Thomas Aumer

Sébastien N Voisin

Thomas Knobloch

Céline Landon

Dr Philippe Bulet
email: philippe.bulet@univ-grenoble-alpes.fr

Impact of an antifungal insect defensin on the proteome of the phytopathogenic fungus Botrytis cinerea

Keywords: Insect defensin, antifungal defensin, Botrytis cinerea, proteomics, fungal infection, heliomicin, mechanism of action

ETD151, an analogue of the antifungal insect defensin heliomicin, is an antifungal peptide active against yeasts and filamentous fungi. In order to decipher the mechanisms underlying its molecular action on the phytopathogenic fungus Botrytis cinerea, a necrotrophic pathogen responsible for gray mold disease, we investigated the changes in three-day old mycelia upon treatment with different concentrations of ETD151. Optical and fluorescent microscopies were used prior to establishing the peptide/protein profiles through two mass spectrometry approaches: MALDI profiling, to generate molecular mass fingerprints as peptide signatures, and a gel-free bottom-up proteomics approach. Our results show that a concentration of ETD151 above the half-maximal inhibitory concentration can alter the integrity of the mycelial structure of B. cinerea. Furthermore, reproducible modifications of the peptide/protein composition were demonstrated in the presence of ETD151 within a 1.5-16 kDa mass range. After the robustness of LC-ESI-MS/MS analysis on B. cinerea mycelial extracts was confirmed, our analyses highlighted 340 significantly modulated proteins upon treatment with ETD151 within a 4.8-466 kDa mass range. Finally, data mapping on KEGG pathways revealed the molecular impact of ETD151 on at least six pathways. In particular, for the effect on oxidative phosphorylation, we clearly demonstrated that ETD151 does not interact directly with the mitochondrial respiratory chain.

INTRODUCTION

Phytopathogenic fungi are a major concern for crops in agriculture. In the last decade, phytopathology experts have alerted the scientific community to several species threatening food security and biodiversity 1 . Every year, fungal pathogens destroy more than 125 million tons of the world's top five major crops [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] : rice (rice blast caused by Magnaporthe oryzae), wheat (rust caused by Puccinia graminis), maize (smut caused by Ustilago maydis), potatoes (late blight caused by Phytophthora infestans) and soybean (rust caused by Phakospora pachyrhizi). These production losses could feed an additional 600 million people per year. Among the phytopathogenic fungi, Botrytis cinerea (teleomorph Botryotinia fuckeliana) is a necrotrophic pathogen responsible for gray mold disease causing significant crop losses in more than 200 plant species worldwide [START_REF] Williamson | The Cause of Grey Mould Disease[END_REF][START_REF] Van Kan | Licensed to Kill: The Lifestyle of a Necrotrophic Plant Pathogen[END_REF] . Ranked scientifically and economically as the second most important fungal pathogen by the international phytopathological community 5 , B. cinerea generates significant costs over a broad host range. Damage occurs in different stages of fruit and vegetable production in open fields or in greenhouses and requires extensive disease control, mainly by fungicide application. Concerns about this fungus have also increased due to the recent discovery of B. cinerea fungicide-resistant strains in field populations [START_REF] Hahn | The Rising Threat of Fungicide Resistance in Plant Pathogenic Fungi: Botrytis as a Case Study[END_REF] . The introduction of site-specific fungicides almost 50 years ago revolutionized crop protection and has remained until now the main approach for reducing fungal disease progression. However, plant pathogenic fungi can adapt, leading to a tremendous loss of plant protection effectiveness. Fungicide resistant pathogens have emerged over time and spread globally [START_REF] Lucas | Chapter Two -The Evolution of Fungicide Resistance[END_REF] . Most important classes of antifungals (e. g. benzimidazoles, strobilurins, hydroxyanilides) are concerned by the development of resistance compromising the efficacy of crop protection. As a new major challenge, it is necessary to propose novel fungicides with innovative mechanisms of action or alternative strategies with non-chemical products [START_REF]Botrytis -the Fungus, the Pathogen and Its Management in Agricultural Systems[END_REF] .

Scientists are investigating the field of antimicrobial peptides (AMPs), looking for new molecules with novel targets, reduced toxicity and broad-spectrum activity for a low-rate use. Also known as host defense peptides (HDPs), AMPs are key components of the innate immune system of all organisms, protecting them against attacks by invading pathogens and stressors [START_REF] Brown | Cationic Host Defense (Antimicrobial) Peptides[END_REF] .

Even if a wide range of AMPs have been identified, they can be globally classified according to their amino acid composition, their structure and the organization of their disulfide bonds. One of these classes, the CSαβ-defensins (Cysteine-stabilized α-helix β-sheet), groups small, cationic, cysteine-rich peptides with specific 3D organization and conserved disulfide bridge arrays [START_REF] Cornet | Refined Three-Dimensional Solution Structure of Insect Defensin A[END_REF][START_REF] Zhu | Phylogenetic Distribution, Functional Epitopes and Evolution of the CSαβ Superfamily[END_REF][START_REF] Shafee | The Defensins Consist of Two Independent, Convergent Protein Superfamilies[END_REF] .

Intriguingly, the CSαβ-defensins have been found in plants and insects. Plant defensins are wellknown and extensively studied for their antifungal activity [START_REF] Vriens | Antifungal Plant Defensins: Mechanisms of Action and Production[END_REF] . On the contrary, only a few insect defensins have been reported to be exclusively antifungal, most of them exhibiting antimicrobial activities (antibacterial and antifungal), with particular efficacy against Gram-positive bacteria [START_REF] Bulet | Insect Antimicrobial Peptides: Structures, Properties and Gene Regulation[END_REF] .

The first antifungal insect defensin to be described was drosomycin [START_REF] Fehlbaum | Insect Immunity: Septic Injury of Drosophila Induces the Synthesis of a Potent Antifungal Peptide with Sequence Homology to Plant Antifungal Peptides[END_REF][START_REF] Landon | Solution Structure of Drosomycin, the First Inducible Antifungal Protein from Insects[END_REF] from the fruit fly Drosophila melanogaster (Diptera). It was followed by heliomicin [START_REF] Lamberty | Insect Immunity: Isolation from the Lepidopteran Heliothis Virescens of a Novel Insect Defensin with Potent Antifungal Activity[END_REF][START_REF] Lamberty | Solution Structures of the Antifungal Heliomicin and a Selected Variant with Both Antibacterial and Antifungal Activities[END_REF] from the tobacco budworm Heliothis virescens (Lepidoptera), termicin [START_REF] Lamberty | Constitutive Expression of a Cysteine-Rich Antifungal and a Linear Antibacterial Peptide in a Termite Insect[END_REF][START_REF] Da Silva | Solution Structure of Termicin, an Antimicrobial Peptide from the Termite Pseudacanthotermes Spiniger[END_REF] from Pseudacanthotermes spiniger (Isoptera), gallerimycin [START_REF] Schuhmann | Cloning and Expression of Gallerimycin, an Antifungal Peptide Expressed in Immune Response of Greater Wax Moth Larvae, Galleria Mellonella[END_REF] from the great wax moth larvae Galleria mellonella (Lepidoptera) and ARD1 [START_REF] Landon | Lead Optimization of Antifungal Peptides with 3D NMR Structures Analysis[END_REF] from the larvae of Archaeoprepona demophon (Lepidoptera).

The promising in vitro antifungal activity of insect defensins against plant or human pathogens, as well as their non-cytotoxicity, highlights their potential as candidates for crop protection strategies [START_REF] Banzet | Expression of Insect Cystein-Rich Antifungal Peptides in Transgenic Tobacco Enhances Resistance to a Fungal Disease[END_REF] and/or therapeutic applications [START_REF] Thevissen | Therapeutic Potential of Antifungal Plant and Insect Defensins[END_REF] . In the 2000s, the French biotechnology company EntoMed SA was the first to develop natural insect AMPs for the treatment of systemic fungal infections affecting immunocompromised patients [START_REF] Andrès | Cationic Antimicrobial Peptides in Clinical Development, with Special Focus on Thanatin and Heliomicin[END_REF] . Heliomicin was the working model of a cutting-edge optimization program based on phylogenic exploration and fine-tuned mutagenesis. The results of their work, a 44 amino acid peptide, called ETD151, revealed enhanced antifungal activity against Candida albicans and Aspergillus fumigatus as well as additional activity against Cryptococcus neoformans, as compared to heliomicin [START_REF] Landon | Lead Optimization of Antifungal Peptides with 3D NMR Structures Analysis[END_REF] . ETD151 was the principal product developed by EntoMed SA for treating antifungal infections [START_REF] Halpern | Entomed mise sur le système immunitaire des insectes pour soigner l'homme[END_REF] .

However, limited data has been gathered on the antifungal mechanism of action of insect defensins, which include the inhibition of spore germination, the alteration of germ tube elongation and the release of cytoplasmic content [START_REF] Banzet | Expression of Insect Cystein-Rich Antifungal Peptides in Transgenic Tobacco Enhances Resistance to a Fungal Disease[END_REF][START_REF] Aerts | The Mode of Antifungal Action of Plant, Insect and Human Defensins[END_REF] . Comparisons based on structural analysis and activity tests on fungal mutants, provided evidence of mechanical commonalities between heliomicin, drosomycin and some antifungal plant defensins such as the radish defensin RsAFP2 [START_REF] Thevissen | Defensins from Insects and Plants Interact with Fungal Glucosylceramides[END_REF][START_REF] Landon | The Active Site of Drosomycin, a Small Insect Antifungal Protein, Delineated by Comparison with the Modeled Structure of Rs-AFP2, a Plant Antifungal Protein[END_REF][START_REF] Gao | Differential Potency of Drosomycin to Neurospora Crassa and Its Mutant: Implications for Evolutionary Relationship between Defensins from Insects and Plants[END_REF] . In contrast to insect defensins, the mechanisms of action of plant defensins have been widely investigated. In most cases, the first step is the interaction with non-protein fungal membrane partners such as sphingolipids or phospholipids [START_REF] Rautenbach | Antifungal Peptides: To Be or Not to Be Membrane Active[END_REF] . Thereafter, plant defensins can either be internalized or remain outside the fungal cells. Cell death can occur via multiple mechanisms, induction of fungal membrane permeabilization, reactive oxygen species (ROS), apoptosis, or mitochondrial functionality loss [START_REF] Cools | Antifungal Plant Defensins: Increased Insight in Their Mode of Action as a Basis for Their Use to Combat Fungal Infections[END_REF] . Effects differ depending on the target organism; the mechanisms of action of the Medicago spp. defensin MtDef4 against the ascomycete fungi Neurospora crassa and Fusarium graminearum [START_REF] El-Mounadi | Antifungal Mechanisms of a Plant Defensin MtDef4 Are Not Conserved between the Ascomycete Fungi Neurospora Crassa and Fusarium Graminearum[END_REF] are not the same. Upon exposure to antifungal plant defensins, fungal cells will induce mechanisms to defend themselves against these adverse effects. A family of conserved kinases, MAPK, regulate tolerance to plant defensins. In yeast, the MAPK pathways are involved in filamentous growth, cell wall integrity (CWI) and the high osmolarity/glycerol (HOG) response [START_REF] Parisi | The Evolution, Function and Mechanisms of Action for Plant Defensins[END_REF] . All of these elements provide a background for studying the mode of action of ETD151, an optimized peptide analogue of the two lepidopteran insect antifungal peptides heliomicin and ARD1.

To decipher the mechanism of action of ETD151 on the fungal phytopathogen B. cinerea, a proteomics approach based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography-mass spectrometry coupled to electrospray ionization (ESI-LC-MS) was used. The elucidation of the B. cinerea genome was a significant breakthrough for the proteomics research community and has provided for the accurate identification of proteins in this fungus, mainly from mycelial samples [START_REF] Amselem | Genomic Analysis of the Necrotrophic Fungal Pathogens Sclerotinia Sclerotiorum and Botrytis Cinerea[END_REF][START_REF] González-Fernández | Proteomic Analysis of Mycelium and Secretome of Different Botrytis Cinerea Wild-Type Strains[END_REF][START_REF] Van Kan | A Gapless Genome Sequence of the Fungus Botrytis Cinerea[END_REF][START_REF] Staats | Genome Update of Botrytis Cinerea Strains B05.10 and T4[END_REF] . In the past ten years, several methodological developments and technological advances in Botrytis proteomics have offered effective new tools for achieving a better understanding of B. cinerea biology and how its proteins are involved in pathogenic biological processes [START_REF] Liñeiro | Contribution of Proteomics Research to Understanding Botrytis Biology and Pathogenicity. In Botrytis -the Fungus[END_REF][START_REF] Fernández-Acero | Proteomic Analysis of Phytopathogenic Fungus Botrytis Cinerea as a Potential Tool for Identifying Pathogenicity Factors, Therapeutic Targets and for Basic Research[END_REF] . Most of this work focused on the secretome or virulence factors produced by the fungus, like cell wall degrading enzymes, considered as key elements in the different steps of the infection cycle of phytopathogenic fungi [START_REF] González-Fernández | Unraveling the in Vitro Secretome of the Phytopathogen Botrytis Cinerea to Understand the Interaction with Its Hosts[END_REF][START_REF] Shah | Comparative Proteomic Analysis of Botrytis Cinerea Secretome[END_REF] .

Due to the dynamic nature of the proteome, the addition of an antifungal compound should affect processes occurring at the surface and / or within the fungal cell [START_REF] Hou | Proteomic Analysis of Fusarium Graminearum Treated by the Fungicide JS399-19[END_REF] . In this work we compare the protein profiles of extracts of fresh mycelia treated with ETD151 vs untreated cultures. This proteomics study gives insight into the mechanism of action of this insect defensin on the fungus B. cinerea.

MATERIALS AND METHODS

Chemical reagents

A liquid minimum medium based on Tanaka's medium B [START_REF] Tanaka | Nutrition of Piricularia Oryzae in Vitro[END_REF][START_REF] Ou | [END_REF] and a Potato Dextrose Broth medium diluted 1:4 in water (25% PDB) were prepared using reagents from Sigma-Aldrich (St. Louis, MO, USA). Potato Dextrose Agar (PDA) was purchased from Oxoid (Basingstoke, UK). The reagents α-cyano-4-hydroxycinnamic acid (4-HCCA), ammonium bicarbonate (ABC), dithiothreitol (DTT), iodoacetamide (IAA), HPLC-grade dimethyl sulfoxide (DMSO) and LC-MS-grade formic acid (FA) were from Sigma-Aldrich. SYTOX Green Nucleic Acid Stain (5 mM in DMSO) was from Thermo Fisher Scientific (Waltham, MA, USA), RapiGest SF surfactant was from Waters (Milford, MA, USA) and sequencing-grade modified trypsin was from Promega (Madison, WI, USA). Acetonitrile (ACN), ethanol absolute anhydrous (EtOH), trifluoroacetic acid (TFA), all LC-MS grade quality or higher, were obtained from Carlo Erba Reagents (Val de Reuil, France). Protein Calibration Standard I (ProtMix) and Peptide Calibration Standard II (PepMix) were purchased from Bruker Daltonics (Germany). For all experiments, MilliQ water was used (Billerica, MA, USA).

Biological material

Spores of Botrytis cinerea isolated from the field and cryopreserved in 10% DMSO were provided by the La Dargoire Research Center (Bayer CropScience, France). All spores of B. cinerea used in this study were derived from the same initial batch. For each experiment, a cryotube was thawed and the stock solution of spores was diluted with sterile MilliQ water resulting in a final concentration of 1.5 x 10 5 spores per mL (sp/mL) in the presence of 0.1% DMSO. In order to have fresh fungal material, Falcon® culture petri dishes (Corning, NY, USA) containing PDA solid medium were inoculated with 150 µL of the appropriate spore suspension.

Culture dishes were left in the dark for seven days in an incubator (Memmert, Schwabach, Germany) maintained at 20°C and at 70% level of hygrometry. These parameters were defined as the culture conditions for all our experiments. To prepare a fresh spore solution, spores were harvested from one seven-day old culture dish using a Falcon® cell scraper (Corning), filtered on a Falcon® nylon 100 µm cell strainer (Corning), counted twice with a Malassez chamber and adjusted to a final concentration of 500 sp/mL in minimal medium.

Antifungal assay

The bioactive peptide ETD151, provided by Dr. Philippe Bulet, was produced by high cell density fermentation of a modified Saccharomyces cerevisiae strain developed by the former company EntoMed S.A. (Illkirch, France) and preserved as a stock solution in saline buffer. ETD 151 was desalted by solid phase extraction using Sep-Pak C 18 Classic Cartridges (Waters) following the manufacturer's instructions. Solutions from 250 to 0.49 µM of ETD151 were prepared in sterile MilliQ water. Antifungal assays on B. cinerea were performed in sterile 96well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland). To assess inhibition of mycelium growth, microplate wells were filled with 135 µL of the fresh suspension at 500 sp/mL prior to incubation in the conditions reported in the Biological material section. Addition of 15 µL of the ETD151 solutions, giving final concentrations of 25 to 0.049 µM, occurred three days later. The plate was gently shaken and incubated for four additional days. Evolution of the assay was monitored daily by absorbance measurement at 630 nm with an ELx808 microplate reader (BioTek, Winooski, VT, USA). The dose-response curve was generated with Microsoft Excel software and XLfit plug-in (release 5.4). Data were adjusted according to the "Dose-response one site -205" model to determine the half-maximal inhibitory concentration (IC50).

Fluorescence microscopy

A fresh spore solution at 1 x 10 4 sp/mL was prepared in a 25% PDB medium as reported in the Biological material section and 200 µL were distributed in several microplate wells. Spores were supplemented with SYTOX Green (final concentration of 30 µM) prior to shaking and incubation for 24 hours. Young mycelia of B. cinerea were treated by addition of ETD151 at final concentrations of 0.78 and 3.12 µM before a new incubation period of 24 hours. Morphological changes were monitored in transmitted light (TransLight50, 30 ms exposure) with an ImageXpress Micro XL fully automated inverted microscope (Molecular Devices, San Jose, CA, USA) equipped with a Plan Fluor ELWD 40x objective (Nikon). SYTOX Green fluorescence was observed with a Green Fluorescent Protein filter (excitation wavelength, 472 nm; emission wavelength, 520 nm; 100 ms exposure).

Sample preparation for proteomic studies

In a 96-well plate (TPP product), 135 µL of the fresh suspension at 500 sp/mL in minimum medium was distributed in six rows of 10 wells each. Mycelia were cultivated by maintaining the plate for three days in the growth conditions defined previously. Subsequently, 15 µL of sterile MilliQ water was added to the first row as an untreated control. The five other rows were treated with five different concentrations of ETD151, ranging from 31.2 µM to 1.95 µM. The experimental plate was maintained for 24 hours in the previously described growth conditions until protein extraction. Each microplate was monitored daily by measuring the absorbance at 630 nm with the ELx808 microplate reader (BioTek).

Protein extraction

For each plate, the fungal material (mycelium) from each of the 60 wells was collected by manual pipetting. For each experimental condition (untreated or treated with different concentrations of ETD151), 10 individual wells of a same row were pooled into a single 1.5 mL Eppendorf tube and maintained on ice until use. The fungal material was pelleted by centrifugation for 4 min at 13,000 rpm and 4°C. The fungal pellet was washed twice by up-anddown pipetting with 1 mL of sterile MilliQ water before adding 1 mL of 70% EtOH. The washed mycelia were stored overnight at -80°C. The fungal material was disrupted using a Mill-Mix 20 system (Domel, Železniki, Slovenia). The mycelia were ground in 2-mL tubes filled with 0.5 mm zirconium oxide beads for 10 min at 30 Hz. After centrifugation for 4 min at 13,000 rpm and 4°C, the pellets were collected and dried under CentriVap vaccum (Labconco, Kansas City, MO, USA). Finally, the proteins were extracted by incubating the dried fungal pellet in the presence of 2 M acetic acid, prepared in 50% ACN, for 4 h at 4°C under gentle shaking. After incubation, the extracts were centrifuged for 4 min at 13,000 rpm and 4°C and the protein supernatants were stored at 4°C until use.

Sample preparation for MALDI mass spectrometry analyses

For MALDI MS analyses, a fresh solution of matrix was prepared by adding, up to saturation, a few mg of 4-HCCA in 1 mL of Biotyper solution (Bruker Daltonics) composed of 50% ACN and 2.5% TFA. The samples to be analyzed (0.5 µL) were spotted and immediately covered with 0.5 µL of the matrix solution on an MTP 96 polished steel MALDI target (Bruker Daltonics).

Calibration of the instrument was performed using PepMix complemented with ProtMix. The sample spots were quickly dried under mild vacuum prior to analysis.

Peptide mass fingerprint of B. cinerea mycelium by MALDI-TOF mass spectrometry

Mass spectra from protein supernatants described in the Protein extraction section were acquired on an AutoFlex III Smartbeam instrument (Bruker Daltonics) using the FlexControl 3.4 software (Bruker Daltonics) in positive linear mode. The instrument was set up with the following parameters: 200 Hz laser at a 40% global attenuation offset, 20 kV source voltage 1, 18.5 kV source voltage 2, 9 kV lens voltage, 1.82 kV linear detector voltage, 140 ns of pulsed ion extraction delay and 600 Da detector gating. MALDI MS spectra were recorded at the mass range 600 Da to 18 kDa by summing two sets of 1,000 laser shots. Data were previewed using the FlexAnalysis 3.4 software (Bruker Daltonics) and reprocessed in mMass software [START_REF] Strohalm | MMass 3: A Cross-Platform Software Environment for Precise Analysis of Mass Spectrometric Data[END_REF] (version 5.5.0) Average spectra were generated from three selected biological replicates (from a pool of six), based on their fingerprint similarities and their intensities (maximum arbitrary intensity > 10,000). The average spectra were cropped from 1,500 to 16,000 m/z, baseline correction was performed with a relative offset of 100 and a precision of 40, along with a smoothing according to the Savitzky-Golay algorithm (window size 5.0 m/z in one cycle). The peaks were automatically picked using a signal-to-noise (S/N) ratio of 2.0, a picking height of 90 and an application of baseline. Finally, the average mass spectra were concomitantly and manually curated to select 100 representative ions.

Sample preparation for bottom-up proteomics

Proteins were digested using the modified procedure reported by Masson and colleagues [START_REF] Masson | Comparative Proteomics Studies of Insect Cuticle by Tandem Mass Spectrometry: Application of a Novel Proteomics Approach to the Pea Aphid Cuticular Proteins[END_REF] .

Briefly, the protein supernatants were dried under vacuum (Labconco), and then suspended with gentle shaking in an ammonium bicarbonate (ABC) buffer (50 mM, pH 7.5) supplemented with 0.1% RapiGest surfactant. The proteins were reduced with 280 mM dithiothreitol in ABC buffer for 30 min at 56°C in the dark prior to alkylation with 480 mM iodoacetamide in ABC buffer for 30 min at room temperature in the dark. Digestions were carried out for 1 h at 37°C by adding 0.5 µg of trypsin (0.1 µg/µL in ABC buffer). Following this initial digestion, 0.5 µg of trypsin was added before overnight incubation at 37°C. To stop proteolysis and cleave the RapiGest surfactant, samples were acidified with 8 µL of a 10% TFA and 20% ACN solution and incubated for 30 min at 37°C. Finally, samples were centrifuged for 10 min at 13,000 rpm at room temperature. The digests were transferred into injection vials (Agilent Technology, France) containing 5 µL of 2% ACN acidified with a solution of 0.1% TFA.

Proteomic analyses by nano-LC-ESI-MS/MS

Nano-LC-ESI-MS/MS instrumentation, software, chromatographic columns and other items were obtained from Thermo Fisher Scientific. Peptides were analyzed using an Ultimate 3000 nanoflow HPLC system coupled to a Q-Exactive Orbitrap high-resolution mass spectrometer, controlled by Xcalibur 2.2, Chromeleon Xpress 6.80 and Thermo QExactive Tune 2.5 software.

Nano-LC separations were performed according to Pisani and colleagues [START_REF] Pisani | Ex Vivo Assessment of Testicular Toxicity Induced by Carbendazim and Iprodione, Alone or in a Mixture[END_REF] . Briefly, the digests were loaded at a flow rate of 10 µL/min for 6 min onto a PepMap 100 C18 precolumn (300 µm x 5 mm, 5 µm) and separated at a flow rate of 300 nL/min onto an Acclaim PepMap 100 C 18 nanoviper column (75 µm × 250 mm, 3 µm) maintained at 35°C. Peptides were eluted using a multi-step linear gradient from 2% to 32% and from 32% to 65% ACN in 0.1% (v/v) formic acid for 94 min and 5 min, respectively. For MS analyses, the mass spectrometer was operating in positive mode and data-dependent acquisition. The instrument acquired a full-range MS scan from 380 to 2000 m/z (70,000 resolution, AGC target 3.10 6 , maximum IT 200 ms) and then fragmented the top ten peptide ions in each cycle (17,500 resolution, AGC target 2.10 5 , maximum IT 100 ms, intensity threshold 4.10 4 , excluding charge-unassigned ions, Normalized Collision Energy 27). Parent ions were then excluded from MS/MS for the next 15 s.

Databases searching and annotation

All MS and MS/MS spectral data were analyzed using Proteome Discoverer software (version 2.2.0.388, Thermo Fisher Scientific). Processing and consensus workflows were adapted from manufacturer's basic settings. The acquired MS/MS spectra were matched using the SEQUEST HT [START_REF] Eng | An Approach to Correlate Tandem Mass Spectral Data of Peptides with Amino Acid Sequences in a Protein Database[END_REF] search algorithm (University of Washington) against the EnsemblFungi [START_REF] Kersey | Ensembl Genomes 2018: An Integrated Omics Infrastructure for Non-Vertebrate Species[END_REF] Botrytis cinerea B05.10 protein sequence database (http://fungi.ensembl.org/Botrytis_cinerea).

The release 42 downloaded on February 16, 2019 contains 13,749 entries. The spectrum identification considered the following parameters: trypsin as proteolytic enzyme with a maximum of two missed cleavages, 6 and 144 amino acids as minimum and maximum peptide lengths, and a tolerance of 10 ppm/0.02 Da for precursor and fragment masses, respectively.

Cysteine carbamidomethylation was defined as a static modification. Methionine/tryptophan oxidation, serine/threonine/tyrosine phosphorylation and C-terminal amidation were defined as dynamic modifications. False discovery rates (FDRs), referred to as q-values, and posterior error probabilities (PEPs) for peptide spectral matches (PSMs) were calculated and filtered in the Percolator node using a decoy database [START_REF] Käll | Semi-Supervised Learning for Peptide Identification from Shotgun Proteomics Datasets[END_REF][START_REF] Käll | Non-Parametric Estimation of q-Values and Posterior Error Probabilities[END_REF] . Peptides and identified proteins were respectively validated based on high-confidence (FDR < 1%) and medium-confidence criteria (FDR < 5%).

Protein grouping was applied to regroup protein candidates covered by the same set or subset of identified peptides under a representative 'master' protein. Annotations were implemented through Proteome Discoverer using Gene Ontology (biological process, molecular function, cellular compartment), protein families from Pfam and biochemical pathway maps from the Kyoto Encyclopedia of Genes and Genomes (KEGG).

Protein quantification and functional analysis

Label-free quantification was performed using the Proteome Discoverer software.

Chromatographic alignment for LC/MS mapping features, created by the Minora Feature

Detector for each individual file, was achieved with a maximum retention time shift of 10 min and a mass tolerance of 10 ppm. Protein abundance was determined on intensity of precursor ions of unique and razor peptides.

Activity of complex I/ III and II/III of the respiratory transport chain

Mitochondrial preparation

Two liters of fresh spores of B. cinerea were cultured in glucose yeast mycological peptone medium and incubated in Erlenmeyer flasks with stirring for 72h at 22°C. Mycelia were filtered through a 200 µm nylon cloth NITEX (Sefar AG, Heiden, Switzerland), before washing with MilliQ ice water, spinning and weighing. The following steps were performed in a cold room (4°C) to preserve functionality of mitochondria. Mycelia were resuspended in a 0.5 M sucrose/1 mM EDTA/0.3% BSA/20 mM MOPS wash buffer adjusted to pH 7.4. After addition of 5 mM cysteine, the mixture was ground with 1 mm glass beads for three cycles of 30 s with 1 min pauses to cool down the suspension. Fungal material was filtered on a 100 µm nylon cloth (Sefar) and beads were rinsed with the washing buffer, prior to centrifugation of the filtrate for 10 min at 1,500 g. The supernatant was filtered on a 150 µm nylon cloth (Sefar) prior to centrifugation for 20 min at 15,000 g. The mitochondrial pellet was resuspended in the wash buffer and fractionated in 2-mL aliquots prior to purification on a 1.8 M/1.2 M sucrose gradient, involving centrifugation for 45 min at 36,000 g followed by slow deceleration. Purified mitochondrial fractions at the sucrose bilayer interface were diluted in the wash buffer and centrifuged for 15 min at 36,000 g. Pellets were combined prior to a new washing step to obtain a single purified fraction of mitochondria.

Measurement of inhibition of complex I/III and II/III by colorimetry

The activity of a molecule as a possible electron transport chain complex inhibitor was measured by a potassium ferricyanide-based colorimetric test [START_REF] Estabrook | Studies of Oxidative Phosphorylation with Potassium Ferricyanide as Electron Acceptor[END_REF] in the presence of α-ketoglutarate and succinate, substrates of complexes I and II, respectively. Viability of fresh mitochondria was evaluated in 96-well microplates (Greiner Bio One). For complex I, 3 μL of the purified fraction was diluted in 400 µL of Mix I solution (Supplementary data S1) before depositing 88 μL of this mixture into 4 control wells and adding the 0.05 M α-ketoglutarate substrate. After a 3-min incubation at room temperature, the absorbance was measured for 5 min at 405 nm (SpectraMax Plus 384, Molecular Devices). Data was converted to a slope using a linear regression model.

Complex II activity was evaluated in the same way using Mix II (Supplementary data S1) and the appropriate substrate. For this assay, the ETD151 peptide diluted in sterile Milli-Q water was compared to two standard inhibitors diluted in DMSO: antimycin A and boscalid. From a dilution range (factor of 3.5) prepared upstream, 2 µL of the molecule to test (50X) was deposited in a well containing 88 µL of fresh mitochondria in Mix I or Mix II, for complex I or complex II, respectively. Immediately for complex I or after 7 min of incubation for complex II, 10 μL of 0.05 M substrate were added before incubation for 3 min at room temperature. After a brief stirring, the mitochondrial activity was measured as previously described.

RESULTS AND DISCUSSION

Fungal growth inhibition and morphological changes in Botrytis cinerea

Several antifungal defensins from insects, like heliomicin [START_REF] Banzet | Expression of Insect Cystein-Rich Antifungal Peptides in Transgenic Tobacco Enhances Resistance to a Fungal Disease[END_REF] and drosomycin [START_REF] Fehlbaum | Insect Immunity: Septic Injury of Drosophila Induces the Synthesis of a Potent Antifungal Peptide with Sequence Homology to Plant Antifungal Peptides[END_REF] , or from plants, such as RsAFP2 [START_REF] Terras | Analysis of Two Novel Classes of Plant Antifungal Proteins from Radish (Raphanus Sativus L.) Seeds[END_REF] and VvAMP2 [START_REF] Nanni | VvAMP2, a Grapevine Flower-Specific Defensin Capable of Inhibiting Botrytis Cinerea Growth: Insights into Its Mode of Action[END_REF] , have been reported to be active against Botrytis cinerea at a micromolar range. We have previously shown that ETD151 exhibits a broad-spectrum of activities against several yeasts and filamentous fungi [START_REF] Landon | Lead Optimization of Antifungal Peptides with 3D NMR Structures Analysis[END_REF] at the micromolar range. However, ETD151 had not been tested on B. cinerea. In this study, the antifungal activity of ETD151 was assessed in vitro against three-day-old mycelia of B. cinerea prepared from a fresh spore solution. In our experimental conditions, growth inhibition reaches a maximum, 78% as measured by optical density, at 6.25 µM of ETD151 (Figure 1A). Based on this value, the halfmaximal inhibitory concentration (IC50) was estimated to be 0.59 µM.

The antifungal action of ETD151was monitored by optical microscopy (Figure 1B). At 0.78 and 3.12 µM, ETD151 is able to promote lysis and the release of intracellular content at some points of the growing mycelium, preferentially at the apical end of the hyphae (Fig. 1B, b-c).

Such a leakage of cytosolic material suggests a loss of the cell wall integrity. An equivalent observation has already been reported for heliomicin and drosomycin on B. cinerea spores, but for these molecules, comparatively weaker effectiveness against the mycelium was recorded [START_REF] Banzet | Expression of Insect Cystein-Rich Antifungal Peptides in Transgenic Tobacco Enhances Resistance to a Fungal Disease[END_REF] . Thus, our data confirm the enhanced activity of ETD151 compared to heliomicin, the reference molecule.

The permeabilization of the fungal membrane has emerged as a trend in the antifungal mode of action of plant defensins [START_REF] Cools | Antifungal Plant Defensins: Increased Insight in Their Mode of Action as a Basis for Their Use to Combat Fungal Infections[END_REF][START_REF] Thevissen | Permeabilization of Fungal Membranes by Plant Defensins Inhibits Fungal Growth[END_REF] . The results obtained on B. cinerea mycelia treated with the insect defensin homologue ETD151 suggest a similar mode of action. Nevertheless, it remains unclear if ETD151 is internalized or if it remains outside the fungal cells. To assess whether ETD151 is able to disturb membrane integrity/permeability, the SYTOX Green nucleic acid stain, a cellimpermeant fluorophore, was used. Compared to the control experiment (Fig. 1B,g), in the presence of ETD151, the dye penetrated locally the hyphae and emitted a green-fluorescent signal revealing a compromised plasma membrane (Fig. 1B, h-i).

In addition, ETD151 reduced hyphal elongation with a concomitant disorder in hyphal branching at both tested concentrations (Fig. 1B, e and f, respectively 0.78 and 3.12 µM).

Development of the B. cinerea mycelium post-treatment highlights the fungistatic aspect of ETD151 activity and may explain the inhibition plateau observed with the fungal growth curve (Fig. 1A). Swelling of hyphae was also noticed with a particularly strong phenotype at 3.12µM (Fig. 1B,f). These morphological changes defined ETD151 as a morphogenic defensin of B. cinerea. This is homologous to observations reported for several plant defensins (RsAFP2, HsAFP1, MsDef1) in filamentous fungi [START_REF] Osborn | Isolation and Characterisation of Plant Defensins from Seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae[END_REF][START_REF] Broekaert | Plant Defensins: Novel Antimicrobial Peptides as Components of the Host Defense System[END_REF][START_REF] Spelbrink | Differential Antifungal and Calcium Channel-Blocking Activity among Structurally Related Plant Defensins[END_REF] and confirms commonalities with the antifungal action of the insect defensins. Results obtained after this first series of analyses showed that there are perturbations of the mycelium physiology and morphology, as the optical microscopy pictures and the uptake of the SYTOX Green nucleic acid stain differed between all treatment conditions.

Thevissen and colleagues [START_REF] Thevissen | Defensins from Insects and Plants Interact with Fungal Glucosylceramides[END_REF] demonstrated through an ELISA-based assay with Pichia pastoris that heliomicin and RsAFP2 interact with fungal membrane lipids called glucosylceramides. All of our structural and biological data support a common mechanism of action for these three defensins, and lead us to suggest that ETD151 interacts with the same fungal partners.

To study the antifungal effects of ETD151 on the intracellular protein content, we used a proteomics study based on a two-step MS workflow, and conducted on B. cinerea mycelial extracts. Firstly, a comparison of mass spectrometry (MS) molecular mass fingerprints was conducted by MALDI-MS. This first step was designed to follow the evolution of the peptide protein profiles within the mass range of 600 Da to 18 kDa. Secondly, a bottom-up proteomics analysis by nano-LC-ESI-MS/MS was performed to identify and characterize the proteins changes. The concentration range from 0.19 to 3.12 µM, which includes the IC50 and covers the entire inhibition profile of ETD151, was adopted for a dose-response approach.

Protocol development to prepare B. cinerea mycelial extracts

Fungal culture, extraction and MALDI-MS analysis protocols have been developed for this study. The extraction protocol is inspired by the work of Schulthess and colleagues [START_REF] Schulthess | Use of the Bruker MALDI Biotyper for Identification of Molds in the Clinical Mycology Laboratory[END_REF] , using 35% formic acid for the BioTyping of clinical molds. The objective was to establish the optimal parameters for recording the most representative MALDI peptide signatures/molecular mass fingerprints (MFPs) of B. cinerea, with or without different concentrations of ETD151. The following parameters were reviewed: the starting concentration of B. cinerea spores, the mycelial culture duration in microplate, the treatment duration with ETD151 and the extraction process to generate mycelial extracts. The development of the protocols was focused on the conditions providing the most reproductible MFPs and with a rich molecular diversity. We settled on a starting concentration of 500 sp/mL prior to three days of growth in microplate and a 24htreatment of the mycelia with ETD151. Peptides/proteins were extracted by grounding the mycelia with zirconium oxide beads, drying the pellet and incubating it in the presence of 2 M acetic acid solution prepared in 50% ACN (personal communication, Victor Masson and Philippe Bulet). The different parameters for MALDI-MS analysis were optimized for an optimal detection (in terms of sensitivity, resolution, reproducibility and accuracy) of molecular ions between 1,500 -12,000 mass m/z of the B. cinerea mycelial extracts.

Peptide mass fingerprint of B. cinerea mycelium in response to ETD151

Once the experimental methodology had been established, MALDI-MS analyses were performed directly on B. cinerea mycelial peptide/protein extracts, and restricted to peptides and small proteins (<20 kDa) as a preliminary approach to assess the antifungal effect of ETD151 at the molecular scale. Three-day-old mycelia were incubated for 24 h with the defensin following the previous concentration range, from 0.19 to 3.12 µM final concentrations. Average spectra (n=3) were generated from biological replicates to first, highlight modifications of the peptide/protein composition in the presence of ETD151 and second, visualize a putative doseresponse effect (Figure 2). For each concentration, a manual selection from automatic peak labeling resulted in a representative peak list of 100 ions. Similarities between MALDI MFPs were calculated following the conservation of representative ions (Supplementary data S2).

In the presence of 0.19 µM of ETD151, no strict molecular change was observed on the MFPs.

Compared to the untreated mycelia, MFPs were very similar with 91% identity (Fig. 2A). This observation is consistent with the very reduced growth inhibition effect (less than 10%) observed at this dose (Fig. 1A). From 0.39 µM of ETD151, the antifungal activity of ETD151 has a significant impact on the MFPs recorded. Several modifications were highlighted on the MFPs compared to untreated mycelia, with identity dropping to 68% (Fig. 2B). Differences in the peptide/protein profiles are significant in particular within the 6,000-9,000-mass range (Supplementary data S3). At 0.39 µM, two sets of peptides/proteins within the 9,000-10,000 and 14,000-17,000 mass ranges emerged; those were not detectable in the control MFP or at an ETD151 concentration of 0.19 µM. At 0.39 µM or higher doses of ETD151, the MALDI-MS spectra showed similar overall MFPs (Fig. 2C). Indeed, the dose of 0.39 µM shares 92%, 84% and 77% identity with the 0.78, 1.56 and 3.12 µM doses, respectively. Several ions within the 9,000-10,000 and 14,000-17,000 mass ranges display intensity variation according to the concentration of the defensin with intensities increasing as the concentration reaches 0.39 µM (Supplementary data S2). These results suggest that MALDI-MS could be a rapid and simple method to assess the impact of an antifungal molecule on its fungal target. The modifications of the MFPs observed between the proteins extracted from the control experiment versus B. cinerea mycelia treated with different doses of ETD151 also revealed that ETD151 modified the peptide/protein profiles of B. cinerea mycelia, in a dose dependent manner up to a concentration that is more than the IC50 (0.59 µM).

Despite the extraction method used, including removal of the culture supernatant and two successive washing steps of the mycelia, the ion observed at m/z 4,839 corresponds to ETD151.

It is worth mentioning that the intensity of this ion does not decrease with two additional washes (unpublished data). This suggests that in our experimental conditions, ETD151 was interacting with the membrane of the altered mycelium of B. cinerea, or was entering the mycelium. Note that the arbitrary intensity (a.i.) of the ETD151 ion increases with the concentration of ETD151 used, from 1.9.10 3 a.i. at 0.19 µM to 174.103 a.i. at 3.12 µM (Supplementary data S2).

As important modifications of the MFPs are observed within the highest molecular mass range detectable in our MALDI-MS conditions, this preliminary approach attests to the antifungal effect of ETD151 on B. cinerea and prompted us to analyze the mycelial extract of B. cinerea treated with ETD151 at the peptide/protein scale using a bottom-up proteomics approach.

Figure 2: MALDI profiling of extracts of B. cinerea mycelia treated with ETD151 456

The MALDI mass spectra show the average MALDI-molecular mass fingerprint (n=3) from mycelial protein extracts treated or not with the antifungal peptide ETD151. The mass spectra are recorded in the 1,500 -12,000 mass (m/z) range, for untreated mycelia (Control) versus mycelia treated with 0.19 µM ETD151 (A) and 0.39 µM ETD151 (B). Zooms show enlarged area at the 4,000-6,000 and 7,000-9,000 mass ranges. The mass spectra recorded when the mycelial cultures were treated with 0.39, 0.78, 1.56 and 3.12 µM ETD151 are also reported (C).

The zoom shows the enlarged mass range (m/z) between 6,000 and 9,000.

Effect of ETD151 on B. cinerea mycelium by bottom-up proteomics

Differential proteomics analysis in response to ETD151

Our MALDI-MS results showed that the MFPs of the mycelial preparations are altered in response to the different concentrations of ETD151 used to treat B. cinerea mycelia. To have a better picture of the molecular response of B. cinerea mycelium upon our ETD151 treatment, we used gel-and detergent-free proteomics to monitor protein changes on mycelial extracts. B. cinerea mycelial peptide/protein acidic extracts prepared for MALDI-MS were dried under vacuum prior to reduction-alkylation-enzymatic digestion. B. cinerea protein digests were then analyzed by LC-ESI-MS/MS analysis as a conventional bottom-up proteomics approach. To evaluate the methodology implemented and validate our data analysis strategy, two sets of experiments were performed (Figure 3) allowing to generate two datasets (I and II). First, three individual cultures were harvested, following the same experimental procedure, from the collection of non-treated or treated cultures with different doses of ETD151 (Fig. 3A,dataset I).

Second, a triplicate experiment was conducted on three individual plates with three-day-old mycelia (Fig. 3B, dataset II) originating from a unique culture of spores. Dataset I allowed the identification of 1,431 typical proteins with molecular masses ranging from 4.8 to 466 kDa. Among these proteins, 1,430 originate from B. cinerea while the last one corresponds to ETD151, detected by LC-ESI-MS/MS with 80% coverage (see raw data). We observed an increase in the number of identified proteins in response to increasing concentrations of ETD151 (Figure 4A, diamond plots). The average number (Fig. 4A, black diamonds) of identified proteins was 532 and 534 for control and 0.19 µM ETD151, respectively. This average number increased to 794 and 899 proteins when the cultures were treated with 0.39 and 3.12 µM ETD151, respectively. The increase in the number of proteins extracted in response to elevated concentrations of ETD151, may reflect a perturbation of the integrity of the cell membrane of the mycelium and/or the stimulation of the expressions of series of genes, and would corroborate microscopic data (see Fig. 1B) that points to an antifungal effect of the defensin on B. cinerea. The reproducibility within dataset I was assessed for each concentration of ETD151 as the average ratio between the number of identified proteins in each experiment and the number of recurrent proteins (Fig. 4A, histograms), as identified in all three experiments. The reproducibility within the control experiment and after a treatment with 0.19 µM ETD151was at 68 ± 11 % and 67 ± 8 % , respectively. For 0.39 µM ETD151, it was 73 ± 6 %, and for higher concentrations of ETD151, the reproducibility was 75 % with a standard deviation ≤ 6 % (Fig. 4A, square plots). Furthermore, a principal component analysis (PCA) was run on proteins of dataset I and showed a clustering of experiments according to the concentration of ETD151 (Figure 4B). Control and 0.19 µM samples were clearly separated from the other tested concentrations by the first component (42.8%). The first component (PC1, Fig. 4B) may reflect the molecular impact of ETD151 on the B. cinerea mycelium. Samples from 0.39 to 3.12 µM of ETD151 could not be separated according to the second component (13.4%). Dataset II, a triplicate of three individual plates, identified 1,468 master proteins from a 4.8 to 466 kDa mass range. The goal of this experimental design was to estimate the impact of the initial culture of spores or of the harvest method on the reproducibility of the data. It should be noted that working simultaneously on three individual microplates significantly complexified the experimental protocol. Treatment of mycelia with ETD151, as well as the manual collection of samples from each microplate, should be completed within a very short period of time. As reported previously for dataset I, the average number of identified proteins increased upon treatment with ETD151 (Figure 4C). As with dataset I, we observed an increase in the number of identified proteins in response to increasing concentrations of ETD151 (Fig. 4C, diamond plots).

The average numbers (Fig. 4C, black diamonds) of identified proteins were 745 and 778 for the control and 0.19 µM ETD151 samples, respectively. This average number increased to 910 and 917 proteins when the cultures were treated with 0.39 and 3.12 µM ETD151 (Fig. 4C, diamond plots). The reproducibility was assessed in the same way as for dataset I (Fig. 4C,histograms). In the control and 0.19 µM samples, the reproducibility was 80 ± 2 % and 75 ± 7 %, respectively.

For higher concentrations of ETD151, the reproducibility ranged from 83 to 84 % with a standard deviation ≤ 3 %. PCA was also run on dataset II (Figure 4D). As observed for dataset I, the first component (53.1 %) separated Control and 0.19 µM samples from the other concentrations. Unlike the PCA on dataset I, the second component (12.6 %) allowed to separate the different samples according to the concentrations of ETD151. This new experimental design improved the robustness of the data as the average reproducibility between dataset I and II increased by 9% while the variability between replicates decreased, as reflected by the reduction in standard deviations. This explains the greater weight in our PCA analyses of the first component, at 43% and 53% in datasets I and II, respectively. Interestingly, in terms of the average number of identified or recurrent proteins, the samples treated with 0.19 µM ETD151 showed values closer to the control experiments (see above), which is supported by the clustering observed for control and 0.19 µM ETD151 replicates on one side within the first component in PCA whereas replicates for higher concentrations aggregated on the other side. Although the results improved in dataset II, samples for 0.19 µM ETD151 were the only ones with a reproducibility below 80% and a standard deviation > 5%. This concentration of ETD151 appeared as an inflection point in the molecular effect of the antifungal peptide as the changes induced by ETD151 barely allow to distinguish control from 0.19 µM samples. Finally, datasets I and II were grouped to consolidate data analysis. Indeed, both dataset shared similar properties on the increase in identified and recurring proteins for identical concentrations of ETD151, from 0.39 to 3.12 µM.

The protein distributions were represented on a multidimensional Venn diagram based on sixexperimental datasets (untreated vs treated with five increasing concentrations of ETD151) (Figure 5). The Venn diagram is a simplified tool, free of statistical test, allowing to classify proteins from large datasets and to highlight groups of interest in our study. The diagram we obtained, allowed to represent the logical relations between the 1,592 summed proteins identified in the two datasets I and II. Among these proteins, 42 were specifically found in the non-treated samples and 21 to 26 of them specifically in each concentration of ETD151 (21 in 0.19 µM, 22 in 0.39 µM, 24 in 0.78 µM and 26 in 1.56 µM). We found the most abundant number of specific proteins (44) when the mycelial culture was treated at 3.12 µM, the highest concentration of ETD151. Interestingly, as previously discussed, at the inflexion point, 82 proteins were found mutually in the control experiments and in samples treated with 0.19 µM ETD151. Overall, 676 proteins were observed to be common to all samples, 120 proteins were found in all treated samples, regardless of the concentration of ETD151, and 185 proteins were specifically found at the concentrations of 0.39 µM ETD151 and above.

Our results demonstrated that ETD151 affected the molecular composition of B. cinerea mycelial samples and confirmed, over a wide mass range (4.8 -466 kDa), the results presented previously by MALDI-MS MFPs up to 18 kDa. This comparative approach allows us to build an overview of the behavior of protein groups following treatment with the peptide and to select by label-free quantification characteristic proteins and metabolic pathways altered upon treatment with ETD151.

Gene ontology and network analysis

Proteins were annotated using ProteinCenter (Proteome Discoverer) to investigate molecular functions, biological processes, and cellular components from the Gene Ontology (GO) database (Figure 7). The molecular function-based categorization showed that among the 340 selected proteins, 168 (42%) could not be classified while the others were classified according to the GO terms as follow: 103 (26%) have catalytic activities (GO:0003824), 92 (23%) have binding properties (GO:0005488), 27 (7%) contribute to the structural integrity of a complex or its assembly within or outside a cell (GO:0005198), 7 (2%) act as transporters (e.g. lipids, proteins, oxygen) (GO:0005215), 4 (1%) are regulators of molecular functions (GO:0098772), and finally 2 (0.5%) were recognized as having antioxidant activity (GO:0016209) (Fig. 7A). Following the biological process-based categorization, 178 (49%) among the 340 proteins could not be classified. The most affected process is metabolism with 125 proteins (34%) (GO:0008152). The rest of the proteins are involved in biological processes such as (i) localization (21, 6%) (GO:0051179), (ii) regulation (15, 4%) (GO:0065007), (iii) cellular component organization (14, 4%) (GO:0071840), (iv) response to a stimulus (7, 2%) (GO:0050896), or (v) cellular process (2, 1%) (GO:0009987) (Fig. 7B). Cellular component annotation was also investigated despite a lack of information for B. cinerea in this GO categorization. Among the 340 selected proteins, 288 (84%) could not be classified (data not shown).

Molecular impact of ETD151 on B. cinerea pathways

Significantly modulated proteins upon treatment with ETD151 were searched in Botrytis cinerea KEGG pathways (117 pathways reported). From the 339 proteins (340 minus ETD151) previously selected, 106 were successfully mapped over 67 different pathways with a varied number of hits, from 1 to 38 matched proteins per map (Supplementary data S5). Pathways with less than five hits were not considered for further investigations, as a low number of matched proteins did not allow us to interpret their relevance in the mechanism of action of ETD151 on B. cinerea. Moreover, most of these maps were included in carbohydrate, lipid or amino acid metabolisms. Direct effects on these pathways had been evaluated by measuring the uptake and incorporation of radiolabeled precursors ( 14 C-glucose, 3 H-phenylalaline and 14 C-acetate) by Botrytis cinerea spores in the presence of ETD151 (data not shown). ETD151 did not interfere with carbohydrate, protein and lipid biosynthesis confirming that modulated proteins in previously mentioned pathways resulted from global stress in response to an antifungal molecule. The following global pathway maps (metabolic pathways (bfu01100), biosynthesis of secondary metabolites (bfu1110), biosynthesis of antibiotics (bfu01130) and carbon metabolism (bfu01200) were also excluded from the analysis in favor of the study of specific maps. By considering the filters previously described, six pathways were impacted by the treatment, namely ribosome (bfu03010), spliceosome (bfu03040), protein processing in endoplasmic reticulum (bfu04141), endocytosis (bfu04144), MAPK signaling pathway (bfu04011), and oxidative phosphorylation (bfu00190). and Golgi apparatus [START_REF] Funato | Vesicular and Nonvesicular Transport of Ceramide from ER to the Golgi Apparatus in Yeast[END_REF] . Further investigation will be necessary to monitor a direct effect of ETD151 on glucosylceramide biosynthesis and/or export.

Effect on endocytosis

Endocytosis is a cellular process in which substances (e.g. nutrients, plasma membrane proteins, lipids) are brought into the cell. Interestingly, research on antifungal plant defensins demonstrated that certain ones, such as MtDef4, Psd1 and NaD1, can be internalized by fungal cells upon interaction with a membrane partner [START_REF] Cools | Antifungal Plant Defensins: Increased Insight in Their Mode of Action as a Basis for Their Use to Combat Fungal Infections[END_REF] . Mechanisms of fungal cell entry are still under investigation, however it is known that MtDef4 and NaD1 internalization are energy dependent and require endocytosis in Neurospora crassa [START_REF] El-Mounadi | Antifungal Mechanisms of a Plant Defensin MtDef4 Are Not Conserved between the Ascomycete Fungi Neurospora Crassa and Fusarium Graminearum[END_REF] and Candida albicans [START_REF] Hayes | The Plant Defensin NaD1 Enters the Cytoplasm of Candida Albicans via Endocytosis[END_REF] , respectively. In our study, 35 proteins, out of a total of 58 reported in this pathway for B. cinerea, were identified (Supplementary data S6). Upon treatment with ETD151, 7 proteins were significantly dysregulated. Three of them were upregulated in the presence of ETD151: Bcin13p05610/CAPZA capping protein (actin filament) muscle Z-line starting at a concentration of 0.19 µM, Bcin11p01640/VPS26 and Bcin11p01920/VPS35 vacuolar protein sortingassociated protein at a concentration of 0.39 µM. In contrast, four were downregulated: Bcin01p06150/Arp2/3 actin related protein 2/3 complex subunit, at a concentration of 0.19 µM only, Bcin03p0497/Epsin membrane protein and Bcin13p04030/ArfGAP stromal membraneassociated protein starting at the concentration of 0.39 µM, and Bcin13p00990/Hrs hepatocyte growth factor-regulated tyrosine kinase substrate at the concentrations of 0.78 and 1.56 µM. These results could be valuable for future research on the mechanism of action of ETD151, and specifically the behavior of the antifungal defensin upon its interaction with the fungal membrane.

Effect on MAPK signaling pathway

Fungi, like other eukaryotes, rely on the rapid transduction of signals through the mitogenactivated protein kinase (MAPK) pathway in order to overcome environmental stresses such as the addition of chemical fungicides and antifungal peptides. At lower concentrations, one or more stress response pathways are activated to respond to the cell damage. At higher concentrations, the fungal stress response leads to the inhibition of cell growth or induction of cell death. Several antifungal plant defensins [START_REF] Parisi | The Evolution, Function and Mechanisms of Action for Plant Defensins[END_REF] (e.g. RsAFP2, Psd1, MsDef1) are known to activate the MAPK pathway, with the cell wall integrity pathway (CWI), inducing changes to cell wall biogenesis, β-glucan synthesis and the organization of the actin cytoskeleton. The Defensin NaD1 activates the high-osmolarity glycerol (HOG) pathway [START_REF] Hayes | The Plant Defensin NaD1 Enters the Cytoplasm of Candida Albicans via Endocytosis[END_REF] , leading to an increase in intracellular glycerol which provides protection against osmotic stress. In our study, 23 proteins, out of a total of 56 reported in the HOG pathway (bfu04011) for B. cinerea, were identified in datasets I and II (Supplementary data S6). Upon treatment with ETD151, three proteins were dysregulated in the CWI pathway: Bcin08p03780/Rom1,2 (down at 3.12 µM), Bcin02p06930/Fks2 (up starting from 0.19 µM) and Bcin16p00490 (up starting from 0.19 µM); one protein was dysregulated in the HOG pathway: Bcin16p04710 (down starting from 0.39 µM). The protein Bcin16p00490/Cdc28, in both CWI and HOG pathways, was also upregulated starting from 0.19 µM.

Effect on oxidative phosphorylation

Oxidative phosphorylation, coupling the cascade of oxidation-reduction of the mitochondrial respiratory chain and the phosphorylation of ADP to ATP, is an essential metabolic pathway for the survival of most aerobic eukaryotic organisms. In our study, 60 out of a total of 72 reported proteins in the B. cinerea bfu00190 pathway were identified (Figure 8A). Upon treatment with ETD151, two subunits of NADH dehydrogenase (complex I) Bcin08p06780/NDUFV1, Bcin08p01490/NDUFA8 were significantly up-regulated starting from a concentration of 0.78 µM of ETD151. Similarly, a subunit of succinate dehydrogenase (complex II) Bcin05p04430 was also up-regulated at 0.19 µM of ETD151. By contrast, a subunit of ubiquinol-cytochrome c reductase (complex III) Bcin03p08370/QCR9 was significantly downregulated at 0.19 µM of ETD151. Finally, several subunits of F-type H+-transporting ATPase (complex V) were significantly modulated upon treatment: Bcin02p02510/epsilon (down at 0.19 µM), Bcin10p01500/c (up starting at 0.39 µM), Bcin02p02250/e (down at 3.12 µM) and Bcin12p02610/k (down starting at 1.56 µM). Our proteomics data suggested a mitochondrial dysfunction in the presence of the antifungal defensin ETD151. To go further, the direct activity of ETD151 on complexes I to III of the mitochondrial respiratory chain was assessed and compared to boscalid and antimycin, two molecules providing reference profiles as inhibitors of succinate dehydrogenase and ubiquinol-cytochrome c reductase, respectively (Figure 8B). A colorimetric assay based on the reduction of ferricyanide allowed to measure combined activities of complexes I and III on the one hand, and complexes II and III on the other hand. The activity of boscalid demonstrated a partial inhibition (50%), when testing complexes I and III together, and a total inhibition, when testing complexes II and III together. The activity of antimycin A was characterized by a total inhibition in both associations of complexes. In the presence of ETD151, no inhibition was measured regardless of the tested complex, using up to 50 µM of the antifungal defensin. This result confirmed that ETD151 does not interact directly with complexes of the mitochondrial respiratory chain. Mitochondrial dysfunction highlighted by our proteomics data may result from mitochondrial membrane perturbation, reflecting a secondary effect of 770 ETD151 disturbing cell membrane integrity. 771

Conclusions & Perspectives

Through this study, we demonstrated that the proposed approach offers an experimental workflow to evaluate changes in the peptide and protein contents of mycelial extracts of B. cinerea in response to different concentrations of the antifungal peptide ETD151. As an initial approach, molecular mass fingerprinting by MALDI-MS allowed to highlight molecular changes in B. cinerea mycelia treated with ETD151. Even with spectra obtained on a partial mass range (<20 kDa), it could be interesting to compare average fingerprints in the presence of ETD151 to spectra generated with other antifungal defensins. This comparison could direct the study on the mechanism of action of ETD151 toward other peptides. As a second step, bottom-up analyses of mycelial extracts provided robust and reproductible data, necessary to study in-depth molecular changes induced by ETD151 on B. cinerea. Thus, we revealed six pathways (ribosome (bfu03010), spliceosome (bfu03040), protein processing in endoplasmic reticulum (bfu04141), endocytosis (bfu04144), MAPK signaling pathway (bfu04011), and oxidative phosphorylation (bfu00190)) impacted by the insect defensin. At least for one of these pathways, we also demonstrated that the observed impact must be indirect. Indeed, we clearly demonstrated that there was no direct effect of ETD151 on the B. cinerea mitochondrial respiration chain. As a conclusion, proteomics could offer a way to improve the research strategy into the resolution of antifungal mechanisms of action by targeting preferentially involved proteins or metabolic pathways. Finally, an additional objective for a deeper understanding of the mechanisms of action of ETD151 is to study the interaction of ETD151 with lipid components of the fungal membrane, such as e.g. glucosylceramides.

Figure 1 :

 1 Figure 1: Growth inhibition and morphogenic alterations induced by ETD151 in B. cinerea

Figure 3 :

 3 Figure 3: Workflow developed for the bottom-up proteomics approach on B. cinerea

Figure 4 :

 4 Figure 4: Protein identification of B. cinerea mycelial extracts and assessment of

Figure 5 :

 5 Figure 5: Multidimensional six-set Venn diagram

Figure 6 :

 6 Figure 6: Significantly dysregulated proteins upon treatment of B. cinerea mycelium with

Figure 7 :

 7 Figure 7: Gene Ontology of dysregulated proteins in B. cinerea mycelial extracts

Figure 8 :

 8 Figure 8: Effect of ETD151 on the B. cinerea mitochondrial chain respiration complex

Acknowledgments & Funding Sources

This work was supported by the Convention Industrielle de Formation par la Recherche CIFRE n°2015/1111 -through the research contract between Bayer SAS, the French National Centre for Scientific Research (CNRS, CBM UPR 4301 and IAB UMR 5309, France) and Research and Development from the Association Plateforme BioPark of Archamps (France). We thank our colleagues: Dr Karim Arafah and Victor Masson, in the MassOmics group of the Association Plateforme BioPark of Archamps, for providing knowledge in LC-ESI-MS/MS and data processing, and in the preparation and processing of mycelial extracts, respectively. We also thank Stephane Peyrard and Catherine Sirven from Computational Life Science group in the La Dargoire Research Center (Bayer SAS, Lyon), for providing their expertise in protein annotation and data processing, respectively.

S6

). Upon treatment with ETD151, 7 proteins were significantly dysregulated. Bcin08p03160/Smd2 and Bcin08p06600/Smd3, small nuclear ribonucleoproteins, and Bcin09p07050/Lsm7, an snRNA-associated Sm-like protein, were upregulated at 0.39 µM of ETD151 and higher concentrations. In contrast, four proteins were downregulated in the presence ofETD151: Bcin09p06610/PPIL1 peptidyl-prolyl cis-trans isomerase-like from 0.19 µM, Bcin08p00660/Prp1 pre-mRNA-processing factor from 1.56 µM, Bcin09p06940/SF3b splicing factor and Bcin13p05240/THOC THO complex subunit at 3.12 µM. There is no clear tendency to interpret the molecular changes induced by ETD151 on this pathway. Moreover, as there is no report in the antifungal defensin literature of a direct effect on the spliceosome, this pathway was not considered for the continuation of the work on the mechanism of action of ETD151.

Effect on protein processing in the endoplasmic reticulum (ER)

The ER is a continuous membrane system found within the cytoplasm of eukaryotic cells allowing for protein synthesis, folding, modification and in some cases transport to the Golgi apparatus through vesicles. In our study, 37 proteins, out of a total of 70 reported in this pathway for B. cinerea, were identified (Supplementary data S6). Upon treatment with ETD151, seven proteins were significantly dysregulated. Bcin13p00700/ERGIC53 and Bcin05p05180/OS9 are upregulated starting a concentration of 0.39 µM. Bcin07p01430/Sec62, Bcin02p05070/Sec24, Bcin01p05490/Bcnpl4 and Bcin10p00510 are downregulated starting at 0.39 µM of ETD151.

These proteins play roles in the transport of proteins from the ER. Proteins that are located in the ER and/or Golgi apparatus, are interesting to highlight, as sphingolipids, including glucosylceramides, fungal partners of RsAFP2 61 and possibly ETD151, are synthesized in the ER

Supporting Information

The following files are available free of charge.

Conflicts of Interest:

The authors declare no conflicts of interest.

Notes

All relevant data are within the paper and its Supporting Information files.

For Table of Contents Only


Dr Anna Pellat

Thomas Walter

Jeremy Augustin

Vincent Hautefeuille

Olivia Hentic

Christine Do Cao

Astrid Lièvre

Romain Coriat

Pascal Hammel

Olivier Dubreuil

Augustin Jérémy

Astrid Lievre

Romain Cohen

Anne Couvelard

Thierry André

Magali Svrcek

Eric Baudin

Pauline Afchain

Chemotherapy in resected neuroendocrine carcinomas of the digestive

Keywords: chemotherapy, neuroendocrine carcinomas, ki-67 index

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

Neuroendocrine neoplasms (NEN) of the digestive tract are rare tumours with a rising incidence due to their better identification [START_REF] Yao | One hundred years after "carcinoid": epidemiology of and prognostic factors for neuroendocrine tumors in 35,825 cases in the United States[END_REF][START_REF] Korse | Incidence and survival of neuroendocrine tumours in the Netherlands according to histological grade: experience of two decades of cancer registry[END_REF][START_REF] Walter | Epidemiology of digestive neuroendocrine tumors with focus on French data[END_REF]. They are classified according to their differentiation and proliferation rate (mitotic count and Ki-67 index) and divided in 3 grades (G): G1 and G2 tumours (Ki-67≤20%) and G3 (Ki-67>20%) [START_REF] Rindi | Pathology of gastrointestinal disorders[END_REF]. The WHO classification has recently been updated for pancreatic NEN integrating a new entity of G3 well differentiated tumours, or NET-G3 [START_REF] Basturk | The high-grade (WHO G3) pancreatic neuroendocrine tumor category is morphologically and biologically heterogenous and includes both well differentiated and poorly differentiated neoplasms[END_REF][START_REF] Coriat | Gastroenteropancreatic Well-Differentiated Grade 3 Neuroendocrine Tumors: Review and Position Statement[END_REF][START_REF] Perren | ENETS Consensus Guidelines for the Standards of Care in Neuroendocrine Tumors: Pathology: Diagnosis and Prognostic Stratification[END_REF][START_REF] Klöppel | Neuroendocrine Neoplasms: Dichotomy, Origin and Classifications[END_REF]. Therefore, the term of neuroendocrine carcinoma (NEC) only applies to G3 poorly differentiated tumours [START_REF] Klöppel | Neuroendocrine Neoplasms: Dichotomy, Origin and Classifications[END_REF].

Among NEN of the digestive tract, about 15% are NEC with values varying from 7 to 21% depending on studies [START_REF] Yao | One hundred years after "carcinoid": epidemiology of and prognostic factors for neuroendocrine tumors in 35,825 cases in the United States[END_REF][START_REF] Korse | Incidence and survival of neuroendocrine tumours in the Netherlands according to histological grade: experience of two decades of cancer registry[END_REF][START_REF] Scoazec | Professional Practices and Diagnostic Issues in Neuroendocrine Tumour Pathology: Results of a Prospective One-Year Survey among French Pathologists (the PRONET Study)[END_REF]. NEC represent the most aggressive subgroup, accounting for 80 % of all G3 NEN and are often diagnosed at a metastatic state [START_REF] Scoazec | Professional Practices and Diagnostic Issues in Neuroendocrine Tumour Pathology: Results of a Prospective One-Year Survey among French Pathologists (the PRONET Study)[END_REF]. Median overall survival (OS) is about 34 months for patients with a localised disease [START_REF] Yao | One hundred years after "carcinoid": epidemiology of and prognostic factors for neuroendocrine tumors in 35,825 cases in the United States[END_REF][START_REF] Dasari | Comparative study of lung and extrapulmonary poorly differentiated neuroendocrine carcinomas: A SEER database analysis of 162,983 cases[END_REF][START_REF] Sorbye | Gastroenteropancreatic highgrade neuroendocrine carcinoma[END_REF] and 5 months for patients with metastatic disease [START_REF] Dasari | Comparative study of lung and extrapulmonary poorly differentiated neuroendocrine carcinomas: A SEER database analysis of 162,983 cases[END_REF][START_REF] Sorbye | Gastroenteropancreatic highgrade neuroendocrine carcinoma[END_REF][START_REF] Sorbye | Predictive and prognostic factors for treatment and survival in 305 patients with advanced gastrointestinal neuroendocrine carcinoma (WHO G3): the NORDIC NEC study[END_REF]. The majority of therapeutic guidelines for NEC derive from studies in small cell lung cancer (SCLC), which is the closest tumour entity [START_REF] Pellat | Therapeutic management of poorly differentiated neuroendocrine lung tumors and neuroendocrine carcinomas of the digestive system[END_REF][START_REF] Garcia-Carbonero | ENETS Consensus Guidelines for High-Grade Gastroenteropancreatic Neuroendocrine Tumors and Neuroendocrine Carcinomas[END_REF][START_REF] Cicin | Extrapulmonary small-cell carcinoma compared with small-cell lung carcinoma: a retrospective singlecenter study[END_REF]. Few retrospective clinical studies have focused on the therapeutic management of localised and resectable NEC of the digestive tract and the role of neoadjuvant or adjuvant chemotherapy is not well known [START_REF] Pellat | Therapeutic management of poorly differentiated neuroendocrine lung tumors and neuroendocrine carcinomas of the digestive system[END_REF]. For localised NEC, international guidelines recommend surgery, followed by adjuvant systemic chemotherapy combining platinum salts with etoposide (VP16) [START_REF] Garcia-Carbonero | ENETS Consensus Guidelines for High-Grade Gastroenteropancreatic Neuroendocrine Tumors and Neuroendocrine Carcinomas[END_REF][START_REF] Strosberg | Prognostic validity of a novel American Joint Committee on Cancer Staging Classification for pancreatic neuroendocrine tumors[END_REF]. There are no recommendations on neoadjuvant chemotherapy.

Surgery is offered when tumours can be entirely resected and after performing a thoracoabdomino-pelvic computerised tomography (CT) and a positron emission tomography (PET) scanner, if available, to evaluate the preoperative node status and research distant metastasis.

In France, adjuvant chemotherapy was proposed as an option before 2016 and is now recommended based on expert consensus. Nevertheless, there are no prospective (randomised or not) studies with adjuvant chemotherapy available in localised SCLC or in NEC of the digestive tract. Trials are difficult to conduct because of the rarity and diversity of these tumours. Minor progress in therapeutic strategy and management has been made in the past 4 decades for NEC, which makes them a priority of research. This is underlined by their poor prognosis and the description of new entities such as NET-G3 or mixed neuroendocrine-non neuroendocrine neoplasms (MINEN) [START_REF] Klöppel | Neuroendocrine Neoplasms: Dichotomy, Origin and Classifications[END_REF]. Moreover, these 2 entities show better prognosis and could benefit from a different therapeutic approach [START_REF] Coriat | Gastroenteropancreatic Well-Differentiated Grade 3 Neuroendocrine Tumors: Review and Position Statement[END_REF][START_REF] Coriat | In Reply[END_REF]. The aim of our study was to evaluate the effect of chemotherapy, perioperative (defined as neoadjuvant +/-adjuvant) or adjuvant chemotherapy, in patients operated for a localised NEC of the digestive tract.

METHODOLOGY

We conducted an observational, retrospective, multicentre, French study. Patients were recruited with the help of the expert network "groupe des tumeurs neuroendocrines" (GTE) and French pathology and oncology departments. Ethical approval was provided by Saint Antoine hospital's ethics committee on November 8th, 2016. Data was recovered for patients treated for NEC of the digestive tract between the 1 st of January 2000 and the 31 st of December 2016.

Inclusion and exclusion criteria

Patients included in the study were adults over 18 years old presenting a localised NEC of the digestive tract, surgically resected, with residual margins R0 or R1 (according to the R residual tumour classification, R1 was defined as a ≤1mm margin). We included both patients receiving chemotherapy (perioperative or adjuvant) or not. Exclusion criteria were as follows: patients with MINEN, well differentiated tumours, presence of distant metastasis at the time of diagnosis, R2 margin, non-digestive NEC.

Histology

Given the rarity of NEN and the difficulties in pathological diagnosis, a specialised French pathology network has been created in 2010 (TENpath network). Sections reviewing by an expert pathologist from the TENpath network is now recommended for difficult cases (NET-G3, MINEN, and NEC with low Ki-67 values) without delaying treatment. Interpretation by a pathologist from the TENpath network was not mandatory for inclusion in our study, even for patients treated after 2010. Similarly, a centralised review for pathology was not performed for practical purposes.

Evaluation criteria

We evaluated the overall survival (OS), defined as the time between histological diagnosis of NEC and time of death. Pathological diagnosis was made on tumour biopsies or surgical specimens. Then, we evaluated disease free survival (DFS) defined as the time between histological diagnosis of NEC and relapse or death from any cause. May 31 st 2017 was the data cut off.

We performed a univariate analysis followed by a multivariate analysis in order to study the impact of 10 potential prognostic factors on OS and DFS: age, sex, administration of chemotherapy (all modalities), of adjuvant chemotherapy alone, of perioperative chemotherapy, size of cells, node status on surgical specimens (pN), Ki-67 index, initial site of NEC and surgical margin (R0 or R1). Toxicity was graduated according to the NCI-CTCAE v4, and only grades 3 and 4 toxicities were reported.

Study flow

Patients data were recovered from medical files in each centre: clinical, pathological and imaging data. Imaging exams performed to evaluate disease staging, mainly thoracoabdomino-pelvic computed tomography (CT), were not centralized for independent review. Finally, we recovered therapeutic data and information on follow-up for each patient.

Statistical analysis

This study was observational and groups were not compared (chemotherapy versus no chemotherapy). Time-related parameters, OS and DFS, were assessed by Kaplan-Meier methods with the associate p-value of log-rank test. Values are expressed as median (range).

Univariate and multivariate analysis were assessed by Cox model. Kaplan-Meier curves were drawn using Medcalc 18.2.1. The association between time-related parameters (OS and DFS) and variables were evaluated using univariate and multivariate Cox proportional hazards regression models (R survival package, R Foundation for Statistical Computing, 3.3.1-R Studio). Variables with a p value <0.05 were selected as covariates for the multivariate analysis. Statistical significance was defined by a p value of 0.05 or less.

RESULTS

Patients characteristics

Ninety patients from 21 medical centres in 18 French cities were screened, and 73 patients were included in the final analysis. Seventeen patients were excluded: 10 patients were misdiagnosed (7 patients had MINEN and 2 had well differentiated tumours; 1 patient had pancreatic adenocarcinoma after reviewing of surgical specimen), 6 patients had metastatic disease, and 1 patient was lost to follow-up just after surgery. Regarding the 59 patients treated after 2010 in our population, 78% were diagnosed by an expert pathologist member of the TEN path network because they were treated in a NEN expert centre.

Patients characteristics are reported in Table 1. Median age was 68 years old with 62% of males. Regarding tumour site, 35 % of patients had colorectal NEC, 34% had pancreatic or ampullary NEC and 31 % had NEC at other sites (oesophagus, stomach, anal, gall bladder).

The majority of patients had large cells NEC (58%) and a positive node status (pN+) after surgery (62%). Median value of Ki-67 was 70% .

In total, 43 patients (59%) received chemotherapy and 37 (86%) received the combination of platinum salts (cisplatin or carboplatin) and etoposide. Six patients (14%) received fluorouracil-based chemotherapy: in neoadjuvant setting, 1 received FOLFOX and 1 received capecitabine along with radiotherapy; the 4 others received capecitabine in adjuvant setting.

Time-related parameters

With a median follow up of 48 months (0-130), median OS for all patients (n=73) was 24 months (Figure 1a). Forty-one patients died (56%). Median DFS was 9 months. (Figure 1b).

There were 48 events (relapse or death), so an events rate for DFS of 66%. The majority of patients presented metastatic relapse: hepatic (54%), bone (15%), brain (6%), often associated with a local node relapse.

Regarding the population who received chemotherapy (all modalities), median OS and DFS were 62 and 13 months respectively (Figures 2 and3). Regarding the 30 patients who did not receive any chemotherapy, median OS and DFS were 19 and 5 months respectively (Figures 2 and3).

Study of potential prognostic factors

We evaluated 10 potential prognostic factors: age ≥ 68 years (median age of the population), male sex, administration of chemotherapy, administration of adjuvant chemotherapy alone, administration of perioperative chemotherapy, large cells NEC, positive node status on surgical specimens (pN+), Ki-67 index, initial site of NEC and surgical margin (R0 or R1). In univariate analysis, a postoperative positive node status (pN+) had a negative impact on both OS (Table 2, HR=3.11, p=0.0046) and DFS (Table 3, HR=2.04, p=0.033). Administration of chemotherapy (all modalities) had a positive impact on both OS and DFS (Table 2, HR=0,38, and Table 3, HR=0,44). Administration of perioperative chemotherapy (n=16) and adjuvant chemotherapy (n=27) also had a positive impact on OS and DFS compared to no chemotherapy (n=30). For Ki-67 we used 3 thresholds for evaluation: 55%, 70% and 80%.

We did not find any impact on OS whichever the threshold used while Ki-67 ≥80% had a negative impact on DFS (p=0.026). Age, sex, large cells and R1 resection margin had no impact on survival. The initial tumour site did not show impact on survival either.

In multivariate analysis (Tables 2 and3), Ki-67≥80%, positive node status on surgical specimens (pN+) and administration of chemotherapy (all modalities, n=43) were independent prognostic factors on OS and DFS. Administration of chemotherapy showed a positive impact on survival for both OS and DFS (aHR=0,32 p=0.00055 and aHR=0,36, p=0.00099) while Ki-67≥80% and pN+ were poor prognostic factors.

Analysis was also performed after exclusion of the 6 patients who had received fluorouracilbased chemotherapy. In multivariate analysis, administration of chemotherapy showed a positive impact on survival for both OS and DFS (p=0,0032 and p=0,0039).

Toxicity

There were no toxic deaths in our study. We reported 16 grades 3 and 4 toxicities for the 43 patients who received chemotherapy (Supplementary table) excluding alopecia. Neutropenia was the most common toxicity. We also observed 3 deaths within the month following surgery for patients with pancreatic NEC (2 cephalic duodenopancreatectomies and 1 splenopancreatectomy).

DISCUSSION

In this retrospective study of resected NEC of the digestive tract, administration of chemotherapy (all modalities) showed better prognosis for OS and DFS. For the whole population, median OS and DFS were 24 and 9 months respectively. There were no toxic deaths with chemotherapy.

This study is one of the largest observational cohort of resected digestive NEC and only a few retrospective studies have been conducted [START_REF] Smith | A retrospective review of 126 high-grade neuroendocrine carcinomas of the colon and rectum[END_REF][START_REF] Shafqat | Survival of patients with neuroendocrine carcinoma of the colon and rectum: a population-based analysis[END_REF]. Conducting studies on these tumours is difficult because of their rarity and their diversity, but also because of the recent description of the new histological entity of well differentiated tumours of grade 3, bearing better prognosis, that must be separated from NEC. European and French guidelines recommend the administration of an adjuvant chemotherapy, by analogy with SCLC, despite a lack of proof of efficacy, and in relation with the bad prognosis of NEC of the digestive tract [START_REF] Perren | ENETS Consensus Guidelines for the Standards of Care in Neuroendocrine Tumors: Pathology: Diagnosis and Prognostic Stratification[END_REF]. To our knowledge, this work is the first focusing on the role of perioperative or adjuvant chemotherapy in resected NEC and no clinical prospective study has ever been conducted comparing adjuvant and perioperative chemotherapy with surgery to surgery alone.

Our study was retrospective, which is an important limitation. Also, we had to include patients treated over 16 years, time during which pathological classifications have evolved.

We did not perform a centralised review for histology and Ki-67, therefore creating a risk of bias in interpretation. Nevertheless, the majority of patients treated after 2010 in our population were diagnosed by an expert pathologist member of the TENpath network, because they were treated in a NEN expert centre. This illustrates the centralised therapeutic management of these tumours in France. Platinum salts and etoposide have been used in these tumours for more than 20 years without new combinations emerging as a new standard. One phase III clinical trial in extensive SCLC found no difference in survival with the association of cisplatin and irinotecan compared to etoposide and platinum salts [START_REF] Lara Pn | Phase III trial of irinotecan/cisplatin compared with etoposide/cisplatin in extensive-stage smallcell lung cancer: clinical and pharmacogenomic results from SWOG S0124[END_REF]. The combination of cisplatin and irinotecan can be administered as first line treatment in extensive SCLC in Japan [START_REF] Lara Pn | Phase III trial of irinotecan/cisplatin compared with etoposide/cisplatin in extensive-stage smallcell lung cancer: clinical and pharmacogenomic results from SWOG S0124[END_REF][START_REF] Moertel | Treatment of neuroendocrine carcinomas with combined etoposide and cisplatin. Evidence of major therapeutic activity in the anaplastic variants of these neoplasms[END_REF]. In our work, 6 patients did not receive the recommended regimen of platinum salts and etoposide, with heterogeneous dose and schedule of platinum salts and etoposide. Also, the decision to administer chemotherapy depended on patient's general condition, which creates a selection bias. Finally, data on post-operative performance status was lacking in our work.

Poorly differentiated neuroendocrine tumours, such as SCLC and NEC of the digestive tract, are chemosensitive to etoposide and cisplatin combination with overall responses ranging from 40% to 67% [START_REF] Mitry | Treatment of poorly differentiated neuroendocrine tumours with etoposide and cisplatin[END_REF][START_REF] Walter | Poorly differentiated gastro-entero-pancreatic neuroendocrine carcinomas: Are they really heterogeneous? Insights from the FFCD-GTE national cohort[END_REF]. This effect is however temporary and patients rapidly progress, often at a distant site, underlining the spreading potential of these tumours. In our population, 66% of patients relapsed with a majority of hepatic or node metastases. Recent data were also obtained by the poorly differentiated neuroendocrine carcinoma (CEPD) French cohort in which 39% of 25 patients with operated NEC had relapsed [START_REF] Walter | Poorly differentiated gastro-entero-pancreatic neuroendocrine carcinomas: Are they really heterogeneous? Insights from the FFCD-GTE national cohort[END_REF].

A review from 1997 on localised small cells NEC of the oesophagus showed higher survival for patients treated with a combined treatment of surgery and chemotherapy vs surgery alone (median OS of 20 months vs 5 months respectively, p<0.001) [START_REF] Casas | Primary small cell carcinoma of the esophagus: a review of the literature with emphasis on therapy and prognosis[END_REF]. The types and protocols of chemotherapy were not described in this study. In Haugvik's work [START_REF] Haugvik | Surgical Treatment as a Principle for Patients with High-Grade Pancreatic Neuroendocrine Carcinoma: A Nordic Multicenter Comparative Study[END_REF], all patients operated for pancreatic NEC had received adjuvant chemotherapy; median OS was 24 months, which is similar to our result. Without consensual recommendations, the therapeutic strategy was heterogeneous in clinical practice. The administration of neoadjuvant chemotherapy could help select patients with less aggressive tumours who could benefit from surgery, like the strategy adopted for borderline and locally advanced pancreatic adenocarcinomas. However, the proportion of patients showing progressive disease during neoadjuvant chemotherapy was not evaluated. For now, little data is available on neoadjuvant and/or perioperative chemotherapy in NEC and its administration is not recommended by guidelines (it is only an option in French recommendations).

Regarding other prognostic factors, we found that a positive nodal status (pN+) was a pejorative factor for both OS and DFS. Current guidelines do not take this factor into account for the therapeutic decision. French guidelines have changed and now recommend, when available, the performance of a PET-18 FDG-CT before surgery in order to improve the detection of preoperative node involvement and/or distant metastases. We did not report the number of patients with a PET-18 FDG-CT before surgery in our work. We did not find any impact on survival with a threshold of 55%. In the NORDIC study, patients with a Ki-67<55% showed lower response rate to chemotherapy combining etoposide and platinum salts but better OS (14 months versus 10 months) [START_REF] Sorbye | Predictive and prognostic factors for treatment and survival in 305 patients with advanced gastrointestinal neuroendocrine carcinoma (WHO G3): the NORDIC NEC study[END_REF]. On the contrary, in Haugvik's work [START_REF] Haugvik | Surgical Treatment as a Principle for Patients with High-Grade Pancreatic Neuroendocrine Carcinoma: A Nordic Multicenter Comparative Study[END_REF], a Ki-67 55% threshold did not show any impact on survival in the 28 patients who underwent surgery for pancreatic NEC. The NORDIC study included patients with metastatic and localised NEC regardless of their histological differentiation status, which might explain this difference. In addition to the Ki-67 threshold, recent studies in colon and other locations suggested that the tumour molecular profile may help to adapt chemotherapy regimen in patients with NEC. A subgroup of NEC are genetically closely related to adenocarcinomas and better respond to chemotherapies used for exocrine tumours [START_REF] Jesinghaus | Colorectal mixed adenoneuroendocrine carcinomas and neuroendocrine carcinomas are genetically closely related to colorectal adenocarcinomas[END_REF][START_REF] Takizawa | Molecular characteristics of colorectal neuroendocrine carcinoma; similarities with adenocarcinoma rather than neuroendocrine tumor[END_REF].These genetic tests were not possible to perform in our study. Initial site of NEC was not a prognostic factor in our study, as in the CEPD cohort [START_REF] Casas | Primary small cell carcinoma of the esophagus: a review of the literature with emphasis on therapy and prognosis[END_REF]. One retrospective study found that exclusive administration of chemotherapy or radiochemotherapy were associated with a similar survival than surgery in a small population of localised anorectal NEC [START_REF] Brieau | Radiochemotherapy Versus Surgery in Nonmetastatic Anorectal Neuroendocrine Carcinoma: A Multicenter Study by the Association des Gastro-Enterologues Oncologues[END_REF].

In conclusion, our work suggests a positive prognostic impact of adjuvant or perioperative chemotherapy on survival, for patients operated for a localised NEC. Patients treated for NEC of the digestive tract should be rigorously selected before surgery and some patients might benefit from an alternative treatment such as radiochemotherapy. This underlines the need for prospective studies comparing these therapeutic strategies in different sites of NEC. We plan to conduct a prospective cohort of localised NEC of the digestive tract evaluating the role of

  Among the 43 patients, 16 received perioperative chemotherapy (22%): 16 patients received neoadjuvant chemotherapy and 8 also received adjuvant chemotherapy. Four patients also received radiotherapy combined with chemotherapy in neoadjuvant setting. Twenty-seven patients (37%) received adjuvant chemotherapy alone (Supplementary Figure). The median number of cycles of chemotherapy per patient was 4. Regarding the 30 other patients: 21 (70%) were not offered chemotherapy; 1 patient refused treatment and the others presented surgery complications or alteration of general state.

  neoadjuvant chemotherapy, followed by surgery or radiochemotherapy, or adjuvant chemotherapy in case of surgery first. The main part of data was provided by Dr Anna Pellat, Pr Thomas Walter, Dr Vincent Hautefeuille, Dr Olivia Hentic, Dr Christine Do Cao, Pr Astrid Lievre, Dr Olivier Dubreuil, Pr Romain Coriat, and Pr Eric Baudin. Statistics were done by Dr Jérémy Augustin. The article was written by Dr Anna Pellat. It was reviewed by Pr Thierry André, Dr Pauline Afchain, Pr Thomas Walter, Dr Vincent Hautefeuille, Dr Olivia Hentic, Pr Pascal Hammel, Dr Jérémy Augustin, Dr Romain Cohen, Pr Anne Couvelard and Pr Magali Svrcek.

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Overall survival and disease-free survivals of the whole population (73 patients)

ACKNOWLEDGMENTS

We thank all members of the GTE for their contribution to this work, including David Tougeron and Come Lepage. We also thank Delphine Lees for editorial assistance.

FUNDING SOURCES

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

STATEMENT OF ETHICS

Ethical approval was provided by Saint Antoine hospital's ethics committee on November 8th, 2016. The authors have no ethical conflicts to disclose?

DECLARATIONS OF INTEREST

The authors declare no following competing interests for this work

AUTHOR CONTRIBUTIONS

The project was initiated by Pr Thierry André, Dr Pauline Afchain and Dr Anna Pellat.