We analyze the mechanical properties of three epithelial/mesenchymal cell lines (MCF-10A, MDA-MB-231, MDA-MB-436) that exhibit a shift in E-, N-and P-cadherin levels characteristic of an epithelial−mesenchymal transition associated with processes such as metastasis, to quantify the role of cell cohesion in cell sorting and compartmentalization. We develop a unique set of methods to measure cell-cell adhesiveness, cell stiffness and cell shapes, and compare the results to predictions from cell sorting in mixtures of cell populations. We find that the final sorted state is extremely robust among all three cell lines independent of epithelial or mesenchymal state, suggesting that cell sorting may play an important role in organization and boundary formation in tumours. We find that surface densities of adhesive molecules do not correlate with measured cell-cell adhesion, but do correlate with cell shapes, cell stiffness and the rate at which cells sort, in accordance with an extended version of the differential adhesion hypothesis (DAH). Surprisingly, the DAH does not correctly predict the final sorted state. This suggests that these tissues are not behaving as immiscible fluids, and that dynamical effects such as directional motility, friction and jamming may play an important role in tissue compartmentalization across the epithelial−mesenchymal transition. transgress even the strong lineage boundaries, invading adjacent tissues. Therefore, a question of immediate practical importance is what changes allow metastatic cells to break through these boundaries, or conversely, what prevents non-metastatic tumour cells from leaving the compartment? Moreover, it is a fundamental question if a solid tumour behaves sufficiently like a fluid that surface tension-like effects hold cancer cells back at the tumour boundary.Metastasis has been attributed to tumour cells losing epithelial characteristics and acquiring a more migratory mesenchymal phenotype [9][10][11]. This change known as the epithelial−mesenchymal transition (EMT) is typically accompanied by a loss of specific types of cellular adhesion. While epithelial cells are closely connected via various types of cell junctions such as adherens junctions and desmosomes that allow them to form organized cell layers in vivo and cell clusters in vitro, mesenchymal cells are less constrained, contacting only through focal points [11]. During EMT, the expression of E-cadherin decreases while the expression of N-cadherin and other cadherins increases [12][13][14]. This might be the molecular origin for the change in adhesiveness, although recent work highlights that different cadherins play vastly different roles in regulating intercellular forces and adhesion [15]. In addition, EMT also causes a down-regulation of the keratin cytoskeleton and a replacement with vimentin [16], which also hinders desmosome formation. This leads to secondary effects that modulate cell-cell adhesion. However, it remains unclear how these processes interact with boundary formation and compartment m...

Mechanical characterization of living cells undergoing substantial external strain promises insights into material properties and functional principles of mechanically active tissues. However, due to the high strains that occur in physiological situations (up to 50%) and the complex structure of living cells, suitable experimental techniques are rare. In this study, we introduce a new system composed of an atomic force microscope (AFM), a cell stretching system based on elastomeric substrates, and light microscopy. With this system, we investigated the influence of mechanical stretch on monolayers of keratinocytes. In repeated indentations at the same location on one particular cell, we found significant stiffening at 25% and 50% strain amplitude. To study the contribution of intermediate filaments, we used a mutant keratinocyte cell line devoid of all keratins. For those cells, we found a softening in comparison to the wild type, which was even more pronounced at higher strain amplitudes.

A critical step in the metastatic cascade is the process wherein a single cell breaks through the basement membrane, leaving the primary tumor and entering the stroma whence it can disseminate. In the case of many solid tumor cancers, increased cell deformability is thought to facilitate this process. However, the ligand density, alignment, and stiffness of the matrix determine which mode of motility will succeed. To investigate the mechanical interplay between the cell and ECM during this process, we created a simple model of stromal invasion using 10-200 mm thick bovine collagen I hydrogels ranging from 0.1-5 kPa in Young's modulus that were seeded at low density with highly metastatic MDA-MB-231 breast cancer cells. Significant population fractions invaded the matrices either partially or fully within 24 h. We then combined confocal fluorescence microscopy and AFM indentation to determine the Young's moduli of individual embedded cells and the pericellular matrix using novel analysis methods for heterogeneous samples. In partially embedded cells, we observe a statistically significant correlation between the degree of invasion and the Young's moduli (~220 Pa/mm; p<0.001), which was up to an order of magnitude greater than that of the same cells measured in 2D. ROCK inhibition returned the cells' Young's moduli to 2D values (~0.5 kPa) and diminished but did not abrogate invasion. This provides evidence that Rho/ROCK-dependent actomyosin contractility is employed for matrix reorganization during initial invasion, and suggests the observed cell stiffening is due to an attendant increase in actin stress fibers. Yet even with MMP and ROCK inhibition, 39% of cells fully invaded the matrix after 6 days, indicating the cells may have alternate motility mechanisms at their disposal.