Proteomic profiling of small extracellular vesicles derived from mouse pancreatic cancer and stellate cells: Role in pancreatic cancer.

Small extracellular vesicles (sEVs) are cell-derived vesicles evolving as important elements involved in all stages of cancers. sEVs bear unique protein signatures that may serve as biomarkers. Pancreatic cancer (PC) records a very poor survival rate owing to its late diagnosis and several cancer cell-derived proteins have been reported as candidate biomarkers. However, given the pivotal role played by stellate cells (PSCs, which produce the collagenous stroma in PC), it is essential to also assess PSC-sEV cargo in biomarker discovery. Thus, this study aimed to isolate and characterise sEVs from mouse PC cells and PSCs cultured alone or as co-cultures and performed proteomic profiling and pathway analysis. Proteomics confirmed the enrichment of specific markers in the sEVs compared to their cells of origin as well as the proteins that are known to express in each of the culture types. Most importantly, for the first time it was revealed that PSC-sEVs are enriched in proteins (including G6PI, PGAM1, ENO1, ENO3, and LDHA) that mediate pathways related to development of diabetes, such as glucose metabolism and gluconeogenesis revealing a potential role of PSCs in pancreatic cancer-related diabetes (PCRD). PCRD is now considered a harbinger of PC and further research will enable to identify the role of these components in PCRD and may develop as novel candidate biomarkers of PC.

Differential effects of physiological agonists on the proteome of platelet-derived extracellular vesicles.

Arterial thrombosis manifesting as heart attack and stroke is the leading cause of death worldwide. Platelets are central mediators of thrombosis that can be activated through multiple activation pathways. Platelet-derived extracellular vesicles (pEVs), also known as platelet-derived microparticles, are granular mixtures of membrane structures produced by platelets in response to various activating stimuli. Initial studies have attracted interest on how platelet agonists influence the composition of the pEV proteome. In the current study, we used physiological platelet agonists of varying potencies which reflect the microenvironments that platelets experience during thrombus formation: adenosine diphosphate, collagen, thrombin as well as a combination of thrombin/collagen to induce platelet activation and pEV generation. Proteomic profiling revealed that pEVs have an agonist-dependent altered proteome in comparison to their cells of origin, activated platelets. Furthermore, we found that various protein classes including those related to coagulation and complement (prothrombin, antithrombin, and plasminogen) and platelet activation (fibrinogen) are attributed to platelet EVs following agonist stimulation. This agonist-dependent altered proteome suggests that protein packaging is an active process that appears to occur without de novo protein synthesis. This study provides new information on the influence of physiological agonist stimuli on the biogenesis and proteome landscape of pEVs.

Transcriptomic analysis and fusion gene identifications of midbody remnants released from colorectal cancer cells reveals they are molecularly distinct from exosomes and microparticles.

Previously, we reported that human primary (SW480) and metastatic (SW620) colorectal (CRC) cells release three classes of membrane-encapsulated extracellular vesicles (EVs); midbody remnants (MBRs), exosomes (Exos), and microparticles (MPs). We reported that MBRs were molecularly distinct at the protein level. To gain further biochemical insights into MBRs, Exos, and MPs and their emerging role in CRC, we performed, and report here, for the first time, a comprehensive transcriptome and long noncoding RNA sequencing analysis and fusion gene identification of these three EV classes using the next-generation RNA sequencing technique. Differential transcript expression analysis revealed that MBRs have a distinct transcriptomic profile compared to Exos and MPs with a high enrichment of mitochondrial transcripts lncRNA/pseudogene transcripts that are predicted to bind to ribonucleoprotein complexes, spliceosome, and RNA/stress granule proteins. A salient finding from this study is a high enrichment of several fusion genes in MBRs compared to Exos, MPs, and cell lysates from their parental cells such as MSH2 (gene encoded DNA mismatch repair protein MSH2). This suggests potential EV-liquid biopsy targets for cancer detection. Importantly, the expression of cancer progression-related transcripts found in EV classes derived from SW480 (EGFR) and SW620 (MET and MACCA1) cell lines reflects their parental cell types. Our study is the report of RNA and fusion gene compositions within MBRs (including Exos and MPs) that could have an impact on EV functionality in cancer progression and detection using EV-based RNA/ fusion gene candidates for cancer biomarkers.

ACAD10 is not required for metformin's metabolic actions or for maintenance of whole-body metabolism in C57BL/6J mice.

AIM: Acyl-coenzyme A dehydrogenase family member 10 (ACAD10) is a mitochondrial protein purported to be involved in the fatty acid oxidation pathway. Metformin is the most prescribed therapy for type 2 diabetes; however, its precise mechanisms of action(s) are still being uncovered. Upregulation of ACAD10 is a requirement for metformin's ability to inhibit growth in cancer cells and extend lifespan in Caenorhabditis elegans. However, it is unknown whether ACAD10 plays a role in metformin's metabolic actions. MATERIALS AND METHODS: We assessed the role for ACAD10 on whole-body metabolism and metformin action by generating ACAD10KO mice on a C57BL/6J background via CRISPR-Cas9 technology. In-depth metabolic phenotyping was conducted in both sexes on a normal chow and high fat-high sucrose diet. RESULTS: Compared with wildtype mice, we detected no difference in body composition, energy expenditure or glucose tolerance in male or female ACAD10KO mice, on a chow diet or high-fat, high-sucrose diet (p ≥ .05). Hepatic mitochondrial function and insulin signalling was not different between genotypes under basal or insulin-stimulated conditions (p ≥ .05). Glucose excursions following acute administration of metformin before a glucose tolerance test were not different between genotypes nor was body composition or energy expenditure altered after 4 weeks of daily metformin treatment (p ≥ .05). Despite the lack of a metabolic phenotype, liver lipidomic analysis suggests ACAD10 depletion influences the abundance of specific ceramide species containing very long chain fatty acids, while metformin treatment altered clusters of cholesterol ester, plasmalogen, phosphatidylcholine and ceramide species. CONCLUSIONS: Loss of ACAD10 does not alter whole-body metabolism or impact the acute or chronic metabolic actions of metformin in this model.

HB-EGF-loaded nanovesicles enhance trophectodermal spheroid attachment and invasion.

The ability of trophectodermal cells (outer layer of the embryo) to attach to the endometrial cells and subsequently invade the underlying matrix are critical stages of embryo implantation during successful pregnancy establishment. Extracellular vesicles (EVs) have been implicated in embryo-maternal crosstalk, capable of reprogramming endometrial cells towards a pro-implantation signature and phenotype. However, challenges associated with EV yield and direct loading of biomolecules limit their therapeutic potential. We have previously established generation of cell-derived nanovesicles (NVs) from human trophectodermal cells (hTSCs) and their capacity to reprogram endometrial cells to enhance adhesion and blastocyst outgrowth. Here, we employed a rapid NV loading strategy to encapsulate potent implantation molecules such as HB-EGF (NVHBEGF ). We show these loaded NVs elicit EGFR-mediated effects in recipient endometrial cells, activating kinase phosphorylation sites that modulate their activity (AKT S124/129, MAPK1 T185/Y187), and downstream signalling pathways and processes (AKT signal transduction, GTPase activity). Importantly, they enhanced target cell attachment and invasion. The phosphoproteomics and proteomics approach highlight NVHBEGF -mediated short-term signalling patterns and long-term reprogramming capabilities on endometrial cells which functionally enhance trophectodermal-endometrial interactions. This proof-of-concept study demonstrates feasibility in enhancing the functional potency of NVs in the context of embryo implantation.

The effect of altered pH growth conditions on the production, composition, and proteomes of Helicobacter pylori outer membrane vesicles.

Gram-negative bacteria release outer membrane vesicles (OMVs) that contain cargo derived from their parent bacteria. Helicobacter pylori is a Gram-negative human pathogen that produces urease to increase the pH of the surrounding environment to facilitate colonization of the gastric mucosa. However, the effect of acidic growth conditions on the production and composition of H. pylori OMVs is unknown. In this study, we examined the production, composition, and proteome of H. pylori OMVs produced during acidic and neutral pH growth conditions. H. pylori growth in acidic conditions reduced the quantity and size of OMVs produced. Additionally, OMVs produced during acidic growth conditions had increased protein, DNA, and RNA cargo compared to OMVs produced during neutral conditions. Proteomic analysis comparing the proteomes of OMVs to their parent bacteria demonstrated significant differences in the enrichment of beta-lactamases and outer membrane proteins between bacteria and OMVs, supporting that differing growth conditions impacts OMV composition. We also identified differences in the enrichment of proteins between OMVs produced during different pH growth conditions. Overall, our findings reveal that growth of H. pylori at different pH levels is a factor that alters OMV proteomes, which may affect their subsequent functions.

Rapid and in-depth proteomic profiling of small extracellular vesicles for ultralow samples.

The integration of robust single-pot, solid-phase-enhanced sample preparation with powerful liquid chromatography-tandem mass spectrometry (LC-MS/MS) is routinely used to define the extracellular vesicle (EV) proteome landscape and underlying biology. However, EV proteome studies are often limited by sample availability, requiring upscaling cell cultures or larger volumes of biofluids to generate sufficient materials. Here, we have refined data independent acquisition (DIA)-based MS analysis of EV proteome by optimizing both protein enzymatic digestion and chromatography gradient length (ranging from 15 to 44 min). Our short 15 min gradient length can reproducibly quantify 1168 (from as little as 500 pg of EV peptides) to 3882 proteins groups (from 50 ng peptides), including robust quantification of 22 core EV marker proteins. Compared to data-dependent acquisition, DIA achieved significantly greater EV proteome coverage and quantification of low abundant protein species. Moreover, we have achieved optimal magnetic bead-based sample preparation tailored to low quantities of EVs (0.5 to 1 µg protein) to obtain sufficient peptides for MS quantification of 1908-2340 protein groups. We demonstrate the power and robustness of our pipeline in obtaining sufficient EV proteomes granularity of different cell sources to ascertain known EV biology. This underscores the capacity of our optimised workflow to capture precise and comprehensive proteome of EVs, especially from ultra-low sample quantities (sub-nanogram), an important challenge in the field where obtaining in-depth proteome information is essential.

Impact of tidal volume strategy at birth on initiating lung injury in preterm lambs.

Tidal ventilation is essential in supporting the transition to air-breathing at birth, but excessive tidal volume (VT) is an important factor in preterm lung injury. Few studies have assessed the impact of specific VT levels on injury development. Here, we used a lamb model of preterm birth to investigate the role of different levels of VT during positive pressure ventilation (PPV) in promoting aeration and initiating early lung injury pathways. VT was delivered as 1) 7 mL/kg throughout (VTstatic), 2) begun at 3 mL/kg and increased to a final VT of 7 mL/kg over 3 min (VTinc), or 3) commenced at 7 mL/kg, decreased to 3 mL/kg, and then returned to 7 mL/kg (VTalt). VT, inflating pressure, lung compliance, and aeration were similar in all groups from 4 min, as was postmortem histology and lung lavage protein concentration. However, transient decrease in VT in the VTalt group caused increased ventilation heterogeneity. Following TMT-based quantitative mass spectrometry proteomics, 1,610 proteins were identified in the lung. Threefold more proteins were significantly altered with VTalt compared with VTstatic or VTinc strategies. Gene set enrichment analysis identified VTalt specific enrichment of immune and angiogenesis pathways and VTstatic enrichment of metabolic processes. Our finding of comparable lung physiology and volutrauma across VT groups challenges the paradigm that there is a need to rapidly aerate the preterm lung at birth. Increased lung injury and ventilation heterogeneity were identified when initial VT was suddenly decreased during respiratory support at birth, further supporting the benefit of a gentle VT approach.NEW & NOTEWORTHY There is little evidence to guide the best tidal volume (VT) strategy at birth. In this study, comparable aeration, lung mechanics, and lung morphology were observed using static, incremental, and alternating VT strategies. However, transient reduction in VT was associated with ventilation heterogeneity and inflammation. Our results suggest that rapidly aerating the preterm lung may not be as clinically critical as previously thought, providing clinicians with reassurance that gently supporting the preterm lung maybe permissible at birth.

Rapid generation of functional nanovesicles from human trophectodermal cells for embryo attachment and outgrowth.

Extracellular vesicles (EVs) are important mediators of embryo attachment and outgrowth critical for successful implantation. While EVs have garnered immense interest in their therapeutic potential in assisted reproductive technology by improving implantation success, their large-scale generation remains a major challenge. Here, we report a rapid and scalable production of nanovesicles (NVs) directly from human trophectoderm cells (hTSCs) via serial mechanical extrusion of cells; these NVs can be generated in approximately 6 h with a 20-fold higher yield than EVs isolated from culture medium of the same number of cells. NVs display similar biophysical traits (morphologically intact, spherical, 90-130 nm) to EVs, and are laden with hallmark players of implantation that include cell-matrix adhesion and extracellular matrix organisation proteins (ITGA2/V, ITGB1, MFGE8) and antioxidative regulators (PRDX1, SOD2). Functionally, NVs are readily taken up by low-receptive endometrial HEC1A cells and reprogram their proteome towards a receptive phenotype that support hTSC spheroid attachment. Moreover, a single dose treatment with NVs significantly enhanced adhesion and spreading of mouse embryo trophoblast on fibronectin matrix. Thus, we demonstrate the functional potential of NVs in enhancing embryo implantation and highlight their rapid and scalable generation, amenable to clinical utility.

Comparative proteomic analysis of three major extracellular vesicle classes secreted from human primary and metastatic colorectal cancer cells: Exosomes, microparticles, and shed midbody remnants.

Cell-derived extracellular vesicles (EVs) are evolutionary-conserved secretory organelles that, based on their molecular composition, are important intercellular signaling regulators. At least three classes of circulating EVs are known based on mechanism of biogenesis: exosomes (sEVs/Exos), microparticles (lEVs/MPs), and shed midbody remnants (lEVs/sMB-Rs). sEVs/Exos are of endosomal pathway origin, microparticles (lEVs/MPs) from plasma membrane blebbing and shed midbody remnants (lEVs/sMB-Rs) arise from symmetric cytokinetic abscission. Here, we isolate sEVs/Exos, lEVs/MPs, and lEVs/sMB-Rs secreted from human isogenic primary (SW480) and metastatic (SW620) colorectal cancer (CRC) cell lines in milligram quantities for label-free MS/MS-based proteomic profiling. Purified EVs revealed selective composition packaging of exosomal protein markers in SW480/SW620-sEVs/Exos, metabolic enzymes in SW480/SW620-lEVs/MPs, while centralspindlin complex proteins, nucleoproteins, splicing factors, RNA granule proteins, translation-initiation factors, and mitochondrial proteins selectively traffic to SW480/SW620- lEVs/sMB-Rs. Collectively, we identify 39 human cancer-associated genes in EVs; 17 associated with SW480-EVs, 22 with SW620-EVs. We highlight oncogenic receptors/transporters selectively enriched in sEVs/Exos (EGFR/FAS in SW480-sEVs/Exos and MET, TGFBR2, ABCB1 in SW620-sEVs/Exos). Interestingly, MDK, STAT1, and TGM2 are selectively enriched in SW480-lEVs/sMB-Rs, and ADAM15 to SW620-lEVs/sMB-Rs. Our study reveals sEVs/Exos, lEVs/MPs, and lEVs/sMB-Rs have distinct protein signatures that open potential diagnostic avenues of distinct types of EVs for clinical utility.

Small extracellular vesicles promote invadopodia activity in glioblastoma cells in a therapy-dependent manner.

PURPOSE: The therapeutic efficacy of radiotherapy/temozolomide treatment for glioblastoma (GBM) is limited by the augmented invasiveness mediated by invadopodia activity of surviving GBM cells. As yet, however the underlying mechanisms remain poorly understood. Due to their ability to transport oncogenic material between cells, small extracellular vesicles (sEVs) have emerged as key mediators of tumour progression. We hypothesize that the sustained growth and invasion of cancer cells depends on bidirectional sEV-mediated cell-cell communication. METHODS: Invadopodia assays and zymography gels were used to examine the invadopodia activity capacity of GBM cells. Differential ultracentrifugation was utilized to isolate sEVs from conditioned medium and proteomic analyses were conducted on both GBM cell lines and their sEVs to determine the cargo present within the sEVs. In addition, the impact of radiotherapy and temozolomide treatment of GBM cells was studied. RESULTS: We found that GBM cells form active invadopodia and secrete sEVs containing the matrix metalloproteinase MMP-2. Subsequent proteomic studies revealed the presence of an invadopodia-related protein sEV cargo and that sEVs from highly invadopodia active GBM cells (LN229) increase invadopodia activity in sEV recipient GBM cells. We also found that GBM cells displayed increases in invadopodia activity and sEV secretion post radiation/temozolomide treatment. Together, these data reveal a relationship between invadopodia and sEV composition/secretion/uptake in promoting the invasiveness of GBM cells. CONCLUSIONS: Our data indicate that sEVs secreted by GBM cells can facilitate tumour invasion by promoting invadopodia activity in recipient cells, which may be enhanced by treatment with radio-chemotherapy. The transfer of pro-invasive cargos may yield important insights into the functional capacity of sEVs in invadopodia.

A Simplified and Effective Approach for the Isolation of Small Pluripotent Stem Cells Derived from Human Peripheral Blood.

Pluripotent stem cells are key players in regenerative medicine. Embryonic pluripotent stem cells, despite their significant advantages, are associated with limitations such as their inadequate availability and the ethical dilemmas in their isolation and clinical use. The discovery of very small embryonic-like (VSEL) stem cells addressed the aforementioned limitations, but their isolation technique remains a challenge due to their small cell size and their efficiency in isolation. Here, we report a simplified and effective approach for the isolation of small pluripotent stem cells derived from human peripheral blood. Our approach results in a high yield of small blood stem cell (SBSC) population, which expresses pluripotent embryonic markers (e.g., Nanog, SSEA-3) and the Yamanaka factors. Further, a fraction of SBSCs also co-express hematopoietic markers (e.g., CD45 and CD90) and/or mesenchymal markers (e.g., CD29, CD105 and PTH1R), suggesting a mixed stem cell population. Finally, quantitative proteomic profiling reveals that SBSCs contain various stem cell markers (CD9, ITGA6, MAPK1, MTHFD1, STAT3, HSPB1, HSPA4), and Transcription reg complex factors (e.g., STAT5B, PDLIM1, ANXA2, ATF6, CAMK1). In conclusion, we present a novel, simplified and effective isolating process that yields an abundant population of small-sized cells with characteristics of pluripotency from human peripheral blood.

Quantitative proteomic landscape of unstable atherosclerosis identifies molecular signatures and therapeutic targets for plaque stabilization.

Atherosclerotic plaque rupture leading to myocardial infarction is a major global health burden. Applying the tandem stenosis (TS) mouse model, which distinctively exhibits the characteristics of human plaque instability/rupture, we use quantitative proteomics to understand and directly compare unstable and stable atherosclerosis. Our data highlight the disparate natures and define unique protein signatures of unstable and stable atherosclerosis. Key proteins and pathway networks are identified such as the innate immune system, and neutrophil degranulation. The latter includes calprotectin S100A8/A9, which we validate in mouse and human unstable plaques, and we demonstrate the plaque-stabilizing effects of its inhibition. Overall, we provide critical insights into the unique proteomic landscape of unstable atherosclerosis (as distinct from stable atherosclerosis and vascular tissue). We further establish the TS model as a reliable preclinical tool for the discovery and testing of plaque-stabilizing drugs. Finally, we provide a knowledge resource defining unstable atherosclerosis that will facilitate the identification and validation of long-sought-after therapeutic targets and drugs for plaque stabilization.

Protocols for the Isolation of Platelets for Research and Contrast to Production of Platelet Concentrates for Transfusion.

Platelets are specialized cellular elements of blood and play a central role in maintaining normal hemostasis, wound healing, and host defense but also are implicated in pathologic processes of thrombosis, inflammation, and tumor progression and dissemination. Transfusion of platelet concentrates is an important treatment for thrombocytopenia (low platelet count) due to disease or significant blood loss, with the goal being to prevent bleeding or to arrest active bleeding. In blood circulation, platelets are in a resting state; however, when triggered by a stimulus, such as blood vessel injury, become activated (also termed procoagulant). Platelet activation is the basis of their biological function to arrest active bleeding, comprising a complex interplay of morphological phenotype/shape change, adhesion, expression of signaling molecules, and release of bioactive factors, including extracellular vesicles/microparticles. Advances in high-throughput mRNA and protein profiling techniques have brought new understanding of platelet biological functions, including identification of novel platelet proteins and secreted molecules, analysis of functional changes between normal and pathologic states, and determining the effects of processing and storage on platelet concentrates for transfusion. However, because platelets are very easily activated, it is important to understand the different in vitro methods for platelet isolation commonly used and how they differ from the perspective for use as research samples in clinical chemistry. Two simple methods are described here for the preparation of research-scale platelet samples from human whole blood, and detailed notes are provided about the methods used for the preparation of platelet concentrates for transfusion.

Preparation of Cryoprecipitate and Cryo-depleted Plasma for Proteomic Research Analysis.

When frozen plasma is slowly thawed in cold conditions (1-6 °C), high-molecular-weight plasma proteins precipitate forming a concentrate known as cryoprecipitate. The concentrate is enriched with several important coagulation proteins, including fibrinogen, antihemophilic factor (factor VIII), von Willebrand factor, fibrin stabilizing factor (factor XIII), fibronectin, and small amounts of other plasma proteins. In current medical practice, clinical-grade preparations of cryoprecipitate are used mostly to correct fibrinogen deficiency caused by acute blood loss or due to functional abnormalities of the fibrinogen protein. In the past, cryoprecipitate was used to treat von Willebrand disease and hemophilia A (factor VIII deficiency), but the availability of more highly purified coagulation factor concentrates or recombinant protein preparations has superseded the use of cryoprecipitate for these coagulopathies. Cryo-depleted plasma (also called cryosupernatant) is the plasma supernatant remaining following removal of the cryoprecipitate from frozen-thawed plasma and contains all the remaining soluble plasma proteins. This protocol describes the research-scale preparation of cryoprecipitate and cryo-depleted plasma suitable for proteomic studies and is based on the procedures used to prepare clinical-grade cryoprecipitate.

An Optimized Data-Independent Acquisition Strategy for Comprehensive Analysis of Human Plasma Proteome.

Cartography of the plasma proteome remains technically challenging, primarily due to the abundance and dynamic range of plasma proteins and their concentrations, exceeding ten orders of magnitude, including low-abundant tissue-derived proteins in the pg/mL range. Data-independent acquisition mass spectrometry (DIA-MS) has seen advances in unbiased mass spectrometry-based proteomic analysis of the plasma proteome. Here, we describe a comprehensive proteomic workflow of human plasma from clinically relevant sample (10 µL) that includes anti-protein immunodepletion and highly sensitive sample preparation workflow, with optimized scheduled isolation DIA-MS and deep learning analysis. This approach results in over 960 proteins quantified from a single-shot analysis of broad dynamic range, across 8 orders of magnitude (8.2 ng/L to 0.67 g/L). We further compare data-dependent acquisition (DDA) MS to highlight the advantage in protein quantification and inter-sample variation. These developments have provided streamlined identification of the human plasma proteome, including low-abundant tissue-enriched proteins, and applications toward understanding the plasma proteome.

Generation of Red Blood Cell Nanovesicles as a Delivery Tool.

Extracellular vesicles (EVs) are natural membranous vesicles with immense potential as drug delivery tools. However, their large-scale production remains a huge technical challenge, is time consuming, and expensive. Thus, EV mimetics (nanovesicles) generated from easily sourced red blood cells (RBCs) have gained vested interest as an effective and scalable drug delivery system. Their surface proteins (e.g., CD47) inherited from parental RBCs also improve their biocompatibility and bioavailability. Here, we outline a step-by-step guide for large-scale production of RBC nanovesicles using one-step extrusion method coupled to rapid density-cushion centrifugation. We also outline protocol for their extensive biophysical characterization (size and morphology using single particle analysis and cryogenic electron microscopy), and in-depth mass spectrometry-based proteome characterization. Finally, we outline two strategies (active loading during extrusion vs. passive loading via diffusion) to incorporate pharmacological compound(s) into nanovesicles and detect their loading using spectrophotometry.

The mechanism of Pseudomonas aeruginosa outer membrane vesicle biogenesis determines their protein composition.

Gram-negative bacteria produce outer membrane vesicles (OMVs) and contain bacterial cargo including nucleic acids and proteins. The proteome of OMVs can be altered by various factors including bacterial growth stage, growth conditions, and environmental factors. However, it is currently unknown if the mechanism of OMV biogenesis can determine their proteome. In this study, we examined whether the mechanisms of OMV biogenesis influenced the production and protein composition of Pseudomonas aeruginosa OMVs. OMVs were isolated from three P. aeruginosa strains that produced OMVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis. We identified that the mechanism of OMV biogenesis dictated OMV quantity. Furthermore, a global proteomic analysis comparing the proteome of OMVs to their parent bacteria showed significant differences in the identification of proteins in bacteria and OMVs. Finally, we determined that the mechanism of OMV biogenesis influenced the protein composition of OMVs, as OMVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome and functional enrichment analysis. Overall, our findings reveal that the mechanism of OMV biogenesis is a main factor that determines the OMV proteome which may affect their subsequent biological functions.

Extracellular vesicles as next generation immunotherapeutics.

Extracellular vesicles (EVs) function as a mode of intercellular communication and molecular transfer to elicit diverse biological/functional response. Accumulating evidence has highlighted that EVs from immune, tumour, stromal cells and even bacteria and parasites mediate the communication of various immune cell types to dynamically regulate host immune response. EVs have an innate capacity to evade recognition, transport and transfer functional components to target cells, with subsequent removal by the immune system, where the immunological activities of EVs impact immunoregulation including modulation of antigen presentation and cross-dressing, immune activation, immune suppression, and immune surveillance, impacting the tumour immune microenvironment. In this review, we outline the recent progress of EVs in immunorecognition and therapeutic intervention in cancer, including vaccine and targeted drug delivery and summarise their utility towards clinical translation. We highlight the strategies where EVs (natural and engineered) are being employed as a therapeutic approach for immunogenicity, tumoricidal function, and vaccine development, termed immuno-EVs. With seminal studies providing significant progress in the sequential development of engineered EVs as therapeutic anti-tumour platforms, we now require direct assessment to tune and improve the efficacy of resulting immune responses - essential in their translation into the clinic. We believe such a review could strengthen our understanding of the progress in EV immunobiology and facilitate advances in engineering EVs for the development of novel EV-based immunotherapeutics as a platform for cancer treatment.