Estrogen increases cancer stem cells in ERα⁺ cell lines. (A) Average percentage of CD44⁺/CD24⁻/ESA⁺ cells in the ERα⁺ cell line MCF7 following 6-d treatment with either 1 nM 17-β-estradiol (E2) or vehicle (EtOH); n = 5 biological replicates. Data are mean ± SEM. (B) MCF7 tumorsphere formation presented as the average number of spheres per 1,000 cells plated ± SEM; n = 3 biological replicates. Representative phase contrast micrographs of MCF7 spheres are shown. (C) Tumor formation of MCF7 cells pretreated with EtOH and DMSO (vehicles), E2, or E2 and PD173074 injected in limiting dilution into ovariectomized mice. *Nonparametric χ² statistic was used to test the expected vs. observed frequencies of tumor formation at limiting dilution with a level of 0.001 (critical value, 10.83). (D) Representative H&E-stained sections of mammary glands injected with EtOH-pretreated or 1 nM estrogen (E2)-pretreated MCF7 cells are shown.

Paracrine factors produced in response to estrogen expand ERα⁻ breast CSCs. (A) Immunofluorescence of sorted cytospun MCF7 cells for ERα (green) and ESA/EpCAM (red) expression, counterstained for nuclei with DAPI (blue). (B) Cytometric plots of ERα expression in CD44⁺/CD24⁺/ESA⁺ cells (red, bulk), and in CD44⁺/CD24⁻/ESA⁺ stem-like cells (green), which comprise 2% of the culture. (C) Average percentage of CD44⁺/CD24⁻/ESA⁺ cells in ERα⁻ SUM149, SUM159, and BT20 cultures following treatment with conditioned media from either ethanol (EtOH) or E2-pretreated MCF7. MCF7 cells are shown for reference, *P < 0.0001, n = 4 biological replicates. Data are mean ± SEM. (D) Tumorsphere-forming potential of SUM149 or SUM159 cultures described in C; n = 4 biological replicates. Data are mean ± SEM.

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.

FGF/Tbx3 signaling is intact human breast cancer cells. (A) Western blot of Tbx3 in MCF7 cultures treated with vehicle (EtOH+DMSO), 1 nM E2, 100 ng/mL FGF9, 100 nM 4OHT, or FGFR inhibitor 10 μM PD173074. (B) Quantitative RT- PCR of Tbx3 expression in the same MCF7 cells assayed in A. Data are represented as average delta (deltaCt) ± SEM; n = 4 experiments. (C) Immunofluorescence of MCF7 cells treated with 1 nM E2 or EtOH vehicle; ESA/EpCAM (green), Tbx3 (red), and DAPI (blue) show nuclear localization of Tbx3. Quantification is shown below. (D) Quantitative RT-PCR analysis of DUSP6 and Tbx3 expression in SUM149, SUM159, and BT-20 cultures treated with FGF9 or PD173074 relative to expression in cultures treated with DMSO. Data are represented as average delta (deltaCt) ± SEM; n = 4 biological replicates. (E) Western blot analysis of Tbx3 expression in SUM149 and SUM159 cells described in D.

Tbx3 is necessary and sufficient for breast CSC expansion. (A) (Left) Average percentage of CD44⁺/CD24⁻/ESA⁺ cells in MCF7 cultures transduced with lentiviruses encoding short hairpins targeting a scrambled sequence (Cntrl), GFP, or Tbx3 and treated with 1 nM 17-β-estradiol (E2) or vehicle (EtOH). *P < 0.0015. (Right) Average percentage of CD44⁺/CD24⁻/ESA⁺ cells in SUM149 and SUM159 cultures transduced with lentiviruses encoding short hairpins targeting Tbx3 and treated with recombinant FGF9. Data are mean ± SEM; n = 4 biological replicates. *P < 0.003; **P < 0.007. (B) Normalized tumorsphere-forming potential of SUM159, SUM149, or MCF7 cultures transduced with hairpins targeting a scramble sequence (Cntrl) or Tbx3. Data as mean ± SEM; n = 4 experiments. *P < 0.001; **P < 0.005; ***P < 0.02. (C) Normalized tumorsphere formation of breast cancer cells isolated from a primary human breast cancer (TUM177) transduced with lentiviruses containing two different short hairpin sequences targeting Tbx3. *P < 0.002; **P < 0.0008. (D) Normalized sphere-forming ability of immortalized human mammary epithelia cells (HMEC) or MCF7 cells ectopically overexpressing human Tbx3; n = 4 experiments, 2 biological replicates. *P = 0.002; **P = 0.003. (E) Tumor formation of MCF7 cells overexpressing Tbx3 or empty vector (EV) injected in limiting dilution into NOD/SCID mice. *Nonparametric χ² statistic was used as described in Fig. 1.