CSC sphere-formation inhibition assay

To assess the effect on CCS sphere-forming ability, the cells (1000 cells per well) derived from the primary CSC cultures were re-seeded into poly-HEMA-coated 96-well plate and treated with or without drug. After 4 days, the spheres were fixed in 1% formalin and the spheres (≥ 100 μm in diameter) were counted under microscope (Nikon, Japan). For comparison, the cytotoxicity to the cells cultured in the attached condition on the plates in same duration were determined using MTS assay. Culture medium supplemented with 10% or 1% FBS was used to minimize the effect of serum proteins in the action of drugs because the defined CSC medium contains minimal proteins.

ALDH assay was performed to quantify putative CSC cells that express high ALDH. After the secondary spheres were cultured with or without SPC-160002 for 48 h, the cells were dissociated and analyzed with Aldeflour assay kit (Stem Cell Technology, Cambridge, MA) followed by flow cytometry. A well-defined ALDH inhibitor, N,N-diethylaminobenzaldehyde (DEAB), was used for negative control.