To address the question of cell–cell interaction and ECM effect on EZH2’s function, we utilized [13C6]-glucose as tracer and stable isotope-resolved metabolomic (SIRM) approach to examine the effect of EZH2 suppression on the metabolic networks in human lung adenocarcinoma A549 cells, grown as 2D cultures or 3D spheroids. Here, EZH2 is linked to lung adenocarcinoma.