Cell Mechanics Cluster
Mechanical Stiffness Grades Metastatic Potential in Patient Tumor Cells and in Cancer Cell Lines
Cancer cells are defined by their ability to invade through the basement membrane, a critical step during metastasis. While increased secretion of proteases, which facilitates degradation of the basement membrane, and alterations in the cytoskeletal architecture of cancer cells have been previously studied, the contribution of the mechanical properties of cells in invasion is unclear. Here, we applied a magnetic tweezer system to establish that stiffness of patient tumor cells and cancer cell lines inversely correlates with migration and invasion through three-dimensional basement membranes, a correlation known as a power law. We found that cancer cells with the highest migratory and invasive potential are five times less stiff than cells with the lowest migration and invasion potential. Moreover, decreasing cell stiffness by pharmacologic inhibition of myosin II increases invasiveness, whereas increasing cell stiffness by restoring expression of the metastasis suppressor TβRIII/betaglycan decreases invasiveness. These findings are the first demonstration of the power-law relation between the stiffness and the invasiveness of cancer cells and show that mechanical phenotypes can be used to grade the metastatic potential of cell populations with the potential for single cell grading. The measurement of a mechanical phenotype, taking minutes rather than hours needed for invasion assays, is promising as a quantitative diagnostic method and as a discovery tool for therapeutics. By showing that altering stiffness predictably alters invasiveness, our results indicate that pathways regulating these mechanical phenotypes are novel targets for molecular therapy of cancer.
V. Swaminathan, K. Mythreye, E. T. O’Brien, A. Berchuck, G. C. Blobe, and R. Superfine, “Mechanical stiffness grades metastatic potential in patient tumor cells and in cancer cell lines.,” Cancer Res., vol. 71, no. 15, pp. 5075–80, Aug. 2011
The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins
How individual cells respond to mechanical forces is of considerable interest to biologists as force affects many aspects of cell behaviour. The application of force on integrins triggers cytoskeletal rearrangements and growth of the associated adhesion complex, resulting in increased cellular stiffness, also known as reinforcement. Although RhoA has been shown to play a role during reinforcement, the molecular mechanisms that regulate its activity are unknown. By combining biochemical and biophysical approaches, we identified two guanine nucleotide exchange factors (GEFs), LARG and GEF-H1, as key molecules that regulate the cellular adaptation to force. We show that stimulation of integrins with tensional force triggers activation of these two GEFs and their recruitment to adhesion complexes. Surprisingly, activation of LARG and GEF-H1 involves distinct signalling pathways. Our results reveal that LARG is activated by the Src family tyrosine kinase Fyn, whereas GEF-H1 catalytic activity is enhanced by ERK downstream of a signalling cascade that includes FAK and Ras.
C. Guilluy, V. Swaminathan, R. Garcia-Mata, E. T. O’Brien, R. Superfine, and K. Burridge, “The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins.,” Nat. Cell Biol., vol. 13, no. 6, pp. 722–7, Jun. 2011.
Enhancement of Nanoparticle Gene Delivery via Oscillating Magnetic Fields
Gene therapy involves the delivery of genes to act as therapeutic agents to treat or cure diseases. In order to be effective for eventual in vivo applications, appropriate vectors and delivery systems must be developed to target the tissues of interest. One way to achieve this is to use magnetizable nanoparticles to apply forces to direct the vectors and stimulate their uptake by cells. We use paramagnetic nanoparticles to transfect antisense oligonucleotides into HeLa EGFP-654 cells. This cell line uses EGFP as a model for the inherited blood disorder β-thalassemia and the oligos serve to correct the splicing mutation which causes the disease. It has been shown that an oscillating magnetic field can enhance nanoparticle uptake into the cells. Data show that uptake enhancement can be maximized at a certain optimal range of frequencies. Studies are being performed to determine the specific endocytic pathways which are most affected by the oscillating field applications.
Mair, L., K. Ford, et al. (2009). “Size-Uniform 200 nm Particles: Fabrication and Application to Magnetofection.” Journal of Biomedical Nanotechnology 5(2): 182-191.
High Throughput Cell Mechanics on Monoptes/Panoptes
The involvement of mechanosensing, a cell’s ability to sense and respond to mechanical signals, has been shown to contribute to the development of many diseases. When cells become cancerous, adhesion and migratory behaviors – characteristics defined by the mechanical structure of a cell’s cytoskeleton – are altered. Traditional tests to explore cell metastasis involve hours of cell tracking in scratch assays or cell counting in invasion assays. Here, we report on the progress towards a parallel array of 12 independently functioning imaging systems capable of gathering mechanical measurements for a 96-well specimen plate via external passive bead rheology. Our results support previously documented work describing the inverse relationship between mechanical stiffness and invasion behavior. This demonstrates the value of our high throughput passive rheology assay as a screening tool for studying specific signaling pathways involved in cancer and mechanotransduction.
Our single and multiple unit magnetic systems, the 3D force microscope (3DFM) and Magnetic High Throughput Systems (MHTS), have proven very useful in probing the mechanical properties of cells and cellular components. Cells maintain their physical integrity and withstand and exert forces via complex interactions involving their cytoskeleton, cytoplasm, plasma membrane components and other structures. The nucleus breaks down, chromosomes condense and divide. Cancer cells change shape and invade healthy tissue. Cells react to external and internal signals by changing shape, changing tension or activity.
By applying forces to cells via magnetic beads of various sizes and surface chemistries, or resisting the forces exerted by cells or organelles, our magnetic systems can assess the forces being exerted by cells, or the responses of cells to various forces, from the thermal floor to tens of nN. We can also apply forces in many different regimes, from steady pulls, to ramps, to oscillations. Data can be recorded with video at rates of up to 200/s, or with laser tracking in 3D at 10,000 points/s.
Cell Mechanics studies also include the manufacture and analysis of micropattened posts and pillars that can be deformed by cells under various conditions and binding regimes. The pillars can be calibrated and thus forces assessed.
Pictured above is an example of a cell mechanics experiment, recently published in Methods in Cell Biology and Biophysical Journal (O’Brien et al 2008) shows 2.8 μm magnetic beads on IMR cells, with the dark shadow a magnetic pole tip. The data, B-D show high resolution tracking of the bead before during and after the application of force. The magenta line shows when the activation of the poles takes place. Links from the beads to the cells take place via antibodies to the actin cytoskeleton via intergrins (B,D), or a GPI-anchored, non-cytoskeletal linker (C). D shows the response of an integrin-linked bead after treatment with latrunculin B, which destabilizes the actin cytoskeleton.
O’Brien, E.T., J. Cribb, D. Marshburn, R.M.T. II, and R. Superfine, Magnetic Manipulation for Force Measurements in Cell Biology, in Methods in Cell Biology. 2008, Elsevier. p. 433-450