3D co-culture system mimicking masld for hepatocellular carcinoma research

Abstract

The present invention relates to a three-dimensional (3D) co-culture system that mimics the MASLD microenvironment and is used to investigate its effects on hepatocellular carcinoma (HCC). The system comprises hepatic stellate cells embedded in a collagen matrix and HCC cells cultured in a metabolic medium that simulates MASLD conditions. This model enables the investigation of the molecular mechanisms of HCC under laboratory conditions and can be used to identify new therapeutic targets.

Description

[0001] DESCRIPTION

[0002] 3D CO-CULTURE SYSTEM MIMICKING MASLD FOR HEPATOCELLULAR CARCINOMA RESEARCH

[0003] Field of the Invention

[0004] The invention relates to a three-dimensional (3D) co-culture system that mimics the microenvironment of metabolic dysfunction-associated steatotic liver disease (MASLD) to investigate its effects on hepatocellular carcinoma (HCC), as well as the method for constructing this system.

[0005] Background of the Invention

[0006] Hepatocellular carcinoma (HCC) is the third most common cancer type worldwide, accounting for approximately 90% of all liver cancers. Metabolic disorders, particularly metabolic dysfunction-associated steatotic liver disease (MASLD), play a key role in HCC development. MASLD is a liver disease associated with metabolic syndrome, obesity, and type 2 diabetes. It is characterized by fat accumulation (steatosis), inflammation, and progressive fibrosis in hepatocytes. MASLD is one of the most common causes of chronic liver disease and, in advanced stages, can progress to nonalcoholic steatohepatitis (NASH). During disease progression, inflammation and fibrosis lead to genetic and epigenetic changes in hepatocytes, which may result in malignant transformation and trigger HCC development [1],

[0007] To better understand the pathophysiology of MASLD, develop new therapeutic strategies, and test the efficacy of drugs, both 2D and 3D in vitro MASLD models have been developed. Due to the complex and multi-factorial nature of MASLD, suitable models that reflect the disease’s natural course and pathogenic mechanisms are needed. These models aim to provide structures that recapitulate the natural microenvironment of MASLD and simulate disease conditions.

[0008] In models used for MASLD-derived HCC research, intercellular signaling and interactions must be assessable. An ideal MASLD model should allow detailed investigation of molecular mechanisms involved in HCC progression, assessment of tumor-related processes such as cell migration and infiltration, and evaluation of new therapeutic strategies. This is essential for understanding the influence of MASLD on HCC and for developing more effective treatments.

[0009] Previous studies, such as that of Bronsard et al., utilized a 3D multicellular liver organoid model for in vitro modeling of MASLD and drug toxicity evaluation. HepaRG cells, primary human macrophages, and hepatic stellate cell line LX-2 were used in that model [2], However, that system could not evaluate interactions between tumor and normal liver cells or observe metastatic potential. Furthermore, steatosis induction was achieved mainly through high-fat content, which does not fully reflect actual nutritional or metabolic conditions.

[0010] Prior art includes 2D monocultures, 2D co-cultures, spheroid models, liver-on-chip, and organoid models [3], Although these systems offer advantages in liver disease modeling, the use of a modified Boyden chamber system in MASLD-related HCC models has not been reported. Consequently, the paracrine interactions between tumor and normal hepatocytes under MASLD-like conditions cannot be effectively studied without direct contact between cells.

[0011] Due to the limitations of existing techniques — insufficient representation of tumor-MASLD interactions, unrealistic steatosis induction, and limited ability to observe bidirectional signaling — a realistic, metabolically relevant 3D in vitro model that closely mimics human MASLD-associated microenvironmental conditions is needed. Short Description and Objectives of the Invention

[0012] The invention provides a three-dimensional co-culture system mimicking the MASLD microenvironment. This system consists of activated hepatic stellate cells embedded in a collagen matrix and HCC cells cultured in a metabolically simulated MASLD medium containing glucose, free fatty acids (oleic and palmitic acids), fructose, and insulin. The MASLD microenvironment provides the necessary metabolic conditions for studying MASLD’s effects on HCC. Tumor and hepatic cells interact via a membrane in a two-chamber Boyden system without direct contact.

[0013] The aim is to explore molecular mechanisms underlying MASLD-driven HCC, identify novel therapeutic targets, discover predictive biomarkers for MASLD-associated HCC, and evaluate drug efficacy under MASLD conditions. Using this model, HCC cell behavior in a MASLD-like microenvironment can be simulated, enabling a detailed study of tumor progression mechanisms under metabolic dysfunction.

[0014] Another objective of the invention is to identify biomarkers that can predict the development of MASLD-associated HCC using this model. In this context, the invention employs the Boyden chamber system, which allows interactions between tumor cells and the environment that mimics MASLD conditions. Through the analysis of genetic, epigenetic, and metabolic alterations induced by factors secreted into the medium, biomarkers or therapeutic target molecules that can predict the development of MASLD-associated HCC can be identified. Furthermore, since the system contains both tumor and liver cells under MASLD conditions, it is possible to add candidate therapeutic drugs to different parts of the system and use it to test the effects of these drugs on both cancer cells and normal liver cells.

[0015] Description of Figures

[0016] Figure 1 :

[0017] (A) Schematic representation of the experimental design of the 3D co-culture system treated with the metabolic medium (MM) and (B) the 3D co-culture system containing the control medium without MM treatment. The figure also shows the PCR (i), and DigiWest, PCR, WB (ii) analyses performed after separating motile and non-motile cells, as well as the time points at which these analyses were conducted and the explanation of symbols used.

[0018] Figure 2:

[0019] Following the application of MM (metabolic medium) and control medium to cells grown in the 3D co-culture system, images showing intracellular lipid droplet accumulation in (A) LX-2 and (B) SNU-449 cells are presented, along with (C) DAPI and (D) Nile Red staining images.

[0020] Figure 3:

[0021] Expression analysis of inflammation- and fibrogenesis-related genes (A) CXCR4, (B) IL1 B, (C) FAS, and (D) TGFB1 following MM (metabolic medium) and control medium application to cells grown in the 3D co-culture system.

[0022] Figure 4:

[0023] Analysis of the proliferation assay performed on SNU-449 cells under control conditions and MM treatment, demonstrating one of the effects of MM application on the cellular behavior of SNU-449 cells.

[0024] Figure 5:

[0025] Analysis of the cell cycle assay in SNU-449 cells exposed to control conditions or MM treatment for 48 hours, showing one of the effects of MM application on cellular behavior, and the numerical (A) and percentage (B) distribution of the cell cycle phases.

[0026] Figure 6:

[0027] Analysis of the motility assay results showing the images (A) and quantitative representation (B) of SNU-449 cell motility under control conditions and after MM treatment, illustrating one of the effects of MM application on cellular behavior.

[0028] Figure 7:

[0029] Analysis of the spheroid formation assay results showing the colony formation capacity of SNU-449 cells under control conditions and MM treatment, with images of the colonies (A) and their quantitative representation (B), demonstrating one of the effects of MM application on cellular behavior.

[0030] Figure 8:

[0031] Verification of the quality and quantity of proteins obtained from experiments using the MASLD-HCC-3D co-culture system for high-throughput proteomic analyses. Comparison of protein lysates from the co-culture system with control lysates obtained from mouse liver at different concentrations (A) 20 pg, (B) 10 pg, (C) 5 pg, (D) 2.5 pg, and (E) 1.5 pg after Coomassie blue staining and detection using the LI-COR system (F-l).

[0032] Figure 9:

[0033] Protein concentration analysis confirms that the protein yield and quality obtained from the MASLD-HCC-3D co-culture system are sufficient for high-throughput proteomic studies.

[0034] Figure 10:

[0035] Schematic representation of the alterations in insulin receptor and mTOR pathway molecules, known to change under MASLD-HCC conditions, as revealed by high-throughput proteomic analyses conducted using the MASLD-HCC-3D co-culture system.

[0036] Figure 11 :

[0037] Expression analysis of a biomarker candidate (IDH2) selected from the proteomic study: Western blot (A), RT-qPCR (B), and flow cytometry (C) analyses in SNU-449 HCC cells under control and MM conditions; Western blot analysis (D) in HuH-7 HCC cells under control and MM conditions; and in silico validation / analysis of the biomarker candidate using the GSE164441 (E) and E-MEXP-3291 (F) datasets.

[0038] Figure 12:

[0039] Microscopic images showing IDH2 immunohistochemical staining results in HCC patient tissues and parenchyma — (A) IDH2-positive, (B) IDH2-negative samples; comparison of IDH2-positive staining ratios between parenchymal and HCC tumor tissues (C), and between tumor samples from patients with or without microvascular invasion (D). Detailed Description of the Invention

[0040] This invention presents a three-dimensional (3D) co-culture system developed using a Boyden chamber configured to mimic the Metabolic Dysfunction-Associated Steatotic Liver Disease microenvironment (MASLD-ME). The system is designed to investigate the effects of MASLD on hepatocellular carcinoma (HCC) and to explore molecular mechanisms underlying MASLD-driven HCC.

[0041] The system is a 3D Boyden-chamber co-culture model that contains: collagen-matrix-embedded hepatic stellate cells to recreate the MASLD microenvironment and to observe cell-cell interactions; a metabolic medium formulated to realistically simulate the microenvironment (containing glucose at 5.5-25 mM, free fatty acids — oleic acid and palmitic acid — at a total of 0.25-2 mM, fructose at 5-20 mM, and insulin at 0.1-100 nM); and HCC cells. The system is arranged in two compartments to monitor cell motility and interactions: tumor cells are seeded in the upper compartment, while hepatic stellate cells are grown in 3D within a gel matrix in the lower compartment. Cells are not in direct contact; rather, the nested culture wells are separated by a porous membrane that permits passage of secreted factors while preventing direct cell-cell contact.

[0042] In another embodiment of the invention, the collagen matrix-embedded hepatic stellate cells used in the model of the invention are composed of LX-2 cells.

[0043] In another embodiment, the HCC cells used in the model are SNU-449 cells.

[0044] The system is established, and experimental data are obtained and analyzed using the following steps:

[0045] 1. Culturing hepatic stellate cells in a collagen matrix.

[0046] 2. Treating the cultured hepatic stellate cells with either the metabolic medium (MASLD conditions) or the control medium.

[0047] 3. Adding hepatocellular carcinoma cells to the upper well of the Boyden chamber. 4. Collecting motile cells and transfering them to a new culture vessel for downstream analyses (see Figure 1).

[0048] In one embodiment of the invention, the hepatic stellate cells used in the construction of the system of the invention and in the analysis of the data obtained from the system are LX-2 cells.

[0049] In one embodiment of the invention, the hepatocellular carcinoma cells used in the construction of the system of the invention and in the analysis of the data obtained from the system are SNU-449 cells.

[0050] In one embodiment of the invention, the metabolic medium used in the construction of the system of the invention and in the analysis of the data obtained from the system contains glucose, free fatty acids (oleic acid and palmitic acid), fructose, and insulin. Using these methods and components, the model provides a robust and comprehensive platform to study the effects of the MASLD microenvironment on HCC. The model enables detailed examination of cellular interactions and changes in gene and protein expression.

[0051] The developed MASLD-mimicking 3D co-culture system was subjected to a variety of experimental tests; key results are summarized below. One goal of the invention — identification of biomarkers that may predict HCC development using the model — yielded two biomarker candidates (IDH2 and a CANDIDATE 2). IDH2 was further tested in the model and subsequently validated clinically.

[0052] In SNU-449 cells cultured under MASLD microenvironment conditions, IDH2 expression was analyzed by RT-PCR and Western blot. MASLD formation was confirmed by observing intracellular lipid droplet accumulation (Figure 2D) — a hallmark of MASLD — together with activation of stellate cells and significant upregulation of inflammation and fibrogenesis-related genes such as CXCR4 (Figure 3A) and interleukin-1 beta (IL1B; Figure 3B), while expression of genes such as FAS (Figure 3C) and TGF-[31 (Figure 3D) were shown to decrease.

[0053] Subsequent cell-behavior analyses assessed proliferation, motility, and invasion capacities. These studies demonstrated that factors present under MASLD conditions promote aggressive cellular behaviors in tumor cells, evidenced by increased proliferation (Figure 4), progression through the cell cycle (increased S and G2 / M phases) (Figures 5A and 5B), enhanced cell motility (Figures 6A and 6B), and increased 3D colony (spheroid) formation capacity (Figures 7Aand 7B).

[0054] Figures 8 and 9 show that sufficient quantity and quality of protein can be obtained from the cancer cells in this system for high-throughput -omics analyses (e.g., proteomics). The regression shown in Figure 9 is: y = 0.00024595x - 0.99831902, R2= 0.99449082. Using these proteins, detailed protein expression analyses were performed by the Digiwest method, and it was demonstrated that, under co-culture conditions with hepatic stellate cells grown in three dimensions in the MASLD environment, there were changes in the activation of signaling molecules such as mTOR, AMPK, and IGF1, which are known to be associated with the development of MASLD, in HCC cells (Figure 10). These results validate that the developed 3D coculture model recapitulates key aspects of the MASLD phenotype.

[0055] DigiWest analyses further revealed, for the first time, that the IDH2 protein level is decreased in HCC cells cultured in the MASLD microenvironment (SNU-449 cell line, Figures 11 A and 11 C; HuH-7 cell line, Figure 11 D). Consistent with protein data, IDH2 mRNA levels were reduced in SNU-449 cells under MASLD conditions as determined by RT-PCR (Figure 11 B). In-silico validation using publicly available NAFLD / NASH and HCC patient datasets (GSE164441 and MEXP-3291) also showed decreased levels of this molecule (Figures 11 E and 11 F). Immunohistochemical (IHC) analysis of HCC patient tissues and paired parenchyma (n = 27) revealed IDH2-positive and IDH2-negative staining examples (Figures 12A and 12B), comparative IDH2 levels in parenchyma versus HCC tumor tissues (Figure 12C), and comparisons of IDH2 positivity in tumor samples from patients with or without microvascular invasion (Figure 12D). These analyses indicate that low IDH2 levels in HCC are associated with the presence of microvascular invasion.

[0056] Taken together, these data demonstrate that the MASLD microenvironment represses IDH2 expression in HCC cells. The potential of this protein as a biomarker for MASLD-associated HCC development and as a predictor of aggressive phenotype was confirmed by WB, RT-PCR, bioinformatics analyses, and IHC.

[0057] The application of the IDH2 findings exemplifies that the claimed co-culture system can reproduce known MASLD-associated biomarker behaviors within the co-culture context and can reveal previously unknown mechanisms by which MASLD contributes to HCC development. The system can therefore be used to discover new biomarkers or therapeutic target molecules involved in MASLD-mediated HCC progression. Accordingly, the data shown in this embodiment support the utility of the developed system for biomarker discovery, for identification of diagnostic candidate molecules, and for testing the early-diagnostic or therapeutic potential of those candidate molecules.

[0058] REFERENCE

[0059] [1] Phoolchund, A. G., & Khakoo, S. I. (2024). Masld and the development of HCC: Pathogenesis and therapeutic challenges. Cancers, 16(2), 259. https: / / doi.Org / 10.3390 / cancersl 6020259

[0060] [2] Bronsard, J., Savary, C., Massart, J., Viel, R., Moutaux, L., Catheline, D., Rioux, V., Clement, B., Corlu, A., Fromenty, B., & Ferron, P. J. (2024). 3D multi-cell-type liver organoids: A new model of non-alcoholic fatty liver disease for drug safety assessments. Toxicology in Vitro, 94, 105728. https: / / doi.Org / 10.1016 / j . tiv.2023.105728

[0061] [3] Ramos, M. J., Bandiera, L., Menolascina, F., & Fallowfield, J. A. (2022). In vitro models for non-alcoholic fatty liver disease: Emerging platforms and their applications. iScience, 25(1), 103549. https: / / doi.Org / 10.1016 / j.isci.2021.103549

Claims

CLAIMS1. A co-culture system that mimics the MASLD microenvironment, consisting of a three-dimensional structure with two separate wells, wherein the compartments are separated by a membrane, hepatocellular carcinoma cells are located in the upper compartment, and stellate cells are located in the lower compartment; characterized in that it comprises:- hepatic stellate cells embedded in a collagen matrix,- a metabolic medium containing glucose (5.5-25 mM / L), free fatty acids (oleic acid and palmitic acid) (0.25-2 mM), fructose (5-20 mM), and insulin (0.1-100 nM), and - hepatocellular carcinoma cells,wherein the collagen-matrix-embedded hepatic stellate cells are placed in the lower chamber of the Boyden system and the hepatocellular carcinoma cells are located in the upper compartment.

2. The system according to claim 1 , characterized in that it is used for the discovery of biomarkers associated with MASLD-related HCC.3.The system according to Claim 1 or 2, characterized in that the collagen-matrix-embedded hepatic stellate cells are LX-2 cells.4.The system according to any one of Claims 1-3, characterized in that the hepatocellular carcinoma cells are SNU-449 cells.5.The system according to any one of Claims 1-4, characterized in that it is used as a model for the discovery of biomarkers and therapeutic targets related to MASLD and for drug efficacy analyses.

6. A method for establishing a three-dimensional co-culture system that mimics the MASLD microenvironment, characterized in that it comprises the steps of:- Culturing hepatic stellate cells within a collagen matrix,- Treating the cultured hepatic stellate cells with a metabolic medium or under control conditions,- Adding hepatocellular carcinoma cells to the upper well of the Boyden chamber system,- Collecting motile cells and transferring them to a new culture vessel for further analyses.7.The method according to Claim 6, characterized in that the collagen-matrix-embedded hepatic stellate cells are LX-2 cells.8.The method according to Claim 6 or 7, characterized in that the hepatocellular carcinoma cells added to the upper well are SNU-449 or HuH-7 HCC cells.9.The method according to any one of Claims 6-8, characterized in that the metabolic medium contains 0.5 mM free fatty acids (oleic acid and palmitic acid at a 2:1 ratio), high glucose concentration (4.5 g / L), fructose (20 mM), and insulin (100 nM).

10. The system according to any one of Claims 1-5, characterized in that it is used for the detection of biomarkers predictive of MASLD-related HCC development and for drug effect analyses.

11. The system according to any one of Claims 1-5, characterized in that it is used for biomarker detection and drug effect analyses aimed at predicting the prognosis of MASLD-related HCC.

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