Size Distribution Of Exosomes Research Articles

Isolation of three different sizes of exosomes in an Asian population with different retinal diseases before and after treatment: preliminary results

ABSTRACT Exosomes are membranous structures measuring between 40–120 nm that are secreted by various cells of the human body into the body fluid system. Exosomes contain proteins, mRNA, miRNA, and signaling molecules, and physiologically they assist in the intercellular transport of proteins and RNA molecules. In this study, we used an immunoaffinity filter paper platform combined with scanning electron microscopy and microfluidic systems to detect the size of exosomes within the aqueous humor. Eight aqueous humor samples showed three distinct sizes of exosomes that were significantly different on scanning electron microscopy(P < 0.01). We further used nanoparticle tracking analysis to assess the size distribution of exosomes within the aqueous humor. We found significantly different distributions of exosomes between patients with three different ocular diseases and patients with normal cataracts as controls. An obvious peak of exomeres(size around 35 nm)was found in the patients with central retinal vein occlusion and vitreous hemorrhage. Flare-ups of large exosomes(size 90–120 nm)were found in the patients with the inflammatory ocular disease pars planitis. No obvious peaks in exomeres or large exosomes were found in the control group. There was a high association between the distribution of exosomes and the pathogenesis of ocular diseases. After intravitreal anti-vascular endothelial growth factor treatment, the aqueous humor from the patients with neovascular diseases showed a significant reduction in exosomes in nanoparticle tracking analysis. These findings suggest that at least three distinct sizes of exosomes exist in the aqueous humor:(1)exomeres:<35 nm;(2)small exosomes:60–80 nm; and (3)large exosomes:90–120 nm. Different sizes of exosomes may have different implications in normal or diseased eyes.

Exosomes Promote Atherosclerosis Progression by Regulating Circ_100696/miR-503-5p/PAPPA Axis-Mediated Vascular Smooth Muscle Cells Proliferation and Migration.

Circular RNAs (circRNAs) are known to play a crucial role in the progression of atherosclerosis (AS). In this study, we aim to explore the function of oxidized low-density lipoprotein (ox-LDL)-induced macrophage-derived exosomal circ_100696 in AS.THP-1 macrophages were induced by ox-LDL to mimic AS cell model. A quantitative real-time polymerase chain reaction (qRT-PCR) assay was applied to determine the expression of circ_100696, microRNA-503-5p (miR-503-5p), and pregnancy-associated plasma protein A (PAPPA). The morphology and size distribution of exosomes were examined by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Western blot assay was performed for protein levels. Cell proliferation was assessed using 5-ethynyl-2'-deoxyuridine (EdU) assay. Flow cytometry analysis was performed to analyze the cell cycle. Wound-healing assay and transwell assay were done to examine cell migration. RNA pull-down assay, dual-luciferase reporter assay, and RNA immunoprecipitation (RIP) assay were employed to analyze the relationship among circ_100696, miR-503-5p, and PAPPA.Circ_100696 level was increased in ox-LDL-induced THP-1 macrophages and ox-LDL-treated THP-1 macrophage-derived exosomes (OM-Exo). OM-Exo promoted the proliferation, cell cycle, and migration of vascular smooth muscle cells (VSMCs). Circ_100696 was upregulated in VSMCs cocultured with OM-Exo. Circ_100696 knockdown reversed the effects of OM-Exo on VSMC proliferation and migration. Circ_100696 was demonstrated to function as the sponge for miR-503-5p, and miR-503-5p directly targeted PAPPA. Circ_100696 overexpression facilitated VSMC proliferation and migration, with miR-503-5p upregulation or PAPPA silencing reversing these effects. Moreover, circ_100696 overexpression promoted PAPPA expression by targeting miR-503-5p.OM-Exo promoted VSMC growth and migration by regulating the circ_100696/miR-503-5p/PAPPA axis, thereby promoting AS progression.

Interaction between astrocytes and neurons in simulated space radiation-induced CNS injury

Purpose Astronauts exhibit neurological dysfunction during long-duration spaceflight, and the specific mechanisms may be closely related to the cumulative effects of these neurological injuries in the space radiation environment. Here, we investigated the interaction between astrocytes and neuronal cells exposed to simulated space radiation. Materials and methods we selected human astrocytes (U87 MG) and neuronal cells (SH-SY5Y) to establish an experimental model to explore the interaction between astrocytes and neuronal cells in the CNS under simulated space radiation environment and the role of exosomes in the interactions. Results We found that γ-ray caused oxidative and inflammatory damage in human U87 MG and SH-SY5Y. The results of the conditioned medium transfer experiments showed that astrocytes exhibited a protective effect on neuronal cells, and neuronal cells influenced the activation of astrocytes in oxidative and inflammatory injury of CNS. We demonstrated that the number and size distribution of exosomes derived from U87 MG and SH-SY5Y cells were changed in response to H2O2, TNF-α or γ-ray treatment. Furthermore, we found that exosome derived from treated nerve cells influenced the cell viability and gene expression of untreated nerve cells, and the effect of exosomes was partly consistent with that of the conditioned medium. Conclusion Our findings demonstrated that astrocytes showed a protective effect on neuronal cells, and neuronal cells influenced the activation of astrocytes in oxidative and inflammatory damage of CNS induced by simulated space radiation. Exosomes played an essential role in the interaction between astrocytes and neuronal cells exposed to simulated space radiation.

Decoding of exosome heterogeneity for cancer theranostics.

Extracellular Vesicles (EVs) discovery establishes a new milestone in cancer research. It explains cancer biology in a new way. EVs classified some significant subclasses such as macrovesicle (originated from plasma membrane), apoptotic bodies (originated from the plasma membrane and endoplasmic reticulum), and exosomes (originated from endosomes).1 The theranostic signature of exosomes in cancer healing is impressive, but its heterogeneity leads most complicated domain of research. Therefore we comment on decoding exosome heterogeneity for future efficiency and effective cancer theranostics development. EVs are the cell-secreted one of the most important entities. The exosome attracted much attention due to its versatile uses. Overall, its participation in intercellular communication and its inner cargos (DNA, RNA, proteins, lipids and glycan)2 can reprogram the recipient cells. In addition, it also shows the healthy or pathologic complication status of the parent cells. In cancer, tumor and exosomes interlink a complex chapter of cancer biology. Exosomes play an important role in accelerated tumor growth, immune cell reprogramming,2 angiogenesis,3 extracellular matrix remodelling,4, 5 metastasis,4, 5 epithelial-mesenchymal transition (EMT),4, 5 organ-specific metastasis,5, 6 drug resistance and cancer stem cell development.3, 4 Clinically aspect, the exosome is the source of the cancer biomarkers.7-9 Exosome heterogeneity1 is evolving into a major conflict in cancer biomarker and therapeutic research.10 The exosome heterogeneity (Figure 1) is based on several facts such as origin, size, quantity, and internal molecular diversity.1 Exosome size distribution is the most complicated side of tumor-derived exosomes (TEXs). Scientific evidence shows that the same cells release differently-sized exosomes. This size-oriented biodiversity is reflected in using different isolation methods.9, 10 The tumor cells release more exosomes compared to healthy cells. The higher release of exosomes from tumor cells depends on several factors, such as drug and therapeutic exposures, hypoxic conditions, and senescence. The functional diversity of exosomes is related to the surface molecular expression and inner cargos of exosomes. All these complicated/complex parameters lead to a challenging ecosystem development of exosome-based cancer theragnostics (combination of biomarkers and therapeutics research). This scenario requires a solution to solve these challenges. Based on several innovative nano platform-based approaches transform these challenges into an opportunity for exosome researcher in a detailed exploration of exosome.11 Exosomes isolation, size and quantity related complication solved via microfluidic device, fluorescence antibody based exosome tracking, Nanoparticle tracking analysis (NTA) (above 60 nm exosome size challenging for its application),12, 13 magnetic and surface plasma resonance principle based single exosome screening,1 advanced sensitive flow cytometry (it is capable of the separation of exosome subpopulation) and electrophoresis chip. Molecular profiling of exosomes is one of the most critical tasks revealing the functional diversity of bioactive cargo molecules of exosomes. Droplet digital polymerase chain reaction (it is sensitive to the detection of rear mutations in tumor-derived exosomes),1 microfluidic technology combined with a chip system, and an electrochemical principle-based micro RNA profiling of exosomes explain the cancer complication of a new dimension.1 The downstream process of exosome profiling needs a muti-omics approach for proper molecular diversity to understand.1 The single cells exosome profiling approach combines with machine learning for more precision cancer marker development.14 All these technological advancements support constructing the next-generation promising cancer theranostic era based on exosomes.15, 16 Finally, decoding exosome heterogeneity opens a new door for exosome-based precision medicine17 and vaccines18 for cancer. We hope this article encourages large-scale EV research to explore a single exosome profiling approach and future exosome-based cancer precision medicine development. Not applicable. The authors declare no conflict of interest. There is no funding for this study.

Plasma‑derived CD16 exosomes and peripheral blood monocytes as correlating biomarkers in head and neck cancer.

Exosomes play an important role in the individual immune regulation in patients with head and neck squamous cell carcinoma (HNSCC) as part of the tumor microenvironment. Patients with HNSCC with advanced tumor stages reveal significantly increased levels of plasma derived CD16+ (FcRIIIA) total exosomes as demonstrated in our previous study. Furthermore, increased individual abundances of peripheral blood CD16+ non-classical monocytes have been shown to correlate with increased monocytic programmed death ligand 1 (PD-L1) and CD4+ T cell disturbances in oropharyngeal cancer. However, the context of plasma derived CD16+ exosomes in patients with HNSCC and their role in the immune-regulation of circulating monocyte subsets has not been investigated so far. In the present study, exosomes were isolated from plasma samples of healthy donors and patients with HNSCC and evaluated for their morphology, size and protein composition using transmission electron microscopy, western blotting and bead-based flow cytometry. Monocyte subset abundances were analyzed in whole blood measurements in terms of the CD14/CD16 cell surface expression patterns, different monocytic adhesion molecules and checkpoint molecule PD-L1 using flow cytometry. Isolated exosomes were positive for the tetraspanins CD63 and CD9 as well as the endosomal marker TSG101, but negative for the non-exosomal marker glucose-regulated protein 94 and apolipoprotein ApoA1. Plasma derived CD16+ exosomes and exosome size distribution were significantly correlated with abundances of CD16+ non-classical monocytes and CD16+ intermediate monocytes, respectively. Furthermore, the data revealed significant correlations between CD16+ plasma derived exosomes and adhesion molecules CD29 (integrin β1) and CX3CR1 on certain monocyte subsets. These data suggested that CD16 positive exosomes and exosome size distribution were potential surrogates to characterize the composition of monocyte subsets in patients with HNSCC. Overall, both CD16-positive exosomes and CD16-positive monocyte subsets are potential liquid biomarkers that could be used to characterize the individual immunological situation of patients with HNSCC.

Improved exosome isolation methods from non-small lung cancer cells (NC1975) and their characterization using morphological and surface protein biomarker methods.

This study has demonstrated improved methods for isolating exosomes from non-small lung cancer cells, which address the problems characterized by exosome morphological and chemical methods. To improve the isolation methods, cells from the NCI 1975 cell line were used as the source for exosomes. The isolation processes were carried out using serial isolation techniques in addition to specific preservation tools. The isolated exosomes were characterized using transmission electron microscopy (TEM), and scanning electron microscopy (SEM) was added for further assurance of the investigation results. The statistical analysis results showed that the size distributions of apoptotic vesicles (APV) 450nm and necrotic bodies (NCB) 280nm (extracellular vesicles) were significantly different from exosomes (P < 0.001). In contrast, the exosome size distribution was not significantly different from the published exosome sizes, as demonstrated by statistical analysis tools. This study confirmed the improved methods for isolating exosomes that make exosomes accessible for use in the diagnosis and prognosis of non-small cell lung cancer (NSCLC).

Circ_0026218 ameliorates oxidized low-density lipoprotein-induced vascular endothelial cell dysfunction by regulating miR-188-3p/TLR4/NF-κB pathway.

Circular RNAs (circRNAs) have shown important regulatory roles in cardiovascular diseases, including atherosclerosis (AS). However, the role and mechanism of circ_0026218 in AS remain unclear. The cell model of AS in vitro was established by stimulating human umbilical vein endothelial cells (HUVECs) with oxidized low-density lipoprotein (ox-LDL). In addition, circ_0026218, microRNA-188-3p (miR-188-3p), and toll-like receptor 4 (TLR4) expression was determined via real-time quantitative polymerase chain reaction (RT-qPCR) in serum samples from AS patients and healthy volunteers. Cell proliferation was assessed using Cell Counting Kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay. Cell apoptosis was measured using flow cytometry. The inflammatory response was assessed using enzyme-linked immunosorbent assay (ELISA). Oxidative stress level was assessed using corresponding kits. Nitric oxide (NO) level was examined using NO detection assay. The interaction between miR-188-3p and circ_0026218 or TLR4 was determined via dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays. Exosomes were observed using transmission electron microscopy (TEM). The size distribution of exosomes was analyzed using nanoparticle tracking analysis (NTA). Ox-LDL treatment caused HUVEC dysfunction by inhibiting cell proliferation and promoting apoptosis, inflammation, and oxidative stress. Circ_0026218 was upregulated in AS serum samples and ox-LDL-treated HUVECs. Knockdown of circ_0026218 attenuated ox-LDL-induced dysfunction in HUVECs. MiR-188-3p acted as a target of circ_0026218, and miR-188-3p downregulation reversed the suppression role of circ_0026218 knockdown on ox-LDL-induced HUVEC disorder. TLR4 was a target of miR-188-3p, and miR-188-3p overexpression alleviated ox-LDL-induced dysfunction in HUVECs by targeting TLR4. Circ_0026218 could deregulate the TLR4/NF-κB pathway by sponging the miR-188-3p. Importantly, circ_0026218 was overexpressed in exosomes from ox-LDL-treated HUVECs and could be delivered via exosomes. Circ_0026218 knockdown attenuated ox-LDL-induced dysfunction in HUVECs via regulating miR-188-3p/TLR4/NF-κB pathway.

Astragaloside IV - mediated endothelial progenitor cell exosomes promote autophagy and inhibit apoptosis in hyperglycemic damaged endothelial cells via miR-21/PTEN axis.

As one of the basic components of Astragalus, Astragaloside IV (AS-IV) has a protective effect on endothelial injury caused by diabetes. AS-IV stimulated endothelial progenitor cells (EPCs) to secrete exosomes loaded with miR-21. This study aimed to investigate the effects of AS-IV-mediated EPCs exosomal miR-21 (EPC-exos-miR-21) on high glucose (HG) damaged endothelial cells. After the isolation of EPCs derived from fetal umbilical cord blood, exosomes of EPCs were obtained by differential centrifugation. The morphology of exosomes was observed by electron microscopy. The particle size distribution of exosomes was detected by Nanoparticle Tracking Analysis. Human umbilical vein endothelial cells (HUVECs) were treated with 33 mM glucose to establish an HG injury model. Flow cytometry and TUNEL assay were used to characterize the surface markers of primary EPCs and the apoptosis of HUVECs. The gene and protein expression were detected by qPCR, immunofluorescence, and Western blotting. A dual luciferase assay was used to verify the targeting relationship of miR-21 with PTEN. HG environment led to time- and dose-dependent inhibition and enhancement of autophagy and apoptosis in HUVECs. AS-IV stimulated EPCs to secrete exosomes loaded with miR-21. Exosomes secreted by EPCs pretreated with AS-IV [EPC-exo(ASIV)] promoted autophagy and inhibited apoptosis in HG-impaired HUVECs. PTEN is a target of miR-21. MiR-21 carried by EPC-exo(ASIV) repressed PTEN expression in HG-impaired HUVECs. In contrast, p-AKT, p-mTOR, p-PI3K, cleaved PARP and PARP levels were upregulated. Compared to the HG group, the expression of autophagy regulatory genes (ATG5, beclin1 and LC3) was enhanced in the EPC-exo(ASIV) group and EPC-exo(ASIV)-miR-21 mimic group. In contrast, apoptosis-positive regulatory genes (Bax, caspase-3 and caspase-9) were attenuated. Further overexpression of PTEN reversed the expression of these genes. AS-IV-mediated EPC-exos-miR-21 could enhance autophagy and depress apoptosis in HG-damaged endothelial cells via the miR-21/PTEN axis.

Exosome-mediated transfer of circ_0063526 enhances cisplatin resistance in gastric cancer cells via regulating miR-449a/SHMT2 axis.

Exosomes are critical mediators of intercellular communication. Exosomal circular RNAs (circRNAs) have been reported to play critical roles in the development of chemoresistance in various tumors, including gastric cancer. However, the role of exosomal circ_0063526 in cisplatin (CDDP) resistance of gastric cancer is still unclear. The expression of circ_0063526, microRNA-449a (miR-449a) and serine hydroxymethyltransferase 2 (SHMT2) mRNA was determined by quantitative real-time PCR (qRT-PCR). Cell viability was assessed by the Cell Counting Kit-8 assay. Cell migration and invasion were evaluated by the transwell assay and wound healing assay. Western blot assay was used to measure the protein expression of light Chain 3 (LC3) I/II, p62 and SHMT2. Exosomes were detected using transmission electron microscopy. The size distribution of exosomes was analyzed by nanoparticle tracking analysis. The interaction between miR-449a and circ_0063526 or SHMT2 was confirmed by a dual-luciferase reporter and RNA pull-down assays. Circ_0063526 expression was increased in gastric cancer tissues and cells and CDDP-resistant cells. Extracellular circ_0063526 could be packaged into exosomes and transmitted to sensitive cells, thus disseminating CDDP resistance. Knockdown of exosomal circ_0063526 inhibited CDDP resistance via suppressing migration, invasion and autophagy in gastric cancer cells. Moreover, circ_0063526 was identified as a molecular sponge of miR-449a to upregulate SHMT2 expression. Further, exosomal circ_0063526 regulated SHMT2 expression to enhance CDDP resistance of gastric cancer cells. Additionally, high expression of exosomal circ_0063526 in serum was associated with poor response to CDDP treatment in gastric cancer patients. Exosomal circ_0063526 facilitated CDDP resistance in gastric cancer via regulating the miR-449a/SHMT2 axis.

Proteomic Profiling of Aqueous Humor Exosomes from Age-related Macular Degeneration Patients.

Purpose: The alteration of the exosomal proteins in the aqueous humor (AH) is linked to the development of eye diseases. The goal of this study was to examine the exosomal protein profile of patients with age-related macular degeneration (AMD) to better understand their role in the pathogenesis of AMD.Methods: Exosomes were isolated from the AH of 28 AMD and 25 control eyes. The quality, concentration, and size distribution of exosomes were measured using a nanoparticle tracking analysis system (NTA). Total exosomal proteins from each sample were purified and digested with trypsin for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.Results: Based on LC-MS/MS analysis, we got 105 exosomal peptides from AMD and control patients. Gene ontology (GO) analysis in the biology process revealed that exosomal proteins of AMD were enriched in the lipoprotein metabolic process. T-test analysis revealed six exosomal proteins in patients with AMD were significantly different from controls. Comparing the exosomal protein profile of AMD patients who were receiving anti-VEGF therapy, we observed the amount of two proteins decreased with the duration of the anti-VEGF treatment time.Conclusions: In this study, we successfully isolated and purified AH exosomes. Our results provide pioneering findings for the exosomal protein profile in AMD development and under therapy. These unique proteins could be the new targets for drug discovery or biological markers for evaluating therapeutic efficacy.

Label-free imaging and biomarker analysis of exosomes with plasmonic scattering microscopy.

Exosome analysis is a promising tool for clinical and biological research applications. However, detection and biomarker quantification of exosomes is technically challenging because they are small and highly heterogeneous. Here, we report an optical approach for imaging exosomes and quantifying their protein markers without labels using plasmonic scattering microscopy (PSM). PSM can provide improved spatial resolution and distortion-free image compared to conventional surface plasmon resonance (SPR) microscopy, with the signal-to-noise ratio similar to objective coupled surface plasmon resonance (SPR) microscopy, and millimeter-scale field of view as a prism-coupled SPR system, thus allowing exosome size distribution analysis with high throughput. In addition, PSM retains the high specificity and surface sensitivity of the SPR sensors and thus allows selection of exosomes from extracellular vesicles with antibody-modified sensor surfaces and in situ analyzing binding kinetics between antibody and the surface protein biomarkers on the captured exosomes. Finally, the PSM can be easily constructed on a popular prism-coupled SPR system with commercially available components. Thus, it may provide an economical and powerful tool for clinical exosome analysis and exploration of fundamental issues such as exosome biomarker binding properties.

Endocytosis-associated patterns in nerve regeneration after peripheral nerve injury.

BackgroundClearance of myelin debris and remyelination of myelin are necessary steps for peripheral nerve remodeling and regeneration. It has yet to be clarified which genes or proteins are involved in endocytosis or exocytosis in the removal of myelin debris during peripheral nerve repair.MethodsFor this project, a rat model of subacute stage of sciatic nerve injury was established first. Subsequently, normal Schwann cells (NSCs) and activated Schwann cells (ASCs) were harvest before and after peripheral nerve injury (PNI). Following methylated DNA immunoprecipitation sequencing (MeDIP-seq) and tandem mass tags (TMT) labeling analysis of NSCs and ASCs, what common biomarkers changes in peripheral nervous systems remain to be elucidated.ResultsA total of 14,770 different expression genes (DEGs) and 3249 different expression proteins (DEPs) were screened between ASCs and NSCs. For the exosomes, the diameter and particles concentration of exosomes were 141.7 ​nm and 2.97 ​× ​107 particles/mL, respectively. The size distribution of exosomes was 50–200 ​nm. ASCs showed higher cellular uptake ability than the NSCs by cellular uptake test. Moreover, RAB7A, ARF6, ARF1, VPS45, RAB11A, DNM3, and NEDD4 were the core markers and may control the molecular mechanism of the Endocytosis pathway.ConclusionThese biomarkers may play significant roles in the initiation phase of demyelination and axon regeneration.The translational potential of this articleThis study explores that the endocytosis-associated patterns of Schwann cells may be new therapeutic strategy for nerve tissue engineering and nerve regeneration.

Plasmatic Exosome Number and Size Distinguish Prostate Cancer Patients From Healthy Individuals: A Prospective Clinical Study.

There is a urgent need for valuable strategy in early and less invasive diagnosis for cancer. Preliminary data have shown that the plasmatic levels of exosomes increase in cancer condition. This study investigates the relevance of plasmatic levels and size distribution of exosomes in 42 individuals with no signs of urological disease (CTR) as compared to 65 prostate cancer patients (PCa). It was used Nanoparticle Tracking Analysis (NTA), a highly reliable and sensitive method for exosomes characterization and quantification. The relation structure among the NTA-derived parameters was assessed by means of Principal Component Analysis, which allowed detecting the global discriminant power of NTA test in terms of Receiver Operating Characteristic (ROC) curve and the selection of cut-off thresholds. The results showed that PCa had significantly higher plasmatic levels of exosomes and that the exosomes were smaller in size as compared to the CTR; the values reached 89% sensitivity and 71% specificity, in distinguishing PCa from CTR. These results propose a new exosome-based non-invasive clinical approach for the clinical follow-up of prostate cancer undergoing surgical treatment; in addition this method may be developed as a new screening test for prostate cancer’s early diagnosis. While this clinical study was performed in prostate cancer, it may represent a proof of concept extendable to virtually all cancers, as it is suggested by both pre-clinical evidence and clinical data obtained with different technical approaches.

Exosomal circ_0007385 enhances non-small cell lung cancer cell proliferation and stemness via regulating miR-1253/FAM83A axis.

Exosomes are critical mediators of intercellular communication in the tumor microenvironment. Exosomal circular RNAs (circRNAs) can act as biomarkers and play crucial roles in many cancers, including non-small cell lung cancer (NSCLC). The aim of this study was to explore the functions and regulatory mechanism of exosomal circ_0007385 in NSCLC. The expression levels of circ_0007385, microRNA-1253 (miR-1253), family with sequence similarity 83, member A (FAM83A) mRNA were determined by quantitative real-time PCR (qRT-PCR). Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (Edu), and colony formation assays were utilized to determine cell proliferation ability. Sphere formation efficiency was determined by sphere formation assay. All protein levels were detected by western blot assay. Exosomes were detected using transmission electron microscopy analysis. Size distribution of exosomes was analyzed by nanoparticle tracking analysis. The interaction between miR-1253 and circ_0007385 or FAM83A was confirmed by dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays. Mice xenograft model was established to verify the function of circ_0007385 in vivo. Circ_0007385 was upregulated in NSCLC tissues and cells. Knockdown of circ_0007385 inhibited NSCLC cell proliferation and stemness, while exosomal circ_0007385 facilitated NSCLC cell proliferation and stemness. In addition, miR-1253 was a direct target of circ_0007385, and miR-1253 reversed the inhibitory effects of circ_0007385 on cell proliferation and stemness in NSCLC cells. Moreover, FAM83A was a direct target of miR-1253, and miR-1253 suppressed NSCLC cell proliferation and stemness by targeting FAM83A. Furthermore, circ_0007385 knockdown inhibited tumor growth in vivo. Exosomal circ_0007385 promoted NSCLC cell proliferation and stemness by regulating miR-1253/FAM83A axis.

Proteomic analysis of Exosomes derived from the Aqueous Humor of Myopia Patients.

Objectives: Myopia is the most common refractive vision disorder. In recent years, several studies have suggested that the alteration of the exosomal protein levels in the aqueous humor (AH) is associated with the development of several eye diseases. Therefore, we aimed to explore the exosomal protein profile of the AH from myopia patients.Methods: Exosomes were isolated from the AH. The quality, concentration, and size distribution of exosomes for each patient were measured using nanoparticle tracking analysis system. Then, the exosomal proteins were purified and digested by trypsin for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.Results: There was no significant difference observed between the myopia and control when comparing the concentration and size distribution of exosomes in the AH for each sample. Based on LC-MS/MS analysis, myopia patients had higher and more complex exosomal peptide content. We found two proteins that were common in AH exosomes and eight proteins that were highly expressed in the myopia group.Conclusions: Our results provide pioneering findings for the exploration of the exosomal protein profile in myopia development. Further studies may provide significant information for the diagnosis, clinical treatment, and prognosis of myopia.

Exosomes isolated from two different cell lines using three different isolation techniques show variation in physical and molecular characteristics

Exosomes are the nanoscopic lipid bi-layered extracellular vesicles with the potential to be utilized as targeted therapeutics. In our investigation, we compared three major exosome isolation techniques that were Total Exosome Isolation reagent (TEI), Protein organic solvent precipitation (PROSPR) and differential ultracentrifugation (UC) based on the biophysical and physicochemical characteristics of exosomes isolated from COLO 205 and MCF-7 cancer cell's conditioned media with an aim to select a suitable method for translational studies. 3D image analysis and particle size distribution of exosomes from their HRTEM images depicted the morphological differences. Molecular and analytical characterization of exosomes using western blotting, Raman and ATR-FTIR spectroscopy and the multivariate analysis on the spectral data obtained, assessed for better molecular specifications and purity of particle. TEI method isolated exosomes with higher exosomal yield, purity, and recovery directly translatable into drug delivery and targeted therapeutics whereas ultracentrifuge had good recovery of particle morphology but showed particle aggregation and yielded exosomes with smaller mean size. PROSPR technique isolated a mixture of EVs, showed lower protein recovery in PAGE and western blotting but higher spectroscopic protein to lipid ratio and distinguishable EV population in multivariate analysis compared to exosomes isolated by TEI and UC. This comparative study should help in choosing a specific exosome isolation technique required for the objective of downstream applications.

Canine colostrum exosomes: characterization and influence on the canine mesenchymal stem cell secretory profile and fibroblast anti-oxidative capacity

BackgroundCanine colostrum milk (CCM) is a specific secretion of the mammary gland that is fundamental for the survival of the newborn. CCM has many described components (immunoglobulins, proteins or fat), but its small vesicles, named exosomes, are largely unknown.ResultsA characterization of CCM exosomes was performed. Exosomes were abundant in CCM and appeared with the characteristic cup-shaped morphology and well-defined round vesicles. The size distribution of exosomes was between 37 and 140 nm, and western blot analysis showed positive expression of specific exosomal markers. Proteomic analysis revealed a total of 826 proteins in exosome cargo. We also found that exosomes modified the proliferation and secretory profiles in canine mesenchymal stem cells derived from bone marrow (cBM-MSCs) and adipose tissue (cAd-MSCs). Additionally, CCM exosomes demonstrated a potent antioxidant effect on canine fibroblasts in culture.ConclusionsOur findings highlight, for the first time, the abundant presence of exosomes in CCM and their ability to interact with mesenchymal stem cells (MSCs). The addition of exosomes to two types of MSCs in culture resulted in specific secretory profiles with functions related to angiogenesis, migration and chemotaxis of immune cells. In particular, the cAd-MSCs secretory profile showed higher potential in adipose tissue development and neurogenesis, while cBM-MSC production was associated with immunity, cell mobilization and haematopoiesis. Finally, exosomes also presented antioxidant capacity on fibroblasts against reactive oxygen species activity within the cell, demonstrating their fundamental role in the development and maturation of dogs in the early stages of their life.

Exosome‐like Vesicles Facilitate Intercellular Communication between Uterine Artery Smooth Muscle Cells and Perivascular Adipose Tissue

IntroductionPerivascular adipose tissue (PVAT) contributes to regulation of vascular tone. We recently reported that PVAT surrounding uterine arteries had anti‐dilatory effects on isolated uterine arteries and contributed to local regulation of uterine blood flow in pregnant rats. The objective of the current study was to investigate the mechanisms underlying the functional interaction between PVAT and uterine arterial wall. Adipose tissue passively secretes exosomes, which are membrane bound vesicles (30 – 150 nm in diameter). It is unknown whether PVAT, which has a different developmental origin from other adipose depots, secretes exosomes, and if exosomes facilitate intercellular communication between PVAT and vascular cells. We hypothesized that uterine PVAT sheds exosome‐like vesicles (Exo‐PVAT) that transfer their cargo into neighboring uterine vascular smooth muscle cells (USMCs), facilitating maternal uterine artery function.MethodsExo‐PVAT and primary USMCs were isolated from pregnant (gestational day 16, term=22–23 days) and non‐pregnant rats. Exosomes were isolated and purified with tissue culture, and ultrafiltration and ultracentrifugation techniques, and USMCs were isolated using enzymatic digestion. Exosome morphology was characterized via transmission electron microscopy, and exosome size distribution and molecular weight were determined via a Malvern Zetasizer and fast protein liquid chromatography (FPLC), respectively. Protein expression of various exosome markers was determined using Western blot analysis. To determine USMC uptake of Exo‐PVAT, uterine Exo‐PVAT were labelled with a membrane‐labeling dye (DiO, Invitrogen) and then co‐cultured with USMCs for 3 hours. Vascular reactivity to acetylcholine (ACh; 10−9 – 3×10−5 M) was measured in uterine arteries using wire myography after exposure to Exo‐PVAT or phosphate‐buffered saline (PBS, control) for 3 hours.ResultsExo‐PVAT expressed TSG101, Alix, and CD9. Pregnancy did not affect Exo‐PVAT size [Median(IQR) (nm), Non‐pregnant: 99.4 (80.5) vs. Pregnant: 46.7 (21.2), p=0.5]. Using FPLC, we identified exosomes of 40–200 kDa in samples from both pregnant and non‐pregnant rats. Samples from pregnant rats had a relatively high expression of exosomes of 2000 kDa when compared to samples from non‐pregnant rats. Using immunocytochemistry, we demonstrated that USMCs were able to take up exosomes derived from their adjacent PVAT. Using a functional bioassay, we found that Exo‐PVAT reduced sensitivity to ACh in uterine arteries from pregnant rats (pEC50, control: 7.46 vs. Exo‐PVATPREG: 7.02) but had the opposite effect in non‐pregnant rats (pEC50, control: 6.91 ± 0.04 vs. Exo‐PVATNP: 7.03 ± 0.06, p&lt;0.01).ConclusionUterine PVAT sheds exosome‐like vesicles that transfer their cargo to the adjascent uterine arterial wall, where they are taken up by USMCs. The interaction between Exo‐PVAT and USMCs is a novel type of intercellular communication that may have important implications in uterine artery function and local regulation of uterine blood flow in pregnancy.Support or Funding InformationUNTHSC Seed Grant

Abstract 606: Stimulation of Cardiomyocyte a- and b-Adrenoceptors Uniquely Regulates Exosome Generation and miRNA Content

Background and Purpose: Influence of adrenoceptor (AR) signaling for the regulation of exosomes secreted from cardiomyocytes is unknown and since catecholamines are increased in heart failure (HF), there is interest in uncovering whether α- or β-ARs can induce specific changes in circulating blood exosomes in HF. In this study, we have evaluated whether α- or β-AR stimulation of neonatal rat ventricle myocytes (NRVMs) can alter the number, size or microRNA (miR) content of secreted exosomes. Methods and Results: The number and size distribution of exosomes from NRVMs were evaluated by Nanosight NS300. Phenylephrine (PE, 10 uM for 3 days), an α-AR agonist, increased exosome number significantly of NRVM compared than vehicle (PBS) treatment group (Veh: 7.82X10 8 ± 5.06X10 7 and PE: 10.09X10 8 ± 15.0 X10 7 / 1X10 6 NRVMs, n=3) but Isoproterenol (ISO, 1uM), a β-AR agonist, did not. Both agonists had no effect on exosome size distribution. After purifying total RNA from secreted exosomes, we evaluated the expression level of 37 candidate miR’s, which were selected from previous microarray data. ISO treatment decreased 14 miR’s (miR-222, -106a, -292a, -181b, -210, -489, -214, -1947, -195, -17, -7b, -93, -532 and -19a) and PE increased 5 miR’s (miR-20a, -210, -17-1, -Let7a, and 146b). Importantly, PE or ISO regulated any identical miR’s. As a control for treatment effects, we found that PE and ISO elevated myocyte mRNA levels of ANP and BNP to similar degrees. Conclusions: Both α- and β-AR agonists are can stimulate cardiomyocyte hypertrophy to similar degrees, however they have distinct regulatory patterns for secreted exosomes. This includes the number produced and more importantly, miR content. These miR’s and exosomes found in circulating blood may be able to distinguish and be biomarkers for different modes of maladaptive cardiac hypertrophy and HF.

Abstract 1341: Magnetic isolation and identification of exosomes using Fe/Au nanowires

Abstract We are developing segmented magnetic nanowires (MNWs) as a new platform for highly specific biolabels. We found that cancer cells internalize MNWs and package them into exosomes, which are then secreted for several days. These MNWs thus enable magnetic isolation of exosomes, which could prove useful in future diagnosis. Our immediate goals are to study how cells internalize MNWs, to determine the timing and reproducibility of exosome secretion, and to improve the magnetic isolation of exosomes. Following this work, we aim to use ferromagnetic resonance (FMR) to identify specific MNW biolabels, similar to radio frequency identification (RFID). In this study, we incubated osteosarcoma (OSCA-8) cells with Fe/Au segmented MNWs with and without PEG coating for 48 hours. Internalization of MNWs as a function of concentration (5-40 µg/ml) was followed by fluorescence and transmission electron microscopy (TEM). We obtained quantitative estimates of MNW internalization by magnetic measurements. Our experiments indicated better internalization of the MNWs using PEG as a capping agent. As we increased the concentration of MNWs, both the number of cells with MNWs and the number of MNWs per cell increased. TEM images show that after uptake by cancer cells, MNWs were predominantly located within lysosomes, and they appeared to be fragmented into small segments of similar size as exosomes. These segments were mostly composed of either Fe or Au, suggesting that fragmentation occurred at or near the boundary of segments. We used two different methods for exosome isolation: non-magnetic isolation (centrifuge and ExoQuick TC) as a control and magnetic isolation. We incubated OSCA-8 cells with different concentrations of MNWs (0 to 35 µg/ml), and exosomes were isolated for up to 11 days. We analyzed the exosome size distribution using a nanoparticle tracking analyzer (NanoSight) and found that exosomes isolated magnetically had similar size distributions as those isolated non-magnetically. A minimum concentration of 25 µg/ml MNWs in 3×105 cells was required for appreciable magnetic isolation. Increasing the concentration of MNWs progressively; however, isolated microvesicles that had wider and more heterogeneous size distributions. Next, we successfully identified two different types of MNW biolabels by FMR. MNWs were fixed in a polymer that was placed onto an RFID chip. Each type of MNW exhibited a unique RF signature as the sample was exposed to an external magnetic field. Our preliminary data show that MNWs appear to allow fast, inexpensive magnetic exosome isolation and is our future goal is MNW identification of exosomes. The methodology developed in this study should be transferable to develop comparable approaches to isolate and identify exosomes from virtually any type of cell. Citation Format: Zohreh Nemati, Thomas Gage, Mohammad Reza Zamani Kouhpanji, Joseph Um, Alicia Donnelly, Daniel Shore, Kelly Makielski, Yali Zhang, Rhonda Franklin, Jaime F. Modiano, Bethanie J. Stadler. Magnetic isolation and identification of exosomes using Fe/Au nanowires [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1341.