Extracellular vesicles and their associated cargoes as potential biomarker in liver diseases

Background Extracellular vesicles (EVs) are a heterogeneous group of lipid-enclosed nanoparticles secreted by eukaryotic and prokaryotic cells. EVs are known for participating to intercellular communication and for contributing to diverse cellular processes, such as immune responses and coagulation. EVs have garnered much interest due to their potential utility both as circulating biomarkers in human diseases and as possible delivery agents in targeted therapies. However, EVs small sizes and heterogeneity pose a number of challenges to their isolation, study and characterization.

Methods NTA (Nanoparticle Tracking Analysis), FACS (fluorescence-activated cell sorting) and western blot were used to characterize EVs circulating in the serum of animal models for liver diseases. EVs functionality following isolation was evaluated by in-vitro uptake assay and monitored fluorometrically. To evaluate EVs cargoes as potential source of biomarkers, EVs-associated microRNAs and cytokines were measured. EVs-associated cytokines were quantified in patients with cholangiocarcinoma (CCC) by using Luminex 8plex, whereas EVs-associated microRNAs in patients with non-alcoholic steatohepatitis (NASH) and auto-immune liver diseases (AIH) were measured by miQPCR.

Results Here we show that the amount and size of EVs circulating in the serum of patients with NASH and AIH are significantly different compared to EVs circulating in sera prepared from healthy donors. Furthermore, the expression of several EVs-associated microRNAs was found to be significantly altered. The levels of miR-142-3p, miR-15a and miR-10a were significantly upregulated in EVs circulating in AIH patients. We also discovered that EVs transport a significant amount of cytokines. Specifically, we showed that EVs isolated from CCC patients contain a significantly higher amount of IL2 and IL8 compared to those from the serum of heathy donors.

Conclusions While this is an exploratory, hypothesis-generating study, it shows that the quantitative analysis of EVs can discriminates between healthy and diseased individuals, whereas miR-142-3p, miR-15a and miR-10a might have a potential utility as biomarkers in AIH. Moreover, our data indicate that the analysis of cytokines in EVs might enable for more experimental flexibility. Overall, our goal is, in combination with future studies, to assess the potential of qualitative and quantitative analyses of EVs as predictive and prognostic biomarkers in liver diseases.

Publication History

Article published online:
04 January 2021