Alcohol Promotes Exosome Biogenesis and Release via Modulating Rabs and miR-192 Expression in Human Hepatocytes

Exosomes are membrane vesicles released by various cell types into the extracellular space under different conditions including alcohol exposure. Exosomes are involved in intercellular communication and as mediators of various diseases. Alcohol use causes oxidative stress that promotes exosome secretion. Here, we elucidated the effects of alcohol on exosome biogenesis and secretion using human hepatocytes. We found that alcohol treatment induces the expression of genes involved in various steps of exosome formation. Expression of Rab proteins such as Rab1a, Rab5c, Rab6, Rab10, Rab11, Rab27a and Rab35 were increased at the mRNA level in primary human hepatocytes after alcohol treatment. Rab5, Rab6 and Rab11 showed significant induction in the livers of patients with alcohol-associated liver disease. Further, alcohol treatment also led to the induction of syntenin, vesicle-associated membrane proteins (VAMPs), and syntaxin that all play various roles in exosome biogenesis and secretion. VAMP3, VAMP5, VAPb, and syntaxin16 mRNA transcripts were increased in alcohol treated cells and in the livers of alcohol-associated liver disease (ALD) patients. Induction in these genes was associated with increases in exosome secretion in alcohol treated hepatocytes. We found that hepatocyte enriched miR-192 and miR-122 levels were significantly decreased in alcohol treated hepatocytes whereas their levels were increased in the cell-free supernatant. The primary transcripts of miR-192 and miR-122 were reduced in alcohol treated hepatocytes, suggesting alcohol partially affects these miRNAs at the transcriptional level. We found that miR-192 has putative binding sites for genes involved in exosome secretion. Inhibition of miR-192 in human hepatoma cells caused a significant increase in Rab27a, Rab35, syntaxin7 and syntaxin16 and a concurrent increase in exosome secretion, suggesting miR-192 regulates exosomes release in hepatocytes. Collectively, our results reveal that alcohol modulates Rabs, VAMPs and syntaxins directly and partly via miR-192 to induce exosome machinery and release.

Secretory Autophagy Forges a Therapy Resistant Microenvironment in Melanoma

Melanoma is the most aggressive skin cancer characterized by high mutational burden and large heterogeneity. Cancer cells are surrounded by a complex environment, critical to tumor establishment and progression. Thus, tumor-associated stromal components can sustain tumor demands or impair cancer cell progression. One way to manage such processes is through the regulation of autophagy, both in stromal and tumor cells. Autophagy is a catabolic mechanism that provides nutrients and energy, and it eliminates damaged organelles by degradation and recycling of cellular elements. Besides this primary function, autophagy plays multiple roles in the tumor microenvironment capable of affecting cell fate. Evidence demonstrates the existence of novel branches in the autophagy system related to cytoplasmic constituent’s secretion. Hence, autophagy-dependent secretion assembles a tangled network of signaling that potentially contributes to metabolism reprogramming, immune regulation, and tumor progression. Here, we summarize the current awareness regarding secretory autophagy and the intersection with exosome biogenesis and release in melanoma and their role in tumor resistance. In addition, we present and discuss data from public databases concerning autophagy and exosome-related genes as important mediators of melanoma behavior. Finally, we will present the main challenges in the field and strategies to translate most of the pre-clinical findings to clinical practice.

Exogenous and Endogenous Dendritic Cell-Derived Exosomes: Lessons Learned for Immunotherapy and Disease Pathogenesis

Immune therapeutic exosomes, derived exogenously from dendritic cells (DCs), the ‘directors’ of the immune response, are receiving favorable safety and tolerance profiles in phase I and II clinical trials for a growing number of inflammatory and neoplastic diseases. DC-derived exosomes (EXO), the focus of this review, can be custom tailored with immunoregulatory or immunostimulatory molecules for specific immune cell targeting. Moreover, the relative stability, small size and rapid uptake of EXO by recipient immune cells offer intriguing options for therapeutic purposes. This necessitates an in-depth understanding of mechanisms of EXO biogenesis, uptake and routing by recipient immune cells, as well as their in vivo biodistribution. Against this backdrop is recognition of endogenous exosomes, secreted by all cells, the molecular content of which is reflective of the metabolic state of these cells. In this regard, exosome biogenesis and secretion is regulated by cell stressors of chronic inflammation and tumorigenesis, including dysbiotic microbes, reactive oxygen species and DNA damage. Such cell stressors can promote premature senescence in young cells through the senescence associated secretory phenotype (SASP). Pathological exosomes of the SASP amplify inflammatory signaling in stressed cells in an autocrine fashion or promote inflammatory signaling to normal neighboring cells in paracrine, without the requirement of cell-to-cell contact. In summary, we review relevant lessons learned from the use of exogenous DC exosomes for immune therapy, as well as the pathogenic potential of endogenous DC exosomes.

Macros to Quantify Exosome Release and Autophagy at the Neuromuscular Junction of Drosophila Melanogaster

Automatic quantification of image parameters is a powerful and necessary tool to explore and analyze crucial cell biological processes. This article describes two ImageJ/Fiji automated macros to approach the analysis of synaptic autophagy and exosome release from 2D confocal images. Emerging studies point out that exosome biogenesis and autophagy share molecular and organelle components. Indeed, the crosstalk between these two processes may be relevant for brain physiology, neuronal development, and the onset/progression of neurodegenerative disorders. In this context, we describe here the macros “Autophagoquant” and “Exoquant” to assess the quantification of autophagosomes and exosomes at the neuronal presynapse of the Neuromuscular Junction (NMJ) in Drosophila melanogaster using confocal microscopy images. The Drosophila NMJ is a valuable model for the study of synapse biology, autophagy, and exosome release. By use of Autophagoquant and Exoquant, researchers can have an unbiased, standardized, and rapid tool to analyze autophagy and exosomal release in Drosophila NMJ.Code available at: https://github.com/IreneSaMi/Exoquant-Autophagoquant

Daratumumab Resistant Natural Killer/T-Cell Lymphoma Exhibit an Addiction to the Exosome Biogenesis Pathway for Survival

A phase 2 clinical trial has demonstrated that daratumumab monotherapy was safe and well tolerated in relapsed/refractory Natural Killer/T-Cell Lymphoma (NKTL). However, no patients achieved complete response, and duration of response was short. Similarly, in Multiple Myeloma (MM), while daratumumab based combinations are approved for front line treatment, responses are heterogeneous and development of treatment resistance inevitable. Therefore, elucidation of mechanisms which can overcome daratumumab resistance is essential for the optimization of therapeutic response in patients. To this end, 2 pairs of isogenic daratumumab resistant and sensitive models of NKTL were developed in vitro via sequential exposure of cell lines to increasing concentrations of daratumumab in the presence of complement serum. A 3rd model was also studied in vivo whereby long-term administration of daratumumab in mice identified a sub-group of tumors which stopped responding to treatment and began to rapidly enlarge ('Resistant') as compared to others which remained similar to or smaller ('Sensitive') than the tumour volume at the initiation of treatment. RNA sequencing was performed on these models and genes commonly upregulated or downregulated analyzed. Differential gene expression analysis highlighted an enrichment for the upregulation of genes involved in exosome biogenesis and secretion in both cell line and mouse-derived daratumumab resistant NKTL models. An additional daratumumab resistant model was developed in an MM cell line to further validate these findings and extend the study in an MM model. Immunoblotting of the 3 pairs of isogenic sensitive and resistant cell line models demonstrate that there is indeed an upregulation of proteins regulating exosomal biogenesis and secretion; Alix, TSG101 and Rab27b in the daratumumab resistant phenotype. This is associated with a concomitant increase in secreted exosomes levels in the tumour microenvironment. The size and quantification of extracellular vesicles (EV) secreted in the media were studied by nanoparticle tracking analysis. Extracellular vesicles ranged in the size of 70-150nM corroborating with the size of exosomes and nanoparticle quantification revealed a higher concentration of exosomes present in the tumour microenvironment of resistant cells as compared to sensitive cells. Subsequently, exosomes were purified via ultracentrifugation and protein expression analysis confirmed elevation of exosome markers CD63 and CD81. To study the role of exosomal-mediated mechanisms in the survival of daratumumab resistant cells, we treated isogenic models with neticonazole and ketoconazole (azoles) which have been identified as selective inhibitors of exosomal biogenesis in a drug repurposing study for advanced cancers. Interestingly, azole treatment demonstrated a selective and more effective suppression of tumour cell viability in daratumumab resistant than sensitive cell lines. Immunoblot analysis showed that azole treatment at identical concentrations resulted in a more extensive downregulation of Alix, Rab27b and CD81 protein expression in resistant than sensitive cells. Additionally, depletion of Alix and Rab27b protein expression via siRNA knockdown induced cell death confirming that daratumumab-resistant cell lines are dependent on exosomal-mediated pathways for survival. Current research is focused mainly on intrinsic or immune cell-mediated mechanisms of daratumumab resistance, but little is known regarding the effect of extrinsic components in the tumour microenvironment. We demonstrate that daratumumab resistant models exhibit a distinct upregulation of proteins mediating exosome biogenesis resulting in enhanced exosome secretion. Daratumumab resistant cells are targeted more efficaciously with exosome biogenesis inhibitors than sensitive counterparts thereby suggesting an addiction to exosome-mediated mechanisms of survival. This is further supported by gene silencing studies. In future, we aim to perform miRNA profiling of exosomes purified from the tumour microenvironment of isogenic daratumumab-resistant and sensitive cell line models as well as from bone marrow plasma of daratumumab treated patients. miRNA which are enriched in the exosomes of resistant phenotypes will be characterized, unique biomarkers of response identified and in depth mechanisms of resistance studied. Disclosures No relevant conflicts of interest to declare.

Blockade of Exosome Release Suppresses Atrial Fibrillation by Alleviating Atrial Fibrosis in Canines With Prolonged Atrial Pacing

Background: Clinical studies have shown that exosomes are associated with atrial fibrillation (AF). However, the roles and underlying mechanisms remain unclear. Hence, this study aimed to investigate the function of exosomes in AF development.Methods: Twenty beagles were randomly divided into the sham group (n = 6), the pacing group (n = 7), and the pacing + GW4869 group (n = 7). The pacing and GW4869 groups underwent rapid atrial pacing (450 beats/min) for 7 days. The GW4869 group received intravenous GW4869 injection (an inhibitor of exosome biogenesis/release, 0.3 mg/kg, once a day) during pacing. Electrophysiological measurements, transmission electron microscopy, nanoparticle tracking analysis, western blotting, RT-PCR, Masson's staining, and immunohistochemistry were performed in this study.Results: Rapid atrial pacing increased the release of plasma and atrial exosomes. GW4869 treatment markedly suppressed AF inducibility and reduced the release of exosomes. After 7 days of pacing, the expression of transforming growth factor-β1 (TGF-β1), collagen I/III, and matrix metalloproteinases was enhanced in the atrium, and the levels of microRNA-21-5p (miR-21-5p) were upregulated in both plasma exosomes and the atrium, while the tissue inhibitor of metalloproteinase 3 (TIMP3), a target of miR-21-5p, showed a lower expression in the atrium. The administration of GW4869 abolished these effects.Conclusions: The blockade of exosome release with GW4869 suppressed AF by alleviating atrial fibrosis in a canine model, which was probably related to profibrotic miR-21-5p enriched in exosomes and its downstream TIMP3/TGF-β1 pathway.

Silver nanoparticle enhances secretion of exosomes in SH-SY5Y cells: Potential therapeutic strategy for human neuroblastoma cancer

Background: Exosomes—a subset of extracellular vesicles (EVs)—are secreted by virtually all cells, including human neuroblastoma cancer (SH-SY5Y) cells, into bodily fluids. Oxidative stress is critically involved in exosome biogenesis and release. Silver nanoparticles (AgNPs) induce cytotoxicity, oxidative stress, and apoptosis in cancer and non-cancer cells. Methods: Here, we studied whether AgNPs-induced oxidative stress could enhance exosome biogenesis and release under low serum conditions in the presence of AgNPs. Although several studies have reported various mechanisms that contribute to EV biogenesis and release from cells, none exists on the involvement of external stimuli by controlling acetylcholinesterase (AChE) and neutral-sphingomyelinase (n-SMase) activities, total protein concentration of exosomes, and exosome count. Results: Owing to cytotoxic and oxidative stresses, AgNPs-treated cells and exosome release were significantly facilitated, which strongly correlated with the AgNPs-induced oxidative stress. Moreover, the expression levels of some important exosome biomarkers were found to be significant under oxidative stress conditions. N-acetylcysteine prevented oxidative stress-induced exosome biogenesis and release. Furthermore, we identified the involvement of the ceramide pathway in exosome functions by inhibiting AChE and n-SMase activities, and exosome protein/counts. These data contribute to the understanding of how AgNPs and intracellular molecular pathways affect exosome biogenesis and release in SH-SY5Y cells. Conclusion: To the best of our knowledge, this is the first study showing that AgNPs stimulate exosome biogenesis and release by inducing oxidative stress and ceramide pathways.

Exosomes: Potential Disease Biomarkers and New Therapeutic Targets

Exosomes are extracellular vesicles released by cells, both constitutively and after cell activation, and are present in different types of biological fluid. Exosomes are involved in the pathogenesis of diseases, such as cancer, neurodegenerative diseases, pregnancy disorders and cardiovascular diseases, and have emerged as potential non-invasive biomarkers for the detection, prognosis and therapeutics of a myriad of diseases. In this review, we describe recent advances related to the regulatory mechanisms of exosome biogenesis, release and molecular composition, as well as their role in health and disease, and their potential use as disease biomarkers and therapeutic targets. In addition, the advantages and disadvantages of their main isolation methods, characterization and cargo analysis, as well as the experimental methods used for exosome-mediated drug delivery, are discussed. Finally, we present potential perspectives for the use of exosomes in future clinical practice.