Fig. 3.

FGFR signaling is necessary for estrogen CSC expansion. (A) Average percentage of CD44⁺/CD24⁻/ESA⁺ cells in MCF7 cultures treated with 1 nM 17-β-estradiol (E2) or E2-conditioned medium in the presence of the FGFR inhibitor PD173074. n = 6 Biological replicates for fresh media, *P < 0.0001; n = 4 biological replicates for conditioned media, **P < 0.005, Data are mean ± SEM. (B) MCF7 cells pretreated E2, FGF9 (100 ng/mL), or E2 and PD173074 were seeded for tumorspheres and resulting spheres, Data are mean ± SEM, n = 4 biological replicates. *P = 0.01 either E2 or FGF9 vs. EtOH. (C) Sphere formation of estrogen-pretreated MCF7 cultures transduced with indicated small hairpins. (D) Flow-cytometric analysis of CD44⁺/CD24⁻/ESA⁺ cells in ERα⁻ SUM149, SUM159, BT20 cultures following treatment with either recombinant human FGF9 or the FGFR inhibitor, PD173074. MCF7 cells treated with E2 are shown as reference. Data are mean ± SEM; n = 4 biological replicates. *P < 0.004 FGF9 vs. vehicle; **P < 0.0005 PD vs. vehicle. (E) Tumor formation of 10⁴ SUM159 cells pretreated with DMSO, FGF9, or PD173074 injected orthotopically into mice; n = 12 for each treatment. *P < 0.02 DMSO vs. PD. Data are mean ± SEM. (F) Tumorsphere formation of breast cancer cells isolated from a primary human breast cancer (TUM177) treated with FGF9 or the FGFR inhibitor PD173074, *P = 0.01 DMSO vs. PD. Data are mean ± SEM.