Multicellular Aligned Bands Disrupt Global Collective Cell Behavior

Mechanical stress patterns emerging from collective cell behavior have been shown to play critical roles in morphogenesis, tissue repair, and cancer metastasis. In our previous work utilizing microcontact printing to geometrically constrain valvular interstitial cell monolayers into specific shapes, we demonstrated that the general patterns of observed cell alignment, size, and apoptosis correlate with predicted mechanical stress fields if nonuniform cell properties are used in the computational models. However, these radially symmetric models did not predict the substantial heterogeneity in cell behavior observed in individual circular aggregates. The goal of this study is to determine how the heterogeneities in cell behavior emerge over time and diverge from the predicted collective cell behavior. Cell-cell interactions in circular multicellular aggregates of valvular interstitial cells were studied with time-lapse imaging ranging from hours to days, and migration, proliferation, and traction stresses were measured. Our results indicate that individual elongated cells create strong local alignment within pre-confluent cell populations on the microcontact printed protein islands. These cells influence the alignment of additional cells to create dense, locally aligned bands of cells which disrupt the global behavior. Cells are highly elongated at the endpoints of the bands yet have decreased spread area in the middle and reduced mobility. Although traction stresses at the endpoints of bands are enhanced, even to the point of detaching aggregates from the culture surface, the cells in dense bands exhibit reduced proliferation, less nuclear YAP, and increased apoptotic rates indicating a low stress environment. These findings suggest that strong local cell-cell interactions between primary fibroblastic cells can disrupt the global collective cellular behavior leading to substantial heterogeneity of cell behaviors in constrained monolayers. This local emergent behavior within aggregated fibroblasts may play an important role in development and disease of connective tissues. ### Competing Interest Statement The authors have declared no competing interest.