Arunkumar Rengaraj

Lauriane Bosc

Paul Machillot

Colin Mcguckin

Clément Milet

Nico Forraz

Philippe Paliard

Denis Barbier

Catherine Picart

Arunkumar Rengaraj Ab#

Lauriane Bosc Ab#

Paul Machillot Ab

Engineering of a Microscale Niche for Pancreatic Tumor Cells Using Bioactive Film Coatings Combined with 3D-Architectured Scaffolds

Keywords: Tissue engineering, biomimetism, niche, pancreatic cancer, growth factors, surface coating, cell adhesion, and proliferation

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

Introduction

Pancreatic cancer is the seventh leading cause of cancer-related death in humans, with a 91% mortality rate within five years of diagnosis. In 2018, nearly 432,000 new deaths related to pancreatic cancer were reported worldwide [START_REF] Bray | GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[END_REF] . This high mortality rate is due to the lack of diagnostic tools and limitations of existing models used to study cancer cell mechanisms. Classical culture plates (TCPS) are unable to mimic the biophysical and chemical conditions of the tumor microenvironment [START_REF] Breslin | Three-Dimensional Cell Culture: The Missing Link in Drug Discovery. Drug Discovery Today[END_REF] . Moreover, these models are not physiological because the cells are cultured on stiff materials such as TCPS or glass. In vivo, the mature tumor is organized in a three-dimensional (3D) architecture and surrounded by an extracellular matrix (ECM). In vivo models have been developed, such as patient-derived xenograft (PDX) mice [START_REF] Zhang | Establishment of a Human Primary Pancreatic Cancer Mouse Model to Examine and Investigate Gemcitabine Resistance[END_REF] and genetically modified mice [START_REF] Lee | Genetically Engineered Mouse Models of Pancreatic Cancer: The KPC Model (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre), Its Variants, and Their Application in Immuno-Oncology Drug Discovery[END_REF] . However, these procedures are lengthy, expensive, and raise many ethical issues related to animal handling. Therefore, there is a need to develop new in vitro platforms that mimic the in vivo tumor microenvironment at all stages of development so that cells can eventually form a three-dimensional (3D) tumor and allow better control and tuning of experimental conditions [START_REF] Suklabaidya | Experimental Models of Pancreatic Cancer Desmoplasia[END_REF] .

Researchers developed various types of 3D tumor models such as spheroids [START_REF] Pape | Tissue-Engineering the Fibrous Pancreatic Tumour Stroma Capsule in 3D Tumouroids to Demonstrate Paclitaxel Response[END_REF] , 3D-printed tumor models [START_REF] Chiellini | Modelling of Pancreatic Ductal Adenocarcinoma in Vitro with Three-Dimensional Microstructured Hydrogels[END_REF] , scaffold-based models [START_REF] Gupta | A Novel Scaffold-Based Hybrid Multicellular Model for Pancreatic Ductal Adenocarcinoma-Toward a Better Mimicry of the in Vivo Tumor Microenvironment[END_REF][START_REF] Totti | A 3D Bioinspired Highly Porous Polymeric Scaffolding System for in Vitro Simulation of Pancreatic Ductal Adenocarcinoma[END_REF] , and microfluidic platforms for preclinical cancer studies [START_REF] Peng | 3D Bioprinting for Drug Discovery and Development in Pharmaceutics[END_REF] . In these methods, it is challenging to obtain a tumor model with uniform size and morphology. For example, in spheroid models, cancer cells are forced to aggregate in a defined space to achieve uniform size and shape [START_REF] Athanasiou | Self-Organization and the Self-Assembling Process in Tissue Engineering[END_REF] . This spatial restriction disrupts the processes of cell adhesion, spreading, and proliferation. In addition, growth factors must be administered in soluble form, which is different from in vivo delivery through the ECM or cell-to-cell. In direct printing of a tumor, the cancer cells are mixed with a bio-ink consisting of polymers and cross-linking agents and 3D printed directly. The cells are thus encapsulated in a polymer matrix to facilitate interactions between them. To solve these problems, it is important to develop a platform that allows the cells to self-organize and proliferate to create the 3D tumor model.

In addition, cancer cells require a suitable environment consisting mainly of ECM proteins for biochemical and structural support, growth factors for cell communication, and stromal cells [START_REF] Li | Tumor Microenvironment: The Role of the Tumor Stroma in Cancer[END_REF] . The ECM enables cancer cells to communicate with each other through specific biochemical and mechanical signals. Its composition and stiffness have a significant impact on tumor mechanisms [START_REF] Köninger | Pancreatic Tumor Cells Influence the Composition of the Extracellular Matrix[END_REF][START_REF] Seewaldt | ECM Stiffness Paves the Way for Tumor Cells[END_REF] . These mechanical and chemical signals from the ECM are converted into biochemical signals involved in cell adhesion and spreading [START_REF] Migliorini | Tuning Cellular Responses to BMP-2 with Material Surfaces[END_REF] . A number of ECM proteins, including fibronectin, interact with cell mechanoreceptors such as integrins, which influence cancer cell processes such as tumor growth, proliferation, migration, and vascularization [START_REF] Jang | CAFs and Mechanical Forces in the Progression of Cancer[END_REF] . Cancer cells can modulate their integrin heterodimer receptors, making them receptive to signals sent through the extracellular matrix, especially its stiffness [START_REF] Jang | CAFs and Mechanical Forces in the Progression of Cancer[END_REF] . Researchers found a large amount of fibronectin on pancreatic ductal adenocarcinomas (PDA), which play an important role in cancer metastasis and chemoresistance [START_REF] Topalovski | Matrix Control of Pancreatic Cancer: New Insights into Fibronectin Signaling[END_REF] . The bone morphogenic protein (BMPs) family is also emerging as an important factor in pancreatic cancer. BMP2 and BMP4, particularly play a role in angiogenesis and are increasingly targeted in cancer therapy.

BMPs bind to specific type I and type II BMP receptors. Upon BMP binding, these receptors form heterodimers and initiate the Smad signaling pathway, which begins with the phosphorylation of Smad1/5 to pSMAD1/5. This phosphorylated pSMAD1/5 forms a complex with Smad4 and is then translocated to the nucleus to initiate transcription of specific genes responsible for regulating cellular mechanisms. In pancreatic cancer, Smad4 mutations are common and have also been cited as factors influencing tumorigenesis. Voornveld et al. also suggested that BMPs enhance the invasive behavior of pancreatic cancer due to the loss of Smad4 [START_REF] Voorneveld | Reduced Expression of Bone Morphogenetic Protein Receptor IA in Pancreatic Cancer Is Associated with a Poor Prognosis[END_REF] . They also found that TGF-β family proteins induce phosphorylation of Smad1/5 [START_REF] Daly | Transforming Growth Factor β-Induced Smad1/5 Phosphorylation in Epithelial Cells Is Mediated by Novel Receptor Complexes and Is Essential for Anchorage-Independent Growth[END_REF] . The above literature shows that cell adhesion and cell spreading significantly affect the Smad signaling pathway.

Polyelectrolyte multilayers (PEM) prepared by the layer-by-layer technique are of interest for tissue engineering due to their composition, which can mimic ECM [START_REF] Gribova | Polyelectrolyte Multilayer Assemblies on Materials Surfaces: From Cell Adhesion to Tissue Engineering[END_REF][START_REF] Detzel | Polyelectrolyte Multilayers in Tissue Engineering[END_REF] . It is possible to tune the film architecture, thickness, chemistry, and mechanical properties [START_REF] Richardson | Technology-Driven Layer-by-Layer Assembly of Nanofilms[END_REF] and develop protein-inspired nanofilms [START_REF] Zhang | Context Dependence of the Assembly, Structure, and Stability of Polypeptide Multilayer Nanofilms[END_REF] , biosensors, biomimetics [START_REF] Tang | Biomedical Applications of Layer-by-Layer Assembly: From Biomimetics to Tissue Engineering[END_REF] , and drug delivery systems [START_REF] Zelikin | Drug Releasing Polymer Thin Films: New Era of Surface-Mediated Drug Delivery[END_REF] , especially for proteins and growth factors. Based on previous studies by our group, we have selected films of hyaluronic acid (HA) and poly(L-lysine) (PLL) whose stiffness can be adjusted by changing the concentration of the EDC crosslinking agent [START_REF] Ren | Polyelectrolyte Multilayer Films of Controlled Stiffness Modulate Myoblast Cell Differentiation[END_REF] . These films can serve as reservoirs to deliver growth factors and proteins to cells locally and over a long period of time [START_REF] Crouzier | Layer-By-Layer Films as a Biomimetic Reservoir for RhBMP-2 Delivery : Controlled Differentiation of Myoblasts to Osteoblasts[END_REF] . In addition, proteins loaded into the films are also protected from aggregation that is commonly observed when they are in solution. This is particularly important as an altered 3D biochemical structure often leads to a loss of protein function.

In this work, our goal was to create a 3D microenvironment in which cancer cells self-organize and proliferate to form an independent 3D microscale tumor tissue that can be further used for highcontent analysis and drug screening. We fabricated a 3D scaffold from the synthetic polymer Ormocomp using two-photon photopolymerization (TPP) to support the structure. Furthermore, the scaffold was functionalized with a multilayer bioactive polyelectrolyte film that mimics simple aspects of ECM (stiffness, protein presentation). (Figure 1). The microscaffold allows cells to migrate and organize their 3D cellular network. Based on our previous studies, we prepared films made of 12 (PLL/HA) layer pairs [START_REF] Crouzier | Layer-By-Layer Films as a Biomimetic Reservoir for RhBMP-2 Delivery : Controlled Differentiation of Myoblasts to Osteoblasts[END_REF] , which were then chemically cross-linked to adjust the stiffness to produce films with an elastic modulus of E0 ~200 kPa and ~400 kPa for soft "EDC30" and stiff "EDC70" films, respectively [START_REF] Ren | Polyelectrolyte Multilayer Films of Controlled Stiffness Modulate Myoblast Cell Differentiation[END_REF][START_REF] Schneider | Polyelectrolyte Multilayers with a Tunable Young's Modulus: Influence of Film Stiffness on Cell Adhesion[END_REF][START_REF] Boudou | Multiple Functionalities of Polyelectrolyte Multilayer Films: New Biomedical Applications[END_REF] . The bioactive coating was built up in 24-well plates on the 3D-architectured patterned scaffold [START_REF] Machillot | Automated Buildup of Biomimetic Films in Cell Culture Microplates for High-Throughput Screening of Cellular Behaviors[END_REF] , proteins were loaded into the films, and cells were seeded (Figure 1.A). In parallel, for studies with high content cancer cell content, we performed initial cell adhesion, spreading, and proliferation on polyelectrolyte films with two different stiffnesses loaded with proteins on films deposited in 96-well plates. This fabrication process was performed using a recently developed automated liquid robot 31 (Figure 1.B) and films loaded with increasing concentrations of the proteins.

The stiffness was chosen to match the average elasticity of normal tissue (252 ± 134 kPa) that of the pancreatic tumor (349 ± 222 kPa) [START_REF] Rubiano | Viscoelastic Properties of Human Pancreatic Tumors and in Vitro Constructs to Mimic Mechanical Properties[END_REF] . To determine the effects of fibronectin (bFN), bone morphogenetic protein 2 (bBMP2), and bone morphogenetic protein 4 (bBMP4), we additionally loaded these proteins onto the slide. Recently, the effect of BMPs on cancer has been widely discussed due to their emerging role in cancer mechanisms [START_REF] Singh | The Yin and Yang of Bone Morphogenetic Proteins in Cancer[END_REF] . We compared the cellular response of primary patient-derived pancreatic cancer cells with PANC-1 cells, a commonly studied cell line.

MATERIALS & METHODS

Scaffold fabrication using two-photon polymerization technique

The 3D scaffold architecture consists of two floors of 8x8 meshes with a width and height of 120 meters. The design of the scaffold was sliced using Simpoly software (Microlight3D, La Tronche, France). The scaffolds were fabricated with Ormocomp polymer (Micro resist technology, Berlin, Germany) using two-photon polymerization process. The TPP system (µFab3D.Advanced by Microlight3D, La Tronche, France) is equipped with a 20x magnification objective and a numerical aperture of 0.75. A 3D piezoelectric actuator allows the laser focal point set on the Ormocomp resin to initiate polymerization in 3D space. The microchip laser generates femtosecond laser pulses at 532 nm that are highly focused inside the monomer. The TPP is then localized at the focal point in a small volume ( 1 µm 3 ) called a voxel. 3D patterning can be achieved by scanning the focal point in a drop of Ormocomp deposited on a Ø14 mm glass coverslip (0.17 mm thick) between two spacers. The laser power was set between 4 and 7.2 mW via software (Lithos, Microlight3D, La Tronche, France). The lateral printing speed was 120 µm/s, and the layer spacing was 300 µm. After polymerization, the residual monomer was removed by washing the sample several times with 90% acetone, leaving the scaffolds on the glass. The scaffolds were designed to maximize cell-cell interaction and molecular diffusion with a minimal hypoxic core.

Polyelectrolyte multilayer film preparation and cross-linking

HA (sodium hyaluronate, MW 360,000 g/mol) was purchased from Lifecore (Chaska, MN, USA). PLL (poly(L-lysine), P2636) and PEI (poly(ethyleneimine), P3143) were purchased from Sigma-Aldrich (St-Quentin-Fallavier, France). PLL and HA were dissolved to 0.5 mg/ml in HEPES-NaCl buffer (20 mM HEPES at pH 7.4, 0.15 M NaCl), both purchased from Sigma-Aldrich (H4034 and S9625, respectively).

For the studies on the 2D substrate, film deposition was performed in a 96-well plate (Greiner, Les Ulis, France) using an automatic liquid robot (EVO100, Tecan, Lyon, France). This robot can automatically aspirate and dispense fluids into multiwell plates, as previously described [36]. Prior the deposit the 12 (PLL/HA) layers pairs, a first anchoring layer of PEI was incubated at 5 mg/ml for 20 min. For 3D studies, the scaffolds were placed in a 24-well plate (662892, Greiner, Les Ulis, France) together with their Ø14 mm glass coverslip. Then, 2.5 mg/ml PEI was added to the wells and incubated under vacuum for 20 min. The robot applied successive layers of PLL and HA until the 12 bilayers were formed. After formation, all films were cross-linked for 18 h at 4 °C with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, E7750) at a concentration of 30 mg/ml or 70 mg/ml and Nhydrosulfosuccinimide (Sulfo-NHS, KRO-12021) at a constant concentration of 11 mg/ml, obtained from Sigma-Aldrich (St-Quentin-Fallavier, France) and Chemrio (Ningbo, China), respectively. Final washing was performed with HEPES-NaCl buffer for 1 h. The films were then dried after a brief rinse with H2O.

The multilayer films are hereafter referred to as EDC30 and EDC70, which stand for soft and stiff films, respectively, and refer to the concentration of the cross-linking agent.

Characterization of the polyelectrolyte film-functionalized structure

Both the Ormocomp scaffold and the Ormocomp scaffold functionalized with PEM were characterized using optical and electronic microscopy. Images were acquired using a Zeiss LSM700 confocal scanning microscope (Zeiss, Le Peck, France) equipped with a 63X oil immersion objective and laser diodes at 405, 488, 555, and 639 nm. Images were acquired using the Zen software by Zeiss.

In addition, 3D reconstructions of the acquired z-stacks were created using ImageJ software (imagej.nih.gov/ij/). For the observation of the polyelectrolyte film, the last layer was a PLL-FITC layer (Sigma-Aldrich, P3069). For analysis of SEM, the scaffolds were coated with a 20 nm thick carbon layer using the Bal-Tec CED030 carbon filament evaporator (Bal-Tec Union Ltd., Liechtenstein) and imaged with a Quanta 250 field emission gun SEM (FEI Company) at 5 kV with a high contrast backscatter detector.

Protein loading on polyelectrolyte films

Fibronectin (11051407001) was obtained from Roche (Basel, Switzerland), BMP2 from the IndoctOs kit from Medtronic (Minneapolis, USA), and BMP4 (120-05ET) from Peprotech (Neuilly-sur-Seine, France). Proteins were dissolved in 1 mM HCl buffer (258148 from Sigma Aldrich) at pH 3. For studies on 2D substrates, proteins were incorporated into the film for 1h30 at 37°C and then rinsed with HEPES-NaCl buffer. They were then dried after a short rinse with H 2 O. For 3D studies on the scaffold, 1 µl of protein solution was applied to the scaffold and incubated for 10 min at RT to allow incorporation. Then the scaffolds were quickly rinsed with H 2 O and dried. Incorporation of proteins into the PEM was confirmed with 10% fluorescent BMP2 coupled with rhodamine (prepared in-house) and observed under the LSM700 confocal microscope. To control for proteins bound to the matrix, we also examined the dynamics of cancer cells exposed to soluble proteins added directly to the cell culture media. We will refer to the proteins loaded into the PEM as matrix-bound proteins (bFN, bBMP2, and bBMP4) and soluble proteins (sFN, sBMP2, and sBMP4). The amount of protein loaded into the film was checked using a QuantiPro™ BCA assay (QPBCA, Sigma-Aldrich), and the results are shown in Table S1. The stiffness previously measured using the AFM nanoindentation technique was not affected by protein loading [START_REF] Gribova | A Material's Point of View on Recent Developments of Polymeric Biomaterials: Control of Mechanical and Biochemical Properties[END_REF] .

Cell culture and cell seeding on the scaffold

Pancreatic cancer cell line PANC1, derived from epithelioid carcinoma in a 56-year-old man, was acquired from ATCC (CRL -1469). The patient-derived cells PAN092 (CTI-214) were developed by CTI-Biotech (Meyzieu, France), from the French cancer consortium IMODI. PAN092 cell was isolated from a patient's biopsy and propagated in mice as patient-derived xenografts (PDX).

It is an invasive and moderately differentiated pancreatic ductal carcinoma (G2) of the pancreatic head were cultured in DMEM-F12 supplemented with 10% FBS (10270-106) and 1% Antibiotic-Antimycotic (15240-062) (all from Thermo Fisher Scientific, Bourgoin-Jallieu, France) and maintained at 37°C and 5% CO 2 . All substrates (plates and scaffolds) were sterilized under UV light for 20 min before seeding the cells. For the 2D studies, cells were seeded at a cell density of 15,000 cells/cm². In the 3D cell culture, cells were seeded at 32,000 cells per scaffold. Cells were seeded in 1 µl on the top of the scaffold, with 1 µl of medium added periodically to prevent desiccation. The wells were filled with medium once the cells were attached to the scaffold after approximately 1 hour.

High content imaging and quantification of cell adhesion and spreading

Cell adhesion and spreading were analyzed after 16 h for PANC1 cells and after 24 h for PAN092 cells due to their low adhesion rate. Cells were fixed with 3.7% paraformaldehyde (252549 from Sigma-Aldrich, Saint-Quentin Fallavier, France) for 20 min. The actin cytoskeleton of the cells was stained by incubation with phalloïdine rhodamine (P1951, from Sigma Aldrich) for 30 min. Cell nuclei were stained by 5-minute incubation with DAPI (D9542, from Sigma Aldrich). Automated imaging was performed using a high-content imager GE InCell 2500 (General Electric, Buc, France) equipped with 4X, 10X, and 20X air objectives. Bright field images were acquired with a 4X objective. Fluorescent cell images were acquired with the 10X objective in eight fields to cover most of the well. Automated analysis was performed using the manufacturer's GE IN-Carta software (General Electric, Buc, France).

The cell count indicates the average number of nuclei counted in 1 mm² of the well surface. Cell spread corresponds to the cell area labeled with phalloïdine rhodamine (targeting the cytoplasm of the cells).

Each experiment was performed at least in triplicate, with three independent samples per condition in each experiment. The average cell number per mm² or the average cell area (µm²) was plotted along with the SEM.

Cell proliferation assay

To assess the proliferation of PANC1 and PAN092 on the 2D substrate, cells were cultured on a polyelectrolyte film with increasing concentrations of bFN, bBMP2, and bBMP4. Cells were lysed and processed using the CyQUANT assay (C7026, Thermo Fisher Scientific) according to the manufacturer's protocol at 38, 76, 114, and 152 h for PANC1 cells and 24, 48, 72, 96, and 120 h for PAN092 cells. Fluorescence was detected using a plate reader (Infinite1000, Tecan, France) with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. All experiments were performed at least in triplicate (i.e; three independent experiments) with three independent samples per condition in each experiment, and the average + SEM was plotted.

pSMAD analysis

For analyzing pSMAD1/5/9, cells were fixed 5 h after seeding to allow adhesion. After fixation and membrane permeabilization, the cells were blocked using 3% w/v BSA (A4503, Sigma-Aldrich, Saint-Quentin Fallavier, France) in PBS. Next, the primary antibody anti-pSMAD1/5/9 (13820S, Cell Signaling, Leiden, Netherlands) was diluted in the ratio of 1:400 in the 3% BSA solution and incubated overnight at 4°C. After overnight incubation, the plates were washed 3 times with PBS to remove an attached primary antibody and then incubated with the fluorescently tagged secondary antibody (A32732, Invitrogen, Paris, France) at room temperature for 1 h. The images were analyzed using GE INCA 2500 (General Electric, Buc, France) imaging system using a 20X objective. Up to 1000 cells were analyzed in each condition for three independent experiments, and graphs were plotted on Origin software with the average pSMAD signal and SEM.

Statistical analysis

Statistical comparisons were performed using OriginPro software version 2020b. The nonparametric Bonferroni test was used to analyze data from two independent groups. ANOVA, followed by an appropriate pairwise comparison or comparison with the control group, was performed (p<0.05 was considered significant). All experiments were repeated at least 3 times with at least two samples per condition in each experiment. The error bars represent the standard errors of the mean.

The asterisk indicates that the value is significantly different from the control condition.

Results

Fabrication of the 3D Ormocomp scaffold

The details of the structure and scaffold design, which was created using computer-aided design software, are shown in Figure 2.A-B. Scaffolds with three different porosities (80 µm, 100 µm, and 120 µm) were fabricated and analyzed using optical microscopy. These different porosities were chosen to allow cell-cell contact, nutrient exchange, and proliferation in the structure [START_REF] Lee | User-Friendly 3D Bioassays with Cell-Containing Hydrogel Modules: Narrowing the Gap between Microfluidic Bioassays and Clinical End-Users' Needs[END_REF] . The bright-field image of the top of the bare scaffold (Figure 2.C) and the 3D reconstruction (Figure 2.D) show that the Ormocomp scaffold is compatible with any optical microscopy analysis technique due to its transparency. The scanning electron micrographs show that the scaffold was fabricated without defects (Figure 2.E). The roughness of the scaffold surface (Figure 2.F) can be adjusted by increasing or decreasing the laser power and writing speed [START_REF] Käpylä | Ormocomp-Modified Glass Increases Collagen Binding and Promotes the Adherence and Maturation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells[END_REF] . Initially, a photo-initiator was added to Ormocomp to improve the polymerization process. However, it caused undesirable autofluorescence and led to the destruction of the scaffold during UV sterilization (data not shown). In addition, photo-initiators are known to exhibit some cell toxicity [START_REF] Zheng | Cucurbit[7]Uril-Carbazole Two-Photon Photoinitiators for the Fabrication of Biocompatible Three-Dimensional Hydrogel Scaffolds by Laser Direct Writing in Aqueous Solutions[END_REF] . Due to these problems, the scaffolds were prepared without this additional photo-initiator. An advantage of the Ormocomp scaffold is that they did not show swelling compared to natural polymers [START_REF] Wang | Novel Biodegradable Porous Scaffold Applied to Skin Regeneration[END_REF] .

Film buildup and protein incorporation on the 3D scaffold

Due to the low interaction between Ormocomp and cells [START_REF] Kidwell | Chemistries for Making Additive Nanolithography in OrmoComp Permissive for Cell Adhesion and Growth[END_REF] , it is crucial to apply an additional coating on the scaffold surface that mimics the ECM [START_REF] Kidwell | Chemistries for Making Additive Nanolithography in OrmoComp Permissive for Cell Adhesion and Growth[END_REF] to allow cell culture and colonization inside the 3D structure. Three different scaffold pore sizes (80, 100, and 120 µm) were tested, and the PEM film coating was characterized by fluorescence microscopy. PLL-FITC was used for the visualization of the film [START_REF] Crouzier | Layer-By-Layer Films as a Biomimetic Reservoir for RhBMP-2 Delivery : Controlled Differentiation of Myoblasts to Osteoblasts[END_REF] . Fluorescence microscopy showed that increasing the pore size leads to a uniform coating of the PEM film on the scaffold (Figure S1). This uniform coating is mainly due to reduced surface tension and facilitated fluid exchange due to larger pores during successive layer deposition. Therefore, we selected the scaffold with a pore size of 120 µm for further experiments.

PEI was precoated on the scaffold as a anchoring layer to improve the deposit of polyelectrolytes: PEI has excellent UV resistance, transparency, mechanical stability, and good biocompatibility [START_REF] Lee | Fundamentals and Applications of 3D Printing for Novel Materials[END_REF] . In the absence of this first PEI layer, the film was not uniformly deposited on the scaffold (Figure S2). Due to the hydrophobicity of the Ormocomp material, incubation was performed under vacuum to enable liquid impregnation into the structure. The confocal fluorescence images at high magnification (63X) showed a uniform coating of  1.5 µm on the hydrated scaffold (Figure 3.A-B), which is previous quantification of film thickness [START_REF] Machillot | Automated Buildup of Biomimetic Films in Cell Culture Microplates for High-Throughput Screening of Cellular Behaviors[END_REF] . The microscopic size of this 3D scaffold being fully compatible with the protocols already established for the polyelectrolyte films in 2D, similar protocols were used to visualize labelled films and proteins. Rhodamine-labelled BMP2 was loaded into the film-coated scaffold and then imaged using confocal microscopy (Figure 3

Pancreatic cancer cell line on PEM film-loaded with FN, BMP2, and BMP4

To design an optimal 3D microtumor model, it is essential to know how signaling molecules affect basic processes such as adhesion, spreading, and proliferation of cells on PEM film [START_REF] Lee | Cell Adhesion and Long-Term Survival of Transplanted Mesenchymal Stem Cells: A Prerequisite for Cell Therapy[END_REF] . It is also known that film stiffness affects the interaction between cancer cells and the matrix-bound biomolecules. We studied these processes on soft (EDC30) and stiff (EDC70) films. Moreover, the extracellular matrix proteins play an important role in cell dynamics and cancer cell survival [START_REF] Dufort | Balancing Forces: Architectural Control of Mechanotransduction[END_REF] . To construct the successful 3D microtumor model, we investigated the effects of the bioactive film loaded with the selected proteins (FN, BMP2 and BMP4) on the two pancreatic cancer cell lines PANC1 and PAN092.

PANC1 response to matrix-bound FN, BMP2, and BMP4

The PANC1 cell line, established from pancreatic ductal carcinoma, is a widely used model of pancreatic cancer and is well characterized. As previously mentioned, adhesion proteins and growth factors significantly influence cellular processes such as cell adhesion, spreading, and proliferation. PANC1 cells cultured on TCPS showed higher cell adhesion, spreading, and proliferation than control films EDC30, EDC70 (Figure S3). The proliferation assay showed that the cells grew linearly on TCPS for up to 160 h. Cell proliferation was higher than that of the control films. This increased proliferation may be due the physico-chemical properties of TCPS to improve cell attachment. EDC30 is also known as a soft film due to its low stiffness. When cultured on EDC30 and EDC70 films, the cells are round and often observed as clamped with each other (Figure S3.A-B). In addition, cell spreading was lower on EDC30 films (Figure S3.C) since a softer substrate allows less interaction between the cell adhesion receptors integrins and the underlying matrix. This lower adhesion favors cell-cell interactions instead of cell-substrate interactions [START_REF] Guo | Substrate Rigidity Regulates the Formation and Maintenance of Tissues[END_REF] , and also to a lower proliferation rate than that on TCPS (Figure S3.D). On EDC30 and EDC70, cells proliferated slightly within 80-120 h, and after 120 h, the cell population decreased. At 120 h, PANC1 cells aggregated and formed organoid-like aggregates on EDC30 and EDC70 films (Figures S4 andS5).

As a control, we added the soluble proteins on cells cultured on TCPS and analyzed cell adhesion and spreading for both cell lines. For the PANC1 cell line, only fibronectin had an effect on cell adhesion and spreading (Figure S6). In PAN092 cells, the proteins had no significant effect on cell attachment, but also had no effect on spreading (Figure S7). Next, we examined the above effect of the proteins in soluble form (sFN, sBMP2 and sBMP4) and matrix-bound form (bFN, bBMP2 and bBMP4) in EDC30 and EDC70. bFN highly promoted adhesion and proliferation of PANC1 cells on both EDC30 and EDC70, but had no effect on cell spreading (Figure 4.A-E). The corresponding fluorescence images are shown in Figures S8 andS9. When a minimal amount of bFN (2.5 µg/ml) was added to the EDC30 film, cell adhesion increased dramatically. The number of attached cells was about 100 per mm², and when 2.5 µg/ml of bFN was present in the film, the cell number increased 5-fold for cells on EDC30 and 15-fold for EDC70 films (Figure 4.C). This significant increase in cell attachment indicates that bFN provides binding sites for PANC1 cells. Interestingly, sFN had no effect on cell adhesion for cells on EDC30 films (Figure S6.A). Indeed, fibronectin increases cell adhesion by interacting with integrins at the cell surface [START_REF] Reuter | Oxidative Stress, Inflammation, and Cancer: How Are They Linked? Free radical biology and medicine[END_REF] .

The proliferation of PANC1 cells was analyzed using a cyQUANT assay. PANC1 cells proliferated on both EDC30 and EDC70 films loaded with bFN (Figure 4.E). In contrast, bBMP2 and bBMP4 showed a much milder effect on cell adhesion, with a dose-dependent increase on cell adhesion (Figure 4.F and 4.I). They did not induce cell spreading (Figure 4.G and 4.J), with here again no distinction between soft and stiff films. However, this may be explained by the epithelial-like nature of pancreatic cells. Indeed, epithelial cells remain morphologically compact and therefore did not spread even when under favorable conditions. Both sBMP2 and sBMP4 showed no effect on the adhesion of PANC1 to TCPS (Figure S6.C and S6.E). Although bBMP2 and bBMP4 slightly increased cell adhesion, they did not affect cell proliferation, except for the lowest bBMP2 concentration (5 µg/ml), regardless of film stiffness (Figure 4.H and 4.K). The cell population is relatively stable and begins to decrease after 114 h for bBMP2. Finally, bBMP4 had no effect on cell proliferation except at the highest concentration on soft films, where the cell number increased significantly after 160 h (Figure 4.K).

Overall, we found that the number of attached cells was lower on EDC70 films in the presence of matrix-bound proteins than on EDC30 loaded with the same proteins. This indicates that the combination of soft films with matrix-bound proteins (bFN, bBMP2, and bBMP4) provided more favorable conditions for PANC1 cell attachment. This suggests that adhesion of PANC1 cells depends on film stiffness independently of the proteins presented. On the other hand, sFN, sBMP2 and sBMP4 show similar trends for cells cultured on slide (Figure S10). However, the number of attached cells in the presence of soluble proteins was lower than with matrix-bound matrix. BMP2 and BMP4 are known to promote epithelial to mesenchymal transition by limiting cell adhesion and proliferation [START_REF] Voorneveld | Reduced Expression of Bone Morphogenetic Protein Receptor IA in Pancreatic Cancer Is Associated with a Poor Prognosis[END_REF] . This was confirmed by the proliferation inhibitory role of BMPs on pancreatic cancer cells, as already highlighted in the literature [START_REF] Ehata | Bi-Directional Roles of Bone Morphogenetic Proteins in Cancer: Another Molecular Jekyll and Hyde?: Bi-Directional Roles of BMPs in Cancer[END_REF] . After 120 h, PANC1 cells aggregated and formed organoid-like aggregates in bFN, bBMP2 and bBMP4 on EDC30 and EDC70 (Figures S4 andS5). This cellular morphology confirms that the bioactive film mimics the ECM by allowing cells to self-organize, to aggregate and form colonies. When the concentration of bFN increased, we obseved that this organoid morphology was disrupted, with cells being mostly present as single cells. At the same time, the organoid morphology on both bBMP2 and bBMP4 was similar to the control at all concentrations. To note, we did not observe this organoid morphology with sFN, SBMP2 and sBMP4 (data not shown).

PAN092 response to matrix-bound FN, BMP2, and BMP4

To compare the results of the immortalized cell line (PANC1) with those of the patient-derived cells (PAN092), a similar experiment was performed (Figure 5). CTI-Biotech maintains these cells as part of the IMODI Cancer Consortium, and the cells were dissociated from a patient-derived pancreatic tumor xenograft. The cell line PAN092 was isolated from an invasive but moderately differentiated pancreatic ductal carcinoma of the pancreatic head. Interestingly, the number of cells was higher on EDC70 than on TCPS and EDC30 (Figure S11.A-B). Importantly, proliferation was also slower than PANC1 cells, but similar to TCPS, EDC30 and EDC70, as expected for a patient-derived tumor population (Figure S11.D). On both soft and stiff films, bFN strongly increased adhesion of PAN092 cells, even at the lowest dose of 2.5 µg/ml (Figure 5.C). bBMP2 showed a gradual increase in adhesion in a dose-dependent manner from 5 to 20 µg/ml, but it was significant only for the lowest dose loaded into the stiff film (Figure 5.F). In contrast, bBMP4 had a negative effect on adhesion to stiff films at the highest dose (Figure 5.I). We also found stiffness-dependent adhesion for bBMP2 and bBMP4, but unlike PANC1, PAN092 cells seemed to prefer stiffer film. Cell spreading was very similar to PANC1 cells and was not affected by the loaded proteins and stiffness, except for the highest dose of bFN on EDC30 (Figure 5.D, 5.G, 5.J). bFN slightly increased cell proliferation, while bBMP2 and bBMP4 had no significant effect (Figure 5.E, 5.H, 5.K). On the other hand, sFN, sBMP2 showed an increase in cell attachment on TCPS and EDC30 (Figures S12,S13). sFN and sBMP2 showed no effect on EDC70 (Figures S12,S13). sBMP4 showed the opposite effect than bBMP4 on TCPS,

EDC30 and EDC70 (Figures S7.E and S13.E).

bBMP2 and bBMP4 did not affect PAN092 proliferation, regardless of film stiffness. This was consistent with the proliferation inhibitory role of BMPs on pancreatic cancer cells reported in the literature [START_REF] Ehata | Bi-Directional Roles of Bone Morphogenetic Proteins in Cancer: Another Molecular Jekyll and Hyde?[END_REF] . Nevertheless, these patient-derived PAN092 cells showed specific differences from the ATCC cell line PANC1. Overall, PAN092 proliferated two time slower than PANC1. This may be explained by the non-immortalization of the cell line compared to immortalized established cell lines and its low adhesion rate, which remains a particular stumbling block in pancreatic cancer research [START_REF] Pham | Isolation of Pancreatic Cancer Cells from a Patient-Derived Xenograft Model Allows for Practical Expansion and Preserved Heterogeneity in Culture[END_REF] .

There was stiffness-dependent cell adhesion in both cell lines, but, unexpectedly, PANC1 and PAN092 responded in opposite ways. In fact, PANC1 cells preferred a softer film regardless of the matrix-bound protein, while PAN092 preferred stiffer films. bFN appeared to override this stiffness-dependent adhesion of PAN092. This may be explained by the presence of RGD motifs in the protein, being a strong adhesive signal for the cells by interacting with the adhesion receptors integrins [START_REF] Akiyama | Fibronectin and Integrins in Invasion and Metastasis[END_REF] .

Bioactivity in response to the matrix-bound proteins in the films

To further investigate the effect of proteins on cellular processes, we assessed the cell response to BMPs via the Smad signaling pathway. Upon interaction of BMP with its receptor at the cell surface, a signaling cascade is triggered in which Smad1/5/9 is phosphorylated. We quantified the phosphorylated form of Smad1/5/9 using immunofluorescence (Figure 6). For PANC1 cells (Figure 6A-E), we observed that there was no pSMAD signal on films with bFN whatever their stiffness. This is consistent with the fact that fibronectin is an adhesion protein that interacts with integrins and not with BMP receptors. However, the pSMAD in response bBMP2 and bBMP4 showed a strong increase for both EDC70 and EDC30 films (Figure 6.A-E). For PAN092 cells (Figure 6.F-J), we did not detect a pSMAD response whatever the bBMP concentration. Thus, PAN092 cells did not respond to BMPs via the Smad signaling pathway. The same proteins administered in soluble form showed the same trends for both cell lines, but with a lower pSMAD intensity (Figure S15). This increased effect of matrix-bound proteins may be related to the increased protection of the proteins from aggregation and conformational changes, and the presentation of the proteins at the ventral side of the cell is also more physiological [START_REF] Liu | Biomaterial-Enabled Delivery of SDF-1α at the Ventral Side of Breast Cancer Cells Reveals a Crosstalk between Cell Receptors to Promote the Invasive Phenotype[END_REF] .

The effects of the proteins on the different cellular processes are summarized in Table 1. cell line and PAN092 cells on stiff or soft films with matrix-bound bFN, bBMP2 and bBMP4. The '+', '++', "+++" and "++++" indicate an increasing level of positive effects, while "-" and "--" indicate a negative effect on the process. "=" indicates that there is no effect on the process

3D cell culture on functionalized scaffolds

To combine scaffold functionalization with cell culture on planar films, cancer cells were cultured on the 3D functionalized scaffolds coated with the protein-loaded films. We followed and studied their growth and proliferation over 20 days. Initial results for these cell cultures in 3D are shown in Figure 7.

Long-term 3D culture of PANC1 cells on fibronectin-loaded soft sheets was followed for up to 21 days by optical imaging (Figure 7 Our team previously reported the fabrication of various types of polyelectrolyte multilayer films mimicking the ECM. We have studied the cellular dynamics in detail using different signaling molecules and several cell lines cultured on different PEM films [START_REF] Liu | Biomaterial-Enabled Delivery of SDF-1α at the Ventral Side of Breast Cancer Cells Reveals a Crosstalk between Cell Receptors to Promote the Invasive Phenotype[END_REF][START_REF] Migliorini | Tuning Cellular Responses to BMP-2 with Material Surfaces[END_REF][START_REF] Dalonneau | The Effect of Delivering the Chemokine SDF-1α in a Matrix-Bound Manner on Myogenesis[END_REF] . In the present work, we studied the adhesion and growth of pancreatic cancer cells (PANC1 and PAN092) on PEM and proved that bioactive PEM can be successfully deposited on 3D Ormocomp scaffold. Advantageously, this approach may be applied to various polymers, ceramics and composites. In a tumor, the stiffness of the ECM is continuously adjusted to ensure the survival of cancer cells. The present approach enables to independently control and tune the 3D scaffold architecture and the 2D biomimetic films coatings, notably its stiffness and the type of loaded proteins (ECM or growth factor), in order to mimic the tumor microenvironment in a simplified and well-controlled manner. We also address another critical problem, regarding protein aggregation and the burst release of proteins from 3D scaffolds. Indeed, the PEM-functionalized Ormocomp structure is stable under cell culture conditions for extended periods of time without mechanical damage or swelling problems.

In most 3D-printed materials, proteins are adsorbed directly on the surface, leading to a burst release during cell culture [START_REF] Koons | Progress in Three-Dimensional Printing with Growth Factors[END_REF] . Microtumor generation with the TPP has significant advantages [START_REF] Zhou | A Review on the Processing Accuracy of Two-Photon Polymerization[END_REF] over available 3D cancer models such as organoids based on passive movement into a scaffold and spheroids based on passive accumulation due to limited direction of culture pathways. The femtosecond laser uses a nonlinear process to create a free-standing 3D structure. Various natural polymers (e.g., collagen, dextran, fibrin, and chitosan), their combination (e.g., collagen-acrylate, alginate-acrylate), and ceramics can be used in TPP [START_REF] Ciuciu | Two-Photon Polymerization of Hydrogels-Versatile Solutions to Fabricate Well-Defined 3D Structures[END_REF] . Natural polymers, such as collagen, are interesting in that they mimic the ECM, but conversely, they naturally induce various signaling pathways in cancer cells. In addition, using natural polymers may need high levels of crosslinking agents or photo-initiators, which can have toxic effects on cancer cells [START_REF] Van Landuyt | Evaluation of Cell Responses toward Adhesives with Different Photoinitiating Systems[END_REF] . Ormocomp is an inert and biocompatible polymer, which is photosensitive and enables high-resolution polymerization when exposed to a specific wavelength [START_REF] Schizas | Mechanical Characteristics of an Ormocomp® Biocompatible Hybrid Photopolymer[END_REF] . Ormocomp-based 3D models have been much discussed in recent research because of their biocompatibility, excellent transparency, and higher mechanical and chemical stability [START_REF] Kidwell | Chemistries for Making Additive Nanolithography in OrmoComp Permissive for Cell Adhesion and Growth[END_REF] . The use of TPP and the low refractive index of Ormocomp (1.520) allowed the scaffold to be fabricated without defect at submicron resolution. Thanks to its inertness, it does not trigger cell signaling pathways, unlike collagen-based hydrogels. However, the drawback is precisely that it needs further surface modification [START_REF] Kidwell | Chemistries for Making Additive Nanolithography in OrmoComp Permissive for Cell Adhesion and Growth[END_REF] . This is possible in view of the fact that it is a silicate-based organicinorganic composite [START_REF] Käpylä | Ormocomp-Modified Glass Increases Collagen Binding and Promotes the Adherence and Maturation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells[END_REF] . The roughness of the Ormocomp framework facilitates the adsorption of polyelectrolytes on the surface. Thus, surface functionalization via the biomimetic layer-by-layer films containing ECM proteins and growth factors can be advantageously used to provide bioactive signals.

The automation of the LbL deposit process ensures an excellent reproducibility in the surface coating of the 3D scaffold. (Figure 2). Altogether, the automated fabrication process and functionalization of the Ormocomp 3D structure ensured a high precision and robustness for large-scale preclinical in vitro studies. We noted that mechanical stability was good over time and that long-term cell culture was possible. We found that cell adhesion was enhanced after coating the scaffold with the PEM film loaded with FN. This is consistent with previous results showing that TU scaffolds coated with fibronectin enabled long-term cell culture [START_REF] Totti | A 3D Bioinspired Highly Porous Polymeric Scaffolding System for in Vitro Simulation of Pancreatic Ductal Adenocarcinoma[END_REF] . Furthermore, we showed that cells can remain in culture for at least 20 days in the 3D scaffold, which is important and will enable future long-term drug testing.

We have previously reported the uniform loading of BMP2 in PEM film [START_REF] Crouzier | Layer-By-Layer Films as a Biomimetic Reservoir for RhBMP-2 Delivery : Controlled Differentiation of Myoblasts to Osteoblasts[END_REF] . Here, using fluorescence confocal microscopy, we showed that protein loading in the film-coated 3D Ormocomp structure was uniform. Micro-BCA analysis confirmed that the three proteins (FN, BMP2 and BMP4)

were loaded in the film at 80 to 90% efficiency.

Here, we studied two types of pancreatic cancer cells, a model cell line PANC1 and a patientderived cell line PAN092, both with a similar number of passages (< 14). PANC1 cells are known for their aggressive growth [START_REF] Deer | Phenotype and Genotype of Pancreatic Cancer Cell Lines[END_REF] , while PAN092 showed slower growth than PANC1. PANC1 cells showed significant stiffness-dependent adhesion and preferred softer substrates. In contrast, PAN092 adhered more to the stiff EDC70 film than to EDC30 ones. The proliferation of PANC1 and PAN092 cells was similar on EDC30 and EDC70 films. In TCPS, PANC1 cells proliferated in an exponential phase even after 120 h. However, on EDC30 and EDC70 films, cell proliferation slowed down after 120 h and we observed an organoid-like morphology of cell aggregates in the film. Conversely, PAN092 cells grew in a steady state over the 5 days of culture and showed a similar morphology. These organoid-like cell assemblies proved that PEM properly supports and maintains the cells to allow 3D expansion. Picollet D'hahan et al. observed a small accumulation of cancerous epithelial prostate cells (PC3) on the polyelectrolyte films, which they explained by the fact that PEM reduces cancer cell migration [START_REF] Picollet-D'hahan | The Modulation of Attachment, Growth and Morphology of Cancerous Prostate Cells by Polyelectrolyte Nanofilms[END_REF] .

We analyzed the effect of signaling molecules, e.g. matrix-bound FN, BMP2 or BMP4 on cancer cells, and compared to their soluble form [START_REF] Virtanen | Bone Morphogenetic Protein -4 and -5 in Pancreatic Cancer-Novel Bidirectional Players[END_REF][START_REF] Amrutkar | Secretion of Fibronectin by Human Pancreatic Stellate Cells Promotes Chemoresistance to Gemcitabine in Pancreatic Cancer Cells[END_REF] . We found that the number of adherent cells in the presence of matrix-bound protein was higher than for cells in contact with soluble proteins. bFN had a similar effect on PANC1 and PAN092 cells, increasing cell adhesion and proliferation for EDC30 and EDC70 films, independently of film stiffness. We found that twice as much PANC1 cells adhered to EDC30 film than to EDC70 films, even if the amount of FN was similar for both EDC30 and EDC70

films. The increased adhesion induced by FN is consistent with cell adhesion being triggered by integrins [START_REF] Reuter | Oxidative Stress, Inflammation, and Cancer: How Are They Linked? Free radical biology and medicine[END_REF][START_REF] Arao | Β1 Integrins Play an Essential Role in Adhesion and Invasion of Pancreatic Carcinoma Cells[END_REF] . Conversely, when fibronectin was added as soluble molecule, it inhibited cell adhesion.

bFN increased PANC1 and PAN092 cell proliferation. It has reported that FN enables the continuous proliferation of cancer cells by inhibiting pro-apoptosis pathways through activating FAK and decreasing cytochrome release [START_REF] Pistritto | Apoptosis as Anticancer Mechanism: Function and Dysfunction of Its Modulators and Targeted Therapeutic Strategies[END_REF] . Although bFN boosted PAN092 proliferation, the rate was still lower compared to PANC1 cells. We also observed that bFN reduced the organoid nature of the tumor and allowed a more homogeneous distribution of cells inside the culture wells.

Regarding BMP2, bBMP2 slightly enhanced cell adhesion for both cell lines, regardless of film stiffness. However, sBMP2 showed only a minor inhibition of adhesion for both PANC1 and PAN092 cells. Although bBMP2 induced a small increase in adhesion, at higher concentrations (10-20 µg/ml) it had almost no effect on PANC1 cell proliferation whatever the film stiffness. In patient-derived PAN092 cells, bBMP2 had a dual effect on cell proliferation: having no effect for cell proliferation on soft films, while enhancing cell proliferation on stiff films. Here, we observed that bBMP2 inhibited proliferation of PANC1 cells at higher concentrations (10-20 µg/ml) and slightly increased proliferation at low concentrations (5 µg/ml) at both stiffness levels (Figure 4). Previous literature highlighted the effect of BMP2 on cancer cell signaling via the Smad pathway. Voorneveld et al. [START_REF] Voorneveld | Reduced Expression of Bone Morphogenetic Protein Receptor IA in Pancreatic Cancer Is Associated with a Poor Prognosis[END_REF] classified cell lines as Smadpositive when BMP2 inhibited proliferation and Smad-negative when BMP2 promoted cell proliferation.

In addition, BMP2 was shown to inhibit proliferation of pancreatic cancer cell lines via the Smad pathway [START_REF] Gordon | Bone Morphogenetic Proteins Induce Pancreatic Cancer Cell Invasiveness through a Smad1-Dependent Mechanism That Involves Matrix Metalloproteinase-2[END_REF][START_REF] Li | Secreted Phosphoprotein 24κD (Spp24) Inhibits Growth of Human Pancreatic Cancer Cells Caused by BMP-2[END_REF] . Previous studies also revealed the effects of ECM stiffness on Smad signaling pathway.

The above results show that film stiffness is very important in the control of Smad signaling in pancreatic cancer.

Regarding bBMP4, it improved both cell adhesion and proliferation of PANC1 cells. However, sBMP4 showed little inhibition of PANC1 cell adhesion. This result also highlighted the importance of film stiffness and delivery mode in the regulation of cell adhesion, spreading and proliferation. However, for PAN092 cells, bBMP4 on EDC30 films inhibited both cell adhesion and proliferation. On stiff EDC70 films, bBMP4 also inhibited of cell adhesion. The above results highlight the bipolar effect of bBMP4 on cancer cells. Virtanen et al. observed that BMP4 significantly reduced cell proliferation in PANC1 and HPAF-II cells by arresting cell growth in the G1 phase [START_REF] Virtanen | Bone Morphogenetic Protein -4 and -5 in Pancreatic Cancer-Novel Bidirectional Players[END_REF] . They also observed that MIA PaCa-2 cells did not respond to BMP4. In our work, bBMP4 slightly increase PANC1 cell proliferation on both EDC30 and EDC70 films. For PAN092 cells, the reverse was observed, with an increase in cell adhesion in response to both sBMP4 and bBMP4.

An interesting aspect is the differential pSMAD response for PANC1 cell versus PAN092 cells, PANC1

being BMP responsive notably with sBMP2 and bBMP2, while PAN092 did not respond to BMPs.

BMP2 interactions with BMP receptors are the initial steps of the BMP signaling response, which was recently quantified using biolayer interferometry [START_REF] Heinecke | Receptor Oligomerization and beyond: A Case Study in Bone Morphogenetic Proteins[END_REF] . The fact that PAN092 were insensitive to BMPs may be due to a mutation of Smad proteins that is common in pancreatic cancer [START_REF] Singh | The Smad Family and Its Role in Pancreatic Cancer[END_REF] . In terms of sensitivity to different BMPs, we observed an increase in pSMAD signaling for bBMP4, but of lower intensity than in response to bBMP2. We also noted that pSMAD response was systematically higher for cells on soft films with bBMPs (Figure 6). Regarding FN alone, we found that it did not induce pSMAD signaling and even decreased the pSMAD background. PANC1 grown on FN-coated films were more responsive to sBMP2 on soft films, while PAN092 were more responsive to sBMP2 on stiff films (Figure S15). In a previous study, we showed that myoblast cells cultured on FN micropatterns can modulate the BMP response [START_REF] Fitzpatrick | Signal Mingle: Micropatterns of BMP-2 and Fibronectin on Soft Biopolymeric Films Regulate Myoblast Shape and SMAD Signaling[END_REF] . Interestingly, our results demonstrated that the combination of the presentation of signaling molecules and film stiffness can affect cancer cell phenotype, proliferation, and the signaling cascade.

Overall, we showed that engineered biomaterials can be used to study pancreatic cancer cell response to a combination of film stiffness and bioactive proteins. We revealed cell-specific responses for PANC1 versus PAN092, stiffness-dependent cellular behaviors, and BMP-dependent cellular responses.

Notably, PAN092 did not respond to BMPs while PAN-1 exhibited a strong BMP-dose dependent BMP signaling, which was stronger for bBMP2 than for bBMP4.

Conclusion and Perspectives

Here, using almost full-automated processes, we engineered a bioactive niche with a controlled 3D microenvironment at the micro-scale in which cells can form a microtumor-like tissue. We have also shown that biomimetic film coating can mimic the biophysical conditions of the tumor microenvironment, including stiffness and delivery of adhesion proteins and growth factors like BMP2 and BMP4. We showed that pancreatic cancer cell adhesion, spreading, proliferation and BMP signaling were influenced by both the biomechanical and biochemical signals in a cell-type specific manner. Notably, we observed that cells derived from PAN092 patients did not respond to BMP2 and BMP4, suggesting that these cells may possess Smad mutations reported in previous studies. Interestingly, the biomimetic films may be applied as coating of various polymers, ceramics or composite materials made using different 3D printing systems, thus opening the range of studied biomaterials. In the future, we plan to perform longer-term studied and drug treatment in order to modulate cancer cell signaling and prevent cancer cell growth and metastasis.

Declaration of Competing Interest

None.

Figure 1 .

 1 Figure 1. Schematic representation of the overall process of microtumor development on 3D bioactive scaffolds. (A) Polyelectrolyte multilayer film coating using a liquid handling robot, protein loading and cell seeding on the 3D Ormocomp scaffold and (B) Polyelectrolyte multilayer coating and protein loading on the 96-well plates for high-throughput cellular assays.

Figure 2 .

 2 Figure 2. Fabrication and characterization of the 3D Ormocomp scaffold. (A) Structure and steps in TPP, (B) with the computer-aided design. Optical image in bright field (C) of the top view and (D) 3D reconstruction. Scanning electronic microscope (SEM) images of (E) the scaffold and (F) its surface.

  .C-D). The uniform red staining around the scaffold bars shows the homogeneous incorporation and distribution of the protein within the film.

Figure 3 .

 3 Figure 3. Characterization of the coated 3D PEM-Ormocomp structure and of protein loading on the scaffold using a confocal scanning microscope. PEM coating labelled with PLL-FITC (green) (A, B) and BMP2 rhodamine (red) on the hybrid structure (C, D).

Figure 4 .

 4 Figure 4. Cell adhesion, spreading and proliferation of PANC1 on soft (EDC30) and stiff (EDC70) PEM film. Fluorescence images of cells with nucleus labelled with DAPI (blue) and actin cytoskeleton labelled with rhodamine (red) on (A) soft film and (B) stiff film with FN, bBMP2 or bBMP4. Cell adhesion

Figure 5 .

 5 Figure 5. Cell adhesion, spreading and proliferation of PAN092 on soft (EDC30) and stiff (EDC70) PEM film. Fluorescence imaging of cells with nucleus labelled with DAPI (blue) and actin cytoskeleton labelled with rhodamine (red) on (A) soft film and (B) stiff film with FN, BMP2 or BMP4. Cell adhesion (C, F, I), cell spreading (D, G, J), and proliferation (E, H, K) were examined in the presence of fibronectin (C-E), BMP2 (F-H), or BMP4 (I-K). The mean of 3 independent experiments is plotted with SEM. * indicates that the value is significantly different from the control condition.

Figure 6 .

 6 Figure 6. pSMAD signaling of PANC1 and PAN092 on soft (EDC30) and stiff (EDC70) PEM film. Fluorescence images of PANC1 cells on (A) EDC30 and (B) EDC70 and PAN092 cells on (F) EDC30 and (G) EDC70, with the nucleus marked in blue, the actin cytoskeleton in red, and pSMAD1/5 in green. The intensity of the fluorescent signal from pSmad1/5 was measured (C-E) in PANC1 cells and (H-J) in PAN092 cells on EDC30 or EDC70 in the presence of bFN, bBMP2, or bBMP4. The average intensity of the signal was plotted along with the SEM. * indicates that the value is significantly different from the control condition.

1 .

 1 Table summarizing the effects of the matrix-bound proteins on the different cellular processes, including cell adhesion, spreading, and proliferation, for the two cell types: PANC1

  .A). After 20 days, a colonization of the scaffold was observed from bottom to top and the formation of a compact cell mass. After 11 days, the nuclei and cytoskeleton were stained to highlight cell colonization inside the scaffold (Figure 7.B). Cells were present between the scaffold bases, demonstrating vertical colonization by PANC1 cells. In addition, a Live and Dead assay with live cells in green and dead cells in red, was performed after 20 days of culture (Figure 7.C). A very high viability of the cells was found within the cell aggregates. No necrotic core was visible in the cell mass. These data provide a first insight into 3D cell culture within the functionalized scaffolds.

Figure 7 .

 7 Figure 7. 3D culture of PANC1 cells on the bioactive scaffold loaded with 5 µg/ml fibronectin. (A) Bright field images of cells after 3, 4, 10 and 20 days of culture. Confocal images of PANC1 cells (B) with nucleus stained blue (DAPI) and cytoskeleton stained red (rhodamine) after 11 days in culture and (C) Live & Dead assay after 20 days in culture with live cells labelled in green and dead cells labelled in red.

Figure S4 .

 S4 Figure S4. Bright field images of PANC1 cells after 120 h of culture on EDC30 loaded with increasing concentrations of either bFN, bBMP2 or bBMP4.

Figure S5 .

 S5 Figure S5. Bright field images of PANC1 cells after 120 h of culture on EDC70, loaded with increasing concentrations of either bFN, bBMP2 or bBMP4.

Figure S6 .

 S6 Figure S6. Adhesion and spreading of PANC1 cells after 16 h of culture on a plastic plate with either sFN, sBMP2 or sBMP4.

Figure S7 .

 S7 Figure S7. Adhesion and spreading of PAN092 cells after 16 h of culture on a plastic plate with either sFN, sBMP2 or sBMP4.

Figure S8 .

 S8 Figure S8. Fluorescence micrographs of PANC1 cells after 16 h of culture on EDC30 with either bFN, bBMP2, or bBMP4.

Figure S9 .

 S9 Figure S9. Fluorescence micrographs of PANC1 cells after 16 h of culture on EDC70 with either bFN, bBMP2, or bBMP4.

Figure S10 .

 S10 Figure S10. Adhesion and spreading of PANC1 cells after 16 h of culture on EDC30 and EDC70 with sFN, sBMP2 and sBMP4.

Figure S11 .

 S11 Figure S11. Adhesion, spreading and proliferation of PAN092 cells on different substrates.

Figure S12 .

 S12 Figure S12. Adhesion and proliferation of PAN092 cells on EDC30 and EDC70 with sFN, sBMP2 and sBMP4.

Figure S13 .

 S13 Figure S13. Fluorescence micrographs of PAN092 cells after 24 h of culture on EDC30 with either bFN, bBMP2 or bBMP4.

Figure S14 .

 S14 Figure S14. Fluorescence micrographs of PAN092 cells after 24 h of culture on EDC70 with either bFN, bBMP2 or bBMP4.

Figure S15. pSmad1/ 5

 5 Figure S15. pSmad1/5 signal intensity in (a) PANC1 cells and (b) PAN092 cells cultured on either EDC30 or EDC70 with sFN, sBMP2 and sBMP4.

Acknowledgments

This work was supported by the Fonds Unique Interministériel (FUI) -Banque Publique de l'Innovation (BPI) France, Metropoles of Lyon and Grenoble, Lyonbiopole, Minalogic and La Région Auvergne-Rhône-Alpes (AAP-23 3D ONCOCHIP, N° DOS0062033/0). A. Rengaraj is a postdoctoral fellow funded by Fondation ARC (PDF20171206771). C. Picart is a senior member of the Institut Universitaire de France. Our team is supported by Fondation pour la Recherche Medicale (DEQ20170336746), ERC POC Bioactivecoatings (GA692924) and SATT Bioactivcoatings (160129).

We would like to acknowledge the French IMODI cancer consortium for providing us with the patientderived cancer cells and the financial support to CTIBIOTECH of BPI France and the French Government (PSPC).

For Table of Contents Only

Supporting Information

Table S1. µBCA analysis of protein loading on PEM film for EDC30 and EDC70.


Dr Marvin Langlais

Dr Nicolas Baulu

Dr Séverin Dronet

François Charlotte Dire

Dr David Albertini

Franck D ' Agosto
email: franck.dagosto@univ-lyon1.fr

Dr Damien Montarnal
email: damien.montarnal@univ-lyon1.fr

Dr Christophe Boisson
email: christophe.boisson@univ-lyon1.fr

François Jean-Baptiste-Dit- Dominique

Dr Franck D'agosto

Dr F Jean-Baptiste

Multiblock Copolymers Based on Highly Crystalline Polyethylene and Soft Poly(ethylene-co-butadiene) Segments: Towards Polyolefin Thermoplastic Elastomers

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Introduction

The properties of thermoplastic elastomers (TPE) are strongly dependent on the structuration at the micro-or nanoscale between a major elastomeric phase and a minor rigid phase playing the role of physical cross-links. Either obtained through reactive processing or self-organization, these materials are not based on chemical cross-linking percolating through the continuous phase which allows them to be (re)transformed like thermoplastics and recycled. This class of materials is thus particularly appealing in the context of circular economy. Consequently, it appears necessary to develop new classes of TPEs to improve their overall environmental impact and broaden their range of usage properties. This can be achieved by controlling the glass transition temperature (Tg) of the soft matrix and increasing the Tg or the melting temperature (Tm) of the rigid domains. [1] A number of TPEs have been developed industrially from the step-growth polymerization of a large variety of building blocks such as polyurethanes, polyether block amides, polyether block esters. [START_REF] Holden | Thermoplastic Elastomers[END_REF] Well-controlled or living chain growth strategies able the control precisely the macromolecular architecture of block copolymers with soft and rigid segments offer however easier handles to fine-tune the morphologies of the materials, that directly impacts their thermomechanical properties. As an example, block copolymers obtained by living anionic polymerization such as polystyrene-b-polybutadiene-bpolystyrene (PS-b-PB-b-PS also called SBS) and polystyrene-bpolyisoprene-b-polystyrene (PS-b-PI-b-PS, SIS) are ubiquitous class of TPEs. [1] Recent developments can be highlighted in the domain of polyesters such as switchable catalysis [START_REF] Stößer | [END_REF], [4] or chainshuttling copolymerization [5] that paves the way to sequencedefined architectures. Polyolefin thermoplastic elastomers (P-TPEs) constitute another attractive class of materials for their high performance and economical sourcing. Different synthesis strategies from coordination polymerization and challenges in this field have been thoroughly discussed in this area in a recent review. [6] A large range of amorphous or crystalline polyolefins can be obtained by (co)polymerizing a very narrow set of easily available and cheap monomers such as ethylene (E), -olefins or conjugated dienes. These materials also combine several highly valuable properties such as high entanglement moduli (above 1 MPa) and low Tg. The living polymerization by coordination-insertion polymerization of olefins (E, propene (P), -olefins) has also been extensively studied to produce block copolymers with remarkable properties. [7] However, only one polymer chain is produced per metallic active center, which considerably increases the overall cost and the amount metallic residues, and thus reduces the possibility of large-scale production of these polymers. Coordinative chain transfer polymerization (CCTP) allows generating a very large number of polymeric chains per metallic active center. This controlled coordination-insertion polymerization technique takes advantage of degenerative chaintransfer between the metallic active center used as a catalyst and an organometallic chain transfer agent (CTA) such as MgR2, ZnR2 or AlR3. [8][9][10][11][12] Diblock and multiblock copolymers have been synthesized via CCTP. At Dow Chemical researchers obtained multiblock copolymers, [13,14] now commercialized by the tradename INFUSE TM , by combining two catalysts with a common CTA, ZnEt2, in the presence of E and octene (Oct). Oct being preferentially copolymerized with E by one of the two catalysts, soft P(E-co-Oct) and rigid PE segments are formed within the same chains. More recently, using a single catalyst, Lee and coll. successfully prepared by CCTP PE-b-P(E-co-P)-b-PE triblock copolymers by successive polymerization of E and a mixture of E and P followed by a coupling reaction. [15] The same group has developed a divalent chain transfer agent based on zinc. [16] PS-b-P(E-co-P)-b-PS triblock copolymers have been obtained by successive pyridyl-amido hafnium catalyst-mediated coordination-insertion copolymerization of E and P and anionic polymerization of styrene. We showed that a new class of elastomers can be formed by copolymerizing E and butadiene (B) with the catalysts {Me2Si(C13H8)2Nd(BH4)2Li(THF)}2/MgR2. [17], [18] Called ethylene butadiene rubbers (EBR), these elastomers feature ethylene units, trans-1,4-and vinyl butadiene units and cyclohexyl motifs formed by intramolecular cyclisation (Scheme 1). More recently, we have highlighted that this catalyst fulfills the requirement of CCTP [19] and is amenable to the design of macromolecular architectures incorporating EBR segments. [20], [21] Satisfactory elastomeric properties include low moduli, high extensibility and high elastic recovery after deformation. [22] In the case of TPEs from block copolymers, such properties are strongly linked with the proper separation of hard segments into noncontinuous domains, and proper anchoring of the soft segments across hard domains thanks to triblock or multiblock architectures. Widely used SBS-or SIS-type TPEs feature high incompatibility between the PS and PB or PI segments, which ensures strong segregation in the melt and easy control of the morphology by tuning the volumic fraction of PS. Polyolefin block copolymers feature low incompatibility in the melt, and often rely on crystallization-induced phase separation from weakly segregated melts. [23] They display in this way an advantageous combination of good mechanical performances and low melt viscosities. The present paper demonstrates that well-defined diblock, triblock and multiblock copolymers featuring soft EBR and crystalline PE segments can be prepared using {Me2Si(C13H8)2Nd(BH4)2Li(THF)}2 (1)/MgR2 as a catalyst according to CCTP. Structural, mechanical and rheological characterization confirms that although these materials are made from more than 80 wt% E overall, the phase separation between the crystalline PE domains and the soft EBR phase leads to remarkable performances as TPEs.

Results and Discussion

Synthesis of diblock copolymers via CCTP

As mentioned in the introduction, the copolymerization of ethylene with butadiene using {Me2Si(C13H8)2Nd(BH4)2Li(THF)}2 ( 1)/(n-Bu)(n-Oct)Mg (BOMAG) catalyst is controlled by a mechanism of CCTP and thus well-adapted to the design of block copolymers. First, diblock copolymers EBR-b-PE (Scheme 1 and Table 1, runs 2,4-6,8) were prepared in a single reactor by changing the feed from an ethylene-butadiene mixture to pure ethylene. The theoretical molar mass of each block is directly monitored by the consumption of monomers during each step (i.e. pressure decay in the ballast), the number of chains being fixed by the amount of BOMAG. Reference EBRs were prepared as models for EBR segments before chain extension (Table 1, runs 1, 3, 7). As already reported, a good control of the copolymerization was confirmed by the good match between theoretical and measured molar masses and rather low dispersity values. For each diblock copolymers (runs 2, 4-6, 8 in Table 1), there is also a good agreement between the theoretical and experimental Mn, although a broadening of the molar mass distribution is observed for the highest PE molar mass targeted (runs 6 and 8 in Table 1). Such broadening could be due to -H elimination during the second step leading to dead EBR-b-PE chains and PE chains after reinitiation. However, PE could not be detected by SEC (Figure S2 andS3) although its formation cannot be completely ruled out. We believe that the broadening could indeed originate from the variety of chain ends of the EBR block, such as alkyl, cyclohexyl-methylene or (vinylcyclohexyl)-methylene depending on the different insertion modes of butadiene and reinsertion of pendant double bonds. [19] Without impeding the formation of block copolymers after chain extension, the corresponding various reactivities towards ethylene insertion during chain extension would broaden the final molar mass distribution. We indeed previously highlighted that the catalytic insertion of ethylene after (vinylcyclohexyl)-methylene terminal groups is significantly slower than after other terminal groups. On the other hand, for polymers with higher molar masses, the viscosity of the polymerization medium increases significantly during the course of polymerization. This may affect chain transfer rates and increase the dispersity values. Diblock copolymers of increasing molar masses (Table 1, runs 2, 5, 8) were synthesized keeping the same fraction of PE (~17 wt%). SEC analyses of the reference EBRs and of the corresponding diblock copolymers showed successful chain extensions (Figure S2). In addition, the three diblock copolymers displayed very similar microstructures as expected (Table S1). A series of diblock copolymers based on a EBR with a targeted Mn of 30 kg mol -1 (EBR30K) with an increasing fraction of PE were also prepared (runs 4-6 in Table 1). As expected, the percentage of ethylene units increases from an EBR to an EBR-b-PE block copolymers (Table S2). Mn theo [a] (g mol -1 )

Mn SEC [b] (g mol -1 )

Đ [b]
Tg [c] (°C)

Tm [c] (°C)

Tc [c] (°C) PE fraction (wt %)

Crystallinity

of PE segments [d] (%) (MesMg(CH2)5MgMes). In association with the complex 1, a selective polymer chain growth on the pentanediyl moiety was demonstrated, the mesityl moiety remaining unreactive. [24] During the course of this work, we realized that the ability of Grignard reagents such as butylmagnesium bromide (BMB) to act both as alkylating agent and CTA was specific to the use of complex 1 (Table S3). Indeed BMB does not activate bispentamethylcyclopentadienyl neodymium precursors such as (C5Me5)2NdCl2Li(OEt2)2 and ((C5Me5)2Nd(BH4)(THF) for ethylene polymerization. The pentanediyl-1,5-di(magnesium bromide) (BrMg(CH2)5MgBr -PDMB) was then anticipated to be an efficient CTA when used in combination with 1 for the synthesis of the targeted PE-b-EBR-b-PE triblock copolymers (Scheme 2). PDMB was prepared by reacting dibromopentane with magnesium in 2methyltetrahydrofuran (MeTHF). After evaporation of the solvent, the product was solubilized in toluene (0.43 M, MeTHF/Mg = 2.15). The control of the copolymerization of E and B by the 1/PDMB catalyst was first demonstrated when forming EBR using various amounts of PDMB. As expected, a decrease of Mn was observed when increasing the concentration of the CTA (runs S3-S6 in Table S3) while dispersity values as low as 1.4 could be obtained, similarly to dispersities previously obtained with 1/BOMAG. PDMB was next implemented to synthesize triblock copolymers by achieving EBR by sequential copolymerization of E with B followed by its chain extension in the presence of E (Scheme 2) in a one-pot process. Two triblock copolymers with overall 17 wt% PE were prepared from EBR blocks of Mn of ca 30 and 60 kg mol -1 (Table 2, runs 9 and 10, respectively) according to the same previously described procedure employed for the synthesis of the EBR-b-PE diblock copolymers with BOMAG. SEC traces of the isolated triblock copolymers (Figure S4) were

PE-b-EBR-b-PE

unimodal showing the formation of the desired triblock copolymers.

The control of the monomer feeds and the pseudo-living nature of the polymerization enable multiple switches from a mixture of E and B to pure E while forming multiblock copolymers with PE and EBR segments. This was indeed achieved up to an heptablock copolymer (Table 2, run 11d). Mn of 10000 and 3000 g mol -1 were targeted for the EBR and PE segments, respectively. In this case, the polymerization was performed at 80°C and aliquots of the reaction medium were withdrawn before each switch of monomer feed. The evolution of the molar mass distribution after each step (initial EBR block, triblock, pentablock and heptablock formation) is given in Figure 1 and shows the excellent control over the successive chain extensions. )

Mn SEC [b] (g mol -1 ) Đ [b]
Tg [c] (°C)

Tm [c] (°C)

Tc [c] (°C) PE fraction (wt%)

Crystallinity

of PE segments [d] (%) Figure 1. SEC chromatograms obtained after each switch of monomer feed during the synthesis of the heptablock copolymer.

Thermomechanical and rheological properties DSC analysis (Table 1, Figures S5 andS6) of the diblock copolymers showed a Tg of EBR segments at about -33°C, a very weak meltng endotherm related to EBR segments around 10°C, and the melting of the PE block at 100-120°C. The normalized crystallinity of PE (See Table 1) is relatively low for short segments on low molar mass copolymers (15% and 33% for EBR15k-b-PE3k and EBR30k-b-PE3k, respectively) and the corresponding melting endotherms are very broad on DSC traces. In all other cases however, melting endotherms were narrow and the relative crystallinity was above 50%. Similar results were found for triblock copolymers PE-b-EBR-b-PE (Table 2, Figure S7), with the notable exception that even short PE3k segments displayed high crystallinities of 60%.

Regarding the multiblock copolymer (Figure S8 2). The heptablock displayed two distinct Tm that we attributed to internal (101.8°C) and external (121.3°C) PE blocks. Indeed, similar drastic changes in the crystallization temperature of internal or external blocks have already been observed in block copolymers based on poly(εcaprolactone) and poly(trimethylene carbonate) segments. [25] Interestingly, the relative PE crystallinity barely changes in the three samples (See Table 2). Extended thermomechanical information was obtained from DMA in tensile mode (Figure 2-a,b, Table S5) that enables to characterize the materials up to their melting point. Similar glass transition temperatures for all samples were confirmed. In the temperature range 0-30°C, the moduli appeared essentially driven by the fraction of PE, and reach at 25°C about 4 MPa for 9 wt% PE (run 4), about 11 MPa for 17 wt% PE (runs 2,5,9,10) and about 25 MPa for 29 wt% PE (runs 6 and 11d). Above 50°C, low molar mass diblocks EBR15k-b-PE3k and EBR30k-b-PE3k, and to a lower extent EBR30k-b-PE6k, softened progressively, thus confirming the broadness of the melting transition. The ratio E'25°C / E'80°C characterizes the conservation of properties in the service temperature range: triblock copolymers, even with short PE segments, performed significantly better than diblock copolymers. The multiblock copolymer, also containing short PE segments, demonstrated the least dissipative performance, with the lowest tan δ value at 80°C While the two distinct melting transitions in the multiblock copolymer 11d can be clearly observed, analysis in tensile mode 15 20 25 Retention time (min)

Block 1 -EBR 10K Block 2 -PE 3K -b-EBR 10K -b-PE 3K Block 3 -EBR 10K -b-PE 3K -b-EBR 10K -b-PE 3K -b-EBR 10K Block 4 -PE 3K -b-EBR 10K -b-PE 3K -b-EBR 10K -b-PE 3K -b-EBR 10K -b-PE 3K
is not adapted for melts, and to complete the monitoring of melting and crystallization we resorted to DMA experiments in a rheometer, using oscillatory shear by plate-plate geometries (Figure S9). The complex modulus G* during cooling-heating steps showed moderate hystereses between crystallization and melting, between 16 and 30°C, indicating fast crystallizations. In the melt, the complex moduli appeared to increase with the total molar mass of the copolymers, with however a surprisingly high value for EBR30k-b-PE12k. This was confirmed by small amplitude oscillatory shear (SAOS) at 150°C (Figure S10) that showed melts in or near terminal flow regime (G'~ω 2 , G''~ω 1 ) except for EBR30kb-PE12k. Finally, zero shear viscosities at 150°C were extrapolated from flow curves (Figure S11) and reported on Figure S12. While most samples obey the power law η0~M 3.4 characteristic of entangled melts, significant deviations are found for EBR15k that appears to be below the critical entanglement molar mass, and for EBR30k-b-PE12k and EBR45k-b-PE9k that may present to a certain extent phase separation in the melt.

Structural analysis

SAXS analyses at 150°C (Figure S13) confirmed that most EBRb-PE block copolymers display featureless patterns, thus corroborating the absence of structuration in the melt. The good compatibility between the blocks could be indeed anticipated from the microstructure of EBR containing about 78 mol% of ethylene units (Table S2). In accordance with melt rheology, EBR45k-b-PE9k displayed however a broad scattering signal, rather distinctive of weak segregation without long range ordering. This could either stem from segregation between the PE and EBR blocks that may have reached a sufficient molar mass, or from the presence of a small amount of PE homopolymer that cannot be ruled out from SEC traces (Figure S2). At room temperature (RT) however, sharp scattering peaks and corresponding 2q* harmonics indicated strong microphase separation and formation of lamellar structures. Simultaneous SAXS/WAXS monitoring during cooling (see selected examples in Figure S14) indicated that this phase segregation is concomitant with crystallization of PE segments, and thus that these copolymers undergo crystallization-induced self-assembly from disordered melts. As a consequence, the morphology and interdomain spacings between crystalline PE lamellae are not necessarily dictated by the molar mass or the copolymers or the volume ratio of PE and EBR blocks. [START_REF] Müller | Block Copolym[END_REF], [START_REF] Van Horn | [END_REF] EBR15k-b-PE3k displayed for example longer interdomain spacing (58 nm) than EBR30k-b-PE6k (45 nm), most probably because of its lower crystallinity. Direct imaging of the structuration was obtained by carefully surfacing bulk samples using ultracryo microtomy and imaging with PeakForce AFM. The same tip was kept for all images to allow for direct comparison of moduli between samples. The modulus images (Figure 3) show for all cases crystalline PE lamellae (in bright), separated with amorphous matrix. Profile analysis (see selected traces in Figure S15) shows interlamellar spacing in good accordance with the primary diffraction peak determined with SAXS. The moduli of crystalline lamellae measured with PeakForce (Figure S15) appears strongly correlated to the relative crystallinity within PE segments (Tables 1 and2), varying typically between 20 MPa for weakly crystalline PE domains in EBR15k-b-PE3k to 200 MPa for highly crystalline PE domains in PE3k-b-EBR30k-b-PE3k. Interestingly, the lamellae appeared also thicker and flatter for triblock copolymers than for the corresponding diblocks (Figure 3). In the multiblock copolymer 11d, no distinction can be made between two types of PE crystalline domains. The lamellae are however much smaller than in triblocks (no more than 200 nm large) and appear tightly packed.

Tensile properties

Tensile properties at RT of block copolymers displayed a variety of behaviors (Figure 2c, Table S5). Sample EBR15k-b-PE3k and EBR30k-b-PE3k had a viscoplastic behaviour that precluded conventional tensile testing. Initially, all materials displayed a rigid behaviour and a yield point at about 50% elongation typical of semicrystalline materials. The Young moduli E were in good accordance with DMA storage moduli at 25°C (See Table S5), and were essentially driven by the overall fraction of crystalline PE. This indicated to some extent a physical percolation between crystalline lamellae as confirmed by the AFM pictures showing intertwined lamellar stacks (Figure 3). The multiblock 11d, also composed of short PE3k segments showed in this regard improved strength (σmax = 3.2 MPa) without compromises on the elongation at break, due to a higher fraction of crystalline materials. Finally, PE6k-b-EBR60k-b-PE6k displayed far better strength with accelerated strain hardening occurring above 900% elongation similarly to chemically crosslinked elastomers and reached the maximal course of our tensile setup without breaking (σmax > 4.4MPa, εb > 1450%).

The tensile characterization was completed by cyclic extensions to 300% to assess the elastic recovery (Figure S16 and Figure 4). This property is indeed critical for TPEs, that must be able to withstand repeated loads while maintaining their dimensional stability. All block copolymers present a significant and irreversible loss of moduli and disappearance of yield behavior after the first stretching (Figure S16). As mentioned above, we attributed this to slip across lamellar stacks. The EBR45k-b-PE9k diblock copolymer performed relatively poorly as expected in absence of chain bridging across PE lamellae, and the corresponding strain recovery decayed continuously with each cycle. Triblock copolymers and especially the PE3k-b-EBR30k-b-PE3k performed significantly better, with strain recoveries up to 93%. Finally, the multiblock 11d offered a very promising combination of high stiffness with excellent strain recovery up to 89%.

Conclusion

The development of polyolefin thermoplastic elastomers by catalytic routes is a major challenge for achieving recyclable polyolefin-based elastomers. We have shown in this paper that combining the control provided by CCTP and the design and use of original chain transfer agents such as bimetallic magnesium bromide allowed to synthesize from diblock to multiblock copolymers incorporating crystalline PE and elastomeric EBR segments in the same chain. Low molar mass copolymers (< 100 kg mol -1 ) displaying low melt viscosities thanks to the absence of phase segregation in the melt underwent crystallization-driven selfassembly below 100°C. Depending on their macromolecular architectures (e.g. triblock or multiblock structures, segment molar mass and overall fraction of crystalline segments), a variety of mechanical properties can be achieved. The most promising material was PE6kb-EBR60k-b-PE6k triblock that demonstrate excellent extensibility above 1400% and strain recovery of 87% after 9 stretching cycles. In comparison, a multiblock ethylene-octene OBCs from Dow Chemical with a comparable content of 18 wt% in crystalline PE segments but a molar mass twice higher displayed similarly a low yield stress about 1.2 MPa, high extensibility up to 1200%, and an efficient elastic recovery of 94% after one cycle. Strain hardening at extensions > 500% is however much stronger due to a higher entanglement and leads to significantly higher ultimate tensile stress, at 14 MPa. [28] The PE3k-b-EBR10kb-PE3k-b-EBR10k-b-PE3k-b-EBR10k-b-PE3k multiblock copolymer also offered an excellent compromise with improved modulus, while also maintaining excellent strain recovery of 89% after 9 stretching cycles. SWING@Soleil, respectively, as well as Fabrice Brunel for performing temperature-dependent SAXS/WAXS experiments.

Scheme 1 .

 1 Scheme 1. Synthesis of EBR-b-PE diblock copolymers via CCTP.

Scheme 2 .

 2 Scheme 2. Synthesis of triblock copolymers using a (1)/PDMB catalyst.

  11d PE3K-b-EBR10K-b-PE3K-b-EBR10K-b-PE3K-b-EBR10K-bmL of toluene, 4 bars, 90°C (80°C for run 11), E/B = 80/20 mol%, [Nd] = 250 µM, [MesMgBr] = 1.5 mM, [PDMB] = 2.5 mM (run 9 and 11), 1.25 mM (run 10) and [a] Mn theo = yield/(nCTA), for the multiblock copolymer the expected Mn was considered. [b] Determined by SEC in 1,2,4-trichlorobenzene at 150°C. [c] Determined by DSC. [d] Determined by DSC ΔH100%PE = 293 J g -1 .

  ), the melting temperature of the PE block decreased when extending the PEb-EBR-b-PE triblock (113.9°C) to the EBR-b-PE-b-EBR-b-PE-b-EBR pentablock (104.4°C) (Table

Figure 2 .

 2 Figure 2. a,b) DMA analysis of block copolymers in tensile mode (1 Hz, 3°C min -1 ). c) Tensile testing (RT, 500 mm min -1 ). 1450% elongation corresponds to the maximal course and indicates that the samples did not break during the test.

Figure 3 .

 3 Figure 3. Modulus mapping through PeakForce AFM of samples a) EBR45k-b-PE9k, b) EBR30k-b-PE6k, c) EBR15k-b-PE3k, d) Multiblock 11d, e) PE6k-b-EBR60k-b-PE6k, f) PE3k-b-EBR30k-b-PE3k. The color scales are in Pa. 0 2 4 6 8 10 12 14A

Figure 4 .

 4 Figure 4. Elastic recovery after cyclic extension of the block copolymers at RT up to 300% elongation.

  b-EBR 60K -b-PE 6K

Table 1 .

 1 Molar masses and thermal properties of EBR and EBR-b-PE diblock copolymers discussed in this manuscript.

	Run	Polymer	[BOMAG] (mmol L -1 )	Yield (g)

Table 2 .

 2 Molar masses and thermal properties of PE-b-EBR-b-PE triblock and multiblock copolymers discussed in the manuscript.

	Run	Polymer	Yield (g)	Mn theo [a] (g mol -1

Acknowledgements

The authors thank the Manufacture Française des Pneumatiques Michelin for support and useful discussion. They thank the NMR Polymer Center of Institut de Chimie de Lyon (FR5223) for access to the NMR facilities and assistance. The authors acknowledge the Consortium Lyon Saint-Etienne de Microscopie (CLYM, FED 4092) for the access to the AFM microscope. The AFM characterization was supported by the LABEX iMUST of the University of Lyon (ANR-10-LABX-0064), created within the "Plan France 2030" set up by the french government and managed by the French National Research Agency (ANR). Julie Bratasanu is acknowledged for the preparation of AFM samples. We acknowledge the European Synchrotron Radiation Facility (ESRF) and SOLEIL for provision of synchrotron radiation facilities and we would like to thank Isabelle Morfin and Thomas Bisien for support in using beamlines BM02@ESRF and

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.

Keywords: polyolefin • thermoplastic elastomers • block copolymer • ethylene • butadiene