Nges in endothelial permeability are closely associated with intravasation[24], so we sought to study similar changes that might occur during extravasation. In addition, by tracking the cells over time, we were able to explore the time-dependent behavior, an important factor that impacts both the survival of the CTCs priorIn Vitro Model of Tumor Cell ExtravasationFigure 3. Optimization of tumor cell seeding density. 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 12926553 of total tumor cells present in each 
ROI (250 mm6250 mm6120 mm) show PHCCC web 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 in vivo 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). doi:10.1371/journal.pone.0056910.gto extravasation as well as their ability to reconfigure the immediate microenvironment prior to transmigration.Confirmation of Endothelial Layer IntegrityThe microfluidic system was designed to model tumor cell extravasation where the tumor cells are introduced into a channel lined with a confluent endothelial monolayer. Using phase contrast microscopy, hMVECs were observed forming a confluent monolayer on the microchannel surfaces and ECM-endothelial channel interface two days after endothelial cell seeding. 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 dextranis introduced, and persists over time as dextran slowly diffuses across the monolayer into the gel (Fig. 2a ). 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 (Fig. 1516647 2c). Quantification and analysis of fluorescence intensity yields values for the endothelial permeability to a 70 kDa dextran (3.7060.59)?1026 cm/s, or roughly one order of magnitude higher than published in vivo values but consistent with previously reported values in in vitro systems [35,38]. The higher values of permeability may be due to a variety of factors present in vivo but missing from the in vitro model. For example, it is well known that the presence of pericytes helps to establish the low permeability of vessels in vivo [38]. In view of our previous work demonstrating that increased permeability correlates with increased rates of 94-09-7 web intravasation [24], to the extent that cells use similar mechanisms for extravasation as intravasation, the presentIn Vitro Model of Tumor Cell Extravasationcadherin staining in red) and extravasated into the gel region (a). The surface view of the confocal scan shows three different possible locations of tumor cells: 1) extravasated and in gel, 2) adhered and on endothelium adjacent.Nges in endothelial permeability are closely associated with intravasation[24], so we sought to study similar changes that might occur during extravasation. In addition, by tracking the cells over time, we were able to explore the time-dependent behavior, an important factor that impacts both the survival of the CTCs priorIn Vitro Model of Tumor Cell ExtravasationFigure 3. Optimization of tumor cell seeding density. 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 12926553 of total tumor cells present in each ROI (250 mm6250 mm6120 mm) 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 in vivo 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). doi:10.1371/journal.pone.0056910.gto extravasation as well as their ability to reconfigure the immediate microenvironment prior to transmigration.Confirmation of Endothelial Layer IntegrityThe microfluidic system was designed to model tumor cell extravasation where the tumor cells are introduced into a channel lined with a confluent endothelial monolayer. Using phase contrast microscopy, hMVECs were observed forming a confluent monolayer on the microchannel surfaces and ECM-endothelial channel interface two days after endothelial cell seeding. 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 dextranis introduced, and persists over time as dextran slowly diffuses across the monolayer into the gel (Fig. 2a ). 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 (Fig. 1516647 2c). Quantification and analysis of fluorescence intensity yields values for the endothelial permeability to a 70 kDa dextran (3.7060.59)?1026 cm/s, or roughly one order of magnitude higher than published in vivo values but consistent with previously reported values in in vitro systems [35,38]. The higher values of permeability may be due to a variety of factors present in vivo but missing from the in vitro model. For example, it is well known that the presence of pericytes helps to establish the low permeability of vessels in vivo [38]. In view of our previous work demonstrating that increased permeability correlates with increased rates of intravasation [24], to the extent that cells use similar mechanisms for extravasation as intravasation, the presentIn Vitro Model of Tumor Cell Extravasationcadherin staining in red) and extravasated into the gel region (a). The surface view of the confocal scan shows three different possible locations of tumor cells: 1) extravasated and in gel, 2) adhered and on endothelium adjacent.
