Incubation Of Exosomes Research Articles

Abstract 7247: Exosome-mediated delivery of RNA and DNA: Enhanced efficiency with polycationic PEI-PEG

Abstract Emerging advances in gene therapy over the past few decades have revolutionized biomedical research. Our lab has pioneered bovine milk and colostrum-derived exosomes (Exo) and devised a nanoplatform for nucleic acid delivery achieving targetability with folic acid (FA) functionalization. We showed that bovine colostrum exosomes complexed with high MW polyethyleneimine (PEI-60K) can efficiently delivery siRNA and plasmid DNA (pDNA) in vitro and in vivo. We hypothesize that lower MW PEI may improve transfection efficiency and enhance oral uptake of EPM-siRNA, in addition to presumably higher degradability in a cellular system. We now report that exosomes and PEI of lower MW (25K)-PEG copolymer is as effective or more effective for delivery of siRNA and pDNA than PEI-60K. Incubation of exosomes, PEI-25K and siRNA in the presence of 5’-32P-labeled siRNA as a tracer showed high siRNA entrapment (>90%) based on the distribution of the radioactivity in EPM-siRNA precipitate compared with the supernatant. The efficacy of the EPM matrix is gene specific. As treatment of A549 lung cancer cells with EPM-(PEI-60K)-siKRAS showed 60% knockdown of target KRAS gene, which was enhanced to 70% by EPM-(PEI-25K)-siKRAS. Moreover, p53-pDNA delivered by EPM-25K resulted in nearly 2-fold higher expression of p53 in p53-null H1299 lung cancer cells compared with EPM-60K. However, still lower MW PEI (PEI-15K) diminished the transfection efficiency (36% KRAS knockdown). EPM formulated with PEI-60K and 25K had similar effect on cell viability of HEK293 cells. Upon in vivo testing, EPM-25K loaded with siKRAS and functionalized with folic acid (FA) showed time-dependent growth inhibition of A549-innoculated orthotopic lung tumors in NOD Scid mice eliciting 76% (p<0.01) tumor growth inhibition. The tumor inhibition was accompanied with 50% (p<0.05) knockdown of the target gene in the tumor tissue, while the adjacent lung tissue showed no modulation. The anti-tumor efficacy of siKRAS delivered by FA-functionalized EPM-25K is the same or somewhat higher than the efficacy reported previously with FA-functionalized EPM-60K.In conclusion, based on our in vitro and in vivo results, lower MW PEI (PEI-25K) is a suitable alternative for PEI-60K with boosted efficacy in terms of knockdown of KRAS. Our proposed technology offers a platform to advance and overcome the obstacles in the clinical translation of gene therapy. Funding: Work supported from funds provided by 3P Biotechnologies and in part Agnes Brown Duggan Funds. Disha Nagesh Moholkar is supported by IPIBS Fellowship, University of Louisville. Citation Format: Disha Nagesh Moholkar, Raghuram Kandimalla, Margaret Wallen, Jeyaprakash Jeyabalan, Wendy Spencer, Farrukh Aqil, Ramesh C. Gupta. Exosome-mediated delivery of RNA and DNA: Enhanced efficiency with polycationic PEI-PEG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7247.

Abstract 5875: Upregulation of the RNA binding protein HuR by cancer-secreted exosome to mediate multidrug resistance

Abstract Background and Aim: Multidrug resistance (MDR) is an unresolved obstacle to cancer chemotherapy. It is usually caused by overexpression of drug efflux transporters including ABCG2. Exosomes are extracellular lipid vesicles released from all cells upon the fusion of multivesicular bodies with plasma membrane. Tumor-derived exosomes have been shown to mediate cell-to-cell transfer of oncogenic materials to promote drug resistance. Human antigen R (HuR) is an extensively studied RNA binding protein that regulates the stability, translation, and nucleus-to-cytoplasm shuttling of mRNAs. Numerous HuR-targeting mRNAs play crucial roles in tumorigenesis, invasion and metastasis. We have previously reported the induction of ABCG2 and MDR phenotype by HuR overexpression in colon cancer. HuR overexpression is linked with high-grade malignancy and poor prognosis in cancer patients. This study investigated the enrichment of HuR inducing microRNAs (miRNAs) in exosomes secreted from MDR colon cancer and its intracellular transfer to drive MDR. Method: Cancer exosome-mediated transfer of miRNAs from ABCG2-overexpressing resistant colon cancer cells S1M1-80 to a few sensitive colon cell lines was evaluated. Exosomes were isolated from S1M1-80 conditioned medium by differential centrifugation method. The uptake of fluorescence-labeled exosome from donor MDR cancer cells by recipient sensitive cells was visualized by fluorescence microscopy. MiRNA microarray was conducted to identify differentially expressed miRNAs in drug sensitive cells after incubation with S1M1-80-secreted exosomes. Western blot and qPCR analysis were conducted to measure HuR and ABCG2 expression. Result: Exosomes were isolated from resistant S1M1-80 cells, whose purity was confirmed by the presence of exosomal markers (Tsg101 and Alix) but absence of cellular marker (GRP78) by Western blot analysis. These exosomes reduced the apoptotic effect of SN-38 or 5-fluorouracil in sensitive colon cancer cell lines (HCT116, S1, and SW480) by inducing both HuR and ABCG2. A common upregulated miRNA (miR-155-5p) was identified by miRNA microarray in the sensitive cell lines after incubation with S1M1-80-derived exosomes. MiR-155-5p was found to induce HuR expression by promoting its translation. MiR-155-5p inhibitor was shown to reverse the upregulation of HuR in HCT116 cells after exosome incubation, which subsequently suppressed ABCG2 expression and the MDR phenotype. Furthermore, inhibition of exosome uptake by dynasore was found to prevent the induction of HuR/ABCG2, and re-sensitize the exosome-treated sensitive cells to SN38. Conclusion: MDR cells-derived exosomes may spread the drug resistance phenotype by transferring a miR-155-5p/HuR/ABCG2 regulatory machinery to sensitive cells. Inhibition of this exosomal transfer of regulatory materials may prevent the spread of MDR and sensitize cancer cells to chemotherapy. Citation Format: Kenneth K.W. To. Upregulation of the RNA binding protein HuR by cancer-secreted exosome to mediate multidrug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5875.

Magnetically Controlled Photothermal, Colorimetric, and Fluorescence Trimode Assay for Gastric Cancer Exosomes Based on Acid-Induced Decomposition of CP/Mn-PBA DSNBs.

The accurate quantification of cancer-derived exosomes, which are emerging as promising noninvasive biomarkers for liquid biopsies in the early diagnosis of cancer, is becoming increasingly imperative. In our work, we developed a magnetically controlled photothermal, colorimetric, and fluorescence trimode aptasensor for human gastric cancer cell (SGC-7901)-derived exosomes. This sensor relied on CP/Mn-PBA DSNBs nanocomposites, created by decorating copper peroxide (CP) nanodots on polyethyleneimine-modified manganese-containing Prussian blue analogues double-shelled nanoboxes (PEI-Mn-PBA DSNBs). Through self-assembly, we attached CD63 aptamer-labeled CP/Mn-PBA DSNBs (Apt-CP/Mn-PBA DSNBs) to complementary DNA-labeled magnetic beads (cDNA-MB). During exosome incubation, these aptamers preferentially formed complexes with exosomes, and we efficiently removed the released CP/Mn-PBA DSNBs by using magnetic separation. The CP/Mn-PBA DSNBs exhibited high photoreactivity and photothermal conversion efficiency under near-infrared (NIR) light, leading to temperature variations under 808 nm irradiation, correlating with different exosome concentrations. Additionally, colorimetric detection was achieved by monitoring the color change in a 3,3',5,5'-tetramethylbenzidine (TMB) system, facilitated by PEI modification, NIR-enhanced peroxidase-like activity of CP/Mn-PBA DSNBs and their capacity to generate Cu2+ and H2O2 under acidic conditions. Moreover, in the presence of Cu2+ and ascorbic acid (AA), DNA sequences could form dsDNA-templated copper nanoparticles (CuNPs), which emitted strong fluorescence at around 575 nm. Increasing exosome concentrations correlated with decreases in temperature, absorbance, and fluorescence intensity. This trimode biosensor demonstrated satisfactory ability in differentiating gastric cancer patients from healthy individuals using human serum samples.

Exosomal miR-221-3p Derived from Bone Marrow Mesenchymal Stem Cells Alleviates Asthma Progression by Targeting FGF2 and Inhibiting the ERK1/2 Signaling Pathway.

Exosomes derived from human bone marrow mesenchymal stem cells (BMSCs) play potential protective roles in asthma. However, the underlying mechanisms remain not fully elucidated. Herein, exosomes were isolated from BMSCs, and the morphology, particle size, and exosome marker proteins were identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot, respectively. Then airway smooth muscle cells (ASMCs) were treated with transforming growth factor-β1 (TGF-β1) to construct a proliferation model and then incubated with BMSCs-derived exosomes. We found that exosome incubation increased miR-221-3p expression and inhibited proliferation, migration, and the levels of extracellular matrix (ECM) proteins including fibronectin and collagen III. Moreover, FGF2 was identified as a target gene of miR-221-3p. FGF2 overexpression reversed the inhibitory effects of exosomal miR-221-3p on ASMC progression. Besides, the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) is inhibited by exosomal miR-221-3p, which was reversed by FGF2 overexpression. And ERK1/2 signaling activator reversed the effects of exosomal miR-221-3p on ASMC progression. Additionally, an ovalbumin (OVA)-induced asthmatic mice model was established, and exosome treatment alleviated airway hyper-responsiveness (AHR), histopathological damage, and ECM deposition in asthmatic mice. Taken together, our findings indicated that exosomal miR-221-3p derived from BMSCs inhibited FGF2 expression and the ERK1/2 signaling, thus attenuating proliferation, migration, and ECM deposition in ASMCs and alleviating asthma progression in OVA-induced asthmatic mice. Our findings may provide a novel therapeutic strategy for asthma.

O-137 Evaluation of the effect of seminal plasma exosomes from unexplained infertile men on the expression of implantation-related genes

Abstract Study question Do exosomes from seminal plasma of men with unexplained infertility affect endometrial receptivity? Summary answer Seminal plasma exosomes from unexplained infertile men alter the expression of genes and signaling pathways related to endometrial receptivity. What is known already Unexplained infertile Men are a challenging group with no obvious cause of infertility. Many Complicated mechanisms play a crucial role in preparing the receptive endometrium for embryo implantation. The exosomes of seminal plasma are a heterogeneous cargo of nucleic acids, proteins, and lipids that participate in sperm maturation and function by binding to sperm. It is now apparent that the seminal plasma and its exosomes play a complex role in controlling the female reproductive system. There is no study investigating the effect of Unexplained seminal plasma exosomes on endometrial receptivity. Study design, size, duration Samples were collected from 10 men with normal semen parameters from couples undergoing infertility treatment and from 10 donor men with proven fertility and analyzed according to WHO -2010 criteria. Endometrial biopsy tissue was collected from 6 women in the proliferative phase of a natural menstrual cycle. Endometrial epithelial cells were cultured immediately after tissue processing and treated with 100 μg/ml seminal exosomes for (6 and 24 hours); then analyzed for gene expression. Participants/materials, setting, methods Exosomes isolated by an ultracentrifuge and characterized size and ultrastructure with dynamic light scattering and transmission electron microscopy. Western blot analysis showed the presence of the exosome markers. After incubation of exosomes with DiI dye, cytoplasmic localization of exosomes detected by fluorescence microscopy. The endometrial receptivity and signaling pathway genes were analyzed by qRT-PCR and immunocytochemistry. Image J and Prism software were used to quantify and analyze the data. P-value <0.05 was considered statistically significant. Main results and the role of chance Compared with the embryo donation group, the mRNA levels of MUC1, LIF, G-CSF, CX3CL1, and VEGF were significantly downregulated in the endometrium treated with unexplained infertile exosomes, especially after 24 hours of treatment. Mean mRNA levels of IL6, IL8, and TGFB genes in the unexplained infertile group changed compared with the embryo donation group but were not significant (p > 0.05). Immunocytochemical analysis showed that MUC1 and LIFR protein levels were increased in endometrial epithelial cells. Limitations, reasons for caution larger sample size and exosomal compositions assessment will be useful to identify the effect of exosomes on the endometrium. Wider implications of the findings Alterations in the expression of genes associated with endometrial receptivity by exosomes of the unexplained infertile men compared with embryo donation could be one of the causes of infertility in these individuals. Presumably, the atypical composition of exosomes has a different effect on sperm functions and endometrial genes expression. Trial registration number Not applicable

Adipose-Derived Stem Cells-Derived Exosomes with High Amounts of Circ_0001747 Alleviate Hypoxia/Reoxygenation-Induced Injury in Myocardial Cells by Targeting MiR-199b-3p/MCL1 Axis.

Recent studies demonstrated that circular RNAs play important roles in exosome-mediated cardio-protective effects after acute myocardial infarction (AMI). A previous study reported that circ_0001747 level is down-regulated in mouse hypoxia/reoxygenation (H/R) injury model. However, its biological role and working mechanism in AMI remain largely unknown.Exosomes were isolated from the culture supernatant of adipose-derived stem cells (ADSCs) using an ExoQuick precipitation kit. We treated mouse myocardial cells HL-1 with H/R to explore the role of exosomal circ_0001747 in AMI pathology. Cell viability, proliferation, apoptosis, and inflammation were analyzed by Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine assay, flow cytometry, and enzyme-linked immunosorbent assay. Dual-luciferase reporter assay and RNA immunoprecipitation assay were conducted to confirm the interaction between microRNA-199b-3p (miR-199b-3p) and circ_0001747 or MCL1 apoptosis regulator, BCL2 family member (MCL1).H/R-induced HL-1 dysfunction was attenuated by the incubation of exosomes derived from ADSCs, especially the exosomes with high amounts of circ_0001747. Circ_0001747 directly targeted miR-199b-3p in HL-1 cells. miR-199b-3p overexpression partly overturned exosomal circ_0001747-mediated protective effects in H/R-induced HL-1 cells. MCL1 was a direct target of miR-199b-3p in HL-1 cells. miR-199b-3p silencing alleviated H/R-induced damage in HL-1 cells partly by up-regulating MCL1. Circ_0001747 can elevate the messenger RNA and protein levels of MCL1 by sequestering miR-199b-3p.Overall, these results indicated that ADSCs-derived exosomes with high amounts of circ_0001747 attenuated H/R-induced HL-1 dysfunction partly by targeting miR-199b-3p/MCL1 signaling.

Exosomal miR-9-5p derived from BMSCs alleviates apoptosis, inflammation and endoplasmic reticulum stress in spinal cord injury by regulating the HDAC5/FGF2 axis

Exosomes derived from human bone marrow mesenchymal stem cells (BMSCs) play potential protective roles in spinal cord injury (SCI). However, the underlying mechanisms remain not fully elucidated. Herein, we isolated exosomes from BMSCs, and exosome morphology and marker protein levels were identified by transmission electron microscopy (TEM) and Western blot, respectively. PC12 cells were treated with lipopolysaccharide (LPS) to construct an injury model, and then incubated with BMSCs-derived exosomes. We found that exosome incubation increased miR-9-5p expression, and inhibited apoptosis and the levels of inflammation cytokines and ER stress marker proteins. Moreover, histone deacetylase 5 (HDAC5) was identified as a target gene of miR-9-5p by dual-luciferase reporter gene assay. Exosomal miR-9-5p upregulated fibroblast growth factor 2 (FGF2) expression by inhibiting HDAC5-mediated FGF2 deacetylation. Then, it was observed that HDAC5 overexpression or FGF2 inhibition reversed the inhibitory effects of exosomal miR-9-5p on apoptosis, inflammation and ER stress in PC12 cells. Additionally, an SCI rat model was established and exosomes were injected for treatment. Exosomal miR-9-5p treatment alleviated locomotor ability, histopathological damage, neuronal apoptosis, inflammation and ER stress in SCI rats. In conclusion, our findings indicated that exosomal miR-9-5p derived from BMSCs promoted FGF2 expression by inhibiting HDAC5-mediated deacetylation, thus inhibiting LPS-induced apoptosis, inflammation, and ER stress in PC12 cells, and alleviating SCI in rat model. Our study may provide a therapeutic direction for SCI.

Exosomal CD73 from serum of patients with melanoma suppresses lymphocyte functions and is associated with therapy resistance to anti-PD-1 agents

BackgroundCD73 is an ectonucleotidase producing the immunosuppressor mediator adenosine. Elevated levels of circulating CD73 in patients with cancer have been associated with disease progression and poor response to immunotherapy. Immunosuppressive...

Exosomes Isolated from Serum of Type 2 Diabetic Mice Increase Proliferation with no Effect on Insulin Stimulated Glucose Uptake of C2C12 Cells

PURPOSE Exosomes are small (30-150 nm) extracellular vesicles that are of interest as a potential diagnostic and therapeutic. C2C12 cells are a murine model of muscle stem cells (MSCs) that proliferate and differentiate to myotubes similarly to the behavior of MSCs during muscle repair. Because muscle repair and insulin-stimulated glucose uptake are known to be impaired in type 2 diabetes, the purpose of this project was to test the hypothesis that exosomes isolated from the serum of diabetic mice (Db-Db mice) impair myoblast proliferation and insulin-stimulated glucose uptake in myotubes of C2C12 cells. METHODS To test this hypothesis, we used CellTrace (ThermoFisher, Waltham, MA), a flow-cytometry based measure of proliferation, and 2-NBDG (ThermoFisher, Waltham, MA), a fluorescent glucose analog that can be used to measure glucose uptake. Exoquick exosome isolation reagent (System Biosciences, Palo Alto, CA) was used to isolate exosomes from serum, and exosome protein was measured using the Bradford method. Proliferation measures were obtained after a 24-hour incubation period with exosomes from either a C57 or Db-Db mouse. To determine if secreted exosomes affected glucose uptake, myotubes were established by incubating C2C12 cells in 2% horse serum (DM). After 5 days in DM, myotubes were incubated for 24 hours with exosomes from either Db-Db mice or C57 control mice. After 24 hours of exosome incubation, myotubes were serum-starved in glucose-free DMEM for 1 hour and then incubated in glucose-free DMEM with 100 µm 2-NBDG with or without 100 nm insulin. Fluorescence was then measured with excitation/emission of 465/540 nm. RESULTS Contrary to our hypothesis, treatment of C2C12 cells with 2 µg/µl of diabetic exosomes significantly increased cell proliferation compared to control cells (One-way ANOVA P-value=0.001) with no change in proliferation observed between treatment with C57 exosomes and untreated controls. Unexpectedly, there was no difference in insulin-stimulated 2-NBDG incorporation (Student's T-test P=0.19) in myotubes incubated with diabetic or control exosomes. However, there was a tendency for an increased basal glucose uptake following incubation with diabetic exosomes (Student's T-test, P=0.09). CONCLUSION Overall, these data suggest that exosomes may modulate MSC proliferation and glucose uptake during muscle repair in the diabetic condition.

Exosome carrying PSGR promotes stemness and epithelial-mesenchymal transition of low aggressive prostate cancer cells

AimProstate-specific G-protein coupled receptor (PSGR) in prostate cancer (Pca) are associated with poor overall survival. However, the effect of exosomal PSGR on PCa metastasis remains unknown. Main methodsThe effect of exosome derived from PSGR-overexpressed PC3 cells (PC3 PSGR+ exosomes) on migration, invasion, epithelial-mesenchymal transition (EMT) and stemness of low invasive cells (LNCaP and RWPE-1) was assessed. Transcriptome sequencing was performed to identify differentially expressed (DE) mRNAs in low invasive cells incubated by PC3 PSGR+ exosomes or negative control (NC) exosomes. Key findingsThe PSGR was stably overexpressed in PC3 cells. The PC3 PSGR+ exosomes were internalized in LNCaP and RWPE-1cells, and significantly promoted cells migration and invasion. The expression of E-cadherin was decreased, and Vimentin, Snail, SOX2 and OCT4a was increased in low invasive cells after PC3 PSGR+ exosome incubation. Additionally, a total of 993 and 1170 DE mRNAs were respectively identified in LNCaP and RWPE-1 cells after PC3 PSGR+ exosome incubation, and 5 upregulated mRNAs and 11 down regulated mRNAs were shared. The DE mRNAs were predominantly implicated in “activation of Rho GTPase activity” and “response to zinc ion” in LNCaP cells, and “extracellular matrix organization” and “patterning of blood vessels” in RWPE-1 cells. The KEGG analysis showed the DE mRNAs were enriched in pathways associated with EMT such as “Adherens junction”, “Cell adhesion molecules (CAMs)” and “Focal adhesion”. SignificanceExosomal PSGR promoted migration, invasion, stemness and epithelial-mesenchymal transitions, and reshaped the mRNAs profiling of LNCaP and RWPE-1 cells.

B7-H3 in Medulloblastoma-Derived Exosomes; A Novel Tumorigenic Role.

(1) Aim: Medulloblastoma is the most common aggressive pediatric cancer of the central nervous system. Improved therapies are necessary to improve life outcomes for medulloblastoma patients. Exosomes are a subset of extracellular vesicles that are excreted outside of the cell, and can transport nucleic acids and proteins from donor cells to nearby recipient cells of the same or dissimilar tissues. Few publications exist exploring the role that exosomes play in medulloblastoma pathogenesis. In this study, we found B7-H3, an immunosuppressive immune checkpoint, present in D283 cell-derived exosomes. (2) Methods: Utilizing mass spectrometry and immunoblotting, the presence of B7-H3 in D283 control and B7-H3 overexpressing exosomes was confirmed. Exosomes were isolated by Systems Biosciences from cultured cells as well as with an isolation kit that included ultracentrifugation steps. Overlay experiments were performed to determine mechanistic impact of exosomes on recipient cells by incubating isolated exosomes in serum-free media with target cells. Impact of D283 exosome incubation on endothelial and UW228 medulloblastoma cells was assessed by immunoblotting. Immunocytochemistry was employed to visualize exosome fusion with recipient cells. (3) Results: Overexpressing B7-H3 in D283 cells increases exosomal production and size distribution. Mass spectrometry revealed a host of novel, pathogenic molecules associated with B7-H3 in these exosomes including STAT3, CCL5, MMP9, and PI3K pathway molecules. Additionally, endothelial and UW228 cells incubated with D283-derived B7-H3-overexpressing exosomes induced B7-H3 expression while pSTAT1 levels decreased in UW228 cells. (4) Conclusions: In total, our results reveal a novel role in exosome production and packaging for B7-H3 that may contribute to medulloblastoma progression.

LncRNA KLF3-AS1 in human mesenchymal stem cell-derived exosomes ameliorates pyroptosis of cardiomyocytes and myocardial infarction through miR-138-5p/Sirt1 axis

AimMyocardial infarction (MI) is a severe disease with increased mortality and disability rates, posing heavy economic burden for society. Exosomes were uncovered to mediate intercellular communication after MI. This study aims to explore the effect and mechanism of lncRNA KLF3-AS1 in exosomes secreted by human mesenchymal stem cells (hMSCs) on pyroptosis of cardiomyocytes and MI.MethodsExosomes from hMSCs were isolated and identified. Exosomes from hMSCs with transfection of KLF3-AS1 for overexpression were injected into MI rat model or incubated with hypoxia cardiomyocytes. Effect of KLF3-AS1 on MI area, cell viability, apoptosis, and pyroptosis was determined. The relationship among miR-138-5p, KLF3-AS1, and Sirt1 was verified by dual-luciferase reporter assay. Normal cardiomyocytes were transfected with miR-138-5p inhibitor or sh-Sirt1 to clarify whether alteration of miR-138-5p or sh-Sirt1 can regulate the effect of KLF3-AS1 on cardiomyocytes.ResultsExosomes from hMSCs were successfully extracted. Transfection of KLF3-AS1 exosome in rats and incubation with KLF3-AS1 exosome in hypoxia cardiomyocytes both verified that overexpression of KLF3-AS1 in exosomes leads to reduced MI area, decreased cell apoptosis and pyroptosis, and attenuated MI progression. KLF3-AS1 can sponge miR-138-5p to regulate Sirt1 expression. miR-138-5p inhibitor transfection and KLF3-AS1 exosome incubation contribute to attenuated pyroptosis and MI both in vivo and in vitro, while transfection of sh-Sirt1 could reverse the protective effect of exosomal KLF3-AS1 on hypoxia cardiomyocytes.ConclusionLncRNA KLF3-AS1 in exosomes secreted from hMSCs by acting as a ceRNA to sponge miR-138-5p can regulate Sirt1 so as to inhibit cell pyroptosis and attenuate MI progression.

Exosomes from N-Myc amplified neuroblastoma cells induce migration and confer chemoresistance to non-N-Myc amplified cells: implications of intra-tumour heterogeneity

ABSTRACTNeuroblastoma accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is a well-established poor prognostic marker for neuroblastoma. Whilst N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressiveness of the disease is poorly understood. Exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. Hence, characterising the exosomal protein components from N-Myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. In this study, a comparative proteomic analysis of exosomes isolated from cells with varying N-Myc amplification status was performed. Label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the neuroblastoma cells. Gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in N-Myc amplified exosomes. Treatment of SH-SY5Y cells with N-Myc amplified SK-N-BE2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. Incubation of exosomes from N-Myc knocked down SK-N-BE2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. These findings suggest that exosomes could play a pivotal role in N-Myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells.Abbreviations: RNA = ribonucleic acid; DNA = deoxyribonucleic acid; FCS = foetal calf serum; NTA = nanoparticle tracking analysis; LC-MS = liquid chromatography–mass spectrometry; KD = knockdown; LTQ = linear trap quadropole; TEM = transmission electron microscopy

Abstract P5-03-10: Circulating exosomal microRNA profiling to depict mechanisms of chemotherapy resistance among triple negative breast cancer

Abstract Background: Chemotherapeutic resistance leads to high mortality among triple negative breast cancer (TNBC) and underlying mechanisms are poorly understood. Exosomes have been new member of liquid biophsy. MicroRNAs (miRNAs), as the most important inclusions in exosomes, making them ideal candidate biomarkers and therapeutical targets. Methods: We isolated exosomes and analyzed exosome-carried miRNAs signatures in several TNBC cells lines sensitive or resistant to adariamycin, docetaxel or cisplatin. The resistance transfer capacity was determined by flow cytometry after sensitive cells incubated 48 hours with exosomes from drug resistant cells. Locked nucleic acid probes and enzyme-labeled fluorescence (LNA-ELF-FISH) was performed to detect exosomal miRNA molecules transfer. Animal mode was constructed to evaluate treatment feasibility using miRNA modified exosomes. Serum exosomes from 40 TNBC stage IV patients underwent chemotherapy before or after progress disease (PD) status were isolated to analyze miRNA profiling for potential biomarkers identification. Results: We successfully isolated and identified exosomes from several drug sensitive and resistant TNBC cell lines and patients. Exosomal miR-222, miR-4443, miR-100, miR-17, miR-210 were found significantly upregulated from chemotherapy resistance cells. Incubation of exosomes from the resistant cells with the sensitive cells resulted in increasing resistant capacity among sensitive cells. Exosomal miRNA molecules transfer were detected using LNA-ELF-FISH. Transfection of synthesized miRNAs competitors into exosomes increased drug sensitivity in vivo. Exosomal miR-222, miR-4443, miR-100, miR-17, miR-210 were also found upregulated significantly from serums of patients after PD status. These five miRNAs were able to differentiate patients with PD status from those with CR or PR status with at least 89% accuracy. Conclusion: Exosomes from chemotherapy resistant TNBC cells could transfer drug resistance to sensitive cells via exosomal miRNAs. A circulating exosomal microRNA profiling was estabilished for potential biomarkers and therapeutical targets identification. Citation Format: He Y, Tang J. Circulating exosomal microRNA profiling to depict mechanisms of chemotherapy resistance among triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-10.

Exosomes in the serum of Acute Myeloid Leukemia patients induce dendritic cell tolerance: Implications for immunotherapy

Exosomes may represent an interesting antigenic pulse for new forms of anti-tumor immunotherapy. We evaluated exosomes from serum of patients with acute myeloid leukemia (AML) as an antigenic source for dendritic cells (DC) and the effects upon antitumor cytotoxicity, assessed by the percentage of specific lysis of K562 leukemic cells in co-cultures. Surprisingly, incubation of exosomes with DCs decreased lysis of K562, which may correspond to a mechanism of tumor evasion in vivo. However, when immature DCs were pulsed with exosomes purified from K562 culture supernatants, the lysis of target cells was notably enhanced, associated with a substantial increase in the expression of the maturation marker CD83. Thus, the development of vaccines using patients’ exosomes would probably add no benefits to the treatment of AML; alternately, exosomes from cultured cells may represent an effective way for maturing DCs into a cytotoxic phenotype, without the immunosuppression observed with patients’ exosomes.

Exosomal FMR1-AS1 facilitates maintaining cancer stem-like cell dynamic equilibrium via TLR7/NF\u03baB/c-Myc signaling in female esophageal carcinoma

BackgroundThough esophageal cancer is three to four times more common among males than females worldwide, this type of cancer still ranks in the top incidence among women, even more than the female specific cancer types. The occurrence is currently attributed to extrinsic factors, including tobacco use and alcohol consumption. However, limited attention has been given to gender-specific intrinsic genetic factors, especially in female.MethodsWe re-annotated a large cohort of microarrays on 179 ESCC patients and identified female-specific differently expressed lncRNAs. The associations between FMR1-AS1 and the risk and prognosis of ESCC were examined in 206 diagnosed patients from eastern China and validated in 188 additional patients from southern China. The effects of FMR1-AS1 on the malignant phenotypes on female ESCC cells were detected in vitro and in vivo. ChIRP-MS, reporter gene assays and EMSA were conducted to identify the interaction and regulation among FMR1-AS1, TLR7 and NFκB.ResultsWe found FMR1-AS1 expression is exclusively altered and closely associated with the level of sXCI in female ESCC patients, and its overexpression may correlate to poor clinical outcome. ChIRP-MS data indicate that FMR1-AS1 could be packaged into exosomes and released into tumor microenvironment. Functional studies demonstrated that FMR1-AS1 could bind to endosomal toll-like receptor 7 (TLR7) and activate downstream TLR7-NFκB signaling, promoting the c-Myc expression, thus inducing ESCC cell proliferation, anti-apoptosis and invasion ability. Exosome incubation and co-xenograft assay indicate that FMR1-AS1 exosomes may secreted from ESCC CSCs, transferring stemness phenotypes to recipient non-CSCs in tumor microenvironment. Furthermore, we also found a correlation between the serum levels of FMR1-AS1 and the overall survival (OS) of the female ESCC patients.ConclusionsOur results highlighted exosomal FMR1-AS1 in maintaining CSC dynamic interconversion state through the mechanism of activating TLR7-NFκB signaling, upregulating c-Myc level in recipient cells, which may be taken as an attractive target approach for advancing current precision cancer therapeutics in female patients.

Abstract P144: Characterization of Vascular Endothelial and Smooth Muscle Exosomes and Their Role in Vascular SignalTransduction

Vascular homeostasis is predicated on the integral interaction between a myriad of cell types. Principle to homeostasis is the interaction between the endothelial cells (ECs) of the intima and the vascular smooth muscle cells (VSMCs) of the media. Disruption of the tightly controlled communication between these cell types can lead to development of pathophysiology. In the past decade, much study has been given to a novel means of intercellular communication in exosomes. Exosomes are nanovesicles derived from endosomal compartments capable of carrying functional proteins, small RNAs and lipids into extracellular space. Initially characterized in the field of immunology, little is known on exosomes within vascular biology. Therefore, we present an in vitro characterization of rat EC and VSMC exosomes and evidence of EC exosomes as initiators of signal transduction within VSMCs. Primary rat aortic EC and VSMC exosomes isolated in serum-free conditions displayed characteristic size, shape and protein content of exosomes as analyzed through nanoparticle tracking analysis, electron microscopy, and western blot. Interestingly, alpha smooth muscle actin was found to be cell-specific for VSMC exosomes. Proteomics identified differential expression between EC and VSMC exosomes suggesting altered cargo between the two populations. Incubation of EC exosomes with VSMC identified a dose- and time-dependent increase in the expression of vascular cell adhesion molecule 1 in VSMC with a maximum at 10 9 EC exosomes and 6 hours through immunoblot analysis. In addition, EC exosomes stimulated expression of early growth response protein 1 at 1-3 hours, suggesting activation of pro-inflammatory gene expression. EC exosomes stimulated a maximal rate of protein synthesis at 3 hours in VSMC (3.53 fold increase over control, p=0.016) determined through puromycin incorporation showing qualitative changes in cell function. Analysis of cell volume identified a hypertrophic response in VSMC after exosome incubation as well, displaying a role for exosomes in vascular hypertrophy. In conclusion, we present a broad characterization of exosomes in vascular biology and a functional crosstalk mechanism between RAEC and VSMC through exosome transfer.

Exosomal miR-126 as a circulating biomarker in non-small-cell lung cancer regulating cancer progression

Lung cancer is one of the leading causes of cancer-related deaths. It is diagnosed mostly at the locally advanced or metastatic stage. Recently, micro RNAs (miRs) and their distribution in circulation have been implicated in physiological and pathological processes. In this study, miR-126 was evaluated in serum, exosome and exosome-free serum fractions in non-small cell lung cancer (NSCLC) patients at early and advanced stages, and compared with healthy controls. Down-regulation of miR-126 was found in serum of advanced stage NSCLC patients. In healthy controls, circulating miR-126 was equally distributed between exosomes and exosome-free serum fractions. Conversely, in both early and advanced stage NSCLC patients, miR-126 was mainly present in exosomes. Different fractions of miR-126 in circulation may reflect different conditions during tumour formation. Incubation of exosomes from early and advanced NSCLC patients induced blood vessel formation and malignant transformation in human bronchial epithelial cells. On the other hand, exosome-enriched miR-126 from normal endothelial cells inhibited cell growth and induces loss of malignancy of NSCLC cells. These findings suggest a role of exo-miRs in the modulation of the NSCLC microenvironmental niche. Exosome-delivered miRs thus hold a substantial promise as a diagnostics biomarker as well as a personalized therapeutic modality.

Hypoxia Inducible Factor-1α Potentiates Jagged 1-Mediated Angiogenesis by Mesenchymal Stem Cell-Derived Exosomes.

Insufficient vessel growth associated with ischemia remains an unresolved issue in vascular medicine. Mesenchymal stem cells (MSCs) have been shown to promote angiogenesis via a mechanism that is potentiated by hypoxia. Overexpression of hypoxia inducible factor (HIF)-1α in MSCs improves their therapeutic potential by inducing angiogenesis in transplanted tissues. Here, we studied the contribution of exosomes released by HIF-1α-overexpressing donor MSCs (HIF-MSC) to angiogenesis by endothelial cells. Exosome secretion was enhanced in HIF-MSC. Omics analysis of miRNAs and proteins incorporated into exosomes pointed to the Notch pathway as a candidate mediator of exosome communication. Interestingly, we found that Jagged1 was the sole Notch ligand packaged into MSC exosomes and was more abundant in HIF-MSC than in MSC controls. The addition of Jagged1-containing exosomes from MSC and HIF-MSC cultures to endothelial cells triggered transcriptional changes in Notch target genes and induced angiogenesis in an in vitro model of capillary-like tube formation, and both processes were stimulated by HIF-1α. Finally, subcutaneous injection of Jagged 1-containing exosomes from MSC and HIF-MSC cultures in the Matrigel plug assay induced angiogenesis in vivo, which was more robust when they were derived from HIF-MSC cultures. All Jagged1-mediated effects could be blocked by prior incubation of exosomes with an anti-Jagged 1 antibody. All together, the results indicate that exosomes derived from MSCs stably overexpressing HIF-1α have an increased angiogenic capacity in part via an increase in the packaging of Jagged1, which could have potential applications for the treatment of ischemia-related disease. Stem Cells 2017;35:1747-1759.

Exosomes from HIV-1-infected Cells Stimulate Production of Pro-inflammatory Cytokines through Trans-activating Response (TAR) RNA

HIV-1 infection results in a chronic illness because long-term highly active antiretroviral therapy can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under highly active antiretroviral therapy frequently develop various metabolic disorders, neurocognitive abnormalities, and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naive cells to HIV-1 infection. This study indicates that exosomes from HIV-1-infected primary cells are highly abundant with TAR RNA as detected by RT-real time PCR. Interestingly, up to a million copies of TAR RNA/μl were also detected in the serum from HIV-1-infected humanized mice suggesting that TAR RNA may be stable in vivo. Incubation of exosomes from HIV-1-infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines, IL-6 and TNF-β, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. The intact TAR molecule was able to bind to PKR and TLR3 effectively, whereas the 5' and 3' stems (TAR microRNAs) bound best to TLR7 and -8 and none to PKR. Binding of TAR to PKR did not result in its phosphorylation, and therefore, TAR may be a dominant negative decoy molecule in cells. The TLR binding through either TAR RNA or TAR microRNA potentially can activate the NF-κB pathway and regulate cytokine expression. Collectively, these results imply that exosomes containing TAR RNA could directly affect the proinflammatory cytokine gene expression and may explain a possible mechanism of inflammation observed in HIV-1-infected patients under cART.