Development of colorectal three-dimensional tumor microenvironment to investigate Immune response

Colorectal cancer (CRC) progression and therapeutic resistance are strongly influenced by the tumor microenvironment (TME), including its biochemical composition, mechanical properties, and immune contexture. Conventional two-dimensional (2D) cell culture models fail to accurately reproduce these complex interactions, limiting their translational relevance. The aim of this study was to develop a biomimetic three-dimensional (3D) in vitro model of the CRC microenvironment using a laminin-based bioink supplemented with patient-derived decellularized extracellular matrix (dECM) powder, Apelin-13, and collagen type I. LoVo colorectal cancer cells and cancer-associated fibroblasts (CAFs) were incorporated into 3D bioprinted scaffolds to recreate stromal–tumor interactions. Cellular proliferation, migration, metabolic activity, and stemness-related gene expression (SOX2 and SOX9) were evaluated. In addition, peripheral blood mononuclear cells (PBMCs) from CRC patients were co-cultured with the constructs to assess T-cell phenotypic remodeling. The optimized dECM concentration enhanced LoVo cell viability without inducing cytotoxicity. Apelin-13 and collagen type I synergistically increased proliferation, directional migration, and upregulation of SOX2 and SOX9 expression. Long-term culture demonstrated progressive metabolic activity and formation of multicellular aggregates within the scaffolds. Importantly, co-culture experiments revealed a shift from naïve and central memory T cells toward effector memory and terminally differentiated phenotypes, accompanied by increased expression of exhaustion-associated markers including PD-1, TIM-3, CD39, loss of CD28, and gain of CD57. Overall, this study establishes a reproducible and physiologically relevant 3D bioprinted CRC model that integrates tumor-derived ECM components and immune interactions. This platform may serve as a valuable tool for mechanistic studies and for the development of personalized therapeutic strategies in colorectal cancer.