Focus on Extracellular Vesicles: Development of Extracellular Vesicle-Based Therapeutic Systems

Many types of cells release phospholipid membrane vesicles thought to play key roles in cell-cell communication, antigen presentation, and the spread of infectious agents. Extracellular vesicles (EVs) carry various proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs), like a “message in a bottle” to cells in remote locations. The encapsulated molecules are protected from multiple types of degradative enzymes in body fluids, making EVs ideal for delivering drugs. This review presents an overview of the potential roles of EVs as natural drugs and novel drug-delivery systems.     

ALCAM/CD166 Is Involved in the Binding and Uptake of Cancer-Derived Extracellular Vesicles

Colorectal cancer (CRC) and ovarian cancer (OvC) patients frequently develop peritoneal metastasis, a condition associated with a very poor prognosis. In these cancers, tumor-derived extracellular vesicles (EVs) cause immunosuppression, facilitate the direct attachment and invasion of cancer cells through the mesothelium, induce the conversion of peritoneal mesothelial cells (PMCs) into cancer-associated fibroblasts (CAFs) and transfer a more aggressive phenotype amongst cancer cells. Although the promoting role of EVs in CRC and OvC peritoneal metastasis is well established, the specific molecules that mediate the interactions between tumor-derived EVs and immune and non-immune target cells remain elusive. Here, we employed the SKOV-3 (ovarian adenocarcinoma) and Colo-320 (colorectal adenocarcinoma) human cell lines as model systems to study the interactions and uptake of EVs produced by ovarian carcinoma and colorectal carcinoma cells, respectively. We established that the adhesion molecule ALCAM/CD166 is involved in the interaction of cancer-derived EVs with recipient cancer cells (a process termed “EV binding” or “EV docking”) and in their subsequent uptake by these cells. The identification of ALCAM/CD166 as a molecule mediating the docking and uptake of CRC and OvC-derived EVs may be potentially exploited to block the peritoneal metastasis cascade promoted by EVs in CRC and OvC patients.     

Critical Review of the Evolution of Extracellular Vesicles’ Knowledge: From 1946 to Today

Extracellular vesicles (EVs) are a family of particles/vesicles present in blood and body fluids, composed of phospholipid bilayers that carry a variety of molecules that can mediate cell communication, modulating crucial cell processes such as homeostasis, induction/dampening of inflammation, and promotion of repair. Their existence, initially suspected in 1946 and confirmed in 1967, spurred a sharp increase in the number of scientific publications. Paradoxically, the increasing interest for EV content and function progressively reduced the relevance for a precise nomenclature in classifying EVs, therefore leading to a confusing scientific production. The aim of this review was to analyze the evolution of the progress in the knowledge and definition of EVs over the years, with an overview of the methodologies used for the identification of the vesicles, their cell of origin, and the detection of their cargo. The MISEV 2018 guidelines for the proper recognition nomenclature and ways to study EVs are summarized. The review finishes with a “more questions than answers” chapter, in which some of the problems we still face to fully understand the EV function and potential as a diagnostic and therapeutic tool are analyzed.     

Stem Cells-Derived Extracellular Vesicles: Potential Therapeutics for Wound Healing in Chronic Inflammatory Skin Diseases

Endosome-derived small extracellular vesicles (EVs), often referred to as exosomes, are produced by almost all, if not all, cell types, and are critical for intercellular communication. They are composed of a lipid bilayer associated with membrane proteins and contain a payload of lipids, proteins and regulatory RNAs that depends on the parental cell physiological condition. By transferring their “cargo”, exosomes can modulate the phenotype of neighboring and distant cells. Stem cells (SC) were widely studied for therapeutic applications regarding their regenerative/reparative potential as well as their immunomodulatory properties. Whether from autologous or allogeneic source, SC beneficial effects in terms of repair and regeneration are largely attributed to their paracrine signaling notably through secreted EVs. Subsequently, SC-derived EVs have been investigated for the treatment of various diseases, including inflammatory skin disorders, and are today fast-track cell-free tools for regenerative/reparative strategies. Yet, their clinical application is still facing considerable challenges, including production and isolation procedures, and optimal cell source. Within the emerging concept of “allogeneic-driven benefit” for SC-based therapies, the use of EVs from allogeneic sources becomes the pragmatic choice although a universal allogeneic cell source is still needed. As a unique temporary organ that ensures the mutual coexistence of two allogeneic organisms, mother and fetus, the human placenta offers a persuasive allogeneic stem cell source for development of therapeutic EVs. Advancing cell-free therapeutics nurtures great hope and provides new perspectives for the development of safe and effective treatment in regenerative/reparative medicine and beyond. We will outline the current state of the art in regard of EVs, summarize their therapeutic potential in the context of skin inflammatory disorders, and discuss their translational advantages and hurdles.     

A Microfluidic Platform to Monitor Real-Time Effects of Extracellular Vesicle Exchange between Co-Cultured Cells across Selectively Permeable Barriers

Exosomes and other extracellular vesicles (EVs) play a significant yet poorly understood role in cell–cell communication during homeostasis and various pathological conditions. Conventional in vitro and in vivo approaches for studying exosome/EV function depend on time-consuming and expensive vesicle purification methods to obtain sufficient vesicle populations. Moreover, the existence of various EV subtypes with distinct functional characteristics and submicron size makes their analysis challenging. To help address these challenges, we present here a unique chip-based approach for real-time monitoring of cellular EV exchange between physically separated cell populations. The extracellular matrix (ECM)-mimicking Matrigel is used to physically separate cell populations confined within microchannels, and mimics tissue environments to enable direct study of exosome/EV function. The submicron effective pore size of the Matrigel allows for the selective diffusion of only exosomes and other smaller EVs, in addition to soluble factors, between co-cultured cell populations. Furthermore, the use of PEGDA hydrogel with a very small pore size of 1.2 nm in lieu of Matrigel allows us to block EV migration and, therefore, differentiate EV effects from effects that may be mediated by soluble factors. This versatile platform bridges purely in vitro and in vivo assays by enabling studies of EV-mediated cellular crosstalk under physiologically relevant conditions, enabling future exosome/EV investigations across multiple disciplines through real-time monitoring of vesicle exchange.     

Extracellular Vesicles in Facial Aesthetics: A Review

Facial aesthetics involve the application of non-invasive or minimally invasive techniques to improve facial appearance. Currently, extracellular vesicles (EVs) are attracting much interest as nanocarriers in facial aesthetics due to their lipid bilayer membrane, nanosized dimensions, biological origin, intercellular communication ability, and capability to modulate the molecular activities of recipient cells that play important roles in skin rejuvenation. Therefore, EVs have been suggested to have therapeutic potential in improving skin conditions, and these highlighted the potential to develop EV-based cosmetic products. This review summarizes EVs’ latest research, reporting applications in facial aesthetics, including scar removal, facial rejuvenation, anti-aging, and anti-pigmentation. This review also discussed the advanced delivery strategy of EVs, the therapeutic potential of plant EVs, and clinical studies using EVs to improve skin conditions. In summary, EV therapy reduces scarring, rejuvenates aging skin, and reduces pigmentation. These observations warrant the development of EV-based cosmetic products. However, more efforts are needed to establish a large-scale EV production platform that can consistently produce functional EVs and understand EVs’ underlying mechanism of action to improve their efficacy.     

Glioblastoma Extracellular Vesicle-Specific Peptides Inhibit EV-Induced Neuronal Cytotoxicity

WHO Grade 4 IDH-wild type astrocytoma (GBM) is the deadliest brain tumor with a poor prognosis. Meningioma (MMA) is a more common “benign” central nervous system tumor but with significant recurrence rates. There is an urgent need for brain tumor biomarkers for early diagnosis and effective treatment options. Extracellular vesicles (EVs) are tiny membrane-enclosed vesicles that play essential functions in cell-to-cell communications among tumor cells. We aimed to identify epitopes of brain tumor EVs by phage peptide libraries. EVs from GBM plasma, MMA plasma, or brain tumor cell lines were used to screen phage-displayed random peptide libraries to identify high-affinity peptides. We purified EVs from three GBM plasma pools (23 patients), one MMA pool (10 patients), and four brain tumor cell lines. We identified a total of 21 high-affinity phage peptides (12 unique) specific to brain tumor EVs. The peptides shared high sequence homologies among those selected by the same EVs. Dose–response ELISA demonstrated that phage peptides were specific to brain tumor EVs compared to controls. Peptide affinity purification identified unique brain tumor EV subpopulations. Significantly, GBM EV peptides inhibit brain tumor EV-induced complement-dependent cytotoxicity (necrosis) in neurons. We conclude that phage display technology could identify specific peptides to isolate and characterize tumor EVs.     

Characterization of Urine Stem Cell-Derived Extracellular Vesicles Reveals B Cell Stimulating Cargo

Elucidation of the biological functions of extracellular vesicles (EVs) and their potential roles in physiological and pathological processes is an expanding field of research. In this study, we characterized USC–derived EVs and studied their capacity to modulate the human immune response in vitro. We found that the USC–derived EVs are a heterogeneous population, ranging in size from that of micro–vesicles (150 nm–1 μm) down to that of exosomes (60–150 nm). Regarding their immunomodulatory functions, we found that upon isolation, the EVs (60–150 nm) induced B cell proliferation and IgM antibody secretion. Analysis of the EV contents unexpectedly revealed the presence of BAFF, APRIL, IL–6, and CD40L, all known to play a central role in B cell stimulation, differentiation, and humoral immunity. In regard to their effect on T cell functions, they resembled the function of mesenchymal stem cell (MSC)–derived EVs previously described, suppressing T cell response to activation. The finding that USC–derived EVs transport a potent bioactive cargo opens the door to a novel therapeutic avenue for boosting B cell responses in immunodeficiency or cancer.     

Proteomic Landscape of Extracellular Vesicles for Diffuse Large B-Cell Lymphoma Subtyping

The role of extracellular vesicles (EVs) proteome in diffuse large B-cell lymphoma (DLBCL) pathology, subclassification, and patient screening is unexplored. We analyzed by state-of-the-art mass spectrometry the whole cell and secreted extracellular vesicles (EVs) proteomes of different molecular subtypes of DLBCL, germinal center B cell (GCB subtype), and activated B cell (ABC subtype). After quality control assessment, we compared whole-cell and secreted EVs proteomes of the two cell-of-origin (COO) categories, GCB and ABC subtypes, resulting in 288/1115 significantly differential expressed proteins from the whole-cell proteome and 228/608 proteins from EVs (adjust p-value < 0.05/p-value < 0.05). In our preclinical model system, we demonstrated that the EV proteome and the whole-cell proteome possess the capacity to separate cell lines into ABC and GCB subtypes. KEGG functional analysis and GO enrichment analysis for cellular component, molecular function, and biological process of differential expressed proteins (DEP) between ABC and GCB EVs showed a significant enrichment of pathways involved in immune response function. Other enriched functional categories for DEPs constitute cellular signaling and intracellular trafficking such as B-cell receptor (BCR), Fc_gamma R-mediated phagocytosis, ErbB signaling, and endocytosis. Our results suggest EVs can be explored as a tool for patient diagnosis, follow-up, and disease monitoring. Finally, this study proposes novel drug targets based on highly expressed proteins, for which antitumor drugs are available suggesting potential combinatorial therapies for aggressive forms of DLBCL. Data are available via ProteomeXchange with identifier PXD028267.     

Hypoxic Conditions Promote the Angiogenic Potential of Human Induced Pluripotent Stem Cell-Derived Extracellular Vesicles

Stem cells secrete paracrine factors including extracellular vesicles (EVs) which can mediate cellular communication and support the regeneration of injured tissues. Reduced oxygen (hypoxia) as a key regulator in development and regeneration may influence cellular communication via EVs. We asked whether hypoxic conditioning during human induced pluripotent stem cell (iPSC) culture effects their EV quantity, quality or EV-based angiogenic potential. We produced iPSC-EVs from large-scale culture-conditioned media at 1%, 5% and 18% air oxygen using tangential flow filtration (TFF), with or without subsequent concentration by ultracentrifugation (TUCF). EVs were quantified by tunable resistive pulse sensing (TRPS), characterized according to MISEV2018 guidelines, and analyzed for angiogenic potential. We observed superior EV recovery by TFF compared to TUCF. We confirmed hypoxia efficacy by HIF-1α stabilization and pimonidazole hypoxyprobe. EV quantity did not differ significantly at different oxygen conditions. Significantly elevated angiogenic potential was observed for iPSC-EVs derived from 1% oxygen culture by TFF or TUCF as compared to EVs obtained at higher oxygen or the corresponding EV-depleted soluble factor fractions. Data thus demonstrate that cell-culture oxygen conditions and mode of EV preparation affect iPSC-EV function. We conclude that selecting appropriate protocols will further improve production of particularly potent iPSC-EV-based therapeutics.     

Extracellular Vesicles in Diagnosing Chronic Coronary Syndromes the Bumpy Road to Clinical Implementation

Coronary artery disease (CAD), comprising both acute coronary syndromes (ACS) and chronic coronary syndromes (CCS), remains one of the most important killers throughout the entire world. ACS is often quickly diagnosed by either deviation on an electrocardiogram or elevated levels of troponin, but CCS appears to be more complicated. The most used noninvasive strategies to diagnose CCS are coronary computed tomography and perfusion imaging. Although both show reasonable accuracy (80–90%), these modalities are becoming more and more subject of debate due to costs, radiation and increasing inappropriate use in low-risk patients. A reliable, blood-based biomarker is not available for CCS but would be of great clinical importance. Extracellular vesicles (EVs) are lipid-bilayer membrane vesicles containing bioactive contents e.g., proteins, lipids and nucleic acids. EVs are often referred to as the “liquid biopsy” since their contents reflect changes in the condition of the cell they originate from. Although EVs are studied extensively for their role as biomarkers in the cardiovascular field during the last decade, they are still not incorporated into clinical practice in this field. This review provides an overview on EV biomarkers in CCS and discusses the clinical and technological aspects important for successful clinical application of EVs.     

Muscle and Adipose Tissue Communicate with Extracellular Vesicles

In numerous body locations, muscle and adipose tissue are in close contact. Both tissues are endocrine organs that release cytokines, playing a crutial role in the control of tissue homeostasis in health and diseases. Within this context, the identification of the signals involved in muscle–fat crosstalk has been a hot topic over the last 15 years. Recently, it has been discovered that adipose tissue and muscles can release information embedded in lipid-derived nanovesicles called ‘extracellular vesicles’ (EVs), which can modulate the phenotype and the homeostasis of neighboring recipient cells. This article reviews knowledge on EVs and their involvement in the communication between adipose tissue and muscle in several body locations. Even if the works are scarce, they have revolutionized our vision in the field of metabolic and cardiovascular diseases.     

Mechanisms Underlying Connexin Hemichannel Activation in Disease

Gap junctions and connexin hemichannels mediate intercellular and extracellular communication, respectively. While gap junctions are seen as the “good guys” by controlling homeostasis, connexin hemichannels are considered as the “bad guys”, as their activation is associated with the onset and dissemination of disease. Open connexin hemichannels indeed mediate the transport of messengers between the cytosol and extracellular environment and, by doing so, fuel inflammation and cell death in a plethora of diseases. The present mini-review discusses the mechanisms involved in the activation of connexin hemichannels during pathology.     

Size-Exclusion Chromatography Separation Reveals That Vesicular and Non-Vesicular Small RNA Profiles Differ in Cell Free Urine

Urinary extracellular vesicles (EVs) and their RNA cargo are a novel source of biomarkers for various diseases. We aimed to identify the optimal method for isolating small (<200 nm) EVs from human urine prior to small RNA analysis. EVs from filtered healthy volunteer urine were concentrated using three methods: ultracentrifugation (UC); a precipitation-based kit (PR); and ultrafiltration (UF). EVs were further purified by size-exclusion chromatography (SEC). EV preparations were analysed with transmission electron microscopy (TEM), Western blotting, nanoparticle tracking analysis (NTA) and an Agilent Bioanalyzer Small RNA kit. UF yielded the highest number of particles both before and after SEC. Small RNA analysis from UF-concentrated urine identified two major peaks at 10–40 nucleotides (nt) and 40–80 nt. In contrast, EV preparations obtained after UC, PR or SEC combined with any concentrating method, contained predominantly 40–80 nt sized small RNA. Protein fractions from UF+SEC contained small RNA of 10–40 nt in size (consistent with miRNAs). These data indicate that most of the microRNA-sized RNAs in filtered urine are not associated with small-sized EVs, and highlights the importance of removing non-vesicular proteins and RNA from urine EV preparations prior to small RNA analysis.     

Direct Cell-Cell Communication via Membrane Pores,Gap Junction Channels,and Tunneling Nanotubes: Medical Relevance of Mitochondrial Exchange

The history of direct cell-cell communication has evolved in several small steps. First discovered in the 1930s in invertebrate nervous systems, it was thought at first to be an exception to the “cell theory”, restricted to invertebrates. Surprisingly, however, in the 1950s, electrical cell-cell communication was also reported in vertebrates. Once more, it was thought to be an exception restricted to excitable cells. In contrast, in the mid-1960s, two startling publications proved that virtually all cells freely exchange small neutral and charged molecules. Soon after, cell-cell communication by gap junction channels was reported. While gap junctions are the major means of cell-cell communication, in the early 1980s, evidence surfaced that some cells might also communicate via membrane pores. Questions were raised about the possible artifactual nature of the pores. However, early in this century, we learned that communication via membrane pores exists and plays a major role in medicine, as the structures involved, “tunneling nanotubes”, can rescue diseased cells by directly transferring healthy mitochondria into compromised cells and tissues. On the other hand, pathogens/cancer could also use these communication systems to amplify pathogenesis. Here, we describe the evolution of the discovery of these new communication systems and the potential therapeutic impact on several uncurable diseases.     

Circulating Extracellular Vesicles: The Missing Link between Physical Exercise and Depression Management?

Depression is associated with an increased risk of aging-related diseases. It is also seemingly a common psychological reaction to pandemic outbreaks with forced quarantines and lockdowns. Thus, depression represents, now more than ever, a major global health burden with therapeutic management challenges. Clinical data highlights that physical exercise is gaining momentum as a non-pharmacological intervention in depressive disorders. Although it may contribute to the reduction of systemic inflammation associated with depression, the mechanisms underlying the beneficial physical exercise effects in emotional behavior remain to be elucidated. Current investigations indicate that a rapid release of extracellular vesicles into the circulation might be the signaling mediators of systemic adaptations to physical exercise. These biological entities are now well-established intercellular communicators, playing a major role in relevant physiological and pathophysiological functions, including brain cell–cell communication. We also reviewed emerging evidence correlating depression with modified circulating extracellular vesicle surfaces and cargo signatures (e.g., microRNAs and proteins), envisioned as potential biomarkers for diagnosis, efficient disease stratification and appropriate therapeutic management. Accordingly, the clinical data summarized in the present review prompted us to hypothesize that physical exercise-related circulating extracellular vesicles contribute to its antidepressant effects, particularly through the modulation of inflammation. This review sheds light on the triad “physical exercise–extracellular vesicles–depression” and suggests new avenues in this novel emerging field.     

Profiling Blood Serum Extracellular Vesicles in Plaque Psoriasis and Psoriatic Arthritis Patients Reveals Potential Disease Biomarkers

Psoriasis vulgaris (PsV) and psoriatic arthritis (PsA) are inflammatory diseases with unresolved pathophysiological aspects. Extracellular vesicles (EVs) play an important role in intercellular communication. We compared the miRNA contents and surface proteome of the EVs in the blood serum of PsV and PsA patients to healthy controls. Size-exclusion chromatography was used to isolate EVs from the blood serum of 12 PsV patients, 12 PsA patients and 12 healthy control subjects. EV samples were characterized and RNA sequencing was used to identify differentially enriched EV-bound miRNAs. We found 212 differentially enriched EV-bound miRNAs present in both PsV and PsA groups—a total of 13 miRNAs at FDR ≤ 0.05. The predicted target genes of these miRNAs were significantly related to lesser known but potentially disease-relevant pathways. The EV array revealed that PsV patient EV samples were significantly enriched with CD9 EV-marker compared to controls. Analysis of EV-bound miRNAs suggests that signaling via EVs in the blood serum could play a role in the pathophysiological processes of PsV and PsA. EVs may be able to fill the void in clinically applicable diagnostic and prognostic biomarkers for PsV and PsA.