A multi-omics study integrating single-cell transcriptomics, spatial transcriptomics, and mendelian randomization...

m5C, an important RNA modification, is closely associated with breast cancer; however, their causal relationship is unknown. We examined the relationship between m5C regulation and breast cancer. Genome-wide association study breast cancer data and expression quantitative trait loci of m5C regulatory factor genes in blood were collected. We explored the causal relationship among m5C regulatory genes, breast cancer susceptibility, and prognosis through Mendelian randomization. Single-cell RNA sequencing data from peripheral blood mononuclear cells of patients with breast cancer and healthy controls were used to determine the differential expression of key genes among different cellular subpopulations. The effects of these genes on specific cellular subpopulations were further explored in single-cell RNAseq data from primary breast cancer using cellular communication, metabolic and cellular function analyses and validated the findings using flow cytometry. We validated the prognostic value of specific cellular subpopulations expressing key genes and assessed the impact of antitumor immunity using spatial transcriptomics, bulk transcriptomics, and immunofluorescence analysis of tissue microarray (120 patients). Mendelian randomization analysis indicated that the expression of the m5C writer tRNA aspartate methyltransferase 1 (TRDMT1) in blood was associated with breast cancer prognosis. TRDMT1 expression in T cells was decreased in patients with breast cancer. TRDMT1 was closely related to T-cell function. Moreover, it may modulate the antitumor immune effects of CD8⁺ T cells by influencing cellular communication and metabolism, impacting the prognosis and immunotherapeutic response of patients with breast cancer. We confirmed the immunotherapy predict value and prognostic value of measuring TRDMT1 levels in CD8⁺ T cells, provided insights into enhancing CD8⁺ T-cell function, and identified potential targets to improve breast cancer prognosis and immunotherapy. Our research may help guide breast cancer treatment decisions and provide a prognostic evaluation option for future breast cancer treatment.