Rapalink 1

Organoids were collected in basis medium and centrifuged for 3 min at 100 rcf, then they were resuspended in TripLE Express and incubated at 37°C with occasional resuspension until completely dissociated. Cell suspension was then washed with basis medium and centrifuged at 300 rcf for 5 min. Cells were resuspended at 175,000 cells/ml in complete PCa organoids medium and seeded in 20 μl volume in a 384-well low-attachment plate, with black walls (Corning). After 48 h, wells with organoids were treated with the appropriate compound, resuspended in 20 μl of PCa organoids medium. The compounds used were Rapalink-1 (10–0.001 μM, ApexBio), rapamycin (10–0.1 μM), everolimus (1–0.1 μM), abiraterone (1 μM, in EtOH), enzalutamide (10 μM), disulfiram (10–0.1 μM, Sigma-Aldrich), DMSO (0.1%), and EtOH (0.1%). Where not stated otherwise, the compounds were from Selleckchem, and were resuspended in DMSO. Wells treated with disulfiram were additionally supplemented with copper-gluconate 1 μM (Sigma-Aldrich). Each condition was assessed in quadruplicates, each experiment was repeated 3 times for the PDX models and 2 times for the patient-derived bone metastasis material.