Fig. 3.

Inhibition of self-renewal of stemness-high cancer cells by BBI608. (A) Spheres derived from DLD1 and HCT116 cell lines were dissociated to single cells and allowed to form spheres in suspension with cancer stem cell medium for 48 h before treatment with BBI608 (2 μM). After 24 h, the drugs were removed and the cells were cultured in fresh cancer stem cell medium for another 24 h. Representative images are shown. (B) SW480 colon cancer cells were isolated by FACS based on Hoechst dye exclusion and were cultured for 72 h in cancer stem cell conditions before the addition of the indicated concentrations of therapeutic agents. Sphere growth was scored by counting the number of spheres possessing >50 cells. SP, side population; NSP, nonside population. (C) CD44^high FaDu cells were isolated by FACS and were cultured for 72 h in cancer stem cell conditions before the addition of the indicated therapeutic agents (400 nM BBI608, 2 μM imatinib, 10 μM sunitinib, 10 μM erlotinib, 100 nM doxorubicin). Sphere growth was scored by counting the number of spheres possessing >50 cells. Representative images are shown. (D) CD34⁺ bone marrow mononuclear cells were treated with either DMSO or BBI608 for 6 h at 37 °C. Cells were then washed and plated in complete Methocult H4434 medium. Colonies of both erythroid and myeloid lineages containing >50 cells per colony were counted. Each treatment was performed in triplicate.