Characterization of Cellular Transition within the Expanding Monolayer
Authors: Cho Youngbin , Gweon Bomi , Notbohm Jacob , Ko Ung Hyun , Jang Hwanseok , Park Yongdoo , Shin Jennifer 
Published: November 2023

INTRODUCTIONCell motility is a fundamental process in wound healing, cancer metastasis, and morphogenesis. While some types of cells, such as fibroblasts and immune cells, tend to migrate individually, cells often migrate as a pack in a collective manner in vivo. Within the cell monolayer, the cells experience forces through adhesions to the substrate and the neighboring cells. In a jammed monolayer with highly packed cells, they maintain stable cellular junctions displaying minimal intercellular motions. When the cells are patterned as monolayer islands, the cells on the outer edge would encounter the free space, triggering the monolayer expansion toward the void. This monolayer expansion accompanies the phenotypic transformation from a stable to a migratory state, so called, epithelial‐mesenchymal‐transition (EMT). Such dramatic transition involves dynamic remodeling of cellular junctions and redistribution of cellular stresses within the monolayer. For decades, many studies have identified the key molecules that regulate the adhesions to promote the transition of cellular phenotypes, which include focal adhesion kinase and associated proteins as well as E‐cadherin and N‐cadherin. These studies lack the dynamic details on how such transition involves the key proteins to redistribute the forces. Here, we aimed to investigate the dynamic EMT phenomenon within the expanding cell island to unravel the correlation between physical stresses and cellular components like cytoskeletons, adhesions, and membrane.METHODSWe patterned MCF‐10A epithelial cells as a circular island (diameter of 700 μm) on a flat hydrogel coated with collagen. Using time lapse microscopy, we obtained the sequential phase images of the monolayer expansion in every 10 minutes. After acquisition of images, we used PIV analysis to measure the velocity of individual cells within the monolayer over time. To compute the forces that cells apply to the substrate (commonly called as traction force), we used traction force microscopy (TFM). TFM calculates the traction force using the displacement of the soft hydrogel, which is the result from the forces applied by the cell.RESULTSAs the monolayer encounters the free space to expand, the cells at the edge of the monolayer acquire the migratory phenotype featuring pronounced lamellipodia with fast migration speed whereas the cells at the center maintained the epithelial phenotype with limited movement. At the edge of the monolayer within a band of 3–4 cell layers along the perimeter, the fast migrating cells generated high inward traction with strong cell‐substrate adhesion, evidenced by mature focal adhesion complex. We denote this band of high motility and strong focal adhesion as the mesenchymal band (MB) whose width does not change within the expanding island over time. The cells in the MB experience the protrusive force from active actin polymerization, inward pulling force from neighboring cells through cell‐cell adhesion, and surface drag force through cell‐substrate adhesion. On the other hand, the cells at the core (C) exhibit opposite traction to balance the strong intercellular adhesion from neighboring cells. Our dynamic adhesion analysis indicates the existence of a single‐celled ring between MB and C; the cells along this intermediate ring exhibits no bias in radial force, making the balance point between two distinct groups of cells.Support or Funding InformationThis work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF‐2013S1A2A2035518, NRF‐2014R1A6A3A04059713)