Nod scid gamma female mice

NOD-SCID gamma (NSG) female mice were obtained from Jackson Labs. Mice were 8 weeks of age upon the start of all experiments. Mice were injected intraperitoneally (IP) with atorvastatin at 2 mg/kg or 10 mg/kg, EdU at 10 mg/kg, or the 2% DMSO vehicle at a volume of 10 μL/mg body weight. Mice were killed by asphyxiation in a chamber with adequate CO₂ flow as per AVMA procedures.

NOD scid gamma (NSG) female mice at age of 6–8 weeks were obtained from Jackson Laboratory (USA) and maintained in pressurized ventilated caging. To sustain tumor growth in MCF-7 models, 17β-estradiol pellets (0.18 mg) were implanted subcutaneously 3 days before BC cells injection. For both MCF-7 and MDA-MB-231 models, cancer cells were injected in the mammary fat pads (MFPs) of mice. For each experimental sample, cell suspensions were mixed with an equal volume of Matrigel (BD Biosciences, USA). For the MCF-7 model, injectable fulvestrant (Faslodex®, AstraZeneca, UK) was given intramuscularly in the tibialis posterior/popliteal muscles (200 mg/kg injection, once a week) for 20 days. Control mice received isotype control (placebo) or PBS injection. Tumor volumes were measured with vernier calipers starting from 14 days after cell implantation. Animals were sacrificed as they reached an experimental endpoint. All procedures and experiments were completed in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees at MSKCC (MSKCC#12-10-016).

All animal experiments were approved by the Institutional Animal Protection Committee (CIPA) of the Centre de Recherche de Centre hospitalier de l’Université de Montréal (CRCHUM) under protocol C17017SHs. Either 5 million MDAMB231 or 2 million MX1/HCC1806 cells were resuspended in 50% Matrigel Matrix Phenol Red Free (#CB40234C, Fisher Scientific), 25% PBS and 25% collagen type 1 (#08-115, Millipore Sigma) solution [33 (link)]. Using 7-week-old NOD-SCID gamma (NSG) female mice (#005557, Jackson Laboratory), 0.2 mL cell suspension was surgically implanted. Once tumours reached an average volume of 150 mm³, mice were randomised into treatment groups based on tumour volume volumes and weight. Our sample size was based on the experiments previously described [34 (link)]. Except for the control group for MDAMB231, which had 14 mice, each treatment group consisted of 8–10 mice. See Supplementary Methods for further details.

PDX samples (HCI-002, HCI-009 and HCI-010) were generated and published by Dr Alana Welm and colleagues at the University of Utah following local institutional review and patient consent. Briefly, donated primary breast tumors and metastatic breast cancer cells were freshly obtained following surgery and transplanted into cleared mammary fat pads of female immunocompromised NOD/SCID mice^{38 (link)}. For this study, we obtained 4-week-old immunocompromised NOD/SCID/gamma female mice purchased from Jackson Laboratory. The viably frozen HCI-002, HCI-009, and HCI-010 tumor samples were transplanted into the cleared inguinal 4R mammary fat pads of NOD/SCID/gamma mice. Tumor growth was monitored daily by caliper measurement in two dimensions. When tumors reached 2 cm in any dimension (HCI-002—after 8 weeks on average, HCI-009 and HCI-010—12 weeks), mice were killed, and tumor and NAT isolated from the 4R gland, and NCT from the 4L gland, and flash-frozen in liquid nitrogen. The protocols described in this section regarding animal studies were approved by the UCSF Institutional Animal Care and Use Committee.

Female NOD scid gamma (NSG) mice, 6–8 weeks (Jackson Laboratory), were housed in a pathogen-free room. All experiments involving mice were carried out with an approved protocol by the University of North Carolina Animal Care and Use Committee. In vivo pharmacokinetic (PK) studies were carried out with 3 single drug ISFI formulations and 3 combination drug ISFI formulations. Liquid ISFI drug formulations were administered subcutaneously with a 19G needle on the shaved back of anesthetized NSG mice (Supplementary Fig. 6). Peripheral blood was collected from mice into capillary tubes coated with EDTA to isolate plasma. All samples were stored at −80 °C until analysis.