SP cells are more chemoresistant in vitro and Dox treatment selects for cells from the SP compartment in vivo. (A) Hoechst 33342 dye–stained 4T1 and NXS2 cells were sorted into 4 separate groups, SP (4T1 and NXS2) and non-SP (4T1 and NXS2), and each was cultured in the presence of DMSO or Dox for 24 hours. The number of live cells remaining in each culture was then determined by MTT assay using the DMSO-treated cells as a baseline value. (B) The same procedure was used as in panel A except that cells were cultured in the presence of CDDP instead of Dox. Data represent the mean and SEM of the percentage of live cells remaining in each culture following chemotherapy. (C-F) BALB/c mice (n = 2) were challenged orthotopically with 8 × 10³ 4T1 tumor cells and (D,F) Dox (10 mg/kg) or (C,E) mock was administered intravenously on days 7, 14, and 21. Single-cell suspensions of tumors were harvested on day 23 and stained with the Hoechst 33342 dye. Cells in panels E-F were coincubated with verapamil. Data are presented as mean and SEM of the percentage of live cells. Each figure represents 1 of 2 experiments. *P < .05. **P < .01.