A novel 3D porous hydrogel model, mimicking the tumor microenvironment (TME), was developed as a physiologically relevant platform to investigate the role of extrachromosomal DNA (ecDNA) in colorectal cancer.We fabricated a tailorable collagen-based hydrogel that overcomes the limitation of 2D cultures by enabling crucial cell-cell and cell-matrix interactions.Methods: First, we validated whether the selected COLO320 cell lines were suitable for investigation of ecDNA in 3D tumor model and confirmed that ecDNA structures were stably maintained under 3D culture conditions by whole genome sequencing (WGS).Additionally, to provide an appropriate environment for colorectal cancer cells, we fabricated collagen-based porous hydrogels using a whipping process that requires no surfactants or sacrificial materials.During this process, we optimized the bioink formulation to achieve extracellular matrix (ECM) stiffness favorable for colorectal cancer cell proliferation, aggregation, stem-like behavior, and epithelial-mesenchymal transition (EMT)-related gene expression.Results: By optimizing the porous structure for enhanced nutrient diffusion and cell infiltration, we successfully maintained ecDNA structures in COLO320 cells.Our optimized porous platform significantly enhanced cellular proliferation, aggregation, and metabolic activity compared to conventional bulk model.We also observed elevated expressions of key oncogenes like MYC and activation of mechanotransduction pathways associated with aggressive tumor phenotypes. Conclusion:This reproducible and effective model accurately reflects ecDNA-driven biological behaviors, making it ideal for long-term ecDNA research and future TME-related studies.
Jo et al. (Mon,) studied this question.