Rapamycin suppresses stem cell population, in vitro cell proliferation and in vivo tumorigenicity. (A) CD24-CD44+ cells of BC0145 xenograft tumors were cultured for mammosphere formation for 7 days, and the number of spheres were counted and shown as the number per 1,000 cells. #P <0.01. (B) Differential sensitivity of breast cancer stem cells (BCSCs) and non-BCSCs to rapamycin in vitro. BCSCs and non-BCSCs were cultured at 1×104 cells/well with rapamycin and cell titers were determined at 48 hours by alamar blue assay. (C) Effects of rapamycin on tumorigenicity of BCSCs in vivo. Groups of three NOD/SCID mice were inoculated with 105 cells of BCSCs in the mammary fat pads and treated with intraperitoneal injections of rapamycin every 2 days at 4 mg/kg for 3 weeks. Tumor volumes were monitored weekly from weeks 4 to 9. #P <0.01. (D) The CD24-CD44+ BC0145 cells were injected into mammary fat pads. Two weeks later, the mice were treated with rapamycin at 2 mg/kg/every 2 days for first week and 1 mg/kg/every 2 days for a further 2 weeks. The therapeutic effect of rapamycin was demonstrated by the reduced tumor formation and tumor volume. The BCSC activity in vehicle-treated or rapamycin-treated tumors was determined by mammosphere forming capability. To avoid the influence of mouse cells, the assay was conducted with H2Kd- tumor cells. #P <0.01. All experiments were repeated independently at least twice and results shown were collected from a representative experiment. CSC, cancer stem cell; DMSO, dimethylsulfoxide.