Fig. 5

The cancer stem cell (CSC)-like characteristics of tumor cells isolated from the secondary enhanced green fluorescent protein (EGFP)⁺ MCF7/HRE-EGFP xenografts. a Generation of secondary MCF7/HRE-EGFP xenografts by re-implantation of sorted EGFP⁺ and EGFP⁻ cells isolated from the primary MCF7/HRE-EGFP xenografts. Tumor cells freshly isolated from the secondary xenografts were sorted into EGFP⁺ and EGFP⁻ populations for (b) fluorescence-activated cell sorting (FACS) analysis of the CD44⁺/CD24⁺ and CD44⁺ populations (n = 5 for EGFP⁺ cells, n = 4 for EGFP⁻ cells; ***p < 0.001, Student’s t test). c Side population (SP) of the secondary xenograft-derived tumor cells. MCF7 tumor cells were isolated from the secondary xenografts derived from EGFP⁺ and EGFP^- tumor cells, respectively, and expanded in vitro for three passages. Cells were stained with Hoechst 33342 for side population analysis by FACS. Verapamil (50 μM) was used to block nuclear export of Hoechst 33342. These results were validated in two independent experiments. d Clonogenic potential of the freshly sorted EGFP⁺ and EGFP⁻ populations from the secondary xenografts (n = 6; ****p < 0.0001, ***p < 0.001, Student’s t test)