Single-cell RNA sequencing uncovers an immunosuppressive program in PDAC mast cells: tumour-driven activation, proliferation, and association with Treg recruitment.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly immunosuppressive tumour microenvironment (TME), which contributes to its resistance to immunotherapy. Although mast cells (MCs) have been implicated in PDAC progression, their functional heterogeneity and candidate signaling models of immune modulation remain poorly understood. Through an integrated analysis of multiple single-cell RNA sequencing (scRNA-seq) datasets, we identified a significant enrichment of MCs in PDAC tissues and established a characteristic gene signature (TPSAB1, TPSB2, CPA3, HPGDS, KIT, LTC4S) for their precise identification. Single-cell RNA-seq analysis categorized MCs in PDAC into resting, activated, and proliferating subpopulations. To functionally validate these transcriptomic predictions, in vitro co-culture experiments showed that pancreatic cancer cells promote the activation and proliferation of MCs. Cell-cell communication analysis suggested that activated MCs preferentially interact with regulatory T cells (Tregs) via the MIF-(CD74/CXCR4) signaling axis. This interaction was associated with an immunosuppressive T-cell landscape, characterized by an expanded population of Tregs exhibiting a highly activated immunosuppressive phenotype. Spatial transcriptomics and immunofluorescence validated the confirmed the spatial proximity of MCs and Tregs in PDAC tissues. Clinically, high expression of MC-Treg signature genes correlated with poor patient survival. Our study suggests that MCs are key orchestrators of immunosuppression in PDAC, predicted to interact with Tregs through the MIF-CD74/CXCR4 axis, offering a novel rationale for targeting the MC-Treg axis in future immunotherapeutic strategies.