Sphere‐forming assay and colony formation assay

For sphere‐forming assay, single‐cell suspensions were re‐suspended in culture medium with 1% N2 supplement (Gibco, Carlsbad, CA, USA), 10 ng/ml bFGF (Invitrogen) and 10 ng/ml EGF (Gibco) according to the manufacturer's instructions and plated in ultra‐low attachment plates (Corning) at a density of 1 × 10³ per well. Inverted microscope and 96‐well plates (NEST Biotechnology Co.LTD.) were used to count the tumour sphere number. To obtain re‐adherent cells, spheres were digested and re‐suspended in DMEM/high glucose with 10% foetal medium and plated in six‐well plates (NEST Biotechnology Co. LTD.). Protein extraction was performed after 72 hrs. For colony formation assay, cells were seeded in 12‐well plates at a density of 200 cells/well with 10% FBS contained‐medium. After 7 days incubation (10 days for SCC25), the colonies were fixed with 4% paraformaldehyde and stained with crystal violet. The numbers of colonies were counted. Each assay was performed in triplicate.