Estradiol

Mice were anesthetized with inhaled isoflurane (2 ± 0.5%) after receiving an analgesic injection (buprenorphine hydrochloride, Buprex) and then ovariectomized bilaterally at 90 ± 1 days of age through a 1 cm dorsal incision that was then closed with surgical clips (Table 1). The day after ovariectomy, mice received a 3 mm pellet containing 0.05 mg estradiol on a 90-day time-release or a placebo (Innovative Research of America, Sarasota, FL, United States). The hormonal dose was chosen from preliminary experiments in which the levels of estradiol in 90-day-old treated animals were 7.2 ± 4.5 pg/ml (mean ± SEM), as determined by radioimmunoassay (RIA) (Architect System, ref #B7K720, Abbot, Germany). These levels are within the range of those observed in cycling animals of the same age at proestrus (20.9 ± 6.0 pg/ml) and estrus (1.8 ± 4.5 pg/ml). The pellets were implanted subcutaneously in the subscapular region using a trocar, following the manufacturer’s instructions, and no inflammation around the implantation area was observed after 3 days. Mice were then housed according to the different types of diet and hormone treatment until the end-point, 90 days later.

All procedures performed were approved by the Institutional Animal Care and Use Committee at Duke University. Cultured BT474, MDA-MB-453, or BT20 breast cancer cells were trypsinized at 80% confluence, washed with PBS, and resuspended in a 1:1 mixture of serum free medium and Matrigel (Corning Inc.). The cells (1–5 × 10⁶) were then orthotopically implanted into the axial mammary fat pads of 6-week old Nu/Nu mice (The Jackson Laboratory). Of note, 48 h prior to the injection of cells, mice receiving BT474 tumors were ovariectomized and implanted with estradiol (0.72 mg/60 days continuous release) treatment pellets (Innovative Research of America) to facilitate tumor initiation. Tumors dimensions were measured three times weekly with calipers, and body weight and behavior were assessed at the time of measurement. Tumor volume was calculated as A × B2 × 0.5, where A is the longer of the perpendicular axes. After reaching 0.5–1 cm³ volume (6–10 weeks depending on cell line), tumors were excised following humane euthanasia, cryopreserved and archived at -80 °C for later use.