A Protocol for Colorectal Tumor Spheroid Culture in Tunable Stiffness Alginate-Based Hydrogels and Subsequent Immunohistochemical Analysis.

Tumor mechanical microenvironment, particularly extracellular matrix stiffness, plays a critical role in regulating cancer cell behavior, including proliferation, quiescence, and drug resistance. Conventional 2D culture or stiff 3D scaffolds fail to recapitulate the physiological soft (normal) or pathologically stiff (tumoral) mechanical niches. Here, we present a detailed protocol for establishing a tunable 3D tumor spheroid culture system using sodium alginate-based hydrogels crosslinked with calcium ions at different concentrations to achieve soft or stiff conditions that mimic normal colon and colorectal cancer tissues, respectively. We describe the step-by-step procedures for fabricating stiffness-tunable hydrogels, culturing colorectal cancer spheroids, releasing spheroids for downstream analysis, and performing immunohistochemical staining on intact spheroids. This protocol enables the reproducible investigation of mechanosensitive pathways and drug resistance mechanisms in a physiologically relevant 3D context. Key features • Tunable stiffness hydrogel system based on sodium alginate and calcium carbonate crosslinking. • 3D tumor spheroid culture that recapitulates normal and tumor mechanical microenvironments. • Gentle spheroid release using sodium citrate chelation, preserving morphology and viability. • Compatible with immunohistochemistry.