Tissue-Specific Isolation of Hepatocyte Extracellular Vesicles From Human Plasma Enables Tissue-Resolved Proteomic and miRNA Profiling.

Tissue-specific resolution remains a critical barrier in liquid biopsy for precision medicine. To address this, we developed NEXPLOR (Novel EXtracellular vesicle PopuLation Omics Revealer), an immunoaffinity platform enabling selective enrichment of circulating tissue-specific extracellular vesicles (TS-EVs), demonstrated here for liver-derived hepatocyte-EVs (h-EVs). Through in silico discovery and orthogonal antibody validation across tissues, we identified a four-marker panel (ASGR1, ASGR2, TFR2, SLCO1B1) that selectively enriched hepatocyte EVs without co-capturing the dominant platelet- and leukocyte-EV background. In a clinical cohort of hepatocellular carcinoma (HCC), non-cancer cirrhosis and healthy donors, NEXPLOR recovered 57% more proteins than bulk EV isolation, with greater reproducibility. By restoring tissue context, h-EVs exposed liver biology that bulk EVs miss, spanning three levels: metabolic pathways (carbon metabolism, HIF-1α signalling, ferroptosis), HCC-defining proteins (PGD, H2AZ1, PRDX5), and progression-tracking candidates (CFB, PCSK9). Small RNA sequencing detected five miRNAs upregulated in HCC versus cirrhosis h-EVs, against 21 in bulk EVs (no overlap); yet only the h-EV miRNAs mapped to HCC-relevant signalling, and hepatocyte-regulatory miR-124-3p appeared exclusively in h-EVs. Together, these results establish tissue-specific EV enrichment as a route to more actionable, multi-omic liquid biopsies in HCC, cirrhosis and other diseases in urgent need of non-invasive biomarkers for patient diagnosis, monitoring and stratification.