Fig 2: Metabolomic analysis of macrophages in breast cancer. (A) Differentiation of THP1 cells into macrophages for metabolomics analysis. THP-1 cells were differentiated after 24 h in PMA to obtain resting (M0) macrophages (n = 3). M0 macrophages were polarized toward M1 (n = 3) or M2 (n = 3) phenotypes through 48 h incubation with lipopolysaccharide and IFN-γ, or with IL-4 and IL-13. For obtaining tumor-associated macrophages (n = 3), M0 macrophages were cultured in MDA-MB-231 cell conditioned medium (MB231-CM) for 48 h. Successful differentiation into tumor-associated macrophages was confirmed through gene expression analysis of specific markers (such as IL1β, IL-6, TNF-α, IL-10, CD163, AGR1, TGF-β1, and CCL22). M1 and M2 macrophages were incorporated as typing controls (Table 1). (B) Percentage of each metabolite class, detected by LC-MS/MS. (C) Levels of metabolites of glycolysis, determined via mass spectrometry (n = 3). (D) Levels of metabolites of the TCA cycle, determined via mass spectrometry. All metabolite levels were normalized to the control. Bar graphs represent the mean ± SD of experimental triplicates (t-test, *P < 0.05, **P < 0.01, ***P < 0.001). (E) Heatmap showing the distinct metabolic landscapes. (F) Metabolic pathway enrichment analysis of differential metabolites with MetaboAnalyst (https://www.metaboanalyst.ca) (t-test, *P < 0.05, **P < 0.01, ***P < 0.001). (G) Schematic of glycolysis and the PPP and the SSP metabolic pathways. (H) Bubble plot illustrating the expression levels of key enzymes in the serine biosynthesis pathway (PHGDH, PSAT1, and PSPH) across immune cell subgroups. Bubble size indicates the proportion of cells in a cell population expressing serine biosynthesis pathway proteins. Color represents the average expression of serine biosynthesis pathway components. (I) UMAP view of immune cell clusters from 2 samples (Table 1). UMAP plot depicting the serine biosynthesis pathway in immune cell clusters. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.

Fig 3: PHGDH cooperates with STAT3 in regulating GLUD1 and GLS2 transcription. (A, B) qRT-PCR and Western blot detection of changes in the glutamine metabolic pathway induced by PHGDH knockdown. (C, D) qRT-PCR and Western blot detection of changes in the glutamine metabolic pathway induced by PHGDH overexpression. (E) Dual-luciferase assay demonstrating that PHGDH knockdown increases the transcriptional activity of GLUD1 and GLS2 (Table 2). (F) ChIP and real-time PCR assays indicating PHGDH binding to the promoter region binding sites of GLUD1 and GLS2. (G) Translocation of PHGDH in THP1 cells after treatment with MB231-CM, detected with confocal microscopy. Scale bar, 10 μm. (H) Translocation of PHGDH in THP1 cells after treatment with MB231-CM and nuclear/cytosolic fragmentation. (I) Co-immunoprecipitation of PHGDH in TAMs. Detection of PHGDH binding to STAT3 with Western blot (t-test, *P < 0.05, **P < 0.01, ***P < 0.001).

Fig 4: Inhibition of PHGDH enhances the M2-like macrophage polarization phenotype and immunosuppressive function. (A, B) Overexpression and knockdown of PHGDH validated at the mRNA level by qRT-PCR and the protein level by Western blot in THP1 cells. (C, D) Validation of overexpression and knockdown of PHGDH at the mRNA level by qRT-PCR and the protein level by Western blot in BMDM cells. (E, F) Effects of PHGDH overexpression or knockdown in tumor-associated macrophages on polarization function and (G, H) immunosuppressive function (t-test, *P < 0.05, **P < 0.01, ***P < 0.001) (Table 1). (I) Correlation analyses of the transcriptional levels of macrophage-associated markers (TGF-β and TNF-α) with PHGDH in human breast cancer, according to data from the TCGA database. (J–L) PHGDH overexpressing BMDMs (OE-PHGDH) and control BMDMs (OE-NC) were mixed with EO771 cells and grafted into C57BL/6 mice (n = 7). (J) Schematic diagram of the cell transfer experiment and tumor image. (K) Growth curve and tumor weight (t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant). (L) Representative immunohistochemistry images of paraffin-embedded xenograft sections. Scale bar, 20 μm.

Fig 5: Inhibition of PHGDH promotes M2 macrophage polarization by activating the glutamine metabolic pathway. (A) Effects of glutamine deprivation on macrophage polarization and immunosuppressive function, determined with qRT-PCR. (B) Schematic overview of the molecular mechanism through which STAT3 regulates M2 genes. (C) Glutamine deprivation reversed the sh-PHGDH-mediated enhancement of macrophage polarization and immunosuppressive function. (D) Function of the STAT3 inhibitor W1131 (3 μM) on cytokine protein mRNA expression in PHGDH knockdown macrophages (Table 1). The results were obtained from three independent experiments. P values were calculated with one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.