All animal studies were performed according to the guidelines and approval of the Animal Care Committee of the University Of Maryland, Baltimore. Female ovariectomized athymic nude mice 4–6 weeks of age were obtained from the National Cancer Institute - Frederick Cancer Research and Development Center (Frederick, MD). The mice were housed in a pathogen-free environment under controlled conditions of light and humidity; received food, and water ad libitum.
The tumor xenografts of MCF-7Ca cells were grown in the mice as previously described (6 (link), 9 (link), 12 (link)-14 (link)). Each mouse received subcutaneous (sc) inoculations in one site per flank with 100μL of cell suspension containing ~ 2.5×107 cells. The mice were injected daily with supplemental Δ4A (100μg/day). Weekly tumor measurements and treatments began when the tumors reached ~ 300 mm3. Mice were assigned to groups for treatment so that there was no statistically significant difference in tumor volume among the groups at the beginning of treatment. Letrozole and Δ4A for injection were prepared using 0.3% HydroxyPropylCellulose (HPC) in 0.9% NaCl solution. Trastuzumab for injection was prepared as 20 mg/ml stock solution in bacteriostatic water for injection which was then diluted in 0.9% NaCl solution to obtain the required concentration. Mice were then injected sc 5 times weekly with the indicated drugs (except trastuzumab was injected intra-peritoneally (ip) twice a week). The doses of trastuzumab (5mg/kg/wk divided in two doses), letrozole (10μg/day) and Δ4A (100μg/day) used are as previously determined and reported (13 (link)).