Xenograft Model of Breast Cancer

All animal studies were performed according to the guidelines and approval of the Animal Care Committee of the University Of Maryland, Baltimore. Female ovariectomized BALB/c 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 and received food and water ad libitum.The tumor xenografts of MCF-7Ca cells were grown in the mice as previously described (1 (link), 16 (link), 17 (link)). Each mouse received subcutaneous inoculations in one site per flank with 100μL of cell suspension containing ~ 2.5×10⁷ cells. The mice were injected daily with 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 in 0.3% HydroxyPropylCellulose (HPC). Trastuzumab for injection was prepared as 20 mg/ml stock solution in bacteriostatic water for injection and then diluted in 0.9% NaCl solution to obtain the required concentrations. Mice were then injected subcutaneously (sc) 5 times weekly with the indicated drugs: 100μg/mouse/day of Δ⁴A plus 10μg/mouse/day of letrozole or 10μg/mouse/day of letrozole plus trastuzumab or 100μg/mouse/day of Δ⁴A plus trastuzumab for indicated time. The doses of letrozole and Δ⁴A used are as previously determined and reported (1 (link), 2 (link)). Mice in the trastuzumab group received 5mg/kg/week of the drug intra-peritoneally (ip) divided in two doses. Mice in the Δ⁴A and trastuzumab group were treated for 7 weeks after which they were sacrificed due to large tumor volumes; by decapitation and the blood was collected for analysis. The other groups (letrozole, trastuzumab plus letrozole, letrozole switched to trastuzumab plus letrozole and letrozole switched to trastuzumab) were sacrificed on week 28.