Larotrectinib

All compounds were dissolved in DMSO at 10 mM (except for cisplatin and carboplatin) and aliquots were stored at −20 °C, 4 °C or room temperature according to the manufacturer’s recommendations. Sorafenib tosylate, lenvatinib mesylate, cabozantinib mesylate, regorafenib, octreotide acetate, lanreotide acetate, etoposide, sunitinib malate, everolimus, entrectinib, larotrectinib: all from Selleckchem; pasireotide ditrifuloroacetate (MedChem Express); cisplatin (Sandoz); carboplatin (Labatec). For drug screenings, organoids were dissociated with 0.25% Trypsin-EDTA (Gibco) and seeded at 1000 cells/well in 384-well plates in organoid expansion medium supplemented with 10% BME2. Two days later, compounds were added in a 2-fold dilution series ranging from 0.02 nM to 10 μM. After 6 days of treatment, cell viability was measured using CellTiter-Glo 3D (Promega). Luminescence was measured on a Synergy H1 Multi-Mode Reader (BioTek Instruments). Results were normalized to vehicle control (100% DMSO or 100% water). All experiments were performed twice. Dose-response curves were calculated using Prism 9.3.1 (GraphPad), nonlinear regression algorithm was used with a constrain of 0 for the bottom and 100 for the top.

Cells were plated on six-well or 6-cm plate, treated for 24, 48, and 72 h at different concentrations of Entrectinib (Bio Vision #1324) and Larotrectinib (Selleckchem LOXO-101 S7960) and harvested for western blot and apoptosis assay. For viability assay cells were treated on 96-well plates for 7 days and viability assay performed using the Cell Titer Glo (Promega, G9242) following the manufacturer instructions.