Small extracellular vesicles in plasma carry luminal cytokines that remain undetectable by antibody-based assays in cancer patients and healthy donors

Abstract

Small (30–150 nm) extracellular vesicles (sEV), also known as exosomes, play a key role in cell-to-cell signaling. They are produced by all cells, circulate freely and are present in all body fluids. Evidence indicates that cytokines are present on the surface and/or in the lumen of sEV. The contribution of intravesicular cytokines to cytokine levels in plasma are unknown. sEV were isolated by ultrafiltration/size exclusion chromatography from pre-cleared plasma obtained from patients with head and neck squamous cell carcinoma (HNSCC) and healthy donors (HDs). Multiplex immunoassays were used to measure cytokine levels in paired untreated and detergent-treated (0.5% Triton X-100) plasma and plasma-derived detergent-treated sEV. Non-parametric tests were used to assess differences in cytokine levels. The presence of cytokines in sEV isolated from patients’ and HDs’ plasma was confirmed by immunoblots and on-bead flow cytometry. sEV-associated cytokines were functional in various in vitro assays. Levels of cytokines in sEV varied among the HNSCC patients and were generally significantly higher than the levels observed in sEV from HDs. Compared to untreated plasma, levels for the majority (40/51) of the evaluated proteins were significantly higher in detergent-treated plasma (P < 0.0001–0.03). In addition, levels of 24/51 proteins in sEV, including IL6, TNFRII, IL-17a, IFNa and IFNg, were significantly positively correlated with the difference between levels detected in detergent-treated plasma and untreated plasma. The data indicate that sEV-associated cytokines account for the differences in cytokine levels measured in detergent-treated versus untreated plasma. Ab-based assays using untreated plasma detect only soluble cytokines and miss cytokines carried in the lumen of sEV. Permeabilization of sEV with a mild detergent allows for Ab-based detection of sEV-associated and soluble cytokines in plasma. The failure to detect cytokines carried in the sEV lumen leads to inaccurate estimates of cytokine levels in body fluids.