Thibault Comont
email: comont.thibault@iuct-oncopole.fr

K Delavigne

P Cougoul

S Bertoli

E Delabesse

P Fenaux

O Beyne-Rauzy

syndromes myélodysplasiques en 2019 : mise au point

Keywords: Syndromes myélodysplasiques, revue, traitement, auto-immunité, chélation myelodysplastic syndromes, review, treatment, auto-immunity, chelation

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

Les syndromes myélodysplasiques (SMD) constituent un ensemble d'hémopathies myéloïdes clonales caractérisé par une hématopoïèse inefficace et se traduisant par des cytopénies périphériques(1) et un risque de progression en leucémie aiguë myéloïdes (LAM) de l'ordre de 30%. Bien que des progrès sur la compréhension des mécanismes physiopathologiques et la caractérisation des différentes entités de SMD aient été observés ces dernières années, la prise en charge thérapeutique reste souvent relativement limitée et le pronostic grave, dans les formes de haut risque. Cette revue a pour objectif de faire le point sur l'état actuel de la prise en charge des SMD.

Épidémiologie

Les SMD représentent 13% de l'ensemble des diagnostics d'hémopathies et la première hémopathie du sujet de plus de 65 ans, touchant préférentiellement les hommes avec un sex ratio de 1,5-2/1 [START_REF] Rollison | Epidemiology of myelodysplastic syndromes and chronic myeloproliferative disorders in the United States, 2001-2004, using data from the NAACCR and SEER programs[END_REF][START_REF] Maynadié | Twenty-five years of epidemiological recording on myeloid malignancies: data from the specialized registry of hematologic malignancies of Cote d'Or (Burgundy, France)[END_REF]. Les formes secondaires représentent 10 à 20% des SMD. L'exposition antérieure à des traitements anticancéreux (chimiothérapie ou radiothérapie) est associée à l'émergence de t-SMD (therapy-related SMD) [START_REF] Smith | Clinicalcytogenetic associations in 306 patients with therapy-related myelodysplasia and myeloid leukemia: the University of Chicago series[END_REF]. Les agents alkylants (cyclophosphamide, chlorambucil) et les inhibiteurs de topo-isomérase II (étoposide, doxorubicine) sont les plus souvent en cause. Certaines expositions professionnelles (benzène, radiations ionisantes, solvants, etc.) peuvent donner lieu à une reconnaissance comme maladie professionnelle [START_REF] Iwanaga | Risk of myelodysplastic syndromes in people exposed to ionizing radiation: a retrospective cohort study of Nagasaki atomic bomb survivors[END_REF]. Une méta-analyse récente confirme l'association entre tabagisme, obésité et SMD [START_REF] Du | Smoking and alcohol intake as risk factors for myelodysplastic syndromes (MDS)[END_REF]. L'incidence des SMD est augmentée chez les patients porteurs de maladies autoimmunes/inflammatoires (MAII) suggérant un lien physiopathologique entre les deux entités [START_REF] Anderson | Risks of myeloid malignancies in patients with autoimmune conditions[END_REF]. En parallèle certains traitements immunosuppresseurs utilisés couramment dans les MAII sont associés à la survenue de SMD, principalement l'azathioprine [START_REF] Ertz-Archambault | Association of Therapy for Autoimmune Disease With Myelodysplastic Syndromes and Acute Myeloid Leukemia[END_REF]. Les SMD peuvent être associés à une prédisposition constitutionnelle. On distingue les maladies génétiques pouvant se compliquer de SMD (trisomie 21, anémie de Fanconi, neurofibromatose de type I, syndrome de Pearson, syndrome de Noonan, maladie de Blackfan-Diamond), des SMD familiaux secondaires à des mutations germinales ponctuelles sur certains gènes myéloïdes (GATA2, RUNX1, TERT/TERC, DDX41, etc.) [START_REF] Liew | Familial myelodysplastic syndromes: a review of the literature[END_REF]. Enfin il faut souligner que les SMD peuvent s'associer aux mastocytoses systémiques (mutation c-kit) [START_REF] Valent | Mastocytosis: 2016 updated WHO classification and novel emerging treatment concepts[END_REF] dont la prise en charge est alors particulièrement délicate.

En pratique : il est important d'identifier le caractère secondaire d'un SMD. Il faudra également rechercher des manifestations extra-hématologiques afin de ne pas méconnaitre une origine génétique (en interrogeant sur des antécédents familiaux de SMD, LAM, aplasie médullaire et/ou une MAII associée).

Circonstances de découvertes

Les SMD sont principalement révélés par des cytopénies périphériques (surtout l'anémie), symptomatiques ou de découverte fortuite. Mais d'autres manifestations, rhumatologiques, dermatologiques ou encore systémiques, peuvent être au premier plan, rendant le diagnostic plus difficile.

Cytopénies

Anémie

L'anémie est présente chez 90% des patients et peut être responsable d'une altération franche de la qualité de vie (en particulier chez les sujets âgés ou avec co-morbidités) [START_REF] Stauder | Health-related quality of life in lower-risk MDS patients compared with age-and sex-matched reference populations: a European LeukemiaNet study[END_REF]. Elle est habituellement normo/macrocytaire et arégénérative. Plus rarement peuvent s'observer des marqueurs d'hémolyse ou une hyperferritinémie pouvant accompagner la dysérythropoïèse ou révéler une anémie hémolytique auto-immune associée.

Thrombopénie

Une thrombopénie peut être présente dans 20-40% des cas, d'origine centrale ou plus rarement périphérique (thrombopénie immunologie (TI) associée). La thrombopénie peut être isolée d'où l'importance de réaliser une exploration médullaire en cas de suspicion de TI chez un patient de plus de 60 ans [START_REF]Protocole National de Diagnostic et de Soins (PNDS)-Purpura thrombopénique immunologique de l'enfant et de l'adulte[END_REF]. Cette thrombopénie expose le patient à un risque hémorragique, surtout chez des patients âgés sous anticoagulants (13).

Neutropénie

Une neutropénie est présente dans plus de 50% des cas, associée à des anomalies morphologiques visibles sur le frottis sanguin (dégranulation des polynucléaires neutrophiles). Un taux de polynucléaires neutrophiles inférieur à 0,5 G/L expose le patient à un risque d'infections bactériennes sévères ou fungiques invasives (aspergillose, mucormycose)(14).

Autres anomalies de l'hémogramme D'autres anomalies permettent d'évoquer un SMD :

-monocytose : elle doit faire évoquer lorsqu'elle est significative (> 1G/L et >10% des leucocytes) et chronique, une leucémie myélomonocytaire chronique (LMMC). Il n'est parfois pas évident de la différencier d'une monocytose réactionnelle, mais l'immunophénotype des monocytes sanguins se positionne comme un outil fiable permettant d'argumenter le diagnostic de LMMC lorsque les monocytes de phénotype CD14+ CD16-représentent plus de 94% des monocytes [START_REF] Selimoglu-Buet | Characteristic repartition of monocyte subsets as a diagnostic signature of chronic myelomonocytic leukemia[END_REF].

-thrombocytose : elle peut être présente dans les formes de SMD avec délétion 5q (del5q) ou dans l'anémie sidéroblastique avec thrombocytose.

Syndrome tumoral

La présence d'un syndrome tumoral n'est pas classique dans les SMD et doit faire évoquer une forme de chevauchement avec une néoplasie myéloproliférative (SMD/SMP), une LMMC ou une anémie sidéroblastique (présence fréquente de mutations de JAK2 ou SF3B1) [START_REF] Patnaik | Refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T): 2017 update on diagnosis, risk-stratification, and management[END_REF].

Manifestations inflammatoires ou auto-immunes

De nombreuses associations entre MAII et SMD ont été rapportée dans la littérature avec une fréquence de 10-28% et un odd-ratio de 4-5 [START_REF] Ustwani | Myelodysplastic syndromes and autoimmune diseases--case series and review of literature[END_REF][START_REF] Mekinian | Systemic inflammatory and autoimmune manifestations associated with myelodysplastic syndromes and chronic myelomonocytic leukaemia: a French multicentre retrospective study[END_REF][START_REF] Seguier | Autoimmune diseases in myelodysplastic syndrome favors patients survival: A case control study and literature review[END_REF][START_REF] Grignano | Autoimmune manifestations associated with myelodysplastic syndromes[END_REF]. Les principales manifestations décrites sont des vascularites, des connectivites comme la polychondrite atrophiante [START_REF] Dion | Relapsing polychondritis: What's new in 2017?[END_REF][START_REF] Moulis | Efficacy and safety of biologics in relapsing polychondritis: a French national multicentre study[END_REF], première association historiquement décrite, des arthropathies inflammatoires et des dermatoses neutrophiliques dont la fréquence peut atteindre 10-15% des patients [START_REF] Beyne-Rauzy | Myelodysplastic syndromes][END_REF]. On observe une association entre la présence d'une trisomie 8 et la survenue d'une maladie de Behçet [START_REF] Wesner | Inflammatory disorders associated with trisomy 8-myelodysplastic syndromes: French retrospective casecontrol study[END_REF]. Certains patients présentent des manifestations inflammatoires inclassées se rapprochant du syndrome d'activation lymphohistiocytaire ou d'une maladie de Still. Les cytopénies auto-immunes restent rares mais peuvent poser des problèmes de diagnostic et de prise en charge. L'azacitidine (AZA) semble avoir une action significative sur ces manifestations et permettre une épargne en immunosuppresseur [START_REF] Fraison | Efficacy of Azacitidine in autoimmune and inflammatory disorders associated with myelodysplastic syndromes and chronic myelomonocytic leukemia[END_REF]. Un essai prospectif est actuellement en cours (NCT02985190) pour valider l'intérêt de l'AZA dans le traitement des MAII cortico-résistantes ou cortico-dépendantes associées aux SMD.

En pratique : un diagnostic de SMD doit être suspecté devant toute cytopénie chronique chez un patient de plus de 60 ans. De plus, il faut savoir évoquer un SMD chez un patient âgé présentant une MAII et des anomalies de l'hémogramme.

Confirmation diagnostique

Les différentes anomalies clinico-biologiques ne sont pas spécifiques et la confirmation diagnostique vient de l'analyse cytologique médullaire après réalisation d'un myélogramme. [START_REF] Greenberg | Revised international prognostic scoring system for myelodysplastic syndromes[END_REF]. Certaines anomalies sont considérées comme de mauvais pronostic (anomalie du chromosome 7, caractère complexe et/ou monosomique). Ces anomalies cytogénétiques peuvent se modifier au cours de l'évolution d'un SMD ce qui nécessite de renouveler l'examen en cas de suspicion de progression.

Biologie moléculaire

La grande majorité des SMD présentent une ou plusieurs mutations impliquées dans divers mécanismes de régulation cellulaire (Figure 1). Le principal intérêt est actuellement pronostique, certaines mutations étant associées à un risque de progression [START_REF] Kennedy | Clinical Implications of Genetic Mutations in Myelodysplastic Syndrome[END_REF]. Cependant, la mise en en évidence de mutation(s) peut avoir également un intérêt thérapeutique (indication d'allogreffe de moelle osseuse, nouveaux traitements comme les inhibiteurs d'IDH1/2, résistance à l'AZA) mais aussi diagnostique, notamment dans les SMD avec sidéroblastes en couronnes (mutation de SF3B1 dans 80% des cas) ou encore devant une dysplasie modérée (cf infra).

Diagnostic précoce d'hémopathie clonale : l'avenir ?

Le diagnostic de SMD peut s'avérer plus difficile lorsque la dysplasie est modérée. On parle alors de cytopénie idiopathique de signification indéterminée (ou ICUS pour idiopathic cytopenia of undetermined significance). Certaines données suggèrent l'intérêt de rechercher le caractère clonal des cytopénies(31) retrouvé dans 40% des cas dans la littérature et dans notre expérience chez 23 patients sur 70 testés (33%). Le terme de cytopénie clonale de signification indéterminée (CCUS pour clonal cytopenia of undetermined significance) est alors utilisé. Ces nouvelles distinctions sont importantes car 25% des ICUS vont développer un SMD ou une LAM et ce risque passe de 9 à 82% à 5 ans en présence de mutations.

En pratique : le diagnostic de SMD en 2019 doit comprendre une analyse cytogénétique conventionnelle et de plus en plus souvent de la biologie moléculaire.

La leucémie myélo-monocytaire chronique, une entité à part

La LMMC se différencie des autres SMD car il s'agit d'une forme frontière avec un syndrome myéloprolifératif caractérisée par une monocytose, ainsi qu'une fréquence élevée de signes généraux, d'un syndrome tumoral clinique, de manifestations auto-immunes, de marqueurs moléculaires telles que les mutations de TET2, AML1 ou RUNX1, ASXL1 et de RAS. Le traitement de la LMMC est mal codifié faisant appel à une simple surveillance dans les formes indolentes, à l'hydroxyurée dans les formes prolifératives ou encore l'AZA qui a l'AMM dans cette indication pour des blastes médullaires supérieurs à 10%(32).

Stratification du risque et détermination du pronostic

Scores pronostiques

Les SMD regroupent un ensemble de pathologies hétérogènes dans leur présentation clinico-biologique mais également dans leur pronostic. Plusieurs scores pronostiques ont été proposés mais le principal utilisé reste le score IPSS (pour International Prognostic Scoring System), publié en 1997 et basé sur le pourcentage de blastes, le nombre de cytopénies et les données cytogénétiques (Tableau 3). Il sépare les SMD de haut risque (SMD-HR, score supérieur ou égal à 1,5) des SMD de faible risque (SMD-LR pour low risk). Cependant, les données plus récentes sur l'importance pronostique de la cytogénétique ont amené à réviser ce score (IPSS-R, Tableau 4), incluant la nouvelle classification cytogénétique détaillée dans le Tableau 2. La signification pronostique du score IPSS-R (survie, risque de transformation en LAM) est résumée dans le Tableau 5. La plupart des mutations somatiques, sauf SF3B1 et TET2, ont une valeur pronostique défavorable, qui augmente avec leur nombre, et qui est souvent indépendante de l'IPSS-R.

Importance des co-morbidités

Les SMD affectent principalement des sujets âgés qui présentent une ou plusieurs comorbidités qui peuvent influencer le pronostic indépendamment des facteurs liés à la maladie. Ces dernières années, différents scores associant données hématologiques et facteurs liés au patient (co-morbidités, fragilité) ont été développés [START_REF] Porta | Risk stratification based on both disease status and extra-hematologic comorbidities in patients with myelodysplastic syndrome[END_REF]. Nous avons repris les données de 129 patients âgés de plus de 75 ans et traités par AZA pour un SMD-HR (45 patients) ou une LAM <30% de blastes (33 patients) ou >30% (51 patients) et montré que l'âge et les co-morbidités constituent dans cette population un facteur pronostique [START_REF] Brechemier | Impact of comorbidities and geriatric assessment in high risk myelodysplastic syndromes over 75 yeayrs treated with azacitidine[END_REF]. Cependant pour les sujets les plus âgés, l'apport d'une évaluation gériatrique standardisée devrait permettre d'apprécier plus finement les facteurs prédictifs de survie et/ou de tolérance aux traitements. Un essai multicentrique est actuellement en cours en France chez les patients ayant un SMD-HR âgés de plus de 70 ans afin de déterminer quels paramètres sont capables de prédire la survie globale et pourraient aider le clinicien à repérer les patients auxquels le traitement va être bénéfique (étude PREDICTOR).

Prise en charge thérapeutique

Principes

La prise en charge thérapeutique est différente entre SMD-LR et SDM-HR (résumé dans la Figure 2). Alors que le traitement des SMD-LR, qui représentent 2/3 des patients, consiste essentiellement à corriger les cytopénies, le traitement des SMD-HR va cibler la prolifération blastique et l'instabilité chromosomique afin de contrôler le clone leucémique.

6.2 Traitement des SMD-LR 6.2.1 Agents stimulants l'érythropoïèse L'anémie et la dépendance aux transfusions sont des facteurs clairement identifiés de morbimortalité et d'altération de la qualité de vie dans les SMD. Un traitement précoce est donc recommandé. L'érythropoïétine (EPO) est le traitement de première intention pour les patients porteurs de SMD-LR avec une anémie significative (< à 10 g/dl et/ou dépendant des transfusions). Les agents stimulant l'érythropoïèse (ASE) améliorent la survie chez les répondeurs. Le taux de réponse varie entre 30 et 75% avec une durée médiane de réponse de 24 mois [START_REF] Park | Predictive factors of response and survival in myelodysplastic syndrome treated with erythropoietin and G-CSF: the GFM experience[END_REF][START_REF] Messa | Effects of erythropoiesis-stimulating agents on overall survival of International Prognostic Scoring System Low/Intermediate-1 risk, transfusion-independent myelodysplastic syndrome patients: a cohort study[END_REF]. Leur introduction précoce semble retarder l'apparition des transfusions. Il existe un protocole thérapeutique temporaire pour les 3 EPO et une extension d'autorisation de mise sur le marché pour l'époïetine alpha. Le dosage de l'EPO sérique (s)EPO est un marqueur prédictif de réponse aux ASE. Un sEPO <100 UI/L est associé à un taux de réponse supérieur à 70% alors qu'un taux >500 UI/L est associé à un taux de réponse < 10% [START_REF] Hellström-Lindberg | A validated decision model for treating the anaemia of myelodysplastic syndromes with erythropoietin + granulocyte colony-stimulating factor: significant effects on quality of life[END_REF]. En cas d'inefficacité initiale, une association avec un facteur de croissance granulocytaire peut être proposée (GCSF) à faible dose, ce médicament ayant démontré in vitro une action synergique sur l'érythropoïèse et permettant d'obtenir 10 à 20% de réponses supplémentaires.

En pratique : l'EPO sera débutée en cas d'anémie <10 g/dL et si le taux de sEPO est <500 UI/L. La dose d'époïetine alpha ou beta se situe entre 20 000 et 80 000 UI par semaine par voie sous-cutanée, à adapter selon le poids et la réponse observée avec un délai habituel d'environ 8 semaines. Pour la darbépoétine, la dose habituelle de départ est de 150 µg par semaine, avec la possibilité d'augmenter à 300 µg par semaine, l'avantage de cette molécule étant de disposer d'un stylo auto-injectable qui permet aux patients d'être autonomes. Dans tous les cas l'objectif est un taux d'hémoglobine compris entre 10,5 et 12 g/dl. En cas de bonne réponse, la dose minimale efficace sera recherchée. A 12 semaines, le traitement doit être interrompu s'il n'est pas efficace. En dehors des effets indésirables en lien avec une réponse thérapeutique excessive (hypertension par exemple) il faut garder en mémoire le risque d'érythroblastopénie par l'apparition d'auto-anticorps anti-EPO (événement particulièrement rare). En cas de perte de réponse, il faudra penser à rechercher une carence en fer associée parfois visible uniquement sur la réduction du VGM.

Lenalidomide

Chez les patients qui présentent une délétion isolée du bras long du chromosome 5 (del5q) le lenalidomide a l'AMM lorsque les patients sont dépendants des transfusions et lorsque les autres options thérapeutiques (en particulier l'EPO) sont insuffisantes ou inappropriées. Il permet de restaurer la lignée érythroïde, avec un taux de réponse obtenu dans 65-70% et de rémission cytogénétique dans 30-40% des cas, grâce à une action ciblée sur le clone porteur de la délétion 5q. La durée médiane de réponse est de l'ordre de deux ans [START_REF] List | Lenalidomide in the myelodysplastic syndrome with chromosome 5q deletion[END_REF]. Il est important de surveiller chez ces patients la survenue d'une mutation de TP 53 qui, si elle est durable, est de mauvais pronostic et doit faire considérer le SMD comme de haut risque Chez les patients ne présentant pas de del5q, le taux de réponse est moindre mais reste intéressant chez les patients en échec des ASE ayant un rythme transfusionnel élevé [START_REF] Toma | Lenalidomide with or without erythropoietin in transfusion-dependent erythropoiesisstimulating agent-refractory lower-risk MDS without 5q deletion[END_REF]. Il semble que l'association lenalidomide+EPO permette dans ce cas de doubler le pourcentage d'indépendance transfusionnelle (IT), y compris chez des patients résistants à l'EPO seule [START_REF] Toma | Lenalidomide with or without erythropoietin in transfusion-dependent erythropoiesisstimulating agent-refractory lower-risk MDS without 5q deletion[END_REF].

En pratique : Le lenalidomide est utilisé à la posologie initiale de 10 mg/j pendant 21 jours par mois. Le principal effet secondaire est la survenue de cytopénies (neutropénie, thrombopénie) qui peuvent être très profondes le premier mois de traitement et nécessitent une surveillance rapprochée de l'hémogramme. Elles peuvent amener à utiliser du GCSF en cas de neutropénie sévère <1G/L, voire dans certains cas conduire à l'interruption et à une adaptation de la posologie (neutropénie<0,5 ou plaquettes<25G/L). La réponse s'observe en 4 à 6 semaines et le traitement est poursuivi tant qu'il est efficace. Le risque thrombotique est peu élevé et ne nécessite pas systématiquement de prophylaxie (à discuter en fonction des facteurs de risque).

Agonistes du récepteur de la thrombopoïétine

Les agonistes des récepteurs de la thrombopoïétine (ARTPO) ont l'AMM au cours de la thrombopénie immunologique et de l'aplasie médullaire. Leur utilisation dans les SMD-LR avec thrombopénie a été évaluée dans plusieurs études [START_REF] Giagounidis | Results of a randomized, double-blind study of romiplostim versus placebo in patients with low/intermediate-1-risk myelodysplastic syndrome and thrombocytopenia[END_REF][START_REF] Oliva | Eltrombopag versus placebo for low-risk myelodysplastic syndromes with thrombocytopenia (EQoL-MDS): phase 1 results of a single-blind, randomised, controlled, phase 2 superiority trial[END_REF]. Les premiers travaux concernant le romiplostim avaient fait émerger une alerte concernant le risque de transformation leucémique et l'apparition de myélofibrose, ayant amené à un arrêt de l'évaluation de cette molécule [START_REF] Brierley | Thrombopoiesis-stimulating agents and myelodysplastic syndromes[END_REF]. Des données plus récentes (suivi à 5 ans) sont rassurantes avec un taux de réponse de 57% et l'absence de sur-risque de LAM [START_REF] Fenaux | Romiplostim monotherapy in thrombocytopenic patients with myelodysplastic syndromes: long-term safety and efficacy[END_REF]. D'autres études ont évalué l'intérêt de l'eltrombopag, montrant des taux de réponse similaires et la diminution des manifestations hémorragiques sans promouvoir l'expansion de clones.

En pratique : l'utilisation d'un ARTPO dans les SMD-LR doit être discutée en cas de thrombopénie symptomatique, en tenant compte des facteurs de risques thrombotiques et après validation en RCP. Il paraît indispensable de se limiter aux patients ayant une blastose médullaire <5% 6.2.4 Les facteurs de croissance granulocytaire L'utilisation de GCSF peut être discutée chez les patients avec un SMD-LR et présentant une neutropénie sévère symptomatique, bien qu'aucune étude n'ait pu montrer un bénéfice en termes de survie.

L'azacitidine

A la différence de l'autorisation européenne, l'azacitidine (AZA) est également indiquée dans les SMD-LR aux États-Unis. Les différentes études publiées montrent un intérêt chez des patients dépendants des transfusions et en échec des autres traitements (ASE, lenalidomide) [START_REF] Filì | Prospective phase II Study on 5-days azacitidine for treatment of symptomatic and/or erythropoietin unresponsive patients with low/INT-1-risk myelodysplastic syndromes[END_REF]. Une étude française du GFM sur 93 patients avec l'AZA n'a toutefois constaté de réponse érythroïde significative que dans 20% des cas environ. Rappelons par ailleurs que la décitabine, autre agent hypométhylant (HMA), n'a pas d'AMM en Europe dans les SMD.

En pratique : L'AZA n'est actuellement pas indiquée en France dans les SMD-LR mais peut être discutée en cas d'impasse thérapeutique chez les patients dépendants des transfusions ou thrombopéniques ou encore lors d'association à des manifestations auto-immunes dans le cadre d'un essai du GFM en cours (AZA-SAID).

Autres approches thérapeutiques

Dans le cadre d'une thrombopénie, le danazol, un androgène permet une réponse dans environ 30% des cas avec les précautions d'utilisation habituelles (éliminer un carcinome prostatique et surveiller le bilan hépatique régulièrement). La posologie est de 600mg/j au maximum, à adapter à la réponse et à la tolérance [START_REF] Marini | Therapeutic efficacy of danazol in myelodysplastic syndromes[END_REF] Certains SMD en particulier des formes hypoplasiques ont des mécanismes physiopathologiques qui se rapprochent de l'aplasie médullaire et peuvent tirer bénéfice de traitements immunosuppresseurs comme le sérum anti-lymphocytaire et la ciclosporine avec, dans des formes très sélectionnées des taux de réponse globale de presque 50% et une IT dans 30%(46).

Allogreffe de cellules souches hématopoïétiques

L'ASCT ne constitue habituellement pas un traitement des SMD-LR. Cependant, des données récentes suggèrent un effet bénéfique sur la survie globale de l'ASCT dans des formes qui comportent certains facteurs de gravité et ne répondent pas aux traitements analysés plus haut [START_REF] Jabbour | Outcome of patients with low-risk and intermediate-1-risk myelodysplastic syndrome after hypomethylating agent failure: a report on behalf of the MDS Clinical Research Consortium[END_REF].

En pratique : l'ASCT doit être discutée en centre expert chez des patients jeunes en échec des autres thérapeutiques, une étude du GFM est en cours.

Luspatercept (ACE-536)

Parmi les nouvelles approches thérapeutiques ayant été évaluées récemment, l'inhibition du TGF-β semble la plus prometteuse dans les SMD-LR. En effet, une des causes de l'inhibition de l'érythropoïèse est l'augmentation de la concentration des ligands de la super famille des TGF-β et parmi ceux-ci le GDF11 et l'activine B, régulateurs négatifs des stades tardifs de l'érythropoïèse. Le luspatercept est décrit comme un «piège» de ces ligands ce qui aboutit à inhiber cette régulation négative. Initialement évalué dans la thalassémie avec des résultats très intéressants, il a par la suite été étudié chez des patients SMD-LR dépendant des transfusions en échec d'EPO, avec des résultats positifs, notamment chez les patients présentant des sidéroblastes en couronne et/ou une mutation de SF3B1. Les résultats d'un essai de phase III dans cette population viennent d'être communiqués à l'ASH 2018 avec l'obtention d'une IT de 8 semaines chez 37,9% des patients [START_REF] Fenaux | The Medalist Trial: Results of a Phase 3, Randomized, Double-Blind, Placebo-Controlled Study of Luspatercept to Treat Anemia in Patients with Very Low-, Low-, or Intermediate-Risk Myelodysplastic Syndromes (MDS) with Ring Sideroblasts (RS) Who Require Red Blood Cell (RBC) Transfusions[END_REF]. De nouveaux essais vont débuter avec cette molécule en 1 e ligne en comparaison à l'EPO.

En pratique : le luspatercept n'est pas disponible pour le moment en France et nous sommes en attente de son AMM. 6.2.9 Soins de supports 6.2.9.1 Transfusions Les transfusions chroniques érythrocytaires (plus rarement plaquettaires) restent très souvent employées, après échec des ASE. Selon la HAS et l'ANSM, il est recommandé ́ de transfuser au-dessous de 8 g/dL d'hémoglobine, ou à ̀ un taux plus élevé ́ en cas de comorbidités ou de retentissement clinique. L'anémie chronique et le traitement transfusionnel érythrocytaire comportent toutefois de nombreux inconvénients : surcharge martiale, fatigue, diminution de la qualité ́ de vie, majoration des complications cardiovasculaires, accidents transfusionnels (hémolyse, surcharge cardiaque chez le sujet âgé).

Chélation

L'effet délétère de l'accumulation tissulaire de fer est bien connu dans les hémoglobinopathies constitutionnelles. Les données sont moins importantes dans les SMD mais des complications comme la cirrhose ou surtout une insuffisance cardiaque sont décrits [START_REF] Killick | Iron chelation therapy in low risk myelodysplastic syndrome[END_REF]. Le taux de surcharge en fer à partir duquel ces complications peuvent survenir est discuté. Si une surcharge asymptomatique hépatique semble survenir dès la transfusion cumulée de 20 CGR, une surcharge cardiaque significative semble n'apparaître généralement qu'après transfusion de 50 CGR au moins. L'IRM cardiaque est un bon examen pour détecter cette surcharge cardiaque, qui vient s'ajouter à la surveillance de la ferritinémie. Une situation particulière est celle des patients potentiellement candidats à une allogreffe, chez qui une surcharge en fer même assez modérée (ferritinémie supérieure à 1000 ng/ml) paraît associée à une surmortalité post greffe. Chez ces patients (c'est-à-dire à priori tous les SMD-LR régulièrement transfusés âgés de moins de 70 ans et sans contreindication évidente à la greffe), une chélation du fer doit être entreprise dès ce taux. Pour les autres patients régulièrement transfusés, le taux devant déclencher le début de la chélation est plus discuté. Certaines études suggèrent que la chélation pourrait améliorer la survie, mais elles ne sont pas randomisées [START_REF] Gattermann | Deferasirox in iron-overloaded patients with transfusion-dependent myelodysplastic syndromes: Results from the large 1-year EPIC study[END_REF][START_REF] Gattermann | Hematologic responses to deferasirox therapy in transfusion-dependent patients with myelodysplastic syndromes[END_REF][START_REF] Rose | Does iron chelation therapy improve survival in regularly transfused lower risk MDS patients? A multicenter study by the GFM (Groupe Francophone des Myélodysplasies)[END_REF][START_REF] Callens | Targeting iron homeostasis induces cellular differentiation and synergizes with differentiating agents in acute myeloid leukemia[END_REF]. Alors que la chélation est indiscutable chez les patients ayant un projet d'allogreffe de moelle osseuse ou les sujets jeunes dépendants des transfusions, le bénéfice exact de celle-ci chez les sujets âgés ayant des comorbidités reste à préciser et son indication sera discutée au cas par cas.

Deux médicaments sont disponibles en France, le deferasirox et la déféroxamine, mais ce dernier est très peu utilisé car il nécessite la voie sous-cutanée prolongée, 8 à 10 heures par jour. Il n'est guère utilisé que s'il existe une insuffisance rénale contre indiquant l'emploi du deferasirox. Le déférasirox est administré par voie orale. La posologie habituelle est de 14 mg/kg/j mais peut-être augmentée selon le rythme transfusionnel. Des troubles digestifs sont fréquents (plus de 50% des patients, surtout à type de diarrhée dans 30% des cas) mais l'arrivée récente d'une nouvelle forme galénique (comprimés pelliculés) devrait améliorer la tolérance de ce médicament [START_REF] Taher | New film-coated tablet formulation of deferasirox is well tolerated in patients with thalassemia or lower-risk MDS: Results of the randomized, phase II ECLIPSE study[END_REF].

En pratique : la chélation martiale doit être envisagée précocement, dès 20 culots érythrocytaire transfusés et/ou un taux de ferritine > 1000 ng/ml, chez des patients potentiellement candidats à une allogreffe si leur SMD s'aggravait. Le moment de son début est plus discuté dans les autres cas. Sous deferasirox (généralement prescrit), il est nécessaire de surveiller initialement les fonctions rénales et hépatiques ; au départ à un rythme hebdomadaire. La réalisation annuelle d'un fond d'oeil et d'un audiogramme est recommandée en raison des risques de rétinopathie, d'opacification du cristallin ou de baisse de l'audition sous ces traitements.

Prophylaxie anti-infectieuse

Le risque infectieux et sa gravité varient selon le type de SMD, le traitement et le terrain du patient. La neutropénie est le principal facteur de risque de complications infectieuses, exposant principalement le patient à des infections bactériennes (bacilles gram négatif, staphylocoques, etc.) et fungiques invasives (aspergillose pulmonaire, mucormycose).

En pratique, il n'existe pas de données sur l'intérêt d'une antibioprophylaxie au long cours, d'autant plus que l'on prend le risque de favoriser l'émergence de résistance. Par contre, il peut se discuter l'instauration d'une prophylaxie antifungique primaire (neutropénie chronique sévère, corticothérapie ou autre traitement immunosuppresseur) ou secondaire (après une infection fungique invasive) par posaconazole 300 mg/j [START_REF] Maertens | European guidelines for primary antifungal prophylaxis in adult haematology patients: summary of the updated recommendations from the European Conference on Infections in Leukaemia[END_REF]. Il est par ailleurs souvent recommandé, chez les patients très neutropéniques, de disposer à domicile d'une association amoxicilline acide clavulanique et fluoroquinolone à débuter au moindre épisode fébrile, même si cette association n'a pas été validée prospectivement 6.3 Prise en charge des syndromes myélodysplasiques de haut risque

Azacitidine

Alors que la chimiothérapie intensive avait montré de mauvais résultats sur la survie globale, les HMA sont devenus le traitement de référence des SMD-HR (IPSS ≥1,5 ou IPSS-R >4,5). Leur mécanisme d'action supposé est de permettre la réexpression de certains gènes suppresseurs de tumeur dont le promoteur hyperméthylé est inactivé. L'essai de phase III, ayant conduit à l'AMM de l'AZA en Europe pour les SMD-HR (et leucémies aiguës entre 20 et 30% de blastes), a démontré un bénéfice médian en survie de 9,4 mois dans le bras AZA comparativement au traitement conventionnel [START_REF] Fenaux | Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study[END_REF]. Le bénéfice est essentiellement en lien avec l'amélioration des cytopénies et la bonne tolérance de cette molécule chez les sujets âgés, car l'effet sur l'histoire naturelle de la maladie n'est que partiel en retardant la survenue de la transformation. En effet le taux de rémission complète (disparition des blastes) est de 10% alors qu'une indépendance transfusionnelle est obtenue dans presque 50% des cas. La réponse est obtenue en médiane après 4 cycles de traitement pendant lesquels on peut observer une aggravation initiale des cytopénies, nécessitant des soins de support.

En pratique : L'AZA est utilisée pour le traitement des SMD-HR à la posologie de 75mg/m 2 /j, 7 jours tous les 28 jours par voie sous-cutanée (schéma thérapeutique particulièrement adapté à l'ambulatoire, l'hospitalisation de jour, voire l'hospitalisation à domicile). L'évaluation de l'efficacité du traitement se fait après 6 cycles. Les principaux effets secondaires sont une hémato-toxicité initiale, pouvant majorer le recours transfusionnel et s'associer à des infections pouvant conduire à des hospitalisations. La constipation (liée à l'utilisation des anti émétiques, car l'AZA est plutôt responsable de diarrhée) est fréquente et parfois sévère et nécessite une prévention systématique. Des rougeurs aux points d'injections sont fréquentes. En cas de toxicité excessive peut se discuter une adaptation du schéma posologique (cycle de 5 jours, espacement des cycles, diminution de la posologie, recours au G-cSF). La coordination entre les professionnels de santé est un enjeu important de la prise en charge des patients âgés co-morbides. La pluridisciplinarité (oncologues, gériatres, internistes, pharmaciens, médecins généralistes) est une caractéristique du parcours de soins de ces patients. 6.3.2 L'allogreffe de cellules souches hématopoïétiques L'ASCT est le seul traitement curatif des SMD-HR, généralement proposé jusqu'à l'âge de 70 ans environ en l'absence de co-morbidités significatives. Ses résultats s'améliorent au fil des années, grâce notamment à l'utilisation des conditionnements atténués et au meilleur traitement des complications post-greffe. De plus en plus de patients peuvent être allogreffés grâce à des donneurs alternatifs (y compris haploidentiques)(57,58).

Perspectives thérapeutiques

Alors que l'AMM de l'AZA a tout juste 10 ans et qu'il s'agissait de la 1 e obtention d'AMM pour un médicament dans cette indication en Europe, aucune association de l'AZA avec un autre médicament n'a démontré pour l'instant sa supériorité par rapport à l'AZA seule, notamment du fait de l'augmentation du profil de toxicité. De plus, le pronostic des SMD-HR est actuellement sombre après échec ou perte de réponse à l'AZA. De nouveaux agents sont cependant prometteurs : avant tout les inhibiteurs d'IDH1 et IDH2, utilisés soit en échec d'AZA soit en association avec AZA lorsque la mutation correspondante est présente ; le venetoclax, un inhibiteur de bcl2, vient d'obtenir une AMM en association avec un HMA dans les LAM du sujet âgé, et est actuellement testé dans les SMD-HR ; enfin l'APR 246 , agent « reconformant » des protéines p53 mutées semble donner de bons résultats en association avec AZA dans les SMD-HR avec caryotype complexe et mutation TP 53. L' apparition de ces traitements « ciblés », même si elle concerne encore un nombre limité de patients, incite à réaliser de façon de plus en plus systématique la recherche de mutations comme IDH1, IDH2 et TP 53 dans les SMD-HR(59).

Conclusion

Les nouveaux outils cytogénétiques et moléculaires, la prise en considération de facteurs non liés à la maladie (tels que les co-morbidités) ou encore l'évaluation gériatrique standardisée pour les patients les plus âgés, vont permettre d'élaborer dans les années à venir une prise en charge personnalisée des SMD. Parallèlement, la collaboration villehôpital est la clé d'un parcours de soin efficace. Bien que de nouveaux traitements aient montré des résultats encourageants, le pronostic des SMD-HR reste sombre chez les patients ne pouvant pas être allogreffés , l'AZA ne donnant que des résultats transitoires. Pour les SMD de faible risque, le problème principal reste celui du traitement de l'anémie, nécessitant très souvent des transfusions itératives malgré les quelques nouveaux traitements dont nous disposons.

Déclarations d'intérêts

Les auteurs déclarent ne pas avoir de conflits d'intérêts en relation avec cet article.

Tableaux et

  Figures Tableau 1 : Classification des syndromes myélodysplasiques selon Arber et al.(26) Proposition d'algorithme de prise en charge des syndromes myélodysplasiques en 2019 SMD-LR : syndrome myélodysplasique de faible risque ; SMD-HR : syndrome myélodysplasique de haut risque, hb : hémoglobine ; sEPO : érythropoïétine sérique ; ASE : agent stimulant l'érythropoïèse ; del5q : délétion du bras long du chromosome 5 ; AHM : agents hypométhylant ; IS : immunosuppresseur ; ASCT : allogreffe de cellules souches hématopoïétiques ; ARTPO : agoniste du récepteur de la thrombopoïétine Tableau 1 : Classification des syndromes myélodysplasiques selon Arber et al.(26) SMD : Syndrome myélodysplasique ; del5q : délétion du bras long du chromosome 5 Valeurs pronostiques des anomalies cytogénétiques d'après Greenberg et al.(29) Tableau 3 : Score IPSS-R (Revised International Prognostic Scoring System) d'après Greenberg et al.(29) Tableau 4 : Pronostic selon le score IPSS-R d'après Greenberg et al.[START_REF] Greenberg | Revised international prognostic scoring system for myelodysplastic syndromes[END_REF]

	Nom Tableau 2 : Groupes selon le caryotype (fréquence %) Groupe pronostique SMD avec atteinte unilignée Très bon (4%) Bon (72%) Score Très bon SMD avec atteinte multilignée Intermédiaire (13%) % blastes Bon médullaires SMD avec Mauvais (4%) Cytogénétique Intermédiaire atteinte unilignée et Très mauvais (7%) Défavorable Hémoglobine (g/dL) sidéroblastes Très bon Lignée(s) dysplasique(s) Anomalies cytogénétiques Cytopénies Survie médiane Sidéroblastes en couronne Survie médiane sanguins (BS) Délai médian d'évolution Blastes médullaires (BM) et Caryotype (années) en LAM (pour 25% des Transformation Nombre de points (ans) leucémique (25%, ans) patients), année conventionnel 1 1 ou 2 < 15% / < 5%* BM < 5% BS < 1% -Y, del(11q) 5,4 Non atteinte Normal, del(5q), del(12p), del(20q), 4,8 9,4 0 point 0,5 point 1 point 1,5 2 points 3 points 4 points points ≤ 1,5 8,8 Non atteinte deux anomalies incluant del(5q) Tous sauf del 5q 2 ou 3 1-3 < 15% / < 5%* BM < 5% BS < 1% Del(7q), +8, +19, i(17q), toute autre anomalie simple ou double dans des 2,7 2,5 ≤ 2% _____ > 2 < 5% _____ 5-10% >10% _____ 2-3 5,3 10,8 clones indépendants Tous sauf del 5q -7, inv(3)/t(3q)/del(3q), deux anomalies incluant -7/del(7q), complexe avec 3 anomalies 1,5 1,7 _____ Bon _____ > 3-4,5 3 3,2 Très Intermédiaire Mauvais mauvais 1 1 ou 2 > 15% / > 5% BM < 5% BS < 1% Tous sauf del 5q Complexe avec plus de 3 anomalies 0,7 0,7 > 4,5-6 1,6 1,4 ≥ 10 _____ 8 < 10 < 8 _____ _____ _____ en couronne Très défavorable > 6 0,8 0,73
	PNN (G/L)	≥ 0,8	< 0,8	_____	_____	_____	_____	_____
	SMD avec
	atteinte multilignée et sidéroblastes en Plaquettes (G/L) ≥ 100 PNN : polynucléaires neutrophiles 2 ou 3 50 -99	1-3 < 50	> 15% / > 5%* _____	_____ BM < 5% BS < 1%	_____ Tous sauf del 5q _____
	couronne
							Del 5q isolée ou
							associée à 1
	SMD avec del 5q isolée	1-3		1-2	aucun	BM < 5% BS < 1%	anomalie excepté -7 ou
							del7q
	SMD avec excès de blastes-1	0-3		1-3	aucun	BM 5-9% BS 2-4%	Toutes les anomalies
	SMD avec excès de blastes-2	0-3		1-3	aucun	BM 10-19% BS 5-19%	Toutes les anomalies
	SMD inclassés	Anomalie cytogénétique isolée, atteinte unilignée avec pancytopénie

SMD : Syndrome myélodysplasique ; del5q : délétion du bras long du chromosome 5 Tableau 2 : Valeurs pronostiques des anomalies cytogénétiques d'après Greenberg et al.(29) Tableau 3 : Score IPSS-R (Revised International Prognostic Scoring System) d'après Greenberg et al.(29) PNN : polynucléaires neutrophiles Tableau 4 : Pronostic selon le score IPSS-R d'après Greenberg et al.(29) Figure 1 : Mutations somatiques retrouvées dans les syndromes myélodysplasiques, d'après James A. Kennedy and Benjamin L. Eber(30) 2 :


Justine Creff

Rémi Courson

Thomas Mangeat

Julie Foncy

Sandrine Assié-Souleille

Christophe Thibault

Arnaud Besson
email: arnaud.besson@univ-tlse3.fr

Laurent Malaquin
email: laurent.malaquin@laas.fr

Sandrine Souleille

Fabrication of 3D scaffolds reproducing intestinal epithelium topography by high-resolution 3D stereolithography

Keywords: 3D printing, scaffold, intestine, crypt, villus, hydrogel

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

Introduction

The primary function of the intestinal tract is the digestion and absorption of nutrients. To maintain optimal function and tissue integrity, the intestinal epithelium is constantly renewed.

The epithelium of the small intestine is organized into a large number of "crypt-villus" units.

Villi are finger-like structures covered by a simple epithelium of differentiated cells projecting into the intestinal lumen to maximize nutrient absorption. The base of each villus is surrounded by several invaginations, called crypts of Lieberkühn. The constant renewal of the epithelium is driven by intestinal stem cells (ISCs) that reside at the crypt base [START_REF] Barker | Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration[END_REF][START_REF] Barker | Identification of stem cells in small intestine and colon by marker gene Lgr5[END_REF][START_REF] Santos | The Intestinal Stem Cell Niche: Homeostasis and Adaptations[END_REF]. ISCs proliferate and produce progenitors, called transit-amplifying cells, localized just above the stem cell zone in the crypt. These progenitors undergo several rounds of division and migrate along the crypt-villus axis while they terminally differentiate into different cell types, including enteroendocrine, tufts and goblet cells, and enterocytes [START_REF] Barker | Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration[END_REF][START_REF] Santos | The Intestinal Stem Cell Niche: Homeostasis and Adaptations[END_REF][START_REF] Clevers | The intestinal crypt, a prototype stem cell compartment[END_REF]. Stem cell self-renewal and progenitor differentiation are supported by the crypt microenvironment, or niche. This microenvironment changes along the crypt-villus axis and determines the balance between proliferative and differentiation signals [START_REF] Santos | The Intestinal Stem Cell Niche: Homeostasis and Adaptations[END_REF]. For instance, the Wnt, Notch and Noggin growth factors are present in decreasing gradients from the crypt bottom [START_REF] He | BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling[END_REF][START_REF] Clevers | Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control[END_REF]. Additional signals provided by the extracellular matrix (ECM), microbiota and immune cells are also important for ISC maintenance and progenitor differentiation [START_REF] Santos | The Intestinal Stem Cell Niche: Homeostasis and Adaptations[END_REF][START_REF] Pageot | Human cell models to study small intestinal functions: recapitulation of the crypt-villus axis[END_REF]. Despite numerous studies, the exact mechanisms governing intestinal homeostasis remain poorly understood, possibly due to the lack of in vitro models to grow and study ISCs and their progeny.

Recent progress in stem cell technology has allowed developing three-dimensional (3D) cultures from primary mouse and human adult ISCs [START_REF] Lancaster | Organogenesis in a dish: modeling development and disease using organoid technologies[END_REF][START_REF] Sato | Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium[END_REF][START_REF] Sato | Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche[END_REF]. In these cultures, cells self-organize into intestinal organoids exhibiting self-renewing crypt compartments, with proliferative cells that differentiate to give flat "villi-like" domains. While these 3D systems recapitulate the heterogeneity of the intestinal tissue in vitro, they present several limitations, notably their spherical architecture and lack of lumen. Moreover, these structures cannot support the formation of signaling gradients and mechanical forces, and therefore do not fully recreate the native microenvironment [START_REF] Yin | Engineering Stem Cell Organoids[END_REF].

To address these limitations, several studies reported the development of new engineered culture systems to study the intestinal epithelium [START_REF] Yu | Three dimensional human small intestine models for ADME-Tox studies[END_REF]. In particular, recent advances in microfabrication technologies and biomaterials made possible the development of new 3D in vitro systems more physiologically relevant to study the intestinal epithelium [START_REF] Yin | Engineering Stem Cell Organoids[END_REF][START_REF] Bhatia | Microfluidic organs-on-chips[END_REF][START_REF] Rossi | Progress and potential in organoid research[END_REF]. Most of these systems use Caco-2 cells as tissue mimic. Caco-2 are human colorectal adenocarcinoma cells that spontaneously differentiate into a polarized epithelium exhibiting characteristics similar to small intestine enterocytes upon reaching confluence [START_REF] Hidalgo | Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability[END_REF]. In order to recreate the architecture of the intestinal epithelium, various 3D micro-patterned scaffolds mimicking intestinal villi [START_REF] Costello | Synthetic small intestinal scaffolds for improved studies of intestinal differentiation[END_REF][START_REF] Costello | March, 3-D intestinal scaffolds for evaluating the therapeutic potential of probiotics[END_REF][START_REF] Yu | In vitro 3D human small intestinal villous model for drug permeability determination[END_REF][START_REF] Chen | Robust bioengineered 3D functional human intestinal epithelium[END_REF][START_REF] Esch | On chip porous polymer membranes for integration of gastrointestinal tract epithelium with microfluidic 'body-on-a-chip' devices[END_REF][START_REF] Sung | Microscale 3-D hydrogel scaffold for biomimetic gastrointestinal (GI) tract model[END_REF] or crypts [START_REF] Wang | Influence of micro-well biomimetic topography on intestinal epithelial Caco-2 cell phenotype[END_REF] have been developed, mostly using molding or lithography technologies.

These studies have clearly demonstrated the interplay between cell differentiation, polarization and 3D microenvironment. Recently, a 3D model reproducing the crypt/villi architecture and biochemical gradients using micro-molded cross-linked collagen was developed to culture primary human intestinal cells [START_REF] Wang | A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium[END_REF][START_REF] Wang | Formation of Human Colonic Crypt Array by Application of Chemical Gradients Across a Shaped Epithelial Monolayer[END_REF]. This model has shown that 3D topology, in addition to other physical and chemical cues, is a key factor to promote cell differentiation and polarization. However, due to limitations in the resolution and/or complexity of the fabrication techniques, many of these 3D models still fail to fully recapitulate or accurately control the topology of crypt/villi compartments. In this work, we investigated the capabilities of 3D printing to improve the resolution and complexity of topographies accessible for scaffold fabrication compared to molding technologies. Recent advances in additive manufacturing technologies such as extrusion printing, inkjet printing or laser assisted printing now allow printing biocompatible materials with full flexibility on the design and shape. An attractive method for bioprinting is stereolithography (SLA) that uses a light-induced polymerization process [START_REF] Murphy | 3D bioprinting of tissues and organs[END_REF][START_REF] Mandrycky | 3D bioprinting for engineering complex tissues[END_REF]. This approach offers the possibility to rapidly create complex objects at high resolution using a large range of photosensitive materials including rigid photoresists or synthetic hydrogels [START_REF] Zhang | Stereolithographic hydrogel printing of 3D culture chips with biofunctionalized complex 3D perfusion networks[END_REF][START_REF] Zhang | Rapid fabrication of complex 3D extracellular microenvironments by dynamic optical projection stereolithography[END_REF]. This technology has been used for cell culture, either for the fabrication of scaffolds or for direct printing of cells encapsulated in ECM [START_REF] Gauvin | Microfabrication of complex porous tissue engineering scaffolds using 3D projection stereolithography[END_REF][START_REF] Hribar | Light-assisted direct-write of 3D functional biomaterials[END_REF]. However, most of the approaches reported so far are still limited by the resolution of the printing technologies, typically above 50 µm. Two-photons based techniques may offer a resolution in the submicrometer range but are still strongly limited by the printing speed and by the maximum sample volume that is accessible. We recently reported a new 3D SLA printing technology that compensates for these limitations [START_REF] Accardo | Direct laser fabrication of meso-scale 2D and 3D architectures with micrometric feature resolution[END_REF]. It combines a resolution of 5 µm in x, y and z and the possibility to print large objects (maximum 10(x) x 10(y) x 5(z) cm 3 ). This resolution enables to print scaffolds mimicking the architecture and dimensions of the intestinal epithelium.

The choice of the material used to create the scaffold is critical. To improve biocompatibility and relevance for bioengineered systems, various materials have been investigated, such as Polydimethylsiloxane (PDMS), SU-8 epoxy resin, collagen, or silk [START_REF] Costello | Synthetic small intestinal scaffolds for improved studies of intestinal differentiation[END_REF][START_REF] Costello | March, 3-D intestinal scaffolds for evaluating the therapeutic potential of probiotics[END_REF][START_REF] Chen | Robust bioengineered 3D functional human intestinal epithelium[END_REF][START_REF] Esch | On chip porous polymer membranes for integration of gastrointestinal tract epithelium with microfluidic 'body-on-a-chip' devices[END_REF][START_REF] Sung | Microscale 3-D hydrogel scaffold for biomimetic gastrointestinal (GI) tract model[END_REF][START_REF] Wang | Influence of micro-well biomimetic topography on intestinal epithelial Caco-2 cell phenotype[END_REF]. While natural materials are very effective to support cell growth and differentiation, they are often not completely defined and contain animal-derived products that preclude clinical applications.

Moreover, ECM such as collagen are mostly biodegradable and the 3D topography is not conserved in long-term cultures [START_REF] Yu | In vitro 3D human small intestinal villous model for drug permeability determination[END_REF]. A common alternative for tissue engineering is the use of synthetic polymers such as poly(ethylene)glycol diacrylate (PEG-DA) or polyacrylamide [START_REF] Phelps | Bioartificial matrices for therapeutic vascularization[END_REF][START_REF] Smithmyer | Hydrogel scaffolds as in vitro models to study fibroblast activation in wound healing and disease[END_REF]. These synthetic hydrogels are more controllable and offer more flexibility to control mechanical and chemical properties in a reproducible manner. Moreover, these materials are compatible with 2D/3D fabrication through photolithography based approaches [START_REF] Hribar | Light-assisted direct-write of 3D functional biomaterials[END_REF][START_REF] Moon | Micropatterning of poly(ethylene glycol) diacrylate hydrogels with biomolecules to regulate and guide endothelial morphogenesis[END_REF][START_REF] Urrios | 3D-printing of transparent bio-microfluidic devices in PEG-DA[END_REF].

Beyond their reproducibility, another advantage of synthetic materials is the possibility to modify them, allowing their tuning with degradation and/or adhesion sites or even growth factors [START_REF] Phelps | Bioartificial matrices for therapeutic vascularization[END_REF][START_REF] Verhulsel | A review of microfabrication and hydrogel engineering for micro-organs on chips[END_REF][START_REF] Lutolf | Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering[END_REF][START_REF] Caliari | A practical guide to hydrogels for cell culture[END_REF]. For instance, to support cell growth and colonization, adhesion ligands such as RGD peptides or laminin can be grafted to the hydrogel [START_REF] Guarnieri | Covalently immobilized RGD gradient on PEG hydrogel scaffold influences cell migration parameters[END_REF]. Synthetic hydrogels based on poly(ethylene)glycol polymer (such as PEG-4MAL or 8arm-PEG4) have been used to replace Matrigel in 3D intestinal organoid cultures to improve the control and reproducibility over matrix physicochemical properties [START_REF] Cruz-Acuna | Synthetic hydrogels for human intestinal organoid generation and colonic wound repair[END_REF][START_REF] Gjorevski | Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture[END_REF]. Here, we developed a novel photosensitive biomaterial based on an intricate network composed of PEG-DA and acrylic acid polymer chains that allows cell adhesion and proliferation. Whereas many material formulations may require complex synthesis or numerous functionalization steps, this material is simple to prepare and can be functionalized easily with ECM proteins to improve cell attachment and proliferation.

Moreover, it combines a high optical transparency for microscopy imaging with tunable stiffness properties. Using high-resolution stereolithography technology, we 3D printed this material with a micrometric resolution and successfully reproduced the topography and dimensions of the different compartments of the mouse intestinal epithelium. Finally, we showed that this culture system supports the growth of Caco-2 cells and allows the formation of a polarized and differentiated epithelium in three dimensions.

Experimental section

Cell culture

Caco-2 cells were cultivated in DMEM 4.5 g/L glucose (Sigma, D6429) supplemented with 20% Fetal Bovine Serum (FBS, Life Technologies, 10270106), 0.1 mM non-essential amino acids (Sigma, M7145) and 2 µg/mL penicillin/streptomycin (Sigma, P4333). SW480 cells were cultivated in RPMI 2 g/L glucose (Sigma, R8758) supplemented with 10% FBS, 0.1 mM nonessential amino acids, 2 µg/mL penicillin/streptomycin and 2.5 g/L D-Glucose (Sigma, G7021).

All cells were grown at 37°C and 5% CO2. For cell culture on 2D hydrogels, Caco-2 or SW480 cells were seeded at a density of 20,000 cells.cm -2 and grown for 6 days with medium changes every 2-3 days. For 3D cultures or control 2D cultures, Caco-2 cells were plated at a density of 75,000 cells.cm -2 in conditioned media (culture supernatant) and kept in culture for up to 3 weeks with medium changed every 2-3 days.

Hydrogel preparation

Hydrogels were prepared as described previously [START_REF] Chen | Synthesis of Nonspherical Microcapsules through Controlled Polyelectrolyte Coating of Hydrogel Templates[END_REF]. The photoinitiator Phenylbis(2,4,6trimethyl-benzoyl)phosphine oxide (Irgacure 819, Sigma, 511447) was dissolved at 0.1% (wt/vol) in PEG-DA MW 700 (Sigma, 455008). To prepare hydrogels, the different components were mixed in a conical tube: PEG-DA 700 (30-60%) (vol/vol) containing the photoinitiator, 30% acrylic acid (Sigma, 147230) (vol/vol) supplemented or not with either 250 µg/ml fibronectin (Corning, 356008), 50 µg/ml laminin (Sigma, L2020) or 1% collagen-1 (Sigma, C4243), the volume was then completed with culture medium (10-40%) (vol/vol). Ultraviolet crosslinking was performed using a UV lamp (365 nm, 12 W) or the Dilase 3D printer (Kloé, France). Polymerized hydrogels were washed overnight in culture medium prior to cell seeding.

3D printing of intestinal scaffold

Intestinal scaffolds were printed by stereolithography with a Multiresolution Dilase 3D setup (Kloé SA, France) (Fig. 1). This equipment and its performances have been described in our previous work [START_REF] Accardo | Direct laser fabrication of meso-scale 2D and 3D architectures with micrometric feature resolution[END_REF]. STL file, made with CAO 3D software, was sliced using the 3D slicer software (Kloé). The first 200 µm (crypt part) were sliced in 50 µm thick layers (four layers) and printed with a 5 µm spot size, while villi were sliced in 20 µm layers (35 layers) and printed with a 20 µm spot size. The total dimensions of the sample were 4x4x0.7 mm 3 for 500µm high villi and 4x4x1.7 mm 3 for 1500µm high villi (Fig. 3). To ensure adhesion of the PEG-DA hydrogel during the 3D printing process, a DS-3000 (DWS, Italy) substrate of 1x1x0.05 cm 3 was prepared by 3D printing using DWS 29J+ or B9 creator® systems. Indeed, just after the development step, the DS-3000 surface (last printed layer) is still reactive because of the uncrosslinked polymer chains that was in contact with the PDMS in the printer vat [START_REF] Accardo | Direct laser fabrication of meso-scale 2D and 3D architectures with micrometric feature resolution[END_REF]. Consequently, PEG-DA can create covalent bonds with pending acrylate functions on the DS-3000 surface.

Regarding the parameters employed for the production of the 3D scaffold, a pedestal of 50 µm was first fabricated with 100% laser power and 15 mm/s writing speed. This pedestal allowed a strong adhesion on substrate and ensured parallelism between the substrate and the top of the tank. Then, a laser power of 100% and a writing speed of 20 mm/s were set to fabricate the rest of the scaffold. Total fabrication time of the structure was 12 h. After laser writing, the sample was removed from the building table and developed by two successive water baths, the sample was kept in water to prevent dehydration and deformation of the structure. Before cell seeding, 3D scaffolds were sterilized using UV light (365 nm, 12 W) for 20 min and washed 3 times with culture medium.

Immunofluorescence

Cells were rinsed with PBS and fixed with 2% PFA for 20 min at 37°C. Cells were permeabilized using PBS-0.2% Triton X-100 for 3 min. After three 5 min washes in PBS, cells were incubated with primary antibodies diluted in PBS + 3% BSA + 0.05% Tween 20 + 0.08% sodium azide for 1 h at 37 °C or with phalloïdin conjugated to FITC or rhodamine (Interchim) at 1/500 for 30 min at 37°C. Primary antibodies used for immunofluorescence were: Mouse anti Pan-cytokeratin (Cell signaling, C11 #4545 [1/100]), Goat anti Villin (Santa Cruz Biotechnology, C-19, sc-7672 [1/50]) and Mouse anti β-catenin (BD Biosciences #610153 [1/200]). After three 5 min washes in PBS, cells were incubated for 30 min at 37°C with Cy2conjugated secondary antibodies (Jackson ImmunoResearch) at 1/400 dilution. For F-actin costaining, phalloïdin was added to secondary antibody solution. Cells were then rinsed three times 5 min in PBS, with the first wash containing Hoechst 33342 (0.1 µg/mL) or DRAQ5 (Thermo Fisher Scientific) at 1/1000 for nuclear DNA staining.

Alkaline phosphatase activity

Alkaline phosphatase activity was assessed with Blue Alkaline Phosphatase (Blue AP) Substrate kit (Vector Laboratories, SK-5300), according to the manufacturer's protocol. Briefly, living cells were rinsed with PBS and incubated for 30 min at 37°C with Blue AP substrate in Tris Buffer (150 mM, pH 8.4), rinsed with PBS and fixed with 4% PFA for 20 min at 37°C.

Atomic Force Microscopy (AFM)

AFM measurements were performed using Borosilicate Glass Particle (10 µm) on silicon nitride cantilevers (PT.BORO.SN.10, Novascan Technologies) with a nominal spring constant of 0.06 N/m mounted on a NanoWizard III AFM (JPK Instruments) coupled to an inverted optical microscope (Axiovert 200, Carl Zeiss). To ensure reproducibility in force application and measurement, the cantilever sensitivity and spring constant were calibrated before each experiment using the JPK Instrument software using the thermal noise method. Force-distance curves were recorded in contact mode in liquid at 2 Hz (1 s per approach-retract force curve) with an applied force of 1 nN. Each hydrogel was tested at 4 locations with about 250 force curves acquired per location and 512 data points per curve.

Surface topography imaging was performed by AFM using a MLCT cantilever (Bruker) with a nominal spring constant of 0.07 N/m on a NanoWizard III AFM (JPK Instrument). Imaging 3D scaffold was realized at the top of the villi.

Image acquisition and analysis

Images of 2D cultures and brightfield images of 3D scaffolds and 2D hydrogels were captured on a Nikon Eclipse 90i microscope using a Nikon DS-Qi2 HQ camera or a DS-U3 color camera (Nikon) and NIS Element BR software. Two-photon confocal imaging was performed using an upright multiphoton Zeiss LSM 7MP microscope equipped with a pulsed laser adjustable from 690 to 1080 nm and a 20X plan-Apo water immersion objective.

Confocal images were acquired on an upright Leica SP8 confocal microscope equipped with 2 PMT detectors and 488 nm, 552 nm and 638 nm diodes and a 25X physio objective.

For Light Sheet Fluorescence Microscopy, samples were first embedded in 1% low-melt agarose and specimens were imaged from the side with a 2X objective and a 561 nm laser. SEM imaging was performed using a Hitachi S-3700 microscope with an acceleration voltage ranging from 1,5 kV to 2 kV. Dynamic Speckle Imaging (DSI) [START_REF] Ventalon | Dynamic speckle illumination microscopy with wavelet prefiltering[END_REF] was performed for whole 3D epithelium high-resolution imaging. This technique produces fast 3D resolution imaging and combines high sectioning properties together with high lateral resolution. The method consists in producing hundreds (200 during the experiments performed in this work) of speckle illumination in each acquisition plane along the depth of the sample. A post treatment combining pre-filtering of each raw image with statistical analysis based on the square root of the variance was made with a homemade Matlab software. This process enables a confocal imaging of the entire biological object and preserves the lateral resolution up to 700 µm depth. Indeed, speckle illumination is known to be poorly sensitive to optical aberrations or scattering [START_REF] Goodman | Speckle phenomena in optics: theory and applications[END_REF] and this reduces problems of working distance (a 0.14 NA microscope lens was used for the collection of fluorescence emission). All images were treated and analyzed with the Fiji Software.

Statistical analyses

Statistical analyses were performed on three independent experiments using the GraphPad Prism 6.0 Software. Differences between groups were considered statistically significant if p < 0.05.

Data were compared using one-way ANOVA followed by Bonferroni correction for multiple comparisons. Data are presented as mean +/-SEM.

Results

Development of a new photosensitive hydrogel

The goal of this study was to develop a new 3D model of intestinal epithelium using highresolution 3D printing (Fig. 1). One of the main challenges was to develop a material compatible with cell culture adapted to a 3D printing process and offering optical properties suitable for fluorescence microscopy imaging. We selected PEG-DA polymer as a base material to form hydrogel through radical polymerization in presence of photoinitiator [START_REF] Accardo | Multiphoton Direct Laser Writing and 3D Imaging of Polymeric Freestanding Architectures for Cell Colonization[END_REF]. PEG-DA hydrogels were previously reported to constitute poor substrates for cellular adhesion due to their protein repelling properties [START_REF] Phelps | Bioartificial matrices for therapeutic vascularization[END_REF][START_REF] Guarnieri | Covalently immobilized RGD gradient on PEG hydrogel scaffold influences cell migration parameters[END_REF]. To improve cellular adhesion, we investigated the addition of acrylic acid to PEG-DA to form composite hydrogel networks. Acrylic acid reacts with PEG-DA and provides free carboxylic groups that negatively charge the resulting hydrogel. These groups were shown to promote protein adhesion at neutral pH (Fig. 2A) [START_REF] Chen | Synthesis of Nonspherical Microcapsules through Controlled Polyelectrolyte Coating of Hydrogel Templates[END_REF]. After polymerization, the remaining acrylic acid species that did not react were extracted from the polymer matrix by several washes in culture medium to reestablish a neutral pH in the scaffold and avoid potential toxicity on cell cultures. Cell adhesion and biocompatibility of PEG-DA hydrogels complemented or not with acrylic acid and/or ECMs were evaluated after 6 days of culture with SW480 colorectal cancer cells (Fig. 2B,C). While the presence of fibronectin in pure PEG-DA hydrogel was not efficient to promote cell adhesion, addition of both acrylic acid and fibronectin dramatically improved cell adhesion and growth, and after 6 days of culture a confluent monolayer was observed (Fig 2B). We investigated the influence of PEG-DA on cell culture using hydrogels containing proportions of PEG-DA 700 varying from 30% up to 60% (Fig. S1).

A minimum concentration of 30% (vol/vol) PEG-DA was necessary to permit Irgacure 819 solubilization and allow polymerization. We found that 40% of PEG-DA provided the best adhesion and proliferation for SW480 cells.

Comparisons of different ECMs, including fibronectin, collagen-1 and laminin were also performed. Results indicated that these three matrices similarly support cell adhesion and proliferation (Fig. 2C). Likewise, the optimal concentration of each matrix within the hydrogel was determined for SW480 cells (Fig. S2). Overall, fibronectin at 250 µg/mL was identified as the most favorable concentration for cell adhesion and was chosen for subsequent experiments.

These results were confirmed with two other cell lines, Caco-2 (Fig. S3) and HeLa cells (data not shown). Importantly, addition of fibronectin to PEG-DA hydrogel in absence of acrylic acid did not efficiently promote cell adhesion, and the presence of acrylic acid with fibronectin or laminin was necessary to allow cell adhesion and support cell growth for 6 days (Fig. S3). Thus, the retained hydrogel formulation for subsequent experiments was 40% PEG-DA 700 + 30% acrylic acid + 250 µg/mL fibronectin + 0.1% Irgacure 819 + 30 % culture medium.

It is difficult to provide a hierarchical ranking of the parameters influencing cell behavior. As a first hypothesis, the variation of the PEG-DA/acrylic acid ratio may influence the density of carboxylic acid groups available that in return promotes protein and cell adhesion. Another hypothesis is that cell proliferation could be affected by the mechanical and viscoelastic properties of the hydrogel scaffold. All these parameters are closely dependent on the material composition and their interplay may strongly influence cell proliferation and differentiation.

Investigations are ongoing to decipher the influence of the PEG-DA/acrylic acid ratio on the material stiffness of the printed scaffold by Atomic Force Microscopy (AFM). Preliminary AFM data indicates a young modulus of 92 +/-9.8 kPa for the selected hydrogel formulation (40% PEGDA, 30% acrylic acid and 250 µg/mL fibronectin). Interestingly, addition of fibronectin induced a significant stiffening of the resulting material with a young modulus value varying from 40 +/-8.7 kPa for the PEG-DA/acrylic acid mix up to 90 kPa with fibronectin. This phenomenon may be explained by the interactions between fibronectin and the carboxylic groups provided by acrylic acid that induce a crosslinking effect in the matrix.

Fabrication of intestinal scaffolds

Three-dimensions scaffolds were printed using high-resolution stereolithography (SLA) 3D printing. The 3D printer (Dilase 3D, Kloe France) uses a 405 nm laser to print objects in a layerby-layer building process [START_REF] Accardo | Direct laser fabrication of meso-scale 2D and 3D architectures with micrometric feature resolution[END_REF]. Each individual scaffold measured 4x4 mm (x,y) with a total thickness of 0.75 mm. The topography was designed to reproduce the architecture and dimensions of the murine intestinal epithelium. Each single villus was surrounded by 6 crypts.

Villi were 500 µm high, with a diameter of 150 µm at the top and 300 µm at the base. Crypt wells were 200 µm deep with a diameter of 50 µm (Fig. 3A). Intestinal scaffolds were printed on a DS3000 photoresist backplane to promote attachment of the hydrogel. We did not observe any noticeable cytotoxic effect of DS3000 on cell culture (data not shown). Scanning electron microscopy (SEM) images showed faithful replication of the design and reproducibility of the printing (Fig. 3B). Dehydration of the hydrogel before SEM imaging resulted in partial collapse of the structures, impeding accurate measurement of the structures' dimensions. As PEG-DA is optically transparent in the visible range, 300 nm diameter fluorescent nanoparticles were added to the hydrogel to measure the 3D scaffold dimensions by two-photon microscopy and Light Sheet Fluorescence Microscopy (LSFM). Both imaging technologies allowed visualization of the villi of the scaffold. Although LSFM has an intrinsic resolution lower than two-photon imaging, it allows the visualization of a larger field including multiple rows of the scaffolds. Measures performed on two-photon images showed dimensions of 574 +/-15 µm (n=10), compared to an initial programmed value of 500 µm. This expansion was confirmed on several samples observed showing a reproducible swelling of approximately 15%. This swelling effect may be related to the variation of pH (from 3.5 to 7.4) occurring between the fabrication process and the development step following printing. However, measures performed by LSFM showed high correspondence to the programmed dimensions (Fig. 3C-D). These differences of measurement may be explained by the fact that in LSFM, images are acquired from the side of the object and not from the top as in two-photon microscopy. Moreover, before imaging with LSFM, the sample was embedded in agarose, which may result in shrinking of the sample. Two-photon microscopy did not allow visualizing the crypt compartments, likely due to optical aberrations. Visualization of the crypt was possible by LSFM, but with a low resolution not allowing accurate measurements (Fig. 3D).

Suitability of 3D scaffolds for tissue culture

Three-dimensional hydrogel scaffolds supported adhesion and growth of Caco-2 cells. After 6 days of culture, a cell monolayer was observed around villi, with no cells inside the gel (Fig. 4A-C, E). LSFM imaging showed that the scaffold surface was not entirely covered by cells (Fig. 4C), indicating that 6 days of culture may not be sufficient for cells to proliferate and migrate to cover the entire structure uniformly. Focus on the crypt compartment by confocal imaging showed that cells also formed a continuous monolayer inside the crypt-like wells of the scaffold (Fig. 4D). However, we only observed cells down to a 72 +/-4 µm depth. This value is significantly lower than the values of crypt depth measured before cell culture. We believe that the swelling effects observed earlier could result in a global compressive force and thus in a decrease of the crypt diameter that limits cell invasion within the structures. This swelling effect could be compensated by adapting the STL file and increasing crypt diameter to obtain the correct dimensions in culture conditions.

Effect of 3D culture on cell differentiation

Caco-2 cells are known to differentiate spontaneously over time. After 21 days of culture they acquire properties similar to intestinal enterocytes and form a highly polarized epithelium with tight junctions [START_REF] Hidalgo | Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability[END_REF][START_REF] Natoli | Good Caco-2 cell culture practices[END_REF]. To study the impact of our 3D model on Caco-2 cell differentiation, we compared cells grown on various substrates for up to 21 days. First, the impact of the hydrogel was evaluated. Caco-2 cells were seeded on 2D hydrogel printed with the Dilase 3D or on glass coverslip, coated or not with fibronectin. F-Actin staining showed that Caco-2 cells formed a confluent monolayer in all three culture conditions (Fig. 5). However, cells grown on glass, coated or not with fibronectin, formed a highly flattened epithelium and exhibited no apico-basal polarization of nuclei or actin (Fig. 5A,B). On the contrary, cells that grew on 2D hydrogels exhibited an elongated morphology with apical actin and basal nuclei (Fig. 5C). Interestingly, cultures on 2D hydrogel reproducibly formed 3D structures, previously described as villi-like structures [START_REF] Kim | Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow[END_REF][START_REF] Kim | Gut-on-a-Chip microenvironment induces human intestinal cells to undergo villus differentiation[END_REF]. For example, Fig. 5C shows a cross section of a 115 µm-high cellular structure displaying strong polarization of the actin network. This improved polarization of Caco-2 cells was not solely due to the presence of fibronectin in the hydrogel since cells that grew on fibronectin coated glass did not exhibit this characteristic morphology (Fig. 5B).

Polarization of Caco-2 cells was then evaluated on 3D intestinal scaffolds. High-resolution dynamic speckle imaging [START_REF] Ventalon | Dynamic speckle illumination microscopy with wavelet prefiltering[END_REF] allowed a complete visualization of the 3D scaffold and after 21 days of culture, cells had colonized it entirely, from the crypt base to the top of villi (Fig. 6A).

Observation of the scaffold at 21 days showed that the structure had maintained its integrity and that the hydrogel was not degraded by cells (Fig. 6B). However, villi dimensions were significantly higher compared to measures obtained directly after printing (villi height = 728 +/-64 µm after 21 days of culture, n=10), suggesting a progressive swelling of the gel over time (Fig. 6B). Monitoring of cellular morphology by F-actin staining showed that at 21 days post seeding, the epithelium was polarized, with apical actin and basal nuclei (visualized on the side of villi), while at 18 days of culture, the F-actin network was not fully organized yet (Fig. 6C,D). Caco-2 cells were previously shown to become more elongated during differentiation [START_REF] Peterson | An in vitro model for the analysis of intestinal brush border assembly. I. Ultrastructural analysis of cell contact-induced brush border assembly in Caco-2BBe cells[END_REF][START_REF] Bazellieres | Apico-basal elongation requires a drebrin-E-EB3 complex in columnar human epithelial cells[END_REF]. Thus, we measured the height of cells cultivated on different substrates on vertical sections. Interestingly, after 6 and 21 days of culture, cells grown on hydrogels (in 3D or 2D) were more elongated compared to cultures on glass coverslips (Fig. 6E), suggesting a better differentiation and confirming morphological observations. The presence of ECM in the hydrogel cannot explain this phenotype, as cells grown on fibronectin-coated glass exhibited poorly organized actin network and were less elongated than on 2D hydrogels (Fig. 6E). Cell length increased over time and was significantly higher in 3D at 21 days of culture compared to 6 days. However, only a slight elongation (< 3 µm) was observed on 2D hydrogels between 6 and 21 days of culture.

Finally, there was a marked increase in cell length when grown on 3D scaffolds compared to 2D hydrogel, suggesting that the 3D topography provides cues promoting cell polarization that are not available in 2D (Fig. 6E).

To further evaluate the importance of 3D topography, we determined the influence of villus length on cell differentiation. For this, scaffolds were printed with 500 µm or µm high villi and seeded with Caco-2 cells for 21 days (Fig. S4). Monitoring of cell morphology with F-actin and β-catenin staining and cell height measurements did not reveal any difference and we observed a polarized epithelium with elongated cells, apical actin and membrane β-catenin in both conditions (Fig. S4 B-D). Thus, villi lengthening from 500 µm to 1500 µm does not appear to improve cell polarization.

Improved differentiation of cells grown on 3D scaffolds may be caused by distinct mechanical properties of the hydrogel, as well as surface topography and roughness. The latter was assessed by AFM and SEM imaging (Fig. S5). By SEM, the surface of the hydrogel was slightly uneven, with a regular motif probably caused by laser writing (Fig. S5 A,B). Measurements of surface topography by AFM revealed that the surface of 3D scaffolds was rougher than that of 2D hydrogels (Fig. S5 C,D). Indeed, measurements performed on cross sections indicated that the Rugosity average (Ra) was 419.3 nm for 3D hydrogel compared to a Ra = 85.78 nm for 2D hydrogel.

Differentiated absorptive enterocytes exhibit strong expression of villin, cytokeratin, phosphatase alkaline and membrane localization of β-catenin. The expression of differentiation markers by

Caco-2 cells grown on 2D or 3D hydrogels was investigated after 21 days of culture (Fig. 7). As previously, cells exhibited strong apical F-actin staining in 3D, as well as extensive lateral junction localization of β-catenin, while these were only observed on limited subsets of cells on 2D hydrogels (Fig. 7A). Similarly, cytokeratin staining gave abundant apicolateral signal on cells grown on 3D scaffolds compared to 2D hydrogels where it was restricted to the lateral junction of only a subpopulation of cells (Fig. 7B). Strikingly, Caco-2 cells grown on 3D scaffolds expressed villin and exhibited alkaline phosphatase activity, while it was absent or gave only weak staining in cells on 2D hydrogel (Fig 7 C,D), indicating that 3D topography provides additional cues that promote cellular differentiation, independently of the adhesion substrate.

Interestingly, in 3D, villin and alkaline phosphatase staining were preferentially located towards the top of villi, suggesting a compartmentalization of the cells, with more differentiated cells located at the top of the villi, as observed in vivo. These results suggest that 3D architecture promotes cell differentiation and organization compared to 2D monolayer cultures.

Discussion

The intestinal epithelium presents a characteristic architecture with high aspect ratio villus and crypt compartments that exhibit distinct and intricate biochemical and biophysical environments.

The existing in vitro systems, either 2D monolayer or 3D organoid cultures, do not faithfully reproduce this complex 3D microenvironment. Thus, over the last few years, intense efforts have been devoted to recreate this complex environment and recapitulate the topography of the small intestine for in vitro culture using microengineering approaches, with varying degrees of success [START_REF] Wang | A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium[END_REF][START_REF] Wang | Bioengineered Systems and Designer Matrices That Recapitulate the Intestinal Stem Cell Niche[END_REF]. In this study, we developed a 3D scaffold mimicking the intestinal architecture using for the first time a PEG-DA/acrylic acid hydrogel enriched with fibronectin using a new 3D printing system [START_REF] Accardo | Direct laser fabrication of meso-scale 2D and 3D architectures with micrometric feature resolution[END_REF].

We found that both the use of a hydrogel material and the micro-structuration of the surface with 3D topology is strongly influencing cell behavior. In fact, we showed that compared to traditional 2D cultures, Caco-2 cells grown on a 3D scaffold exhibit an improved differentiation and acquisition of an epithelial morphology with expression of enterocyte markers. These results indicate that the 3D morphology of the scaffold strongly impacts cell differentiation in our model, highlighting the importance of the 3D topography on cellular behavior. Other studies performing cultures on 3D scaffolds made with other materials or providing gradient biochemical stimulation similarly found that a 3D microenvironment promotes cell differentiation and offer a more physiologically relevant model to study the intestinal epithelium [START_REF] Costello | Synthetic small intestinal scaffolds for improved studies of intestinal differentiation[END_REF][START_REF] Yu | In vitro 3D human small intestinal villous model for drug permeability determination[END_REF][START_REF] Sung | Microscale 3-D hydrogel scaffold for biomimetic gastrointestinal (GI) tract model[END_REF][START_REF] Wang | A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium[END_REF].

As a proof of concept, the scaffold was made with a single hydrogel and a uniform environment. However, multiple gradients (growth factors, ECMs, oxygen, inflammatory signals…) have been described in vivo along the length of the crypt/villus axis [START_REF] Wang | Bioengineered Systems and Designer Matrices That Recapitulate the Intestinal Stem Cell Niche[END_REF]. For instance, concentrations of ECM proteins vary and adopt a specific distribution depending on the compartment. Indeed, different laminin subtypes are regionally expressed along the crypt/villus axis: laminin α2 is enriched in the crypts, while laminin α5 and α3 are expressed in villi [START_REF] Meran | Intestinal Stem Cell Niche: The Extracellular Matrix and Cellular Components[END_REF][START_REF] Teller | Interactions between laminin and epithelial cells in intestinal health and disease[END_REF]. Laminin α5 was shown to play a crucial role in the maintenance of the mucosa and architecture, suggesting a relationship between ECM deposition and functional intestinal cell differentiation [START_REF] Mahoney | Laminin alpha 5 influences the architecture of the mouse small intestine mucosa[END_REF]. Other ECM proteins are also distributed regionally, including fibronectin, which is expressed preferentially in the crypt basement membrane [START_REF] Benoit | RGD-Dependent Epithelial Cell-Matrix Interactions in the Human Intestinal Crypt[END_REF]. Changes in ECM composition suggest variations of stiffness, although it has not been directly measured in vivo due to technical limitations, and ECM mechanical properties are known to be important in the regulation of stem cell proliferation and fate [START_REF] Guilak | Control of stem cell fate by physical interactions with the extracellular matrix[END_REF]. A recent study using defined matrices for intestinal organoid cultures highlighted the importance of biochemical and biophysical roles of ECM in intestinal stem cell proliferation and differentiation [START_REF] Gjorevski | Designer matrices for intestinal stem cell and organoid culture[END_REF]. Using customized PEG hydrogels, it was demonstrated that high matrix stiffness promotes ISCs expansion through YAP/Hippo pathway signaling, while soft and degradable matrices and the presence of laminin α1β1γ1 were needed for cell differentiation [START_REF] Gjorevski | Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture[END_REF][START_REF] Gjorevski | Designer matrices for intestinal stem cell and organoid culture[END_REF]. For these reasons, it would be interesting to print 3D scaffolds using several hydrogels to recreate the different ECM composition to mimic these gradients along the crypt/villus axis and recapitulate more accurately the intestinal environment.

Moreover, synthetic materials such as PEG-DA hydrogels are chemically and physically tunable, allowing their modification to modulate regional stiffness. In this work, we investigated the impact of both concentrations of PEG-DA and acrylic acid. We believe that the composition of the material may influence both the surface roughness and the mechanical properties, in particular the material's stiffness that could in return impact cell adhesion and proliferation.

AFM measurements are currently in progress to investigate the correlation between cell culture results and the local stiffness sensed by the cells individually or when forming an epithelium.

Finally, gradients of soluble growth factors in the intestine are critical to control cell specification and proliferation. Among these, the Wnt/β-catenin pathway plays a crucial role for the maintenance and proliferation of intestinal stem cells [START_REF] Pinto | Canonical Wnt signals are essential for homeostasis of the intestinal epithelium[END_REF], Wnt ligands are secreted by several crypt cells including Paneth cells and mesenchymal cells surrounding the crypts [START_REF] Gregorieff | Expression pattern of Wnt signaling components in the adult intestine[END_REF][START_REF] Kabiri | Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts[END_REF].

Other signaling pathways that are essential in the crypt include Notch, BMPs and Eph/Ephrin [START_REF] Clevers | The intestinal crypt, a prototype stem cell compartment[END_REF]. Most of these signals are secreted by the mesenchymal compartment, which contains multiple cell populations [START_REF] Powell | Mesenchymal cells of the intestinal lamina propria[END_REF]. These reports underscore the importance of the cellular niche in the intestinal environment and to fully recreate a physiological in vitro model, this cellular niche should be integrated. With recent progress in bioprinting, it is now possible to print 3D matrices containing cells [START_REF] Madden | Bioprinted 3D Primary Human Intestinal Tissues Model Aspects of Native Physiology and ADME/Tox Functions[END_REF]. With adequate materials and 3D printing technology, we can now envision printing 3D scaffolds mimicking the architecture of intestinal epithelium and containing supporting mesenchymal cells.

Conclusion

Herein, we describe a new 3D culture model mimicking the architecture of the intestinal epithelium. First, we developed a PEG-DA/acrylic acid hydrogel mix that supports cell adhesion and growth. This material is optically transparent, it exhibits a good compatibility with cell culture and can be easily tailored with a large variety of matrices. We then showed that this hydrogel mix can be advantageously combined with stereolithography to generate 3D scaffolds accurately recapitulating the intestinal topography. In addition, we showed by image analyses that compared to 2D cultures, cell culture on these 3D hydrogel scaffolds improved differentiation of Caco-2 intestinal epithelial cells. This 3D model could constitute an in vitro platform for studying fundamental mechanisms that drive intestinal homeostasis and regeneration. Beyond its application to intestinal tissues, the proposed approach provides a generic fabrication method that directly benefits from the flexibility of 3D printing technologies for the fabrication of 3D architectures with a resolution at the sub-cellular level. It opens new routes for the fabrication of in vitro microenvironment models with controlled topography either devoted to fundamental studies or used as standardized platforms for drug testing. 3D intestinal scaffolds were printed using a Dilase 3D high-resolution printer. The object is printed layer by layer of 20 to 50µm using a 405nm laser (A-B). Scaffolds were then seeded with colorectal cancer cells that were grown for up to 21 days (C). Representative phase-contrast images of SW480 cells cultured for 6 days on various hydrogels.

Bar graphs show the number of cells counted per field (4 fields with a 20x objective). Cell were grown on 30%, 40%, 50% and 60% PEG-DA 700 hydrogels ± acrylic acid ± fibronectin. Scale bars = 50 µm. Images and graph for the 40% condition are reproduced from Fig. 2B.

Figure 1 :

 1 Figure 1: Schematic of the experimental strategy.

Figure 2 :

 2 Figure 2: Development of a new photosensitive hydrogel for tissue culture. A. A mixture consisting of 40% PEG-DA 700 (v/v) and 30% acrylic acid (v/v) and Fibronectin in presence of Irgacure 819 was polymerized with UV light to form a hydrogel. B. Representative phase-contrast images of SW480 cells grown for 6 days on PEG-DA hydrogels containing or not acrylic acid and/or fibronectin. The bar graph shows the number of adherent cells per field (4 fields with a 20x objective counted from three independent experiments). C. Representative phase-contrast images of SW480 grown for 6 days on hydrogels containing different ECMs. Quantification was performed as in B. Scale bars = 50 µm.

Figure 3 :

 3 Figure 3: 3D printing and imaging of intestinal scaffold. A. CAD design and schematic of the 3D intestinal scaffold mimicking the topography and dimensions of murine intestinal epithelium. Numbers are in µm. Scaffolds were printed using a photopolymerizable hydrogel composed of 40% PEG-DA 700, 30% acrylic acid and 250 µg/ml fibronectin, fluorescent beads were added to visualize the scaffolds by fluorescence microscopy. B. SEM images of the 3D scaffold. C. 3D reconstructions (left and middle) and xz section (right) of the scaffold image by two-photon microscopy. D. Side view of the scaffold image (left) with Light Sheet Fluorescence Microscopy and 3D reconstruction (right). Scale Bars = 100 µm.

Figure 4 :

 4 Figure 4: Cell culture on intestinal scaffolds. Representative images of Caco-2 cells seeded on intestinal scaffolds. Cells were grown for 6 days fixed and then stained for DNA (Hoechst 33342, blue) and F-actin (FITC-Phalloidin, green). A. Two-photon imaging of the top, middle and base of the villi. Scale bars = 50 µm B. xz section of a single villus imaged by two-photon microscopy. C. Side view of the scaffold (top) and 3D reconstruction (bottom) of Light Sheet Fluorescence Microscopy acquisition. Scale bars

Figure 5 :

 5 Figure 5: PEG-DA hydrogel induces apico-basal polarization of Caco-2 cells. Cells were plated on uncoated glass coverslips (A), glass coverslips coated with fibronectin (B), or on 2D hydrogel (C). After 21 days, cells were fixed and stained for F-actin with Phalloïdin-FITC (green) and nuclear DNA with DRAQ5 (blue). A-B. xy and xz view of Caco-2 cells cultured on uncoated glass coverslip (A) or fibronectin coated coverslips (B) for 21 days. C. Cellular morphology of Caco-2 cells grown on 2D PEG-DA/acrylic acid/fibronectin hydrogel for 21 days, 3D reconstruction of Z-stack imaging (lower left) and xz section (lower right) by confocal imaging. These experiments were realized on 3 independent samples. Scale bars = 50 µm.

Figure 6 :

 6 Figure 6: Caco-2 cells form a polarized epithelium on 3D scaffold. Cells were plated on 3D scaffold and after 18 or 21 days, cells were fixed and stained for nucleic acid (DRAQ5, blue) and F-actin (Phalloidin-FITC, green). A. 3D reconstruction of the scaffold by high-resolution dynamic speckle illumination imaging (left), focus on crypt level (middle) and xz section (right). B. Confocal imaging of villi xy section (left) and xz sections of the middle and base of a single villus (middle and right, respectively). C-D. Cellular morphology and actin polarization of cells cultured on 3D scaffolds for 18 and 21 days, respectively. These experiments were realized on 3 independent scaffolds. E. Average height of Caco-2 cells grown on 2D hydrogels or 3D scaffolds. Scale bar = 50 µm.

Figure 7 :

 7 Figure 7: Caco-2 cells differentiates in 3 dimensions Cells were seeded on 3D scaffolds or 2D hydrogels printed with the Dilase 3D. After 21 days of culture, cells were fixed and stained for F-actin, β-catenin, Pan-cytokeratin or villin. Nuclear DNA was stained with DRAQ5 (blue). A. Confocal imaging of β-catenin/F-actin co-staining on 2D or 3D hydrogels. B. Confocal imaging of Pan-cytokeratin staining (green) of Caco-2 grown on 2D or 3D hydrogels. C. xy and xz view of Caco-2 cells grown on 2D or 3D hydrogels stained for villin. D. Brightfield images of staining for alkaline phosphatase activity (blue) of Caco-2 grown on 2D or 3D hydrogels. Scale bars = 50 µm

Figure S2 :

 S2 Figure S2: Optimization of ECM concentrations in hydrogels for tissue culture Determination of the optimal concentrations of fibronectin (A), collagen-1 (B) or laminin (C) in 40% PEG-DA/30% acrylic acid hydrogels using SW480 cell cultured for 6 days on hydrogels. Graphs show the number of cells counted per field from 4 fields with a 20X objective. Scale bars = 50 µm.

Figure S3 :

 S3 Figure S3: Confirmation of the hydrogel with another colorectal cell line. Representative fluorescence images of Caco-2 cells cultured on various hydrogels. Cells were seeded at 20,000 cells/cm 2 , after 6 days of culture, cells were fixed and stained for F-actin with phalloïdin-FITC and nuclear DNA was stained with Hoechst 33342. The bar graph shows the quantification of cells number per field (40X, 4 fields counted). Scale bars = 25 µm.

Figure S4 :

 S4 Figure S4: Influences of villi length on cellular behavior (A) SEM images of 3D scaffolds of 500 µm and 1500 µm high villi. (B, C) Representative confocal images of Caco-2 cells grown on 500 µm (B) or 1500 µm (C) high villi. Cells were grown for 21 d, fixed and stained for F-actin (Phalloidin-rhodamine, red), β-catenin (green) and nuclear DNA (DRAQ5, blue). (D) Average height of Caco-2 cells grown on 3D scaffolds with 500 µm or 1500 µm high villi.

Figure S5 :

 S5 Figure S5: Surface topography and roughness of the hydrogel (A, B) Representative SEM images of 2D (A) or 3D (B) hydrogels. (C, D) AFM imaging of 2D (C) or 3D (D) hydrogels. Cross section and 3D view is presented for each sample.

Acknowledgements J.C. was supported by a studentship from the Region Midi-Pyrénées and Université Paul Sabatier -Toulouse III. This project was supported by funds from the Ligue Nationale Contre le Cancer, Fondation ARC pour la Recherche sur le Cancer and an "FRM Equipes" grant (DEQ20170336707) from the Fondation pour la Recherche Médicale to A.B. This work was supported by the French RENATECH network. It was partly supported as part of the MuliFAB project funded by FEDER European Regional Funds and French Région Occitanie (grant agreement number 16007407/MP0011594) and by the HoliFAB project funded by the European Union's Horizon 2020 research and innovation program (grant agreement No 760927). The authors thanks Sophie Allart and Danielle Daviaud (CPTP, Toulouse) for assistance with multiphoton imaging, Jacques Rouquette and Lise Teyssedre (ITAV, Toulouse) for LSFM imaging acquisitions and Brice Ronsin and Stephanie Bosch (CBI, Toulouse) for confocal imaging.