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Article History

Received: 13 November 2025

Accepted: 27 March 2026

First Online: 10 April 2026

Declarations

:

: The authors declare no competing interests.

: To establish the 4T1-induced breast cancer model, it was confirmed that all animal care and experimental procedures conducted in this study adhered to the ethical guidelines approved by Urmia University of Medical Sciences, in alignment with ARRIVE guidelines (ARRIVE 2.0) 61 . All authors acknowledge the ethical considerations relevant to this manuscript. Ethical approval for this research was obtained from the Ethics Committee of Urmia University of Medical Sciences under approval code [IR.UMSU.AEC.1401.004]. Mice were maintained under standard housing conditions with a controlled temperature of 22°C, following a 12-h light/dark cycle. Food and water were available at all times, and appropriate air conditioning was implemented to minimize potential harm or distress. All necessary measures and precautions were employed to ensure the well-being of the animals. At the conclusion of the experiment, in accordance with IACUC guidelines, all euthanasia procedures for the mice were conducted using carbon dioxide (CO2) gas at a flow rate of 3 L/min until respiration ceased 62 . Prior to this procedure, the animal received anesthesia to minimize distress, which involved administering a mixture of ketamine (80 mg/kg) and xylazine (5 mg/kg) via intraperitoneal injection. Euthanasia was subsequently performed using CO₂ asphyxiation, followed by the cervical dislocation method, which involved placing a pen directly behind the ears at the base of the skull and, while lifting the base of the tail, dislocating the cervical vertebrae. In this study, female BALB/c mice, weighing between 21 and 23 g and aged 6–8 weeks, were obtained from the Pasteur Institute in Tehran, Iran. Upon reaching the logarithmic growth phase, 4T1 cell lines were trypsinized (using Gibco), resuspended, and a suspension containing 1 × 10 5 4T1 cells in 100 μL of sterile phosphate-buffered saline (PBS) was injected into the mammary fat pad of the mice. Eight days post-implantation, the mice were evaluated for tumor growth and were randomly assigned to treatment and control groups. The experimental groups consisted of eight categories: Control or Untreated, Alp (Alpelisib treated), TRPM8 ant (TRPM8 antagonist treated), CB1 ant (CB1 antagonist treated), Alp/CB1 ant (Alpelisib & CB1 antagonist treated), TRPM8/CB1 ant (TRPM8 & CB1 antagonist treated), Alp/TRPM8 ant (Alpelisib & TRPM8 antagonist treated), and Alp/CB1/TRPM8 ant (Alpelisib & CB1 & TRPM8 antagonist treated), with n = 5 mice in each group. In total, n = 50 mice were utilized for tumor induction, resulting in n = 4–5 mice in each experimental group. Mice that did not develop a tumor mass or exhibited a tumor mass significantly larger than the others by day 8 of induction were excluded from the study. Additionally, 4–5 mice were randomly allocated to each cage (Large cage Size (W × L × H), 7.5″ × 11.75″ × 5″) for each experimental group.