EMT phenotype in isolated CTCs

To evaluate EMT phenotype in CTCs, samples were stained using the protocol described in the previous section but targeting different biomarkers. At the time of antibody addition, cells were stained with 100 μL of 10 μg/mL anti-CD45 conjugated with biotin (Clone HI30, Biolegend) for 45 min. Then, cells were incubated with 100 μL of 10 μg/mL Streptavidin-Alexa Fluor 594 (Biolegend), 10 μg/mL of anti-cytokeratin conjugated with FITC (CK, Clone CAM5.2, BD) and 10 μg/mL of anti-vimentin conjugated with Alexa Fluor 647 (Clone W16220A, Biolegend) in 0.02% Tween-20 (Research Products) for 45 min. Fluorescence micrographs were acquired using a LSM 710 META inverted confocal microscope and analyzed using Image J software. To determine the EM phenotype, we used the cell lines MDA-MB-231 and MCF-7 to set the appropriate exposure time for these biomarkers. These two cell lines were used as a positive and negative control due to the strong expression of EMT independently. The E phenotype in CTCs was determined by using the following criteria: (1) positive for cytokeratin, (2) negative for vimentin, (3) negative for CD45 and (4) positive for DAPI (nuclear staining). However, the M phenotype in CTCs was determined by using the following criteria: (1) negative for cytokeratin, (2) positive for vimentin, (3) negative for CD45 and (4) positive for DAPI (nuclear staining). The samples were imaged using 20x magnification and 5 pictures were taken per sample at random locations. The cell number was then scaled up by the frame area divided by the slide area. The number of CTCs expressing either epithelial or mesenchymal markers were enumerated using confocal pictures and the percentages were calculated.