Exosome-free Media Research Articles

Feline coronavirus influences the biogenesis and composition of extracellular vesicles derived from CRFK cells.

Coronavirus (CoV) has become a public health crisis that causes numerous illnesses in humans and certain animals. Studies have identified the small, lipid-bound structures called extracellular vesicles (EVs) as the mechanism through which viruses can enter host cells, spread, and evade the host's immune defenses. EVs are able to package and carry numerous viral compounds, including proteins, genetic substances, lipids, and receptor proteins. We proposed that the coronavirus could alter EV production and content, as well as influence EV biogenesis and composition in host cells. In the current research, Crandell-Rees feline kidney (CRFK) cells were infected with feline coronavirus (FCoV) in an exosome-free media at a multiplicity of infection (MOI) of 2,500 infectious units (IFU) at 48 h and 72 h time points. Cell viability was analyzed and found to be significantly decreased by 9% (48 h) and 15% (72 h) due to FCoV infection. EVs were isolated by ultracentrifugation, and the surface morphology of isolated EVs was analyzed via Scanning Electron Microscope (SEM). NanoSight particle tracking analysis (NTA) confirmed that the mean particle sizes of control EVs were 131.9 nm and 126.6 nm, while FCoV infected-derived EVs were 143.4 nm and 120.9 nm at 48 and 72 h, respectively. Total DNA, RNA, and protein levels were determined in isolated EVs at both incubation time points; however, total protein was significantly increased at 48 h. Expression of specific protein markers such as TMPRSS2, ACE2, Alix, TSG101, CDs (29, 47, 63), TLRs (3, 6, 7), TNF-α, and others were altered in infection-derived EVs when compared to control-derived EVs after FCoV infection. Our findings suggested that FCoV infection could alter the EV production and composition in host cells, which affects the infection progression and disease evolution. One purpose of studying EVs in various animal coronaviruses that are in close contact with humans is to provide significant information about disease development, transmission, and adaptation. Hence, this study suggests that EVs could provide diagnostic and therapeutic applications in animal CoVs, and such understanding could provide information to prevent future coronavirus outbreaks.

Abstract 832: Exosome-based hybrid nanosystem for targeted TNBC therapy

Abstract Treatment of metastatic Triple-Negative Breast Cancer (TNBC) remains a challenge despite an increasing number of newer drugs being approved in recent years. The major challenge for improved treatment outcomes for metastatic cancer originates from the fact that the drugs are unable to reach the targeted site and hence are not able to elicit the desired response. Recently, natural mesenchymal stem cells (MSC) cells and their exosomes have been shown to have tumor-homing properties. By exploiting these homing properties, we propose the development of an exosome-polymeric hybrid nanosystem (EPHN) by using exosomes for their targeting ability to enhance the targetability and thus the therapeutic efficacy of the drug against TNBC. In this study, we bioengineered MSC exosome-coated drug-loaded polymeric nanoparticles to deliver our chemotherapeutic drug in a targeted fashion. To isolate the MSC exosomes, we grew the cells in exosome-free media and used the ultracentrifugation standard method. The MSC exosomes had a size of approximately 70 ± 5 nm and a PDI of 0.3. Our exosome sample was positive for exosomal proteins and negative for all other extracellular vesicles. The optimal doxorubicin-loaded PLGA nanoparticles were prepared using a microfluidics method resulting in a higher entrapment efficiency of 42 ± 3% and a smaller size of 70 ± 3 nm and a PDI of 0.2. To make the exosome-coated NPs, we used the extrusion method. After extrusion, the zeta potential of our EPHN was closer to -11mV compared to PLGA NPs (-50mV), which demonstrates that our NPs were coated with an exosomal membrane. We were able to make MSC exosome-coated drug-loaded polymeric nanoparticles and show that they were stable and uniform in size. Microfluidics-assisted doxorubicin-loaded PLGA nanoparticles show uniform size and higher entrapment efficiency. Release kinetics showed a strong burst phase release over the first 8 hours and subsequent sustained release up to 72 hours. In vivo biodistribution of EPHN shows better tumor accumulation compared to PLGA NP. The next stage in this project is in vivo tumor efficacy testing in breast cancer tumor models. This targeted NP system can serve as a novel therapeutic platform for developing improved treatments for metastatic TNBC. Citation Format: Rohan Joshi, Jana Lampe, Jamboor K. Vishwanatha, Amalendu P. Ranjan. Exosome-based hybrid nanosystem for targeted TNBC therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 832.

Canine Coronavirus Infection Modulates the Biogenesis and Composition of Cell-Derived Extracellular Vesicles.

Coronavirus (CoV) has persistently become a global health concern causing various diseases in a wide variety of hosts, including humans, birds, and companion animals. However, the virus-mediated responses in animal hosts have not been studied extensively due to pathogenesis complexity and disease developments. Extracellular vesicles (EVs) are widely explored in viral infections for their intercellular communication, nanocarrier, and immunomodulatory properties. We proposed that coronavirus hijacks the host exosomal pathway and modulates the EV biogenesis, composition, and protein trafficking in the host. In the present study, Crandell-Rees feline kidney (CRFK) cells were infected with canine coronavirus (CCoV) in an exosome-free medium at the multiplicity of infection (MOI) of 400 infectious units (IFU) at various time points. The cell viability was significantly decreased over time, as determined by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Post-infection EVs were isolated, and transmission electron microscopy (TEM) showed the presence of small EVs (sEVs) after infection. NanoSight particle tracking analysis (NTA) revealed that EV sizes averaged between 100 and 200 nm at both incubation times; however, the mean size of infection-derived EVs was significantly decreased at 48 h when compared to uninfected control EVs. Quantitative analysis of protein levels performed by dot blot scanning showed that the expression levels of ACE-2, annexin-V, flotillin-1, TLR-7, LAMP, TNF-α, caspase-1, caspase-8, and others were altered in EVs after infection. Our findings suggested that coronavirus infection impacts cell viability, modulates EV biogenesis, and alters cargo composition and protein trafficking in the host, which could impact viral progression and disease development. Future experiments with different animal CoVs will provide a detailed understanding of host EV biology in infection pathogenesis and progression. Hence, EVs could offer a diagnostic and therapeutic tool to study virus-mediated host responses that could be extended to study the interspecies jump of animal CoVs to cause infection in humans.

Intravenous Infusion of Exosomes Derived from Human Adipose Tissue-Derived Stem Cells Promotes Angiogenesis and Muscle Regeneration: An Observational Study in a Murine Acute Limb Ischemia Model.

Recent studies have demonstrated that adipose tissue-derived stem cell (ADSC) transplantation could promote neoangiogenesis in various ischemic diseases. However, as whole cells, ADSCs have some disadvantages, such as shipping and storage issues, high costs, and controversies related to the fates of grafted cells in the recipients. Therefore, this study aimed to investigate the effects of intravenously infused exosomes purified from human ADSCs on ischemic disease in a murine hindlimb ischemia model. ADSCs were cultured in exosome-free medium for 48h before the conditioned medium was collected for exosome isolation by ultracentrifugation. The murine ischemic hindlimb models were created by cutting and burning the hindlimb arteries. Exosomes were intravenously infused into murine models (ADSC-Exo group), with phosphate-buffered saline (PBS) used as a placebo (PBS group). Treatment efficacy was determined using a murine mobility assay (frequency of pedaling in water per 10s), peripheral blood oxygen saturation (SpO2 index), and the recovery of vascular circulation by trypan blue staining. The formation of blood vessels was shown by X-ray. Expression levels of genes related to angiogenesis and muscle tissue repair were quantified by quantitative reverse-transcription polymerase chain reaction. Finally, H&E staining was used to determine the histological structure of muscle in the treatment and placebo groups. The rates of acute limb ischemia in the PBS and ADSC-Exo injection groups were 66% (9/16 mice) and 43% (6/14 mice), respectively. The mobility of the limbs 28days after surgery was significantly different between the ADSC-Exo treatment group (41±1 times/10s) and the PBS group (24±1 times/10s; n=3; p<0.05). Peripheral blood oxygen saturation 21days after treatment was 83.83%±2.02% in the PBS group and 83%±1.73% in the ADSC-Exo treatment group, and the difference was not statistically significant (n=3, p>0.05). On day 7 after treatment, the time required to stain the toes after trypan blue injection was 20.67±12.5s and 85±7.09s in the ADSC-Exo and PBS groups, respectively (n=3, p<0.05). On day 3 after the operation, the expression of genes promoting angiogenesis and muscle remodeling, such as Flk1, Vwf, Ang1, Tgfb1, Myod, and Myf5, was increased 4-8 times in the ADSC-Exo group compared with the PBS group. No mice in either group died during the experimental period. These results revealed that intravenous infusion of human ADSC-derived exosomes is a safe and effective method to treat ischemic disease, especially hindlimb ischemia, by promoting angiogenesis and muscle regeneration.

Critical Role of Hodgkin Lymphoma Cells-Derived Large and Small Extracellular Vesicles in the Regulation of the Immune-Inflammatory Microenvironment

Introduction: Classical Hodgkin Lymphoma (cHL) is characterized by a low number of CD30+ malignant cells and an extensive immune-inflammatory microenvironment. However, this unique histopathological picture and its pathogenesis are still partially understood. Extracellular vesicles (EVs) are submicron particles delimited by a lipid bilayer that promote (tumor) intercellular communication and carry bioactive molecules that can influence the immune system. Based on size and biogenesis mechanism, Small (S-) and Large (L-) EVs (30-200 nm and 0.2-10 µm, respectively) have been identified. Extracellular Adenosine Triphosphate (eATP), a key component of the tumor microenvironment, plays a role in biogenesis and dynamics of EVs. Here we aim to elucidate the role of cHL cell-derived EVs in the education of the immune microenvironment. Methods: cHL L-428 cell line was cultured in exosome-free medium for 48 hours at 37°C in the presence or absence of eATP (1 mM). L-428 cell blebbing has been detected by fluorescence microscopy (DAPI and FITC-Cholera Toxin B subunit (CTxB) labeling). L-EVs and S-EVs were isolated from the cell culture supernatant by differential ultracentrifugation (2.800g for 2.8K and 20.000g for 20K L-EVs, and 100.000g for 100K S-EVs) and purified by iodixanol density gradient. L- and S-EVs were then characterized by Tunable Resistive Pulse Sensing (TRPS), single EV-flow cytometry, and MACSPlex (bead-based antigen binding array). Functionally, normal peripheral blood mononuclear cells were in vitro cultured with titrated amount of isolated L- and S-EVs (from 1:5 to 1:20) for 24 hours. The intracytoplasmic interleukin-1β and interferon-γ production by CD14+ monocytes and CD3+/CD4+/CD8+ T cells was assessed by flow cytometry. Results: Our results showed that human cell line L-428 is able to produce L-EVs through membrane blebbing (Figure 1A). According to EV size distributions, both 2.8K and 20K L-EVs displayed a size range from 1 to 4 µm whereas S-EVs were enriched in particles of 150-200 nm diameter. Interestingly, the amounts of S-EVs shed by L-428 cells were significantly higher compared to the number of L-EVs. Considering the EV surface protein phenotype, S-EVs express all the EV-related tetraspanin proteins (CD9, CD63, and CD81). By contrast, L-EV express CD63 and CD81 only. Additionally, in L-EVs we observed an enrichment of cancer-related markers including SSEA-4, CD44, CD24, CD29 and CD133-1 in comparison with the S-EV counterpart. Of note, the expression of selected immune-related molecules such as CD86 and CD40 was higher in the 2.8K and 20K L-EVs as compared with that of the 100K S-EVs. The HLA-II molecules were expressed by all fractions, with the highest expression in 2.8K L-EVs. Along with PD-L1, the CD30 HL-associated marker was expressed by all EV fractions at the same extent. When we tested the effects of eATP on EVs produced by L-428 cells, we found a highly decline in the expression of EV-surface proteins from all EV sub-fractions, being most noticeable in L-EVs. Importantly, only PD-L1 expression was increased in the 20K L-EVs after eATP treatment. Functionally, at variance with S-EVs (Figure 1B), L-EVs highly stimulate the intracytoplasmic production of interleukin-1β by monocytes. Conversely, L-EVs derived from e-ATP treated L-428 cells fail to stimulate the intracytoplasmic production of interleukin-1β by CD14+ monocytes, which instead was promoted by the e-ATP treated S-EV counterparts. Of note, neither L-EVs nor S-EVs stimulated interferon-γ production by CD14+ monocytes. No intracytoplasmic production of the selected inflammatory cytokines has been observed when T-cells treated with EVs were analyzed. Conclusions: These results demonstrate that L-EVs from HL cells display a cancer- and immune-related phenotype and play a role in the modulation of the immune microenvironment by promoting the production of inflammatory cytokines by monocytes. This might be partially due to the high expression of CD44 on the surface of L-EVs, since it has been previously demonstrated that cancer-derived soluble CD44 may trigger monocyte/macrophage interleukin-1β production. Interestingly, eATP plays a role in shaping the immune modulatory ability of L- and S-EVs. These findings shed light on the role of HL-derived EVs in the establishment of the immune-inflammatory microenvironment and provide novel insight into clinically relevant EV driven mechanisms in HL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Exosomes from human adipose-derived stem cells promoted the expression of angiogenic factors in endothelial cells

Introduction: In regenerative medicine, human adipose tissue–derived mesenchymal stem cells (hadMSCs) have exhibited tremendous promise as an efficient approach to chronic cardiovascular diseases. Noble effects of mesenchymal stem cells (MSCs) have been acknowledged to contribute to not only cellular engraftment and response to the site of injury but also paracrine action via the release of extracellular vesicles, notably exosomes. This study aimed to investigate the effects of exosomes from hadMSCs (hadMSC-Exo) on the angiogenesis of endothelial cells. Methods: Firstly, hadMSCs were characterized and cultured at a density of 500,000 cells in exosome-free medium for 48 hours. The conditioned medium was ultracentrifuged for exosome isolation. Obtained exosomes were characterized with the expression of CD9, CD63, and CD81 markers by flow cytometry and imaged with a TEM microscope. Exosomes were added to a culture medium of human umbilical vein endothelial cells (HUVECs) to understand the effects of exosomes on HUVECs. The expression of some angiogenic genes in HUVECs was identified by RT-qPCR. Results: The concentration of total protein in isolated exosomes by Bradford assay was 0.51 ± 0.04 mg/mL. Exosomes were successfully isolated and characterized by their morphology and immunophenotype. The results showed that exosomes significantly increased the mRNA expression of TGF (11.56 ± 2.03, P &lt; 0.05), Flk-1 (4.04 ± 0.01, P &lt; 0.05), VE-cadherin (7.25 ± 2.30, P &lt; 0.05), and CD31 (3.02 ± 1.13, P &lt; 0.05). Conclusion: This study suggested that exosomes promoted the angiogenesis of endothelial cells in vitro.

Depletion of serum-derived exosomes aggravates heat stress-induced damage of bovine mammary epithelial cells.

Exosomes are involved in intercellular communication, affecting many physiological and pathological process. The present study evaluated the effects of serum exosomes on the function of bovine mammary epithelial cells (BMECs) and milk synthesis under heat stress. We cultured the BMECs in fetal bovine serum (FBS) or exosome-free FBS medium and examined, their viability using CCK-8 kit. The results showed that culturing the cells in an exosome-free medium decreased viability and increased the levels of reactive oxygen species. The BMECs cultured in the exosome-free medium had reduced mitochondrial membrane potential, decreased manganese superoxide dismutase activity, and disrupted mitochondrial dynamics. They exhibited apoptosis due to upregulated Drp1, Fis1, Bax and HSP70. Lastly, we observed downregulation of milk fat and lactoprotein-related genes: mTOR, PPARγ, p-mTOR and ADD1 and SREBP1, ELF5, and CSN2, respectively, after culturing the cells in an exosome-free medium. These negative effects of the exosome-free medium on the BMECs could be further reinforced under heat stress. Our results demonstrated that exosomes from serum are critical for maintaining the normal function of BMECs.

Poster 155: Muscle-Derived Beige Adipose Precursors Secrete Promyogenic Exosomes that Treat Rotator Cuff Muscle Degeneration in Mice and are Identified in Humans by Single-Cell RNA Sequencing

Objectives:Muscle atrophy, fibrosis, and fatty infiltration are common to a variety of sports-related and degenerative conditions and are thought to be irreversible. Fibroadipogenic progenitors (FAPs) are multipotent resident muscle stem cells with the capacity to differentiate into fibrogenic as well as white- and beige adipose tissue (WAT; BAT). FAPs that have assumed a BAT differentiation state (FAP-BAT) have proven efficacious in treating muscle degeneration in a variety of injury models. The purpose of this study was to characterize the subpopulation of murine FAPs with FAP-BAT activity, determine whether their pro-myogenic effect is mediated via exosomes, and analyze human FAPs for an analogous pro-myogenic, exosome-rich subpopulation. Given their non-myogenic differentiation capacity, it was hypothesized that FAP-BAT exerts a pro-myogenic effect on the injured muscle environment through the secretion of exosomes.Methods:FAPs from UCP1-reporter mice were isolated via FACS and sorted according to the differential intensity of the UCP1 signal observed: negative for UCP1 (UCP1-), intermediate intensity (UCP1+), and high intensity (UCP1++). Bulk RNAseq was performed on UCP1-, UCP1+, and UCP1++ FAPs to evaluate distinct characteristics of each population. Exosomes were harvested from UCP1++ (Exo-FB) and UCP1- (Exo-nFB) cells using cushioned-density gradient ultracentrifugation and used to treat C2C12 cells and MEFs in vitro, and the myotube fusion index was assessed. Exo-FB and Exo-nFB were then used to treat WT C57B/L6J mice that had undergone massive rotator cuff tear. At 6 weeks mice were sacrificed and supraspinatus muscles were harvested and analyzed for muscle atrophy, fibrosis, fatty infiltration, and UCP1 expression. Single-cell RNA sequencing (scRNAseq) was then performed on FAPs isolated from human rotator cuff muscle that were treated with mirabegron or standard media to assess for the presence of a parallel promyogenic subpopulation of FAP-BAT cells in humans.Results:Flow cytometry analysis of sorted UCP1-reporter mouse FAPs revealed a trimodal distribution of UCP1 signal intensity, which correlated with three distinct transcriptomic profiles characterized with bulk RNAseq. UCP1++ cells were marked by high mitochondrial gene expression, BAT markers, and exosome surface makers (Table 1). UCP1- cells were marked by fibrogenic markers and UCP1+ cells were characterized differential enrichment of WAT markers (Table 1). Exo-FB treatment of C2C12 cells resulted in robust myotube fusion, with a myotube fusion index of 33.3 ± 6.3% compared to 7.1 ± 3.2% with Exo-nFB (p <0.001) and 8.8 ± 3.3% with exosome-free media (p = 0.0014), while treatment of MEFs resulted in transdifferentiation into myotubes (Fig. 1). Mice that were treated with Exo-FB at the time of rotator cuff injury demonstrated markedly reduced muscle atrophy and FI compared to treatment with Exo-nFB or PBS (Fig. 2). ScRNAseq of human FAPs from the rotator cuff revealed 6 distinct subpopulations of human FAPs, with differential expression of mitochondrial genes, certain BAT markers, and exosome surface markers noted within one of the subpopulations (Fig. 3).Conclusions:FAP-BAT cells comprise a subpopulation of FAPs with upregulated mitochondrial gene expression and exosome production that mediates promyogenic effects in vitro and in vivo, and are present as a transcriptomically similar subpopulation of FAPs in humans.Table 1.Highly Differentially Expressed Genes of UCP1++ FAPsFigure 1.C2C12 cells and MEFs cultured for 14 days with Exo-FB(2 x 109), Exo-nFB(2 x 109), or exosome-free media, and stained for myosin heavy chain (MF20)j scale bar = 50µm (A), Myotube fusion index was calculated as the percentage of nuclei per field contained within an MF20+ myotube. n-3 replicates per condition; ** indicates p<0.005, *** indicates p<0.001.Figure 3.Light microscoay and immunofluorescence of human FAPs grown in standard media (top row) or mirabegron treated media (bottom row); scale bar =50µm; red arrows indicates predominant perilipin expression, green arrow indicates predominant UCP1 expression, yellow arrow indicates expression of both UCP1 and perilipin (A) UMAP plot of human scRNAseq data demonstrating 5 distinct clusters of human FAPs identified with Seurat (B) UMAP plot demonstrating distribution of human FAPs by in vitro treatment conditions: standard media or mirabegron (c). Dot plot of top 20 differentially expressed genes by treatment condition based upon adjusted p-value; “Average Expression” denotes z-score from mean overaal gene expression (D). Dot plot demonstrating unifrom increase in mitochondrial gene expression with mirabegron (E). Dot plot showing 13 genes that are significantly differentially expressed in both murine UCP1++ FAPs and human FAP subpopulations (F). Dot plot demonstrating additional characterstic genes of interest stratified by cluster. Red arrows indicates populations favorted to contain FAP-BAT cells at varying differentiations stages; green arrow highlights Adrb2 receptor expression; blue arrow highlights exosome surface markers CD63 and CD81(G).

Human Adenovirus Serotype 3 Infection Modulates the Biogenesis and Composition of Lung Cell-Derived Extracellular Vesicles.

Adenovirus (Ad) is a major causal agent of acute respiratory infections. However, they are a powerful delivery system for gene therapy and vaccines. Some Ad serotypes antagonize the immune system leading to meningitis, conjunctivitis, gastroenteritis, and/or acute hemorrhagic cystitis. Studies have shown that the release of small, membrane-derived extracellular vesicles (EVs) may offer a mechanism by which viruses can enter cells via receptor-independent entry and how they influence disease pathogenesis and/or host protection considering their existence in almost all bodily fluids. We proposed that Ad3 could alter EV biogenesis, composition, and trafficking and may stimulate various immune responses in vitro. In the present study, we evaluated the impact of in vitro infection with Ad3 vector on EV biogenesis and composition in the human adenocarcinoma lung epithelial cell line A549. Cells were infected in an exosome-free media at different multiplicity of infections (MOIs) and time points. The cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and fluorometric calcein-AM. EVs were isolated via ultracentrifugation. Isolated EV proteins were quantified and evaluated via nanoparticle tracking, transmission electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting assays. The cell viability significantly decreased with an increase in MOI and incubation time. A significant increase in particle mean sizes, concentrations, and total EV protein content was detected at higher MOIs when compared to uninfected cells (control group). A549 cell-derived EVs revealed the presence of TSG101, tetraspanins CD9 and CD63, and heat shock proteins 70 and 100 with significantly elevated levels of Rab5, 7, and 35 at higher MOIs (300, 750, and 1500) when compared to the controls. Our findings suggested Ad3 could modulate EV biogenesis, composition, and trafficking which could impact infection pathogenesis and disease progression. This study might suggest EVs could be diagnostic and therapeutic advancement to Ad infections and other related viral infections. However, further investigation is warranted to explore the underlying mechanism(s).

Pancreatic cancer-derived exosomal microRNA-19a induces β-cell dysfunction by targeting ADCY1 and EPAC2.

New-onset diabetes mellitus has a rough correlation with pancreatic cancer (PaC), but the underlying mechanism remains unclear. This study aimed to explore the exosomal microRNAs and their potential role in PaC-induced β-cell dysfunction. The pancreatic β cells were treated with isolated exosomes from PaC cell lines, SW1990 and BxPC-3, before measuring the glucose-stimulated insulin secretion (GSIS), validating that SW1990 and BxPC-3 might disrupt GSIS of both β cell line MIN6 and primary mouse pancreatic islets. The difference in expression profiles between exosomes and exosome-free medium of PaC cell lines was further defined, revealing that miR-19a secreted by PaC cells might be an important signaling molecule in this process. Furthermore, adenylyl cyclase 1 (Adcy1) and exchange protein directly activated by cAMP 2 (Epac2) were verified as the direct targets of exogenous miR-19a, which was involved in insulin secretion. These results indicated that exosomes might be an important mediator in the pathogenesis of PaC-DM, and miR-19a might be the effector molecule. The findings shed light on the pathogenesis of PaC-DM.

Overexpression Of PGC-1α In Human Primary Myotubes Increases Regulators Of Exosome Biogenesis And Secretion

Skeletal muscle functions as an endocrine organ. Exosomes, small vesicles containing mRNAs, miRNAs, and proteins, are secreted from muscle cells and facilitate cell-to-cell communication. Our recent work found greater exosome release from oxidative compared to glycolytic muscle. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key driver of mitochondrial biogenesis, a characteristic of oxidative muscle. It was hypothesized that PGC-1α regulates exosome biogenesis and secretion in skeletal muscle. PURPOSE: To determine if PGC-1α regulates skeletal muscle exosome biogenesis and secretion. METHODS: On day 4 of differentiation, primary myotubes from vastus lateralis biopsies from lean donors (BMI < 25.0 kg/m2) were exposed to adenovirus encoding human PGC-1α or GFP control. On day 6 of differentiation, culture media was replaced with exosome-free media. On day 8, cells were collected for mRNA and protein analysis. RESULTS: Overexpression of PGC-1α increases regulators of exosome biogenesis in the endosomal sorting complexes required for transport (ESCRT) pathway: Alix (GFP: 2.9 ± 1.0 vs. PGC-1α: 7.6 ± 1.4), TSG-101 (GFP: 1.8 ± 0.1 vs. PGC-1α: 7.3 ± 2.1), CD63 (GFP: 2.6 ± 0.3 vs. PGC-1α: 3.7 ± 0.4), Clathrin (GFP: 3.5 ± 0.1 vs. PGC-1α: 11.6 ± 2.5), and the secretion pathway: Rab27b (GFP: 2.6 ± 0.7; PGC-1α: 3.2 ± 0.3). CONCLUSION: Overexpression of PGC-1α increases gene expression of regulators of exosome biogenesis and secretion in human primary myotubes.

Effects of Pseudomonas aeruginosa on Microglial-Derived Extracellular Vesicle Biogenesis and Composition.

The packaging of molecular constituents inside extracellular vesicles (EVs) allows them to participate in intercellular communication and the transfer of biological molecules, however the role of EVs during bacterial infection is poorly understood. The goal of this study was to examine the effects of Pseudomonas aeruginosa (P. aeruginosa) infection on the biogenesis and composition of EVs derived from the mouse microglia cell line, BV-2. BV-2 cells were cultured in exosome-free media and infected with 0, 1.3 × 104, or 2.6 × 104 colony forming units per milliliter P. aeruginosa for 72 h. The results indicated that compared with the control group, BV-2 cell viability significantly decreased after P. aeruginosa infection and BV-2-derived EVs concentration decreased significantly in the P. aeruginosa-infected group. P. aeruginosa infection significantly decreased chemokine ligand 4 messenger RNA in BV-2-derived infected EVs, compared with the control group (p ≤ 0.05). This study also revealed that heat shock protein 70 (p ≤ 0.05) and heat shock protein 90β (p ≤ 0.001) levels of expression within EVs increased after P. aeruginosa infection. EV treatment with EVs derived from P. aeruginosa infection reduced cell viability of BV-2 cells. P. aeruginosa infection alters the expression of specific proteins and mRNA in EVs. Our study suggests that P. aeruginosa infection modulates EV biogenesis and composition, which may influence bacterial pathogenesis and infection.

Exosome Production, Isolation and Characterization from A549 Epithelial Carcinoma Cells

Exosomes are natural nanoparticles that special features have been started to be used for recent years in the treatment of cancer as a natural, homogeneous, nanosized, targeted vesicles (~ 50- 100 nm). They have high avidity (many conformational attachment) to attach onto targeted cancer cell surfaces. They are composed of bioactive double-layered lipid layers in which their original nature has the adhesive proteins interacting with the cancer cell membrane easily. In this study, the exosomes of non-small cell lung cancer A549 epithelial carcinoma cells were investigated for their potential to be the natural or synthetic drug carrier. Firstly, exosomes of A549 cell line were produced in exosome-free media. Immediately after, isolation of their exosomes were realized by using ultracentrifugation procedure. SEM image, particle size, zeta potential, exosomal RNA analysis and Protein Content by Bradford assays were performed. Findings (Size: 168 nm; zeta: -16mV) on the properties of A549 cell exosomes proved their potential to be used as the drug carrier for cancer cell therapy.

CLL-Derived Exosomes Function As Trojan Horses That Induce SMAD6-Dependent Apoptosis of Normal B-Cells

Studies from recent years unraveled the role of monocytes and T-cells in the pathogenesis of chronic lymphocytic leukemia (CLL). The role of other immune cells in the pathobiology of CLL is less known. Specifically, whether B-cells, the normal counterpart of CLL cells play a role in CLL is unknown. Nevertheless, since both CLL cells and wild type B-cells reside in lymphatic organs and travel in blood, they either share or compete over common environmental resources. According to the cell competition theory, a sensing mechanism measures the relative fitness of a cell and ensures the elimination of cells deemed to be less fit then their neighbors. Since constitutive activation of intracellular pathways protect CLL cells from apoptosis, the cell competition theory predicts that compared with normal B-cells these cells are sensed as "super fit" and B-cells, the less fit counterparts, are eliminated. Yet, what delivers this massage across a population of cells is unknown. Exosomes are nanosized particles that are secreted by various types of cells. Exosomes carry a cargo of proteins and different types of RNA. They travel in body fluids and are taken up by cells in their vicinity. Since cancer cells including CLL cells secrete exosomes, we have formulated our hypothesis, namely, that exosomes derived from CLL cells are the vehicles that carry a death massage to wild type B-cells. To test this hypothesis, we isolated CLL cells from 3 previously untreated patients with CLL. We then grew these cells in exosome free media for 72 hours and harvested the exosomes by ultracentrifugation. We used NanoSight tracking analysis, Western immunoblotting for CD63, a common exosomal marker, and electron microscopy imaging studies to ensure that our pellet include the typical 100nm exosomal particles. Subsequently, we subjected normal B-cells derived from healthy volunteers to CLL derived exosomes stained by FM-143 dye. Using flow cytometry we found that exosomes are taken up by normal B-cells in a dose- and time- dependent manner. Double staining of the recipient B-cells to Annexin/PI revealed that exosomes induce apoptosis of these cells in a dose- and time- dependent manner. We then used RNA-seq to trace the changes in the molecular makeup of B-cells after exosomal uptake?? they took up exosomes. We found 24 transcripts that were differentially expressed (11 that were upregulated and 13 that were downregulated). We then verified the array results by quantitative real-time PCR for four of these genes. Among the top transcripts that were upregulated in exosome-positive B-cells is SMAD6. Because the upregulation of the SMAD family members including SMAD6 is associated with the induction of apoptosis in various malignant and non-malignant cells we wondered whether the upregulation of SMAD6 also induces apoptosis in normal B-cells. To test this, we transfected normal B-cells with SMAD6 containing vector and verified by RT-PCR that level of SMAD6 transcript were upregulated and by Western immunoblotting that levels of SMAD6 protein are upregulated as well. As expected, the rate of apoptosis was higher, and the rates of viable cells and proliferating cells were significantly lower in SMAD6-transfected B-cells. Taken together, we show here that CLL cells secrete exosomes that function as "Trojan horses". Once they are taken up by normal B-cells they induce SMAD6-dependent apoptosis. In this way the neoplastic cells may actively eliminate their competitors and take over the common environmental resources. Disclosures No relevant conflicts of interest to declare.

1444P - Pulmonary tumour-draining vein exosomal lincRNA-p21 levels impacts non-small cell lung cancer prognosis

BackgroundIn resected non-small cell lung cancer (NSCLC) considerable recurrence rates has been reported (30%). Tumor secretes small extracellular vesicles known as exosomes, which plays a role in the metastasis process. Pulmonary tumor-draining vein blood has proved to be better source than peripheral vein to analyze the prognosis impact of exosomes (Navarro A, et al. Cancers 2019). LincRNA-p21 is a long non-coding RNA which upregulation in tumor tissue induced by hypoxia was related to poor prognosis in NSCLC (Castellano JJ. et al. JTO 2016). We aimed to evaluate the presence of lincRNA-p21 in exosomes and its potential as prognosis biomarker in resected NSCLC patients. MethodsH23 and H1299 NSCLC cell lines were cultured in exosome-free medium 48h under normoxic/hypoxic conditions. LincRNA-p21 was inhibited using a siRNA and quantified using a TaqMan assay. Exosomes were purified by ultracentrifugation and characterized by electron microscopy, western blot and Nanosight. LincRNA-p21 was analyzed in exosomes from pulmonary tumor-draining vein, obtained during surgery, from 54 NSCLC patients. ResultsLincRNA-p21 was present in exosomes showing 18 times more representation than in their origin cells. Under hypoxic conditions exosomal lincRNA-p21 was upregulated in both cell lines (p<0.05). Cellular lincRNA-p21 silencing resulted in a reduction in exosomal lincRNA-p21 (p=0.04) indicating a relation between cellular and exosomal levels. The analysis in patient samples showed that lincRNA-p21 was present in patient exosomes obtained from tumor-draining vein. The survival analysis showed that high exosomal lincRNA-p21 levels were associated with shorter time to relapse (TTR) (37 vs 56 months; p=0.03) and shorter overall survival (OS) (46 vs 66 months; p=0.04). In the multivariate analysis exosomal lincRNA-p21 emerged as an independent prognostic marker for TTR (HR: 3.1; 95%CI: 1.02-9.4; p=0.04) and OS (HR: 5.3; 95%CI: 1.6-17.2; p=0.006) together with disease stage. ConclusionsTumor exosomes carry LincRNA-p21, which increases under hypoxia, and its analysis in tumor-draining vein arises as a promising prognosis marker. Legal entity responsible for the studyThe authors. FundingSAF2017-88606-P (AEI/FEDER, UE). DisclosureAll authors have declared no conflicts of interest.

Alcohol Exposure Impacts the Composition of HeLa-Derived Extracellular Vesicles.

Extracellular vesicles are nanosized vesicles that are under intense investigation for their role in intercellular communication. Extracellular vesicles have begun to be examined for their role in disease protection and their role as disease biomarkers and/or vaccine agents. The goal of this study was to investigate the effects of alcohol exposure on the biogenesis and composition of extracellular vesicles derived from the cervical cancer line, HeLa. The HeLa cells were cultured in exosome-free media and were either mock-treated (control) or treated with 50 mM or 100 mM of alcohol for 24 h and 48 h. Our results demonstrated that alcohol significantly impacts HeLa cell viability and exosome biogenesis/composition. Importantly, our studies demonstrate the critical role of alcohol on HeLa cells, as well as HeLa-derived extracellular vesicle biogenesis and composition. Specifically, these results indicate that alcohol alters extracellular vesicles’ packaging of heat shock proteins and apoptotic proteins. Extracellular vesicles serve as communicators for HeLa cells, as well as biomarkers for the initiation and progression of disease.

Alcohol Modulates the Biogenesis and Composition of Microglia-Derived Exosomes.

Exosomes are small extracellular vesicles that have emerged as an important tool for intercellular communication. In the central nervous system, exosomes can mediate glia and neuronal communication. Once released from the donor cell, exosomes can act as discrete vesicles and travel to distant and proximal recipient cells to alter cellular function. Microglia cells secrete exosomes due to stress stimuli of alcohol abuse. The goal of this study was to investigate the effects of alcohol exposure on the biogenesis and composition of exosomes derived from microglia cell line BV-2. The BV-2 cells were cultured in exosome-free media and were either mock treated (control) or treated with 50 mM or 100 mM of alcohol for 48 and 72 h. Our results demonstrated that alcohol significantly impacted BV-2 cell morphology, viability, and protein content. Most importantly, our studies revealed that exosome biogenesis and composition was affected by alcohol treatment.

Circulating exosomal and Argonaute-bound microRNAs in preeclampsia

IntroductionmicroRNAs (miRNAs) play important role in the regulation of placental development, and abnormal miRNA expression is associated with preeclampsia (PE). miRNAs are released from trophoblast cells to maternal blood flow, where they are highly stable, being encapsulated inside extracellular vesicles, like exosomes or bound to Argonaute proteins. In PE, placental dysfunction leads to aberrant extracellular miRNA secretion. hsa-miR-210 is a hypoxia-sensitive miRNA found to be upregulated in PE; however, it is unknown whether it is the cause or the consequence of the disease. ObjectiveOur aim was to analyze the expression of several miRNAs, including hsa-miR-210 in placenta, exosome and Ago-bound fractions comparing normal (N) and PE pregnancies. We performed in vitro analyses of extracellular hsa-miR-210 secretion of trophoblast cell cultures (of villous and extravillous origin) under hypoxic condition. MethodsPE and N placenta samples were collected from C-sections, and blood samples were drawn from each pregnant woman in the third trimester. HTR-8 and JAR cell lines were cultured in exosome-free media and treated with hypoxia-mimetic agents. Exosome and Ago-bound fractions were isolated by membrane affinity spin column method from plasma and cell media. Short RNAs were extracted from exosomes and vesicle-free fractions, and total-RNA was isolated from the placenta samples. The RNA purity and concentration were measured by spectrophotometry. Expression analysis was carried out by qPCR with specific primers to target and reference miRNAs. ResultsThe level of hsa-miR-210 was significantly higher in PE placentas, which could cause a minor increase of exosomal and a high elevation of Ago-bound miR-210 in circulation. Hypoxia lead to intracellular hsa-miR-210 upregulation in trophoblast cell lines. In extravillous cell (HTR-8) media, only the level of exosomal hsa-miR-210 was increased but no change in Ago-bound hsa-miR-210 level was observed. In contrast, in villous cell (JAR) media, the level of exosomal hsa-miR-210 was increased and enhanced release of Ago-bound hsa-miR-210 was also observed. ConclusionBased on our data, we postulate that in PE, exosomal hsa-miR-210 is secreted actively from the trophoblast, and by intercellular communication, it may have a role in disease etiology. In addition, there is a passive release of Ago-bound hsa-miR-210 into the circulation, which may represent by-products of cell-death and is thereby a possible consequence of the disease.

Fetoplacental endothelial exosomes modulate high d-glucose-induced endothelial dysfunction

INTRODUCTIONGestational diabetes mellitus (GDM) is associated with fetoplacental endothelial dysfunction, which may be induced by hyperglycemia. We hypothesized that endothelial exosomes, which are extracellular nanovesicles affecting endothelial function, play a role in the high glucose (HG)-induced endothelial dysfunction. METHODSExosomes were isolated from HUVECs incubated with basal glucose (5.5 mmol/L; HUVEC- BG; exo-BG) and from HUVECs incubated with HG for 24 h (25 mmol/L; HUVEC-HG; exo-HG) in exosome-free medium. Exosomes were isolated and characterized by ultracentrifugation, sucrose gradient, electron microscopy, nanotracking analysis and Western blotting. HUVEC-BG and HUVEC-HG were exposed to exo-BG and exo-HG in two different concentrations: 5 μg and 1 μg exosome protein/mL. The exosomal effect on endothelial cell function was determined by wound healing assay, expression of endothelial nitric oxide synthase (eNOS), human cationic amino acid transporter type 1 (hCAT-1), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule type 1 (ICAM-1) by Western blotting, qPCR or flow cytometry. RESULTSHG increased the exosomal release from HUVECs, endothelial wound healing and expression of phosphorylated (P∼Ser1177)-eNOS, hCAT-1, VEGF and ICAM-1. Exo-HG also increased endothelial cell wound healing, P∼Ser1177-eNOS, hCAT-1 and ICAM-1 expression in HUVEC-BG. Exo-BG reverted the effect of HG on endothelial cell wound healing and hCAT-1 mRNA expression to normal values. DISCUSSIONOur results show that HG may induce endothelial dysfunction in HUVECs and that exosomes from HUVEC-HG mimicked some of the effects of HG. This study contributes to the unraveling of the mechanism by which hyperglycemia affects the fetoplacental vasculature in GDM.

Toll‐like Receptor‐4 Activation Improves the Immunosuppressive Properties of Cancer Cells‐Derived Exosomes

Tumor cells actively produce and release exosomes to promote tumor growth and progression. Upon the contact with target cells, exosomes alter phenotypic and functional features of recipients, reprogramming them into active contributors to metastasis and immunosuppression. Overexpression of Toll‐like receptor‐4 has been implicated in the development of several types of cancer, since its activation increase their tumorigenic potential of cancer cells.The purpose of this study is to investigate the immunosuppressive properties of cancer cell‐derived exosomes released upon TLR4 activation by LPS.Cancer cell lines (primary colon SW480, metastatic colon SW620, metastatic breast MDA‐MG‐231 and glioma U87) were treated with LPS (1μg/ml) for 24h and then cultured in exosome‐free medium for 48h. Exosomes were isolated from cells supernatants by polymer precipitation method (Exoquick), quantified by determining the activity of Acetyl‐CoA Esterase (Exocet test) and validated by exosome‐specific tetraspanins expression by western blot and flow cytometry analysis. The expression of immunosuppressive factors on exosomes surface was evaluated by flow cytometry and Multiplex ELISA. Exosomes immuneregulatory properties were assessed by in vitro functional assays on immune cell subpopulations isolated from healthy donor's blood.We demonstrated that the treatment with LPS enhances the immunosuppressive potential of cancer cells‐derived exosomes, although differences were observed depending on the origin of the tumor. The expression of TGFβ and MICA/B was increased on surface of exosomes released from LPS‐stimulated cells. Moreover, exosomes became able to inhibit the proliferation of activated CD3+ cells, increase T cells apoptosis and promote the conversion of CD4+ in regulatory T cells.Our data suggest that the activation of TLR4 contributes to tumor progression by promoting the release of more effective immunosuppressive exosomes, which allow tumor cells to escape immune surveillance.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.