Collagen polymerization (Fig. 1C) in part aided by inverting the device upside-down during the first 7 min of collagen polymerization [8]. In vitro collagen matrices are substantially different than those in vivo, specifically being more simple in composition, less dense in collagen, and more spatially homogeneous. A high collagen density (similar to that of breast tumor in vivo) cannot be usedfor in vitro assays, since tumor cell migration is suppressed by the dense and spatially homogenous biomatrix network. However, it has been shown that the in vivo tumor microenvironment is highly heterogeneic in terms of spatial distribution of the collagen fibers. Multiphoton imaging of breast tumor cell migration in mouse model shows that fast and persistent tumor cell migration is often associated with three factors ?the lack of dense collagen network, amoeboid motion, and the contact of cells with ECM fibers [35,36]. It is thus important to place tumor cells in the context of a collagen matrix for in vitro studies, and processes like tumor cell chemoinvasion can be mimicked with close proximity to the in vivo situation in such matrices [24,29,37]. Cell movements were characterized in real time and space by taking a time sequence of images of migrating cells, andRoles of Two Cytokines in Tumor Cell MigrationFigure 3. Chemoinvasive and chemokinetic behavior of tumor cells to linear SDF-1a gradients. A. 11089-65-9 chemical information Average cell velocity x along the V +C . B. Average SDF-1a gradient as a function of the SDF-1a gradient +C. Solid line is a fit to the ligand ?receptor binding kinetics Vx A 2 avg zKD ?cell speed as a function of the SDF-1a concentration gradient. C. Average persistence length along the gradient direction Px as a function of SDF-1a concentration gradient. D. Average persistence length P as a function of SDF-1a concentration gradient. The stars were obtained using a nonparametric t-test compared to the control group (Mann-Whitney test with * for 0.01,p,0.05, ** for 0.001,p,0.01, and *** for p,0.001). doi:10.1371/journal.pone.0068422.gsubsequently tracking the cell positions at various time points. Examples of cell tracking process are shown in Movie S1 and Movie S2. Figure 1D shows cell trajectories of 16 h duration under four different chemical gradient conditions. These polar plots were formed by placing the first cell position of each cell track at the center coordinate. It should be noted that although the experiments were conducted in 3D, the cell tracking was carried out using images taken at a fixed 2D (or x ) plane. Figure 1D shows that tumor cells migrated randomly under control conditions (no chemical gradients), were chemoinvasive towards SDF-1a gradients, and displayed chemokinesis (or enhanced motility) in the presence of EGF gradients as well as in the presence of both EGF and SDF-1a gradients.flowing buffer or chemokine through the two side channels, the 115103-85-0 manufacturer majority of motile cells (,80 ) had aspect ratios less than 3 (See Figure 2B, D). Figure 2D shows that the aspect ratio of the cells decreased with time. Note that we exclude the potential stress under the microscope based on the observations that cells are motile as shown in Movie S1 and Movie S2. In addition, we found that i) the cells without flow in the side channels maintained a high aspect ratio in the microscope stage; and ii) the majority of the cells with flow in the side channels for the initial culture period (24 h) in a conventional incubator showed a similar decreas.Collagen polymerization (Fig. 1C) in part aided by inverting the device upside-down during the first 7 min of collagen polymerization [8]. In vitro collagen matrices are substantially different than those in vivo, specifically being more simple in composition, less dense in collagen, and more spatially homogeneous. A high collagen density (similar to that of breast tumor in vivo) cannot be usedfor in vitro assays, since tumor cell migration is suppressed by the dense and spatially homogenous biomatrix network. However, it has been shown that the in vivo tumor microenvironment is highly heterogeneic in terms of spatial distribution of the collagen fibers. Multiphoton imaging of breast tumor cell migration in mouse model shows that fast and persistent tumor cell migration is often associated with three factors ?the lack of dense collagen network, amoeboid motion, and the contact of cells with ECM fibers [35,36]. It is thus important to place tumor cells in the context of a collagen matrix for in vitro studies, and processes like tumor cell chemoinvasion can be mimicked with close proximity to the in vivo situation in such matrices [24,29,37]. Cell movements were characterized in real time and space by taking a time sequence of images of migrating cells, andRoles of Two Cytokines in Tumor Cell MigrationFigure 3. Chemoinvasive and chemokinetic behavior of tumor cells to linear SDF-1a gradients. A. Average cell velocity x along the V +C . B. Average SDF-1a gradient as a function of the SDF-1a gradient +C. Solid line is a fit to the ligand ?receptor binding kinetics Vx A 2 avg zKD ?cell speed as a function of the SDF-1a concentration gradient. C. Average persistence length along the gradient direction Px as a function of SDF-1a concentration gradient. D. Average persistence length P as a function of SDF-1a concentration gradient. The stars were obtained using a nonparametric t-test compared to the control group (Mann-Whitney test with * for 0.01,p,0.05, ** for 0.001,p,0.01, and *** for p,0.001). doi:10.1371/journal.pone.0068422.gsubsequently tracking the cell positions at various time points. Examples of cell tracking process are shown in Movie S1 and Movie S2. Figure 1D shows cell trajectories of 16 h duration under four different chemical gradient conditions. These polar plots were formed by placing the first cell position of each cell track at the center coordinate. It should be noted that although the experiments were conducted in 3D, the cell tracking was carried out using images taken at a fixed 2D (or x ) plane. Figure 1D shows that tumor cells migrated randomly under control conditions (no chemical gradients), were chemoinvasive towards SDF-1a gradients, and displayed chemokinesis (or enhanced motility) in the presence of EGF gradients as well as in the presence of both EGF and SDF-1a gradients.flowing buffer or chemokine through the two side channels, the majority of motile cells (,80 ) had aspect ratios less than 3 (See Figure 2B, D). Figure 2D shows that the aspect ratio of the cells decreased with time. Note that we exclude the potential stress under the microscope based on the observations that cells are motile as shown in Movie S1 and Movie S2. In addition, we found that i) the cells without flow in the side channels maintained a high aspect ratio in the microscope stage; and ii) the majority of the cells with flow in the side channels for the initial culture period (24 h) in a conventional incubator showed a similar decreas.
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