2.13. Animal Study

Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Debrecen and the National Board for Animal Experimentation (1/2015/DEMÁB) and were performed according to the NIH guidelines (Guide for the care and use of laboratory animals) and applicable national laws. Animal studies are reported in compliance with the ARRIVE guidelines.

Experimental animals were BALB/c female mice (14–16 weeks of age, 20–25 g). Animals were randomized for all experiments. Mice were bred in the “specific pathogen-free” zone of the Animal Facility at the University of Debrecen and kept in the “minimal disease” zone during the experiments. No more than 5 mice were housed in each cage (standard block shape 365 × 207 × 140 mm, surface 530 cm²; 1284 L Eurostandard Type II. L from Techniplast). Cages were changed once a week on the same day. Animals had paper tubes to enrich their environment. The dark/light cycle was 12 h, and temperature was 22 ± 1 °C. Mice had ad libitum access to food and water (sterilized tap water). The animal facility was overseen by a veterinarian. A total of 20 female mice were used in the study: 10 randomly selected control and 10 IPA-fed mice.

4T1 cells were suspended (2 × 10⁶/mL) in ice-cold PBS-Matrigel (1:1, Sigma-Aldrich) at a 1:1 ratio. Twenty female BALB/c mice received 50 µL injections to their second inguinal fat pads on both sides (10⁵ cells/injection site). Tumor growth and animal wellbeing was controlled daily.

IPA treatment was administered by oral gavage at the dose of 1 nmol/g (0.18921 mg/kg) body weight once a day on each day of the experiment. The dose was planned not to exceed the serum reference concentration of IPA [40,49,50]. Animals received single daily oral IPA treatment. IPA stock was prepared in sterilized tap water at 100 × concentration (15 mM) for storage at −20 °C. IPA stock was diluted each day to a working concentration of 150 µM in sterile tap water before treatment. Animals received a daily oral dose of 100 µL/30 g bodyweight from the IPA solution (10 mice) or vehicle (sterilized tap water, 10 mice). Researchers involved in IPA and vehicle solution administration were blinded. Treatment was administered every day at the same time between 9:00 and 11:00. Animals were sacrificed on day 14 post grafting by cervical dislocation; then, primary tumors and metastases were collected for subsequent analysis.

Upon autopsy, primary tumors were visually evaluated and scored based on their infiltration rate into surrounding tissues and the macroscopic appearance of the tumor [27,28]. Tumors were classified as a “low infiltration” class if the primary tumor remained in the mammary fat pads without any attachment to muscle. A “medium infiltration” tumor means that the tumor mass attached to the muscle tissue, but it did not penetrate to the abdominal wall. In case the tumor grew into the muscle tissue and totally penetrated the abdominal wall, it was scored as a “high infiltration” tumor. For the assessment of primary tumor infiltration rate, the researchers were blinded. Both primary and metastatic tumor masses were removed from the animals and were measured on analytical balance in preweighed Eppendorf tubes. From the sections of HE-stained, formalin-fixed, paraffin-embedded tumor tissues tumor-infiltrating lymphocyte (TIL) content was determined as the number of TILs per 100 tumor cells.