Viscoelastic hydrogel potentiates cancer cell stemness and drug resistance through Piezo1‐dependent mechanotransduction

Abstract The dynamic mechanical properties of the tumor microenvironment are crucial for cancer progression, yet their specific effects independent of matrix stiffness remain unclear. Here, we develop a hydrogel system with tunable viscoelasticity based on reversible host–guest crosslinking that decouples network dynamics from stiffness, to compare high‐dynamic (HDy) and low‐dynamic microenvironments. Using HepG2 hepatocellular carcinoma cells, we found that the HDy hydrogel promotes rapid spheroid formation and upregulates stemness‐related genes. The HDy hydrogel matrix suppresses integrin–FAK–RhoA signaling and cell–matrix adhesion, while activating the Piezo1‐mediated calcium influx and PI3K/AKT pathway to stabilize β‐catenin. The enhanced nuclear translocation of stabilized β‐catenin subsequently elevates Wnt‐dependent transcriptional activity and reinforces E‐cadherin‐mediated cell–cell junctions, facilitating cohesive spheroid assembly. Functionally, the HepG2 spheroids derived from HDy hydrogels exhibit increased resistance to multiple chemotherapeutic agents and form larger, more aggressive xenograft tumors in vivo. These findings establish the matrix dynamics as a key biophysical regulator of cancer stemness and drug resistance, mediated through the coordinated downregulation of integrin adhesion and activation of Piezo1–Wnt/β‐catenin axis. This dynamic hydrogel provides a 3D culture platform with defined chemical composition and tunable biomechanics for probing tumor mechanobiology and developing therapeutic strategies against drug‐resistant cancer cells.