Mitochondrial respiratory capacity assays
Tumor cells from both 2D and 3D MTSs were digested to prepare single cell suspensions. Oxygraph-2k mitochondrial function analyzer (Oroboros, Innsbruck, Austria) was used to measure the cellular oxygen consumption rate (OCR) under the following four conditions: Routine, normal cell respiration level; Leak, ATP synthase was inhibited by 1 μL 4 mg/ml oligomycin (Sigma-Aldrich, San Luis, MO, USA); Electron transfer system (ETS): the oxygen consumption level reached the maximum, with supplement of 1 μL 1 mM FCCP (Carbonylcyanide p-trifluoromethoxyphenylhydrazone, Abcam, Cambridge, UK) to destroy the proton gradient and mitochondrial membrane potential; Residual oxygen consumption (ROX): the residual intracellular oxygen consumption was measured by adding1 μL 1mM rotenone (Abcam, Cambridge, UK) and 1 μL 1mM antimycin A (Abcam, Cambridge, UK) to inhibit mitochondrial complex I/III and mitochondrial respiration. Basal respiration: the OCR difference value between Routine and ROX; ATP production: the OCR difference value between Routine and Leak; Maximal respiration: the OCR difference value between ETS and ROX; Spare respiratory capacity: the OCR difference value between ETS and Routine; Non-mitochondrial respiration: the OCR value of ROX.