Abstract

Collective cell migration plays a critically regulatory role in both physiological and pathological processes, e.g. , embryonic development, wound healing, cancer progression and metastasis. Nevertheless, spatiotemporal progression of epithelial and mesenchymal cell migration remains poorly understood. Here, we report an epithelial-mesenchymal transition (EMT)-modulated cell phenotype phase separation (CPPS) within spatially confined microenvironments, where the mesenchymal cells display enhanced boundary-directed colonization in a phenotype-dependent manner. Subsequently, we reveal that the CPPS process correlates with the degree of EMT and cytoskeletal inhibition. With the aid of a boundary attraction potential (BAP), we further develop a reaction-diffusion-based model that can quantitatively capture the spatiotemporal evolution of the EMT-related collective migration. Our findings imply that phenotypic heterogeneity among cells is a major determinant of phase separation in collective migration. This work not only uncovers the CPPS phenomenon and its underlying biophysical mechanism in collective cell migration, but also provides a new perspective for dissecting EMT-regulated intratumoral phenotypic heterogeneity. Statement of Significance Collective cell migration drives development, wound healing, and cancer metastasis, yet how phenotypically heterogeneous populations self-organize remains unclear. Using micropatterned substrates and live-cell tracking, we report cell phenotype phase separation (CPPS) during epithelial-mesenchymal transition (EMT)-mediated collective migration. Inspired by the classical Turing’s theory, we develop a reaction-diffusion model with a boundary attraction potential that recapitulates the spatiotemporal dynamics of CPPS across micropattern geometries, substrate stiffnesses, and cell compositions. Our findings not only reveal a comprehensive biophysical mechanism linking EMT status to collective migration, which is crucial for dissec