Abstract Body: Type 1 Diabetes Mellitus (T1DM) is the most severe form of diabetes, triggered by genetic and environmental factors causing an autoimmune attack on insulin-producing β-cells in the pancreatic islets. This leads to reduced insulin production and hyperglycemic episodes. The only clinically available biomarkers are circulating autoantibodies against β-cell autoantigens, which appear late in disease progression and do not reliably indicate β-cell loss. Further, T1DM is strongly associated with increased cardiovascular morbidity and mortality. There is an urgent need for non-invasive biomarkers that can detect T1DM at an earlier stage, enabling timely intervention. Extracellular vesicles (EVs) have emerged as promising candidates for biomarker discovery. These circulating particles carry cargo that reflects the state of the source cell, making them excellent candidates for non-invasive biomarkers.

We hypothesize that SERPINA3-containing EVs are derived from β-cells and contain distinct RNA and protein cargo representative of β-cell health, thereby serving as biomarkers for early-stage T1DM diagnosis.

We isolated EVs using size-exclusion chromatography from conditioned media of EndoC-βH1 cells and primary human islets, both untreated and treated with a pro-inflammatory cytokine cocktail to mimic the T1DM environment. Proteomic analysis identified 4,532 proteins, with 402 shared between EVs from EndoC-βH1 cells and human islets. Among 57 membrane-enriched proteins, SERPINA3 was selected for immunoprecipitation due to its membrane localization and pancreatic enrichment. Next, an immunoaffinity pull-down targeting SERPINA3 was developed. We isolated SERPINA3+ EVs from EndoC-βH1 cells, human islets, human plasma, and early-onset T1DM patient serum. We then extracted RNA from both SERPINA3+ and total EVs and performed RNA sequencing; several β-cell-related genes were enriched, including SCN3A, involved in insulin secretion, and PLD1, essential for β-cell exocytosis. These findings will be validated using plasma samples from the NIDDK TEDDY study.

EVs are a promising source of minimally invasive biomarkers for the early detection of T1DM. These findings provide proof-of-principle that β-cell EVs can reveal islet dysfunction prior to clinical onset, offering a novel strategy to overcome the limitations of current available biomarkers and paving the way for earlier diagnosis and intervention in T1DM, which may prevent common downstream cardiovascular complications.