Fulvestrant

Female 5–6-week-old NRG mice (Jackson labs stock #007799) were implanted with HCI-011 patient-derived xenograft (PDX) tumor fragments using a previously described protocol with estrogen supplementation (42 (link)). When tumors reached approximately 100 mm³, mice were randomized into treatment or control groups. Mice received either vehicle control (15% captisol IP, 3×/week for 5 weeks), birinapant (20 mg/kg IP, 3×/week for 5 weeks), fulvestrant (200 mg/kg subQ, 1×/week for 5 weeks), or the combination of birinapant and fulvestrant at the same doses as single agents. For birinapant treatment, birinapant (Medchem Express, HY-16591) was dissolved in 15% captisol at a concentration of 2 mg/mL (made fresh every time), and 100 μL/10 g mouse body weight was injected intraperitoneally. For fulvestrant treatment, 250 mg of fulvestrant (Selleck Chemicals, S1191) was dissolved in 0.5 mL DMSO, incubated at 37°C for 15 minutes and then diluted 1:20 with corn oil to give a final concentration of fulvestrant of 25 mg/mL (made fresh every time). 80 μL/10 g mouse body weight was injected subcutaneously. Animal experiments were all conducted in compliance with institutional guidelines and regulations after approval from the University of Utah Institutional Animal Care and Use Committee, under protocol 21-06008.

The MCF7 cell line (ATCC HTB-22) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) with 10 % fetal bovine serum (FBS, Gibco Life Technologies) and penicillin streptomycin (Solarbio). Before treatment, cells were transferred from 10-cm plates to 6-well plates and were serum-starved for 24 h with DMEM without phenol red (Gibco Life Technologies). Treatments included β-estradiol (E2, Sigma-Aldrich), insulin-like growth factor-1 (IGF-1, Genescript) and insulin (Solarbio) in the same medium for 24 h or 15 min. Anti-estrogen drugs include tamoxifen and doxorubicin from Thermo, as well as raloxifene and fulvestrant from Selleckchem. In addition, chemical inhibitors included U0126-EtOH (MEK inhibitor), fulvestrant (ICI 182,780, estrogen receptor-α inhibitor) and MK2206 2HCl (AKT inhibitor), and SP00125 (JNK Inhibitor) and OSI-906 (IGF-1 receptor inhibitor), which were also purchased from Selleckchem, while monensin was from Beyotime.