Extracellular Vesicles Biogenesis Research Articles

Extracellular Vesicles, Circadian Rhythms, and Cancer: A Comprehensive Review with Emphasis on Hepatocellular Carcinoma

This review comprehensively explores the complex interplay between extracellular vesicles (ECVs)/exosomes and circadian rhythms, with a focus on the role of this interaction in hepatocellular carcinoma (HCC). Exosomes are nanovesicles derived from cells that facilitate intercellular communication by transporting bioactive molecules such as proteins, lipids, and RNA/DNA species. ECVs are implicated in a range of diseases, where they play crucial roles in signaling between cells and their surrounding environment. In the setting of cancer, ECVs are known to influence cancer initiation and progression. The scope of this review extends to all cancer types, synthesizing existing knowledge on the various roles of ECVs. A unique aspect of this review is the emphasis on the circadian-controlled release and composition of exosomes, highlighting their potential as biomarkers for early cancer detection and monitoring metastasis. We also discuss how circadian rhythms affect multiple cancer-related pathways, proposing that disruptions in the circadian clock can alter tumor development and treatment response. Additionally, this review delves into the influence of circadian clock components on ECV biogenesis and their impact on reshaping the tumor microenvironment, a key component driving HCC progression. Finally, we address the potential clinical applications of ECVs, particularly their use as diagnostic tools and drug delivery vehicles, while considering the challenges associated with clinical implementation.

Endosomal protein DENND10/FAM45A integrates extracellular vesicle release with cancer cell migration

BackgroundMounting evidence shows that tumor-derived extracellular vesicles (EVs) are critical constituents in the tumor microenvironment. The composition and function of EVs often change during cancer progression. However, it remains less clear how cancer cells modulate their own EV biogenesis to promote tumor development. The release of EVs is closely linked to the endolysosome system residing within the cell. The current study aims to decipher the role of endosomal protein DENND10 in cancer development.ResultsBioinformatics data mining showed that DENND10 expression is significantly associated with poor prognosis in multiple cancer types and up-regulated in metastatic breast cancer cell lines. DENND10 knockout (DENND10-KO) in breast cancer cells led to defective EV biogenesis due to impaired endolysosomal trafficking. Intriguingly, DENND10-KO cells exhibited reductions in cell spreading, migration, invasion, and metastatic potential in vivo. These deficiencies in cell motility were associated with compromised cytoskeleton organization. Importantly, wild-type conditioned medium or EVs restored the migratory ability and cytoskeletal organization of DENND10-KO cells. Global proteomic profiling revealed that DENND10 depletion led to a distinct EV compositional landscape with remodeled profiles of extracellular matrix (ECM) and adhesion molecules. Consistently, exogenous application of ECM molecules rescued the spreading and migration of DENND10-KO cells.ConclusionsIn summary, our study unveiled DENND10 as an intrinsic regulator of cell migration that modifies the tumor microenvironment through autocrine EV release, which could be exploited for developing targeted therapies for tumor metastasis.

Extracellular vesicles involved in the crosstalk between apoptosis and ferroptosis revealed via a viscosity-sensitive fluorescent probe targeting lysosomes and nucleoli

Advancements in cancer therapy have emerged through the combination of multiple signaling pathways, minimizing drug resistance and off-target effects. Therefore, diverse drug design strategies have been developed, including hybrid apoptosis/ferroptosis pathways. However, not much is known regarding their crosstalk, particularly regarding the details of intracellular and intercellular communication. Extracellular vesicles (EVs) play a crucial role in signal transduction and matter exchange among cells and are categorized into ectosomes, exosomes, and apoptotic bodies of varying sizes. The biogenesis and microenvironment of EVs are crucial for understanding their biological functions; however, they are still under debate. Herein, we used a fluorescent probe with viscosity sensitivity that targets lysosomes and nucleoli to detect viscosity alterations during apoptosis and ferroptosis, resulting in triggered green emission. Further fluorescent imaging of cells revealed crosstalk between apoptosis and ferroptosis, detecting apoptotic bodies originating from lysosomes and condensed nucleoli as EVs in hybrid apoptosis/ferroptosis pathways.

Extracellular vesicles involved in the crosstalk between apoptosis and ferroptosis revealed via a viscosity-sensitive fluorescent probe targeting lysosomes and nucleoli

Advancements in cancer therapy have emerged through the combination of multiple signaling pathways, minimizing drug resistance and off-target effects. Therefore, diverse drug design strategies have been developed, including hybrid apoptosis/ferroptosis pathways. However, not much is known regarding their crosstalk, particularly regarding the details of intracellular and intercellular communication. Extracellular vesicles (EVs) play a crucial role in signal transduction and matter exchange among cells and are categorized into ectosomes, exosomes, and apoptotic bodies of varying sizes. The biogenesis and microenvironment of EVs are crucial for understanding their biological functions; however, they are still under debate. Herein, we used a fluorescent probe with viscosity sensitivity that targets lysosomes and nucleoli to detect viscosity alterations during apoptosis and ferroptosis, resulting in triggered green emission. Further fluorescent imaging of cells revealed crosstalk between apoptosis and ferroptosis, detecting apoptotic bodies originating from lysosomes and condensed nucleoli as EVs in hybrid apoptosis/ferroptosis pathways.

Extracellular Vesicles and Immune System Function: Exploring Novel Approaches to Colorectal Cancer Immunotherapy

This review explores the emerging role of extracellular vesicles (EVs) in modulating immune system function and their application in novel cancer immunotherapy strategies, with a focus on colorectal cancer (CRC). EVs, as carriers of bioactive molecules, have shown potential in enhancing immune responses and overcoming the limitations of traditional therapies. We discuss the biogenesis, types, and functional roles of immune cell-derived EVs, their interactions with cancer cells, and their implications in antitumor immunity. Challenges such as tumor heterogeneity and immune evasion are addressed, alongside the promising therapeutic prospects of EV-based strategies. This comprehensive analysis underscores the transformative potential of EVs in cancer treatment paradigms.

Tipifarnib Reduces Extracellular Vesicles and Protects From Heart Failure.

Heart failure (HF) is one of the leading causes of mortality worldwide. Extracellular vesicles, including small extracellular vesicles or exosomes, and their molecular cargo are known to modulate cell-to-cell communication during multiple cardiac diseases. However, the role of systemic extracellular vesicle biogenesis inhibition in HF models is not well documented and remains unclear. We investigated the role of circulating exosomes during cardiac dysfunction and remodeling in a mouse transverse aortic constriction (TAC) model of HF. Importantly, we investigate the efficacy of tipifarnib, a recently identified exosome biogenesis inhibitor that targets the critical proteins (Rab27a [Ras associated binding protein 27a], nSMase2 [neutral sphingomyelinase 2], and Alix [ALG-2-interacting protein X]) involved in exosome biogenesis for this mouse model of HF. In this study, 10-week-old male mice underwent TAC surgery were randomly assigned to groups with and without tipifarnib treatment (10 mg/kg 3 times/wk) and monitored for 8 weeks, and a comprehensive assessment was conducted through performed echocardiographic, histological, and biochemical studies. TAC significantly elevated circulating plasma exosomes and markedly increased cardiac left ventricular dysfunction, cardiac hypertrophy, and fibrosis. Furthermore, injection of plasma exosomes from TAC mice induced left ventricular dysfunction and cardiomyocyte hypertrophy in uninjured mice without TAC. On the contrary, treatment of tipifarnib in TAC mice reduced circulating exosomes to baseline and remarkably improved left ventricular functions, hypertrophy, and fibrosis. Tipifarnib treatment also drastically altered the miRNA profile of circulating post-TAC exosomes, including miR 331-5p, which was highly downregulated both in TAC circulating exosomes and in TAC cardiac tissue. Mechanistically, miR 331-5p is crucial for inhibiting the fibroblast-to-myofibroblast transition by targeting HOXC8, a critical regulator of fibrosis. Tipifarnib treatment in TAC mice upregulated the expression of miR 331-5p that acts as a potent repressor for one of the fibrotic mechanisms mediated by HOXC8. Our study underscores the pathological role of exosomes in HF and fibrosis in response to pressure overload. Tipifarnib-mediated inhibition of exosome biogenesis and cargo sorting may serve as a viable strategy to prevent progressive cardiac remodeling in HF.

Redox signalling and biogenesis of extracellular vesicles (EVs): a perspective for exercise-induced muscle adaptation.

Redox signalling and biogenesis of extracellular vesicles (EVs): a perspective for exercise-induced muscle adaptation.

Cell-Specific Impacts of Surface Coating Composition on Extracellular Vesicle Secretion.

Biomaterial properties have recently been shown to modulate extracellular vesicle (EV) secretion and cargo; however, the effects of substrate composition on EV production remain underexplored. This study investigates the impacts of surface coatings composed of collagen I (COLI), fibronectin (FN), and poly l-lysine (PLL) on EV secretion for applications in therapeutic EV production and to further understanding of how changes in the extracellular matrix microenvironment affect EVs. EV secretion from primary bone marrow-derived mesenchymal stromal cells (BMSCs), primary adipose-derived stem cells (ASCs), HEK293 cells, NIH3T3 cells, and RAW264.7 cells was characterized on the different coatings. Expression of EV biogenesis genes and cellular adhesion genes was also analyzed. COLI coatings significantly decreased EV secretion in RAW264.7 cells, with associated decreases in cell viability and changes in EV biogenesis-related and cell adhesion genes at day 4. FN coatings increased EV secretion in NIH3T3 cells, while PLL coatings increased EV secretion in ASCs. Surface coatings had significant effects on the capacity of EVs derived from RAW264.7 and NIH3T3 cells to impact in vitro macrophage proliferation. Overall, surface coatings had different cell-specific effects on EV secretion and in vitro functional capacity, thus highlighting the potential of substrate coatings to further the development of clinical EV production systems.

Extracellular vesicles in cancer: Golden goose or trojan horse.

Intercellular communication can be mediated by direct cell-to-cell contact and indirect interactions through secretion of soluble chemokines, cytokines, and growth factors. Extracellular vesicles (EVs) have emerged as important mediators of cell-to-cell and cell-to-environment communications. EVs from tumor cells, immune cells, and stromal cells can remodel the tumor microenvironment and promote cancer cell survival, proliferation, metastasis, immune evasion, and therapeutic resistance. Most importantly, EVs as natural nanoparticles can be manipulated to serve as a potent delivery system for targeted cancer therapy. EVs can be engineered or modified to improve their ability to target tumors and deliver therapeutic substances, such as chemotherapeutic drugs, nucleic acids, and proteins, for the treatment of cancer. This review provides an overview of the biogenesis and recycling of EVs, discusses their roles in cancer development, and highlights their potential as a delivery system for targeted cancer therapy.

Preclinical and Clinical Studies on the Use of Extracellular Vesicles Derived from Mesenchymal Stem Cells in the Treatment of Chronic Wounds.

To date, the widespread implementation of therapeutic strategies for the treatment of chronic wounds, including debridement, infection control, and the use of grafts and various dressings, has been time-consuming and accompanied by many challenges, with definite success not yet achieved. Extensive studies on mesenchymal stem cells (MSCs) have led to suggestions for their use in treating various diseases. Given the existing barriers to utilizing such cells and numerous pieces of evidence indicating the crucial role of the paracrine signaling system in treatments involving MSCs, extracellular vesicles (EVs) derived from these cells have garnered significant attention in treating chronic wounds in recent years. This review begins with a general overview of current methods for chronic wound treatment, followed by an exploration of EV structure, biogenesis, extraction methods, and characterization. Subsequently, utilizing databases such as Google Scholar, PubMed, and ScienceDirect, we have explored the latest findings regarding the role of EVs in the healing of chronic wounds, particularly diabetic and burn wounds. In this context, the role and mode of action of these nanoparticles in healing chronic wounds through mechanisms such as oxygen level elevation, oxidative stress damage reduction, angiogenesis promotion, macrophage polarization assistance, etc., as well as the use of EVs as carriers for engineered nucleic acids, have been investigated. The upcoming challenges in translating EV-based treatments for healing chronic wounds, along with possible approaches to address these challenges, are discussed. Additionally, clinical trial studies in this field are also covered.

Predicting Cell Type-Specific Extracellular Vesicle Biology Using an Organism-Wide Single Cell Transcriptomic Atlas - Insights from the Tabula Muris

Background: Extracellular vesicles (EVs) like exosomes are functional nanoparticles traffcked between cells and found in every biofluid. An incomplete understanding of which cells, from which tissues, are traffcking EVs in vivo has limited our ability to use EVs as biomarkers and therapeutics. However, recent discoveries have linked EV secretion to expression of genes and proteins responsible for EV biogenesis and found as cargo, which suggests that emerging “cell atlas” datasets could be used to begin understanding EV biology at the level of the organism and possibly in rare cell populations. To explore this possibility, here we analyzed 67 genes that are directly implicated in EV biogenesis and secretion, or carried as cargo, in ~44,000 cells obtained from 117 cell populations of the Tabula Muris. Methods: We obtained normalized count (FPKM) data from 44,779 FACS-sorted cells described in the Tabula Muris. These data were freely obtained from the Tabula Muris web portal operated by the Chan-Zuckerberg Biohub. We selected 67 EV genes based on their direct role in the regulation of endosome biogenesis, protein sorting, secretion or cargo. These genes include the endosome sorting complexes required for transport (ESCRT), multiple Rab GTPases, tetraspanins and syndecans. To identify the most abundant EV genes, we plotted mean expression (FPKM) across all cells against the fraction of cells with gene expression > 50 FPKM. To measure gene expression variance, we calculated %CV for each gene within each cell population and across all cell populations. For gene co-expression analysis, we performed linear regressions comparing selected EV genes (Cd9, Cd63, Cd81, Sdc4 and Sdcbp) to all other genes, within each cell population. Results: We found that the most abundant EV cargo proteins (tetraspanins (Cd9, Cd63 and Cd81) and syndecans (Sdc4 and Sdcbp)) are also the most abundant EV genes expressed across all cell populations. However, the expression of these genes varied greatly among cell populations. For example, Cd81 is most abundant in brain glial populations and multiple endothelial cell populations. By contrast, Cd9 expression is highest in "barrier" cells that support innate immunity while Cd63, a classic marker of exosomes, is most abundantly expressed by stem cells. Expression variance analysis was used to compare the transcriptional behavior of these abundant EV genes (Cd9, Cd63, Cd81, Sdc4 and Sdcbp). We found that Cd63 is the most dynamic EV gene while others are more constitutively expressed. This finding may help explain why Cd63 is typically found either within the cytoplasm or outside of the cell on EVs, whereas Cd9 and Cd81 are abundant both on the cell surface and on EVs. Finally, we used EV gene co-expression analysis to define cell population-specific transcriptional networks. We discovered that EV gene co-expression networks are heavily dependent on the EV cargo gene examined with different cell populations driving clustering. Conclusions: Our analysis has provided at least four independent observations which advance our understanding of EV biology. These are that: 1) abundant EV cargo genes are differentially expressed among cell populations, 2) skeletal muscle stem cells display the highest expression of both Cd63 and Sdc4, 3) Cd81 may be a primary EV cargo traffcked in the brain whiand 4) that rare cell populations can secrete EVs and thereby influence tissue biology. This analysis is the first, to our knowledge, to predict tissue- and cell type-specific EV biology at the level of the organism. As such, we expect this resource to be the first of many valuable tools for predicting the endogenous impact of specific cell populations on EV function in health and disease. National Institutes on Aging (R01AG078859). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The complexity of extracellular vesicles: Bridging the gap between cellular communication and neuropathology.

Brain-derived extracellular vesicles (EVs) serve a prominent role in maintaining homeostasis and contributing to pathology in health and disease. This review establishes a crucial link between physiological processes leading to EV biogenesis and their impacts on disease. EVs are involved in the clearance and transport of proteins and nucleic acids, responding to changes in cellular processes associated with neurodegeneration, including autophagic disruption, organellar dysfunction, aging, and other cell stresses. In neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, etc.), EVs contribute to the spread of pathological proteins like amyloid β, tau, ɑ-synuclein, prions, and TDP-43, exacerbating neurodegeneration and accelerating disease progression. Despite evidence for both neuropathological and neuroprotective effects of EVs, the mechanistic switch between their physiological and pathological functions remains elusive, warranting further research into their involvement in neurodegenerative disease. Moreover, owing to their innate ability to traverse the blood-brain barrier and their ubiquitous nature, EVs emerge as promising candidates for novel diagnostic and therapeutic strategies. The review uniquely positions itself at the intersection of EV cell biology, neurophysiology, and neuropathology, offering insights into the diverse biological roles of EVs in health and disease.

A chemical inhibitor of IST1-CHMP1B interaction impairs endosomal recycling and induces noncanonical LC3 lipidation

The endosomal sorting complex required for transport (ESCRT) machinery constitutes multisubunit protein complexes that play an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, including cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair, and autophagy. Given the versatile functionality of ESCRTs, and the intricate organizational structure of the ESCRT machinery, the targeted modulation of distinct ESCRT complexes is considerably challenging. This study presents a pseudonatural product targeting IST1-CHMP1B within the ESCRT-III complexes. The compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting, or biogenesis of extracellular vesicles, it rapidly inhibits transferrin receptor recycling in cells, resulting in the accumulation of transferrin in stalled sorting endosomes. Stalled endosomes become decorated by lipidated LC3, suggesting a link between noncanonical LC3 lipidation and inhibition of the IST1-CHMP1B complex.

Schwann cell-derived extracellular vesicles promote memory impairment associated with chronic neuropathic pain.

The pathogenesis of memory impairment, a common complication of chronic neuropathic pain (CNP), has not been fully elucidated. Schwann cell (SC)-derived extracellular vesicles (EVs) contribute to remote organ injury. Here, we showed that SC-EVs may mediate pathological communication between SCs and hippocampal neurons in the context of CNP. We used an adeno-associated virus harboring the SC-specific promoter Mpz and expressing the CD63-GFP gene to track SC-EVs transport. microRNA (miRNA) expression profiles of EVs and gain-of-function and loss-of-function regulatory experiments revealed that miR-142-5p was the main cargo of SC-EVs. Next, luciferase reporter gene and phenotyping experiments confirmed the direct targets of miR-142-5p. The contents and granule sizes of plasma EVs were significantly greater in rats with chronic sciatic nerve constriction injury (CCI)than in sham rats. Administration of the EV biogenesis inhibitor GW4869 ameliorated memory impairment in CCI rats and reversed CCI-associated dendritic spine damage. Notably, during CCI stress, SC-EVs could be transferred into the brain through the circulation and accumulate in the hippocampal CA1-CA3 regions. miR-142-5p was the main cargo wrapped in SC-EVs and mediated the development of CCI-associated memory impairment. Furthermore, α-actinin-4 (ACTN4), ELAV-like protein 4 (ELAVL4) and ubiquitin-specific peptidase 9 X-linked (USP9X) were demonstrated to be important downstream target genes for miR-142-5p-mediated regulation of dendritic spine damage in hippocampal neurons from CCI rats. Together, these findings suggest that SCs-EVs and/or their cargo miR-142-5p may be potential therapeutic targets for memory impairment associated with CNP.

Calix[6]arene dismantles extracellular vesicle biogenesis and metalloproteinases that support pancreatic cancer hallmarks

Many challenges are faced in pancreatic cancer treatment due to late diagnosis and poor prognosis because of high recurrence and metastasis. Extracellular vesicles (EVs) and matrix metalloproteinases (MMPs), besides acting in intercellular communication, are key players in the cancer cell plasticity responsible for initiating metastasis. Therefore, these entities provide valuable targets for the development of better treatments. In this context, this study aimed to evaluate the potential of calix[6]arene to disturb the release of EVs and the activity of MMPs in pancreatic cancer cells. We found a correlation between the endocytic-associated mediators and the prognosis of pancreatic cancer patients. We observed a more active EV machinery in the pancreatic cancer cell line PANC-1, which was reduced three-fold by treatment with calix[6]arene at subtoxic concentration (5 μM; p 〈0,001). We observed the modulation of 186 microRNAs (164 miRNAs upregulated and 22 miRNAs downregulated) upon calix[6]arene treatment. Interestingly, some of them as miR-4443 and miR-3909, regulates genes HIF1A e KIF13A that are well known to play a role in transport of vesicles. Furthermore, Calix[6]arene downmodulated matrix metalloproteinases (MMPs) -2 and − 9 and disturbed the viability of pancreatic organoids which recapitulate the cellular heterogeneity, structure, and functions of primary tissues. Our findings shed new insights on calix[6]arene's antitumor mechanism, including its intracellular effects on vesicle production and trafficking, as well as MMP activity, which may harm the tumor microenvironment and contribute to a reduction in cancer cell dissemination, which is one of the challenges associated with high mortality in pancreatic cancer.

Inhibition of ESCRT-independent extracellular vesicles biogenesis suppresses enterovirus 71 replication and pathogenesis in mice

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease (HFMD), neurological complications, and even fatalities in infants. Clinically, the increase of extracellular vesicles (EVs) in EV71 patients' serum was highly associated with the severity of HFMD. EV71 boosts EVs biogenesis in an endosomal sorting complex required for transport (ESCRT)-dependent manner to facilitate viral replication. Yet, the impact of EVs-derived from ESCRT-independent pathway on EV71 replication and pathogenesis is highly concerned. Here, we assessed the effects of EV71-induced EVs from ESCRT-independent pathway on viral replication and pathogenesis by GW4869, a neutral sphingomyelinase inhibitor. Detailly, in EV71-infected mice, blockade of the biogenesis of tissue-derived EVs in the presence of GW4869 restored body weight loss, attenuated clinical scores, and improved survival rates. Furthermore, GW4869 dampens EVs biogenesis to reduce viral load and pathogenesis in multiple tissues of EV71-infected mice. Consistently, GW4869 treatment in a human intestinal epithelial HT29 cells decreased the biogenesis of EVs, in which the progeny EV71 particle was cloaked, leading to the reduction of viral infection and replication. Collectively, GW4869 inhibits EV71-induced EVs in an ESCRT-independent pathway and ultimately suppresses EV71 replication and pathogenesis. Our study provides a novel strategy for the development of therapeutic agents in the treatment for EV71-associated HFMD.

An inside out journey: biogenesis, ultrastructure and proteomic characterisation of the ectoparasitic flatworm Sparicotyle chrysophrii extracellular vesicles

BackgroundHelminth extracellular vesicles (EVs) are known to have a three-way communication function among parasitic helminths, their host and the host-associated microbiota. They are considered biological containers that may carry virulence factors, being therefore appealing as therapeutic and prophylactic target candidates. This study aims to describe and characterise EVs secreted by Sparicotyle chrysophrii (Polyopisthocotyla: Microcotylidae), a blood-feeding gill parasite of gilthead seabream (Sparus aurata), causing significant economic losses in Mediterranean aquaculture.MethodsTo identify proteins involved in extracellular vesicle biogenesis, genomic datasets from S. chrysophrii were mined in silico using known protein sequences from Clonorchis spp., Echinococcus spp., Fasciola spp., Fasciolopsis spp., Opisthorchis spp., Paragonimus spp. and Schistosoma spp. The location and ultrastructure of EVs were visualised by transmission electron microscopy after fixing adult S. chrysophrii specimens by high-pressure freezing and freeze substitution. EVs were isolated and purified from adult S. chrysophrii (n = 200) using a newly developed ultracentrifugation-size-exclusion chromatography protocol for Polyopisthocotyla, and EVs were characterised via nanoparticle tracking analysis and tandem mass spectrometry.ResultsFifty-nine proteins involved in EV biogenesis were identified in S. chrysophrii, and EVs compatible with ectosomes were observed in the syncytial layer of the haptoral region lining the clamps. The isolated and purified nanoparticles had a mean size of 251.8 nm and yielded 1.71 × 108 particles · mL−1. The protein composition analysis identified proteins related to peptide hydrolases, GTPases, EF-hand domain proteins, aerobic energy metabolism, anticoagulant/lipid-binding, haem detoxification, iron transport, EV biogenesis-related, vesicle-trafficking and other cytoskeletal-related proteins. Several identified proteins, such as leucyl and alanyl aminopeptidases, calpain, ferritin, dynein light chain, 14–3–3, heat shock protein 70, annexin, tubulin, glutathione S-transferase, superoxide dismutase, enolase and fructose-bisphosphate aldolase, have already been proposed as target candidates for therapeutic or prophylactic purposes.ConclusionsWe have unambiguously demonstrated for the first time to our knowledge the secretion of EVs by an ectoparasitic flatworm, inferring their biogenesis machinery at a genomic and transcriptomic level, and by identifying their location and protein composition. The identification of multiple therapeutic targets among EVs' protein repertoire provides opportunities for target-based drug discovery and vaccine development for the first time in Polyopisthocotyla (sensu Monogenea), and in a fish-ectoparasite model.Graphical

Extracellular Vesicles: Translational Agenda Questions forThree Protozoan Parasites.

The protozoan parasites Plasmodium falciparum, Leishmania spp. and Trypanosoma cruzi continue to exert a significant toll onthe disease landscape of the human population in sub-Saharan Africa and Latin America. Control measures have helped reduce the burden of their respective diseases-malaria, leishmaniasis and Chagas disease-in endemic regions. However, the need for new drugs, innovative vaccination strategies and molecular markers of disease severity and outcomes has emerged because of developing antimicrobial drug resistance, comparatively inadequate or absent vaccines, and a lack of trustworthy markers of morbid outcomes. Extracellular vesicles (EVs) have been widely reported to play a role in the biology and pathogenicity of P. falciparum, Leishmania spp. and T. cruzi ever since they were discovered. EVs are secreted by a yet to be fully understood mechanism in protozoans into the extracellular milieu and carry a cargo of diverse molecules that reflect the originator cell's metabolic state. Although our understanding of the biogenesis and function of EVs continues to deepen, the question of how EVs in P. falciparum, Leishmania spp. and T. cruzi can serve as targets for a translational agenda into clinical and public health interventions is yet to be fully explored. Here, as a consortium of protozoan researchers, we outline a plan for future researchers and pose three questions to direct an EV's translational agenda in P. falciparum, Leishmania spp. and T. cruzi. We opine that in the long term, executing this blueprint will help bridge the current unmet needs of these medically important protozoan diseases in sub-Saharan Africa and Latin America.

Abstract 3386: Changes in extracellular vesicle miRNAs from three-dimensional ovarian cancer cell models reflect physiological changes and cancer survival

Abstract Ovarian Cancer (OC) is the most common gynaecological malignancy and the eighth most diagnosed cancer in females worldwide. Currently, it is the fifth leading cause of cancer-related mortality among patients globally, largely due to delayed diagnosis, chemotherapy resistance, high metastasis rates, and subtype heterogeneity. Recent OC research highlights extracellular vesicles (EVs) as pivotal contributors to intercellular communication and disease progression, elucidating the intricacies of OC pathology. EVs, diverse membrane-derived vesicles released by most cells, carry molecular cargoes containing proteins and nucleic acids. Studies indicate that the biogenesis, packaging, and release of EVs are highly dependent and sensitive to the cellular microenvironment and depend on the in-vitro and in-vivo modelling systems. Labs use varied model systems—2D monolayers, animal models, and innovative 3D models—to investigate EVs' ovarian cancer roles. Therefore, in this study we aimed to compare the miRNA profiles associated with EVs in 3D ovarian cancer cell models, and to identify the pathophysiological relevance of the EVs isolated from these models to the patient derived EVs.In this study, two OC epithelial cell lines, SKOV-3 and OVCAR-, were initially cultured as 2D monolayers and embedded within Gelatin Methacryloyl hydrogels. Over nine days, multiple assays observed spheroid formation within the cell-laden hydrogels. EVs isolated from the cell-conditioned media were characterized per MISEV 2018 guidelines. Small RNA sequencing identified statistically significant miRNAs, subject to gene ontology and gene rank analyses. EVs from a cohort of 60 OC patients were used to identify survival-associated miRNA profiles.Our findings in the cell-laden hydrogels demonstrated OC cell growth, proliferation, and aggregation into spheroidal structures, establishing an ideal 3D model. The isolated EVs were characterized for size, concentration, morphology, and surface markers. Small RNA sequencing revealed 18 significantly different EV-associated miRNA species across 3D vs 2D models, influencing apoptosis, angiogenesis, migration, and proliferation in ovarian cancer. Notably, 3D model-derived EV-associated miRNAs mirrored patient-derived EV-associated miRNA profiles, indicating their pathophysiological relevance.This study establishes a robust 3D OC model in hydrogels, showcasing growth, proliferation, and aggregation capabilities. Differential miRNA profiles between 3D and 2D model EVs highlight the critical roles of these miRNAs in essential ovarian cancer processes. Moreover, similarities between 3D model and patient-derived EV-associated miRNA profiles emphasize the clinical relevance of our model. Citation Format: Nihar Godbole, Akhilandeshwari Ravichandran, Dominic Guanzon, Andrew Lai, Flavio Carrion, Priyakshi Kalita de Croft, Lewis Perrin, John Hooper, Laura Bray, Carlos Salomon. Changes in extracellular vesicle miRNAs from three-dimensional ovarian cancer cell models reflect physiological changes and cancer survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3386.

Corrected and republished from: "Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal Virulence".

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants expected to lack NOP16 expression, we observed a reduced EV production. Whole-genome sequencing, RNA-Seq, and cellular proteomics revealed that, contrary to our initial findings, these mutants expressed Nop16 but exhibited altered expression of 14 genes potentially involved in sugar transport. Based on this observation, we designated these mutant strains as Past1 and Past2, representing potentially altered sugar transport. Analysis of the small molecule composition of EVs produced by wild-type cells and the Past1 and Past2 mutant strains revealed not only a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the Past1 and Past2 mutant strains were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the mutant cells in G. mellonella. These results connect EV biogenesis, cargo, and cryptococcal virulence.