Pierre Jean Le Reste

Raphael Pineau

Konstantinos Voutetakis

Juhi Samal

Gwenaele Jegou

Stéphanie Lhomond

Adrienne M Gorman

Afshin Samali

John B Patterson

Qingping Zeng

Gwénaële Jégou

Abhay Pandit

Marc Aubry

Nicolas Soriano

Amandine Etcheverry

Aristotelis Chatziioannou

Jean Mosser

Tony Avril

Eric Chevet
email: eric.chevet@inserm.fr

Local intracerebral inhibition of IRE1 by MKC8866 sensitizes glioblastoma to irradiation/chemotherapy in vivo

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Glioblastoma multiforme (GBM) is the most severe primary brain cancer and represents more than 15% of primary brain tumors. Despite an aggressive treatment comprising surgical resection and a combination of irradiation and chemotherapy ("Stupp protocol"), patient's survival post-diagnosis remains short with a median overall survival of 15 months [START_REF] Stupp | Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[END_REF]. The lack of efficacy of the current treatments is mostly due to the intratumor heterogeneity at diagnosis with the presence of multiple driver alterations [START_REF] Körber | Evolutionary Trajectories of IDHWT Glioblastomas Reveal a Common Path of Early Tumorigenesis Instigated Years ahead of Initial Diagnosis[END_REF], as well as to different tumor cell types that exhibit various sensitivity to anti-cancer agents. Finally, it is due to the diffuse nature of GBM tumors that precludes from performing complete resection. Another limitation for the discovery of new improved therapeutic approaches is the lack of a relevant animal model that extensively recapitulates current GBM patient disease and standard of care.

In the past couple of years, we have demonstrated that the Unfolded Protein Response (UPR) plays an instrumental role in GBM development [START_REF] Drogat | IRE1 signaling is essential for ischemia-induced vascular endothelial growth factor-A expression and contributes to angiogenesis and tumor growth in vivo[END_REF][START_REF] Auf | Inositol-requiring enzyme 1alpha is a key regulator of angiogenesis and invasion in malignant glioma[END_REF][START_REF] Dejeans | Autocrine control of glioma cells adhesion and migration through IRE1alpha-mediated cleavage of SPARC mRNA[END_REF][START_REF] Pluquet | Posttranscriptional regulation of PER1 underlies the oncogenic function of IREalpha[END_REF][START_REF] Obacz | Endoplasmic reticulum proteostasis in glioblastoma-From molecular mechanisms to therapeutic perspectives[END_REF][START_REF] Jabouille | Glioblastoma invasion and cooption depend on IRE1α endoribonuclease activity[END_REF]. More precisely, we have shown that IRE1, the most conserved UPR sensor, signals in tumor cells to remodel the tumor microenvironment, to favor tumor growth and most likely to alter the response to treatment. We demonstrated that the IRE1/XBP1s (spliced XBP1) axis exhibits pro-oncogenic properties, whereas Regulated IRE1 Dependent Decay of RNA (RIDD) dampens tumor angiogenesis and tumor cell migration/invasion. This observation was then used to stratify GBM patients, where we showed that survival of patients with tumors exhibiting high XBP1s and low RIDD was significantly reduced compared to that of patients with low XBP1s and high RIDD tumors [START_REF] Jabouille | Glioblastoma invasion and cooption depend on IRE1α endoribonuclease activity[END_REF]. These observations point toward the IRE1/XBP1s axis as a potentially relevant therapeutic target which, when inhibited in XBP1s high tumors would i) slow down tumor growth by impairing the pro-tumoral remodeling of the tumor stroma and ii) sensitize tumor cells to the current treatments [START_REF] Lhomond | Dual IRE1 RNase functions dictate glioblastoma development[END_REF].

To further test the potential impact of the pharmacological targeting of IRE1/XBP1, we propose to use an IRE1 RNase inhibitor, MKC8866 [START_REF] Volkmann | Potent and selective inhibitors of the inositol-requiring enzyme 1 endoribonuclease[END_REF], already shown to be effective in other cancer models [START_REF] Logue | Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy[END_REF], and in preclinical models of GBM. However, the current preclinical GBM models poorly recapitulate the clinical handling of GBM patients, therefore would not be appropriate to test the therapeutic potential of IRE1 inhibition. In the present study, we developed a novel immunocompetent GBM model that involved tumor surgery and a radio/chemotherapy regimen, resembling the Stupp protocol. Furthermore, we used this

Materials and methods

Reagents -All reagents were purchased from Sigma-Aldrich (St Quentin Fallavier, France), unless otherwise stated. For immunohistochemistry staining, primary antibodies against vimentin (rabbit monoclonal, used at 1 in 250 dilution) were obtained from Abcam (Paris, France); against CD31 (rabbit polyclonal, used at 1/50 dilution) from Bioss Inc. (Cliniscience, Nanterre, France); against IBA-1 (rabbit polyclonal, used at 1/50 dilution) from Wako (Sobioda, Montbonnot, France); against activated caspase-3 from Cell Signaling (#9661, used at 1/500 dilution) and against KI67 from Novusbio (NB600-1252, used at 1/500 dilution). For Western blot experiments, primary antibodies against ATF4 and phospho-eIF2alpha (PeIF2a) were purchased from Cell Signaling Technology (Ozyme, St Quentin-en-Yvelines, France), anti-Calnexin (CNX) antibodies were a kind gift from JJM Bergeron (McGill University, Montreal, Qc, Canada).

Cell lines -The GL261 murine glioblastoma cell line was a kind gift from Dr Clavreul (University of Angers, France). These cells were transfected with the plasmid pGL4-Luc and the luciferase-expressing cells stably selected using G418 (the selected line was polyclonal and renamed GL261-Luc). The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Thermo Fisher Scientific, Courtaboeuf, France) with the addition of 10% FBS (Thermo Fisher Scientific), and 100 μg/ml of G418 (Thermo Fisher Scientific). The cells were grown in a humidified incubator at 37°C with 5% CO 2 . The human U87 and derivates (expressing IRE1 variants) GBM cells were grown in DMEM containing 10% FBS.

The human U251 GBM cells (used to test the MKC8866-containg gels in vitro, Figure S1) were grown in DMEM containing 10% FBS.

Genome analyses -Whole Genome Sequencing of the GL261 cell line was performed by

Integragen (https://www.integragen.com/fr/). The coverage was 25X for 75% of the mouse genome with a mean depth of 33X. An in-house post-processing was used to filter out candidate germline mutations more consistent with artifacts (Mapping Quality, Base Quality, R e v i s e d m a n u s c r i p t 5 Strand Bias, Variant Quality). Ensembl VEP (Variant Effect Predictor, release 87) program processed variants for further annotation using data available in dbSNP (dbSNP146). We detected copy-number variants and allelic imbalances in GL261 using the Control-FREEC algorithm (FREEC-11.5) with the C57BL_6.bam file downloaded from the SANGER FTP server (ftp://ftp-mouse.sanger.ac.uk/current_bams/) as a non-tumor reference sample.

Transcriptome analyses -In order to quantify the transcript-level abundances of implanted GL261-derived tumors for the seven experimental conditions (NR:no resected tumors; R:resected tumors without treatment; MKC:treatment with MKC8866 inhibitor; STUPP protocol; STUPP_MKC: STUPP protocol including MKC8866 inhibitor; PARENTAL:GL261 parental; STUPP_GEL: STUPP protocol including gel implantation lacking MKC8866 inhibitor) RNA sequencing was performed by Integragen (https://www.integragen.com/fr/). FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used for qualitative evaluation of reads with 95% of bases scoring Q30 and above. Transcript-level quantification data were produced using Salmon (v.1.0.0) program [START_REF] Patro | Salmon provides fast and bias-aware quantification of transcript expression[END_REF], installed in Linux server environment (Ubuntu 16.04.6 LTS) by performing mapping of sequence reads to the reference mouse transcriptome (Release M23 -GRCm38.p6). Transcriptome index and quantitation was determined using Salmon (https://salmon.readthedocs.io/en/latest/salmon.html#using-salmon) and the tximport R/Bioconductor package [START_REF] Soneson | Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences[END_REF] was used to summarize the estimated transcript abundance.

For the conversion of mouse gene symbols (mgi_symbol) to human gene symbols (hgnc_symbol) the getLDS() function of biomaRt R package was used retrieving homologous genes after linking of "mmusculus_gene_ensembl" and "hsapiens_gene_ensembl" datasets.

Transcriptome comparisons with other lines -Since mice do not show structural homologs for CXCL8 and MMP1 human genes, Cxcl1 and Mmp1a & Mmp1b were used instead, because of their functional similarity [START_REF] Foley | Matrix metalloprotease-1a promotes tumorigenesis and metastasis[END_REF][START_REF] Girbl | Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis[END_REF][START_REF] Shea-Donohue | Mice deficient in the CXCR2 ligand, CXCL1 (KC/GRO-alpha), exhibit increased susceptibility to dextran sodium sulfate (DSS)-induced colitis[END_REF]. The expression profile of U87-MG human glioblastoma cells (WT-wild type) expressing different IRE1 mutants (A414T, P336L, Q780Stop, S769F) and a dominant negative form of IRE1 (DN), under normal and stress conditions (tn; tunicamycin treated), deposited in GEO (Gene Expression Omnibus) [START_REF] Edgar | Gene Expression Omnibus: NCBI gene expression and hybridization array data repository[END_REF] (GSE107859 accession number) was compared to that of GL261 parental cell line after crossplatform harmonization, and evaluation of the IRE1 activity signature [START_REF] Lhomond | Dual IRE1 RNase functions dictate glioblastoma development[END_REF]. For the cross-R e v i s e d m a n u s c r i p t 6 platform normalization process, the Shambhala algorithm [START_REF] Borisov | Shambhala: a platform-agnostic data harmonizer for gene expression data[END_REF] was performed in R package HARMONY (https://github.com/oncobox-admin/harmony) for a set of 6546 genes in common between the two platforms (Affymetrix microarrays and RNA sequencing). For the exploration of relationships between the samples cluster dendrograms were built using R package ComplexHeatmap [START_REF] Gu | Complex heatmaps reveal patterns and correlations in multidimensional genomic data[END_REF] and the statistical significance of hierarchical clustering was assessed with bootstrapping method using the R package pvclust [START_REF] Suzuki | Pvclust: an R package for assessing the uncertainty in hierarchical clustering[END_REF]. Sample distance matrices to assess the overall similarity between samples were produced using the R function pheatmap() of R pheatmap package [START_REF] Kolde | R package version 1.0.12-pheatmap: Pretty Heatmaps[END_REF] with calculation of Euclidean distance between samples and minimization of the total within-cluster variance by Ward's minimum variance method (ward.D2) [START_REF] Szekely | Hierarchical Clustering via Joint Between-Within Distances: Extending Ward's Minimum Variance Method[END_REF]. Finally, principal component analysis (PCA) on harmonized gene expression data was conducted as previously described [START_REF] Wickham | ggplot2: Elegant Graphics for Data Analysis[END_REF].

Cell culture and treatments -For survival assays, GL261 cells were cultured in 96-well plates at 5,000 cells/well in the presence of increasing amounts of temozolomide (TMZ) (0 to 1000 µM), MKC8866 (0 to 200 µM) with two treatments of radiation at 5 Gy (at day 1 and day 3).

After 5 days (for TMZ) or 6 days (for MKC8866) of culture, 20 µl of the WST1 reagent were added to each well. After 4 hours at 37°C, optical densities (OD) were analyzed using spectrophotometry at 450 nm and 595 nm. Specific OD were given by the difference between the OD observed at 450 nm and the OD at 595 nm. Cell viability was calculated by the ratio of the specific OD observes with cells incubated in the presence of different concentration of TMZ and the specific OD observed with cells cultured in medium alone, this information allowed us to determine IC50 values using Prism software (GraphPad). Each experimental point represents at least a triplicate. For protein and RNA extractions, GL261

cells were seeded at a density of 10 6 cells per dish (6 cm diameter), with two dishes per time point. Cells were treated as described above for 2, 4, 8 and 24 hours. Each experimental point represents at least a triplicate. For protein extraction, cells were lysed using 100 µl of lysis buffer (30 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1.5% CHAPS and Complete™) per plate.

Clarified lysates were complemented with 20 µl of 5 times reducing Laemmli sample buffer prior heat-denaturation and used in Western blot experiments with the indicated antibodies (at 1/1000 dilutions). For RNA preparation, cells were lysed using 1ml of Trizol reagent per plate and RNA was extracted according to the manufacturer's instructions (Thermo Fisher Scientific) and PCR analyses performed using previously described protocols [START_REF] Sicari | A guide to assessing endoplasmic reticulum homeostasis and stress in mammalian systems[END_REF].

R e v i s e d m a n u s c r i p t

Tumor cell orthotopic implantation -Tumor cells (GL261-Luc) were implanted into the brain of immunocompetent C57BL/6rJ, 8 weeks old male mice (Janvier, Laval, France). All animal procedures met the European Community Directive guidelines (Agreement B35-238-40

Biosit Rennes, France/ No DIR 13480) and were approved by the local ethics committee and ensuring the breeding and the daily monitoring of the animals in the best conditions of wellbeing according to the law and the rule of 3R (Reduce-Refine-Replace). GL261-Luc cells were implanted in the mouse brain by intracerebral injection followed by tumor growth monitoring using bioluminescence. The mice were anesthetized intraperitoneally (i.p.) and then fixed on a stereotactic frame. This framework makes it possible to manipulate the brains of living animals, and to reach isolated areas of the brain precisely relative to markings visible to the naked eye through the use of three-dimensional coordinates. After incising the scalp, the stereotaxic coordinates were calculated for injection of tumor cells into a specific point of the brain, and reproducible for all the mice used. In the study, the tumor cells (2.5 x 10 4 cells per mice in 1 μL) were injected at 2.2 mm to the left of the bregma and 3.2 mm deep to perform the implantation at the level of the striatum.

Bioluminescence -Mice were assessed with in vivo bioluminescence imaging every 3 days, for the first week, and weekly onward for tumor progression and followed for signs of neurologic deterioration daily. Mice were injected i.p. with 100 μl of luciferin (Promega, Charbonnières-les-Bains, France). The luciferin was allowed to circulate for 10 min before the mice were anesthetized with a mix of O 2 and isoflurane (2.5%). Mice that showed an increase in tumor burden based on imaging during the first week after tumor implantation were included in the study. Bioluminescence analysis was used to determine the window of resection (day 14).

Resection -The mice were anesthetized by the intraperitoneal route with 90 μl of anesthetic Stupp-like treatment applied to mice -Three days after the resection, we proceeded to apply the Stupp-like protocol, consisting in two steps including (i) concomitant radio/chemotherapy: mice were anesthetized with Isoflurane, and placed in a lead dome to cover the entire animal to prevent ionizing radiation from reaching the healthy tissues of the animals. A hole of a few centimeters was made in the lead dome, corresponding to the resection area of the tumor, in order to irradiate only the area of interest. The animals were subjected to a radiation of 5 times 2Gy 3 days after resection along with 25 mg/kg/day of TMZ delivered intraperitoneally followed by (ii) stand-alone chemotherapy. In the latter procedure animals were subjected to the standard procedure of chemotherapy, namely 30-50 mg/kg of TMZ on 5 days with 2 or 3 days of rest between each treatment. Survival was measured as the time between implantation and sacrifice, which was performed by cervical dislocation in case of critical clinical signs or loss of weight >15%.

Immunohistochemistry -The immunochemistry experiments were performed on 5 µm tumor sections which were incubated at room temperature for 1 hour with primary antibodies.

Immunostaining was performed using BenchMarkXT-Ventana Medical Systems with kit OMNIMAP (system " biotin-free" using multimer technology) with antigen retrieval for all (Tris/borate/EDTA pH 8). To perform the analysis, glass slides were converted on to digital slides with the scanner Nanozoomer 2.0-RS Hamamatsu. The quantification of the necrosis was carried out from HES using the NIS-Elements software (Nikon). Immunohistochemistry R e v i s e d m a n u s c r i p t 9 was performed with anti IBA1 (macrophages and microglial cells), anti CD31 (endothelial cells) antibodies as previously described [START_REF] Lhomond | Dual IRE1 RNase functions dictate glioblastoma development[END_REF]. Cell death and proliferation were determined using anti activated caspase-3 and anti KI67 antibodies, respectively.

Manufacturing of the gel implant containing the IRE1 RNase inhibitor MKC8866 and cellbased testing -Human fibrinogen was dissolved in deionized water at a concentration of 30 mg/ml followed by dialysis against tris-buffered saline (TBS) overnight. MKC8866 was resuspended in the fibrinogen component of fibrin microgels at a final concentration of 10 and 100 µM. The gel-forming fraction containing MKC8866 was then dispensed as 7 μl droplets on a hydrophobic surface (commercial Teflon® tape) to create a micro-droplet. The gel fractions were then crosslinked by human thrombin (40 U/ml) and incubated for 20 minutes at 37 °C for stable cross-linking. To test the gel in vitro, U251 cells were seeded in 12-well plates at 10,000 cells/well or 20,000 cells/well. After 24h, the cells were cultivated in the absence (no gel) or presence of fibrin microgel containing different concentrations of MKC8866 (0 to 100 µM). After seven days, the cells were treated for 5h with tunicamycin at 5 µg/mL to activate IRE1 pathway and XBP1 RNA splicing. They were then lysed using 0.5 ml of Trizol reagent per well and RNA was extracted according to the manufacturer's instructions (Thermo Fisher Scientific). PCR was performed for total XBP1 splicing, and the resulting amplified DNA products were resolved using a 4% agarose gel. The upper band (unspliced XBP1) and lower band (spliced XBP1) were then quantified using the ImageJ software and the ratio between the unspliced (U) and spliced (S) forms of XBP1 were calculated by the following formula: S/(U+S) *100. For in vivo studies, gel implantation was carried out at the moment of the surgery. Briefly preformed microgels were deposited in the resection cavity prior to implanting a teflon plate and sewing the skin.

Statistical analyses -All the statistical analyses were performed using the GraphPad Prism software and the Mann-Whitney test.

Results

Qualification of GL261 murine GBM as a relevant model for in vivo studies with IRE1

inhibitors R e v i s e d m a n u s c r i p t 10 Since we previously showed that activation of IRE1 signaling (and in particular of the IRE1/XBP1 arm) was linked to GBM aggressiveness by promoting tumor-supporting immune cells, angiogenesis and migration/invasion of the tumor cells [START_REF] Jabouille | Glioblastoma invasion and cooption depend on IRE1α endoribonuclease activity[END_REF], we reasoned that a relevant in vivo mouse model should i) use GBM cells exhibiting high IRE1 activity, ii) be immunocompetent and iii) be subjected to a treatment similar to the standard of care in human. As such our first objective was to identify a mouse GBM cell line that could be considered as relevant. To this end we investigated the activation status of the IRE1 pathway in the mouse GL261 line. We compared the mouse line to the human U87 line expressing various mutants of IRE1 and treated or not with the ER stressor tunicamycin (Figure 1A) [START_REF] Jabouille | Glioblastoma invasion and cooption depend on IRE1α endoribonuclease activity[END_REF].

A principal component analysis revealed that GL261 co-clustered with U87, overexpressing the wild type IRE1 or the P336L mutant (in which IRE1 is constitutively activated), treated or not with tunicamycin, thereby indicating that the IRE1 pathway might be constitutively activated in GL261 cells as previously observed in other cell lines [START_REF] Jabouille | Glioblastoma invasion and cooption depend on IRE1α endoribonuclease activity[END_REF]. This result was confirmed by the use of the IRE1 activity signature [START_REF] Jabouille | Glioblastoma invasion and cooption depend on IRE1α endoribonuclease activity[END_REF]. Using hierarchical clustering, we confirmed that GL261 cells exhibited an IRE1 activation profile similar to XBP1+/RIDD+ when comparing to the profiles previously established with human primary GBM lines [START_REF] Jabouille | Glioblastoma invasion and cooption depend on IRE1α endoribonuclease activity[END_REF] (Figure 1B,C). To further document the genetic characteristics of the GL261 line, we focused on 86 GBM related genes (including variants, Tables S1-S3), including 79 genes recently reported [START_REF] Lee | Human glioblastoma arises from subventricular zone cells with lowlevel driver mutations[END_REF], and 7 additional genes (two Stupp protocol response predictors Mgmt and Dgki; the canonical Endoplasmic Reticulum stress sensors Eif2ak3 (Perk), Atf6 and Ern1 (Ire1); the genes Pi3k and Tert frequently mutated in IDH-wild type GBM). We found that 17 of these genes were harboring one or more mutations with a predicted functional impact (Table 1).

Some of those mutated genes are known oncogenes (Kras, Met, Pi3kca) or tumor suppressor genes (Nf1 and Trp53). Cancer genes were also affected by DNA gains (Egfr, Mdm2, Pdgfra for instance) or losses (Cdkn2c and Pten). RNAseq of the parental GL261 cells further indicated that oncogenic mutations were present and sometimes enriched at the mRNA level in those cells (Kras, Nf1, Pik3ca and Trp53 for instance, Table 1, Figure 1D, Tables S1-S3). Interestingly, the Kras mutation is found in 2% of the human GBM tumors and the pathway is activated in more than 50% of the tumors [START_REF] Jabouille | Glioblastoma invasion and cooption depend on IRE1α endoribonuclease activity[END_REF]. These results indicated that GL261 cells express known GBM cancer driver genes, can be used in syngeneic models (in C57BL/6 mice), exhibit a high basal IRE1 signaling and therefore represent a suitable cell line for the development of an in vivo model relevant to test the impact of IRE1 inhibition as an adjuvant R e v i s e d m a n u s c r i p t therapeutic strategy. Of note, GL261 exhibited some features of the mesenchymal sub-type of GBM when their properties (at both genome and transcriptome levels) were analyzed and compared to U87 cells or other primary GBM lines [START_REF] Jabouille | Glioblastoma invasion and cooption depend on IRE1α endoribonuclease activity[END_REF].

Novel preclinical GBM mouse model including surgery, irradiation and chemotherapy

Due to lack of a murine preclinical model recapitulating GBM patients' handling, we sought to develop a novel model that would comprise surgical resection combined with radio/chemotherapy, comparable to the clinical setup (Figure 2A, human vs. mouse). We designed an experimental approach relying on the mouse GBM line GL261 that we modified for stable expression of luciferase, in order to allow live tumor imaging. These cells were then orthotopically injected in C57BL/6 mouse brain and tumors allowed to grow for 12 to 13 days. At this stage, tumors were surgically resected using fluorescein as a tumor tracer (Movie S1) and the mice were allowed to recover from surgery for 1 week before undergoing Stupp-like treatment (Figure 2B). The Stupp-like protocol included radiation ( 2Gy/day, 5 exposures) combined with chemotherapy (TMZ, 25 mg/kg, 5 applications) and followed by TMZ treatment alone (30-50 mg/g, 5 applications followed by 2 days without treatment for 4 weeks). Following the completion of Stupp-like procedure, described in Figure 2, mice were sacrificed at first clinical signs. Brains were collected, fixed in paraformaldehyde (PFA 4%), embedded in paraffin prior to immunohistochemical analysis using anti-vimentin antibodies. This revealed the impact of surgery on tumor size and aggressiveness (Figure 3A), as well as the effects of irradiation/TMZ treatment, which appeared to reduce tumor size and ability to infiltrate surrounding parenchyma (Figure 3A, SIT). Mouse survival was then evaluated upon these treatments (Figure 3B), and showed that while surgery did not have any significant impact, Stupp-like treatment doubled mouse survival, thereby recapitulating observations made in patients following this protocol.

Moreover, we analyzed the impact of surgery on tumor angiogenesis by staining the recurring tumor sections with anti-CD31 antibodies (Figure 3C, arrows). Quantitation of the staining revealed that there was no significant difference in blood vessel density between the non-resected and resected conditions (Figure 3D), however more work should be carried out to provide an in depth analysis of the nature of the blood vessels analyzed in both conditions even though tumor angiogenesis appeared abnormal. Furthermore, we analyzed the ability of tumor cells to invade the neighboring parenchyma in primary or post-surgery R e v i s e d m a n u s c r i p t recurring tumors, by analyzing the vimentin staining (Figure 3E, arrows). Quantitation of this staining revealed that surgical resection of tumors enhanced the capacity of tumor cells to infiltrate non-tumor tissue (Figure 3F).

Impact of IRE1 RNase inhibitor MKC8866 in vitro and in vivo

Once this model was established, we next sought to evaluate the impact of pharmacological IRE1 inhibition on the parameters mentioned above. Since GL261 appear to express MGMT [START_REF] Tao | TGF-β/Smads Signaling Affects Radiation Response and Prolongs Survival by Regulating DNA Repair Genes in Malignant Glioma[END_REF] in an IRE1-independent manner, this might explain the high concentration of TMZ needed to kill them (IC50 ~ 800 µM). First, to document the effects of MKC8866 on GL261 cells in vitro, we evaluated whether MKC8866 blocked ER stress induced by tunicamycin (TUN, an inhibitor of protein N-glycosylation and inducer of ER stress). As expected MKC8866 treatment impaired IRE1 signals as assessed by the inhibition of XBP1 mRNA splicing upon treatment with the N-glycosylation inhibitor tunicamycin (TUN; Figure 4A, top). We next evaluated how TMZ affected XBP1 mRNA splicing using the same approach and did not detect any impact of the drug (TMZ; Figure 4A, bottom). These results were also observed at the level of XBP1s protein, whose expression was undetectable upon TMZ treatment (8h) but increased upon TUN (8h; and dampened upon MKC8866 treatment). To further document these results, we evaluated the expression of a genuine XBP1s target gene, ERdj4. ERdj4 expression increased dramatically upon TUN treatment with kinetics compatible with the activation of XBP1s and attenuated upon MKC8866 treatment (Figure 4B). No major change was observed upon TMZ treatment. MKC8866 did not affect TUNinduced ATF4 expression (Figure 4C, top panel) indicating that MKC8866 did not impact on PERK signaling, another ER stress sensor. We next evaluated the stress induced upon TMZ treatment (performed at the EC50) and how MKC8866 impacted TMZ-induced UPR signaling (Figure 4C-D). This showed that TMZ treatment impacted on ATF4 expression and that this was altered in the presence of MKC8866 (Figure 4C, bottom panel), likely reflecting the impact of DNA damaging agents on RIDD activity as previously reported [START_REF] Dufey | Genotoxic stress triggers the activation of IRE1α-dependent RNA decay to modulate the DNA damage response[END_REF]. Notably, activation of three UPR branches IRE1, PERK and ATF6 also appeared to be altered in response to TMZ, since the expression of UPR-target gene was modified (Figure 4D). Finally, since treatment of GL261 cells with MKC8866 appeared to impact on the response of these cells to different stressors, we evaluated the impact IRE1 RNase inhibition on cell sensitivity to TMZ. To do so, TMZ IC50 was determined for GL261 and evaluated to be of about 800 µM R e v i s e d m a n u s c r i p t 13 (Figure 4E), and did not vary upon combined radiation. GL261 cells' sensitivity to TMZ was increased upon treatment with MKC8866, phenomenon further amplified with combined irradiation (Figure 4E). These results suggest that TMZ-induced damages to the cells promote the activation of IRE1 dependent adaptive signals that if blocked by MKC8866, decrease the cell ability to cope with TMZ treatment. Consequently, these data suggest that combining Stupp-like treatment with MKC8866 might be an interesting approach to enhance treatment efficacy.

A major hurdle in using MKC8866 for in vivo applications in the brain lies in the fact that this IRE1 inhibitor, previously demonstrated to impact tumor growth in other cancer models [START_REF] Logue | Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy[END_REF], does not pass the blood brain barrier (BBB) and as such would not be suitable for intervention in brain tumors. To overcome this problem, we reasoned that we could take advantage of the surgical resection step for selective intra-operative delivery of IRE1targeting drugs, provided that proper biomaterial scaffolds would be used to allow controlled release of the drugs to the tumor. The procedure was successfully set in place using a drug containing fibrin microgel. The plugs of 2 mm 3 were appropriate in terms of size and conformation, they adjusted perfectly to the resection cavity. This drug-containing device alone neither impacted on tumor development (without Stupp-like treatment) nor on mouse survival following resection when containing MKC8866 (not shown). To test whether the adjuvant use of MKC8866 could impact on mouse survival, we evaluated the combination of this inhibitor with Stupp-like treatment using the fibrin-collagen gel plug as delivery tool. Surgery and drug implant were carried out and the first line of treatment including radiation/chemotherapy was done in the previous series of experiments (Figure 5A). Mouse survival was evaluated and brains were collected as sacrifice. In the first instance In addition, tumor infiltration by macrophage or microglial cells was significantly reduced in tumors exposed to Stupp treatment in the presence of MKC8866 (Figure 6C). These results are consistent with our previous results suggesting the pro-inflammatory role of IRE1 signaling in tumor cells [START_REF] Lhomond | Dual IRE1 RNase functions dictate glioblastoma development[END_REF], however, whether macrophage infiltrate is tumor promoting or tumor suppressive remains to be clearly established.

At last, three tumors from each group collected at sacrifice were dissociated and dissociated cells grown in culture to isolate GL261-derived lines following various treatments. These lines were then evaluated for their ability to proliferate (Figure 7A, bottom panel). This showed that except for cells derived from non-resected tumors which proliferated faster, none of the other lines showed any variation compared to the parental line, this result was similar to that observed with the sensitivity of cells to MKC8866 (Figure 7A, top panel). In contrast, all GL261-derived lines exhibited a higher sensitivity to TMZ than the parental line with the most striking observation with the lines derived from tumors To investigate if this heterogeneity was reflected at the level of the transcriptome, lines were analyzed using RNAseq and then clustered based on IRE1 activity signature (Figure 7B). This revealed that treatment of the animals does not seem to impact on the basal IRE1 activity in tumor-derived GL261 cells grown in culture. This result might suggest that the alterations resulting from the treatment do not directly impact on basal IRE1 activity thereby suggesting that global functional analysis of these cells' transcriptomes would be relevant to analyze further to identify the affected pathways.

Discussion

Herein, we established a novel murine preclinical model of GBM that recapitulates standard therapy of human patients. This model includes surgical resection of the tumor and Stupplike treatment combining irradiation and TMZ-based chemotherapy. Our model relies on the use of GL261 cells that exhibit a genome harboring the majority, if not all mutations, driving gliomagenesis. These mutations and/or copy number variations were observed in genes involved in the 3 main pathways affected in GBM (the receptor tyrosine kinase (RTK), P53, RB1 pathways, Figure 1 [START_REF] Brennan | The somatic genomic landscape of glioblastoma[END_REF]). Furthermore, GBM related genes harboring one SNV in GL261 and two DNA copies (Tables S1-S3) had variable mutation allele frequency (MAF) values ranking between 6 and 100%. This MAF distribution strongly suggests that multiple, different clones compose the parental GL261 line (up to 5). Altogether these GL261 characteristics recapitulate molecular features observed for primary GBM at diagnostic with the presence of multiple drivers and/or multiple GBM clones [START_REF] Körber | Evolutionary Trajectories of IDHWT Glioblastomas Reveal a Common Path of Early Tumorigenesis Instigated Years ahead of Initial Diagnosis[END_REF]. Importantly, we shown that GL261 displayed a constitutive activation of IRE1, likely resembling some primary GBM lines previously described [START_REF] Körber | Evolutionary Trajectories of IDHWT Glioblastomas Reveal a Common Path of Early Tumorigenesis Instigated Years ahead of Initial Diagnosis[END_REF]. Therefore, this cell line very likely constitutes a valuable GBM model for preclinical studies in mice, and in particular to study IRE1 inhibitors.

One of the main advantage of our model is the implantation of a very standardized surgical procedure very close to human surgery. This step brought some constraints in the choice of the mouse model. We chose a syngeneic model allowing an immune response R e v i s e d m a n u s c r i p t rather than the widely used U87 xenograft. Furthermore, U87 cell lines produce well-defined tumors with low invasive properties, making them non relevant for a pertinent surgical model, where GL261 form tumors with a macroscopic and invasive behavior very close to human glioblastoma. PDX models appear as an interesting option allowing a more realistic modeling of human glioblastomas than the strictly murine GL261 model. However, the tuning of these models is usually longer and more complex than for the syngeneic ones, and the growth of the tumors appear significantly slower. Furthermore, the GL261 cell line is widely available, making the reproducibility of this model easier. Now that our model is well calibrated, the next steps of development will include PDX models to take into account the great heterogeneity of human glioblastoma. One last alternative to our mice model would have been the use of genetically engineered models for a more physiological tumor environment, but these models do not provide standardized tumor localization which is a major feature for surgical accessibility and prognosis.

The use of such a model in the context of our discovery regarding the role of IRE1 signaling in GBM, has allowed us to test the impact of IRE1 pharmacological inhibition in enhancing the efficacy of Stupp-like treatment as already demonstrated in other contexts, for instance for the sensitization of Triple Negative Breast Cancers (TNBC) to paclitaxel therapy [START_REF] Logue | Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy[END_REF][START_REF] Zhao | Pharmacological targeting of MYC-regulated IRE1/XBP1 pathway suppresses MYC-driven breast cancer[END_REF]. In contrast with the approaches carried out with TNBC which included systemic injection of MKC8866 [START_REF] Logue | Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy[END_REF][START_REF] Zhao | Pharmacological targeting of MYC-regulated IRE1/XBP1 pathway suppresses MYC-driven breast cancer[END_REF], the available pharmacological inhibitors did not pass the blood brain barrier and as such we had to design a novel type of therapeutic strategy which would allow the controlled release of the drug during Stupp-like treatments. As such, we took advantage of the surgical step to use the peri-surgical implantation of a biocompatible graft containing the drug. This was achieved by using a fibrin/collagen graft Collectively, our work provides the proof of concept that the pharmacological inhibition of IRE1 could be used as an adjuvant to enhance the efficacy of combined radiation and chemotherapy in the context of GBM. However, one still must evaluate the impact of these treatments on the characteristics of the recurrent tumors regarding their treatment resistance properties. Also, of note is to investigate whether specific strata from cohorts of GBM patients (e.g., depending on their IRE1 signaling characteristics) would derive more benefit and should be preferentially targeted.

R e v i s e d m a n u s c r i p t 4 unique

 4 model to test the therapeutic relevance of IRE1 inhibition on tumor microenvironment and on treatment efficacy.

( 1 .

 1 5 mg/kg of ketamine and 150 μg/kg of xylazine). Then they were attached to the stereotaxic frame and the scalp was incised to expose the convexity of the skull and the injection point. The opening of the bone was performed with thin forceps allowing minimal craniectomy. Visualization of the tumor was enhanced by using an injection of intravenous fluorescein at the beginning of the procedure. Thanks to a UV source, this fluorophore R e v i s e d m a n u s c r i p t makes possible to visualize the zones of rupture of blood-brain barrier characteristic of glioblastoma. This visual aid makes it possible to delineate the tumor borders and thus minimizes the lesions on healthy parenchyma. The resection was performed using a surgical microscope, and the tumor was aspirated with the help of a small suction. The extent of resection was mainly driven by the depth of the tumor. Once the resection was performed, the hole left by the tumor was filled with a fibrin-collagen gel containing the inhibitor of interest. The craniectomy was closed with the help of a teflon patch (Neuropatch®) slid under the skull, fixed by the application of biocompatible acrylic glue. This step allows maintenance of intracranial pressure and prevents tumors from developing in the subcutaneous space. Finally, the skin was sewn with non-resorbable monofilament sutures at the end of the procedure (see Movie S1).

R e v i s e d m a n u s c r i p t 14 survival

 14 we evaluated the impact of MKC8866, Stupp-like treatment and combined therapy on tumor morphology at sacrifice. Tumors developing in the presence of the MKC8866 containing plug and upon Stupp-like treatment presented a totally different phenotype from those developing under Stupp-like treatment alone. This phenotype corresponded to much smaller tumors with tumor escape significantly delayed (Figure5B). Furthermore, when necrosis was measured in tumors collected at sacrifice (H&E staining, Figure5C, bottom), we observed that the combination of Stupp-like treatment and MKC8866 yielded much important necrosis, reflecting treatment efficacy (Figure5C). At last, we tested whether combined MKC8866 and Stupp-like treatment impacted on mouse survival. This allowed us to establish curves (Figure5D) that demonstrate the positive impact of MKC8866 contained fibrin/collagen graft (1-2 µl) either at a 10 µM (Figure5D, green) or 100 µM (Figure5D, orange) concentration on the efficiency of Stupp-like treatment. We found that MKC8866 yielded a significant increase of mice survival (of about 20%) compared to Stupp alone, however with no significant impact of the drug concentration used in the gel (Figure5D) whereas it was observable in cell culture (FigureS1). This suggested that the main limitation in our experimental design was the capacity of the fibrin/collagen gel to control the delivery of the drug over time. Collectively, our results show that IRE1 RNase inhibition might represent an appealing approach to enhance the efficiency of current treatments towards GBM and that further work is needed to improve drug delivery to the tumor site. We next analyzed how tumors responded to the different treatments using immunohistochemical analyses of apoptosis (activated caspase 3, Figure6A), proliferation (KI67, Figure6B), tumor infiltration by macrophages and microglial cells (IBA1, Figure6C), tumor vascularization (CD31, Figure6D). An representative image of the staining observed in each condition is provided (Figure6A-D, left panels). All tumor sections analyzed (9 fields on 3 independent tumor sections) were quantified as described in the Methods section and quantitation is provided (Figure6A-D, right panels). Our results show that in our experimental conditions, the only significant effect with MKC8866 was an increased apoptosis in response to Stupp treatment. This is consistent with the observed increased tissue necrosis shown in Figure5.

R e v i s e d m a n u s c r i p t 15 relapsing

 15 after resection (Figure7A, middle panel). It is interesting to note that lines derived from tumors treated with Stupp-like and MKC8866 showed great heterogeneity of response, a result contrasting with other lines. The tumor cell selection process post-resection in those conditions (STUPP and STUPP+MKC) at sacrifice might induce a strong bias in the nature of the cells collected.

( 1 - 2 5 )

 125 µl) containing 10 µM (Figure 5D, green) or 100 µM (Figure 5D, orange) of MKC8866, corresponding to non-toxic concentrations in cell culture and in animals. The grafts were implanted at the time of surgery and mice were then exposed to Stupp-like treatments. Both macroscopic exploration, immunohistochemistry and Kaplan-Meier survival curves (Figure indicated that combination of MKC8866 with Stupp-like treatment enhanced its efficacy, leading to increased necrosis within the tumor and to prolonged mouse survival. At last, the observed effects of TMZ on the activation of the UPR arms (ATF4, XBP1s and UPR target genes) might reflect the impact of DNA damaging agents on UPR activation [11, 29], and will R e v i s e d m a n u s c r i p t require an in-depth investigation to be fully characterized. The impact of MKC8866 on TMZinduced UPR signals also suggests important cross-talks between the different arms.

Table 1 :Figure 1 :

 11 Figures legendsFigure 1: Selection of GL261 as a relevant model for testing the relevance of IRE1 inhibition in GBM. A) Biplot projection of PC3 and PC4 components of PCA analysis on harmonized gene expression data of GL261 parental and U87 IRE1_WT, IRE1_DN and IRE1 mutant clones under normal and stress conditions. B) Hierarchical clustering heatmap of GL261 parental and U87 IRE1_WT, IRE1_DN and IRE1 mutant cell lines with tunicamycin (tn) and without stress based on the harmonized gene expression profile of IRE1 signature. Pearson correlation coefficient was used for sample distance correlation and the average agglomeration method was used for sample clustering. U87 cells in normal and stress conditions are represented with pink and yellow bars to the top of the heatmap while the GL261 parental cell line is represented with the grey box. C) Sample distance matrix of GL261 parental and U87 IRE1_WT, IRE1_DN and IRE1 mutant cell lines under normal and stress conditions, based on the harmonized gene expression profile of IRE1 signature using the Euclidean distance between samples. For sample hierarchical clustering Ward's minimum variance method (ward.D2) was used. D) Sequence mutations and copy number changes are depicted as grey boxes for mutations, red boxes for gain of DNA copy and blue boxes for loss of DNA copy. Mutation allele frequency in the gene and the transcript (in parentheses) or DNA copy number are indicated under the corresponding gene name.

Figure 2 :

 2 Figure 2: Preclinical development of a novel GBM model in immunocompetent mice. This model comprises surgical resection followed by radio/chemotherapy that recapitulates the current GBM patient standard of care (Stupp protocol) in mice. A) Schematic representation of the experimental design and comparison between human and the mouse model. B)Schematic representation of the mouse model with the 3 major steps including "Injection" of GL261 cells (orthotopic approach), 14 days later "Resection" of tumors using fluorescein labelling, and finally Stupp-like "Treatment". During resection, the skull was removed and the tumor contour was defined after injecting fluorescein and applying UV source on the tumor site. After aspiration of the tumor material, the resection cavity was filled with hemostatic materials before implanting a teflon prosthesis (replacing the removed bone) and the skin sewn. The Stupp treatment combined irradiation and chemotherapy (temozolomide/TMZ) -the surgical procedure is also reported in Movie S1.

R e v i s e d m a n u s c r i p t

Figure 3 :

 3 Figure 3: Development and analysis of the establishment of a Stupp-like protocol in mice. A) At sacrifice, brains were collected, fixed in PFA, embedded in paraffin prior to be analyzed using immunohistochemistry using anti-vimentin antibodies (CTR: no injection; NS: no surgery; S: surgery only; SIT:surgery/irradiation/TMZ). B) Kaplan-Meier representation of mouse survival under this regiment (green: non-resected, blue: surgical resection, orange: surgical resection + radio/chemotherapy). C-D) Immunohistochemical analysis and quantitation of tumor angiogenesis post-resection. Abnormal vessels are indicated by arrows. E-F) Immunohistochemical analysis and quantitation of tumor infiltration/invasion post-resection. The migration distance of tumor cells from the tumor core in the nonresected and resected conditions is indicated by arrows.

Figure 4 :

 4 Figure 4: Evaluation of the impact of IRE1 inhibition by MKC8866 on GL261 cell response to stress. A) Impact of MKC8866 treatment on IRE1 signaling as monitored using RT-qPCR analysis of XBP1 mRNA splicing upon time-course treatments with TUN (2 µg/ml) or TMZ (800 µM) (top). Expression of XBP1s protein following 8h of TUN or TMZ treatment in the presence or not of MKC8866 as detected using Western blotting (bottom). B) Impact of MKC8866 treatment on the expression of the XBP1s target gene ERdj4 as monitored using RT-qPCR. C) Impact of MKC8866 on TUN-and TMZ-induced ATF4 expression and the phosphorylation of eIF2α as monitored using Western blot. D) Impact of MKC8866treatment on other arms of UPR signaling induced by TMZ as monitored using RT-qPCR analysis of HERPUD1, GRP94 (both reporting for ATF6 signaling) and CHOP (reporting for PERK signaling) mRNA expression. E) Impact of MKC8866 treatment on the sensitivity of GL261 cells to TMZ treatment in the presence or absence of radiation.

Figure 5 :

 5 Figure 5: Impact of IRE1 inhibition on tumor development under Stupp-like treatment. A) Schematic representation of the experimental procedure and mouse survival. B) Macroscopic images of the brains following injection of GL261-Luc cells, surgical resection and treatment with ctrl plug (blue), MKC8866-containing fibrin microgels (grey); ctrl plug + Stupp (red) and MKC8866-containing fibrin microgels + Stupp (green). C) Quantification of tumor necrosis upon different treatments. D) Impact of IRE1 inhibition on tumor development under Stupp-like treatment -Kaplan-Meier representation. p value between Stupp and Stupp+MKC8866 (0.0146 -10 µM (green) and 0.0118 -100 µM (orange)).

R e v i s e d m a n u s c r i p t 24 Figure 6 :

 246 Figure 6: Immunohistochemical analysis of GL261-derived tumors following in vivo treatments. A) Activated Caspase 3 (apoptosis) immunostaining sections from tumors collected at sacrifice. B) Ki67 (proliferation) immunostaining sections from tumors collected at sacrifice. C) IBA1 (macrophages and microglial cells) immunostaining sections from tumors collected at sacrifice. D) CD31 (endothelial cells) immunostaining sections from tumors collected at sacrifice. In each case at least 5 tumors per condition and at least 2 fields per tumor were analyzed. NS: non-significant.

Figure 7 :

 7 Figure 7: Analysis of GL261-derived cell lines following in vivo treatments. A) Characterization of the derived lines regarding their sensitivity to MKC8866 (top) or TMZ (middle), and regarding their proliferation ability. Twelve lines were analyzed in multiple experiments (n > 4). Shown is the average ±SD. B) RNAseq analysis of the different derived cell lines and representation as heat map. Hierarchical clustering was performed as described in the methods section.

R e v i s e d m a n u s c r i p t

FIGURE 1 R e v i s e d m a n u s c r i p t

 1 FIGURE 1

FIGURE 2 R e v i s e d m a n u s c r i p t

 2 FIGURE 2

FIGURE 3 R e v i s e d m a n u s c r i p t

 3 FIGURE 3

FIGURE 4 R e v i s e d m a n u s c r i p t 29 FIGURE 5 R e v i s e d m a n u s c r i p t 30 FIGURE 6 R e v i s e d m a n u s c r i p t 31 FIGURE 7

 4295306317 FIGURE 4

Acknowledgements

We thank the BIOSIT H2P2 platform and Florence Jouan for immunohistochemistry and the BIOSIT Animal facility ARCHE (https://biosit.univ-rennes1.fr/). This work was funded by grants from the French National Cancer Institute (INCa, PLBIO2017, 2019, 2020), the Fondation pour la recherche Médicale (FRM ; équipe labellisée 2018) to EC and EU H2020 MSCA ITN-675448 (TRAINERS) and MSCA RISE-734749 (INSPIRED) grants to AS, AC and EC.

The work was also supported in part by research grant from Science Foundation Ireland (SFI), co-funded under the European Regional Development Fund under Grant number 13/RC/2073.

Authors' contributions

Conceptualization


Waffa Rakhrour

Djamel Selloum
email: selloumdjamel@gmail.com

Abdellah Henni

Noureddine Cherrad

Imene Chikouche

Mokhtar Benalia

Sarra Mouffok

Mabrouk Djedid

Nacira Bouzar

Sophie Tingry

Electrochemical synthesis of an organometallic material based on polypyrrole/MnO2 as high-performance cathode

Keywords: composite electrodes, polypyrrol, manganese dioxide, electrodeposition, oxygen reduction

We report herein the design of promising composite material as a cathode in a rechargeable battery by combining the properties of polypyrrole (PPy) and MnO2 particles. The composites were prepared by electropolymerization of the pyrrole monomer followed by electrodeposition of a manganese salt suspension by two methods. The first method involved the formation of MnO2 in situ in the PPy/ITO electrode, while the second method involved first a preliminary adsorption of Mn 2+ ions in the polymer, followed by an electrochemical of the electrode. The morphology of the resulting composite materials (PPy/MnO2) was studied by Energy-dispersive X-ray spectroscopy (EDS), X-ray diffractometry (XRD), scanning electron microscopy (SEM), Fourier-transform infrared (FTIR). The investigations of the electrochemical properties of the composite electrodes show that the presence of MnO2 play an important role in improving the surface of polymer films, which leads to lower charge transfer resistance and higher electrode activity. For the optimal synthesis method, the electrode generates a maximum current of up to -7.9 mA.cm -2 for the oxygen reduction reaction, which is eight times the current density delivered by the electrode without PPy.

Introduction

Energy requirements have led to the intensification of electrochemical generators and batteries [1][2][3][4], whose electrochemical performance depends mainly on the nature and structure of the electrode materials (anode and cathode electrodes) [5,6]. The efficiency of these conventional batteries is still limited by a low cyclability [7,8]. Transition metal oxides have been extensively investigated as electrode materials for batteries [9][10][11][12][13][14][15]. A great attention has been devoted to manganese dioxide (MnO2) as cathode material in batteries [16][17][18][19] and supercapacitors [20][21][22][23], that exhibits high theoretical capacity and can be produced in a cheap and sustainable way [24][25][26][27][28][29][30][31]. However, MnO2 has a low electronic conductivity that is undesirable in electrochemical devices [32,33], associated with an irreversible reduction in its capacitance at high voltage or high temperature and over several charge-discharge cycles [34][35][36][37]. In this regard, considerable research efforts have been devoted to improve the electrochemical performance of MnO2-based electrodes by combining MnO2 with highly conductive materials such as metallic nanostructures, carbon nanotubes, graphene or conductive polymers (by polypyrrole, polyaniline, and polythiophene) [38][39][40][41][42][43]. In a rechargeable battery, the energy storage results from charging the electrical double layer at the electrode and from faradic reactions at the electrolyte interface. To improve the accessibility of electrolytic doping ions to the electrode surface, large-area electro-active electrodes must be constructed, hence the use of conductive polymers to provide a stable conductive environment for the MnO2 [44][45][46][47][48][49][50][51][52][53]. Among them, the conductive properties of polypyrrole (PPy), as well as its low cost, high theoretical specific capacity and good mechanical stability [38,[54][55][56][57], have been used for the development of high-performance electrodes in supercapacitors, where MnO2 has been fixed by hydrothermal process [58], by chemical oxidative polymerization in-situ [38,40], or used in the form of nanowires as a matrix [59].

In this work, we choose to insert MnO2 into PPy films grown by electrochemical polymerization on ITO substrate. We report herein the assessment of two different methods to insert the particles into the polymer matrix. The first method consisted of forming MnO2 in situ within the PPy/ITO electrode, while the second method involved first a preliminary adsorption of Mn 2+ ions in the polymer, followed by a subsequent electrochemical reduction step at the electrode. The structural morphology of the resulting composite materials MnO2/PPy/ITO was carried out by XRD and SEM studies. We performed cyclic voltammetry (CV), LSV (Linear sweep voltammetry) curves and electrochemical impedance spectroscopy (EIS) studies to evaluate oxygen reduction due to the great importance of active cathodes in rechargeable batteries.

Experimental

Chemicals

The monomer pyrrole (99%), MnCl2, KCl, LiCLO4 and acetonitrile were purchased from Sigma-Aldrich.

Synthesis of PPy and composite film preparation

Electropolymerization of pyrrole monomer was performed onto ITO substrates in a three-electrode cell, with a Pt wire as counter electrode, a saturated calomel electrode (SCE) as the reference electrode and ITO sample (1x2 cm 2 ) as working electrode, previously cleaned successively with HNO3, acetone, ethanol and distilled water. The electropolymerization was carried out by cyclic voltammetry over 20 cycles between -1.0 to 1.2 V vs. SCE at 20 mV.s -1 , in acetonitrile containing 0.1 M of LiClO4 and 0.1 M of Pyrrole [60]. The resulting electrode (estimated area of 1 cm 2 ), named as PPy/ITO, was thoroughly rinsed with water solution to remove any remaining monomeric traces. Two different methods have been used to incorporate MnO2 particles into PPy film.

First method: MnO2 was electrodeposited directly on a PPy / ITO electrode by performing 10 cycles between 0.4 and 1.5 V (vs SCE) in an aqueous solution of 0.4 M KCl and 0.05 M MnCl2., which we called the "direct method" (d-MnO2).

Second method: MnCl2 was first adsorbed after simple immersion of a PPy / ITO electrode in an aqueous solution containing 10 -2 M KCl and 0.05 M MnCl2 for different immersion times (5 and 10 minutes) allowing by electrostatic interactions the exchange of manganese ions with the counterions initially present in the PPy film. A subsequent step of electrochemical reduction resulted in the formation of MnO2 in the PPy/ITO electrode by cyclic voltammetry (10 cycles) in the potential range of 0.2 -1.5 V vs SCE in 0.1 M KCl. The second method was called "indirect method" (i-MnO2).

The resulting composite electrodes were called MnO2/PPy/ITO.

Electrode characterization

The XRD were examined by X'Pert Pro MPD X-ray diffractometer using Cu-Kα radiation (λ=0.154059 nm). The FTIR were traced by Nicolet 6700. The electrochemical behavior of the MnO2/PPy/ITO electrodes with respect to O2 reduction was evaluated by linear sweep voltammetry (LSV). LSV was conducted from OCP value of 1.4 V to 0 V vs. SCE at a scan rate of 25 mV.s -1 in 0.1 M KCl. Electrochemical impedance spectroscopy (EIS) was conducted using Metrohm Autolab (PGSTAT302N) by scanning the frequencies between 100 kHz to 0.1 Hz (10 mV amplitude), at Eappl = 0.2 V vs. SCE.

Results and discussion

Characterization of the composite film MnO2/PPy/ITO

The growth of PPy on ITO is shown on the typical cyclic voltammograms in Fig. 1, characterized by a pair of redox peaks located at 0.5 V vs. SCE and attributed to the electrochemical transition from the reduced form of PPy to the oxidized state [28][29]. Inset in Fig. 1 shows the formation of a three-dimensional polymer network observed by SEM characterization. The MnO2/PPy/ITO composite films were synthesized by entrapping the metal salt to give metal particles in a subsequent reduction step, using two methods. In the direct method (d-MnO2), MnO2 was formed in situ on the PPy/ITO electrode from an aqueous solution containing MnCl2 after ion-exchange subjected to ten successive scans in the potential range starting from the open potential of 0.4 to 1.5 V vs. SCE in KCl (Fig. 2a). The CV profile (Fig. 2b) shows the oxidation peak of Mn(+II) in Mn(+IV) located at 1.25 V vs. SCE and the reduction peak of Mn(+IV) in Mn(+II) at 0.65 V vs. SCE, according to the one-step process related to the reaction (1) [61][62][63]. In the indirect method (i-MnO2), MnO2 was formed within the PPy/ITO electrode in two successive steps: (i) adsorption of Mn 2+ ions in the polymer according to two different times of soaking: 5 and 10 minutes, and (ii) formation of metal particles in a subsequent step of electrochemical reduction at the electrode in KCl solution. The resulting cyclic voltammetry, in Fig. 2c, shows an intense peak at 0.9 V corresponding to the oxidation of the incorporated Mn(II) in Mn(IV) on the surface of the PPy/ITO electrode. The lower value of oxidation potential for the second method reflects the easier transformation of Mn 2+ ions to MnO2, which are more readily available in the matrix after the absorption step. Furthermore, according to the current density values on the first cyclic voltammetry curves in Figure 2d, a soak time of 5 min for the electrode in the MnCl2 solution allows the incorporation of increased MnO2 into the polymer. It can therefore be proposed that MnO2 acts as electron transfer mediators in the nucleation, formation and growth of the PPy film on the ITO surface.

𝑀𝑛 2+ + 2𝐻 2 𝑂 ⇆ 𝑀𝑛𝑂 2 + 4𝐻 + + 2𝑒 -…………………………………….……(1)
The morphology of the as-fabricated materials, obtained by cyclic voltammetry, was analyzed by SEM. The nucleation and growth of PPy occurs randomly, forming irregularly shaped particles with defined boundaries, which are typical morphologies of polypyrroles. This morphology depends on the polymerization charge and the nature of the counter anions.

Contrary to PPy films characterized by an irregular morphology with remaining particle sizes as nucleation sites varying from 0.10 to 5.0 µm (inset SEM images in Fig. 1), the composites electrodes present different morphologies (Fig. 3). For the d-MnO2 method, the surface morphology of MnO2/PPy/ITO is characterized (Fig. 3a) by a homogeneous cauliflower-like structure. For the i-MnO2 method, the surface morphology of MnO2/PPy/ITO (Fig. 3a andb) is rather characterized by a smooth and dense layer surrounded by grains. According to the soaking time, the PPy/ITO electrode in Mn 2+ solution, the grains seem to be localized on the surface of the film for the soaking time of 5 minutes, and rather embedded within the film for the longer soaking time of 10 minutes. EDX spectra of the representative electrodes (Fig. 3a',b',c'), qualitatively shows the successful existence of MnO2 in the PPy matrix with the presence of intense rays at 5.9 and 6.5 keV. The amount of MnO2 varies with the method of synthesis: with the d-MnO2 method, it seems that a higher amount is obtained due to the relatively good conductivity of PPy film. In addition, EDX spectra show the presence of carbon, nitrogen, characteristic of the polymer PPy, and the presence of chlorine and oxygen comes from the doping of PPy with perchlorate ions. The presence of Si, In, O and Sn atoms are characteristic of the ITO substrate.

The presence of MnO2 in the composite electrodes was also confirmed by XRD analyses.

Electrochemical characterization of MnO2/PPy/ITO

EIS experiments were conducted for further characterizing the PPy/MnO2/ITO electrodes. Fig. 5 shows the Nyquist plots with are characterized by a typical semicircle in the high frequency region for all samples, which corresponds to the charge transfer resistance (Rct) of the electrodes. For all samples, the semicircle starts at the same value, indicating that the resistance of the electrolyte remains unchanged. For a given electrode, the lower Rct is, the higher is the electrical conductivity. The results gathered in Table 1 show that Rct = 175, 75, 13 and 8  cm 2 for PPy, PPy/d-MnO2, PPy/i-MnO2

(5 min) and ITO/PPy i-MnO2 (10 min), respectively. In the presence of MnO2, the Rct values decrease drastically, inducing a lower interfacial charge transfer resistance [72] and thus higher electrical conductivity of the electrodes compared to PPy. Rct is even lower when MnO2 was incorporated in the polymer by the indirect method compared to the direct method. The modelling of EIS data by a representative equivalent electrical circuit enables the determination of QCPE (constant phase element), from the equation QCPE ={(Ciω) α } -1 . The α values are between 0.66 and 0.83 for all composite electrodes, suggesting the uniformity and the homogeneity of the materials.

Table 1. Impedance parameters of the composite electrodes.

Given the great importance of the development of electrode materials for O2 reduction in batteries, the behavior of the composite electrodes has been studied by LSV from 1.4 to 0 V vs. SCE in 1M KCl. As shown in Fig. 6, the oxygen reduction current starts without overpotential at 1.4 V vs. SCE, and the current densities show a semi-plateau, resulting from the control of the electrocatalytic reaction by diffusion of the oxygen to the electrode surface.

The MnO2/ITO electrode delivers the lowest current density about -0.9 mA.cm -2 , confirming the crucial role of PPy in improving electrode performance. The maximal current density of O2 reduction for d-MnO2/PPy/ITO, i-MnO2/PPy/ITO(10 min) and i-MnO2/PPy/ITO(5 min) electrode is -4.9, -7.6 and -7.9 mA.cm -2 , respectively. These values reveal the importance of the synthesis technique used in the design of efficient MnO2/PPy composites. In the indirect method, we assume that the amount of MnO2

in PPy affords a close contact between the electrode material and the electrolyte. The maximal current density delivered by i-MnO2/PPy/ITO(5 min) electrode is -7.9 mA/cm 2 , which is eight times the current density delivered by the electrode without PPy. This result shows the advantage of possible applications of MnO2 and PPy to prepare efficient electrodes. In comparison with literature, our composite polypyrrole/MnO2 cathodes have a high and competitive efficiency for oxygen reduction reaction (ORR) with the reported MnO2 cathodes.

As an example, efficient MnO2-based electrocatalysts have been synthesized for ORR, such as multi-walled MnO2/PPy/MnO2 nanotubes [39], or α-MnO2 nanowires prepared in the presence of an ionic liquid as structure-directing agent [73], able to generate current densities closed to 7 mA.cm -2 . The introduction of nanoparticles into MnO2 materials has enable ORR to be boosted with delivered current densities close to 6 mA.cm -2 , such as example with Ag in amorphous MnO2 mixed with carbon nanotubes [74], or with Co3O4 attached to the surface of MnO2 nanorods [75].

Conclusion

In this study, MnO2/PPy electrodes were successfully prepared and examined.

Fig. 1

 1 Fig. 1 Cyclic voltammetry of Pyrrole (0.1 M) at the electrode ITO performed over 20 cycles between -1 and 1.2 V vs. SCE at scanning rate 20 mV.s -1 , in the electrolyte 0.1 M LiClO4 in acetonitrile. Insert: SEM image of PPy/ITO sample.

Fig. 2 . 2 - 1 . 5 V

 2215 Fig. 2.(a,b) Cyclic voltammograms of PPy/ITO electrode over 10 scans in the potential

Fig. 3

 3 Fig.3 SEM images and EDS of (a,a') d-MnO2/PPy/ITO, (b,b') i-MnO2/PPy/ITO(5 min) and (c,c') i-MnO2/PPy/ITO (10 min).

Figure 4

 4 Figure 4 presents the pattern of pure PPy, with a broad peak centered at 2θ = 25° assigned to

Fig. 4 .

 4 Fig. 4. a: XRD diffraction patterns of MnO2, PPy/ITO, PPy/MnO2/ITO (i-MnO2 and tsoaking = 5 min), PPy/MnO2 (i-MnO2 and tsoaking = 10 min), and PPy/MnO2/ITO (d-MnO2). b: FTIR spectra of PPy and PPy/MnO2 films.

Fig. 5 .

 5 Fig.5. Complex-plane Nyquist impedance plots at Eappl = 0.2 V vs. SCE

Fig. 6 .

 6 Fig. 6. LSV of the composite electrodes ITO/PPy/MnO2 in aqueous 1 M KCl electrolyte

Fig. 1 Fig. 2 .Fig. 3

 123 Fig. 1 Cyclic voltammetry of Pyrrole (0.1 M) at the electrode ITO performed over 20 cycles between -1 and 1.2 V vs. SCE at scanning rate 20 mV.s -1 , in the electrolyte 0.1 M LiClO4 inacetonitrile. Insert: SEM image of PPy/ITO sample

Fig. 5 .Fig. 6 .

 56 Fig.5. Complex-plane Nyquist impedance plots at Eappl = 0.2 V vs. SCE

Table 1 . Impedance parameters of the composite electrodes.

 1 The insertion of MnO2 in the polymer matrix was confirmed by DRX and EDX. SEM micrographs showed cauliflower-like growth on the deposited substrate. MnO2 grains of similar size were homogeneously distributed in the polymer. The composite electrode showed a decrease in charge transfer resistance and excellent electrochemical stability. Electrochemical experiments revealed that the composite materials prepared by the direct incorporation of MnO2 gave the highest performance for O2 reduction. This study contributes toward advances in the design of efficient MnO2/PPy composites suitable for rechargeable battery electrodes.

	Sample	ZCPE (F /cm 2 ) Rct(kcm 2 )	
	ITO	137.9	222.6	0.83
	ITO/PPy	138.7	174.7	0.78
	ITO/PPy/d-MnO2	153.4	75.0	0.66
	ITO/PPy/i-MnO2(5 min)	178.6	13.1	0.67
	ITO/PPy i-MnO2(10 min)	194.1	8.2	0.74