In vivo experiments using patient-derived xenografts

All in vivo studies were carried out in accordance with the UK Home Office (Scientific Procedures) Act 1986 under project licence PPL40/3645. NOD scid gamma (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ, NSG) and athymic Nude (Foxn1^nu) mice (Charles River) were used in the following experiments. Small fragments of HBCx34 (early) and BB3RC31 (metastatic) BC PDX tumors were implanted subcutaneously into the flanks of 8–12-week-old female NSG mice. These two preclinical models are estrogen-dependent, so animals were administered with 8 μg/ml 17-beta estradiol (Sigma-Aldrich, #E2758) in drinking water at least 4 days previously to implantation until the end of the experiment. HBCx34 PDX model and their endocrine resistant variant (tamoxifen-resistant, TAMR) were kindly provided by Dr Elisabetta Marangoni (Institut Curie, Paris) [19]. In vivo experiments with HBCx34 TAMR PDX model were carried out using Nude mice in the Institut Curie. For clinical details of BB3RC31 refer to [12, 20].

When average PDX tumor volume reached 200–300 mm³, mice were randomized between treatment groups ensuring that all groups had similar starting mean tumor volumes. All in vivo experiments were performed with a minimum of n = 3 mice per condition. Tumor size and animal weight were recorded twice weekly in a blinded manner. SFX-01 (Evgen Pharma PLC, 300 mg/kg/day) and tamoxifen citrate (Sigma-Aldrich, #T9262, 10 mg/kg/day) were administered by oral gavage (0.1 ml/dose) on a 5 day from 7 day basis (weekends excluded) for 14 or 56 days; whereas Fulvestrant (200 mg/kg/week, Astrazeneca) was injected subcutaneously (0.1 ml/dose) on a weekly basis for 56 days. SFX-01 and tamoxifen citrate were made in 1% carboxymethylcellulose (Sigma-Aldrich, #C9481) dissolved in distilled water. SFN levels were measured in the mice plasma achieving a concentration of about 2 µM (data not shown), which is an appropriate approximation of the in vitro dose used (5 µM) since SFN is rapidly metabolized in vivo [40]. Upon termination, PDX tumors were collected in ice-cold DMEM media and processed as described in [12] for further downstream analyses. Mouse lungs were formalin-fixed paraffin-embedded for histological assessment of metastatic disease.

In vivo limiting dilution assays were performed to evaluate tumor initiation ability of HBCx34 PDX after 2 weeks of in vivo treatment. Xenografts treated with either SFX-01, tamoxifen citrate, combination or vehicle control for 14 days were collected and digested using collagenase-hyaluronidase (Stem Cell Technologies) to obtain single-cell suspensions. Serial limiting dilution of PDX-derived cells (500,000; 100,000; 20,000; 4,000 cells) were resuspended in mammosphere media:Matrigel (1:1) and subcutaneously injected into the flank of NSG mice (n = 4 per condition). The 90-day slow-release estrogen pellets were implanted subcutaneously 2 days prior to cell injection (0.72 mg, Innovative Research of America). At day 90 after cell injection, positive tumor growth was considered in mice bearing a tumor greater than 75 mm³. The tumor-initiating cell frequency was calculated using Extreme Limiting Dilution Analysis software (The Walter and Eliza Hall Institute of Medical Research) with a 95% confidence interval. p values were obtained by Chi-squared statistical analysis.