Fig. 3: Enhanced nucleotide synthesis and phosphorylation in 3D-spheroids.

A A heat map illustrating the comparison of metabolites between 2D- and 3D-cultured IGROV1 cells (Cutoff applied: p-value < 0.01). B A volcano plot revealing distinct metabolite signatures between 2D- and 3D-cultured IGROV1 cells. The volcano plot was generated to identify differentially enriched metabolites (Cutoff criteria: |Difference (Log₂ Fold Change) of group means | > 1, and -Log₁₀ (p-value) > 1). C Comparison of the levels of phosphocreatine in 2D- and 3D-cultured IGROV1 cells after 24 h incubation (n = 3 independent experiments). D Schematic illustrating the substrates and products of the creatine kinase reaction. E Comparison of intermediate levels for nucleotides and intermediates in 2D- and 3D-cultured IGROV1 cells after 24 h of incubation. The ¹³C isotopologue distribution of the metabolites is also illustrated. (n = 3 independent experiments). F Comparison of CTP/CMP, UTP/UMP, TTP/TMP, ATP/AMP, and GTP/GMP ratios in 2D- and 3D-cultured IGROV1 cells after 24 h incubation. (n = 3 independent experiments). G Schematic of 3D-spheroids activating the TCA cycle at higher levels than glycolysis, resulting in increased nucleotide production, phosphorylation, and energy substrate availability. The red circles indicate carbon. “m + 0” represents the carbon contribution from sources other than ¹³C₆-glucose, and “m + 1,” “m + 2,” etc. represent the carbon contribution from ¹³C₆-glucose. PRPP phosphoribosylpyrophosphate, IMP inosine monophosphate. Graph data were presented as mean ± SD of multiple experiments.