2.8. Xenograft mouse models

Empty vector or miR‐126/126*‐transduced A375M cells were subcutaneously injected into the flanks of 5‐week‐old female nude mice (minimum n = 5 mice per group; Harlan, Udine, Italy) at a dose of 10⁶ cells mixed 1 : 1 with Matrigel (BD Biosciences) in 200 μL PBS. Treatments were initiated when xenografted tumors reached 30–60 mm³ and were administered daily intraperitoneally. Mice were randomly allocated to four treatment arms: PIK‐75 (2 mg·kg⁻¹ DMSO : H₂O), vemurafenib (20 mg·kg⁻¹ DMSO : PBS), the combination (2 mg·kg⁻¹ PIK‐75 plus 20 mg·kg⁻¹ vemurafenib), and DMSO : H₂O : PBS (control). Since PIK‐75 and vemurafenib are reversible inhibitors, they were administered five times per week to maintain stable inhibition. Tumor growth was monitored twice a week and diameters measured with calipers. Volumes were calculated using the following formula: volume (mm³) = (width)² × length/2, where length represents the largest tumor diameter and width the perpendicular. Percentage of tumor growth inhibition (TGI) was calculated as the difference between the median tumor volume (MTV) of a treated group and the control group, using the following formula: %TGI = [(MTV_control − MTV_treated)/MTV_control] × 100. During the experiments, doxycycline was administered to the mice by daily oral gavage (10 mg·mL⁻¹; Sigma‐Aldrich). Relative tumor growths were calculated by normalizing over initial tumor volume. Animal experiments were performed at the Istituto Superiore di Sanità (Rome, Italy) according to Italian law and institutional guidelines.