Female immunodeficient nude mice

To establish the BCSC-induced tumor model, female immunodeficient nude mice (Charles River Laboratory, Yokohama, Japan) were used as described elsewhere.50 (link),51 (link) β-Estradiol 17-cypionate was subcutaneously injected into each mouse at a dose of 3 mg/kg once a week as a supplement for BCSC tumor growth, because the growth of MCF-7 tumors depends on the estrogen concentration in the body. Each group of mice was treated with DPBS (control, n=3) or various formulations of GEM (free GEM, L-GEM, or HA-L-GEM, 25 mg/kg, n=6) twice a week for 3 weeks. The size of the tumors was measured using a vernier caliper (Mitutoyo Co., Kanagawa, Japan), and the animals were weighed twice weekly. Tumor volume was calculated using the following equation:
$\text{Tumor volume}({\text{mm}}^3)=\frac{1}{2}(\text{Length}\times {\text{Width}}^2)$