Vesicle Biogenesis Research Articles

Biogenesis of Fungal Extracellular Vesicles: What Do We Know?

So far, extracellular vesicles (EVs) have been described in 15 genera of fungi. They carry molecules that contribute to the interaction of fungal cells with the host. Although the number of studies on fungal EVs has increased, the mechanisms involved in their biogenesis are still poorly understood. The current knowledge of EV biogenesis shows us that they can originate both in the cytoplasm and at the plasma membrane. In this chapter, we will focus on these two cellular sites to review what is known about the biogenesis of fungal EVs.

Plastic Surgery Update on the Mesenchymal Stem-Cell Derived Extracellular Vesicles towards Cell-Free Therapeutic Applications

In the biogenesis of extracellular vesicles (EVs), exosomes and other lipid-lined vesicles are released upon fusion of multivesicular bodies with the cell membrane of stem cells. EVs contain a diverse number of growth factors, cytokines and bioactive molecules of proteins, lipids, microRNA, and mRNA that mediate cell-cell communications for homeostasis, immune signaling, angiogenesis, anti-inflammation, senescence, proliferation, and differentiation. To further explore its potential usages, plastic surgeons are beginning to show an increased interest in this novel cell-free therapy to partially explain the paracrine effects of cell-based therapies on cell repair, tissue engineering, and aesthetic rejuvenation. The burgeoning preclinical and clinical experience appears to be promising, but current in vitro studies, translational research, and IRB-registered investigations emphasize the need to clarify product identification/purity, attributed biologic functions, standardized protocols, and applications to advance basic science findings and provide beneficial safe clinical outcomes. Since the specialty of Plastic Surgery is committed to advancing evidence-based stem cell studies in compliance with FDA regulations, an updated review of EVs is timely to provide insights to achieve these goals.

Phosphoproteomics identify arachidonic-acid-regulated signal transduction pathways modulating macrophage functions with implications for ovarian cancer.

Arachidonic acid (AA) is a polyunsaturated fatty acid present at high concentrations in the ovarian cancer (OC) microenvironment and associated with a poor clinical outcome. In the present study, we have unraveled a potential link between AA and macrophage functions.Methods: AA-triggered signal transduction was studied in primary monocyte-derived macrophages (MDMs) by phosphoproteomics, transcriptional profiling, measurement of intracellular Ca2+ accumulation and reactive oxygen species production in conjunction with bioinformatic analyses. Functional effects were investigated by actin filament staining, quantification of macropinocytosis and analysis of extracellular vesicle release.Results: We identified the ASK1 - p38δ/α (MAPK13/14) axis as a central constituent of signal transduction pathways triggered by non-metabolized AA. This pathway was induced by the Ca2+-triggered activation of calmodulin kinase II, and to a minor extent by ROS generation in a subset of donors. Activated p38 in turn was linked to a transcriptional stress response associated with a poor relapse-free survival. Consistent with the phosphorylation of the p38 substrate HSP27 and the (de)phosphorylation of multiple regulators of Rho family GTPases, AA impaired actin filament organization and inhibited actin-driven macropinocytosis. AA also affected the phosphorylation of proteins regulating vesicle biogenesis, and consistently, AA enhanced the release of tetraspanin-containing exosome-like vesicles. Finally, we identified phospholipase A2 group 2A (PLA2G2A) as the clinically most relevant enzyme producing extracellular AA, providing further potentially theranostic options.Conclusion: Our results suggest that AA contributes to an unfavorable clinical outcome of OC by impacting the phenotype of tumor-associated macrophages. Besides critical AA-regulated signal transduction proteins identified in the present study, PLA2G2A might represent a potential prognostic tool and therapeutic target to interfere with OC progression.

Sensory Cilia Act as a Specialized Venue for Regulated Extracellular Vesicle Biogenesis and Signaling

Extracellular vesicles play major roles in intercellular signaling, yet fundamental aspects of their biology remain poorly understood. Ciliary EV shedding is evolutionary conserved. Here we use super resolution, real time imaging of fluorescent-protein tagged EV cargo combined with in vivo bioassays to study ciliary EVs in C. elegans. We find that neuronal sensory cilia shed the TRP polycystin-2 channel PKD-2::GFP-carrying EVs from two distinct sites - the ciliary tip and the ciliary base. Ciliary tip shedding requires distal ciliary enrichment of PKD-2 by the myristoylated coiled-coil protein CIL-7. Kinesin-3 KLP-6 and intraflagellar transport (IFT) kinesin-2 motors are also required for ciliary tip EV shedding. Blocking ciliary tip shedding results in excessive EV shedding from the base. Finally, we demonstrate that C. elegans male ciliated neurons modulate EV cargo composition in response to sensory stimulation by hermaphrodite mating partners. Overall, our study indicates that the cilium and its trafficking machinery act as a specialized venue for regulated EV biogenesis and signaling.

Cooperation and Interplay between EGFR Signalling and Extracellular Vesicle Biogenesis in Cancer.

Epidermal growth factor receptor (EGFR) takes centre stage in carcinogenesis throughout its entire cellular trafficking odyssey. When loaded in extracellular vesicles (EVs), EGFR is one of the key proteins involved in the transfer of information between parental cancer and bystander cells in the tumour microenvironment. To hijack EVs, EGFR needs to play multiple signalling roles in the life cycle of EVs. The receptor is involved in the biogenesis of specific EV subpopulations, it signals as an active cargo, and it can influence the uptake of EVs by recipient cells. EGFR regulates its own inclusion in EVs through feedback loops during disease progression and in response to challenges such as hypoxia, epithelial-to-mesenchymal transition and drugs. Here, we highlight how the spatiotemporal rules that regulate EGFR intracellular function intersect with and influence different EV biogenesis pathways and discuss key regulatory features and interactions of this interplay. We also elaborate on outstanding questions relating to EGFR-driven EV biogenesis and available methods to explore them. This mechanistic understanding will be key to unravelling the functional consequences of direct anti-EGFR targeted and indirect EGFR-impacting cancer therapies on the secretion of pro-tumoural EVs and on their effects on drug resistance and microenvironment subversion.

Extracellular vesicles and oncogenic signaling.

In recent years, extracellular vesicles (EVs) emerged as potential diagnostic and prognostic markers for cancer therapy. While the field of EV research is rapidly developing and their application as vehicles for therapeutic cargo is being tested, little is still known about the exact mechanisms of signaling specificity and cargo transfer by EVs, especially invivo. Several signaling cascades have been found to use EVs for signaling in the tumor-stroma interaction. These include potentially oncogenic, verbatim transforming, signaling cascades such as Wnt and TGF-β signaling, and other signaling cascades that have been tightly associated with tumor progression and metastasis, such as PD-L1 and VEGF signaling. Multiple mechanisms of how these signaling cascades and EVs interplay to mediate these complex processes have been described, such as direct signal activation through pathway components on or in EVs or indirectly by influencing vesicle biogenesis, cargo sorting, or uptake dynamics. In this review, we summarize the current knowledge of EVs, their biogenesis, and our understanding of EV interactions with recipient cells with a focus on selected oncogenic and cancer-associated signaling pathways. After an in-depth look at how EVs mediate and influence signaling, we discuss potentially translatable EV functions and existing knowledge gaps.

Multiple factors are involved in regulation of extracellular membrane vesicle biogenesis in Streptococcus mutans.

Streptococcus mutans, a major etiological agent of human dental caries, produces membrane vesicles (MVs) that contain protein and extracellular DNA. In this study, functional genomics, along with in vitro biofilm models, was used to identify factors that regulate MV biogenesis. Our results showed that when added to growth medium, MVs significantly enhanced biofilm formation by S. mutans, especially during growth in sucrose. This effect occurred in the presence and absence of added human saliva. Functional genomics revealed several genes, including sfp, which have a major effect on S. mutans MVs. In Bacillus sp. sfp encodes a 4'-phosphopantetheinyl transferase that contributes to surfactin biosynthesis and impacts vesiculogenesis. In S. mutans, sfp resides within the TnSmu2 Genomic Island that supports pigment production associated with oxidative stress tolerance. Compared to the UA159 parent, the Δsfp mutant, TW406, demonstrated a 1.74-fold (p<.05) higher MV yield as measured by BCA protein assay. This mutant also displayed increased susceptibility to low pH and oxidative stressors, as demonstrated by acid killing and hydrogen peroxide challenge assays. Deficiency of bacA, a putative surfactin synthetase homolog within TnSmu2, and especially dac and pdeA that encode a di-adenylyl cyclase and a phosphodiesterase, respectively, also significantly increased MV yield (p<.05). However, elimination of bacA2, a bacitracin synthetase homolog, resulted in a >1.5-fold (p<.05) reduction of MV yield. These results demonstrate that S. mutans MV properties are regulated by genes within and outside of the TnSmu2 island, and that as a major particulate component of the biofilm matrix, MVs significantly influence biofilm formation.

Shedding Light on Extracellular Vesicle Biogenesis and Bioengineering.

Extracellular vesicles (EVs) are biocompatible, nano‐sized secreted vesicles containing many types of biomolecules, including proteins, RNAs, DNAs, lipids, and metabolites. Their low immunogenicity and ability to functionally modify recipient cells by transferring diverse bioactive constituents make them an excellent candidate for a next‐generation drug delivery system. Here, the recent advances in EV biology and emerging strategies of EV bioengineering are summarized, and the prospects for clinical translation of bioengineered EVs and the challenges to be overcome are discussed.

A Budding Relationship: Bacterial Extracellular Vesicles in the Microbiota-Gut-Brain Axis.

The discovery of the microbiota-gut-brain axis has revolutionized our understanding of systemic influences on brain function and may lead to novel therapeutic approaches to neurodevelopmental and mood disorders. A parallel revolution has occurred in the field of intercellular communication, with the realization that endosomes, and other extracellular vesicles, rival the endocrine system as regulators of distant tissues. These two paradigms shifting developments come together in recent observations that bacterial membrane vesicles contribute to inter-kingdom signaling and may be an integral component of gut microbe communication with the brain. In this short review we address the current understanding of the biogenesis of bacterial membrane vesicles and the roles they play in the survival of microbes and in intra and inter-kingdom communication. We identify recent observations indicating that bacterial membrane vesicles, particularly those derived from probiotic organisms, regulate brain function. We discuss mechanisms by which bacterial membrane vesicles may influence the brain including interaction with the peripheral nervous system, and modulation of immune activity. We also review evidence suggesting that, unlike the parent organism, gut bacteria derived membrane vesicles are able to deliver cargo, including neurotransmitters, directly to the central nervous system and may thus constitute key components of the microbiota-gut-brain axis.

Peptidylarginine deiminases and extracellular vesicles: prospective drug targets and biomarkers in central nervous system diseases and repair.

Peptidylarginine deiminases are a family of calcium-activated enzymes with multifaceted roles in physiological and pathological processes, including in the central nervous system. Peptidylarginine deiminases cause post-translational deimination/citrullination, leading to changes in structure and function of a wide range of target proteins. Deimination can facilitate protein moonlighting, modify protein-protein interaction, cause protein dysfunction and induce inflammatory responses. Peptidylarginine deiminases also regulate the biogenesis of extracellular vesicles, which play important roles in cellular communication through transfer of extracellular vesicle-cargo, e.g., proteins and genetic material. Both peptidylarginine deiminases and extracellular vesicles are linked to a number of pathologies, including in the central nervous system, and their modulation with pharmacological peptidylarginine deiminase inhibitors have shown great promise in several in vitro and in vivo central nervous system disease models. Furthermore, extracellular vesicles derived from mesenchymal stem cells have been assessed for their therapeutic application in central nervous system injury. As circulating extracellular vesicles can be used as non-invasive liquid biopsies, their specific cargo-signatures (including deiminated proteins and microRNAs) may allow for disease “fingerprinting” and aid early central nervous system disease diagnosis, inform disease progression and response to therapy. This mini-review discusses recent advances in the field of peptidylarginine deiminase and extracellular vesicle research in the central nervous system, focusing on several central nervous system acute injury, degeneration and cancer models.

&lt;p&gt;Role of Sortilin and Matrix Vesicles in Nϵ-Carboxymethyl-Lysine-Induced Diabetic Atherosclerotic Calcification&lt;/p&gt;

Background and AimsTo investigate the role of Sortilin and matrix vesicles (MVs) in Nε-Carboxymethyl-lysine (CML)-induced diabetic atherosclerotic calcification (AC).MethodsAt human level, the correlation between Sortilin and CD9 (marker proteins of MVs) in serum MVs and CML in serum was explored by enzyme-linked immunosorbent assay (ELISA) detection and Pearson correlation analysis. After a diabetic apoE-/- mouse model was constructed, the calcification of aorta and the expressions of related proteins under CML and MVs injection were observed by calcification staining, immunofluorescence staining, and Western blot. MVs levels released by smooth muscle cells (SMCs) under different treatments was detected by nanometer tracking analysis (NTA). After treating SMCs with MVs and Anti-Sortilin, cell calcification was observed by Alizarin red staining.ResultsSerological analysis of patients showed that the concentrations of Sortilin and CD9 in serum MVs were positively correlated with the concentration of CML in serum. Animal experiments showed that CML could promote the progression of diabetic AC and the high expression of Sortilin in plaques. Diabetic apoE-/- mouse tail vein injection of CML-induced SMCs-derived MVs obviously aggravated AC. Cell experiment results showed that a high concentration of CML significantly promoted the release of MVs from SMCs. MVs from this source could markedly worsen cell calcification, while the administration of GW4869 (a widely used extracellular vesicles biogenesis inhibitor) significantly reduced cell calcification. Finally, treatment of high concentrations of CML could also promote the recruitment of Sortilin to MVs, and administration of Anti-Sortilin could markedly reduce cell calcification caused by MVs.ConclusionWe proved that CML not only affects the release of MVs from SMCs but also affects the recruitment of Sortilin to MVs, thereby promoting diabetic AC. This discovery may provide a new strategy for targeted prevention of vascular calcification in diabetes.

Tetraspanins, More than Markers of Extracellular Vesicles in Reproduction.

The participation of extracellular vesicles in many cellular processes, including reproduction, is unquestionable. Although currently, the tetraspanin proteins found in extracellular vesicles are mostly applied as markers, increasing evidence points to their role in extracellular vesicle biogenesis, cargo selection, cell targeting, and cell uptake under both physiological and pathological conditions. In this review, we bring other insight into the involvement of tetraspanin proteins in extracellular vesicle physiology in mammalian reproduction. We provide knowledge regarding the involvement of extracellular vesicle tetraspanins in these processes in somatic cells. Furthermore, we discuss the future direction towards an understanding of their functions in the tissues and fluids of the mammalian reproductive system in gamete maturation, fertilization, and embryo development; their involvement in mutual cell contact and communication in their complexity.

Tracking exocytosis of a GPI-anchored protein in Aspergillus nidulans.

Secretion of the glycosylphosphatidylinositol-anchored protein (GPI-AP) EglC was investigated in the filamentous fungus Aspergillus nidulans, exploiting a sucrose-inducible promoter to conditionally express the protein in cells blocked at different steps of exocytosis. EglC is delivered to the cell surface in a polarized fashion, but appears to redistribute rapidly toward apico-distal regions. Inactivation of SarASar1 mediating COPII vesicle biogenesis resulted in the accumulation of EglC in the endoplasmic reticulum (ER) but, rather than concentrating in ER-exit-sites, the reporter labeled the ER uniformly. Abnormal posttranslational modifications of EglC were detected in sarAts and sed5ts mutants, suggesting that blocking COPII biogenesis or traffic in the ER/Golgi interface might affect GPI remodeling. EglC delivery to the plasma membrane requires, besides Golgi function, the TRAPPII complex mediating the biogenesis of RAB11 secretory vesicles at the TGN, but is unaffected by the absence of RAB5, the key regulator of early endosome biogenesis/maturation. Thus, unlike the soluble extracellular enzyme inulinase, EglC is directly delivered from the TGN to the plasma membrane without involvement of endosomes. We conclude that in A. nidulans, GPI-APs follow a direct secretory pathway from the ER to the plasma membrane.

Glycometabolic Regulation of the Biogenesis of Small Extracellular Vesicles

The biogenesis of small extracellular vesicles (sEVs) is regulated by multiple molecular machineries generating considerably heterogeneous vesicle populations, including exosomes and non-exosomal vesicles, with distinct cargo molecules. However, the role of carbohydrate metabolism in generating such vesicle heterogeneity remains largely elusive. Here, we discover that 2-deoxyglucose (2-DG), a well-known glycolysis inhibitor, suppresses the secretion of non-exosomal vesicles by impairing asparagine-linked glycosylation (N-glycosylation) in mouse melanoma cells. Mechanistically, 2-DG is metabolically incorporated into N-glycan precursors, causing precursor degradation and partial hypoglycosylation. N-glycosylation blockade by Stt3a silencing is sufficient to inhibit non-exosomal vesicle secretion. In contrast, N-glycosylation blockade barely influences exosomal secretion of tetraspanin proteins. Functionally, N-glycosylation at specific sites of the hepatocyte growth factor receptor, a cargo protein of non-exosomal vesicles, facilitates its sorting into vesicles. These results uncover a link between N-glycosylation and unconventional vesicle secretion and suggest that N-glycosylation facilitates sEV biogenesis through cargo protein sorting.

Molecular players in biogenesis of mineralization-competent matrix vesicles

Molecular players in biogenesis of mineralization-competent matrix vesicles

EPSIN1 and MTV1 define functionally overlapping but molecularly distinct trans-Golgi network subdomains in Arabidopsis

The plant trans-Golgi network (TGN) is a central trafficking hub where secretory, vacuolar, recycling, and endocytic pathways merge. Among currently known molecular players involved in TGN transport, three different adaptor protein (AP) complexes promote vesicle generation at the TGN with different cargo specificity and destination. Yet, it remains unresolved how sorting into diverging vesicular routes is spatially organized. Here, we study the family of Arabidopsis thaliana Epsin-like proteins, which are accessory proteins to APs facilitating vesicle biogenesis. By comprehensive molecular, cellular, and genetic analysis of the EPSIN gene family, we identify EPSIN1 and MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1) as its only TGN-associated members. Despite their large phylogenetic distance, they perform overlapping functions in vacuolar and secretory transport. By probing their relationship with AP complexes, we find that they define two molecularly independent pathways: While EPSIN1 associates with AP-1, MTV1 interacts with AP-4, whose function is required for MTV1 recruitment. Although both EPSIN1/AP-1 and MTV1/AP-4 pairs reside at the TGN, high-resolution microscopy reveals them as spatially separate entities. Our results strongly support the hypothesis of molecularly, functionally, and spatially distinct subdomains of the plant TGN and suggest that functional redundancy can be achieved through parallelization of molecularly distinct but functionally overlapping pathways.

Abstract 2969: Senescent stromal cells promote cancer resistance via Sirt1 loss-potentiated biogenesis of small extracellular vesicles

Abstract Cellular senescence is a potent tumor-suppressive program that prevents neoplastic events. Paradoxically, senescent cells develop an inflammatory secretome, termed the senescence-associated secretory phenotype (SASP) and implicated in age-related pathologies including cancer. Here we report that senescent cells actively synthesize and release small extracellular vesicles (sEVs) with a distinctive size distribution. Mechanistically, SIRT1 loss supports accelerated sEV production despite enhanced proteome-wide ubiquitination, a process correlated with ATP6V1A downregulation and defective lysosomal acidification. Senescent stromal cell-derived sEVs carry a special set of cargoes, including small RNA species such as microRNAs (miRNAs) whose spectrum markedly differ from that of proliferating cell-secreted sEVs. Once released, senescent stromal sEVs significantly alter the expression profile of recipient cancer cells and enhance their aggressiveness, specifically drug resistance mediated by expression of ATP binding cassette subfamily B member 4 (ABCB4). Targeting SIRT1 with an agonist SRT2104 prevents development of cancer resistance through restraining sEV production by senescent stromal cells. In clinical oncology, sEVs in peripheral blood of posttreatment cancer patients are readily detectable by routine biotechniques, presenting a novel biomarker to monitor therapeutic efficacy and predict long term outcome. Together, our study identifies a distinct mechanism supporting pathological activities of senescent cells, and provides a novel avenue to circumvent advanced human malignancies by co-targeting cancer cells and their surrounding microenvironment, which contributes to drug resistance via secretion of sEVs from senescent stromal cells. Citation Format: Liu Han, Qilai Long, Eric Lam, Yu Sun. Senescent stromal cells promote cancer resistance via Sirt1 loss-potentiated biogenesis of small extracellular vesicles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2969.

Molecular characteristics and functional differences of anti-PM/Scl autoantibodies and two other distinct and unique supramolecular structures known as "EXOSOMES".

The term "exosome" has been applied to three distinct supramolecular entities, namely the PM/Scl autoantibodies or "RNA exosomes", transforming DNA fragments termed "DNA exosomes", and small size extracellular vesicles knows as "exosomes". Some of the molecular components of the "PM/Scl exosome complex" or "RNA exosome" are recognized by specific autoantibodies present in the serum from some Systemic Sclerosis (SSc), polymyositis (PM) and polymyositis SSc (PM/Scl) overlap syndrome patients. On the other hand, one of the most active focuses of laboratory investigation in the last decade has been the biogenesis and role of extracellular vesicles known as "exosomes". The remarkable ability of these "exosome" vesicles to alter the cellular phenotype following fusion with target cells and the release of their macromolecular cargo has revealed a possible role in the pathogenesis of numerous diseases, including malignant, inflammatory, and autoimmune disorders and may allow them to serve as theranostic agents for personalized and precision medicine. The indiscriminate use of the term "exosome" to refer to these three distinct molecular entities has engendered great confusion in the scientific literature. Here, we review the molecular characteristics and functional differences between the three molecular structures identified as "exosomes". Given the rapidly growing scientific interest in extravesicular exosomes, unless a solution is found the confusion in the literature resulting from the use of the term "exosomes" will markedly increase.

The Role of Cathepsins in Memory Functions and the Pathophysiology of Psychiatric Disorders.

Cathepsins are proteases with functions in cellular homeostasis, lysosomal degradation and autophagy. Their role in the development of neurodegenerative diseases has been extensively studied. It is well established that impairment of proper cathepsin function plays a crucial role in the pathophysiology of neurodegenerative diseases, and in recent years a role for cathepsins in mental disorders has emerged given the involvement of cathepsins in memory function, hyperactivity, and in depression- and anxiety-like behavior. Here we review putative cathepsin functions with a special focus on their role in the pathophysiology of psychiatric diseases. Specifically, cathepsins are crucial for maintaining cellular homeostasis, particularly as part of the autophagy machinery of neural strategies underlying acute stress response. Disruption of cathepsin functions can lead to psychiatric diseases such as major depressive disease (MDD), bipolar disorder, and schizophrenia. Specifically, cathepsins can be excreted via a process called secretory autophagy. Thereby, they are able to regulate extracellular factors such as brain-derived neurotrophic factor and perlecan c-terminal fragment LG3 providing maintenance of neuronal homeostasis and mediating neuronal plasticity in response to acute stress or trauma. In addition, impairment of proper cathepsin function can result in impaired synaptic transmission by compromised recycling and biogenesis of synaptic vesicles. Taken together, further investigations on cathepsin functions and stress response, neuroplasticity, and synaptic transmission will be of great interest in understanding the pathophysiology of psychiatric disorders.

Berberine inhibits cancer cells growth by suppressing fatty acid synthesis and biogenesis of extracellular vesicles

AimsBerberine is an isoquinoline alkaloid extracted from the root, rhizome and stem bark of Coptidis Rhizoma. Previous studies have revealed the anti-tumor potential of berberine against various types of cancer cells. However, the underlying mechanisms are not yet fully understood. In this study, we focused on the effects of berberine on fatty acid synthesis and extracellular vesicles formation in cancer cells, and revealed the internal mechanism of berberine inhibition on cancer cell proliferation. Materials and methodsAnti-proliferative activity of berberine was determined by cell counting and microscope observation and cell cycle analysis. Activities of AMPK and ACC, expression of extracellular vesicles markers were detected by western blotting. 13C labeling metabolic flux analysis was used for determination of de novo synthesis of fatty acids. The excreted extracellular vesicles in culture mediums were separated by both polyethylene glycol enrichment of extracellular vesicles and differential centrifugation separation. Key findingsAmong our early experiments, 5–10 μmol/L berberine exhibited the substantial anti-proliferative effect against human colon cancer cell line HCT116, cervical cancer cell line HeLa and other cancer cells. It was also revealed that, through activating AMPK, berberine inhibited ACC activity then suppressed intracellular fatty acid synthesis, finally decreased the biogenesis of extracellular vesicles. Moreover, supplement with citrate acid, palmitic acid, as well as exogenous extracellular vesicles, could rescue the inhibitory effect of berberine on cell proliferation, suggesting that inhibited ACC activity, suppressed fatty acid synthesis and decreased extracellular vesicles production were important mechanisms account for berberine inhibiting cancer cell proliferation. SignificanceOur study indicates that berberine suppresses cancer cell proliferation through inhibiting the synthesis of fatty acids and decreasing biogenesis and secretion of extracellular vesicles, suggests that berberine is a promising candidate for the development of new therapies for cancer.