Analysis Of Exosomes Research Articles

Culprits of PDAC resistance to gemcitabine and immune checkpoint inhibitor: Tumour microenvironment components.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer with a dismal five-year survival rate of 11%. Despite remarkable advancements in cancer therapeutics, PDAC patients rarely benefit from it due to insurmountable treatment resistance. Notably, PDAC is pathologically characterized by an extensive desmoplastic reaction and an extremely immunosuppressive tumour microenvironment (TME). The PDAC TME consists of cell components (e.g., tumour, immune and stromal cells) and noncellular components (e.g., extracellular matrix), exhibiting high complexity and their interplay resulting in resistance to chemotherapeutics and immune checkpoint inhibitors. In our review, we shed light on how crosstalk of complex environmental components modulates PDAC drug resistance, and we summarize related clinical trials. Moreover, we extend our discussion on TME exploration and exosome analysis, providing new insights into clinical applications, including personalized medicine, disease monitoring and drug carriers.

Sequential Method for Analysis of CTCs and Exosomes from the Same Sample of Patient Blood.

Circulating tumor cells (CTCs) and exosomes, both released from the primary tumor into peripheral blood, are a promising source of cancer biomarkers. They are detectable in the blood and carry a large diversity of biological molecules, which can be used for the diagnosis and monitoring of minimally invasive cancers. However, due to their intrinsic differences in counts, size, and molecular contents, studies have focused on only one type of vesicle. Herein, we have developed an integrated system to sequentially isolate CTCs and exosomes from a single patient blood sample for further profiling and analysis. The CTCs are isolated using a commercial filtration method and then the remaining blood is processed using multiple cycles of ultracentrifugation to isolate the exosomes. The method uses two available technologies where the eluent from CTC isolation is usually discarded and interfaces them, so that the eluent can be interfaced to exosome isolation methods. The CTCs are identified based on fluorescence staining of their surface markers, while the exosomes are analyzed using transmission electron microscopy, nanosight tracking analysis, and mass spec proteomic analysis. This analysis showed CTCs detected by their surface markers for metastatic hepatocellular carcinoma (HCC), while essentially none were detected for cirrhosis. The exosome analysis resulted in the identification of ∼500–1000 exosome proteins per sample confirmed by detection of exosome surface markers CD9, CD63, CD81, and TSG101 in addition to proteins related to cancer progression. Proteins enriched in HCC exosomes were shown to be involved in the immune response, metastasis, and proliferation.

Tandem Mass Tag (TMT) Quantitative Proteomic Analysis of Serum Exosomes in Cerebral Small-vessel Disease (CSVD) Patients With Depressive Symptoms.

Depressive symptoms are one of the main clinical features of the cerebral small-vessel disease (CSVD). However, the pathogenesis of depressive symptoms of CSVD has not been fully studied, and a lack of effective diagnostic methodseffective diagnostic methods exists. Recently, the emerging body of evidence regarding exosomes has rendered them potentially key players in the neuropsychiatric disease theragnostic. This study's aim was to investigate serumexosome proteomic expression in CSVD patients with depressive symptoms and to screen and analyze potential biomarkers for clinical diagnosis. Serum samples were collected from 36 CSVD patients, including 18 cerebral small-vessel disease (CSVD+D) patients with depressive clinical manifestations and 18 cerebral small-vessel disease patients that did not present depression-related clinical manifestations (CSVD-D). This investigation employed tandem mass tag (TMT) combined with mass spectrometry for sample detection and quantitative analysis of proteins. The differential proteins with significant dysregulated expression levels in patient plasma exosomes were screened and analyzed through bioinformatics techniques. This investigation focused on a global collection of 659 quantifiable proteins. Compared to the CSVD-D group, 7 up-regulated and 30 down-regulated proteins were identified in the CSVD+D group (P < 0.05). Gene ontology (GO) enrichment analyses revealed proteomic expression profile dysregulations within serum exosomes in patients with depression, such as desmosomes and keratins, rendering them as potential biomarkers. Kyoto encyclopedia of genes and genomes (KEGG) database investigations revealed the differentially expressed proteins to be highly aggregated within the estrogen signaling pathway. This investigation pioneered TMT proteomic evaluation of serum exosomes within CSVD patients suffering from depression and reveals the shifts in proteomic expression profiles by serum exosomes within such patients. This study identified several important molecular / signal pathway abnormalities related to depression. These results provide a possible means to further clarify the pathogenesis of depressive symptoms of cerebrovascular disease and its diagnosis and treatment in the future.

Artificial Intelligent Label-Free SERS Profiling of Serum Exosomes for Breast Cancer Diagnosis and Postoperative Assessment.

Breast cancer subtypes have important implications of treatment responses and clinical outcomes. Exosomes have been considered as promising biomarkers for liquid biopsies, but the utility of exosomes for accurate diagnosis of distinct breast cancer subtypes is a grand challenge due to the difficulty in uncovering the subtle compositional difference in complex clinical settings. Herein, we report an artificial intelligent surface-enhanced Raman spectroscopy (SERS) strategy for label-free spectroscopic analysis of serum exosomes, allowing for accurate diagnosis of breast cancer and assessment of surgical outcomes. Our deep learning algorithm trained with SERS spectra of cancer cell-derived exosomes is demonstrated with a 100% prediction accuracy for human patients with different breast cancer subtypes who do not undergo surgery using SERS spectra of serum exosomes. Furthermore, when combined with similarity analysis by principal component analysis, our approach is able to evaluate the surgical outcomes of breast cancer of distinct molecular subtypes.

Microfluidic Technology for the Isolation and Analysis of Exosomes.

Exosomes are lipid-bilayer enclosed vesicles with diameters of 30–150 nm, which play a pivotal role in cell communication by transporting their cargoes such as proteins, lipids, and genetic materials. In recent years, exosomes have been under intense investigation, as they show great promise in numerous areas, especially as bio-markers in liquid biopsies. However, due to the high heterogeneity and the nano size of exosomes, the separation of exosomes is not easy. This review will deliver an outline of the conventional methods and the microfluidic-based technologies for exosome separation. Particular attention is devoted to microfluidic devices, highlighting the efficiency of exosome isolation by these methods. Additionally, this review will introduce advances made in the integrated microfluidics technologies that enable the separation and analysis of exosomes.

Feasibility of Leukemia-Derived Exosome Enrichment and Co-isolated dsDNA Sequencing in Acute Myeloid Leukemia Patients: A Proof of Concept for New Leukemia Biomarkers Detection

Simple SummaryThe present pilot study aimed at investigating the feasibility of a leukemia-derived exosome enrichment approach followed by exosomal dsDNA target re-sequencing for adult Acute Myeloid Leukemias (AML) marker detection. To our knowledge, this is the first time that a proof-of-concept combining a leukemia-derived exosome enrichment strategy based on a commercial CE-IVD kit and next-generation sequencing was applied in a cohort of adult AML patients. The reported approach is easy, quick and user friendly and gives the possibility of obtaining a good quantity of exosomal dsDNA (composed of exosomal cargo and surrounding DNA) suitable for further analysis. The time-effective procedure opens up future effective clinical applications. This pilot study presents the potential of a proof-of-concept based on exosome analysis to be applied in clinical practice, as well as the feasibility of this kind of investigations using a certified kit, avoiding many additional analyses. It may encourage further studies regarding extracellular vesicles in myeloid neoplasia.Exosomes are extracellular vesicles playing a pivotal role in the intercellular communication. They shuttle different cargoes, including nucleic acids from their cell of origin. For this reason, they have been studied as carriers of tumor markers in different liquid biopsy approaches, in particular for solid tumors. Few data are available concerning exosomes as markers of myeloid neoplasia. To better understand their real potential and the best approach to investigate leukemic exosomes, we present the results of a pilot feasibility study evaluating the application of next-generation sequencing analysis of dsDNA derived from exosomes isolated in 14 adult patients affected by acute myeloid leukemias. In particular, leukemia-derived exosome fractions have been analyzed. The concentration of dsDNA co-extracted with exosomes and the number and types of mutations detected were considered and compared with ones identified in the Bone Marrow (BM) and Peripheral Blood (PB) cells. Exosomal DNA concentration, both considering the cargo and the DNA surrounding the lipid membrane resulted in a linear correlation with leukemic burden. Moreover, exosomal DNA mutation status presented 86.5% of homology with BM and 75% with PB. The results of this pilot study confirmed the feasibility of a leukemia-derived exosome enrichment approach followed by exosomal dsDNA NGS analysis for AML biomarker detection. These data point to the use of liquid biopsy in myeloid neoplasia for the detection of active leukemic cells resident in the BM via a painless procedure.

Exosomal microRNA-1 and MYO15A as a target for therapy and diagnosis in renal cell carcinoma

Exosomes are 40–100 nm nano-sized extracellular vesicles and are receiving increasing attention as novel structures that participate in intracellular communication. We previously found that miRNA-1 (miR-1) functions as a tumor suppressor in renal cell carcinoma (RCC). In this study, we investigated the function of exosomal miR-1 and the possibility that the exosome constitutes a tumor maker in RCC. First, we established the method to collect exosomes from cell lysates and human serum by a spin column-based method. Next, we assessed exosomes using Nanosight nanoparticle tracking analysis and Western blot analysis with exosome marker CD63. We confirmed that exosomes labeled with PKH26 fused with recipient cells. Moreover, miR-1 expression was elevated in RCC cells treated with exosomes derived from miR-1-transfected cells. Functional analyses showed that exosomal miR-1 significantly inhibited cell proliferation, migration and invasion compared to control treatment. Our analyses with TCGA database of RCCs showed that miR-1 expression was significantly downregulated in clinical RCC samples compared to that in normal kidney samples, and patients with low miR-1 expression had poorer overall survival in comparison to patients with high expression. Furthermore, RNA sequence analyses showed that expression levels of several genes were altered by exposure to exosomal miR-1. The analyses with TCGA database indicated that high expression of MYO15A was associated with a poorer outcome in RCC. In addition, RT-qPCR analysis of exosomes from clinical patients’ sera showed that MYO15A was significantly upregulated in RCC patients compared to that in healthy controls. This study showed that treatment with exosomal miR-1 might be an effective approach to treating RCCs. In addition, exosomal MYO15A could be a diagnostic tumor marker in RCCs.

Ultrasensitive and preprocessing-free electrochemical biosensing platform for the detection of cancer-derived exosomes based on spiky-shaped aptamer-magnetic beads

Cancer-derived exosomes, as liquid biopsy markers, have been shown to play an important role in the early screening, diagnosis, and prognosis of cancer. However, existing detection methods have shortcomings such as long-time consumption and low sensitivity. Herein, a sandwich-type electrochemical sensing platform based on Prussian blue/graphene oxide (GO/PB) and spiky Au@Fe3O4 nanoparticles was successfully designed and constructed to detect tumor-derived exosomes with high sensitivity and no preprocessing. In this strategy, nanospike structures were introduced on magnetic beads to form spiky Au@Fe3O4, which was used to enrich exosomes from serum, avoiding the extraction and purification processes of previous detections. The enrichment and signal amplification of spiky Au@Fe3O4 could also greatly improve the detection sensitivity of the sensing platform. Consequently, the concentration of exosomes could be directly quantified by monitoring the electroactive molecules of PB. Therefore, the limit of detection (LOD) of the proposed biosensor was 80 particles·μL−1. Furthermore, this proposed biosensor could realize the high sensitivity analysis of exosomes and effectively save detection time, and provide an effective assistant diagnostic tool for the early diagnosis of cancer.

Determination of the Loading Capacity and Recovery of Extracellular Vesicles Derived from Human Embryonic Kidney Cells and Urine Matrices on Capillary-Channeled Polymer (C-CP) Fiber Columns

Extracellular vesicles (EVs) are 50–1000 nm membranous vesicles secreted from all cells that play important roles in many biological processes. Exosomes, a smaller-sized subset of EVs, have become of increasing interest in fundamental biochemistry and clinical fields due to their rich biological cargos and their roles in processes such as cell-signaling, maintaining homeostasis, and regulating cellular functions. To be implemented effectively in fundamental biochemistry and clinical diagnostics fields of study, and for their proposed use as vectors in gene therapies, there is a need for new methods for the isolation of large concentrations of high-purity exosomes from complex matrices in a timely manner. To address current limitations regarding recovery and purity, described here is a frontal throughput and recovery analysis of exosomes derived from human embryonic kidney (HEK) cell cultures and human urine specimens using capillary-channeled polymer (C-CP) fiber stationary phases via high performance liquid chromatography (HPLC). Using the C-CP fiber HPLC method for EV isolations, the challenge of recovering purified EVs from small sample volumes imparted by the traditional techniques was overcome while introducing significant benefits in processing, affordability (~5 $ per column), loading (~1012 particles), and recovery (1011–1012 particles) from whole specimens without further processing requirements.

Systemic proteomics and miRNA profile analysis of exosomes derived from human pluripotent stem cells

BackgroundIncreasing studies have reported the therapeutic effect of mesenchymal stem cell (MSC)-derived exosomes by which protein and miRNA are clearly characterized. However, the proteomics and miRNA profiles of exosomes derived from human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) remain unclear.MethodsIn this study, we isolated exosomes from hESCs, hiPSCs, and human umbilical cord mesenchymal stem cells (hUC-MSCs) via classic ultracentrifugation and a 0.22-μm filter, followed by the conservative identification. Tandem mass tag labeling and label-free relative peptide quantification together defined their proteomics. High-throughput sequencing was performed to determine miRNA profiles. Then, we conducted a bioinformatics analysis to identify the dominant biological processes and pathways modulated by exosome cargos. Finally, the western blot and RT-qPCR were performed to detect the actual loads of proteins and miRNAs in three types of exosomes.ResultsBased on our study, the cargos from three types of exosomes contribute to sophisticated biological processes. In comparison, hESC exosomes (hESC-Exos) were superior in regulating development, metabolism, and anti-aging, and hiPSC exosomes (hiPSC-Exos) had similar biological functions as hESC-Exos, whereas hUC-MSCs exosomes (hUC-MSC-Exos) contributed more to immune regulation.ConclusionsThe data presented in our study help define the protein and miRNA landscapes of three exosomes, predict their biological functions via systematic and comprehensive network analysis at the system level, and reveal their respective potential applications in different fields so as to optimize exosome selection in preclinical and clinical trials.

Autophagy-dependent Na+-K+-ATPase signalling and abnormal urate reabsorption in hyperuricaemia-induced renal tubular injury

Increasing evidence indicates that hyperuricaemia (HUA) is not only a result of decreased renal urate excretion but also a contributor to kidney disease. Na+-K+-ATPase (NKA), which establishes the sodium gradient for urate transport in proximal tubular epithelial cells (PTECs), its impairment leads to HUA-induced nephropathy. However, the specific mechanism underlying NKA impairment-mediated renal tubular injury and increased urate reabsorption in HUA is not well understood. In this study, we investigated whether autophagy plays a key role in the NKA impairment signalling and increased urate reabsorption in HUA-induced renal tubular injury. Protein spectrum analysis of exosomes from the urine of HUA patients revealed the activation of lysosomal processes, and exosomal expression of lysosomal-associated membrane protein-2 was associated with increased serum levels and decreased renal urate excretion in patients. We demonstrated that high uric acid (UA) induced lysosome dysfunction, autophagy and inflammation in a time- and dose-dependent manner and that high UA and/or NKA α1 siRNA significantly increased mitochondrial abnormalities, such as reductions in mitochondrial respiratory complexes and cellular ATP levels, accompanied by increased apoptosis in cultured PTECs. The autophagy inhibitor hydroxychloroquine (HCQ) ameliorated NKA impairment-mediated mitochondrial dysfunction, Nod-like receptor pyrin domain-containing protein 3 (NLRP3)-interleukin-1β (IL-1β) production, and abnormal urate reabsorption in PTECs stimulated with high UA and in rats with oxonic acid (OA)-induced HUA. Our findings suggest that autophagy plays a pivotal role in NKA impairment-mediated signalling and abnormal urate reabsorption in HUA-induced renal tubular injury and that inhibition of autophagy by HCQ could be a promising treatment for HUA.

Distinct functions of dimeric and monomeric scaffold protein Alix in regulating F-actin assembly and loading of exosomal cargo

Alix is a ubiquitously expressed scaffold protein that participates in numerous cellular processes related to the remodeling/repair of membranes and the actin cytoskeleton. Alix exists in monomeric and dimeric/multimeric configurations, but how dimer formation occurs and what role the dimer has in Alix-mediated processes are still largely elusive. Here, we reveal a mechanism for Alix homodimerization mediated by disulfide bonds under physiological conditions and demonstrate that the Alix dimer is enriched in exosomes and F-actin cytoskeleton subcellular fractions. Proteomic analysis of exosomes derived from Alix−/− primary cells underlined the indispensable role of Alix in loading syntenin into exosomes, thereby regulating the cellular levels of this protein. Using a set of deletion mutants, we define the function of Alix Bro1 domain, which is solely required for its exosomal localization, and that of the V domain, which is needed for recruiting syntenin into exosomes. We reveal an essential role for Cys814 within the disordered proline-rich domain for Alix dimerization. By mutating this residue, we show that Alix remains exclusively monomeric and, in this configuration, is effective in loading syntenin into exosomes. In contrast, loss of dimerization affects the ability of Alix to associate with F-actin, thereby compromising Alix-mediated cytoskeleton remodeling. We propose that dimeric and monomeric forms of Alix selectively execute two of the protein’s main functions: exosomal cargo loading and cytoskeleton remodeling.

Plasma exosomal DOK3 reflects immunological states in lung tumor and predicts prognosis of gefitinib treatment.

To identify liquid biomarkers that predict clinical outcomes of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), we enrolled patients with EGFR gene mutation-positive non-small-cell lung cancer who were intended to receive gefitinib treatment. Using plasma samples obtained prior to gefitinib treatment from 12 enrolled patients, we performed comprehensive proteomic analysis of plasma exosomes to explore proteins correlating with tumor reduction rate (TRR), progression-free survival (PFS), or overall survival (OS). Of the detected 1769 proteins, 119, 130, or 119 proteins demonstrated a strong correlation (|r| > 0.5) with TRR, PFS, or OS, respectively. Interestingly, 34 (29%), 41 (32%), or 27 (23%) of them, respectively, were functionally involved in the regulation of the immune response. CD8α chain was consistently listed as a molecule positively correlated with PFS and OS, suggesting that the long-lasting effects of gefitinib may be due to the antitumor effects of CD8+ T cells, as well as the induction of immunogenic apoptosis of tumor cells by blocking the EGFR signaling pathway. Notably, Doking Protein 3 (DOK3), a molecule involved in B-cell receptor signaling, and some immunoglobulin and complement molecules exhibited a clear correlation with PFS longevity of gefitinib treatment. Indeed, the strong expression of DOK3 in B cells was confirmed within tertiary lymphoid structures of lung cancer tissues derived from patients with long PFS. These findings suggest that the patients with active B-cell and T-cell immunity as a host immunological feature are more likely to benefit from gefitinib therapy. Circulating exosomal DOK3 has the potential as a predictive marker of response to gefitinib indicating this immunological feature.

CXCR6 Mediates Pressure Overload-Induced Aortic Stiffness by Increasing Macrophage Recruitment and Reducing Exosome-miRNA29b.

Aortic stiffness is an independent risk factor for aortic diseases such as aortic dissection which commonly occurred with aging and hypertension. Chemokine receptor CXCR6 is critically involved in vascular inflammation and remodeling. Here, we investigated whether and how CXCR6 plays a role in aortic stiffness caused by pressure overload. CXCR6-/- and WT mice underwent transverse aortic constriction (TAC) surgery for 8weeks. CXCR6 deficiency significantly improved TAC-induced aortic remodeling and endothelial dysfunction by decreasing CD11c+ macrophage infiltration, suppressing VCAM-1 and ICAM-1, reducing collagen deposition, and downregulating MMP12 and osteopontin in the aorta. Consistently, blocking the CXCL16/CXCR6 axis also reduced aortic accumulation of CD11c+ macrophages and vascular stiffness but without affecting the release of TNF-α and IL-6 from the aorta. Furthermore, pressure overload inhibited aortic release of exosomes, which could be reversed by suppressing CXCR6 or CXCL16. Inhibition of exosome release by GW4869 significantly aggravated TAC-induced aortic calcification and stiffness. By exosomal microRNA microarray analysis, we found that microRNA-29b was significantly reduced in aortic endothelial cells (AECs) receiving TAC. Intriguingly, blocking the CXCL16/CXCR6 axis restored the expression of miR-29b in AECs. Finally, overexpression of miR-29b significantly increased eNOS and reduced MMPs and collagen in AECs. By contrast, antagonizing miR-29b in vivo further enhanced TAC-induced expressions of MMP12 and osteopontin, aggravated aortic fibrosis, calcification, and stiffness. Our study demonstrated a key role of the CXCL16/CXCR6 axis in macrophage recruitment and macrophage-mediated aortic stiffness under pressure overload through an exosome-miRNAs-dependent manner.

Differential proteomic analysis of plasma-derived exosomes as diagnostic biomarkers for chronic HBV-related liver disease

Hepatitis B virus (HBV) infection is still a major public health problem worldwide. We aimed to identify new, non-invasive biomarkers for the early diagnosis of chronic HBV-related diseases, reveal alterations in the progression of chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Here, exosomes were isolated and characterized through size exclusion chromatography and nanoparticle tracking analysis. Profiles of differentially expressed proteins (DEPs) were analyzed through liquid chromatography-tandem mass spectrometry (LC–MS/MS), Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes analyses. Results showed that the DEPs, including CO9, LBP, SVEP1, and VWF levels in extracellular vesicles (EVs) were significantly higher in CHB than in healthy controls (HCs). VWF expression levels in EVs were significantly lower in CHB than in those with LC. KV311 expression levels in EVs were significantly higher, whereas LBP levels were significantly lower in patients with CHB than in those with HCC. All biomarkers seemed to exhibit a high diagnostic capacity for HBV-related liver disease. Patients with HBV-induced chronic liver disease exhibit characteristic protein profiles in their EVs. Thus, serum exosomes may be used as novel, liquid biopsy biomarkers to provide useful clinical information for the diagnosis of HBV-related liver diseases at different stages.

Intraocular RGD-Engineered Exosomes and Active Targeting of Choroidal Neovascularization (CNV).

Purpose: To assess the transretinal penetration of intravitreally injected retinal multicell-derived exosomes and to develop exosome-based active targeting of choroidal neovascularization (CNV) by bioengineering with ASL, which is composed of a membrane Anchor (BODIPY), Spacer (PEG), and targeting Ligands (cyclic RGD peptide). Methods: Retinal multicell-derived exosomes were recovered from a whole mouse retina using differential ultracentrifugation. Their size, number, and morphology were characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Exosome markers were confirmed using an exosome detection antibody array. Intravitreal injection of fluorescent (PKH-26)-labeled or engineered ASL exosomes (1 × 106 exosomes/μL) were given to the wild-type mouse or laser-induced CNV mouse model. Retinal uptake of exosomes was assessed by in vivo retinal imaging microscopy and histological staining with DAPI, GSA, and anti-integrin αv for retinal sections or choroid/RPE flat mounts. Active targeting of CNV was assessed by comparing retinal uptake between areas with and without CNV and by colocalization analysis of ASL exosomes with integrin αv within CNV. Staining with anti-F4/80, anti-ICAM-1, and anti-GFAP antibodies on retinal sections were performed to identify intracellular uptake of exosomes and immediate reactive retinal gliosis after exosome treatment. Results: An average of 2.1 × 109 particles/mL with a peak size of 140 nm exosomes were recovered. Rapid retinal penetration of intravitreally injected exosomes was confirmed by retinal imaging microscopy at 3 and 24 h post-injection. Intravitreally delivered PKH-26-labeled exosomes reached inner and outer retinal layers including IPL, INL, OPL, and ONL at 1 and 7 days post-injection. Intravitreally injected ASL exosomes were predominantly delivered to the area of CNV including ONL, RPE, and choroid in laser-induced CNV mouse models with 89.5% of colocalization with integrin αv. Part of exosomes was also taken intracellularly to vascular endothelial cells and macrophages. After intravitreal injection, neither naive exosomes nor ASL exosomes induced immediate reactive gliosis. Conclusions: Intravitreally delivered retinal multicell-derived exosomes have good retinal penetration, and ASL modification of exosomes actively targets CNV with no immediate reactive gliosis. ASL exosomes have a great potential to serve as an intraocular drug delivery vehicle, allowing an active targeting strategy.

Magnetic bead-based adsorption strategy for exosome isolation.

Exosomes, one type of extracellular vesicle (EV) secreted by cells, participate in intercellular communication and other biological processes as carriers of lipids, functional proteins, mRNAs, miRNAs, lncRNAs, and DNA fragments. Their presence in biofluids makes them attractive candidates as innovative clinical diagnostic tools. However, the conventional isolation and analysis of high-purity exosomes in clinical application is challenging, with traditional methods facing a number of shortcomings, including low yield or purity, long periods of processing, high cost, and difficulties in standardization. In this study, we provide an overview of commonly used exosome isolation approaches with a focus on magnetic bead-based capture, an ideal methodology with high purity and integrality of exosomes. The current challenges on exosome isolation methods are also described to highlight areas for future research and development.

Current status and outlook of advances in exosome isolation.

Exosomes are extracellular vesicles with a diameter ranging from 30 to 150 nm, which are an important medium for intercellular communication and are closely related to the progression of certain diseases. Therefore, exosomes are considered promising biomarkers for the diagnosis of specific diseases, and thereby, treatments based on exosomes are being widely examined. For exosome-related research, a rapid, simple, high-purity, and recovery isolation method is the primary prerequisite for exosomal large-scale application in medical practice. Although there are no standardized methods for exosome separation and analysis, various techniques have been established to explore their biochemical and physicochemical properties. In this review, we analyzed the progress in exosomal isolation strategies and proposed our views on the development prospects of various exosomal isolation techniques.Graphical abstract

Metastasis suppressor NME1 in exosomes or liposomes conveys motility and migration inhibition in breast cancer model systems.

Tumor-derived exosomes have documented roles in accelerating the initiation and outgrowth of metastases, as well as in therapy resistance. Little information supports the converse, that exosomes or similar vesicles can suppress metastasis. We investigated the NME1 (Nm23-H1) metastasis suppressor as a candidate for metastasis suppression by extracellular vesicles. Exosomes derived from two cancer cell lines (MDA-MB-231T and MDA-MB-435), when transfected with the NME1 (Nm23-H1) metastasis suppressor, secreted exosomes with NME1 as the predominant constituent. These exosomes entered recipient tumor cells, altered their endocytic patterns in agreement with NME1 function, and suppressed in vitro tumor cell motility and migration compared to exosomes from control transfectants. Proteomic analysis of exosomes revealed multiple differentially expressed proteins that could exert biological functions. Therefore, we also prepared and investigated liposomes, empty or containing partially purified rNME1. rNME1 containing liposomes recapitulated the effects of exosomes from NME1 transfectants in vitro. In an experimental lung metastasis assay the median lung metastases per histologic section was 158 using control liposomes and 15 in the rNME1 liposome group, 90.5% lower than the control liposome group (P = 0.016). The data expand the exosome/liposome field to include metastasis suppressive functions and describe a new translational approach to prevent metastasis.

Abstract P1038: Exosomal RNAs And Proteins From Non-invasively Derived Human Induced Mesenchymal Stem Cells Protect Cardiomyocytes From Death

Background: Current stem cells therapy for ischemic cardiomyopathy is effective only about 2-4% in improving cardiac function. In search of novel therapy, we have developed a safe mRNA-based reprogramming of non-invasively derived human urinary epithelial cells into induced pluripotent stem cells (iPSC), and subsequently differentiated them into mesenchymal stem cells (iMSC). We examined therapeutic potential of iMSC in comparison with adult umbilical cord mesenchymal stem cells (MSC). Hypothesis: Autologous iMSC and their exosomes possess an enhanced cardioprotective characteristics compared to MSC. Methods and Results: The generated iMSC and their exosomes (ExoQuick reagent) offered an enhanced protection of human iPSC-derived cardiomyocytes (iCMC) from injuries of (i) angiotensin-II (Ang, 10 μM for 24 h) and (ii) 6 hrs of 1% hypoxia and 24 hrs of reoxygenation, in comparison with their MSC controls. The cardiomyocyte protection was studied through measurement of mitochondrial membrane potential (JC1 dye), intracellular reactive oxygen species (CM-H2DCFDA), in situ cell death by apoptosis, and qRT-PCR expression of survival and proinflammatory genes (BCL2, BAD, TNFA and IL6). Treatment with iMSC-derived exosomes alone enhanced the expression of NRF2, a major regulator of cytoprotective genes in protecting Ang-challenged iCMC. The specific role of iMSC exosomal RNAs and proteins in mediating cardioprotection against Ang-induced injury was identified through in situ cell death assay using the exosomes treated with either RNase A (0.5 μg/μl for 20 min at 37 0 C) or proteinase K (0.05 μg/μl for 10 min at 37 0 C) (Fig.1) . Exosomal noncoding RNA analysis by qRT-PCR revealed the presence of inflammation-associated small nucleolar RNAs such as SNORD32A, SNORD33, SNORD34 and SNORD35A. The transfer of exosomal RNAs into the host cardiac cells was augmented in presence of Ang, identified using 0.5 mM ethynyl uridine-labelled exosomes and detected using Click-iT RNA imaging reagents. All experiments were carried out in triplicates. In vivo studies are in progress. Conclusion: In comparison with adult MSC, non-invasively derived autologous iMSC and their exosomes elicit enhanced cardioprotection and may provide an alternative source of cells or cell-free therapy for treating ischemic cardiomyopathy.