Mammary tumor induction

Mammary tumors were induced in F1 an F2 female offspring by administration of medroxyprogesterone acetate (MPA; 15 mg/100 µl, subcutaneously) to 6 weeks of age female offspring, followed by 3 weekly doses of 1 mg 7,12-dimethylbenz[a]anthracene (DMBA; Sigma, St. Louis, MO) dissolved in peanut oil by oral gavage⁴³. Tumors were detected by palpation once per week, starting at week 2 after the last dose of DMBA. Tumor growth was measured using a caliper, and the width and height of each tumor were recorded.

In the F1 generation, mammary tumors were harvested when reaching approximately 40 mm² in volume and used for mammary tumor transplantation surgery, as described in the next section. In the F2 generation, tumor development was monitored for a total of 20 weeks post-DMBA administrations. Animals in which tumor burden reached approximated 10% of total body weight were euthanized before the end of the monitoring period, as required by our institution. Tumor growth was analyzed using two-way ANOVA (group and time), followed by post-hoc analyses. Kaplan–Meier survival curves were used to compare differences in tumor incidence, followed by the log-rank test. Differences in tumor latency and mortality were analyzed using two-way ANOVA.

All DMBA-induced tumors in F1 and F2 females were blindingly evaluated by a consultant pathologist, Dr. Susana Galli. Only mammary carcinomas were included in the final analysis or used in mammary tumor transplantation below.