Ovariectomized Mice for Breast Cancer

Swiss nu/nu (7 weeks, n = 18) female mice were purchased from Charles River Laboratories-France. Prepubertal mice were ovariectomized under isoflurane anesthesia to remove endogenous ovarian hormone production. Four days before cancer cell injection, mice were implanted s.c. either with a 1.7 mg/60 days MP or with a ME2/60 days RI, or were sham operated (untreated control group). Human adenocarcinoma MCF7 cells were used as previously described [14 (link)]. Tumor cells (1 × 10⁶ cells suspended in 200 μl of Matrigel) were injected s.c. to both flanks of mice. Tumors were measured every 2 to 3 days with digital caliper and tumor volume was calculated as V (mm³) = a x b²× 0.52 (n = 12 tumors per experimental group). At sacrifice, tumors were immediately resected and weighed. The bladder, uterus and kidneys were also dissected, fixed using 4% formalin and embedded in paraffin for histological analysis. Sections were cut at 6 μm, deparaffinised in xylene and rehydrated through graded alcohols, then stained with hematoxylin/eosin. We systematically checked that E2-untreated ovariectomized mice had an atrophied uterus (<10 mg) and that those implanted with an E2-releasing pellet had a significant increase of uterine weight.