Optimized matrix composition for cancer related studies

Abstract

It is provided a biodegradable gel mimetic matrix for cells seeding, the mimetic matrix composed of a functionalized alginate and a functionalized biodegradable polymer with at least one peptide, such as PEG. The PEG carries vinyl-sulfone groups that enable it to interact with the peptides and alginate. Alginate controls the gelation and stiffness of the mimetic matrix. It is further provided a method of culturing cells onto the matrix, providing a model to study epithelial cell behaviour toward a pathological state like inflammatory bowel disease (IBD) or cancer, like colorectal cancer.

Description

OPTIMIZED MATRIX COMPOSITION FOR CANCER RELATED STUDIES CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application is claiming priority from U.S. Provisional Application No. 63 / 764140 filed February 27, 2025, and from U.S. Provisional Application No.63 / 837329 filed July 2, 2025, the content of which is hereby incorporated by reference in their entirety.TECHNICAL FIELD

[0002] It is provided a biodegradable gel mimetic matrix for normal and cancerous organoids seeding.BACKGROUND

[0003] Strategically located between gut epithelial and immune cells, subepithelial mesenchymal (SEM) cells are pivotal in inflammatory bowel disease (IBD) pathogenesis. They establish a natural microenvironment, influencing epithelial behaviour through the secretion of soluble factors, extracellular vesicles (EVs), extracellular matrix (ECM) proteins and stiffness. Recent studies show that SEM cells play a key role in remodelling the physical and cellular microenvironment during intestinal damage and repair.

[0004] IBD, including ulcerative colitis (UC) and Crohn’s disease (CD), pose a growing health and financial burden for Canadians, with an estimated 1.1% of the population projected to be affected by 2035. Chronic inflammation in IBD significantly contributes to the development of Colitis-Associated Cancer (CAC) in approximately 60% of patients. Through their strategic location, subepithelial mesenchymal (SEM) cells play pivotal roles in IBD pathogenesis. These cells shape their surroundings by secreting gradients of soluble factors, extracellular vesicles (EVs), and extracellular matrix (ECM) proteins, all together controlling stiffness and establishing a microenvironment that supports the epithelium. Epithelial cell behaviour, influenced by this microenvironment, is shaped by 2 major mechanisms: diffusible factors (WNT, BMP, BMP inhibitors) and the less-explored physical contacts provided by the ECM. Understanding how the physical communication of the microenvironment supports and influences epithelial behaviour is critical for developing novel therapeutics against Gl diseases.

[0005] Key microenvironment gradients, beyond soluble ligands, could arise as pivotal in novel therapeutic approaches for gastrointestinal (Gl) diseases through their downstream targets. Currently, there is no approved treatment targeting SEM cells or their key products impacting epithelial cell biology during IBD and cancer IBD. Existing treatments predominantly target immune cells, with some in clinical stages targeting the epithelium, but these promote remission at best with no cure. Recognizing the interdependence of the epithelium on its microenvironment might represent a novel opportunity to identify novel therapeutical targets in treating IBD, fibrosis and by extension, preventing further damage that leads to CAC.

[0006] Colorectal cancer (CRC) is the second most deadly cancer in Canada, largely due to late detection. Understanding the mechanisms driving CRC progression is crucial for developing more effective treatments. Intestinal stem cells (ISCs), located at the base of crypts, are influenced by their microenvironment, forming a regulatory network with the epithelium, stromal cells, and the extracellular matrix (ECM). The signaling pathways between the ECM and cells primarily involve WNT, NOTCH, and BMP. Studies on ECM changes in gastrointestinal diseases have focused mainly on the chemical regulators released by the ECM. The diversity of ECM proteins and stiffness levels significantly influence gastrointestinal function, yet this is often underestimated in research on digestive diseases.

[0007] The functional complexity of the colonic epithelium is shaped by its interaction with the ECM, where stiffness influences mechanotransduction and cellular phenotypes linked to diseases like IBD and cancer. Organoids provide a powerful model for studying ECM biodynamics in 3D models.

[0008] There is thus a need to provide minimal mimetic matrices with organoids as a model of how ECM deregulations drive epithelial cell behaviour toward a pathological state.SUMMARY

[0009] It is provided a biodegradable gel mimetic matrix for cells seeding comprising functionalized alginate, and functionalized biodegradable polymers with at least one peptide.

[0010] In an embodiment, alginate is functionalized with cysteines.

[0011] In another embodiment, the biodegradable polymers are polyalkylene glycol, polyalkylene glycol copolymers, polyoxazoline, polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyamides, polyvinyl alcohols, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, polyhydroxy ethyl acrylate, polyhydroxy ethyl methacrylate, or a combination thereof.

[0012] In a further embodiment, the polyalkylene glycol is polyethylene glycol (PEG).

[0013] In another embodiment, PEG is functionalized with an electrophilic group or a nucleophilic group.

[0014] In another embodiment, PEG is functionalized with azide, cyano, trifluoromethyl, vinyl groups or vinyl-containing groups.

[0015] In a further embodiment, PEG is functionalized with vinyl sulfone or maleimide.

[0016] In an embodiment, the gel mimetic matrix comprises functionalized PEG with a molecular weight greater than 2kDa.

[0017] In a further embodiment, the molecular weight of the functionalized PEG is of between 2 kDa and 100 kDa.

[0018] In another embodiment from the core matrisome and / or the associated matrisome proteins (secreted factors), the at least one peptide is a collagen peptide, as a fibronectin peptide, a laminin peptide, a thrombospondin peptide, a BMP or a S100A peptide, or a combination thereof.

[0019] In an additional embodiment, the collagen peptide is Collagen I (CL-I), Collagen IV (CL-IV), Collagen VI (CL-VI), Collagen X (CL-X) or a combination thereof.

[0020] In an embodiment, the laminin peptide is Laminin B1 (LAMB1), Laminin B6 (LAMB6), Laminin A4 (LAMA4), Laminin C1 (LAMC1) ora combination thereof.

[0021] In another embodiment, the S100A peptide is a S100A9 peptide, a S100A10 peptide, or combination thereof.

[0022] In another embodiment, the BMP peptide is a BMP7 peptide derived from the knuckle epitope, a BPM7 peptide derived from the wrist epitope, or combination thereof.

[0023] In an embodiment, the matrix comprises between 0.25 to 2% w / v functionalized alginate.

[0024] In an additional embodiment, the matrix comprises alginate-CYS mixed with PEG- vinyl sulfone-peptides.

[0025] In an embodiment, the matrix comprises 8-arm polyethylene glycol-vinyl sulfone (PEG-VS) macromers.

[0026] In another embodiment, the cells are derived from an organoid.

[0027] In an embodiment, the cells are epithelial cells.

[0028] In a further embodiment, the cells are subepithelial mesenchymal (SEM) cells.

[0029] In an embodiment, the cells are derived from a patient with a gut disease or liver disease.

[0030] In another embodiment, the cells are derived from a patient with an inflammatory bowel disease (IBD), fibrosis, neoplasia, ora cancer.

[0031] In an embodiment, the cancer is colorectal cancer (CRC) or endometrial cancer.

[0032] It is also provided a method of forming the gel mimetic matrix as provided herewith, comprising the steps of grafting cysteines onto alginate, grafting at least one peptide onto functionalized biodegradable polymers, mixing the alginate-CYS with the grafted biodegradable polymers- at least one peptide, and adding an ionic cross-linking agent to the mixture of alginate-CYS-grafted biodegradable polymers-at least one peptide producing the gel mimetic matrix.

[0033] It is also provided a method of culturing cells comprising, providing the gel mimetic matrix as defined herewith, seeding the cells on top of the gel mimetic matrix, and culturing said seeded cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Reference will now be made to the accompanying drawings.

[0035] Fig. 1 illustrates in A) the results of an evaluation of PEG functionalization with peptides by uHPLC. (N=3); and in B) the assessment of alginate (Alg) functionalization with L-cysteine (CYS) by quantification of free thiols (N=8).

[0036] Fig. 2 illustrates a graphical representation of storage modulus at a frequency of 10 Hz for an PEG 5%-alginate 0.5% hydrogel (w / v), Matrix, and Matrigel (N=5-7) in accordance with an embodiment.

[0037] Fig. 3 illustrates representative images of mouse colon organoids at 24h and 96h after seeding in mimetic matrix (or Matrigel) in proliferative medium (N =3).

[0038] Fig. 4 illustrates qPCR analysis of mRNA transcript associated with stem cells marker (LGR5), revival cells markers (murin CLU1 and Anxal), axin 2 gene (AXIN2) and YAP / TAZ signaling associated gene (CCN1). (N=3).

[0039] Fig. 5 illustrates Western blot analysis and densitometry analysis of mouse colon organoids treated with 1 pM Verteporfin for 48 hours confirming that increased matrix stiffness and subsequent activation of YAP signalling regulate CLU+ expression in organoids seeded on PEG-Alginate and within the matrix (N=3).

[0040] Fig. 6 illustrates qPCR analysis and Western blot analysis showing early neoplasia-associated genes Annnexin A in mouse colon organoids seeded on mimetic matrix, or PEG-alginate.

[0041] Fig. 7 illustrates representative images of reseeded mouse colon organoids that had been previously cultured for 96 hours on functionalized matrix, PEG-alginate, or Matrigel® in fresh Matrigel®.DETAILED DESCRIPTION

[0042] In accordance with the present description, there is provided a biodegradable gel mimetic matrix for cells, notably normal and cancerous organoids seeding.

[0043] The mimetic matrix is composed of a PEG part and an alginate (Alg) part. The PEG carries vinyl-sulfone groups that enable it to interact with the peptides and Algbearing cysteine residues (Alg-CYS). The percentage of Alg-CYS controls the gelation via calcium ions and stiffness of the mimetic matrix.

[0044] The functionalization of alginate (Alg) is verified by detection of the attached cysteine (CYS). Functionalized CYS have free thiol groups which react with Ellman's reagent (DTNB, 5,5-dithio-bis-(2-nitrobenzoic acid). The functionalization status of PEG is determined by the presence of ungrafted free peptides and grafted peptides on PEG measured by uHPLC (Fig. 1). The presence of ungrafted peptides is also quantified by measuring free thiol groups using DTNB.

[0045] In an embodiment, the mimetic matrix comprises 0.25 to 2% (w / v) Alg-CYS mixed with 5% (w / v) PEG-VS-Peptides, wherein PEG has a molecular weight greater than 10 kDa, such as between about 10 kDa and 100 kDa.

[0046] As exemplified herein, the mimetic matrices were composed of alginate (Alg) and 8-arm polyethylene glycol-vinyl sulfone macromers (PEG-VS) functionalized, respectively with cysteine (CYS) residues and ECM-mimicking peptides, including fibronectin (p-Fibro), laminin 111 (p-Lamb1), collagen I (p-Coll I) and collagen IV (p-Coll IV).

[0047] In an embodiment, PEG-VS can be functionalized with peptides which include, without be limited to, collagens (e.g. Collagen I (CL-I), Collagen IV (CL-IV), Collagen VI (CL-VI), or Collagen X (CL-X)), fibronectin (FN), thrombospondin, laminin (e.g. Laminin B1 (LAMB1), Laminin B6 (LAMB6), Laminin A4 (LAMA4), Laminin C1 (LAMC1)), BMP derived ligands (e.g. BMP7) or S100A derived peptides (e.g. S100A9 and S100A10).

[0048] The mimetic matrices provided herewith includes biodegradable polymers such as, but are not limited to, polyalkylene glycol such as polyethylene glycol (PEG) and polyalkylene glycol copolymers such as PEG copolymers, polyoxazoline, polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyamides, polyvinyl alcohols, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, polyhydroxy ethyl acrylate, polyhydroxy ethyl methacrylate. In some embodiments, copolymers of PEG or other polymers described above may be used to make the mimetic matrices.

[0049] Preferably, the mimetic matrices provided herewith include PEG. Typically, PEG has a molecular weight greater than 2 kDa, such as between about 2 kDa and100 kDa. It is encompassed that PEG has a molecular weight between about 5 kDa and about 90 kDa, between about 5 kDa and about 80 kDa, between about 5 kDa and about 70 kDa, between about 5 kDa and about 60. Preferably, PEG has a molecular weight of about 50 kDa, about 40 kDa, about 30 kDa, about 20 kDa or about 10 kDa. Preferably, the molecular weight of the PEG is about 20 kDa or about 40 kDa.

[0050] As encompassed herein, the term “functionalize” refers to modifying in a manner that results in the attachment of a functional group or moiety.

[0051] As described herein, PEG are branched and functionalized with a first linking moiety. The first linking moiety is typically a reactive group such as a vinyl sulfone group. In some embodiments, the reactive group can be an electrophilic group or a nucleophilic group. Exemplary electrophilic groups include, but are not limited to, azide, cyano, trifluoromethyl, vinyl groups or vinyl-containing groups (e.g., vinyl sulfone), maleimide. In some embodiments, the electrophilic group is vinyl sulfone or maleimide.

[0052] The composition of the mimetic matrix was designed to replicate the extracellular matrix (ECM) and stiffness changes observed in the Bmpr1a-Foxl1+mouse colon. Functionalization of Alg by cysteine residues (Alg-CYS) and PEG-VS by ECM derived peptides were confirmed by uHPLC or free thiol quantification (Fig. 1). Mimetic matrices’ stiffness was measured by dynamic mechanical analysis (Fig. 2A and B). Wild type mouse organoids were seeded on the mimetic matrices and cultured for 24 or 96h in the organoid growth media (Fig. 3). At 96h, qPCR was used to analyze proliferation, stem cell and differentiation markers, with Matrigel® as a control (Fig. 4).

[0053] Matrigel® is a commercial product used to provide the 3D scaffold for the growth of organoids of all cell types. Matrigel® is derived from a membrane extracellular matrix (ECM)-rich mouse sarcoma. It is known that Matrigel® batch-to-batch variability leads to inconsistent cell behaviors. Further, ECM is known to be implicated in the regulation of tissue development and function. The specific roles of ECM factors are difficult to ascertain using Matrigel® given that its molecular components cannot be readily manipulated.

[0054] Chemical characterization of the mimetic matrix revealed CYS grafting onto the Alg chain at ~190 pmol / g, while PEG-VS was functionalized with the four targeted peptides at concentrations ranging from 0.25 to 1.5 mM. Mimetic matrices’ stiffness exceeded 5 kPa, mimicking the fibrosis and / or early neoplasia microenvironment of thegut. The results presented herein show that mouse colon organoids seeded on these mimetic matrices survived and proliferated similarly to those seeded on Matrigel®, outperforming moderately those seeded on PEG-alginate alone (see Fig. 3). Organoids on mimetic matrix or PEG-alginate exhibited an opaque lumen unlike the open lumen of those grown on Matrigel®. This suggests a mechanosensitive response likely related to stress involving colonic stem cells grown in a stiffer matrix with a specific combination of extracellular matrix peptides (Fig. 3).

[0055] Notably, MYC mRNA levels were significantly higher in mouse colon organoids on the mimetic matrix, indicating increased proliferation and stress. However, LGR5, a stem cell marker, was not significantly expressed on the composite matrix in contrast to Matrigel®. Differentiation analysis revealed the absence of MUC2 mRNA but a marked increase in Chromogranin A mRNA on the mimetic matrix compared to Matrigel®. Revival stem cells (revSCs), marked by CLU1+expression and associated with the YAP / TAZ signalling pathway, are quiescent cells that serve as a reserve for epithelial regeneration during cellular stress or injury. Notably, mouse colon organoids seeded onto a mimetic matrix, or PEG-alginate, exhibited stress-related macroscopic features. Based on these observations, it is observed a potential shift in the dominant stem cell population-from LGR5+stem cells in organoids cultured on Matrigel® to CLU1+cells in those grown on stiffer substrates (see Fig. 4). RT-qPCR analysis revealed a significant difference in the stem cell population between organoids seeded on a mimetic matrix or PEG-alginate and those on Matrigel®. These organoids demonstrated a marked loss of LGR5+stem cells after 24 hours of culture, accompanied by the emergence and expansion of CLU1+RevSCs over 96 hours. In addition, their presence was confirmed by ANXA1 expression, a second RevSCs marker. Furthermore, it was observed a corresponding shift in the signalling pathways associated with each stem cell population. The WNT signalling pathway target gene AXIN2 was highly expressed in organoids cultured on Matrigel®, where the LGR5+stem cell population expanded. In contrast, the YAP target gene CCN1 was upregulated in organoids grown on mimetic matrix or PEG-alginate, correlating with the presence of CLU1+RevSCs (Fig. 4).

[0056] To confirm that CLU1+expression in RevSCs depends on YAP signalling, which is activated by increased cellular microenvironmental stiffness, the YAP inhibitor Verteporfin was used. Verteporfin inhibits YAP function by upregulating chaperone proteins that sequester YAP in the cytoplasm, thereby preventing its nuclear activity. Mouse colon organoids were treated with 1 pM Verteporfin for 48 hours, after whichproteins were extracted. Western blot analysis and densitometry confirmed that increased matrix stiffness and subsequent activation of YAP signalling regulate CLU+ expression in organoids seeded on PEG-Alginate and within the matrix (Fig. 5). Although functionalization with peptides is not absolutely necessary to activate YAP signalling or induce CLU+expression, it likely contributes to creating a more favorable microenvironment for organoid development. Importantly, organoid survival and growth were significantly improved when cultured on the matrix compared to PEG-Alginate alone, indicating that matrix composition, potentially including peptide functionalization, offers additional advantages beyond stiffness alone.

[0057] The stress-related macroscopic features observed in mouse colon organoids seeded on mimetic matrix, or PEG-alginate, indicated a potential development of preneoplastic changes. To further investigate, a qPCR analysis was performed targeting early neoplasia-associated genes. Notably, a significant upregulation of ANXA10 mRNA was detected, marker known to emerge in colorectal preneoplasia within 3-7 days following the transition from LGR5+stem cells to CLU+RevSCs, in organoids grown on both mimetic matrix and PEG-alginate, as compared to those cultured on Matrigel®. Western blot analysis corroborated these findings at the protein level, demonstrating that matrix functionalization is important for the expression of preneoplastic markers (Fig. 6).

[0058] To assess the regenerative capacity of mouse colon organoids seeded on either the functionalized matrix or PEG-alginate matrix, both of which promote a transition from the highly proliferative LGR5+stem cell state to the slow-cycling, quiescent CLU1+RevSCs population from mouse colon organoids. Organoids grown previously on Matrigel® were expected to persist in the LGR5+stem cell state, whereas those cultured on the functionalized matrix or PEG-alginate were anticipated to shift to the CLU1+RevSCs state. Upon reseeding these pre-conditioned organoids into fresh Matrigel® (Fig. 7), all organoids, regardless of their initial substrate, were able to reinitiate organoid formation (24h) and grow (72h). These results demonstrate that mouse colon organoids cultured on the functionalized matrix retain regenerative potential, comparable to a certain extent to that in Matrigel® despite transitioning to a quiescent state.

[0059] The present results indicate that the provided minimal mimetic matrix supports the survival and growth of colonic organoids. Its pathological stiffness and high levels of the core matrisome proteins fibronectin (FN), type I and IV collagens (CL-I and CL-IV) drive a disease-associated stress signature in the epithelium. This model offers a more precise tool than Matrigel® for studying the role of the microenvironment in gut diseases.

[0060] Accordingly, as described herein, a biofunctionalized mimetic matrix was developed to assess the impact of changes in the core and associated proteins of the matrisome on cancer progression. These ECM-mimetic matrix, designed with synthetic composite hydrogels (PEG / alginate), allow better control over the diversity of ECM ligands and the rigidity of the environment. This minimal matrix could serve as a more effective tool than Matrigel® for studying how the surrounding environment influences gut diseases.

[0061] SEM cells play a crucial role in orchestrating physical and biochemical cues within the microenvironment, influencing epithelial behavior during both homeostasis and disease. This interaction, often overlooked, has the potential to shape pathogenesis and contribute to treatment resistance. The ECM, a complex network of proteins, influences mechanical and biochemical stimuli vital for Gl mucosa homeostasis. These stimuli include the physical (porosity, fibre orientation), biochemical (composition), and biomechanical (stiffness) properties of the ECM. The crude protein extract features a complex protein network known as the complete matrisome, which comprises essential extracellular matrix (ECM) proteins collectively referred to as the core matrisome. In addition to these core proteins, the matrisome also includes associated proteins such as ECM regulators and secreted factors. This matrix also functions as a dynamic reservoir for critical growth factors and cytokines, such as TGFp, BMP, SA100, FGF, and WNT, allowing the ECM to sequester and release these molecules in a localized manner. This interaction with the associated matrisome proteins also influences neighboring cells. Comprising roughly 300 different proteins, the ECM includes various components such as collagens (CL), proteoglycans, and glycoproteins, including laminin (LAMB) and fibronectin (FN). Notably, the composition of the ECM undergoes significant alterations in response to various gastrointestinal (Gl) diseases, highlighting the importance of this specific group of proteins that make up the core matrisome. It is known that the comprehensive profiling of the microenvironment matrisome in a TCFoxL1+defective BMP signaling mouse model, unravelled novel targets for investigation. Beyond composition, the physical properties of the ECM, particularly stiffness, have emerged as critical determinants of cellular response.

[0062] Tissue stiffness is a dynamic and adaptive process during homeostasis and stromal remodelling and can be the determinant for IBD, endometrial cancer and colorectal cancer (CRC). Recent studies highlighted the importance of ECM mechanical properties in governing ISC maintenance and differentiation. Through mechanosensing mechanisms, involving cell-surface proteins such as integrins and growth factor receptors, epithelial cells respond to forces by modifying their shape and their behaviour.

[0063] SEM cells, positioned at the interface of immune and epithelial cell compartments, act as a potential communication nexus influencing gut physiology.

[0064] BmpR7aAFoxL1+mice, under stress and subsequent recovery, exhibited altered fibroblasts-myofibroblasts ratio, ECM changes, and delayed wound healing. Similar results occurred in fibroblasts with impaired BMP signaling. Using quantitative proteomics and ex vivo gut tissue deconstruction technique, the matrisome of 90-day BmpR7aAFoxL1+mouse colon was analyzed, revealing quantitative up-regulation of CLI and IV, fibronectin (FN), SERPIN H1, laminin B3 (LNB3), all proteins from the core matrisome and S100A9, from the associated matrisome proteins among others. From these, mimetic matrices as provided herewith were generated, comprising functionalizing alginate with CYS at the density of 193.1 + / - 44 pmol CYS / g of polymers (N=11). Thiol group quantification assays, UHPLC, and FITR analyses verified PEG-VS functionalization with peptides from CLI, CLIV, FN LN and / or S100A9 alone or in different combinations. Results demonstrate the vast potential for controlled matrix composition with various peptide combinations. Matrix stiffness can be modulated by the % of alginate, type and concentration of calcium solution used for the ionotropic gelation.

[0065] Hydrogels from alginate are forms by the addition of an ionic cross-linking agent, such as a divalent cation. A calcium solution (e.g., calcium chloride) is added to induce alginate ionotropic gelation.

[0066] Heterodimeric a|3 integrin receptors on the cell membrane mediate bidirectional transduction of mechanotransduction and biochemical signals. These receptors, categorized by ligands into leukocyte cell adhesion, CL (GFOGER)-binding, Arg-Gly-Asp (RGD)-binding, and LAMB-binding integrins involve downstream signaling through focal adhesion kinase (FAK), Src-family protein tyrosine kinases and integrin-linked kinase (ILK). In the BmpR7aAFoxL1+mouse colon, upregulated a5 integrin subunitsand ECM proteins (FN and CL) correlate with increased tissue stiffness. Composite matrices (alginate-PEG-VS-Peptides) as provided herein can be prepared to mimic these changes that will involve specific integrins.

[0067] In an embodiment, organoids are embedded in composite matrices with hemostasis stiffness (1 kPa) containing peptides (pLAMB, pColl, pS100A9 at 0.25 mM) and varying concentrations of p-Fibro or pColl (0.75 to 3 mM). Negative peptides (Np-Fibro and Np-Coll) maintain similar free PEG-VS groups between experimental conditions and assess the influence of targeted integrins a5pi and av|33 or a2pi on organoids mechanotransduction. Matrigel® and a synthetic PEG-VS-Peptide mimicking Matrigel® effects serve as additional controls. Both FAK and ILK have been identified as essential linkages of integrin-mediated fibrosis. The ILK expression is studied within 24h of plating by immunoprecipitation (IP) and western blots (WB) analyses to identify ILK binding partners (Parvin, Pinch and Paxillin). WB and / or immunofluorescence (IF) analyses verify FAK phosphorylation states (Y397, Y576-576, Y925) and assess actin processing (cofilin) linked to cytoskeleton remodeling.

[0068] Integrins, pivotal in cell-ECM interaction mediating mechanotransduction, also influence responses to soluble factors (cytokines, growth factors) impacting cellular processes like survival, proliferation, and differentiation. Downstream targets of ILK including Akt, glycogen synthase kinase-3 beta (GSK3P) and p-catenin crucial to Wnt3A signaling are explored on organoids response to Wnt3A and BMP to verify the impact of matrix composition and targeted integrins. Intracellular signaling pathways, including total and pGSK3p, p-catenin, MAPK pathways (ERK1 / 2), Smad2 / 3, and Smad1 / 5 / 9 are analyzed through IP, WB and IF analyses. Organoid’s ability to survive and proliferate are assessed using CellTiter-Glo® 3D Cell Viability assay; cleaved caspase 3 and Ki67.

[0069] BmpR 7aAFoxlL1+mice exhibit impaired mucus layers and altered goblet cells glycobiology contributing to increased susceptibility to IBD. The impact on matrix composition on YAP / TAZ pathways are assessed by analyzing the cytoplasmic / nuclear shuttling of YAP / TAZ and measuring the expression of target genes (AXL, CTGF, CYR61) using qRT-PCR, to measure the impact of the whole transcriptome level. Phosphorylated YAP at Ser-127, which inhibits nuclear translocation and is required for suppressing p-catenin signaling are assessed by IF. Changes in matrix stiffness seen in the BmpR7aAFoxL1+mouse colon can potentially affect goblet cell behaviours, impacting secretion and mucus quality as shown in the pancreas and lungs. Assessingmatrix stiffness and composition on goblet cell maturation and differentiation involve electron microscopy to assess mucin vesicles’ shape and organization. The expression of MUC2 and KLF4 are examined through IF and WB. Goblet cell functionality is explored by investigating post-translational modifications of mucins, specifically their glycosylation patterns. YAP’s role in goblet cell differentiation is confirmed using verteporfin to inhibit YAP nuclear translocation.

[0070] The mimetic matrix thus provides a means to investigate how altered ECM composition and stiffness influence gut diseases, such as IBD, fibrosis, or cancer, including but not limited to colorectal cancer (CRC). The biofunctionalized mimetic matrix replicates the toxic microenvironment of the Bmp / '7aAFoxl1+mouse model, which promotes gut fibrosis and early neoplasia. The provided minimal mimetic matrix provides a mean to study the impact of ECM changes on epithelial cell behaviour.

[0071] The matrix provided herewith is not restricted to gut diseases but can also be used to investigate liver-related diseases.

[0072] As described herein, it is provided a method of forming the gel mimetic matrix as described herein, comprising the steps of grafting cysteines onto alginate; grafting at least one peptide onto functionalized biodegradable polymers; mixing the alginate-CYS with the grafted biodegradable polymers-at least one peptide; and adding an ionic cross-linking agent to the mixture of alginate-CYS-grafted biodegradable polymers-at least one peptide producing the gel mimetic matrix.

[0073] Also encompassed is a method of culturing cells comprising providing the gel mimetic matrix as described herein, seeding the cells on top of the gel mimetic matrix, and culturing the seeded cells. In an embodiment, the cells are derived from an organoid. The cells can be epithelial cells, more specifically, subepithelial mesenchymal (SEM) cells. Preferably, the cells are derived from a patient with a gut disease or liver disease, such as, but not limited to, an inflammatory bowel disease (IBD), fibrosis, neoplasia, or a cancer, more preferably, but not limited to, endometrial and colorectal cancer (CRC).EXAMPLE IMatrices formation and characterization

[0074] The composite matrices encompassed herein are created by combining alginate with cysteine residues (CYS) with 8-arm polyethylene glycol-vinyl sulfone (PEG-VS) macromers containing adhesion peptides.

[0075] In Stepl. -110 kDa alginate (2:1 guluronic acid to mannuronic acid ratio) is used and CYS is grafted onto alginate chains via amide bond formation. Alginate is mixed with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) to activate the carboxylic acid groups, followed by L-CYS addition. Non-grafted reagents are removed, and verification includes Fourier Transform Infrared Spectroscopy (FTIR) analyses and thiol quantification to confirm CYS grafting.

[0076] In Step 2, adhesion peptides are sequentially grafted onto 20 kDa PEG-VS macromers via Michael-type addition. PEG-VS macromers are incubated with each peptide (0.25 to 3 mM). Salts are removed. Peptides quantification relies on thiol groups for non-grafted and grafting utilizes FTIR and Ultra High-Performance Liquid Chromatography (UHPLC) with mass spectroscopy.

[0077] In Step 3, lyophilized alginate-CYS (0.25 to 2% w / v) is mixed with 5% (w / v) PEG-VS-Peptides and filtered. Sterile calcium chloride (50 mM) is added to induce alginate ionotropic gelation.

[0078] The measurements were performed with a DMA instrument (Mettler-Toledo DMA 1+) equipped with a compression measurement module. A maximum deformation of 20 pm and a maximum force of 0.2 N, oscillating at frequencies between 0.1 Hz and 1000 Hz.

[0079] The stiffness and high levels of FN, LAMB1, CL-I, and CL-IV peptides create stress in the mimetic surface layer. When LGR5+ stem cells are cultured on this mimetic matrix, they transform into a revival population known as Clu1+.

[0080] While the present description has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations and including such departures from the present disclosure as come within known or customary practice within the art and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A biodegradable gel mimetic matrix for cells seeding comprising:functionalized alginate; andfunctionalized biodegradable polymers with at least one peptide.

2. The gel mimetic matrix of claim 1, wherein alginate is functionalized with cysteines.

3. The gel mimetic matrix of claim 1 or 2, wherein the biodegradable polymers are polyalkylene glycol, polyalkylene glycol copolymers, polyoxazoline, polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyamides, polyvinyl alcohols, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, polyhydroxy ethyl acrylate, polyhydroxy ethyl methacrylate, or a combination thereof.

4. The gel mimetic matrix of claim 3, wherein the polyalkylene glycol is polyethylene glycol (PEG).

5. The gel mimetic matrix of claim 4, wherein PEG is functionalized with an electrophilic group or a nucleophilic group.

6. The gel mimetic matrix of claim 5, wherein PEG is functionalized with azide, cyano, trifluoromethyl, vinyl groups or vinyl-containing groups.

7. The gel mimetic matrix of claim 5 or 6, wherein PEG is functionalized with vinyl sulfone or maleimide.

8. The gel mimetic matrix of any one of claims 4-7, wherein the gel mimetic matrix comprises functionalized PEG with a molecular weight greater than 2kDa.

9. The gel mimetic matrix of claim 8, wherein the molecular weight of the functionalized PEG is of between 2 kDa and 100 kDa.

10. The gel mimetic matrix of any one of claims 1-9, wherein the at least one peptide is a collagen peptide, a fibronectin peptide, a laminin peptide, a thrombospondin peptide, a BMP peptide or a S100A peptide, or a combination thereof.

11. The gel mimetic matrix of claim 10, wherein the collagen peptide is Collagen I (CL-I), Collagen IV (CL-IV), Collagen VI (CL-VI), Collagen X (CL-X) or a combination thereof.

12. The gel mimetic matrix of claim 10, wherein the laminin peptide is Laminin B1 (LAMB1), Laminin B6 (LAMB6), Laminin A4 (LAMA4), Laminin C1 (LAMC1) or a combination thereof.

13. The gel mimetic matrix of claim 10, wherein the S100A peptide is a S100A9 peptide, a S100A9 peptide, or combination thereof.

14. The gel mimetic matrix of claim 10, wherein the BMP peptide is a BMP7 peptide derived from the knuckle epitope, a BPM7 peptide derived from the wrist epitope, or combination thereof.

15. The gel mimetic matrix of any one of claims 1-14, comprising between 0.25 to 2% w / v functionalized alginate.

16. The gel mimetic matrix of any one of claims 1-15, comprising alginate-CYS mixed with PEG- vinyl sulfone-peptides.

17. The gel mimetic matrix of any one of claims 1-16, comprising 8-arm polyethylene glycol-vinyl sulfone (PEG-VS) macromers.

18. The gel mimetic matrix of any one of claims 1-17, wherein the cells are derived from an organoid.

19. The gel mimetic matrix of claim 18, wherein the cells are epithelial cells.

20. The gel mimetic matrix of any one of claims 1-19, wherein the cells are subepithelial mesenchymal (SEM) cells.

21. The gel mimetic matrix of any one of claims 1-20, wherein the cells are derived from a patient with a gut disease or liver disease.

22. The gel mimetic matrix of any one of claims 1-21, wherein the cells are derived from a patient with an inflammatory bowel disease (IBD), fibrosis, neoplasia, or a cancer.

23. The gel mimetic matrix of claim 22, wherein the cancer is endometrial cancer or colorectal cancer (CRC).

24. A method of forming the gel mimetic matrix of any one of claims 1-23, comprising the steps of:a) grafting cysteines onto alginate;b) grafting at least one peptide onto functionalized biodegradable polymers; c) mixing the alginate-CYS with the grafted biodegradable polymers- at least one peptide; andd) adding an ionic cross-linking agent to the mixture of alginate-CYS-grafted biodegradable polymers-at least one peptide producing the gel mimetic matrix.

25. A method of culturing cells comprising:a) providing the gel mimetic matrix of any one of claims 1-23;b) seeding the cells on top of the gel mimetic matrix; andc) culturing said seeded cells.

Images