Soler from the Light Microscopy facility of Multimodal Imaging Center (CNRS UAR2016/Inserm US43/Institut Curie/Université Paris-Saclay), for IncuCyte imaging assistance, Christophe Alberti and Elodie Belloir for mouse colony management, Hua Yu for mouse model monitoring and Sophie Heinrich from the RadExp platform for irradiation experiments assistance. We also thank the ICEP platform of IPC/CRCM for their help in immunohistochemistry experiments.

This work was supported by research funding from charities (RESOP, La Marie-Do, AROU, La Compagnie Après la Pluie, Société Française des Cancers de l'Enfant, Courir pour la Vie - Courir pour Curie, Ligue Contre le Cancer - Corse Sud) and institutions (Cancer Research UK, Canceropôle PACA, Institut National du Cancer and Région Sud). Sequencing was performed by the GenomEast platform, a member of the ‘France Génomique’ consortium (ANR-10-INBS-0009).

Supplementary material associated with this article can be found in the online version at doi: 10.1016/j.ebiom.2022.104149 .