Application of extracellular vesicles in tumor liquid biopsy

Extracellular vesicles (EVs) are released by living cells and play important roles in transporting information between cells. EVs contain physiological and pathological information of the parent cell and can dynamically transmit disease signals. According to different biological pathways, EVs are often classified into apoptotic bodies, microvesicles, exosomes, etc. Due to the size overlap between different vesicles and the inability to determine their origin based on size, accurate differentiation between exosomes and microvesicles is highly challenging. To avoid misunderstandings, the International Society for Extracellular Vesicles (ISEV) recommends using small and large vesicles to distinguish them from each other. EVs have become a new star in the field of tumor liquid biopsy, due to the high variability of biomarkers available. However, the rapid and accurate separation of specific EVs from complex body fluids or cell culture supernatants is an urgent problem. Many researchers are committed to developing new and efficient methods and technologies for EV separation, including size-based ultracentrifugation, size exclusion chromatography, and ultrafiltration; polymer precipitation method based on hydrophobic interactions; immunoaffinity method based on antigen-antibody affinity; affinity methods based on DNA aptamers, microfluidic methods based on size and immune affinity; and so on. With the development of EV isolation and characterization methods, researchers have developed various EV nucleic acid and protein analysis techniques and conducted clinical trials. In recent years, several products have been commercialized, transforming scientific research into productivity. DNA, RNA, and proteins are the most commonly used targets for analysis, such as nucleic acid testing during epidemics and influenza, which uses qRT-PCR technology to analyze virus DNA or RNA and tumor marker screening during physical examinations, which analyzes broad-spectrum cancer-related proteins such as alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), and carbohydrate antigens (CA). EVs are rich in DNA, RNA, and proteins. By analyzing a single component or a combination of multiple components, real-time detection of tumor occurrence, metastasis, and prognosis can be achieved. Nanoscale EVs have played a role in enriching biomarkers, with higher sensitivity than plasma detection. Moreover, the membrane of EVs can effectively prevent RNA and other biomarkers from being degraded by various enzymes present in plasma, further enhancing the sensitivity of EV biomarker detection. Encouraged by the latest research progress, we review the application of EVs in tumor liquid biopsy, introducing the application and progress of EV DNA, RNA, protein, and other biomarkers.Although EVs have great potential, the road ahead is full of difficulties. To achieve large-scale application of EVs in clinical practice, the following problems must be solved. The first is the separation problem. Currently, there are several methods for separating EVs, but none can efficiently separate EVs or even EV subgroups produced by specific cells without introducing impurities. The second is large-scale production. Currently, the main way to obtain EVs is through the separation of cell culture and culture medium. For adherent cells, the surface area of the culture environment limits the number of EVs produced. The third is standardization. ISEV and the Chinese Society for Extracellular Vesicles (CSEV) have developed guidelines and group standards for the naming, acquisition, isolation, characterization, and reporting of EVs. However, there is still no consensus on how to ensure inter-batch stability, absolute quantification, yield, and quality control of EVs, which makes clinical translation difficult. We look forward to these issues being resolved as soon as possible, taking EV research and application to a new level, and thereby benefiting humanity.