Toward large-scale clinical implementation of extracellular vesicle profiling for precision oncology

Extracellular vesicles (EVs) are nanosized, membrane-bound particles released by all cell types. They carry proteins, nucleic acids, and lipids reflective of their cellular origin and have emerged as promising non-invasive biomarkers for cancer diagnosis and monitoring of therapy response. However, the clinical translation of EV-based assays remains limited by heterogeneous isolation methods, a lack of standardization of the clinical workflows, and insufficient validation in large patient cohorts. To address these challenges, our group at the National Center for Tumor Diseases in Heidelberg, Germany, has developed an EV profiling framework compliant with MISEV2023 recommendations. We systematically benchmark isolation and pre-processing procedures to ensure reproducibility and compatibility with high-throughput liquid biopsy workflows within the prospective EValuate study (S-773/2021). Blood samples are collected via the NCT Cell and Liquid Biobank, processed within 30 minutes, and stored as serum and plasma aliquots at -80 °C for longitudinal analyses. To enable large-cohort EV analyses, we also characterized a miniaturized size-exclusion chromatography protocol using low-volume serum or plasma, which requires no specialized equipment and complements conventional differential centrifugation workflows. To demonstrate the feasibility of the pipeline, we collected and analyzed a total of 125 serum samples - 109 from 24 patients with hepatocellular carcinoma (HCC) undergoing immune checkpoint inhibitor therapy and 16 quality-control samples from four healthy donors. EVs were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis, Western blotting, quantitative protein assays, and subsequently profiled proteomically to identify EV-derived protein signatures associated with disease course, radiological treatment response (RECIST), and survival. This standardized, high-throughput EV workflow bridges biobanking, analytical validation, and clinical correlation, providing a robust and scalable framework for integrating EV-based liquid biopsy assays into precision oncology.