2.10. In Vivo Experiments

Animal experiments were approved by the Local Ethics Committee in Krakow in Poland (no. 12/2018 with modifications 208A/2018 and 212/2018). In total, 5 × 10⁶ RH30 cells were injected subcutaneously into 6- to 8-week-old NOD-SCID mice. Each experimental group contained in total eight animals. The experiments were repeated twice, using a group containing four animals for each condition. Tumor size was evaluated using a caliper. Tumor volume was estimated using the formula V = D × d² × 0.5 (where V is the tumor volume, D is the largest dimension, and d is the smallest dimension). After 23 days, the mice were euthanized, and their tumors were harvested. Following the evaluation of tumor weight, the tumor sections were fixed in formalin and stained with hematoxylin-eosin using Dako EnVision Detection Systems (Dako Polska Sp. z o.o., Poland) to visualize tumor morphology. After deparaffinization the tumor sections were stained immunohistochemically with anti-Ki67 primary mouse monoclonal antibody to evaluate tumor proliferation (clone MIB-1; 1: 75, DakoCytomation, Denmark, UK), and anti-CD31 antibody to visualize tumor vascularization (1:50, Abcam, ab28364), as described previously [13].