Isolation and Characterization of Stem-like Cancer Cells

Human PE01, PE04, PEA1, PEA2, and Kuramochi cells were detached and labeled by the ZIP4 antibody (AF7315; R&D Systems, 1:50) at room temperature for 2 h, followed by incubation with a APC-labeled donkey anti-goat IgG secondary antibody (F0108; R&D Systems, 1:500). ALDH activity was measured using the ALDEFLOUR Kit (STEMCELL Technologies, Vancouver, Canada) as we described previously [15 (link)] and detected using the green fluorescence channel (520–540 nm). FACS-based sorting and analysis of markers ZIP4 and ALDH were conducted using the BD FACSAria cell sorter system (Becton-Dickinson, Franklin Lakes, NJ, USA) and BD LSR Fortessa Analyzer (BD Biosciences, San Jose, CA, USA), and data analyzed by FlowJo V10 (BD Biosciences, San Jose, CA, USA). For the self-renewal assay, FACS-sorted ZIP4⁺ cells (100% pure) were collected and seeded into triple wells (1 × 10⁴ cells per well), cultured under differentiation conditions (DMEM medium with 10% FBS) for 48 h, trypsinized using 0.25% trypsin and washed twice in PBS, then cells were relabeled with ZIP4/APC antibodies before the renew/differentiation assays.