S1250

The two OS cell lines with varying metastatic potentials, SaOS-2 and SaOS-LM2, and HT-1080, a highly metastatic chondrosarcoma cell line, were plated in 60 mm dishes at 10⁶ cells and serum-starved for 24 h prior to their harvest for cell migration assays. Treated cells had their respective IC₅₀ concentrations of enzalutamide (Selleck Chemical LLC, Cat.# S1250) added to the serum-free media during this 24-h incubation. After 24 h of serum-starvation, with or without enzalutamide treatment, cells were plated in triplicate on 6.5 mm transwell permeable supports at a density of 5 × 10⁴ cells, in 24-well plates (Corning, Cat.# 07200174). The supports (upper chamber) held a final total volume of 150 µl serum-free, cell suspension. The (lower chamber) wells of the 24-well plate held 800 µl of the cell’s respective media either with or without FBS. Wells with serum-free media in the lower chambers served as discrete negative controls for each group of the study. Conditions were run in triplicate. Cells were incubated at 37 °C (5% CO₂) for 18 h, at which point the inserts were stained with crystal violet to terminate the migration assay. This migration assay was repeated four times.

Enzalutamide (Selleck, S1250) (10 mg/kg; the vehicle contained 1% carboxymethyl cellulose, 0.1% Tween 80, and 5% DMSO) was administered intragastrically to castrated mice daily for 10-30 days according to this study^{22 (link)}.

Animal experiments were performed in accordance with protocol approved by the Service de la Consommation et des Affaires vétérinaires of Canton de Vaud (VD 1865.3, VD 1865.4). NOD. Cg‐Prkdc^scid Il2rg^tm1Wjl/SzJ mice (Charles River), BalbC, and C57Bl6 breeders were purchased from Jackson Laboratories. Mammary glands were isolated from 18‐ to 35‐week‐old mice, lymph nodes removed, minced with surgical blades, and digested in DMEM/F12, 1% penicillin/streptomycin B with collagenase A 0.25 mg/ml (Roche). Tissue was concomitantly stimulated with hormones for 1, 2, or 6 h and centrifuged at 652 g for 5 min. Fat was removed, and pellet containing organoids washed with phosphate‐buffered saline (PBS), resuspended 5 min in 3 ml red cell blood lysis buffer and washed twice with PBS. Hormones and drug used: LNG, Sigma L0551000‐30MG; GSN, Sigma SML0292, DSG, Sigma SML0356‐5MG; DSP, Sigma SML0147‐10MG; CMA, Sigma C5145‐1G; CPA, Sigma C3283000‐30MG, R5020, Perkin Elmer NLP004005, bicalutamide, Toronto research laboratory B382000, enzalutamide, Selleck Chemicals S1250.