Extracellular matrix physical properties regulate cancer cell morphological transitions in 3D hydrogel microtissues

Solid tumor 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 determining which morphological state a cell adopts remain poorly understood. We used a combined theoretical and experimental framework to study how extracellular matrix (ECM) mechanics regulate solid tumor cell morphology in three-dimensional (3D) environments. We developed a theoretical mechanical energy model based on the minimum energy principle, which suggests 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. We confirmed the model's results 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 hydrogel-based ECM's physical, not chemical, properties, as confirmed using chemically distinct yet mechanically matched composite matrices. These new insights have implications for solid tumor cell invasion modeling in vitro. STATEMENT OF SIGNIFICANCE: We study the fundamental question of how solid tumor cells adapt their morphology in response to the physical characteristics of the extracellular matrix. This work establishes a robust experimental platform for studying cellular markers in triple-negative breast cancer (TNBC) cells, followed by a biophysical modeling of the cell invasion. Cell clustering was observed in stiffe ECMs, while an elongated morphology was observed in soft ECMs. Our theoretical modeling revealed how the biophysical properties of the matrix can impact cell morphology and invasion behavior. This work can contribute to personalized medicine by making more effective, tailored cancer models.