Fig. 3: Tumor and muscle tissues compete for exogenous Ser availability during cancer progression.

A, B Total tumor mass. Mass quantification of tumors obtained from HT29 low-PHGDH (A) and CT26 (B) tumor-bearing mice treated as in Fig. 2. Mass values were calculated based on tumor weight measured at the endpoint of the experiment. Each dot represents a plasma sample derived from a single mouse. Data are represented as mean ± SEM, Student’s t test. C, D Intra-tumor Ser levels. Relative Ser levels quantification in tumor tissue-derived samples from HT29 low-PHGDH (C) and CT26 (D) tumor-bearing mice treated as in Fig. 2. Metabolites from tissue samples were extracted as described in the “Materials and methods” section and tissue Ser content was quantified by GC-MS analysis. Each dot represents a plasma sample derived from a single mouse. Data are represented as mean ± SEM, Student’s t test. E PHGDH protein levels in CRC cell lines under Ser starvation. HCT-116, CACO2, and RKO cells were incubated in a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and Gly for 24 h before protein extraction. Cell lysates were analyzed by western blotting with the anti-PHGDH antibody. An anti-actin antibody was used to ensure equal protein loading. Data are expressed as relative to +Ser+Gly condition. Student’s t-test (n = 3). Each dot represents a single experiment. F Representative images of western blotting analysis evaluating PHGDH expression in HCT-116, CACO2, and RKO cells incubated in +Ser +Gly or -Ser -Gly medium for 24 h. G PHGDH protein levels in C2C12 myotubes under Ser starvation. C2C12 myotubes were incubated in a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and Gly for 6, 12, and 24 h before protein extraction. Cell lysates were analyzed by western blotting with the anti-PHGDH antibody. An anti-actin antibody was used to ensure equal protein loading. Data are expressed as relative to +Ser+Gly condition. Student’s t-test (n = 3). Each dot represents a single experiment. H Representative images of western blotting analysis evaluating PHGDH expression in C2C12 myotubes incubated in +Ser +Gly or -Ser -Gly medium for 24 h. I Ser synthesis pathway (SSP) activity analysis by [U-¹³C]-glucose labeling assay. Relative incorporation of [U-¹³C]-glucose-derived carbons in Ser. C2C12, CACO2, RKO, and HCT116 cells were incubated in a (-Ser -Gly) medium containing [U-¹³C]-Glucose for 3 h. Metabolite abundance and labeling enrichment were evaluated by GC-MS analysis. One-way ANOVA with Dunnett post hoc test (n = 3). Each dot represents a single experiment. J Competition for Ser availability between tumor and muscle cells. C2C12 myotubes were pre-incubated with a medium containing ¹³C₁-Ser for 24 h before replacing growth medium with standard serum-starved-DMEM and adding tumor cells in the upper compartment of a Boyden chamber (pores diameter 0.4 µm). The fate of C2C12-derived ¹³C₁-Ser was assessed by GC-MS analysis 24 h after tumor cell’s introduction. K Workflow for the ¹³C₁-Ser labeling experiment described in J. One-way ANOVA with Tukey’s post hoc test (n = 3). Each dot represents a single experiment. Data are represented as mean ± SEM of n independent experiments, ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001.