Female athymic nude mice (Foxn1nu) (The Jackson Laboratory, Bar Harbor, ME) at 5 to 7 weeks of age were used for the xenograft studies. E2 pellets (Innovative Research of America, Sarasota, FL) were implanted 7 days before cancer cell injection to augment the endogenous E2 production. The cultured MCF-7 cells in the logarithmic phase were collected and diluted to 3 × 107 cells/ml. Then, the cancer cell suspension (in Matrigel) was inoculated into the second mammary fat pad on the right side of mice (100 μl per injection). Approximately 14 days after inoculation, the animals were randomly assigned to treatment groups including vehicle only controls, poly(lactic-co-glycolic acid) nanoparticles of C23 (3 mg/kg, intraperitoneally) (18), Dox (1.70 mg/kg, intraperitoneally, five times per week every 2 weeks), or C23 + Dox (n = 10). Electronic calipers were used to determine the length and width of each tumor at intervals after initiation of Dox administration. The following equation was used to calculate tumor volumes: volume = 0.5(length × width2). At the end of the experiment, the animals were sacrificed and the tumors were harvested. The fresh tumor tissues were instantaneously placed in 4% paraformaldehyde for further IHC analysis.

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