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 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) composed of 4-arm-PEGDA, Gelatin Methacryloyl, and Hyaluronic acid Methacryloyl to establish compact 3D cell spheres that mimic TME. The GHP 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 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-based 3D cultures and traditional 2D models. These findings established the GHP as an effective candidate for the 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.