Human breast cancer xenograft mice

Five-week-old female athymic nude mice (NCRNU-F, Taconic Farms, Inc.) were used for the in vivo study. The animal research protocol was approved by and performed per the policies and guidelines of the University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee (Institute IACUC Protocol #100861–14-025-SSH). Mice were kept in a conventional animal facility, with a 12-h on-off light cycle in standard rectangular mouse cages, with 5 mice per cage. Cages were lined with adsorbent bedding and enriched with crinkle paper for nesting. Mice had continuous access to water and food. One week prior to breast cancer cell inoculation the mice were randomly divided into two groups, a standard diet consisting of 0.45% n-3 fatty acids (PicoLab Rodent Diet-20 #5053, LabDiet), a fish oil diet consisting of 7.5% n-3 fatty acids (Envigo TD.110647), and fed with the assigned diet during the whole duration of the experiment. Three days before cell inoculation the mice were supplemented with 17β estradiol in the form of a 0.36 mg/pellet, 60-day release (SE-121, Innovative Research of America) implanted subcutaneously in the intrascapular region. The MCF7 miR-23b and miR-27b knockout or control cells were inoculated at a density of 5 × 10⁶ in 100 μl PBS containing 5 μg/ml Matrigel (BD Biosciences) subcutaneously into the mammary fat pad (n = 6 mice per group). Mouse weight and tumor size measurements were initiated 7 days post inoculation and monitored three times per week. Tumor measurements were taken using digital calipers. Tumor volume (v) was measured using the following formula: v = (l × w²) × 0.5, where l is the length and w is the width of the tumor measured in mm. Tumor weight (W, in grams) was estimated by the following formula: W (g) = v (mm³) × 0.001. When the estimated tumor weight reached or exceeded 10% of the body weight, the mouse was euthanized by CO₂ asphyxiation. No adverse events were observed. Tumor tissues were dissected, fixed in 10% buffered formalin phosphate, followed by paraffin embedding, sectioning, hematoxylin, and eosin (H/E) staining. Survival analysis was conducted in GraphPad Prism (version 7). The date of death was entered for mice within each group over the entire study period. The survival curves were compared using the Log-rank test (Mantel-Cox).