3D Traction Forces in Cancer Cell Invasion

Cell invasion through a dense three-dimensional (3D) matrix is believed to depend on the ability of cells to generate traction forces. To quantify the role of cell tractions during invasion in 3D, we present a technique to measure the elastic strain energy stored in the matrix due to traction-induced deformations. The matrix deformations around a cell were measured by tracking the 3D positions of fluorescent beads tightly embedded in the matrix. The bead positions served as nodes for a finite element tessellation. From the strain in each element and the known matrix elasticity, we computed the local strain energy in the matrix surrounding the cell. We applied the technique to measure the strain energy of highly invasive MDA-MB-231 breast carcinoma and A-125 lung carcinoma cells in collagen gels. The results were compared to the strain energy generated by non-invasive MCF-7 breast and A-549 lung carcinoma cells. In all cases, cells locally contracted the matrix. Invasive breast and lung carcinoma cells showed a significantly higher contractility compared to non-invasive cells. Higher contractility, however, was not universally associated with higher invasiveness. For instance, non-invasive A-431 vulva carcinoma cells were the most contractile cells among all cell lines tested. As a universal feature, however, we found that invasive cells assumed an elongated spindle-like morphology as opposed to a more spherical shape of non-invasive cells. Accordingly, the distribution of strain energy density around invasive cells followed patterns of increased complexity and anisotropy. These results suggest that not so much the magnitude of traction generation but their directionality is important for cancer cell invasion

NEDD9 Stabilizes Focal Adhesions, Increases Binding to the Extra-Cellular Matrix and Differentially Effects 2D versus 3D Cell Migration

The speed of cell migration on 2-dimensional (2D) surfaces is determined by the rate of assembly and disassembly of clustered integrin receptors known as focal adhesions. Different modes of cell migration that have been described in 3D environments are distinguished by their dependence on integrin-mediated interactions with the extra-cellular matrix. In particular, the mesenchymal invasion mode is the most dependent on focal adhesion dynamics. The focal adhesion protein NEDD9 is a key signalling intermediary in mesenchymal cell migration, however whether NEDD9 plays a role in regulating focal adhesion dynamics has not previously been reported. As NEDD9 effects on 2D migration speed appear to depend on the cell type examined, in the present study we have used mouse embryo fibroblasts (MEFs) from mice in which the NEDD9 gene has been depleted (NEDD9 −/− MEFs). This allows comparison with effects of other focal adhesion proteins that have previously been demonstrated using MEFs. We show that focal adhesion disassembly rates are increased in the absence of NEDD9 expression and this is correlated with increased paxillin phosphorylation at focal adhesions. NEDD9−/− MEFs have increased rates of migration on 2D surfaces, but conversely, migration of these cells is significantly reduced in 3D collagen gels. Importantly we show that myosin light chain kinase is activated in 3D in the absence of NEDD9 and is conversely inhibited in 2D cultures. Measurement of adhesion strength reveals that NEDD9−/− MEFs have decreased adhesion to fibronectin, despite upregulated α5β1 fibronectin receptor expression. We find that β1 integrin activation is significantly suppressed in the NEDD9−/−, suggesting that in the absence of NEDD9 there is decreased integrin receptor activation. Collectively our data suggest that NEDD9 may promote 3D cell migration by slowing focal adhesion disassembly, promoting integrin receptor activation and increasing adhesion force to the ECM

Displacements and strain energy density around non-invasive and invasive cells.

<p>a–e: Displacement fields (projected to the x-y plane, normalized to the largest displacements) around invasive and non-invasive carcinoma cells. Non-invasive cells contract the gels more isotropically. Invasive cells generate highly anisotropic displacement fields with large displacements at the cell poles and a region of comparatively small displacements near the cell center. f–j: 3D displacement fields of the same cells as shown in a–e. k–o: Strain energy density around the same cells as shown in a–e. A closed isosurface of strain energy density (30% of maximum value) is shown with cuts projected to the coordinate planes. The strain energy density is distributed more isotropically around non-invasive cells with almost spherical isosurfaces, and is distributed more anisotropically around invasive cells with complex isosurface shapes.</p

Time-lapse measurements of non-invasive and invasive cells.

<p>a: Time course of the total strain energy around a non-invasive vulva carcinoma cell (A-431) in a 3D collagen gel. The zero energy reference was measured after cytochalasin D-induced tension release. The total strain energy fluctuates by about ±25% around a high mean value. b: Time course of the total strain energy around an invasive breast carcinoma cell (MDA-MD-231) in a 3D collagen gel. The strain energy shows large fluctuations of about ±50% around the mean. c–j: Time series of brightfield images of a non-invasive vulva carcinoma cell (A-431) (c–f) and an invasive MDA-MB-231 breast carcinoma cell (g–j) in a 3D collagen gel. Superimposed are contour lines showing relative changes in strain energy density between two subsequent time steps. Blue colors indicate regions where tension is relaxed between successive time steps, and red colors indicate regions where tension is increased. The cell shape is outlined in white for clarity. Dark blue arrows indicate the direction of cell movement between the two time steps (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033476#pone.0033476.s013" target="_blank">Fig. S13</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033476#pone.0033476.s014" target="_blank">S14</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033476#pone.0033476.s015" target="_blank">S15</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033476#pone.0033476.s016" target="_blank">S16</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033476#pone.0033476.s017" target="_blank">S17</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033476#pone.0033476.s020" target="_blank">Movies S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033476#pone.0033476.s021" target="_blank">S4</a> for more details).</p

Strain energy and anisotropy of non-invasive and invasive cells.

<p>a: Invasion profiles of A-125 and A-549 lung, MDA-MB-231 and MCF-7 breast and A-431 vulva carcinoma cells. Cells were plated on the surface of collagen gels and allowed to spread and invade for 3 days. The invasion profile vs. gel depths describes the cumulative probability of finding a cell below a given depth. Invasive cells were characterized by their ability to invaded deep into the gels. b: Strain energy of non-invasive and invasive carcinoma cells. Invasive lung (n = 36) and breast (n = 33) carcinoma cells generate significantly higher strain energies compared to non-invasive lung (n = 49) and breast (n = 31) carcinoma cells. Non-invasive vulva carcinoma cells (n = 35), however, generate the largest strain energy of all cell lines tested (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033476#pone.0033476.s010" target="_blank">Fig. S10</a>). c: Anisotropy of cell shape. Non-invasive cells are significantly rounder compared to invasive cells. d: Anisotropy of the strain energy density is significantly higher in invasive compared to non-invasive cells. Because the strain energy and anisotropy values from different cells follow a log-normal distribution (Supporting Information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033476#pone.0033476.s022" target="_blank">Text S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033476#pone.0033476.s010" target="_blank">Fig. S10</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033476#pone.0033476.s011" target="_blank">S11</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033476#pone.0033476.s012" target="_blank">S12</a>), the geometric mean ± geometric standard error are shown in Fig. b–d.</p

Incorporation of zeolite and silica nanoparticles into electrospun PVA/collagen nanofibrous scaffolds: The influence on the physical, chemical properties and cell behavior

Cartilage is under extensive investigation in tissue engineering research. Herein, we evaluated scaffolds prepared by composites of polyvinyl alcohol (PVA) and collagen incorporated with zeolite and silica nanoparticles (nZe and nSi). The scaffolds were prepared by the electrospinning method. The mean diameters of nanofibers were 0.61 ± 0.34 μm for PVA/collagen versuss 0.62 ± 0.22 μm and 0.66 ± 0.25 μm for the PVA/collagen/nZe and the PVA/collagen/nSi scaffolds, respectively. DAPI staining results revealed that cell proliferations on the PVA/collagen/nZe and PVA/collagen/nSi were strikingly higher than on the pure PVA/collagen. The results encouraged further investigation of PVA/collagen/nSi scaffolds as biomimetic platform for chondrocyte cells in tissue engineering

Paxillin phosphorylation is increased in NEDD9 −/− MEFs.

<p>A. Paxillin (pax) and phospho-paxillin (ppax) immunostaining. Merged images show colour overlays of paxillin (green) and phospho-paxillin (red). Right hand panels show ratio images of paxillin phosphorylation. Red hues reflect regions of highest phosphorylated paxillin. Boxed insets shows magnified focal adhesions. B. Ratio of phosphorylated paxillin at focal adhesions in WT (n = 197 from 10 individual cells) and NEDD9 −/− MEFs (n = 201 from 10 individual cells). ***p<0.0001, Students' <i>t</i>-test.</p