Abstract:
Aberrant glycosylation is a defining feature of cancer and extends beyond tumour cells to tumour-derived extracellular vesicles (TDEVs), which are increasingly recognised as key mediators of intercellular communication within the tumour microenvironment. However, the contribution of tumour-associated glycans to the immunomodulatory functions of TDEVs remains poorly understood. The sialyl-Tn (STn) antigen, a truncated O-glycan frequently expressed in triple-negative breast cancer (TNBC), has been associated with poor prognosis, increased tumour aggressiveness, and the establishment of an immunosuppressive microenvironment. Despite this, its role in extracellular vesicle-mediated immune regulation has not been fully elucidated.
In this study, we investigated the functional impact of STn-positive extracellular vesicles on dendritic cell (DC) biology. Using glycoengineered MDA-MB-231 TNBC cells overexpressing the glycosyltransferase ST6GalNAc-I, we generated extracellular vesicles enriched in STn (STn? EVs). These vesicles were isolated and characterised, and their immunomodulatory effects were evaluated using human monocyte-derived dendritic cells.
Functionally, STn? EVs significantly impaired DC maturation, as demonstrated by reduced expression of key activation and antigen presentation markers, including HLA-DR, CD40, CD86, and CCR7. This immature phenotype translated into a marked functional deficit, with reduced capacity to prime CD4? and CD8? T cells in mixed lymphocyte reactions. Quantitatively, STn? EV-conditioned DCs suppressed CD4? T cell proliferation by approximately 55% and CD8? T cell proliferation by up to 70%, while promoting a substantial increase in regulatory T cell expansion, indicative of a tolerogenic immune shift.
Mechanistically, we demonstrate that STn? EVs act as vehicles for horizontal transfer of both the STn glycan and its biosynthetic enzyme, ST6GalNAc-I, to recipient dendritic cells. Western blot and flow cytometry analyses confirmed the acquisition of ST6GalNAc-I and surface expression of STn in DCs exposed to STn? EVs, revealing a previously underappreciated mechanism by which tumour cells can reprogram immune cell glycosylation . Importantly, enzymatic removal of terminal sialic acids from EVs using sialidase completely abrogated these immunosuppressive effects, restoring DC maturation and T cell activation, thereby demonstrating that the observed immune dysfunction is critically dependent on STn-associated sialylation.
Collectively, these findings uncover a novel glycan-mediated mechanism of immune evasion, in which tumour-derived extracellular vesicles actively reshape dendritic cell phenotype and function through glycosylation-dependent signalling. This study expands the current understanding of extracellular vesicle cargo beyond proteins and nucleic acids, highlighting glycans as functional regulators of immune responses. From a translational perspective, STn-positive EVs emerge as promising biomarkers of immune dysfunction and potential therapeutic targets for restoring anti-tumour immunity in triple-negative breast cancer.
