Intra-tibial inoculation of mice with breast cancer cells

Intra-tibial inoculation of mice with 4T1 BC cells was conducted using a procedure described previously (20). After an acclimation period of 3 weeks and feeding period of 16 weeks, 4T1 metastatic mammary gland tumor cells (1 × 10³) expressing luciferase were transplanted intratibially. Therefore, our study of metastatic progression began in animals at 23 weeks (5.75 months) of age. Tumor progression was monitored by BLI every 4 days. The mice remained on their treatment diets for an additional 23 days postinjection prior to sacrifice.