Figure 3.
NOTCH4 Inhibition using RO4929097 Abrogates Tamoxifen and Fulvestrant Enrichment of CSC Activities
(A–C) Early (HBCx34) and metastatic (Met) (BB3RC31) PDX tumors treated in vivo for 14 days with tamoxifen (10 mg/kg/day, oral gavage) or fulvestrant (200 mg/kg/week, subcutaneous injection) in the presence or absence of the NOTCH4 inhibitor RO4929097 (3 mg/kg/day, oral gavage). (A) MFE (%). (B) Percentage of ALDH-positive cells. (C) Secondary tumor formation. 100,000 cells of metastatic (BB3RC31) PDX were re-implanted subcutaneously in NSG mice with 90-day slow-release estrogen pellets. Tumor growth (>100 mm3) was determined at day 90 after cell injection.
(D and E) MCF-7, T47D, and ZR-75-1 cells were pre-treated in adherence with 10−6 M tamoxifen (red bars) and 10−7 M fulvestrant (blue bars) with RO4929097 (10 μM; hatched bars) or DMSO (filled bars) for 72 hr. (D) MFE and (E) percentage of ALDH-positive cells were assessed after pre-treatments.
(F–H) MCF-7 cells were pre-treated in adherence for 6 days in the presence of RO4929097 (10 μM; hatched bars) or DMSO (filled bars). (F) In vivo experiments were carried out in NSG mice with 90-day slow-release estrogen pellets. Tumor growth (>100 mm3) was assessed at day 60 and is represented as mice positive for growth/mice tested for each cell number tested. ELDA of tumor-initiating cell frequency is shown. (G) Expression of HEY1 and HES1 by real-time PCR was compared to the control. (H) HES1 protein expression levels determined by western blot.
Data are represented as mean ± SEM. p values refer to hatched bars compared to filled control bars. ∗p < 0.05; ∗∗p < 0.01.
See also Figures S3 and S4.