Heterogeneity in environmental stiffness alone can guide cells and shape tissues

While topographical and chemical cues are well known to regulate cell shape and function, the role of stiffness heterogeneity has remained unclear. Here, we demonstrate – for the first time to our knowledge – that cells can be guided solely by the stiffness heterogeneity of their environment. To that end, we engineered a cell-guiding platform with abrupt, subcellular stiff and soft domains, whose flatness and uniform chemistry eliminated confounding cues. Cells elongate and align along stiff regions, sensing soft domains as barriers when wider than 2 microns. Perturbated myosin activity, cortical tension, and elasticity contrast reveal distinct biomechanical contributions, while a probabilistic model integrating adhesion, contractility, and cortical tension extracts key mechanical parameters characterizing the cellular state. Finally, experiments and dissipative particle dynamics demonstrate collective stiffness-based contact guidance. This work identifies stiffness heterogeneity as a fundamental regulator of cell and tissue organization and provides a framework for designing mechanoregulatory biomaterials.