Jack R. Staunton

Mismatch in Mechanical and Adhesive Properties Induces Pulsating Cancer Cell Migration in Epithelial Monolayer

The mechanical and adhesive properties of cancer cells significantly change during tumor progression. Here we assess the functional consequences of mismatched stiffness and adhesive properties between neighboring normal cells on cancer cell migration in an epithelial-like cell monolayer. Using an in vitro coculture system and live-cell imaging, we find that the speed of single, mechanically soft breast carcinoma cells is dramatically enhanced by surrounding stiff nontransformed cells compared with single cells or a monolayer of carcinoma cells. Soft tumor cells undergo a mode of pulsating migration that is distinct from conventional mesenchymal and amoeboid migration, whereby long-lived episodes of slow, random migration are interlaced with short-lived episodes of extremely fast, directed migration, whereas the surrounding stiff cells show little net migration. This bursty migration is induced by the intermittent, myosin II-mediated deformation of the soft nucleus of the cancer cell, which is induced by the transient crowding of the stiff nuclei of the surrounding nontransformed cells, whose movements depend directly on the cadherin-mediated mismatched adhesion between normal and cancer cells as well as α-catenin-based intercellular adhesion of the normal cells. These results suggest that a mechanical and adhesive mismatch between transformed and nontransformed cells in a cell monolayer can trigger enhanced pulsating migration. These results shed light on the role of stiff epithelial cells that neighbor individual cancer cells in early steps of cancer dissemination.

Correlating confocal microscopy and atomic force indentation reveals metastatic cancer cells stiffen during invasion into collagen i matrices

Mechanical interactions between cells and their microenvironment dictate cell phenotype and behavior, calling for cell mechanics measurements in three-dimensional (3D) extracellular matrices (ECM). Here we describe a novel technique for quantitative mechanical characterization of soft, heterogeneous samples in 3D. The technique is based on the integration of atomic force microscopy (AFM) based deep indentation, confocal fluorescence microscopy, finite element (FE) simulations and analytical modeling. With this method, the force response of a cell embedded in 3D ECM can be decoupled from that of its surroundings, enabling quantitative determination of the elastic properties of both the cell and the matrix. We applied the technique to the quantification of the elastic properties of metastatic breast adenocarcinoma cells invading into collagen hydrogels. We found that actively invading and fully embedded cells are significantly stiffer than cells remaining on top of the collagen, a clear example of phenotypical change in response to the 3D environment. Treatment with Rho-associated protein kinase (ROCK) inhibitor significantly reduces this stiffening, indicating that actomyosin contractility plays a major role in the initial steps of metastatic invasion.

Independent Control of Topography for 3D Patterning of the ECM Microenvironment.

Biomimetic extracellular matrix (ECM) topographies driven by the magnetic‐field‐directed self‐assembly of ECM protein‐coated magnetic beads are fabricated. This novel bottom‐up method allows us to program isotropic, anisotropic, and diverse hybrid ECM patterns without changing other physicochemical properties of the scaffold material. It is demonstrated that this 3D anisotropic matrix is able to guide the dendritic protrusion of cells.

In vivo tissue has non-linear rheological behavior distinct from 3D biomimetic hydrogels, as determined by AMOTIV microscopy.

Variation in matrix elasticity has been shown to determine cell fate in both differentiation and development of malignant phenotype. The tissue microenvironment provides complex biochemical and biophysical signals in part due to the architectural heterogeneities found in extracellular matrices (ECMs). Three dimensional cell cultures can partially mimic in vivo tissue architecture, but to truly understand the role of viscoelasticity on cell fate, we must first determine in vivo tissue mechanical properties to improve in vitro models. We employed Active Microrheology by Optical Trapping InVivo (AMOTIV), using in situ calibration to measure in vivo zebrafish tissue mechanics. Previously used trap calibration methods overestimate complex moduli by ∼ 2-20 fold compared to AMOTIV. Applying differential microscale stresses and strains showed that hyaluronic acid (HA) gels display semi-flexible polymer behavior, while laminin-rich ECM hydrogels display flexible polymer behavior. In contrast, zebrafish tissues displayed different moduli at different stresses, with higher power law exponents at lower stresses, indicating that living tissue has greater stress dependence than the 3D hydrogels examined. To our knowledge, this work is the first vertebrate tissue rheological characterization performed in vivo. Our fundamental observations are important for the development and refinement of in vitro platforms.

A comparison of methods to assess cell mechanical properties

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.This Analysis compares and contrasts methods for measuring the mechanical properties of cells by applying the different approaches to the same breast cancer cell line.

In situ calibration of position detection in an optical trap for active microrheology in viscous materials

In optical trapping, accurate determination of forces requires calibration of the position sensitivity relating displacements to the detector readout via the V-nm conversion factor (β). Inaccuracies in measured trap stiffness (k) and dependent calculations of forces and material properties occur if β is assumed to be constant in optically heterogeneous materials such as tissue, necessitating calibration at each probe. For solid-like samples in which probes are securely positioned, calibration can be achieved by moving the sample with a nanopositioning stage and stepping the probe through the detection beam. However, this method may be applied to samples only under select circumstances. Here, we introduce a simple method to find β in any material by steering the detection laser beam while the probe is trapped. We demonstrate the approach in the yolk of living Danio rerio (zebrafish) embryos and measure the viscoelastic properties over an order of magnitude of stress-strain amplitude.