EV Diagnostics: Extracellular Vesicles as Diagnostic Biomarkers in Cancer: From Technological Advances to Clinical Translation
DOI: https://doi.org/10.47184/tev.2025.01.04 Extracellular vesicles (EVs) are emerging as powerful tools for non-invasive cancer diagnostics. Released by virtually all cell types, EVs carry molecular cargo—RNA, DNA, proteins, and lipids—that reflects the physiological and pathological state of their cells of origin. In oncology, tumor-derived EVs are abundant in biofluids and can cross biological barriers, offering stable, information-rich biomarkers for early detection, molecular profiling, and longitudinal disease monitoring. This review outlines the unique advantages of EVs compared to more established liquid biopsy analytes, such as circulating tumor cells or cell-free DNA, and examines how EVs can provide complementary or even superior diagnostic insights. We highlight recent technological innovations enabling more sensitive and specific EV analysis including microfluidic devices and single – vesicle imaging system. Recent advancements in microfluidic EV devices enable the high-throughput purification and on-chip analysis of EVs. When merged with single-vesicle imaging systems, they provide nanoscale characterization of EVs, addressing long-standing challenges such as EV population heterogeneity and the detection of rare tumor-specific signals. In addition, we discuss key efforts to standardize EV-based assays, including the MISEV guidelines and EV-TRACK, and analyze regulatory and practical considerations that currently limit clinical adoption. While EV diagnostics are not yet routine, continued advances in methodology, standardization, and validation are rapidly paving the way for broader clinical integration in precision oncology.
Liquid Biopsy, EVs, Cancer, Technological innovations
Introduction
Cancer remains a major global health challenge, making the development of improved diagnostic tools a priority. Liquid biopsy approaches, which analyze tumor-derived materials in body fluids, have attracted considerable interest for early detection and monitoring of cancer [1,2]. Among emerging liquid biopsy targets, extracellular vesicles (EVs) have attracted particular attention. EVs are lipid bilayer-enclosed particles released by cells that carry diverse molecular cargo (proteins, RNAs, DNA, lipids) from their cell of origin. They include subtypes such as exosomes (50-150 nm endosomal vesicles) and microvesicles (50-5000 nm plasma membrane-derived vesicles), although distinguishing them in practice is challenging [3]. Tumor-derived EVs play a role in cancer progression by facilitating intercellular communication and metastasis and, critically, by encapsulating tumor biomarkers in a stable form. This stability protects the EV contents from enzymatic degradation, allowing tumoral DNA and RNA within EVs to remain in the circulation longer than freely circulating nucleic acids [4]. In addition, EVs are abundant in biofluids and can cross biological barriers, enabling real-time sampling of tumor-derived information from blood, urine, and other accessible fluids [4, 5]. Taken together, these properties position EVs as promising biomarker carriers for cancer diagnostics and disease monitoring. However, the translation of EV-based diagnostics into clinical practice is complex. Technical hurdles in the isolation and analysis of EVs, coupled with the need for rigorous validation, have so far limited the routine clinical use of EV biomarkers. In this review, we examine the role of EVs as cancer biomarkers and review recent technological innovations that aim to overcome these challenges.
EVs as biomarkers in cancer diagnostics
EVs circulate in virtually all body fluids and reflect the physiological or pathological state of the cells of origin. In cancer patients, a subset of circulating EVs is derived from tumor cells; these tumor-derived EVs carry oncogenic cargo (DNA mutations, tumor-specific RNAs, oncoproteins) that can serve as a fingerprint of the cancer. Studies have shown that EVs can reveal the presence of cancer and even reflect tumor characteristics such as mutational status. For example, DNA in EVs isolated from melanoma patients has been found to harbor mutations that closely match those in the primary tumor tissue [6, 7]. EVs also contain tumor-associated RNAs like microRNAs (miRNAs) and proteins that differ from those in EVs shed by normal cells. Because EVs package these biomarkers in a protective vesicle, protecting them from degradation, long RNAs within EVs remain intact despite RNases in the blood. The inherent stability of EV-packaged RNAs enhances the sensitivity and reliability of EV-based assays compared to freely circulating nucleic acids, offering improved sensitivity for tumor biomarker detection and monitoring in biofluids8. Importantly, EVs can complement other liquid biopsy analytes such as circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), by capturing different molecular aspects of tumor biology.
