This review article presents a detailed examination of the integral role that electrochemical detection of extracellular vesicles (EVs) plays, particularly focusing on the potential application for early disease diagnostics through EVs biomarker analysis. Through an exploration of the benefits and challenges presented by electrochemical detection vetted for protein, lipid, and nucleic acid biomarker analysis, we underscore the significance of these techniques. Evidence from recent studies renders this detection modality imperative in identifying diverse biomarkers from EVs, leading to early diagnosis of diseases such as cancer and neurodegenerative disorders. Recent advancements that have led to enhanced sensitivity, specificity and point-of-care testing (POCT) potential are elucidated, along with equipment deployed for electrochemical detection. The review concludes with a contemplation of future perspectives, recognizing the potential shifts in disease diagnostics and prognosis, necessary advances for broad adoption, and potential areas of ongoing research. The objective is to propel further investigation into this rapidly burgeoning field, thereby facilitating a potential paradigm shift in disease detection, monitoring, and treatment toward human health management.
Pancreatic cancer is a highly lethal and metastatic malignancy, mainly because it often remains undetected until advanced stages due to the limitations of current diagnostic methods, rendering currently available therapies ineffective. Therefore, it is imperative to identify useful biomarkers for early diagnosis and new therapeutic targets for pancreatic cancer. Recently, extracellular vesicles have emerged as promising biomarkers for the diagnosis and prognosis of pancreatic cancer. Given their presence in various bodily fluids, extracellular vesicles offer a non-invasive approach through liquid biopsy to detect and monitor cancer progression. In this review, we comprehensively examine the multifaceted roles of extracellular vesicles in the progression of cancer, while also exploring their potential as diagnostic, prognostic, and therapeutic biomarkers in the context of pancreatic cancer.
Aim: Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have been reported to hold great potential as cell-free therapies due to their low immunogenicity and minimal toxicity. However, the large doses of MSC-EVs that are required for their clinical application highlight the urgency of finding a large-scale system for MSC-EV manufacture. In this study, we aimed to set up a hollow fiber bioreactor system for the continuous homogenous production of functional and high-quality MSC-EVs.
Methods: MSC lines from two donors were immortalized (iMSC) and inoculated into hollow fiber bioreactors. Throughout 4 weeks, conditioned medium was daily harvested. iMSC-EVs were purified and characterized for content, immunophenotype, size, and functionality and compared to 2D cultured iMSC.
Results: The iMSC inoculated into the bioreactor remained viable during the whole culture period, and they maintained their MSC phenotype at the end of EV production. Our results showed that the bioreactor system allows to obtain 3D-cultured iMSC-derived EVs (3D-EVs) that are comparable to flask (2D)-cultured iMSC-derived EVs (2D-EVs) in terms of protein and lipid content, size, and phenotype. We also confirm that 3D-derived EVs exhibit comparable functionality to 2D-EVs, showing pro-angiogenic potential in a dose-dependent manner.
Conclusions: These findings suggest that setting up a hollow fiber bioreactor system inoculating immortalized MSC lines facilitates the large-scale, functional, and high-quality production of iMSC-EVs. Our results emphasize the great potential of this production methodology to standardize EV production in the pursuit of clinical applications.
Aim: This study aimed to systematically compare four casein micelle removal methods on the particle and protein characteristics of the isolated human milk EVs.
Methods: The defatted milk was treated with 1% sodium citrate, 20 mM ethylenediaminetetraacetic acid (EDTA), 1% acetic acid, or 1% chymosin/calcium chloride for 30 min at 4 °C to remove casein micelles. EV isolation was performed using qEV size exclusion chromatography. Milk turbidity at the optical density 350 nm and dot immunoblot with casein antibody were applied to monitor the qEV fractions. Particle analyses were performed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The enrichment of human milk EV markers, i.e., tetraspanins, Alix, lactadherin, butyrophilin, and xanthine dehydrogenase, and casein depletion capabilities were evaluated by proteomics and immunoblotting.
Results: Compared to the untreated condition, sodium citrate and EDTA decreased milk turbidity by disrupting casein micelles, while acetic acid and chymosin removed them by inducing precipitation/coagulation. All treatments shifted casein immunoreactivity in the qEV fractions from large micelles (the exclusion volume) to small molecular sizes (gel-infiltrated fractions). Acidification affected human milk EV morphology, while EDTA, acetic acid, and chymosin methods slightly altered EV particle numbers. Different casein micelle removal methods confer different degrees of human milk EV marker enrichment and casein depletion. The method performances could be ranked as follows: chymosin > EDTA > acetic acid > sodium citrate.
Conclusion: Our findings suggest that chymosin and EDTA should be considered as the method of choice for casein micelle removal in future studies involving human milk EV isolation and characterization.
Dysregulated intercellular communication is a key feature driving cancer progression. Recently, extracellular vesicles (EVs) have added a new channel to this dense communication network. Despite solid evidence that EVs are central mediators of dysregulated signaling in onco-pathological settings, this has yet to be translated into clinically actionable strategies. The heterogeneity of EV cargo molecules, plasticity of biogenesis routes, and large overlap with their role in physiological communication, complicate a potential targeting strategy. However, recent work has linked EV biology to perhaps the "most druggable" proteins - G protein-coupled receptors (GPCRs). GPCR targeting accounts for ~60% of drugs in development and more than a third of all currently approved drugs, spanning almost all areas of medicine. Although several GPCRs have been linked to cancer initiation and progression, relatively few agents have made it into oncological regimes, suggesting that their potential is underexploited. Herein, we examine the molecular mechanisms linking GPCRs to EV communication in cancer settings. We propose that GPCRs hold potential in the search for EV-targeting in oncology.
Aim: Tumor-infiltrating macrophages are tumor-promoting and show activation of the unfolded protein response (UPR). The transcription factor X-box binding protein 1 (XBP1) is a conserved element of the UPR. Upon activation, the UPR mediates the transcriptional activation of pro-inflammatory cytokines and immune suppressive factors, hence contributing to immune dysregulation in the tumor microenvironment (TME). miR-214 is a short non-coding miRNA that targets the 3’-UTR of the Xbp1 transcript. Here, we tested a new method to efficiently deliver miR-214 to macrophages as a potential new therapeutic approach.
Methods: We generated miR-214-laden extracellular vesicles (iEV-214) in a murine B cell and demonstrated that iEV-214 were enriched in miR-214 between 1,500 - 2,000 fold relative to control iEVs.
Results: Bone marrow-derived macrophages (BMDM) treated with iEV-214 for 24 h underwent a specific enrichment in miR-214, suggesting transfer of the miR-214 payload from the iEVs to macrophages. iEV-214 treatment of BMDM markedly reduced (> 50%) Xbp1 transcription under endoplasmic reticulum stress conditions compared to controls. Immune-related genes downstream of XBP1s (Il-6, Il-23p19, and Arg1) were also reduced by 69%, 51%, and 34%, respectively.
Conclusions: Together, these data permit to conclude that iEV-214 are an efficient strategy to downregulate the expression of Xbp1 mRNA and downstream genes in macrophages. We propose miRNA-laden iEVs are a new approach to target macrophages and control immune dysregulation in the TME.
Aim: The objective of the present study was to determine the variability of platelet lysate-derived extracellular vesicles (pEV), in terms of characteristics and functionality through wound healing assays, when isolated either from platelet concentrates (PC, obtained from 5 donors) or from multiple PC (MPC, that is 50 donors).
Methods: pEV were isolated under GMP-like conditions in a clean room using Size Exclusion Chromatography (SEC). The differential characteristics between pEV obtained from PC (PC-EV) or MPC (MPC-EV) were evaluated by means of protein concentration, Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), and flow cytometry using the MACSPlex™ arrays for surface analysis profiling of EV. The functionality of the isolated pEV was determined in cell culture by metabolic activity and LDH activity determination and through a wound healing assay after 24 h treatment.
Results: No significant differences were observed in the pEV characteristics evaluated, whether isolated from PC or MPC. As regards functionality, a higher wound closure percentage was obtained in those pEV pools isolated from PC (5 donors). No differences in the coefficient of variation (CV) were found when comparing all the evaluated variables of pEV derived either from PC (5 donors) or from MPC (50 donors).
Conclusion: Our findings challenge the necessity of a larger donor pool for pEV isolation, revealing no significant variations in the analyzed variables of MPC-EV and PC-EV. Notably, our results suggest that, unlike platelet concentrates, a high number of donors is not required to reduce the variability of pEV, showing that the pool of only 5 donors can provide a consistent and reliable therapeutic product.
Mitochondria dysfunction is increasingly recognized as a critical factor in various pathogenic processes. The mechanism governing mitochondrial quality control serves as an adaptive response, ensuring the preservation of mitochondrial morphology, quantity, and overall function, crucial for cell survival. The generation of
Arthritis, a diverse group of inflammatory joint disorders, poses great challenges in early diagnosis and targeted treatment. Timely intervention is imperative, yet conventional diagnostic methods are not able to detect subtle early symptoms. Hence, there is an urgent need for specific biomarkers that discriminate between different arthritis forms and for early diagnosis. The pursuit of such precise diagnostic tools has prompted a growing interest in extracellular vesicles (EVs). EVs, released by cells in a regulated fashion, are detectable in body fluids, including synovial fluid (SF), which fills the joint space. They provide insights into the intricate molecular landscapes of arthritis, and this has stimulated the search for minimally invasive EV-based diagnostics. As such, the analysis of EVs in SF has become a focus for identifying EV-based biomarkers for joint disease endotyping, prognosis, and progression. EVs are composed of a lipid bilayer and a wide variety of different cargo types, of which proteins and RNAs are widely investigated. In contrast, membrane lipids of EVs, especially the abundance, presence, or absence of specific lipids and their contribution to the biological activity of EVs, are largely overlooked in EV research. Furthermore, the identification of specific combinations of different EV components acting in concert in EVs can fuel the definition of composite biomarkers. We here provide a state-of-the-art overview of the knowledge on
Host-bacteria and bacteria-bacteria interactions can be facilitated by extracellular vesicles (EVs) secreted by both human and bacterial cells. Human and bacterial EVs (BEVs) propagate and transfer immunogenic cargos that may elicit immune responses in nearby or distant recipient cells/tissues. Hence, direct colonization of tissues by bacterial cells is not required for immunogenic stimulation. This phenomenon is important in the feto-maternal interface, where optimum tolerance between the mother and fetus is required for a successful pregnancy. Though the intrauterine cavity is widely considered sterile, BEVs from diverse sources have been identified in the placenta and amniotic cavity. These BEVs can be internalized by human cells, which may help them evade host immune surveillance. Though it appears logical, whether bacterial cells internalize human EVs or human EV cargo is yet to be determined. However, the presence of BEVs in placental tissues or amniotic cavity is believed to trigger a low-grade immune response that primes the fetal immune system for ex-utero survival, but is insufficient to disrupt the progression of pregnancy or cause immune intolerance required for adverse pregnancy events. Nevertheless, the exchange of bioactive cargos between human and BEVs, and the mechanical underpinnings and health implications of such interactions, especially during pregnancy, are still understudied. Therefore, while focusing on the feto-maternal interface, we discussed how human cells take up BEVs and whether bacterial cells take up human EVs or their cargo, the exchange of cargos between human and BEVs, host cell (feto-maternal) inflammatory responses to BEV immunogenic stimulation, and associations of these interactions with fetal immune priming and adverse reproductive outcomes such as preeclampsia and preterm birth.
Extracellular vesicles (EVs) contribute to the development of cancer in various ways. Non-Hodgkin lymphoma (NHL) is a cancer of mature lymphocytes and the most common hematological malignancy globally. The most common form of NHL, diffuse large B-cell lymphoma (DLBCL), is primarily treated with chemotherapy, autologous stem cell transplantation (ASCT), and/or chimeric antigen receptor T-cell (CAR-T) therapy. With NHL disease progression and its treatment, extracellular vesicles play remarkable roles in influencing outcomes. This finding can be utilized for therapeutic intervention to improve patient outcomes for NHL. This review focuses on the multifaceted roles of EVs with NHL and its potential for guiding patient care.