TGF-ß induces changes in breast cancer cell deformability.

Mechanical properties of cells are shown to regulate cell behaviors leading to phenotypic changes that may aid in the development and progression of disease. In this study, we used atomic force microscopy (AFM) indentation with a spherical probe to characterize the elastic and viscoelastic properties of invasive (MDA-MB-231) and noninvasive (MCF-7) breast cancer cells treated with transforming growth factor-ß (TGF-ß). We also used confocal fluorescence imaging to investigate the sub-membrane cytoskeletal structure of the cells. Results showed significant alterations in moduli of both cell types after 24 h TGF-ß treatment which had a context dependent response; moduli for MDA-MB-231 decreased whereas MCF-7 demonstrated stiffening response. Stress relaxation tests showed increased fluid-like nature of MDA-MB-231 following TGF-ß treatment and lower fluidity for MCF-7 cells. We also observed significant alterations in the expression and orientation of actin stress fibers with TGF-ß treatment which correlated with the changes in cell mechanics. Less invasive MCF-7 cells had a delayed overall increase in cell deformability after 48 h exposure to TGF-ß; a similar trend was observed for MDA-MB cells. These changes may be important to facilitate migration, for instance, during metastasis of cancer cells through submicron sized spaces.

Traction cytometry: regularization in the Fourier approach and comparisons with finite element method.

Traction forces exerted by adherent cells are quantified using displacements of embedded markers on polyacrylamide substrates due to cell contractility. Fourier Transform Traction Cytometry (FTTC) is widely used to calculate tractions but has inherent limitations due to errors in the displacement fields; these are mitigated through a regularization parameter (γ) in the Reg-FTTC method. An alternate finite element (FE) approach computes tractions on a domain using known boundary conditions. Robust verification and recovery studies are lacking but essential in assessing the accuracy and noise sensitivity of the traction solutions from the different methods. We implemented the L2 regularization method and defined a maximum curvature point in the traction with γ plot as the optimal regularization parameter (γ*) in the Reg-FTTC approach. Traction reconstructions using γ* yield accurate values of low and maximum tractions (Tmax) in the presence of up to 5% noise. Reg-FTTC is hence a clear improvement over the FTTC method but is inadequate to reconstruct low stresses such as those at nascent focal adhesions. FE, implemented using a node-by-node comparison, showed an intermediate reconstruction compared to Reg-FTTC. We performed experiments using mouse embryonic fibroblast (MEF) and compared results between these approaches. Tractions from FTTC and FE showed differences of ∼92% and 22% as compared to Reg-FTTC. Selection of an optimum value of γ for each cell reduced variability in the computed tractions as compared to using a single value of γ for all the MEF cells in this study.

Advancing Edge Speeds of Epithelial Monolayers Depend on Their Initial Confining Geometry.

Collective cell migrations are essential in several physiological processes and are driven by both chemical and mechanical cues. The roles of substrate stiffness and confinement on collective migrations have been investigated in recent years, however few studies have addressed how geometric shapes influence collective cell migrations. Here, we address the hypothesis that the relative position of a cell within the confinement influences its motility. Monolayers of two types of epithelial cells--MCF7, a breast epithelial cancer cell line, and MDCK, a control epithelial cell line--were confined within circular, square, and cross-shaped stencils and their migration velocities were quantified upon release of the constraint using particle image velocimetry. The choice of stencil geometry allowed us to investigate individual cell motility within convex, straight and concave boundaries. Cells located in sharp, convex boundaries migrated at slower rates than those in concave or straight edges in both cell types. The overall cluster migration occurred in three phases: an initial linear increase with time, followed by a plateau region and a subsequent decrease in cluster speeds. An acto-myosin contractile ring, present in the MDCK but absent in MCF7 monolayer, was a prominent feature in the emergence of leader cells from the MDCK clusters which occurred every ~125 µm from the vertex of the cross. Further, coordinated cell movements displayed vorticity patterns in MDCK which were absent in MCF7 clusters. We also used cytoskeletal inhibitors to show the importance of acto-myosin bounding cables in collective migrations through translation of local movements to create long range coordinated movements and the creation of leader cells within ensembles. To our knowledge, this is the first demonstration of how bounding shapes influence long-term migratory behaviours of epithelial cell monolayers. These results are important for tissue engineering and may also enhance our understanding of cell movements during developmental patterning and cancer metastasis.