Small extracellular vesicles in plasma carry cytokines that remain undetectable by antibody-based assays

Small extracellular vesicles (sEVs), commonly referred to as exosomes, have emerged as key players in cell-to-cell communication. These tiny vesicles, ranging from 30 to 150 nanometers in size, are produced by virtually all cells and can be found freely circulating in the body’s various fluids. While it’s known that sEVs carry cytokines on their surface or within their interior, their precise contribution to cytokine levels in the bloodstream has remained elusive. However, a recent study sheds light on this phenomenon, offering novel insights into the role of sEV-associated cytokines in intercellular signaling.

Researchers from the University of Pittsburgh School of Medicine isolated sEVs from the plasma of patients with head and neck squamous cell carcinoma (HNSCC) as well as from healthy donors using a combination of ultrafiltration and size exclusion chromatography. They employed multiplex immunoassays to measure cytokine levels in both untreated plasma samples and plasma-derived sEVs, comparing them with samples treated with a mild detergent (0.5% Triton X-100) to permeabilize the vesicles. Statistical tests were then used to analyze differences in cytokine levels between the various sample groups.

The study confirmed the presence of cytokines within sEVs through immunoblotting and flow cytometry analysis. Furthermore, the researchers found that these sEV-associated cytokines were biologically active, exerting their effects in various in vitro assays. Interestingly, levels of cytokines within sEVs differed significantly between HNSCC patients and healthy donors, with patients generally exhibiting higher levels. Additionally, the study revealed that levels of many cytokines in sEVs correlated positively with the difference in cytokine levels between detergent-treated and untreated plasma samples.

These findings have significant implications for our understanding of cytokine signaling in the body. By demonstrating that sEV-associated cytokines contribute to differences in cytokine levels observed between treated and untreated plasma samples, the study highlights the importance of considering exosomal cytokines in cytokine profiling assays. Traditionally, antibody-based assays only detect soluble cytokines, overlooking those carried within sEVs. However, by permeabilizing sEVs with a mild detergent, the researchers were able to detect both soluble and vesicle-associated cytokines accurately. This underscores the necessity of incorporating sEV analysis into cytokine studies to obtain more comprehensive and accurate measurements of cytokine levels in biological fluids.

This study provides compelling evidence of the critical role played by sEVs in cytokine signaling and intercellular communication. By revealing the presence and functional significance of cytokines within sEVs, these researchers have opened new avenues for understanding the complexities of immune regulation and disease pathogenesis. Moving forward, integrating sEV analysis into cytokine research may lead to more precise diagnostic tools and therapeutic strategies for a wide range of diseases, including cancer and inflammatory disorders.

Hong CS, Diergaarde B, Whiteside TL. (2024) Small extracellular vesicles in plasma carry luminal cytokines that remain undetectable by antibody-based assays in cancer patients and healthy donors. BJC Rep [Epub ahead of print]. [article]

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