Blocking reagent

Flow cytometry analysis was performed on a FACSCanto II and FlowJo software (both from BD Biosciences). Dead cells were excluded using the fixable viability-dye-eFluor780 (Thermo Fisher Scientific), and nonspecific FcR-mediated staining was blocked with blocking reagent (Miltenyi). Stainings were performed for 15 minutes at 4 °C with fluorophore-labeled antibodies as listed in Supplementary Table S1 (Supplementary Material). Fluorescence minus one or isotype-matched antibodies were used as controls. For intracellular staining, Cytofix/Cytoperm kit (BD Biosciences) was used according to the manufacturer’s instructions.

To ensure more reliable isolation of CICs, cells were labeled with one or several common markers of stemness conjugated with different fluorescent dyes, including anti-human CD133/2-APC (clone 293C3) and CD133/1-PE (clone AC133; Miltenyi Biotec, CA, USA); CD166-PE (clone 105902; R&D Systems, MN, USA); CD44-FITC (clone F10-44-2), CD44-PE (clone F10-44-2; Invitrogen/Biosources, USA); CD44v6-FITC (clone 2F10; R&D Systems, USA), EpCAM-FITC (Biosource, CA, USA), Pan-Keratin (C11) -Alexa Fluor 488 (Cell Signaling) and all the isotype controls (Chemicon). Antibodies were diluted in buffer containing 5% BSA, 1 mM EDTA and 15–20% blocking reagent (Miltenyi Biotec) to inhibit unspecific binding to non-target cells. After 15 min incubation at 4°C, stained cells were sorted and analyzed with multiparametric flow cytometer BD FACSAria (Becton Dickinson, CA). Alternatively, dissociated cells were centrifuged at 950 g for 5 min at 4°C, rinsed with sterile MACS buffer (Miltenyi Biotec, CA) and labeled with CD133 Abs directly or indirectly conjugated with ferromagnetic beads (Miltenyi Biotec, CA) as recommended by manufacturer.