Mouse 4T1 tumor model

0.1ml DMEM containing 10⁶ 4T1 cells/μL was injected into the right flank of each mouse. Bodyweight and tumor volume of all animals were measured every other day from beginning until the end of the study. Tumor volume of each mice was quantified using a digital caliper with the bellow equation.

The sample size of each group is also computed according to the following formula²¹ in which “E” is the degree of freedom of analysis of variance (ANOVA).

E = Total number of animals − Total number of groups.

After tumor induction, the animals were randomly grouped into four groups, each contains four mice. Following by tumor volume reached to the following sizes; 60-100 mm³ (early stage), 100-300 mm³ (intermediate stage), 300-500 mm³ (advanced stage), 500-1000 mm³ (end-stage), they were removed by surgery and divided into two parts. One of them kept into RNA latter for later molecular analysis and the second part preserved in formalin for further pathological analysis in order to keep safe without any changing.