Chemotherapy treatments were performed by incubating 3D cultures in growth medium with the indicated dosage of carboplatin (NDC: 61703-360-22; NOVAPLUS, Irving, TX, USA) diluted to 5–2560 μM, paclitaxel (NDC: 61703-342-22; Hospira Worldwide, Inc. Lake Forest, IL, USA), etoposide (NDC: 0703-5653-01; Teva Pharmaceuticals, Irvine, CA, USA) diluted to 1–100 μM, or gemcitabine (NDC: 0781-3282-75; Eli Lily and Company, Indianapolis, IN, USA) diluted to 0.01 to 1000 μM. Chemotherapy agents were allowed to incubate for 72 hours with the exception of caroboplatin incubations which were conducted for a total of 96 hours with a refresh at 48 hours. The incubation times chosen for this study are consistent with previous reports and reflect the observation that 3D tumor models typically require a longer period of drug exposure relative to traditional monolayer cultures and these treatment schedules more closely resemble in vivo therapeutic assessment scenarios19 (link)40 (link)42 (link). All chemotherapy drugs were clinical grade formulations purchased from the central pharmacy at the Massachusetts General Hospital. Treatment response was assessed immediately after removal of the chemotherapeutic agent.