Enzalutamide

For cell culture assays, RD162, a nonsteroidal antiandrogen was used (Merck, Oss, Netherlands). It is closely related to and was selected from the same drug‐screen as MDV3100 (enzalutamide). In vitro and in vivo, it has equal potency in AR‐antagonism as enzalutamide and no significant difference in bioavailability in preclinical testing.31 For AR binding assays, enzalutamide (Axon Medchem, Groningen, The Netherlands) was used because of its current use in clinical practice.
Steroids were obtained from Steraloids (Newport, RI) and dissolved in ethanol. RD162, enzalutamide, TAK700 (Millennium Pharmaceuticals, Cambridge) or abiraterone (Johnson & Johnson, New Brunswick) were all dissolved in dimethyl sulfoxide (DMSO). Similar amounts of DMSO (0.1%) were added to control cells. Concentrations used were based on levels reported in Belanger et al24, 25 and Taplin et al26 (summarized in Table S1).

Depending on the originating PDX model, organoids formed after four to fourteen days of in vitro culture. Preassembled PDXOs were retrieved from the hydrogel domes by gently dissolving the thermo-sensitive hydrogel with ice-cold medium, and then passing them through a 100 μm cell strainer to eliminate large organoids and reduce size heterogeneity at the start of the experiment. Pooled PDXOs were carefully resuspended in Noviogel-P5K (medium-gel ratio 64.5:35.5) and seeded as eight μL hydrogel domes in 96-well plates, with an organoid density of 5000–10,000 per dome. Four days later, drug exposure was initiated: the culture medium was exchanged for medium containing the appropriate concentration of docetaxel (microtubule stabilizer; logarithmic dose range: 0.01–30 nM; Sanofi, Paris, France), cabazitaxel (microtubule stabilizer; logarithmic dose range: 0.01–30 nM; Sanofi), enzalutamide (anti-androgen; logarithmic dose range: 0.1–30 μM; Axon Medchem, Groningen, The Netherlands, cat. no. 1613), a vehicle (for negative controls) or staurosporine (broad-spectrum protein kinase inhibitor; 1 μM for positive controls; Selleckchem, Houston, TX, USA, cat. no. S1421). PDXOs were exposed for a total duration of 10 days, followed by viability assessment or confocal live-cell imaging.

Methyltrienolone was obtained from Perkin Elmer, enzalutamide was obtained from Axon Medchem and bicalutamide was obtained from Sigma chemicals. Dihydrotestosterone, dimethyl sulfoxide (DMSO) were obtained from Sigma chemicals (Dorset, UK). The PBK inhibitor HI-TOPK-032 (PBKi) was purchased from InterBioScreen, Russia. Life Technologies (Carlsbad, CA) provided cell culture media, fetal bovine serum (FBS), antibiotics etc.