In vitro activity: Napabucasin downregulates stemness gene expression driven by Stat3 and cancer stemness properties, and effectively inhibits self-renewal of stemness-high cancer cells with IC50 ranged from 0.291~1.19 μM, on inhibition on normal stem cells.

Cell Assay: For cancer stem cells (U87-MG, U118, COLO205, DLD1, SW480, HCT116, FaDu, ACHN, SNU-475, Huh7, HepG2, H1975, A549, H460, CAOV-3, SW-626, PaCa2.) spheres are dissociated and plated under cancer stem cell culture conditions on coated 96-well plates. After 72 h of culture, wells are dosed with the indicated compounds. Seventy-two hours or 24 h after dosing, CellTiter-Glo 2.0 is added to each well, and the luminescence is measured as described by the manufacturer. IC50 values are calculated by fitting a four parameter dose–response curve to normalized data using GraphPad Prism software. For bulk cells, cells are plated at 5,000 cells per well on 96-well plates. Twenty-four hours after plating, cells are treated with the indicated compounds. Viability is determined at 72 h as described above.