Developing blood extracellular vesicle protein biomarkers for hepatocellular carcinoma: mechanisms, methods, and translation.

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality globally, with late-stage diagnoses predominantly driven by the absence of early clinical symptoms and the inadequate sensitivity of current diagnostic tools, highlighting a critical unmet need in liver cancer management. Circulating extracellular vesicles (EVs), which naturally carry proteins and nucleic acids, serve as both mediators of tumor-specific biology and reporters of early pathophysiological changes, offering a transformative avenue for noninvasive liquid biopsy. This review integrates mechanistic advances in understanding EV-associated proteins involved in HCC pathogenesis with state-of-the-art proteomic methodologies for EV biomarker discovery, validation, and clinical translation. We highlight how functional and mechanistic insights into EV proteins guide the identification of clinically relevant biomarkers and examine recent progress in analytical approaches for isolating and profiling blood-derived EVs in large patient cohorts. Additionally, we discuss the development of high-throughput detection platforms, such as biochip-based assays, which are accelerating the translation of EV protein biomarkers from research to routine clinical practice. Critical challenges, including the lack of standardized analytical workflows, limited demonstration of clinical utility, and the need for multicenter validation, are addressed, with a focus on strategies to ensure robust and reproducible biomarker performance. By bridging mechanistic understanding with analytical innovation and clinical application, we aim to accelerate the deployment of EV proteomics for earlier HCC detection, risk stratification, and treatment monitoring, ultimately advancing precision oncology.