Bioinspired 3D hydrogel scaffold to mimic tumor microenvironment for investigating into the anoikis resistance mechanisms in colorectal cancer

Anoikis resistance constitutes a critical pathophysiological mechanism driving metastatic progression in colorectal cancer. While in vitro models are essential for mechanistic studies, conventional 2D cultures inadequately replicate tumor microenvironment (TME) complexity. In this study, we developed a biomimetic composite hydrogel (GHP_4a) composed of 4-arm-PEGDA, Gelatin Methacryloyl, and Hyaluronic acid Methacryloyl to establish compact 3D cell spheres that mimic TME. The GHP_4a demonstrates superior biocompatibility, suitable mechanical strength at 600–700 Pa, and biomimetic properties that promote cell proliferation and differentiation when co-culturing Caco-2 cells. The cell anchorage and survival for anoikis resistance are enhanced compared to traditional 2D cell incubation due to a well-organized 3D formation akin to the extracellular matrix (ECM). The biomimetic mechanism may be attributed to the fact that the GHP_4a promoted the activation of key pro-survival pathways, including FAK and PI3K/Akt signaling, and suppressed caspase-mediated apoptosis. Single-cell RNA sequencing revealed distinct transcriptional profiles within the proliferating T cell population, suggesting a novel regulatory mechanism of T cell-mediated anti-tumor immunity in the TME. Additionally, radiomic analysis identified significant differences in tumor heterogeneity and texture between GHP_4a-based 3D cultures and traditional 2D models. These findings established the GHP_4a as an effective candidate for the in vitro tumor anoikis resistance model, providing a unique approach for studying anoikis resistance in colorectal cancer and offering a robust tool for the development of advancing cancer diagnosis and therapy strategies.