Spatiotemporal Drug Dynamics in MCTS

HIPEC-like treated and untreated control MCTS were prepared as described above. Cells from the outer, intermediate, and core regions of these MCTS were fractionated using serial trypsinization27 (link). Small molecules were then extracted and nickel chloride (Sigma, St. Louis, MO) was added as the internal standard. The mixture was then derivatized with DDTC, and extracted with ethyl acetate/ n-hexane (EA/Hex, 1:1, v/v)17 . To analyze drug efflux, cell culture medium was collected at different time points and small molecules were then extracted and derivatized using the same sample pretreatment procedure. All treatment conditions were performed with four replicates. For quantification of total Pt concentration, fractionated cells, spiked with the internal standard, were wet-ashed with concentrated nitric acid (HNO₃)18 (link), followed by DDTC derivatization and EA/Hex extraction.