I4409

The MCF-10A, MCF-7, and MDA-MB-231 cells were suspended in serum-free DMEM/F12 (1:1) culture medium supplemented with B27. One hundred cells per well were seeded in an ultra-low attachment 96-well plate (Corning Inc., Lowell, MA, USA). The breast cancer chemo therapeutic drug registrant characteristic of mammospheres were evaluated by treating fulvestrant (5 µM) (I4409, Sigma-Aldrich, St. Louis, MO, USA tamoxifen (10 µM) (Sigma-Aldrich), and paclitaxel, (50 and 100 µM) (Sigma-Aldrich) with or without E2 (100 pg/mL) on the day of seeding cells. The mammospheres were photomicrographed and analyzed at Days 7 and 10.

Prednisone (P6254, Sigma-Aldrich) was resuspended in DMSO (D4540, Sigma-Aldrich) at 5 mg/mL each week. Daily treatment was 1 mg/kg body weight prednisone in 50 μL PBS given every day via i.p. injection. Vehicle treatment was the equivalent amount of DMSO proportional to body weight in 50 μL PBS. Weekly treatment was 1 day of prednisone treatment (1 mg/kg prednisone in 50 μL PBS) and 6 subsequent days of vehicle treatment (DMSO in 50 μL PBS). Unanesthetized mice were injected daily at 7 am with sterile BD Micro-Fine IV Insulin Syringes (14-829-1A, Thermo Fisher Scientific). Mice were weighed weekly and body weight was used for dosing calculations. Short-term (4-week) treatments consisted of 5 weekly prednisone injections total, with the final injection given 2 days prior to sacrifice. Long-term (38-week) treatments consisted of 39 weekly prednisone injections total, with the final injection given 2 days prior to sacrifice. AR activity was suppressed via the competitive antagonist flutamide (F9397, Sigma-Aldrich), which was administered at 15 mg/kg in 90% corn oil/10% ethanol. ER activity was suppressed via the competitive antagonist fulvestrant (I4409, Sigma-Aldrich), which was administered at 5 mg/kg in 95% corn oil/5% DMSO. Sex steroid receptor antagonists were administered daily as 100-μL i.p. injections.