Tumor Xenograft Study.

The MCF7 cells harboring either gAAVS1 or gCSK infected with lentiviruses encoding a firefly luciferase (68) were injected s.c. into the left and right posterior flanks of 5-wk-old, ovariectomized, nude female mice (Charles River Laboratories) in the presence of estrogen. Mice were assigned randomly (day 7) to continued estrogen supplementation [E2, 0.1 mg/kg of 17 β-estradiol valerate (Sigma) once a week] or estrogen withdrawal (−E2). Bioluminescence (BIL) signal was measured once a week, and tumor volumes were calculated by volume = width² × length/2. When the tumor reached ∼150–200 mm³, the mice were randomized for drug treatments [Veh (10% PEG400/Tween-80/PVP-K30 at a ratio of 90:5:5, 15% d-α-tocopheryl polyethylene glycol succinate (vitamin E-TPGS), and 75% hydroxypropylcellulose [0.5%] in 50 mM citrate buffer [pH 3.0]), fulvestrant (5 mg⋅wk⁻¹, s.c. [formulated in arachis oil]), and FRAX597 (60 mg/kg, p.o.)]. FRAX597 was formulated in 10% PEG400/Tween-80/PVP-K30 at a ratio of 90:5:5, 15% vitamin E-TPGS, and 75% hydroxypropylcellulose (0.5%) in 50 mM citrate buffer (pH 3.0), and administered by oral gavage. The fold change in BLI signal was normalized to the first measurement (percentage relative BIL) when treatments were initiated for each tumor. The duration of treatment was 28 d for all of the tumor xenografts. The efficacy of FRAX597 and fulvestrant was also assessed in a PDX model of breast cancer that may better represent the response in patients. Nonobese diabetic/SCID gamma mice bearing tumor fragments of the TM00386 PDX breast cancer tumor were sourced from The Jackson Laboratory. The TM00386 PDX model was derived from an ER⁺/PR⁺/HER2⁻ invasive ductal carcinoma. When the tumor reached ∼150–200 mm³ (80 d postimplantation), mice were randomized into treatment groups (n = 8 mice per group) and administered Veh [10% PEG400/Tween-80/PVP-K30 at a ratio of 90:5:5, 15% vitamin E-TPGS, and 75% hydroxypropylcellulose (0.5%) in 50 mM citrate buffer (pH 3.0)], fulvestrant (5 mg⋅wk⁻¹, s.c.), and/or FRAX597 (60 mg/kg, p.o.). FRAX597 was formulated in 10% PEG400/Tween-80/PVP-K30 at a ratio of 90:5:5, 15% vitamin E-TPGS, and 75% of hydroxypropylcellulose (0.5%) in 50 mM citrate buffer (pH 3.0). Tumor size and body weight were measured twice per week throughout the duration of the study (35 d). All mouse experiments were carried out at the Dana–Farber Cancer Institute Lurie Family Imaging Center. The animal experiments were carried out under the Lurie Center Institutional Animal Care and Use Committee (IACUC) protocol and were in accordance with the IACUC standards for the welfare of animals.