Liver fibrosis is a major global health concern: its evolution into liver cirrhosis is followed by the death of over a million of people every year worldwide. The transdifferentiation of hepatic stellate cells (HSCs, the main collagen-producing cells) from a quiescent into an activated, pro-fibrotic status plays a key role in liver fibrogenesis, making these cells interesting from both a therapeutic and a diagnostic perspective.

Can membrane-bound nanovesicles shuttled between HSCs become a diagnostic tool for liver fibrosis? The biochemical complexity of extracellular vesicles (EVs) and their role in intercellular communication make them an attractive tool to look for biomarkers that might become a valid alternative to highly invasive liver biopsies.

We developed robust sets of methods to isolate and characterize EVs from differently treated LX-2 (human HSCs cell line) in vitro, and we investigated the biological effect they exert onto naïve cells, proving that EVs do play an active role in affecting HSCs’ status. Different purification methods (size exclusion chromatography, SEC, and asymmetrical flow field-flow fractionation, AF4) revealed EV subpopulations with different physicochemical behaviors. Proteomic data from our samples was mined for EV-associated proteins whose expression correlated with HSCs treatment. Consequently, the secreted protein acidic and cysteine rich (SPARC) emerged as a candidate protein to explore the feasibility of using fluorescence nanoparticle tracking analysis (f-NTA) as a non-destructive tool for the determination of HSCs’ physiological state based on EVs.

We could thus use EVs to directly evaluate the efficacy of treatments with polyenylphosphatidylcholines (PPC)-rich lipid S80, with and without experimental drugs elafibranor and obeticholic acid, proving that S80 (but neither of the drugs) greatly reduces relative SPARC-positive EVs’ abundance, while pro-fibrotic treatment with transforming growth factor β1 (TGF) does the opposite.

Here, for the first time, we could measurably correlate the cellular response to PPC-treatment to the relative presence of SPARC on the generated EVs.

Our results pave the way for more precise and less invasive ex vivo analyses: markers related to diseased and healthy states, as well as proteins that are tissue specific or preferentially expressed by specific cells could all be conceivably checked within one EV sample if appropriately selected.