Extended Data Figure 2. Nutrient metabolism in H460 spheroid culture.

a, Cell proliferation and doubling times of H460 cells cultured under monolayer and spheroid conditions (n=4 cultures days 1–4; n=3 cultures days 5–6). b, Effect of glucose or glutamine deprivation on cell counts in monolayer and spheroid culture (n=4 cultures from a representative experiment). c, Rates of glucose consumption and lactate excretion in monolayer and spheroid culture (n=4 cultures from two experiments). d, Rates of glutamine consumption; glutamate and ammonia excretion; ratio of ammonia excretion to glutamine consumption; and rate of excretion of ¹⁵NH₄⁺ originating from [γ-¹⁵N]glutamine in monolayer and spheroid culture (n=3 cultures from a representative experiment). e, Citrate mass isotoplogue analysis in H460 cells in monolayer culture, aggregated into spheroids (P1), or disaggregated from spheroids then permitted to re-aggregate (P2) (n=2 cultures from a representative experiment). f, Right, protein levels of phosphorylated PDHα (pPDH, Ser293), total PDHα (tPDH) and PDK1 in monolayer and spheroid culture with or without 2mM dichloroacetate (DCA). Left, citrate mass isotopologue analysis in H460 spheroids cultured with [U-¹³C]glucose or [U-¹³C]glutamine, and treated with 2mM DCA (n=3 cultures from a representative experiment). *p<0.05, Welch's unequal variances t-test (a, c, d and f), or Welch's unequal variances t-test, followed by multiple-comparison correction (b). All experiments were repeated 3 times or more.