In vivo tumorigenesis assay

A murine B16F10 (H2b) variant of the B16 melanoma cell line originated from C57Bl6 mice was used in the experiments. Cell viability was determined by the Trypan Blue exclusion test, which showed that >95% of the cells were viable. B16F10 cells (8×10⁵/100 µL) were suspended in R10 and injected subcutaneously into flank regions of mice to induce tumor formation. To assess the effects of treatments on early steps of melanoma development, 3 days after injection of cells, the animals were treated with saline, DMSO (AcE vehicle, 2%), LNC (1×10¹² particles/day), AcE (50 mg/kg/day), AcE-LNC (50 mg/kg/day of AcE; 1×10¹² particles/day), or CCT (30 µL plus 220 µL of saline), according to each experimental goal, by intraperitoneal or oral route, and treatments were continued for 7 days. Tumor size was measured daily using a caliper-like instrument and converted to tumor volume by the equation: volume = (the shortest diameter)2 × (the longest diameter) ×0.5, and expressed in cubic millimeters.43,44 Additionally, the weight of animals and food intake were measured daily during all treatments.