Extended data figure 5. Metastatic nodules exhibited increased enrichment of labeled serine and glycine as compared to subcutaneous tumours.

In vivo isotope tracing of uniformly ¹³C-labelled (a) lactate (M+3), (b) 3-phosphoglycerate (M+3), (c) serine (M+3), and (d) glycine (M+2) in subcutaneous tumours versus metastatic nodules from the same mice (UT10, n=3–4 mice per time point in 2 independent experiments). The fractional enrichment of labeled lactate, and 3-PG did not significantly differ among plasma, subcutaneous tumours, or metastatic tumours at any time point. In contrast, the fractional enrichment of labeled serine and glycine were significantly higher in metastatic as compared to subcutaneous tumors. This is consistent with increased de novo serine synthesis in metastatic tumors but could also reflect altered serine/glycine exchange with circulating serine/glycine pools in metastatic as compared to subcutaneous tumors. e) NADPH/NADP ratios in subcutaneous tumours and metastatic nodules from the same mice shown in Figure 2d and 2e. f and g) Western blot of ALDH1L2 (f) and MTHFD1 (g) protein after shRNA knockdown in melanoma cells. Uncropped western blots are shown in Supplementary Figure 1. h and i) Amount of GSH (h) and GSSG (i) per mg of subcutaneous or metastatic tumour as measured by LC-MS (M405, M481 and UT10, n=2–3 mice/melanoma in 2 independent experiments). All data represent mean±sd. Statistical significance was assessed using two-tailed Student’s t-tests (e, h, and i) and one-way analyses of variance (ANOVAs) followed by Dunnett’s tests for multiple comparisons (a – d; *, p<0.05; ***, p< 0.0005). (j) Schematic of the folate pathway including NADPH generating (green box) and NADPH consuming (red box) reactions.