Detection of CTCs in the blood samples of cancer patients

CTCs in the peripheral blood samples of cancer patients were detected as previously described with slight modifications.¹⁰ Briefly, cells recovered from the 7.5 ml blood samples of patients with NSCLC were incubated with 2.3 × 10⁸ VP of rAd-GFP and 1 × 10⁹ VP of rAdF35-142T-GFP at 37 °C. Following a 24-hour incubation, the cells were washed and stained with anti-human CD45 antibody (clone HI30; Biolegend, San Diego, CA), then observed by fluorescence microscopy. CTCs and false-positive cells were defined as GFP⁺/CD45⁻ and GFP⁺/CD45⁺ cells, respectively. The procedures for obtaining peripheral blood from patients with lung cancer were approved by the Institutional Review Board at the Juntendo University School of Medicine (No. 2013067). All patients provided written informed consent.