(A–E) PHGDH-depleted HCT116 or SW480 cells were rescued with rPHGDH WT or K146R. These cells were supplemented with or without SAM (25 μM). Transwell migration assays were performed (A). Intracellular SAM levels were examined (B). These cells were injected into spleens (C and D), or HCT116-derived tumors were implanted into cecum (E), of randomized BALB/c nude mice. The metastatic nodules in the livers were counted and statistically analyzed (C). Data represent the mean ± SD of 6 mice (2-tailed t test). Kaplan-Meier survival analysis was performed (D). Upward tick mark represents censored (alive) mice. Eight weeks after the implantation, bioluminescence imaging of the dissected livers was performed (E). Representative images of hepatic metastasis were shown (top). Data represent mean ± SD of luciferase intensities from 6 mice (bottom, 2-tailed t test). (F) Transwell migration assays were performed with HCT116 cells treated with increasing doses of SAM. (A, B, and F) Data represent the mean ± SD of 3 biologically independent experiments (1-way ANOVA with Tukey’s multiple-comparison test). (G and H) HCT116 cells were injected into spleens (G), or HCT116-derived tumors were implanted into cecum (H), of randomized BALB/c nude mice, followed by SAM administration (20 mg/kg body weight). The metastatic nodules in dissected livers were counted and statistically analyzed (G). Data represent the mean ± SD of 6 mice (2-tailed t test). Bioluminescence imaging of the dissected liver tissues was performed (H). Representative images of hepatic metastasis were shown (top). Data represent mean ± SD of luciferase intensities from 6 mice (bottom, 2-tailed t test). (I) SAM levels were compared between primary tumors and paired metastatic tumors from 12 patients with CRC (paired t test, 2 tailed). (J) SAM levels in blood samples were compared between patients with CRC with or without metastatic recurrence. Data represent the mean ± SD (2-tailed t test). *P < 0.05, **P < 0.01. See also Supplemental Figure 4.