GlycoStamp-seq: Single-cell nascent sialoglycometabolomics captures sialylation dynamics in immune cells of an oxaliplatin-treated bladder cancer mouse model

Dynamic remodeling of immune cell-surface glycans reflects cellular state and immune activity. Despite advances in single-cell glyco-profiling, current methods primarily capture pre-existing glycan epitopes, providing limited information on dynamic sialoglycans. Here, we present GlycoStamp-seq, a method to profile nascent sialoglycan dynamics by coupling Ac₄ManNAz-based metabolic glycoengineering with single-cell RNA/TCR sequencing (scRNA/TCR-seq) and scDyna-seq. We apply GlycoStamp-seq by injecting Ac₄ManNAz into oxaliplatin-treated mice with subcutaneous bladder tumors, days before extracting tumor-derived single-cell suspensions for sequencing. Following oxaliplatin treatment, a subset of CD8⁺ T cells in the tumor immune microenvironment exhibits elevated nascent sialoglycan activity, enhanced effector programs, and increased clonal expansion. Furthermore, elevated nascent sialoglycan levels mark antigen-reactive CD8⁺ T cells with increased anti-tumor efficacy in vitro and in vivo. GlycoStamp-seq may thus serve as a versatile platform for profiling nascent sialoglycan dynamics in complex tissues, thereby expanding the single-cell multi-omics toolkit and providing insights into sialoglycan dynamics during therapeutic response. Glycosylation is critical for an effective immune response, but immune cell glycan dynamics are not fully understood. Here, the authors present GlycoStamp-seq to track sialoglycan dynamics, and apply GlycoStamp-seq in an oxaliplatin-treated subcutaneous bladder cancer mouse model.