Confirmation of endothelial monolayer integrity.

<p>The integrity of the endothelial monolayer was confirmed by both fluorescence imaging of the dextran distribution and confocal microscopy of fixed and labeled cells. An intact endothelial monolayer gives rise to an abrupt intensity drop between the channel and the gel region once the fluorescently-labeled dextran is introduced. Three hours after dextran injection, a sharp drop in fluorescence intensity is seen across the endothelial layer demonstrating its function as a barrier to macromolecules (a). Fluorescence intensity is quantified using Matlab (b). The dashed arrow in (a) the location and direction for the quantification.The intensity value drops to 15% of is peak value due to the barrier effect. The endothelial monolayer is located near the 400 µm point on the plot (shown with dashed line). Samples fixed on the third day after cell seeding and stained for VE-cadherin and nuclei (DAPI-blue) exhibit well-defined junctions with no apparent gaps in the confluent monolayer (c). The confocal image shows the front view of the microfluidic device.</p

Optimization of tumor cell seeding density.

<p>The tumor cell seeding density was optimized to have only a limited number of tumor cells in ROI while maintaining as many experimental ROIs as possible that contain at least one tumor cell so tumor cell events can be observed. Histograms of number of total tumor cells present in each ROI (250 µm×250 µm×120 µm) show different trends in distribution of tumor cells for three different tumor seeding densities: 20,000 cells/ml, 50,000 cells/ml, and 200,000 cells/ml (a). The average value and the histogram can be used for choosing the optimal tumor seeding condition (b). Seeding density of 50,000 cells/ml was chosen as a compromise between mimicking the low number of tumor cells of the <i>in vivo</i> of extravasation condition and increasing the chance to have at least one tumor cell to analyze in any given ROI. The statistical significance was tested with one way ANOVA (p<0.05).</p

General schematic of the device.

<p>Microfluidic system consisting of three independently addressable media channels, separated by chambers into which an ECM-mimicking gel can be injected (a). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056910#pone-0056910-g001" target="_blank">Figure 1b</a> shows the inside view of the device with endothelial monolayer (blue) covering the center channel. This channel acts as cell channel where both endothelial cells and cancer cells are introduced to form monolayer and transmigrate respectively (b). The green region indicates the 3D space filled with collagen gel and the pink regions indicate the channel filled with medium. Cancer cells which adhere to endothelial monolayer can extravasate into the collagen gel region as shown in (c).</p