Extracellular Vesicles in Pathophysiology: A Prudent Target That Requires Careful Consideration.

Extracellular vesicles (EVs) are membrane-bound particles released by cells to perform multitudes of biological functions. Owing to their significant implications in diseases, the pathophysiological role of EVs continues to be extensively studied, leading research to neglect the need to explore their role in normal physiology. Despite this, many identified physiological functions of EVs, including, but not limited to, tissue repair, early development and aging, are attributed to their modulatory role in various signaling pathways via intercellular communication. EVs are widely perceived as a potential therapeutic strategy for better prognosis, primarily through utilization as a mode of delivery vehicle. Moreover, disease-associated EVs serve as candidates for the targeted inhibition by pharmacological or genetic means. However, these attempts are often accompanied by major challenges, such as off-target effects, which may result in adverse phenotypes. This renders the clinical efficacy of EVs elusive, indicating that further understanding of the specific role of EVs in physiology may enhance their utility. This review highlights the essential role of EVs in maintaining cellular homeostasis under different physiological settings, and also discusses the various aspects that may potentially hinder the robust utility of EV-based therapeutics.

Vesiclepedia 2024: an extracellular vesicles and extracellular particles repository.

Vesiclepedia (http://www.microvesicles.org) is a free web-based compendium of DNA, RNA, proteins, lipids and metabolites that are detected or associated with extracellular vesicles (EVs) and extracellular particles (EPs). EVs are membranous vesicles that are secreted ubiquitously by cells from all domains of life from archaea to eukaryotes. In addition to EVs, it was reported recently that EPs like exomeres and supermeres are secreted by some mammalian cells. Both EVs and EPs contain proteins, nucleic acids, lipids and metabolites and has been proposed to be implicated in several key biological functions. Vesiclepedia catalogues proteins, DNA, RNA, lipids and metabolites from both published and unpublished studies. Currently, Vesiclepedia contains data obtained from 3533 EV studies, 50 550 RNA entries, 566 911 protein entries, 3839 lipid entries, 192 metabolite and 167 DNA entries. Quantitative data for 62 822 entries from 47 EV studies is available in Vesiclepedia. The datasets available in Vesiclepedia can be downloaded as tab-delimited files or accessible through the FunRich-based Vesiclepedia plugin.

The E3 ubiquitin ligase NEDD4 regulates chemoresistance to 5-fluorouracil in colorectal cancer cells by altering JNK signalling.

Colorectal cancer (CRC) is the second leading cause of cancer deaths. Though chemotherapy is the main treatment option for advanced CRC, patients invariably acquire resistance to chemotherapeutic drugs and fail to respond to the therapy. Although understanding the mechanisms regulating chemoresistance has been a focus of intense research to manage this challenge, the pathways governing resistance to drugs are poorly understood. In this study, we provide evidence for the role of ubiquitin ligase NEDD4 in resistance developed against the most commonly used CRC chemotherapeutic drug 5-fluorouracil (5-FU). A marked reduction in NEDD4 protein abundance was observed in a panel of CRC cell lines and patient-derived xenograft samples that were resistant to 5-FU. Knockout of NEDD4 in CRC cells protected them from 5-FU-mediated apoptosis but not oxaliplatin or irinotecan. Furthermore, NEDD4 depletion in CRC cells reduced proliferation, colony-forming abilities and tumour growth in mice. Follow-up biochemical analysis highlighted the inhibition of the JNK signalling pathway in NEDD4-deficient cells. Treatment with the JNK activator hesperidin in NEDD4 knockout cells sensitised the CRC cells against 5-FU. Overall, we show that NEDD4 regulates cell proliferation, colony formation, tumour growth and 5-FU chemoresistance in CRC cells.

Proteomic analysis of the small extracellular vesicles and soluble secretory proteins from cachexia inducing and non-inducing cancer cells.

Cancer cachexia is a wasting syndrome characterised by the loss of fat and/or muscle mass in advanced cancer patients. It has been well-established that cancer cells themselves can induce cachexia via the release of several pro-cachectic and pro-inflammatory factors. However, it is unclear how this process is regulated and the key cachexins that are involved. In this study, we validated C26 and EL4 as cachexic and non-cachexic cell models, respectively. Treatment of adipocytes and myotubes with C26 conditioned medium induced lipolysis and atrophy, respectively. We profiled soluble secreted proteins (secretome) as well as small extracellular vesicles (sEVs) released from cachexia-inducing (C26) and non-inducing (EL4) cancer cells by label-free quantitative proteomics. A total of 1268 and 1022 proteins were identified in the secretome of C26 and EL4, respectively. Furthermore, proteomic analysis of sEVs derived from C26 and EL4 cancer cells revealed a distinct difference in the protein cargo. Functional enrichment analysis using FunRich highlighted the enrichment of proteins that are implicated in biological processes such as muscle atrophy, lipolysis, and inflammation in both the secretome and sEVs derived from C26 cancer cells. Overall, our characterisation of the proteomic profiles of the secretory factors and sEVs from cachexia-inducing and non-inducing cancer cells provides insights into tumour factors that promote weight loss by mediating protein and lipid loss in various organs and tissues. Further investigation of these proteins may assist in highlighting potential therapeutic targets and biomarkers of cancer cachexia.

Extracellular Vesicles Biogenesis, Cargo Sorting and Implications in Disease Conditions.

Extracellular vesicles (EVs) are small packages that contain proteins, lipids and nucleic acids and are released by various cell types [...].

Extracellular Vesicles as Drug Targets and Delivery Vehicles for Cancer Therapy.

Extracellular vesicles (EVs) are particles that are released from cells into the extracellular space both under pathological and normal conditions. It is now well established that cancer cells secrete more EVs compared to non-cancerous cells and that, captivatingly, several proteins that are involved in EV biogenesis and secretion are upregulated in various tumours. Recent studies have revealed that EVs facilitate the interaction between cancer cells and their microenvironment and play a substantial role in the growth of tumours. As EVs are involved in several aspects of cancer progression including angiogenesis, organotropism, pre-metastatic niche formation, fostering of metastasis, and chemoresistance, inhibiting the release of EVs from cancer and the surrounding tumour microenvironment cells has been proposed as an ideal strategy to treat cancer and associated paraneoplastic syndromes. Lately, EVs have shown immense benefits in preclinical settings as a novel drug delivery vehicle. This review provides a brief overview of the role of EVs in various hallmarks of cancer, focusing on (i) strategies to treat cancer by therapeutically targeting the release of tumour-derived EVs and (ii) EVs as valuable drug delivery vehicles. Furthermore, we also outline the drawbacks of the existing anti-cancer treatments and the future prospective of EV-based therapeutics.

Pep2Graph: A standalone tool to analyse proteolytic cleavages by proteases from gel-based mass spectrometry data.

Proteases are enzymes that regulate substrates via proteolytic activation and coordinate essential cellular functions including DNA replication, DNA transcription, cell proliferation, differentiation, migration and apoptosis. However, techniques to identify proteolytic events in a high-throughput manner is limited. PROtein TOpography and Migration Analysis Platform (PROTOMAP) is a technique that relies on mass spectrometry-based proteomics to globally identify the shifts in the in-gel migration of proteins and their corresponding fragments that are obtained by proteolysis. However, user-friendly software tool to analyse the proteomic data to identify proteolytic events is needed. Here, we report Pep2Graph, a user-friendly standalone tool that integrates peptide sequence information from in-gel proteomics and presents the data as two-dimensional peptographs with in-gel migration, sequence coverage and MS/MS spectra counts. Pep2Graph (http://www.mathivananlab.org/Pep2Graph) allows users to utilize in-gel proteomics data to study proteolytic events that may play a significant role in normal physiology and pathology.

Repurposing of Antibiotic Sulfisoxazole Inhibits Lipolysis in Pre-Clinical Model of Cancer-Associated Cachexia.

Clinical management of cancer-associated cachexia, a multi-organ wasting syndrome, has been challenging without effective treatment strategies. An effective treatment that directly targets cancer-induced wasting is desperately needed to improve the quality of life and the survival of cancer patients. Recently, an antibiotic SFX was shown to have anti-tumour and anti-metastatic effects in mouse models of breast cancer. Hence, in this study, we examined the efficacy of SFX in the treatment of cancer-induced cachexia. C26 cachexic mice models were administered with SFX, and the tumour volume and body weight were regularly measured. Blood glucose, skeletal muscles, and adipose tissue were examined at the endpoint. Contrary to a previous study, SFX did not reduce the tumour volume in mice bearing C26 cells. Administration of SFX neither revealed any survival benefit nor rescued C26 cachectic mice from muscle wasting. Interestingly, SFX administration partially rescued (~10%) tumour-induced weight loss by preserving both the subcutaneous and intestinal fat mass. Together, these results suggest that the administration of SFX could partially rescue cancer-induced weight loss by inhibiting lipolysis. As anti-cachexia therapies are scarce, the results could facilitate the design of combinatorial therapies involving SFX, standard-of-care chemotherapeutics, and drugs that inhibit muscle atrophy for the treatment of cancer cachexia.

Arrdc4-dependent extracellular vesicle biogenesis is required for sperm maturation.

Extracellular vesicles (EVs) are important players in cell to cell communication in reproductive systems. Notably, EVs have been found and characterized in the male reproductive tract, however, direct functional evidence for their importance in mediating sperm function is lacking. We have previously demonstrated that Arrdc4, a member of the α-arrestin protein family, is involved in extracellular vesicle biogenesis and release. Here we show that Arrdc4-mediated extracellular vesicle biogenesis is required for proper sperm function. Sperm from Arrdc4-/- mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as observed by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and two-cell embryo production. We found a significant reduction in extracellular vesicle production by Arrdc4-/- epididymal epithelial cells, and further, supplementation of Arrdc4-/- sperm with additional vesicles dampened the acrosome reaction defect and restored zona pellucida binding. These results indicate that Arrdc4 is important for proper sperm maturation through the control of extracellular vesicle biogenesis.

Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis.

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.

Temporal Quantitative Proteomics Analysis of Neuroblastoma Cells Treated with Bovine Milk-Derived Extracellular Vesicles Highlights the Anti-Proliferative Properties of Milk-Derived Extracellular Vesicles.

Neuroblastoma (NBL) is a pediatric cancer that accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc occurs in 20% of NBL patients and is considered high risk as it correlates with aggressiveness, treatment resistance and poor prognosis. Even though the treatment strategies have improved in the recent years, the survival rate of high-risk NBL patients remain poor. Hence, it is crucial to explore new therapeutic avenues to sensitise NBL. Recently, bovine milk-derived extracellular vesicles (MEVs) have been proposed to contain anti-cancer properties. However, the impact of MEVs on NBL cells is not understood. In this study, we characterised MEVs using Western blotting, NTA and TEM. Importantly, treatment of NBL cells with MEVs decreased the proliferation and increased the sensitivity of NBL cells to doxorubicin. Temporal label-free quantitative proteomics of NBL cells highlighted the depletion of proteins involved in cell metabolism, cell growth and Wnt signalling upon treatment with MEVs. Furthermore, proteins implicated in cellular senescence and apoptosis were enriched in NBL cells treated with MEVs. For the first time, this study highlights the temporal proteomic profile that occurs in cancer cells upon MEVs treatment.

Introduction to the Community of Extracellular Vesicles.

Since the discovery that extracellular vesicles (EVs) mediate intercellular communication, there is an exponential increase in the interest on EVs, especially in pathological settings. EVs are membranous vesicles that are secreted by various cell types and the release of EVs is conserved in every prokaryotic and eukaryotic organism tested to date. These vesicles were initially thought to be garbage disposal vehicles and subsequent studies over the past 4 decades have attributed several functional roles to EVs, some of which are critical for homeostasis. The molecular cargo of nucleic acids, proteins, lipids and metabolites packaged in EVs often mirror the host cells phenotypic status. EVs can be taken up by recipient cells and upon uptake, EVs through its molecular cargo, can induce a cascade of signal transduction events in recipient cells. EVs are categorised into several subtypes depending on their biogenesis and secretion. Due to several subtypes, differing sizes within a subtype and varying cargo, EVs are heterogenous in nature and the biophysical and biochemical properties of EVs often overlap between EV subtypes. Hence, it is important to be cautious when selecting the method of EV isolation and characterisation. This chapter provides a brief introduction to EVs and their subtypes.

Are Dietary Extracellular Vesicles Bioavailable and Functional in Consuming Organisms?

It has been well established that diet influences the health status of the consuming organism. Recently, extracellular vesicles (EVs) present in dietary sources are proposed to be involved in cross-species and kingdom communication. As EVs contain a lipid bilayer and carry bioactive cargo of proteins and nucleic acids, they are proposed to survive harsh degrading conditions of the gut and enter systemic circulation. Following the bioavailability, several studies have supported the functional role of dietary EVs in various tissues of the consuming organism. Simultaneously, multiple studies have refuted the possibility that dietary EVs mediate cross-species communication and hence the topic is controversial. The feasibility of the concept remains under scrutiny primarily owing to the lack of significant in vivo evidence to complement the in vitro speculations. Concerns surrounding EV stability in the harsh degrading gut environment, lack of mechanism explaining intestinal uptake and bioavailability in systemic circulation have impeded the acceptance of their functional role. This chapter discusses the current evidences that support dietary EV-based cross species communication and enlists several issues that need to be addressed in this field.

Pannexin-1 channel regulates nuclear content packaging into apoptotic bodies and their size.

Apoptotic bodies (ApoBDs), which are large extracellular vesicles exclusively released by apoptotic cells, possess therapeutically exploitable properties including biomolecule loadability and transferability. However, current limited understanding of ApoBD biology has hindered its exploration for clinical use. Particularly, as ApoBD-accompanying cargoes (e.g., nucleic acids and proteins) have major influence on their functionality, further insights into the mechanism of biomolecule sorting into ApoBDs are critical to unleash their therapeutic potential. Previous studies suggested pannexin 1 (PANX1) channel, a negative regulator of ApoBD biogenesis, can modify synaptic vesicle contents. We also reported that trovafloxacin (a PANX1 inhibitor) increases proportion of ApoBDs containing DNA. Therefore, we sought to define the role of PANX1 in regulating the sorting of nuclear content into ApoBDs. Here, using flow cytometry and label-free quantitative proteomic analyses, we showed that targeting PANX1 activity during apoptosis, via either pharmacological inhibition or genetic disruption, resulted in enrichment of both DNA and nuclear proteins in ApoBDs that were unexpectedly smaller in size. Our data suggest that PANX1, besides being a key regulator of ApoBD formation, also functions as a negative regulator of nuclear content packaging and modulator of ApoBD size. Together, our findings provide further insights into ApoBD biology and form a novel conceptual framework for ApoBD-based therapies through pharmacologically manipulating ApoBD contents.

FunRich enables enrichment analysis of OMICs datasets.

High-throughput methods to profile the genome, transcriptome, proteome and metabolome of various systems has become a routine in multiple research laboratories around the world. Hence, to analyse and interpret these heterogenous datasets user-friendly bioinformatics tools are needed. Here, we discuss FunRich tool that enables biologists to perform functional enrichment analysis on the generated datasets. Users can perform enrichment analysis with a variety of background databases and have complete control in updating or modifying the content in most of the databases. Specifically, users can download and update the background database from UniProt at any time thereby allowing a robust background database that can support annotations from >18 taxonomies. Users can create customizable Venn diagrams, pie charts, bar graphs and heatmaps of publication quality for their datasets using FunRich (http://www.funrich.org). Overall, FunRich tool is user-friendly and enables users to perform various analysis on their datasets with minimal or no aid from bioinformaticians.

TREML4 receptor regulates inflammation and innate immune cell death during polymicrobial sepsis.

Sepsis is a biphasic disease characterized by an acute inflammatory response, followed by a prolonged immunosuppressive phase. Therapies aimed at controlling inflammation help to reduce the time patients with sepsis spend in intensive care units, but they do not lead to a reduction in overall mortality. Recently, the focus has been on addressing the immunosuppressive phase, often caused by apoptosis of immune cells. However, molecular triggers of these events are not yet known. Using whole-genome CRISPR screening in mice, we identified a triggering receptor expressed on myeloid cells (TREM) family receptor, TREML4, as a key regulator of inflammation and immune cell death in sepsis. Genetic ablation of Treml4 in mice demonstrated that TREML4 regulates calcium homeostasis, the inflammatory cytokine response, myeloperoxidase activation, the endoplasmic reticulum stress response and apoptotic cell death in innate immune cells, leading to an overall increase in survival rate, both during the acute and chronic phases of polymicrobial sepsis.

Milk-Derived Extracellular Vesicles in Inter-Organism, Cross-Species Communication and Drug Delivery.

Milk is considered as more than a source of nutrition for infants and is a vector involved in the transfer of bioactive compounds and cells. Milk contains abundant quantities of extracellular vesicles (EVs) that may originate from multiple cellular sources. These nanosized vesicles have been well characterized and are known to carry a diverse cargo of proteins, nucleic acids, lipids and other biomolecules. Milk-derived EVs have been demonstrated to survive harsh and degrading conditions in gut, taken up by various cell types, cross biological barriers and reach peripheral tissues. The cargo carried by these dietary EVs has been suggested to have a role in cell growth, development, immune modulation and regulation. Hence, there is considerable interest in understanding the role of milk-derived EVs in mediating inter-organismal and cross-species communication. Furthermore, various attributes such as it being a natural source, as well as its abundance, scalability, economic viability and lack of unwarranted immunologic reactions, has generated significant interest in deploying milk-derived EVs for clinical applications such as drug delivery and disease therapy. In this review, the role of milk-derived EVs in inter-organismal, cross-species communication and in drug delivery is discussed.

Extracellular vesicles containing oncogenic mutant ß-catenin activate Wnt signalling pathway in the recipient cells.

Mutations in ß-catenin, especially at the residues critical for its degradation, render it constitutively active. Here, we show that mutant ß-catenin can be transported via extracellular vesicles (EVs) and activate Wnt signalling pathway in the recipient cells. An integrative proteogenomic analysis identified the presence of mutated ß-catenin in EVs secreted by colorectal cancer (CRC) cells. Follow-up experiments established that EVs released from LIM1215 CRC cells stimulated Wnt signalling pathway in the recipient cells with wild-type ß-catenin. SILAC-based quantitative proteomics analysis confirmed the transfer of mutant ß-catenin to the nucleus of the recipient cells. In vivo tracking of DiR-labelled EVs in mouse implanted with RKO CRC cells revealed its bio-distribution, confirmed the activation of Wnt signalling pathway in tumour cells and increased the tumour burden. Overall, for the first time, this study reveals that EVs can transfer mutant ß-catenin to the recipient cells and promote cancer progression.

Extracellular vesicles secreted by Saccharomyces cerevisiae are involved in cell wall remodelling.

Extracellular vesicles (EVs) are membranous vesicles that are released by cells. In this study, the role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the biogenesis of yeast EVs was examined. Knockout of components of the ESCRT machinery altered the morphology and size of EVs as well as decreased the abundance of EVs. In contrast, strains with deletions in cell wall biosynthesis genes, produced more EVs than wildtype. Proteomic analysis highlighted the depletion of ESCRT components and enrichment of cell wall remodelling enzymes, glucan synthase subunit Fks1 and chitin synthase Chs3, in yeast EVs. Interestingly, EVs containing Fks1 and Chs3 rescued the yeast cells from antifungal molecules. However, EVs from fks1∆ or chs3∆ or the vps23∆chs3∆ double knockout strain were unable to rescue the yeast cells as compared to vps23∆ EVs. Overall, we have identified a potential role for yeast EVs in cell wall remodelling.