Tracking TNBC Cell Intravasation

TNBC intravasation from the tissue compartment, across the EC barrier, into the vascular channels was monitored by fluorescence imaging. For this purpose, CellTracker CM-DiI dye, which is non-toxic, well retained, and allows for multigenerational tracking of cellular movements (Krishnamurthy et al., 2008 (link)), was used. Briefly, TNBC cells were stained with CM-DiI dye before they were introduced into the tissue compartment. Meanwhile, HUVECs were stained with CellTracker Green CMFDA dye for better visualization of TNBC intravasation. Fluorescence images were acquired using the Olympus IX71 microscope system, as explained above. Images were then analyzed in cellSens Dimension software for quantifying intravasation. This was achieved using the “Count and Measure” function in cellSens Dimension software, where ROIs were drawn in different areas of the image as shown in Supplementary Figure S3. The obtained cell numbers were then analyzed and quantified (Detailed steps are given in Supplementary Material).