Cabazitaxel

Resistant sublines were established by continuous exposure to increasing concentrations of the respective drugs, as described before [13 (link)]. The docetaxel-resistant tumor cells were exposed to 2.5 ng/mL docetaxel (Sanofi, Paris, France), and cabazitaxel-resistant tumor cells were exposed to 2.5 ng/mL cabazitaxel (Sanofi, Paris, France) three times a week.
amygdalin from apricot kernels (Sigma-Aldrich, Taufkirchen, Germany) was freshly dissolved in cell culture medium and added to tumor cells at 10 mg/mL based on earlier studies [14 (link)]. Controls remained untreated. In all experiments, treated tumor cell cultures were compared to nontreated cultures. To assess toxic effects of amygdalin and/or docetaxel and cabazitaxel, cell viability was determined by trypan blue (Gibco/Invitrogen).

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.