Could fluorescence resonance energy transfer (FRET) be harnessed to enable the real-time measurement and uptake of specific EVs in living cells?
That was a key question addressed by Bano et al. (2023),who described a FRET-based sensor named ‘ExoSen’ which was implemented to detect EVs from the A549 lung cancer cell line. Referring to the particular EVs as ‘A549 exosomes’ in a study published in Analytical Chemistry, the group sought to detect and measure EV cellular uptake.
As with all FRET, ExoSen gives off a signal when two fluorescent molecules (a donor and acceptor) are close together. When the donor is excited by a light source, it can transfer its energy to the acceptor, causing a detectable emission of light.
In the case of ExoSen, this pair of fluorescent molecules (ECFP and Venus) were conjugated to mitogen-inducible gene 6 (MIG6). These fluorophores were close, as they were on the same protein, but not close enough to exchange energy from donor to acceptor. The authors report that upon binding of MIG6 to EGFR (which is overexpressed in cancer), a conformational change was induced which brought the fluorophores close enough together to react and emit fluorescence which could be detected using a microplate reader. Ultimately, ExoSen could be used to detect the uptake of EGFR-bearing EVs.
As part of the characterisation work, Tunable Resistive Pulse Sensing (TRPS) was used to provide an in-depth overview of particle size, concentration and zeta potential of the qEV-isolated EVs.1
Given the limitations of 2D cell culture models, there has been a strong focus in recent years to develop improved, more physiologically relevant 3D cell culture models. In a study published in Nano Convergence, Kang et al (2023) describe a gut-liver organ-on-a-chip model and various responses of the respective tissues when cultured with microbiota-derived EVs. With the aim of establishing a model for studying the interaction between microbiota and host cells, the group assessed measures of cell viability and liver functions. With Tunable Resistive Pulse Sensing as the method of choice for characterising the physical characteristics of bacterial EVs, the Exoid was used to measure the particle size distribution and concentration of vesicles isolated from the supernatant of two different bacterial cultures. The authors concluded that these microbiota-derived EVs had a positive effect on liver cell (hepG2 spheroids) survival and liver function, and had the ability to alter albumin and urea secretion in recipient cells.2
In a pilot study published in Pediatric Research, researchers explored the proteomic composition of EVs in healthy children and children with viral pneumonia. Through the use of mass spectrometry-based label-free proteomic analysis, significant differences in the proteomic features of urinary EVs were identified. Differential expression of particular proteins suggested an enrichment of biological pathways primarily associated with neutrophil degranulation, carbohydrate metabolism, and endocytosis in children with viral pneumonia. In addition, 8 proteins were identified as biomarker candidates. Through the use of Tunable Resistive Pulse Sensing, the authors could confirm that the urinary preparations did contain abundant levels of EVs, with size and concentration values being comparable across the groups.3
How might synovial fluid EVs influence the progression of rheumatoid arthritis? To provide insight into this question, Nakamachi et al. (2023) collected fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis and osteoarthritis, cultured the cells and transfected a portion of them with miR-124-3p (a miRNA with known regulatory roles in cell differentiation). They then isolated the EVs secreted by these cells. Through proteomics analyses and studies of EV-miRNA – and EV characterisation via Tunable Resistive Pulse Sensing – the group was able to compare EVs across the different groups, leading them to propose novel mechanisms for EVs in the pathogenesis of rheumatoid arthritis.4
Note that this article has been shared as a preprint only – this means it has not yet been subject to peer review and should be interpreted accordingly.
Tunable Resistive Pulse Sensing featured in a recent and fascinating preprint, where Hansen et al. (2023) describe a new therapeutic strategy aimed at enhancing the efficacy of cancer immunotherapy. While huge progress has been made in developing immunotherapies – a class of therapy which harnesses the body’s own immune system to launch an antitumour response – many patients eligible for treatment do not respond the way researchers hoped that they would. It is thought that high immunological tumour activity could be a prerequisite for an effective response to immunotherapy. To induce high immunological tumour activity, researchers are considering novel approaches to triggering relevant inflammatory pathways. In the current study, Hansen et al. (2023) looked to EVs derived from activated CD4+ T cells, and demonstrated their ability to sensitise macrophages to the activation of the STING pathway. Standing for STimulator of INterferon Genes, STING had already been proposed as a strong candidate for a promoting the inflammatory response in cancer. TRPS was used to determine the size and concentration of the EVs.5