The study of ECM hardness and mechanical pressure promoting TSCC progression by activating RET in a 4D culture system

• Combine sodium alginate hydrogel with magnetic nanoparticles to build a magnetically responsive 4D dynamic culture model. • Use the 4D model to simulate the mechanical pressure and ECM stiffness during TSCC occurrence and development. • Mechanical pressure and ECM stiffness promote the malignant transformation of the phenotype of TSCC. • Mechanical pressure and ECM stiffness activate RET, regulating the RET/AKT/GSK-3β/β-catenin/c-Myc signaling pathway. This study developed a 4D dynamic culture model (a 3D hydrogel system integrated with time-dependent mechanical stimuli via magnetic nanoparticles) to simulate tongue movement and investigate the roles of ECM stiffness and mechanical pressure in tongue squamous cell carcinoma (TSCC) progression. The model demonstrated excellent biocompatibility and stability. Results showed that ECM stiffness and mechanical pressure significantly enhanced TSCC cell proliferation, migration, and invasion by activating the RET/AKT/GSK-3β/β-catenin/c-Myc pathway. Specifically, mechanical pressure induced RET phosphorylation at Y1062, triggering downstream signaling. Silencing RET or inhibiting PI3K/AKT suppressed these effects. The 4D model provides a physiologically relevant platform for studying TSCC mechanobiology, highlighting RET as a potential therapeutic target. These findings underscore the importance of mechanical cues in TSCC progression and offer insights for novel treatment strategies.