2.8. MCF-7 Breast Cancer Xenograft Model

All animal experiments were conducted with approval from the national regulatory authority, Direção Geral de Alimentação e Veterinária (DGAV) (ref. 0421/000/000/2017) and the Institutional Ethics Committee (CEFCM 07/2016) and performed according to their guidelines and under the rules of the Federation for Animal Science Associations (FELASA). Five-week-old female NOD scid gamma (NSG) female mice (Charles River Laboratories) were used for this study and maintained under pathogen-free conditions. Four days prior to tumor cell implantation, estrogen pellets (0.36 mg/pellet 90 day release, Innovative Research of America, 0.36 mg/pellet 90-day release, Innovative Research of America, Sarasota, FL, USA, #NE-121) were implanted subcutaneously (s.c.) in the mice dorsal neck region using a sterilized trocar. Xenografts were initiated by s.c. implanting 2 × 10⁶ MCF-7 cells in 100 µL of PBS:matrigel (1:1 ratio, BD Biosciences, #356230) into the mammary fat pad of each tested mouse (2 mammary fat pad injections/mouse) using an established protocol [27]. After fifteen days, mice were weighed and injected intraperitoneally (i.p.) with Dl1.72, Ctr Ab (each at 10 mg/kg of mice body weight (BW), n = 5 mice per each group) or an equal volume of vehicle PBS (n = 6 mice). Treatment was repeated twice a week for 12 weeks. Animals were monitored daily for clinical symptoms and adverse effects. Tumor growth and mouse weight were determined weekly. Tumor volume was estimated by measuring tumor length (L) and width (W) with a digital caliper and by applying the following formula: (π × L × W2)/6. Twelve weeks after the beginning of treatment, mice were sacrificed and their blood, tumor, liver and kidney were collected for analysis.