Extracellular Matrix Physical Properties Regulate Cancer Cell Morphological Transitions in 3D Hydrogel Microtissues

Abstract Cancer cells can adopt a range of morphological states linked to distinct functional behaviors during tumor progression. Some remain in a proliferative state, forming tight clusters; others detach and elongate into an invasive state; and some retain a rounded amoeboid form with minimal matrix adhesion. However, factors that determine which morphological state a cell adopts remain poorly understood. Using a combined theoretical and experimental framework, we showed that extracellular matrix (ECM) mechanics regulate cancer cell morphology in three-dimensional (3D) environments. We developed a theoretical model based on the principle of minimum energy, which predicts that a cell will adopt the morphological state—rounded, elongated, or clustered— that minimizes the total energy of the cell-ECM system. Using MDA-MB-231 breast cancer cells, we established a reliable protocol for encapsulating cells into 3D naturally-derived hydrogels with controlled stiffness and pore size. We validated the model’s predictions in vitro over an extended culture period. In soft ECMs, cells transitioned over time to an elongated morphology, while in stiff ECMs, cells favored clustered configurations. These transitions were governed by the physical— not chemical—properties of the hydrogel-based ECM, as confirmed by using chemically distinct yet mechanically matched composite matrices. These new insights have implications for cancer invasion modeling and potential drug screening. Competing Interest Statement The authors have declared no competing interest.