Alexa 647

Using identical blood draws from the same group of patients (n = 30), an additional PB sample (10 mL in EDTA) was available from 45 matched samples from different time points for CTC isolation using the ISET technology (Rarecells Diagnostics, France). CTCs were captured in the ISET filters according to the manufacturer’s instructions and were then triple stained by immunofluorescence for CK/VIM/CD45 according to a validated protocol and analyzed using the Confocal laser Scanning microscopy (LEICA), as previously described^{60 (link)}. Specifically, for cytokeratins (CK) staining, two different antibodies were used as a cocktail: the A45-B/B3 anti-mouse Ab recognizing the CKs 8/18/19 (Micromet Munich, Germany) and an anti-mouse Ab against CK7 (Abcam, Cambridge, UK). Alexa 488 (Invitrogen Carlsbad, CA, USA) anti-mouse was used as a secondary antibody. Anti-CD45 antibody conjugated with Alexa 647 (Novus Biologicals, USA) was also added. Spots were stained with Vimentin antibody (Santa Cruz, Santa Cruz, CA, USA). Finally, slides were stained with DAPI conjugated with antifade (Invitrogen, Carlsbad, CA, USA).