Extravasation of tumor cells analysis

Tumor cells were divided into two subclasses: intravascular or extravascular, based on their location relative to the vasculature. To determine the location of a cell relative to the vasculature, the images of the vasculature at each time point (t = 8, 16, 24, 32, 40, 48, 56, and 64 h) were analyzed and co-registered with the corresponding prior time point using Adobe Photoshop CC 2015. The tumor cells overlapping with vasculature were considered to be intravascular. Cells not overlapping with the vasculature were considered as having extravasated. Tumor cells were excluded if their localization could not be accurately resolved. For extravascular tumor cells, we were also able to identify three different fates over time: 1) tumor cell death, 2) survival as a single and solitary tumor cell, or 3) growth into micro-metastases. We identified tumor cell death by the appearance of cellular debris (apoptotic bodies) in the field of view. We determined a tumor cell to have survived as a single tumor cell when we observed it remain a single cell in the same field of view over time. Finally, we determined a tumor cells to have formed micro-metastases when we observed cells to have increased in area by larger than that of a single cell.