Views of the JW 74 confocal scan from other samples show three different possible locations of tumor cells 1 day after the seeding: 1) extravasated and in gel, 2) adhered and located onFigure 4. Observation of extravasation and I-BRD9 site permeability of endothelium. The extravasation event is observed in a sample, which is fixed 1 day after tumor cells are introduced. The region of interest is captured in one confocal image scan and shows one cancer cell (green) that has transmigrated across the endothelium (denoted by VE-In Vitro Model of Tumor Cell ExtravasationFigure 5. Beyond extravasation. The tumor cell extravasation is observed for up 
to 3 days after tumor cell seeding 12926553 and compared to the ones fixed and analyzed on day 1. The total number of tumor cells present in region of interest (ROI) increases significantly from 8 cells on day 1 to 13.5 cells on day 3 while the tumor seeding density as well as other experimental condition remained the same between devices (a). The total number of tumor cells are further subdivided into 2 groups depending on their location, either 1) extravasated and in the gel or 2) adherent to the endothelium adjacent to gel (b). 72 of ROIs exhibited at least 1 extravasated cancer cell (denoted extravasation occurrence) by day 1 after introducing tumor cells, and 79 of ROIs included extravasation event by day 3, which the difference is not significant (c). The images show number of tumor cell increase (d). The phase contrast image shows the top view of the region of interest on day 1 after tumor seeding. The tumor cells (green) have proliferated from day 1 to day 3 (shown by arrows). The confocal image shows both the tumor cells and endothelium lining. All images are from the same ROI (VE-cadherin: red, nucleus: DAPI-blue, tumor cell: GFP-green). doi:10.1371/journal.pone.0056910.gendothelium adjacent to gel region, and 3) in the channel not near the gel (Fig. 4b). The surface and sectional views of the device shown in Fig. 4b. All three scenarios of where tumor cells could be are observed here. There are cases where all tumor cells present in the ROI extravasated as well as cases where none of the tumor cells crossed the endothelium. However, it is more common to find regions that contain both extravasated and non-extravasated cells as in Fig. 4b. This is seen quantitatively in Fig. 4c where 51 of ROIs exhibited tumor cells with contrasting fate. The graph shows how many tumor cells have extravasated, as shown by dots, among the total tumor cells present in the each region of interest. The tumor cells are categorized as having extravasated only when they have clearly passed the endothelial monolayer into the gel region. Measuring permeability of the endothelium is one method for quantifying the quality of 15755315 endothelial monolayer. In addition, thepermeability serves as a metric to quantify the change in endothelium when tumor cells are added to the system and interact via physical attachment to the endothelial surface. Leakiness of the vessel with or without tumor cells provides a possible explanation for events leading to cancer extravasation in that signaling by the tumor CTCs could impair barrier function. Alternatively, the increase in permeability could be a result of tumor cell transmigration. From the present experiments, it is not possible to distinguish between these two scenarios. In this experiment, the permeability changed significantly with addition 
of tumor cells compared to the permeability change occurri.Views of the confocal scan from other samples show three different possible locations of tumor cells 1 day after the seeding: 1) extravasated and in gel, 2) adhered and located onFigure 4. Observation of extravasation and permeability of endothelium. The extravasation event is observed in a sample, which is fixed 1 day after tumor cells are introduced. The region of interest is captured in one confocal image scan and shows one cancer cell (green) that has transmigrated across the endothelium (denoted by VE-In Vitro Model of Tumor Cell ExtravasationFigure 5. Beyond extravasation. The tumor cell extravasation is observed for up to 3 days after tumor cell seeding 12926553 and compared to the ones fixed and analyzed on day 1. The total number of tumor cells present in region of interest (ROI) increases significantly from 8 cells on day 1 to 13.5 cells on day 3 while the tumor seeding density as well as other experimental condition remained the same between devices (a). The total number of tumor cells are further subdivided into 2 groups depending on their location, either 1) extravasated and in the gel or 2) adherent to the endothelium adjacent to gel (b). 72 of ROIs exhibited at least 1 extravasated cancer cell (denoted extravasation occurrence) by day 1 after introducing tumor cells, and 79 of ROIs included extravasation event by day 3, which the difference is not significant (c). The images show number of tumor cell increase (d). The phase contrast image shows the top view of the region of interest on day 1 after tumor seeding. The tumor cells (green) have proliferated from day 1 to day 3 (shown by arrows). The confocal image shows both the tumor cells and endothelium lining. All images are from the same ROI (VE-cadherin: red, nucleus: DAPI-blue, tumor cell: GFP-green). doi:10.1371/journal.pone.0056910.gendothelium adjacent to gel region, and 3) in the channel not near the gel (Fig. 4b). The surface and sectional views of the device shown in Fig. 4b. All three scenarios of where tumor cells could be are observed here. There are cases where all tumor cells present in the ROI extravasated as well as cases where none of the tumor cells crossed the endothelium. However, it is more common to find regions that contain both extravasated and non-extravasated cells as in Fig. 4b. This is seen quantitatively in Fig. 4c where 51 of ROIs exhibited tumor cells with contrasting fate. The graph shows how many tumor cells have extravasated, as shown by dots, among the total tumor cells present in the each region of interest. The tumor cells are categorized as having extravasated only when they have clearly passed the endothelial monolayer into the gel region. Measuring permeability of the endothelium is one method for quantifying the quality of 15755315 endothelial monolayer. In addition, thepermeability serves as a metric to quantify the change in endothelium when tumor cells are added to the system and interact via physical attachment to the endothelial surface. Leakiness of the vessel with or without tumor cells provides a possible explanation for events leading to cancer extravasation in that signaling by the tumor CTCs could impair barrier function. Alternatively, the increase in permeability could be a result of tumor cell transmigration. From the present experiments, it is not possible to distinguish between these two scenarios. In this experiment, the permeability changed significantly with addition of tumor cells compared to the permeability change occurri.
