2.8. Tumor Induction

After extracting ascitic fluid from mice infected with Ehrlich ascites carcinoma (EAC), it was diluted in normal saline to create a suspension, with each 0.1 mL containing 2.5 × 10⁶ of EAC cells [27]. Viability of the cells was confirmed using a Trypan blue dye exclusion experiment [28]. On the first day of the experiment, 0.2 mL of the Ehrlich’s ascites carcinoma suspension was subcutaneously injected into the mammary fat pad on the upper left ventral side of mice. Tumor growth was confirmed ten days post-injection using a caliper, and tumor volume was calculated using the formula: Tumor Volume = length × width 2 × 0.52. Treatment commenced once solid tumors (50–100 mm³) became visibly evident, ten days post-inoculation.