2.1. Animal Experiment

We used the wild type (WT) Her2/neu [FVB/N-Tg (MMTVneu)202Mul], a spontaneous mammary tumor mouse model, in this study. The female mice of this model can develop focal hyperplastic and dysplastic mammary tumors with the expression of the WT Her2/neu gene, which has also been deeply implicated in human breast cancer genesis. The Her2/neu mice develop ER-negative mammary tumors at early age (~20 weeks) (median latency is around 30 weeks)[15]. The breeder mice (4 weeks of age) were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred from 10 weeks of age to obtain adequate colonies for follow-up experiments. A standard PCR analysis with tail DNA of mice at 3 weeks of age was used to identify the Tag genotypes according to a previous study[16]. All the mice were housed in the Animal Resource Facility of the University of Alabama at Birmingham and were maintained under the following conditions: 12 h dark/12 h light cycle, 24 ± 2°C temperatures, and 50 ± 10% humidity. We used an online Power and Sample Size Calculator (http://powerandsamplesize.com/) to determine the power and sample size by 2-proportion comparison[17].

A customized BSp diet (TestDiet, St. Louis, MO) was prepared with AIN-93G diet base and adjusted for macronutrient content as used previously[17]. The modified AIN-93G contained 26% (w/w) BSp, which was obtained from Natural Sprout Company (Springfield, MO). Detailed dietary ingredients and nutritional profile are provided in Supplementary data (Fig. S1). To provide maximum protection against possible changes, diets were stored in airtight containers and were kept away from light under refrigeration (2°C for up to six months and −20°C for long term life).

Her2/neu female mice (10 mice) were fed the BSp diet from weaning (3 weeks of age) to 10 weeks of age. The treated female mice were then mated with nontreated Her2/neu male mice and were assumed to be pregnant when a vaginal plug was expelled. Pregnant mice were treated with control AIN-93G diet from their conception throughout gestation and the lactation period. The offspring were weaned at postnatal 21 days of age (PD21). 21 female offspring were selected and randomly assigned into 3 cages. These mice were maintained with control AIN-93G diet until the termination of the experiment. Mammary tumor observation and tissue samples collection were only performed with the female offspring; (Fig. 1)

The upper bar represents different life stages in the maternal and female offspring lifecycle. Transgenic mice (Her2/neu) were administered the BSp diet under different exposure time points: (i) control: female mice were fed ad libitum with the control AIN-93G diet throughout the study; (ii) pregestational maternal BSp treatment (P-BSp): The BSp diet was given to the mother from postnatal 3 weeks of age until mating (10 wks); (iii) long-term maternal BSp treatment (L-BSp): the BSp diet was given to the mother from postnatal 3 weeks of age until the weaning of their offspring (PD21); (iv) postnatal adult BSp treatment (A-BSp): The BSp diet was given to female offspring from 10 weeks of age until the termination of the experiment. Mice were mated at 10 weeks of age followed by 3 weeks of gestation. Offspring were weaned at 3 weeks of age. Two age-matched controls were applied in this study. The long-term control (L-Control) was used in the P-BSp and the L-BSp treatments. The adult control (A-Control) was used as the control of the A-BSp group. In the P-BSp and L-BSp groups, weaned female offspring were maintained on the control AIN-93G diet until the termination of the experiment and monitored for tumor growth weekly. For the A-BSp treatment, female mice were born to a mother administered with the control AIN-93G diet and monitored for tumor growth weekly after weaning. C, conception or gestation day 0; B, birth; PD21, postnatal day 21; 10 wks, onset of adulthood; T, termination of the experiment. The experiments were terminated when all of the control mice developed tumors and had an average tumor diameter exceeding 1.0 cm.

Her2/neu female mice (10 mice) were fed the BSp diet from weaning (3 weeks of age). The treated female mice were mated with nontreated Her2/neu male mice at 10 weeks of age and were assumed to be pregnant when a vaginal plug was expelled. Pregnant mice were sequentially treated with the BSp diet throughout their gestation and lactation periods. Offspring were weaned at postnatal 21 days of age (PD21). 21 female offspring were selected and randomly assigned into 3 cages. These mice were maintained on the control AIN-93G diet until the end of the experiment. Breast tumor observation and tissue samples collection were only performed with the female offspring; (Fig. 1)

Her2/neu female mice (14 mice) were treated with the BSp diet from 10 weeks of age which was continued until termination; (Fig. 1)

Her2/neu female mice were fed ad libitum with the control AIN-93G diet throughout the study. Two age-matched controls were applied in this study. The long-term control (L-Control) was used in the P-BSp and the L-BSp treatments. The adult control (A-Control) was used as the control of the A-BSp group.

The Her2/neu mouse model can develop mammary tumors at early age (around 20 weeks of age). Tumor size and latency were measured and recorded weekly. Tumor volume was determined by the following formula: tumor volume (cm³) = 0.523 × [length (cm) × width² (cm²)][18]. The experiments were terminated when all of the control mice developed tumors and had an average tumor diameter exceeding 1.0 cm. The mammary tumors were then excised, weighed and preserved in liquid nitrogen for follow-up analysis. Animal procedures were reviewed and approved by the UAB Institutional Animal Care and Use Committee (IACUC; Animal Project Numbers: 10088 and 20653). All mouse-related operations were carried out in accordance with the guidelines of the IACUC at UAB.