d-serine as a metabolic immune checkpoint in the tumour microenvironment.

Abstract

d-amino acids (D-AAs), the enantiomers of proteinogenic l-amino acids, are detectable in mammals, yet their biological roles in cancer immunity remain largely unexplored. Whether specific D-AAs modulate tumour progression or influence responsiveness to immunotherapy in gastrointestinal cancer is unknown. We aimed to determine how D-AAs, particularly d-serine (D-ser), shape the tumour immune microenvironment and affect clinical outcomes in gastrointestinal cancers. Mechanistic studies were conducted using murine MC38 tumours and orthotopic gastric cancer (GC) organoid allografts with or without D-AAs supplementation. Immune landscape alterations were assessed using single-cell RNA sequencing of tumour-infiltrating immune cells, flow cytometry, ex vivo macrophage-T cell co-culture assays, and microbiome manipulation experiments. D-AAs concentrations in plasma, urine, and stool were quantified in healthy controls (HCs; n = 87) and patients with GC across three cohorts (Cohort 1, n = 14; Cohort 2, n = 108; Cohort 3, n = 28). Associations between plasma D-ser levels, disease stage, immune cell infiltration, and clinical outcomes following anti-PD-1 antibody therapy were analysed. D-ser promoted tumour progression by suppressing CD8⁺ T cell immunity and enhancing SPP1-associated immunosuppressive macrophage signalling in murine model. Across all clinical cohorts, the plasma, urine, and stool levels of several D-AAs, most prominently D-ser, were significantly elevated in patients with GC compared to HCs. Plasma concentration of D-ser strongly correlated with disease stage (I-IV). Elevated plasma D-ser is associated with an immunosuppressive tumour microenvironment and poor response to anti-PD-1 monotherapy in patients with advanced gastric cancer. This study identifies D-ser as a previously unrecognised immunosuppressive metabolite that promotes tumour immune evasion by increased macrophages and reduced CD8⁺ T cell effector function, thereby shifting the tumour microenvironment toward an immunosuppressive phenotype. Clinically, D-ser is a potential metabolite to predict cancer progression and immunotherapy resistance. This work was supported by The Japan Science and Technology Agency (JST) Fusion Oriented Research for Disruptive Science and Technology (FOREST)[JPMJFR210P], Grants-in-Aid from the Japanese Society for the Promotion of Science (JSPS) (25K10430, 21K18272, 23H02899, 23K27590, 25K22627), KGRI challenge grant, Sakaguchi Memorial Foundation, Japan Agency for Medical Research and Development (CREST 21gm1510002h0001), and Miyarisan Pharmaceutical Grant.