Ex Vivo 3D Tumor Spheroid Assay

Ex vivo cultures were prepared as described previously^{38 (link)}. Briefly, patient tumor tissues were mechanically disaggregated and treated for 1 h with 125 U/mL collagenase and 2.5 mg/mL DNAse (both from Sigma-Aldrich). To remove aggregates and debris the cell suspensions were filtered through 100 μM nylon Cell Strainer (BD Falcon, Franklin Lakes, NJ) and washed with ice-cold PBS. Red blood cells were removed using ACK lysing buffer (Lonza, Basel, Switzerland). Live cells were seeded out at density of 15–20 × 10⁵ cells/well in Nunc™ 96-Well Polystyrene Round Bottom Microwell plates (Thermo Fisher Scientific, Waltham, MA) in RPMI++ medium supplemented with 100 units/mL penicillin and 0.1 mg/mL streptomycin (both from Lonza) and allowed to form 3D spheroids. Drugs were immediately added to the cells and left for five days before viability was assessed using the CTG Luminescent Cell Viability Assay (Promega) and analyzed by Fluoroscan Ascent Fl (Thermo Fisher Scientific). The ex-vivo assay was performed once for each patient sample, with at least three technical replicates per condition.