Fig. 1: Identifying 6PGD as a metabolic vulnerability in human breast tumor myeloid suppressor cells regulated by tumor STAT3 signaling.

Oxidative PPP and PGD gene expression were compared by stage (A) and by subtype, triple-negative breast cancer (TNBC) and non-TNBC patients (B) using the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort dataset. n = 1903; Two-tailed t test. The tSNE plot (C) and PPP gene expression (D) of immune cell populations in primary human TNBC samples from the published single-cell RNA sequencing dataset (GSE161529). n = 8. The t-SNE plot of myeloid cell clusters (E) and oxidative PPP gene expression in myeloid cell compartments (F) of human TNBC samples. G Vectra multispectral imaging of human TNBC demonstrates expression of 6PGD (purple) in myeloid cells. Representative of one TNBC tumor. H Oxidative PPP checkpoints were blocked by shRNA in mice bone marrow (BM) derived MDSCs, then CD11b⁺Ly6C⁺Ly6G⁻ (M-MDSCs) were sorted. Their suppressive capacity was measured in co-culture with activated T cells at 1:8 (MDSC: T cell) ratio at 72 h. From two experiments with n = 3; One-way ANOVA. I The cells were generated as (H). Expression of arginase-1 was measured by flowcytometry. From two experiments with n = 4; One-way ANOVA. J CD11b⁺Ly6C⁺Ly6G⁻F4/80⁻ (M-MDSCs) were sorted from AT3 tumors and BM of tumor-free mice and co-cultured with activated T cells at 1:8 (MDSC: T cell) ratio at 72 h. Representative of two independent experiments, n = 3; One-way ANOVA. K M-MDSCs were prepared as in (J). Pgd expression was evaluated by Real-time PCR. Representative of two independent repeats. n = 3; Two-tailed t test. L, M Mouse BM cells were treated with IL-6 (40 ng/mL) and pSTAT3-Tyr705 phosphorylation was examined (L). pSTAT3 binding to Pgd promoter region was determined by ChIP-qPCR 30 minutes post IL-6 stimulation (M). Anti-histone H3 and IgG served as positive and negative controls, respectively. Representative of 3 independent experiments. N Pgd expression was evaluated by Real-time PCR on BM cells 48 h. post activation with IL-6 (40 ng/mL) with or without STAT3 inhibitor (JSI-124; 1 µM) treatment. Representative of 3 independent repeats. n = 3, two-tailed t test. All data are shown as mean ± SEM.