Motility-limited aggregation of mammary epithelial cells into fractal-like clusters

Abstract

Migratory cells transition between dispersed individuals and multicellular collectives during development, wound healing, and cancer. These transitions are associated with coordinated behaviors as well as arrested motility at high cell densities, but remain poorly understood at lower cell densities. Here, we show that dispersed mammary epithelial cells organize into arrested, fractal-like clusters at low density in reduced epidermal growth factor (EGF). These clusters exhibit a branched architecture with a fractal dimension of Df=1.7, reminiscent of diffusion-limited aggregation of nonliving colloidal particles. First, cells display diminished motility in reduced EGF, which permits irreversible adhesion upon cell–cell contact. Subsequently, leader cells emerge that guide collectively migrating strands and connect clusters into space-filling networks. Thus, this living system exhibits gelation-like arrest at low cell densities, analogous to the glass-like arrest of epithelial monolayers at high cell densities. We quantitatively capture these behaviors with a jamming-like phase diagram based on local cell density and EGF. These individual to collective transitions represent an intriguing link between living and nonliving systems, with potential relevance for epithelial morphogenesis into branched architectures.

The epithelial-mesenchymal transition and the cytoskeleton in bioengineered systems

“…HGF, EGF) Straightforward experimental technique, which can be integrated with 2D traction measurements. However, it is difficult to control cluster size, and cell migration occurs in random directions [ 83 – 87 , 89 ] “Wound Healing” Assay (2D) Plate cells at confluent density on planar 2D substrate; observe how these monolayers collectively migrate into unoccupied regions, either by mechanically “scratching” to remove cells or by removing a barrier Also straightforward experimentally, and cells migrate in a preferred direction, which may not require time-lapse imaging. However, “scratching” can leave cell debris or damage soft substrates, and this assay biases towards collective migration [ 9 , 88 , 90 , 91 ] Micropatterned substrates (2.5D) Analogous to cell scattering or wound healing assays, but incorporating topographical features on the substrate Mimics ECM architecture and can affect directional migration via contact guidance, as well as EMT/MET.…”

“…Subsequently, leader cells transiently guide migration to link clusters together into spanning networks. Reproduced from [ 89 ] with permission …”

“…Leggett et al [ 89 ] showed that mammary epithelial cells in reduced EGF exhibit multicellular aggregation into clusters (analogous to a reverse MET), followed by leader cell formation. Since nontransformed mammary epithelial cells (MCF-10A) are growth factor dependent, their proliferation and migration was slowed when cultured at low exogenous EGF concentrations.…”

“…Experiments on planar 2D substrates have shown that Allows direct measurement of invasive cancer cells in a human patient tumor, but occurs as a two-dimensional snapshot [159] increased motility in response to HGF is sufficient to rupture cell-cell junctions without associated downregulation of E-cadherin [85]. Similarly, slowed cell motility in reduced EGF results in multicellular cluster formation and collective behaviors [89], reminiscent of an MET. Existing traction force microscopy methods are only capable of inferring cell-cell forces from cell-matrix tractions for small cell clusters, where all cells are in contact with the underlying 2D substrate.…”

Mechanophenotyping of 3D multicellular clusters using displacement arrays of rendered tractions

“…EGFR Inhibition with Gefitinib Increases Heterogeneity of Transitory Clusters. Lastly, cluster mechanophenotypes were perturbed by treatment with the EGFR inhibitor gefitinib, which ordinarily inhibits proliferation in epidermal growth factor (EGF)dependent MCF-10A cells (43). Previously, cell lines expressing epithelial biomarkers were shown to be more sensitive to gefitinib and proliferated less in vitro and in vivo, while EMT was associated with resistance to gefitinib (44)(45)(46).…”