Analysis Of Exosomes Research Articles

Detection of Cell-Derived Exosomes Via Surface-Enhanced Raman Scattering Using Aggregated Silver Nanoparticles.

Exosomes are small extracellular vesicles that contain RNA, lipids, and proteins and can act as cellular messengers, carrying information to cells and tissues in the body. Thus, sensitive, label-free, and multiplexed analysis of exosomes may help in early diagnosis of important diseases. Here, we describe the process of pretreatment of cell-derived exosomes, preparation of surface-enhanced Raman scattering (SERS) substrates, and label-free SERS detection of exosomes using sodium borohydride aggregators. This method can enable the observation of SERS signals of exosomes that are clear and stable and have a good signal-to-noise ratio.

Proteomic analysis of exosomes derived from human mature milk and colostrum of mothers with term, late preterm, or very preterm delivery.

The growth and development of the human brain is a long and complex process that requires a precise sequence of genetic and molecular events. This begins in the third week of gestation with the differentiation of neural progenitor cells and extends at least until late adolescence, possibly for life. One of the defects of this development is that we know very little about the signals that modulate this sequence of events. The first 3 years of life, during breastfeeding, is one of the critical periods in brain development. In these first years of life, it is believed that neurodevelopmental problems may be the molecular causes of mental disorders. Therefore, we herein propose a new hypothesis, according to which the chemical signals that could modulate this entire complex sequence of events appear in this early period, and the molecular level study of human breast milk and colostrum of mothers who give birth to children in different gestation periods could give us information on proteins influencing this process. In this work, we collected milk and colostrum samples (term, late preterm and moderate/very preterm) and exosomes were isolated. The samples of exosomes and complete milk from each fraction were analyzed by LC-ESI-MS/MS. In this work, we describe proteins in the different fractions of mature milk and colostrum of mothers with term, late preterm, or very preterm delivery, which could be involved in the regulation of the nervous system by their functions. We describe how they differ in different types of milk, paving the way for the investigation of possible new neuroregulatory pathways as possible candidates to modulate the nervous system.

Integrated separation and detection of exosomes via a label-free magnetic SERS platform.

A label-free magnetic surface enhanced Raman scattering (SERS) platform was constructed, which was composed of superparamagnetic Fe3O4 nanoparticles as cores for separation and Au layers as shells for label-free SERS detection. Our method could effectively distinguish exosomes from different cell sources for cancer diagnosis and showed high sensitivity and specificity within a 95% confidence interval. As a low-cost and efficient exosome analysis method, the designed integrated platform for separation and detection has promising applicability in clinical diagnostics.

ITRAQ-Based Proteomic Analysis of APP Transgenic Mouse Urine Exosomes.

Alzheimer's disease (AD) is a common dementia disease in the elderly. To get a better understanding of the pathophysiology, we performed a proteomic analysis of the urine exosomes (U-exo) in AD model mice (J20). The polymer precipitation method was used to isolate U-exo from the urine of 3-month-old J20 and wild-type (WT) mice. Neuron-derived exosome (N-exo) was isolated from U-exo by immunoprecipitation. iTRAQ-based MALDI TOF MS/MS was used for proteomic analysis. The results showed that compared to WT, the levels of 61 and 92 proteins were increased in the J20 U-exo and N-exo, respectively. Gene ontology enrichment analysis demonstrated that the sphingolipid catabolic process, ceramide catabolic process, membrane lipid catabolic process, Aβ clearance, and Aβ metabolic process were highly enriched in U-exo and N-exo. Among these, Asah1 was shown to be the key protein in lipid metabolism, and clusterin, ApoE, neprilysin, and ACE were related to Aβ metabolism and clearance. Furthermore, protein-protein interaction analysis identified four protein complexes where clusterin and ApoE participated as partner proteins. Thus, J20 U-exo and N-exo contain proteins related to lipid- and Aβ-metabolism in the early stages of AD, providing a new insight into the underlying pathological mechanism of early AD.

Comprehensive analysis of M2 macrophage-derived exosomes facilitating osteogenic differentiation of human periodontal ligament stem cells

BackgroundThe role of periodontal ligament stem cells (PDLSCs) and macrophage polarization in periodontal tissue regeneration and bone remodeling during orthodontic tooth movement (OTM) has been well documented. Nevertheless, the interactions between macrophages and PDLSCs in OTM remain to be investigated. Consequently, the present study was proposed to explore the effect of different polarization states of macrophages on the osteogenic differentiation of PDLSCs.MethodsAfter M0, M1 and M2 macrophage-derived exosomes (M0-exo, M1-exo and M2-exo) treatment of primary cultured human PDLSCs, respectively, mineralized nodules were observed by Alizarin red S staining, and the expression of ALP and OCN mRNA and protein were detected by RT-qPCR and Western blotting, correspondingly. Identification of differentially expressed microRNAs (DE-miRNA) in M0-exo and M2-exo by miRNA microarray, and GO and KEGG enrichment analysis of DE-miRNA targets, and construction of protein–protein interaction networks.ResultsM2-exo augmented mineralized nodule formation and upregulated ALP and OCN expression in PDLSCs, while M0-exo had no significant effect. Compared to M0-exo, a total of 52 DE-miRNAs were identified in M2-exo. The expression of hsa-miR-6507-3p, hsa-miR-4731-3p, hsa-miR-4728-3p, hsa-miR-3614-5p and hsa-miR-6785-3p was significantly down-regulated, and the expression of hsa-miR-6085, hsa-miR-4800-5p, hsa-miR-4778-5p, hsa-miR-6780b-5p and hsa-miR-1227-5p was significantly up-regulated in M2-exo compared to M0-exo. GO and KEGG enrichment analysis revealed that the downstream targets of DE-miRNAs were mainly involved in the differentiation and migration of multiple cells.ConclusionsIn summary, we have indicated for the first time that M2-exo can promote osteogenic differentiation of human PDLSCs, and have revealed the functions and pathways involved in the DE-miRNAs of M0-exo and M2-exo and their downstream targets.

Anticarcinogenic impact of extracellular vesicles (exosomes) from cord blood stem cells in malignant melanoma: A potential biological treatment.

Incidence of Malignant Melanoma has become the 5th in the UK. To date, the major anticancer therapeutics include cell therapy, immunotherapy, gene therapy and nanotechnology-based strategies. Recently, extracellular vesicles, especially exosomes, have been highlighted for their therapeutic benefits in numerous chronic diseases. Exosomes display multifunctional properties, including inhibition of cancer cell proliferation and initiation of apoptosis. In the present in vitro study, the antitumour effect of cord blood stem cell (CBSC)-derived exosomes was confirmed by the CCK-8 assay (p < 0.05) on CHL-1 melanoma cells and improve the repair mechanism on lymphocytes from melanoma patients. Importantly, no significant effect was observed in healthy lymphocytes when treated with the exosome concentrations at 24, 48 and 72 h. Comet assay results (OTM and %Tail DNA) demonstrated that the optimal exosome concentration showed a significant impact (p < 0.05) in lymphocytes from melanoma patients whilst causing no significant DNA damage in lymphocytes of healthy volunteers was 300 μg/ml. Similarly, the Comet assay results depicted significant DNA damage in a melanoma cell line (CHL-1 cells) treated with CBSC-derived exosomes, both the cytotoxicity of CHL-1 cells treated with CBSC-derived exosomes exhibited a significant time-dependent decrease in cell survival. Sequencing analysis of CBSC exosomes showed the presence of the let-7 family of miRNAs, including let-7a-5p, let-7b-5p, let-7c-5p, let-7d-3p, let-7d-5p and two novel miRNAs. The potency of CBSC exosomes in inhibiting cancer progression in lymphocytes from melanoma patients and CHL-1 cells whilst causing no harm to the healthy lymphocytes makes it a potential candidate as an anticancer therapy.

Noninvasive Multiplexed Analysis of Bladder Cancer-Derived Urine Exosomes via Janus Magnetic Microspheres.

Bladder cancer greatly endangers human health, and its early diagnosis is of vital importance. Exosomes, which contain proteins and nucleic acids related to their source cells, are expected to be an emerging biomarker for bladder cancer detection. Here, we propose a novel system for multiplexed analysis of bladder cancer-derived urine exosomes based on Janus magnetic microspheres as barcoded microcarriers. The microcarriers are constructed by droplet-templated coassembly of colloidal silica nanoparticles and magnetic nanoparticles under a magnetic field. The microcarriers possess one hemisphere with structural color and the other hemisphere with magneto-responsiveness. Benefiting from the unique structure, these Janus microcarriers could serve as barcodes and could move controllably in a sample solution, thus realizing the multiplex detection of exosomes with high sensitivity. Notably, the present platform is noninvasive since a urine specimen, as an ideal source of bladder cancer-derived exosomes, is employed as the sample solution. This feature, together with the good sensitivity, specificity, low sample consumption, and easy operation, indicates the great potential of the platform for bladder cancer diagnosis in clinical applications.

Proteomics Analysis of Plasma-Derived Exosomes Unveils the Aberrant Complement and Coagulation Cascades in Dermatomyositis/Polymyositis.

Dermatomyositis and polymyositis (DM/PM) are systemic autoimmune diseases characterized by proximal muscle weakness. The underlying pathogenetic mechanism of this disease remains under-researched. Here, using proteomics analysis, a great overlap of differentially expressed plasma exosomal proteins involved in the complement and coagulation cascade pathway, including FGA, FGB, FGG, C1QB, C1QC, and VWF, was identified in DM/PM patients versus healthy controls. Correlation analysis showed that the expression levels of complement-associated proteins (C1QB and C1QC) correlated positively with CRP, ESR, and platelet count. ROC curve analysis demonstrated that complement and coagulation cascade-associated proteins could be strong predictors for DM/PM. In addition, we also identified several other proteins that were differentially expressed in DM and PM. The selected candidate proteins were further validated by parallel reaction monitoring (PRM) and enzyme-linked immunosorbent assay (ELISA). Together, our findings indicate that these exosome-derived proteins might participate in microvascular damage in DM/PM through the activation of the complement and coagulation cascade pathway and function as biomarkers for the clinical diagnosis of DM/PM.

Quantitative proteomic analysis of exosomes from umbilical cord mesenchymal stem cells and rat bone marrow stem cells.

Exosomes derived from mesenchymal stem cells (MSCs) have been used for cancer treatment, however, an in-depth analysis of the exosomal proteomes is lacking. In this manuscript, we use the diaPASEF (parallel accumulation serial fragmentation combined with the data-independent acquisition) method to quantify exosomes derived from human umbilical cord mesenchymal stem cells (UCMSCs) and rat bone marrow stem cells (BMSCs), resulting in identification of 4200 human proteins and 5362 rat proteins. Comparison of human exosomal proteins and total cellular proteins reveals that some proteins exist in the exosomes exclusively that can be served as potential markers for exosomes. Quantitative proteomic analysis of exosomes from different passages of BMSCs shows that the proteins involved in TGF-β signaling pathway are regulated in abundance, which could be markers for the therapeutic ability of BMSC exosomes. Collectively, the data presented by this study can be a resource for further study of exosome research.

Matrix-assisted laser desorption/ionization-Fourier-transform ion cyclotron resonance-mass spectrometry analysis of exosomal lipids from human serum.

Exosomes contain biomarkers such as proteins and lipids that help in understanding normal physiology and diseases. Lipids, in particular, are infrequently studied using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) for biomarker discovery. In this study, MALDI was equipped with a high-resolution MS to investigate exosomal lipids from human serum. Exosomal lipids were profiled using MALDI with Fourier-transform ion cyclotron resonance (FTICR)-MS. Four matrices (i.e., α-cyano-4-hydroxycinnamic acid [CHCA], 2,5-dihydroxybenzoic acid, sinapinic acid, and graphene oxide [GO]) and three sample preparation methods (i.e., dried droplet, thin layer, and two layer) were compared for the number of lipid species detected and the relative abundance of each lipid from human serum and human serum exosomes. In sum, 172 and 89 lipid species were identified from human serum and human serum exosomes, respectively, using all the methods. The highest number of exosome lipid species, 69, was detected using the CHCA matrix, whereas only 8 exosome lipid species were identified using the GO matrix. Among the identified lipid species, phosphatidylcholine was identified most frequently, probably due to the use of a positive ion mode. Exosomes and human serum showed comparable lipid profiles as determined using MALDI-FTICR-MS. These findings provide a new perspective on exosomal lipidomics analysis and may serve as a foundation for future lipidomics-based biomarker research using MALDI-FTICR-MS.

NKG2D-mediated cytotoxicity improves after primary surgery for high-grade serous ovarian cancer.

Tumors compromise the patients' immune system to promote their own survival. We have previously reported that HGSC exosomes play a central role, downregulating NKG2D cytotoxicity. Primary surgery's effect on tumor exosomes and NKG2D cytotoxicity in HGSC patients has not been studied before. The overall objective of this study was to explore the effect of surgery on the exosome-induced impairment of NKG2D cytotoxicity in HGSC. Paired pre- and post-operative blood samples were subjected to cell and exosome analyses regarding the NKG2D receptor and ligands, and NKG2D-mediated cytotoxicity. Lymphocytes were phenotyped by immunoflow cytometry. Exosomes, isolated by ultracentrifugation, and characterized by nanoparticle tracking analysis, transmission and immune electron microscopy and western blot were used in functional cytotoxic experiments. HGSC explant culture-derived exosomes, previously studied by us, were used for comparison. HGSC exosomes from patients' sera downregulated NKG2D-mediated cytotoxicity in NK cells of healthy donors. In a subgroup of subjects, NKG2D expression on CTLs and NK cells was upregulated after surgery, correlating to a decrease in the concentration of exosomes in postoperative sera. An overall significantly improved NKG2D-mediated cytotoxic response of the HGSC patients' own NK cells in postoperative compared to preoperative samples was noted. Surgical removal of the primary tumor has a beneficial effect, relieving the exosome-mediated suppression of NKG2D cytotoxicity in HGSC patients, thus boostering their ability to combat cancer.

Mitochondrial DNA copy number changes, heteroplasmy, and mutations in plasma-derived exosomes and brain tissue of glioblastoma patients

Glioblastoma is the most common malignant tumor of the central nervous system (CNS) in adults. Glioblastoma cells show increased glucose consumption associated with poor prognosis. Since mitochondria play a crucial role in energy metabolism, mutations and copy number changes of mitochondrial DNA may serve as biomarkers. As the brain is difficult to access, analysis of mitochondria directly from the brain tissue represents a challenge. Exosome analysis is an alternative (still poorly explored) approach to investigate molecular changes in CNS tumors.We analyzed brain tissue DNA and plasma-derived exosomal DNA (exoDNA) of 44 glioblastoma patients and 40 control individuals. Quantitative real-time PCR was performed to determine mtDNA copy numbers and the Kruskal-Wallis and Mann-Whitney U test were used for statistical analysis of data. Subsequently, sequencing libraries were prepared and sequenced on the MiSeq platform to identify mtDNA point mutations.Tissue mtDNA copy number was different among controls and patients in multiple comparisons. A similar tendency was detected in exosomes. Based on NGS analysis, several mtDNA point mutations showed slightly different frequencies between cases and controls, but the clinical relevance of these observations is difficult to assess and likely less than that of overall mtDNA copy number changes. Allele frequencies of variants were used to determine the level of heteroplasmy (found to be higher in exo-mtDNA of control individuals).Despite the suggested potential, the use of such biomarkers for the screening and/or diagnosis of glioblastomas is still limited, thus further studies are needed.

Ultramultiplex NaLnF4 Nanosatellites Combined with ICP-MS for Exosomal Multi-miRNA Analysis and Cancer Classification.

Quantification of exosomal multi-miRNA can reveal the initiation, progression, and metastasis of tumors, which is conducive to the noninvasive early diagnosis of cancer. However, low-sensitivity and single-plex detection characteristics of traditional methods seriously hinder the accuracy and specificity of exosomal miRNAs in cancer diagnosis. Herein, we design an ultramultiplexing strategy that enables simultaneous and sensitive detection of multiple exosomal miRNAs by nanosatellites (magnetic beads (MBs) @ NaLnF4) and catalytic hairpin assembly (CHA) amplification in combination with inductively coupled plasma-mass spectrometry (ICP-MS) to diagnose cancer accurately. The competitive binding of target exosomal miRNAs with the recognition sequences on nanosatellites triggers the drop of NaLnF4 from MBs, followed by a CHA reaction that releases more NaLnF4 labels for ICP-MS detection. This method is used to detect ten types of miRNAs simultaneously with a detection limit of 0.01 fM, which is one order of magnitude lower than the quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. Linear discriminant analysis as a machine learning algorithm is subsequently applied to analyze the signals of exosomal multi-miRNA, and the discrimination accuracy of ten cell exosomes reaches 98.6%. In a clinical cohort of 42 patients, including five cancer types and healthy controls, exosomal multi-miRNA analysis achieves accurate cancer diagnosis and classification with 100% accuracy. Our results show that the combination of nanosatellites, CHA, and ICP-MS provides a universal biosensing platform for simultaneous and ultrasensitive detection of multiple targets.

Exosome Structures Supported by Machine Learning Can Be Used as a Promising Diagnostic Tool.

Principal component analysis (PCA) as a machine-learning technique could serve in disease diagnosis and prognosis by evaluating the dynamic morphological features of exosomes via Cryo-TEM-imaging. This hypothesis was investigated after the crude isolation of similarly featured exosomes derived from the extracellular vehicles (EVs) of immature dendritic cells (IDCs) JAWSII. It is possible to identify functional molecular groups by FTIR, but the unique physical and morphological characteristics of exosomes can only be revealed by specialized imaging techniques such as cryo-TEM. On the other hand, PCA has the ability to examine the morphological features of each of these IDC-derived exosomes by considering software parameters such as various membrane projections and differences in Gaussians, Hessian, hue, and class to assess the 3D orientation, shape, size, and brightness of the isolated IDC-derived exosome structures. In addition, Brownian motions from nanoparticle tracking analysis of EV IDC-derived exosomes were also compared with EV IDC-derived exosome images collected by scanning electron microscopy and confocal microscopy. Sodium-Dodecyl-Sulphate-Polyacrylamide-Gel-Electrophoresis (SDS-PAGE) was performed to separate the protein content of the crude isolates showing that no considerable protein contamination occurred during the crude isolation technique of IDC-derived-exosomes. This is an important finding because no additional purification of these exosomes is required, making PCA analysis both valuable and novel.

Re-Analysis of Published Datasets in Search of Novel Urogenital Diseases Biomarkers.

Exosome research is a current trend in functional proteomics as it provides important data on the pathophysiology and pathogenesis of diseases. The scientific outputs regarding these topics often only approach disease-protein/peptide/exosome or mechanismprotein/ peptide/exosome association. Approaching all three aspects could be the key to a better understanding of the pathophysiology and uncovering novel biomarkers for urogenital diseases. The focus of this work is to study exosome datasets to understand the possible role of underlying proteins in disease manifestation. We also attempt to link 4 different diseases that affect renal functions and are genetically inherited. For this purpose, the existing literature is consulted to understand the importance of exosomes in disease prediction, diagnosis and therapy. Available biotechnological methods of exosome analysis and the tools of proteomic analysis, data mining and visualization are discussed. The database PRIDE is selected to query the information of several datasets related to urinary exosome analysis. We have obtained a list of 19 proteins/genes involved in the mentioned diseases. On this list, we found a proteomic fingerprint consisting of Rab-7a, PDCD6, and CDC42, among others, and we are exploring their biological significance and underlying processes. APOA1, CD59, CD9, IGHG1, RAB7A, RAP1A, SEMG1 and SEMG2 are common in four urogenital diseases, and are involved in interactions with podosomes and endosomes, remodeling of chylomicrons, regulation of interleukin production, regulation of endopeptidase activity, and establishment of apical/basal polarity of epithelial cells.

Metabolomic Analysis of Exosomes Derived from Lung Cancer Cell Line H460 Treated with SH003 and Docetaxel

Exosomes released from tumor cells treated with cancer-targeting drugs reflect altered metabolic processes within the cells. Therefore, metabolites in exosomes can be used as markers to predict the therapeutic response or identify therapeutic targets. In this study, metabolite changes in exosomes were investigated by co-administration of the herbal extract SH003 and docetaxel (DTX), which exert a synergistic anti-cancer effect on lung cancer cells. Exosomes released from cells treated with SH003 and DTX were purified, and untargeted metabolic profiling was performed by liquid chromatography-tandem mass spectrometry. Analysis of altered metabolic-based pathways showed that the combined treatment synergistically increased pyrimidine metabolism compared with single-drug treatment. Additionally, xenobiotic metabolism by cytochrome P450 was specifically increased in cells treated with the combination. However, the released exosomes and increased metabolites in exosomes did not affect the anti-cancer effect of SH003 and DTX. Therefore, our study suggests that metabolite profiling can be used to evaluate the efficacy of combined treatments. Furthermore, such exosome-based metabolism may facilitate understanding the physiological endpoints of combination therapy in human biofluids.

DNA-functionalized covalent organic framework capsules for analysis of exosomes

Exosomes have been deemed as hopeful liquid biopsy biomarkers for malignant tumor identification. However, proposing detection methods with high sensitivity still meets challenges. Herein, we have fabricated an electrochemical biosensor in this work via constructing nucleic acid functionalized covalent organic framework (COF) capsules for sensitive detection of exosomes. Firstly, nucleic acid functionalized COF capsules are prepared by modifying azide-labeled DNA onto alkynyl-containing COF capsules. Then, the capsules are used to fabricate an electrochemical biosensor for exosomes quantification. In this design of the sensor, the modification of DNA strands on COFs can greatly improve the binding efficiency, while the loading of plentiful horseradish peroxidase (HRP) facilitates signal amplification. So, the fabricated biosensor has effectively enhanced the sensitivity and lowered the detection limit to 87 particles/μL. Further studies show that the biosensor can be used for the identification of exosomes from non-small cell lung cancer. Moreover, by simply replacing the DNA sequences, the proposed strategy can generally be used to fabricate different DNA-COF capsules with diverse functions for broader biomedical applications.

Proteomic analysis of vitreal exosomes in patients with proliferative diabetic retinopathy.

To determine the proteomic profiles of exosomes derived from vitreous humour (VH) obtained from proliferative diabetic retinopathy (PDR) patients and non-diabetic controls with idiopathic macular hole/epiretinal membrane. Vitreal exosomes were isolated using differential ultracentrifugation, followed by characterisation performed using different techniques. A label-free proteomic analysis was conducted to determine the protein profiles of the exosomes. A parallel reaction monitoring (PRM) analysis was performed to verify the identified proteins and associated functional annotations were derived by gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Receiver operating characteristic (ROC) analysis was utilised to evaluate the diagnostic value of target proteins in distinguishing PDR from controls. Exosomes were successfully isolated from VH, and were well characterised by various techniques. The results of proteomic analysis showed that a total of 758 proteins were identified and 10 proteins were screened as differentially expressed proteins, significantly changed in the PDR group containing 4 elevated proteins and 6 reduced proteins. GO analysis indicated that these differential proteins were mainly involved in many metabolic pathways, including nicotinamide adenine dinucleotide metabolism, adenosine diphosphate metabolic process and glycolytic process. The KEGG analysis enriched the top five pathways including glycolysis/gluconeogenesis, fructose and mannose metabolism, biosynthesis of amino acids, hypoxia-inducible factor 1 signalling pathway and carbon metabolism. The differential proteins, namely, lactate dehydrogenase A, ficolin 3, apolipoprotein B and apolipoprotein M, were further verified by PRM and showed a consistent trend with label-free proteomic analysis. The ROC analysis identified these proteins as promising biomarkers for PDR diagnosis. Vitreal exosomes from patients with PDR contained few proteins unique to PDR; thus, exosomal proteins have great potential as disease biomarkers and therapeutic targets for PDR.

Pancreatic Cancer-Derived Exosomes Promote the Proliferation, Invasion, and Metastasis of Pancreatic Cancer by the miR-3960/TFAP2A Axis.

Background The microRNAs (miRNAs) in cancer-derived exosomes have the ability to change tumor microenvironment. This study aims to investigate the role of miRNA in cancer-derived exosomes in pancreatic cancer (PC). Methods Based on the analysis of PC-derived and healthy exosomes by bioinformatics analysis and quantitative real-time PCR validation, the miR-3960 was identified to be the most significantly different miRNA, and TFAP2A proved as its potential target gene. Besides, the exosomes were isolated from PANC-1 cells and identified. After that, PANC-1 cells were treated with the isolated exosomes or transfected with miR-3960 mimics or si-TFAP2A, the effect of PC-derived exosomes, as well as the miR-3960/TFAP2A axis in PC cells, were assessed by the CCK-8, EDU staining, Transwell, cell colony formation, and flow cytometry assays. Furthermore, the effects of exosomes and the miR-3960/TFAP2A axis on PC tumor growth were observed in tumor-bearing mice by the measurement of tumor weight and volume, and hematoxylin-eosin staining. Moreover, the expressions of TFAP2A/PTEN/AKT signaling proteins were detected by Western blot. Results PC-derived exosomes were isolated successfully and proved to have promotion effects on the proliferation, metastasis, and invasion of PC cells both in vitro and tumor growth in vivo. Also, the PC-derived exosomes upregulated the TFAP2A, Bcl-2, and p-AKT/AKT protein levels, and inhibited PTEN and Bax levels and PANC-1 cell apoptosis. Overexpression of miR-3960 antagonized the promotion effect of exosomes on PC cells and the TFAP2A/PTEN/AKT signaling pathway, inhibiting the growth of tumors. Besides, si-TFAP2A enhanced the inhibitory effect of miR-3960 in PC. Conclusion MiR-3960 antagonizes the promotion effect of tumor-derived exosomes on the proliferation, invasion, and metastasis of PC via suppressing TFAP2A.

Exo-III Enzyme and DNAzyme-Assisted Dual Signal Recycles for Sensitive Analysis of Exosomes by Using Personal Glucose Meter.

Exosome plays a crucial role in regulating intercellular communication during atherosclerosis development. However, sensitive and portable exosome detection remains a huge challenge. Herein, a personal glucose meter (PGM)-based exosomes detection approach has been proposed that allows detection of exosomes with a high sensitivity and reproducibility. In this method, a catch probe, which is composed of CD63 aptamer and blocker sequence, is utilized for the specific identification of exosomes. The blocker sequence binds with H probe to initiate the Exo-III-assisted signal recycles to generate numerous DNAzyme sequences. Under the assistance of the substrate, DNAzyme forms its active secondary structure to generate gap site in substrate, releasing a linker to conjugate sucrase to streptavidin magnetic beads (SMBs). After removing unbound sucrase, the SMB-linker-sucrase complex is used to catalyze sucrose to glucose, which can be read by PGMs. Based on this, the method exhibits a wide detection range and a low limit of detection, holding a promising prospect for the analysis of exosomes and screening atherosclerosis.