Establishment of the breast cancer orthotopic injection model

A total of 18 specific pathogen-free BALB/c nude mice (5 weeks old; female; weighing 18–20 g) were provided by the Experimental Animal Center at the West China School of Medicine, Sichuan University (Sichuan, China). All mice were housed singly with environmental conditions maintained at 21±1°C with a relative humidity of 50±10% and 15 air charges/h under a 12-h light cycle (12 h of light followed by 12 h of dark). Sawdust and wood shavings were used as bedding. The mice were randomly divided into two groups with 9 mice in each group. Each group was randomly divided into an experimental subgroup (6 mice) and a control group (3 mice). Mice were allowed free access to food and water, and had a 12 h light/dark cycle. With an implanted 17 β-estradiol (E2) pellet (0.36 mg/mouse; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), digested MCF-7 and MCF-7-MEK5 cells were diluted to 1×10⁷/ml with Hank's balanced salt solution (14025092; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and orthotopically injected under the mammary fat pads of mice (0.1 ml/mouse). The mice were raised in fan-filtered units. When the tumor grew to 100 mm³, the E2 pellets were removed and 160 mg/d/kg cerulenin (BML-G237-0025; Biomol GmbH, Hamburg, Germany) was administered by intraperitoneal injection to mice in the experimental group for 10 consecutive days. All mice were carefully observed throughout the experimental period and should signs consistent with severe suffering have been detected, those mice would have been sacrificed immediately. At 5 days following the last injection, the mice were sacrificed by cervical dislocation and their tumor tissues were extracted.