Cell phenotype phase separation in epithelial-mesenchymal transition-mediated collective cell migration

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 dissecting cellular spatial self-organization in tumors and tissue development, but also suggest that CPPS may serve as a potential target for therapeutic intervention and drug screening.