Dependency of cellular behavior of collective migration on the substrate stiffness and cluster size

Collective migration is a crucial mechanism guiding cell movement in developmental processes and disease progression. Understanding the migration behavior of cell clusters is key to advancing our knowledge of morphogenesis, wound healing, and collective cancer invasion. Despite the understanding of the response of single cells to environmental physical cues, the collective behavior of cells in response to different levels of extracellular matrix stiffness is yet to be fully understood. Here, we present a quantitative investigation into how substrate stiffness and cell cluster size modulate the collective behavior and migration dynamics of NIH 3T3 fibroblasts. With the variation of PDMS and curing agent concentrations, two contrasting soft and stiff substrates with different stiffness were developed. Using a combination of atomic force microscopy (AFM) to precisely characterize substrate elastic moduli and time-lapse microscopy for tracking migration parameters, we demonstrate that substrate mechanics and cluster geometry synergistically govern collective behavior. Fibroblast migratory characteristics were greatly improved with increased stiffness and cluster size. Large clusters on stiff substrates exhibited greater circularity (~ 0.8), migration distance, displacement (135.6 µm), directionality (0.81), and velocity (24 µm/h) compared to single cells and small clusters on soft and stiff substrates. Moreover, detailed analysis of cytoskeletal reorganization via actin staining revealed the mechanotransductive pathways that convert physical cues into migratory behavior. These findings provide important insights into how substrate stiffness influences collective cell migration, offering potential applications in elucidating the mechanisms of morphogenesis and the dynamics of collective cell invasion during tumor progression.