ABSTRACT
In the relentless pursuit of innovative diagnostic tools for cancer, this review illuminates the cutting-edge realm of extracellular vesicles (EVs) and their biomolecular cargo detection through advanced optical biosensing techniques with a primary emphasis on their significance in cancer diagnosis. From the sophisticated domain of nanomaterials to the precision of surface plasmon resonance, we herein examine the diverse universe of optical biosensors, emphasizing their specified applications in cancer diagnosis. Exploring and understanding the details of EVs, we present innovative applications of enhancing and blending signals, going beyond the limits to sharpen our ability to sense and distinguish with greater sensitivity and specificity. Our special focus on cancer diagnosis underscores the transformative potential of optical biosensors in early detection and personalized medicine. This review aims to help guide researchers, clinicians, and enthusiasts into the captivating domain where light meets cellular secrets, creating innovative opportunities in cancer diagnostics.
ABSTRACT
Cellular crosstalk between hematopoietic stem/progenitor cells and the bone marrow (BM) niche is vital for the development and maintenance of myeloid malignancies. These compartments can communicate via bidirectional transfer of extracellular vesicles (EVs). EV trafficking in acute myeloid leukemia (AML) plays a crucial role in shaping the BM microenvironment into a leukemia-permissive niche. Although several EV isolation methods have been developed, it remains a major challenge to define the most accurate and reliable procedure. Here, we tested the efficacy and functional assay compatibility of four different EV isolation methods in leukemia-derived EVs: (1) membrane affinity-based: exoEasy Kit alone and (2) in combination with Amicon filtration; (3) precipitation: ExoQuick-TC; and (4) ultracentrifugation (UC). Western blot analysis of EV fractions showed the highest enrichment of EV marker expression (e.g., CD63, HSP70, and TSG101) by precipitation with removal of overabundant soluble proteins [e.g., bovine serum albumin (BSA)], which were not discarded using UC. Besides the presence of damaged EVs after UC, intact EVs were successfully isolated with all methods as evidenced by highly maintained spherical- and cup-shaped vesicles in transmission electron microscopy. Nanoparticle tracking analysis of EV particle size and concentration revealed significant differences in EV isolation efficacy, with exoEasy Kit providing the highest EV yield recovery. Of note, functional assays with exoEasy Kit-isolated EVs showed significant toxicity towards treated target cells [e.g., mesenchymal stromal cells (MSCs)], which was abrogated when combining exoEasy Kit with Amicon filtration. Additionally, MSC treated with green fluorescent protein (GFP)-tagged exoEasy Kit-isolated EVs did not show any EV uptake, while EV isolation by precipitation demonstrated efficient EV internalization. Taken together, the choice of EV isolation procedure significantly impacts the yield and potential functionality of leukemia-derived EVs. The cheapest method (UC) resulted in contaminated and destructed EV fractions, while the isolation method with the highest EV yield (exoEasy Kit) appeared to be incompatible with functional assays. We identified two methods (precipitation-based ExoQuick-TC and membrane affinity-based exoEasy Kit combined with Amicon filtration) yielding pure and intact EVs, also suitable for application in functional assays. This study highlights the importance of selecting the right EV isolation method depending on the desired experimental design.
ABSTRACT
Various types of cells secrete extracellular vesicle (EVs) which contain proteins, lipids and nucleic acids and play important roles in inter-cellular signalling and pathological processes to impact the recipient cells. EVs have demonstrated their potential as biomarkers for disease and as therapeutic agents in regenerative medicine. In recent times, EVs derived from mesenchymal stem cells (MSCs), which are widely used as a promising medicinal product in many clinical applications, are being tested in many preclinical trials. However, the lack of standardization of MSC-derived EV isolation and analysis methods, restricts the utility of MSC-derived EVs in the clinical setting. Here, we focused on optimising the isolation method for EVs derived from MSCs. Four samples of EVs were isolated from human adipose derived MSC culture medium by differential ultracentrifugation with three different ultracentrifuge durations to investigate the influence of ultracentrifuge time on quality and quantity of MSC-derived EVs. Additionally, we used a commercial kit to extract EVs from MSC cultured medium and compared it with the ultracentrifugation method. The EV samples were then characterised for particle concentration, protein concentration, size distribution and the presence of known EV protein markers, by western blot and flow cytometry. A comparison of these results for the five samples demonstrated that 1 h of differential ultracentrifugation was optimal to isolate high quality and quantity of MSC-derived EVs from MSC cultured medium. Additionally, fluorescence imaging of the freshly isolated vs frozen EVs showed that freshly isolated EVs are taken up by cells more efficiently than frozen EVs. These finding establish a simple and reliable method of EV isolation from MSCs.