Pharmacological tools to selectively modulate extracellular vesicle (EV) secretion are scarce. Here, we identify the ALK5 (TGF-β receptor I) inhibitor SD-208 as a potent suppressor of small EV (sEV) secretion that acts independently of its canonical anti-fibrotic activity. SD-208 not only reversed myofibroblast activation but also markedly inhibited sEV secretion. Strikingly, this inhibitory effect persisted in non-activated cardiac fibroblasts and non-fibrotic HEK293 cells, demonstrating that SD-208 regulates EV secretion through mechanisms uncoupled from TGF-β/Smad signalling. Mechanistic analyses revealed that SD-208 disrupts vesicle trafficking rather than EV biogenesis. Reduced secretion of CD63+ EVs was accompanied by intracellular accumulation of CD63+ structures and their selective diversion into LAMP1+ lysosomes. Proteomic profiling of SD-208-treated and control HEK293 cells and cardiac fibroblasts revealed dysregulation of vesicle trafficking pathways, enrichment of ubiquitin ligase complexes, and enhanced endosome-to-lysosome transport. Together, these findings demonstrate that SD-208 diverts CD63+ multivesicular bodies (MVBs) from a secretory fate toward lysosomal degradation. This work identifies SD-208 as a small-molecule tool to interrogate the secretory-versus-degradative fate of MVBs and uncovers a new regulatory link between lysosomal pathways and EV trafficking. Beyond its established role as an anti-fibrotic agent, SD-208 provides mechanistic and therapeutic opportunities for the control of EV secretion in diseases such as fibrosis, cardiac remodelling, hypertrophic cardiomyopathy, and cancer.

ABSTRACT Background Pancreatic ductal adenocarcinoma (PDAC) is a particularly lethal malignancy with few treatment options available. Extensive remodelling of extracellular matrix (ECM) generates a highly fibrotic tumour landscape, which impairs therapeutic response. Objective We investigated whether stromal priming via the highly specific Focal Adhesion Kinase (FAK) inhibitor narmafotinib (AMP945) in combination with the two major standard-of-care chemotherapies in PDAC, gemcitabine/Abraxane and FOLFIRINOX, reduces fibrosis and enhances treatment efficacy. Design 3D organotypic matrices, intravital imaging, and in vivo subcutaneous and orthotopic PDAC models were used to provide a rationale for a first-line priming regimen of narmafotinib prior to chemotherapy. Results Neoadjuvant chemotherapy induces fibrosis in PDAC indicating a need for upfront first-line priming of the ECM to normalise the stroma for optimal treatment response. Narmafotinib is a new potent small molecule FAK inhibitor. Phase I safety data shows excellent safety, tolerability, and pharmacokinetics following oral administration in humans. We reveal that narmafotinib treatment during early ECM remodelling (‘priming’) reduces fibrosis, while limiting subsequent PDAC invasion. Moreover, intravital imaging demonstrates real-time FAK inactivation and cell cycle stalling, leading to improved chemotherapeutic efficacy upon narmafotinib priming in vivo . Long-term assessment in patient-derived models shows that narmafotinib priming prior to gemcitabine/Abraxane or FOLFIRINOX reduces PDAC progression and extends survival in both chemotherapy settings. Conclusions Our results using these Phase II-ready drug combinations strongly support the clinical assessment of narmafotinib in PDAC. Narmafotinib is currently in Phase Ib/IIa trials, assessing a pulsed dosing regimen prior to gemcitabine/Abraxane, and warrants further clinical assessment in combination with FOLFIRINOX. SIGNIFICANCE OF THIS STUDY What is already known on this topic Pancreatic cancer (PC) is one of the most lethal malignancies and is characterised by a dense, fibrotic stroma, which impairs chemotherapy efficacy. The non-receptor tyrosine kinase FAK is known to promote cancer fibrosis and therefore represents a therapeutic target to normalise the PC stroma and to improve chemotherapy performance. What this study adds Neoadjuvant chemotherapy induces early fibrosis indicating a need for upfront first-line priming of the ECM to blunt or normalise stromal fibrosis for optimal response to therapy. The small molecule inhibitor narmafotinib (which is currently under Phase Ib/IIa clinical trial assessment) shows high specificity towards FAK as well as desirable pharmacokinetics and pharmacodynamics in healthy human volunteers. Early short-term narmafotinib priming reduces fibrosis and improves the efficacy of subsequent standard-of-care gemcitabine/Abraxane chemotherapy. FOLFIRINOX (oxaliplatin, irinotecan, leucovorin and 5-fluorouracil) is a multi-agent chemotherapy preferentially used in PDAC patients with good performance status. Our results demonstrate that narmafotinib priming also improves FOLFIRINOX efficacy, leading to extended survival in patient-derived PDAC models. How this study might affect research, practice, or policy This study supports the clinical development of narmafotinib in combination with both gemcitabine/Abraxane (ACCENT trial) and further FOLFIRINOX standard-of-care chemotherapies for PDAC patient treatment. The first-line priming strategy and early ECM normalisation used in this study may also be applicable to other combination therapy settings and warrants further investigation in ongoing clinical studies.

Intermittent fasting (IF) has emerged as a powerful dietary intervention with profound metabolic benefits, yet the tissue-specific molecular mechanisms underlying these effects remain poorly understood. In this study, we employed comprehensive proteomics and transcriptomics analysis to investigate the systemic and organ-specific adaptations to IF in male C57BL/6 mice. Following a 16 hr daily fasting regimen (IF16) over 4 months, IF reduced blood glucose, HbA1c, and cholesterol levels while increasing ketone bodies, indicative of enhanced metabolic flexibility. Proteomic profiling of the liver, skeletal muscle, and cerebral cortex revealed tissue-specific responses, with the liver exhibiting the most pronounced changes, including upregulation of pathways involved in fatty acid oxidation, ketogenesis, and glycan degradation, and downregulation of steroid hormone and cholesterol metabolism. In muscle, IF enhanced pyruvate metabolism, fatty acid biosynthesis, and AMPK signaling, while suppressing oxidative phosphorylation and thermogenesis. The cerebral cortex displayed unique adaptations, with upregulation of autophagy, PPAR signaling, and metabolic pathways, and downregulation of TGF-beta and p53 signaling, suggesting a shift toward energy conservation and stress resilience. Notably, Serpin A1c emerged as the only protein commonly upregulated across all three tissues, highlighting its potential role in systemic adaptation to IF. Integrative transcriptomic and proteomic analyses revealed partial concordance between mRNA and protein expression, underscoring the complexity of post-transcriptional regulation. Shared biological signaling processes were identified across tissues, suggesting unifying mechanisms linking metabolic changes to cellular communication. Our findings reveal both conserved and tissue-specific responses by which IF may optimize energy utilization, enhance metabolic flexibility, and promote cellular resilience.

Apoptotic cells communicate to phagocytic cells through releasing soluble factors and apoptotic cell-derived extracellular vesicles. However, whether there are additional factors that remain attached at the site of cell death to signal to phagocytic cells is currently unknown. Here we show that apoptotic cell retraction generates a membrane-encased, F-actin-rich 'footprint' tightly anchored to the substrate that marks the site of cell death, coined 'the FOotprint Of Death' or FOOD. Formation of FOOD is observed frequently across many different cell types, apoptotic stimuli and surface composition. Mechanistically, FOOD formation is regulated by the protein kinase ROCK1. 3D time-lapse microscopy studies revealed that FOOD vesicularises into distinct large extracellular vesicles. These extracellular vesicles expose the 'eat-me' signal phosphatidylserine and can function to 'flag' the site of cell death to neighbouring phagocytes for efferocytosis. Under a viral infection setting, FOOD can harbour viral proteins and virions, and propagate infection to healthy cells. Together, this study has revealed another route of apoptotic cell-phagocyte communication.

Endothelial cells (ECs) act as gatekeepers and signalling hubs that coordinate communication between blood vessels and surrounding tissues by regulating vascular tone, immune responses and numerous other physiological processes. During vascular inflammation commonly associated with aging, atherosclerosis, diabetes and autoimmunity, a range of biological, environmental and physical stressors can induce activation and apoptosis of ECs. Apoptotic bodies (ApoBDs) are large ( 1–5 μm), membrane‑bound extracellular vesicles generated solely through apoptotic cell disassembly, that are increasingly recognised as mediators of intercellular communication via the transfer of bioactive molecules to target cells. Although EC apoptosis is a central feature of vascular inflammatory disorders, the formation of EC‑derived ApoBDs and their immunomodulatory roles when formed in an inflammatory environment, remains poorly defined. This study aimed to characterise the functional properties of EC‑derived ApoBDs generated under inflammatory conditions in vitro. A proteomics analysis of EC‑derived ApoBDs revealed that EC‑ApoBDs generated during inflammation (‘iApoBDs’) were enriched in inflammatory cytokines/chemokines, adhesion molecules and antigen presentation machinery compared with non-inflammatory (‘ApoBD’) controls. Functionally, iApoBDs promoted monocyte chemotaxis via the release of MCP-1, while altered expression of the adhesion molecule ICAM-1 enhanced efferocytosis by macrophages in vitro and in vivo. Furthermore, iApoBDs generated from antigen-pulsed HUVECs promoted IFN‑𝛾 expression by peptide specific CD8 T cells in an in vitro model of antigen presentation. These findings demonstrate that within an inflammatory setting, apoptotic ECs can participate in continued communication with their environment via the generation of ApoBDs, thereby modulating innate and adaptive immune processes. The formation of ApoBDs by ECs may serve as a target for therapeutic interventions in inflammatory vascular diseases.

Small extracellular vesicles (sEVs) are small membrane vesicles of endocytic origin secreted into the extracellular environment by all cell types and are known to play a crucial role in intercellular and intracellular communication. These vesicles contain proteins, nucleic acids and lipids and their molecular content reflect the normal and pathophysiological conditions of the host cells. Hence, there is a significant interest in cataloguing the molecular content of sEVs in various conditions as this would aid researchers in understanding the biological roles and altered cellular processes under various diseases and healthy state. Here we report ExoCarta (https://www.exocarta.org), a freely accessible web-based compendium of studies that encompasses DNA, RNA, proteins, and lipids that are detected in sEVs. ExoCarta catalogues both published and unpublished sEV studies. Current version of ExoCarta contains data from 1249 sEV studies, 119,489 protein entries, 15,868 RNA entries, 3,946 lipid entries and quantitative data for 24,073 entries. QUANT, a quantitative plugin, enables users to compare protein abundance between conditions in a study for gene/protein of interest in real time. ExoCarta provides Gene Ontology (GO) annotations and reactome pathways along with dynamic protein-protein interaction networks, for sEV proteins. A tab delimited file containing the most identified sEV proteins is available for users to download. The integration of sEV DNA studies, QUANT and regularly updated dataset of the ExoCarta database makes it an invaluable resource for small extracellular vesicles researchers across the globe.

Exercise improves cognitive function in Alzheimer’s Disease (AD), via mechanism that are not fully clear. Here, we first examined the effect of voluntary exercise training (VET) on energy metabolism and cognitive function in the APP/PS1 transgenic mouse (Tg) model of familial AD. Next, we profiled extracellular vesicles (EVs) and examined whether they may play a role in the protective effects of VET via intranasal administration of EVs, purified from the blood of sedentary (sEV) and/or acutely exercised (eEV) donor wild-type mice into APP/PS1Tg mice. We show that VET reduced resting energy expenditure (REE) and improved cognition in APP/PS1 Tg mice. Administration of eEV, but not sEV, also reduced REE, but had no effect on cognition. Taken together, these data show that exercise is effective intervention to improve symptoms of AD in APP/PS1Tg mice. In addition, eEVs mediate some of these effects, implicating EVs in the treatment of age-related neurodegenerative diseases.

Diverse functions of probiotic extracellular vesicles (EVs) have been extensively studied over the past decade, proposing their role in inter-kingdom communication. Studies have explored their therapeutic role in pathophysiological processes ranging from cancer, immunoregulation, and ulcerative colitis to stress-induced depression. These studies have highlighted the significant and novel potential of probiotic EVs for therapeutic applications, offering immense promise in addressing several unmet clinical needs. Additionally, probiotic EVs are being explored as vehicles for targeted delivery approaches. However, the realization of clinical utility of probiotic EVs is hindered by several knowledge gaps, pitfalls, limitations, and challenges, which impede their wider acceptance by the scientific community. Among these, limited knowledge of EV biogenesis, markers and regulators in bacteria, variations in cargo due to culture conditions or EV isolation method, and lack of proper understanding of gut uptake and demonstration of in vivo effect are some important issues. This review aims to summarize the diverse roles of probiotic EVs in health and disease conditions. More importantly, it discusses the significant knowledge gaps and limitations that stand in the way of the therapeutic utility of probiotic EVs. Furthermore, the importance of addressing these gaps and limitations with technical advances such as rigorous omics has been discussed.

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.

Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.

Metastatic triple-negative breast cancer (TNBC) has a low 5-year survival rate of below 30% with systemic chemotherapy being the most widely used treatment. Bovine milk-derived extracellular vesicles (MEVs) have been previously demonstrated to have anti-cancer attributes. In this study, we isolated bovine MEVs from commercial milk and characterised them according to MISEV guidelines. Bovine MEVs sensitised TNBC cells to doxorubicin, resulting in reduced metabolic potential and cell-viability. Label-free quantitative proteomics of cells treated with MEVs and/or doxorubicin suggested that combinatorial treatment depleted various pro-tumorigenic interferon-inducible gene products and proteins with metabolic function, previously identified as therapeutic targets in TNBC. Combinatorial treatment also led to reduced abundance of various STAT proteins and their downstream oncogenic targets with roles in cell-cycle and apoptosis. Taken together, this study highlights the ability of bovine MEVs to sensitise TNBC cells to standard-of-care therapeutic drug doxorubicin, paving the way for novel treatment regimens.

Cancer-associated cachexia is a wasting syndrome that results in dramatic loss of whole-body weight, predominantly due to loss of skeletal muscle mass. It has been established that cachexia inducing cancer cells secrete proteins and extracellular vesicles (EVs) that can induce muscle atrophy. Though several studies examined these cancer-cell derived factors, targeting some of these components have shown little or no clinical benefit. To develop new therapies, understanding of the dysregulated proteins and signaling pathways that regulate catabolic gene expression during muscle wasting is essential. Here, we sought to examine the effect of conditioned media (CM) that contain secreted factors and EVs from cachexia inducing C26 colon cancer cells on C2C12 myotubes using mass spectrometry-based label-free quantitative proteomics. We identified significant changes in the protein profile of C2C12 cells upon exposure to C26-derived CM. Functional enrichment analysis revealed enrichment of proteins associated with inflammation, mitochondrial dysfunction, muscle catabolism, ROS production, and ER stress in CM treated myotubes. Furthermore, strong downregulation in muscle structural integrity and development and/or regenerative pathways were observed. Together, these enriched proteins in atrophied muscle could be utilized as potential muscle wasting markers and the dysregulated biological processes could be employed for therapeutic benefit in cancer-induced muscle wasting.

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

Milk is a complex biological fluid that has high-quality proteins including growth factors and also contains extracellular vesicles (EVs). EVs are a lipid bilayer containing vesicles that contain proteins, metabolites and nucleic acids. Several studies have proposed that EVs in cow milk can survive the gut and can illicit cross-species communication in the consuming host organism. In this study, we isolated and characterized extracellular vesicles from the raw milk of the four species of the Bovidae family, namely cow, sheep, goat and buffalo, that contribute 99% of the total milk consumed globally. A comparative proteomic analysis of these vesicles was performed to pinpoint their potential functional role in health and disease. Vesicles sourced from buffalo and cow milk were particularly enriched with proteins implicated in modulating the immune system. Furthermore, functional studies were performed to determine the anti-cancer effects of these vesicles. The data obtained revealed that buffalo-milk-derived EVs induced significantly higher cell death in colon cancer cells. Overall, the results from this study highlight the potent immunoregulatory and anti-cancer nature of EVs derived from the milk of Bovidae family members.

Cell culture-conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM-derived EVs (CCM-EV). The CCM-EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell-specific recommendations may need to be established for non-mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM-EV research.

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.

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.