Amount Of Exosomes Research Articles

Exosomes in hypoxia-induced remodeling of the tumor microenvironment.

Exosomes are structurally and functionally pleiotropic nano-sized (~30-150nm in diameter) extracellular vesicles (EVs) with endosomal origin. These vesicles are secreted by almost all cells and play a significant role in intercellular communication and bio-waste disposal. To a great extent, exosomes represent biological "snapshot" of parent cells, and their cargos (protein, nucleotides, lipids, and metabolites) are loaded uniquely under different pathophysiological conditions. For example, most cancerous cells secrete a higher amount of exosomes loaded with distinct cargos under stressful low oxygen condition i.e. hypoxia, a key characteristic of solid tumors responsible for disease aggressiveness and poor survival. Exosomes secreted under hypoxia (ExoHypoxic) play a vital role in aiding cancer cells crosstalk with its microenvironment constituents to create conditions advantageous for cancer growth and metastatic spread. In this review article, we have highlighted the effects of ExoHypoxic on various tumor microenvironment components involved in angiogenesis, survival, proliferation, pre-metastatic niches preparation, immunomodulation, epithelial-to-mesenchymal transition, invasion, metastasis, and drug resistance. We have also described key ExoHypoxic cargos (miRNA, proteins, etc) and their targets in the receipt cells, responsible for various biological effects. Finally, we have emphasized the applicability of ExoHypoxic as a biomarker of tumor hypoxia and disease prognosis.

Comparison of miRNA Cargo within Wharton's Jelly mesenchymal stem cell-derived exosomes manufactured in FBS and hPLT expansion media

Background & Aim Wharton's Jelly Mesenchymal Stem Cell (WJMSCs)-derived exosomes have been studied extensively in animal models of degenerative and chronic diseases. Thus, the development of a large-scale manufacturing procedure to harvest clinical-grade exosomes is required for the translation of exosome to the clinic. We have developed an exosome production technique to manufacture large quantities of exosomes using the quantum bioreactor (Terumo BCT) with both fetal bovine serum (FBS) and human platelet lysate (hPLT) supplemented expansion media. AWe aimed to compare the miRNA cargo of exosomes isolated from FBS-expanded and PLT-expanded WJMSC to determine if growth media is a significant contributor to exosome product preparation. Methods, Results & Conclusion WJMSCs were isolated from Wharton's Jelly and expanded to passage 1. After harvest, 30 × 106 cells were loaded into the quantum bioreactor and expanded for 1 week to achieve maximum confluence. Expansion media was washed and replaced with serum-free aMEM media. Conditioned media was collected every 24 hours for 120 hours and exosomes were precipitated by sequential centrifugation and ultracentrifugation. NanoSight analysis of the final products revealed a production of 15.0 × 1012± 0.32 × 1012 total particles with an average mode size of 111±4.78 nm. Surface protein analysis revealed positive expression of exosome markers CD9, CD63, and CD81. Exosome products were subjected to miRNA isolation, sequencing, and miRNA identification for bioinformatics analysis with the Qiagen IPA software. miRNA analysis was completed using 3 WJMSC donors grown in 20% FBS or 5%PLT supplemented media. Sequencing results identified 157 miRNAs in the PLT group and 154 miRNAs in the FBS group (with a 100-copy minimum expression cut off). FBS and PLT comparison revealed 142 common miRNAs with 15 unique to PLT and 12 unique to FBS. Further RNA target analysis revealed 68 miRNAs that target 1024 mRNA in the PLT group and 64 miRNA that target 1048 mRNA in the FBS group. The two groups share 998 common mRNA targets with 27 unique to PLT and 51 unique to FBS. Canonical pathway analysis of the unique targets reveals the top pathway hits, serotonin receptor and JAK/Stat signaling for PLT and IL-7 and RANK signaling for FBS. We found both expansion medias to be effective for large scale expansion and downstream exosome production. miRNA sequencing revealed small variations in the total number of predicted mRNA targets, suggesting a conserved therapeutic effect between the two groups. Wharton's Jelly Mesenchymal Stem Cell (WJMSCs)-derived exosomes have been studied extensively in animal models of degenerative and chronic diseases. Thus, the development of a large-scale manufacturing procedure to harvest clinical-grade exosomes is required for the translation of exosome to the clinic. We have developed an exosome production technique to manufacture large quantities of exosomes using the quantum bioreactor (Terumo BCT) with both fetal bovine serum (FBS) and human platelet lysate (hPLT) supplemented expansion media. AWe aimed to compare the miRNA cargo of exosomes isolated from FBS-expanded and PLT-expanded WJMSC to determine if growth media is a significant contributor to exosome product preparation. WJMSCs were isolated from Wharton's Jelly and expanded to passage 1. After harvest, 30 × 106 cells were loaded into the quantum bioreactor and expanded for 1 week to achieve maximum confluence. Expansion media was washed and replaced with serum-free aMEM media. Conditioned media was collected every 24 hours for 120 hours and exosomes were precipitated by sequential centrifugation and ultracentrifugation. NanoSight analysis of the final products revealed a production of 15.0 × 1012± 0.32 × 1012 total particles with an average mode size of 111±4.78 nm. Surface protein analysis revealed positive expression of exosome markers CD9, CD63, and CD81. Exosome products were subjected to miRNA isolation, sequencing, and miRNA identification for bioinformatics analysis with the Qiagen IPA software. miRNA analysis was completed using 3 WJMSC donors grown in 20% FBS or 5%PLT supplemented media.

Exosome Production and Regenerative Potential is Influenced by Mechanical Stimulus and Cell Origin

INTRODUCTIONExosomes are nanosized membrane bound vesicles (30–150 nm) secreted by cells. Exosomes contain nucleic acids and proteins used to signal and influence the cells around them. It has been shown that mesenchymal stem cells (MSCs) influence tissue regeneration and healing through their paracrine secretion of pro‐regenerative factors. Many of these factors are contained within exosomes suggesting that exosome suspensions may be an effective cell‐free therapy for tissue regeneration and repair. Previously, our group has found that mechanical stimulus can be used to rescue muscle derived stem cells from an aged mouse model (progeria mice), indicating that physical environment influences MSC function. With this knowledge, we hypothesized that myoblasts subjected to mechanical stimulation would improve exosome quantity and function. Given the fact that several studies have also shown muscle to bone cross talk improves fracture repair, we further hypothesized that optimized myoblast exosomes could improve osteoblast cell function.METHODSC2C12 myoblasts were cultured using the FlexCell FX‐5000TT bioreactor. Two exercise regimens were programmed using biaxial or uniaxial strain: 1) low intensity regimen (LIR) (0–15% strain at 0.5 Hz for 24 hours) 2) high intensity interval regimen (HIIR) (12–22% strain at 1 Hz for 10 minutes followed by 50 minutes of rest repeated for 24 hours). Unexercised, control cells were cultured in parallel. MC3T3‐E1 osteoblasts were cultured for 24 hours for exosome collection. Exosomes were isolated using Invitrogen Total Exosome Isolation Reagent. System Bioscience’s ExoELISA‐ULTRA CD81 Kit and a BCA assay were used to quantify exosome number and protein concentration. 20 ug of exosomes (~5e9 exosomes) were used for treatment in proliferation and differentiation assays. Proliferation was assessed using PrestoBlue (Thermo Fisher). Myogenic differentiation was evaluated using image analysis (15 images per group).ResultsAll exercise regimens resulted in increased exosome concentrations in myoblasts. Biaxial strain and HIIR resulted in the greatest exosome production. Further, we found increased functionality of exosomes from exercised cells resulting in greater proliferation and differentiation of myoblasts. Interestingly, myoblast exosomes also significantly increased proliferation in osteoblast cells. However, in contrast, exosomes isolated from osteoblasts significantly decreased proliferation when treated on myoblasts but significantly increased proliferation when treated back upon themselves.DISCUSSION and CONCLUSIONOur results indicate that mechanical stimulus and cell origin directly influence exosome production. We found that exercise can be optimized to improve the production and regenerative capacity of myoblast exosomes. Further, we found a positive effect of myoblast exosomes on osteoblasts, but the converse, osteoblast exosomes on myoblasts, had a negative effect on cell proliferation. Coupling this with findings to indicate exosomes remain stable at −80° C for at least 4 months, we see exosomes as a potential cell‐free therapy for tissue regeneration following musculoskeletal traumas.Support or Funding InformationBorgen Family Foundation(A) Myoblast Cells secrete more exosomes when exercised (flex) compared to non‐exercised (static) cells. (* = p < 0.05, n = 8) (B) Exercise protocol attenuates exosome production. (**** = p < 0.0001, n = 4) (C–F) Exosomes from exercised cells increase myogenic differentiation. (* = p < 0.05, *** = p < 0.001, n = 4)Figure 1(A) Exosomes from exercised cells increase proliferation. (* = p < 0.05, *** = p < 0.001, n = 4) (B) Exosomes effect on proliferation is dependent on cell origin. (*** = p < 0.001, n = 3)Figure 2

GOLPH3 Regulates Exosome miRNA Secretion in Glioma Cells.

We aimed to examine whether golgi protein GOLPH3 could affect the secretion of glioma cell-derived exosomes. The exosomes were extracted by ultra-centrifugation from the supernatant of U251 and U87 cell cultures and identified by transmission electron microscopy (TEM), Malvern analyzer, and western blot. The quantity of exosomes was examined by measuring the total protein levels and the number of multiple vesicle bodies (MVBs), the source of exosomes. The exosome miRNAs were analyzed by high-throughput sequencing followed by GO and KEGG analysis, and validated by qRT-PCR. GOLPH3 could not affect the total protein levels of exosomes and the number of MVBs. However, we found 149 differentially expressed miRNAs in exosomes between vector and GOLPH3 over-expression group, and 14 miRNAs were only examined in GOLPH3 over-expression cells. The predicted target genes of these miRNAs had functions in binding and catalytic activity, which were enriched in the pathways of endocytosis, RNA transportation, thyroid hormone signaling and miRNAs in cancer. GOLPH3 could not affect the quantity of exosomes, but rather contribute to miRNA expression in exosomes, which may play some functions in the promotion effect of GOLPH3 on glioma development.

Characterization of Human NK Cell-Derived Exosomes: Role of DNAM1 Receptor In Exosome-Mediated Cytotoxicity Against Tumor.

Despite the pivotal role of natural killer (NK) cells in defenses against tumors, their exploitation in cancer treatment is still limited due to their reduced ability to reaching tumor sites and the inhibitory effects of tumor microenvironment (TME) on their function. In this study, we have characterized the exosomes from IL2- or IL15-cultured human NK cells. Both cytokines induced comparable amounts of exosomes with similar cargo composition. Analysis of molecules contained within or exposed at the exosome surface, allowed the identification of molecules playing important roles in the NK cell function including IFN-γ, Lymphocyte Function-Associated Antigen (LFA-1), DNAX Accessory Molecule-1 (DNAM1) and Programmed Cell Death Protein (PD-1). Importantly, we show that DNAM1 is involved in exosome-mediated cytotoxicity as revealed by experiments using blocking antibodies to DNAM1 or DNAM1 ligands. In addition, antibody-mediated inhibition of exosome cytotoxicity results in a delay in target cell apoptosis. We also provide evidence that NK-exosomes may exert their cytolytic activity after short time interval and even at low concentrations. Regarding their possible use in immunotherapy, NK exosomes, detectable in peripheral blood, can diffuse into tissues and exert their cytolytic effect at tumor sites. This property offers a clue to integrate cancer treatments with NK exosomes.

Melatonin affects the release of exosomes and tau-content in in vitro amyloid-beta toxicity model

BackgroundRecent studies have been revealed that oxidative damage is the main cause of aging and age-related neurodegenerative diseases like Alzheimer's disease (AD). Melatonin is secreted from the pineal gland and its secretion has been found to be altered in AD. In the last decade the role of exosomes in spreading toxic proteins and inducing the propagation of diseases like AD has been discussed. However, it is not known how melatonin affects the amount of exosomes released from the cells and the content of the exosomes. ObjectiveHerein, we investigated the possible role of melatonin treatment in the releasing of exosomes and exosomal tau content in an in vitro Aβ toxicity model. MethodSH-SY5Y cell line was used. The optimum concentration of Aβ was determined by cell viability and cell proliferation tests. Melatonin (100 µM) was applied before and after Aβ application. Total exosomes isolated from cell culture media were immunoprecipitated. The amount of released exosomes and their tau content were analyzed by Western blots. ResultsOur data demonstrated for the first time that melatonin treatment clearly affected the amount of released exosomes. It would decrease the amyloid beta load and toxicity by inhibiting exosome release. We also demonstated that melatonin also affected the level of tau carried by exosomes depending on whether melatonin was applied before or after Aβ application. ConclusionIt is considered that the effect of melatonin in the release of exosomes and exosomal tau content would contribute the development of therapeutic strategies in AD and related disorders.

Isolation and Characterization of Exosomes from Mouse Feces.

Exosomes secreted by colonic epithelial cells are present in feces and contain valuable epigenetic information, such as miRNAs, proteins, and metabolites. An in-depth study of this information is conducive to the diagnosis or treatment of relevant diseases. A crucial prerequisite of such a study is to establish an efficient isolation method, through which we can obtain a relatively more significant amount of exosomes from feces. This protocol is designed to effectively isolate a large number of exosomes from contaminants and other particles in feces by a combined method with fast filtration and sucrose density gradient ultracentrifugation. Exosomes generated by this method are suitable for further RNA, protein, and lipid analysis.

Rapid and sensitive exosome detection with CRISPR/Cas12a.

Numerous studies have shown that exosomes are closely related to the pathogenesis of various diseases, especially cancers. Therefore, a rapid and sensitive method for exosome detection will be of great importance for the diagnosis and prognosis of diseases. We report here a method for exosome detection based on the CD63 aptamer and clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system. This method consists mainly of exosomal membrane protein recognition based on the CD63 aptamer and signal amplification based on CRISPR/Cas12a. The CD63 aptamer, as an easily adaptable nucleic acid strand, is responsible for the conversion of the amounts of exosomes into nucleic acid detection, whereas CRISPR/Cas12a is responsible for highly specific nucleic acid signal amplification. The detection range of the method was determined as 3 × 103-6 × 107 particles per microliter. Additionally, we successfully applied this method to detect exosomes in clinical samples from both healthy individuals and patients with lung cancer, and the results were highly consistent with those obtained by nanoparticle tracking analysis. In general, this method provides a highly sensitive and specific method for the detection of exosomes and offers an avenue toward future exosome-based diagnosis of diseases.

Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation.

Exosomes are attractive nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and of their ability to penetrate physiological barriers that are impermissible to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs (mRNAs), into cell-secreted exosomes leads to low yields. Here, we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs, and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared to bulk electroporation and to other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and more than a 103-fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic PTEN-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced tumour-growth inhibition, and increased animal survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.

Anti-Leukemia Effects of NK Cell-Derived Exosomes

Introduction: Exosomes are 30-150 nm-sized extracellular vesicles originating from the endocytic compartment of parent cells. The exosome molecular cargo reflects the content of its cells of origin and is delivered to recipient cells in a protective glycol-lipid bilayer without degradation. Because of their small size, exosomes freely circulate within the body, can reach the bone marrow, and can cross biological barriers. Natural killer (NK) cells play a critical role in the innate immune response through their capacity to lyse malignant cells without prior antigen-specific priming. Importantly, NK cell activity is reduced in patients with acute myeloid leukemia (AML) relative to that in healthy donors. To overcome the decreased NK cell activity in AML, several therapeutic strategies have been evaluated for safety and efficacy, both in transplant and non-transplant settings, using autologous and allogeneic activated NK cells. Since NK cell-derived exosomes acquire tumor-killing abilities from the parent NK cells, we hypothesize that NK cell-derived exosomes by transferring exosome content to leukemia blasts can induce the death of these target cells. In the current study, we evaluated the in vitro anti-leukemia effects of NK cell-derived exosomes. Methods: Exosomes were isolated from the supernatants of NK cells obtained from healthy donors (n=12) using mini-size exclusion chromatography (mini-SEC). Protein levels, number and size (qNano), and exosome morphology using transmission electron microscopy were determined. The exosome cargo was studied by Western blots and on-bead flow cytometry for NK cell activating and inhibitory receptors, immune inhibitory molecules, and for perforin and granzyme B. Cytotoxicity of the NK cell-derived exosomes for AML cell lines (Kasumi, MLL-1) and primary leukemia blasts was measured using flow cytometry-based assays. Results: Activated human NK cells produced large quantities of exosomes. Transmission electron microscopy showed the presence of vesicles that were uniform in size (30-150nm in diameter) by NanoSight measurement. Confocal imaging of labeled NK cell-derived exosomes interacting with leukemia cells showed that they are rapidly internalized by leukemic targets. NK cell-derived exosomes carried activating NK cell receptor NKG2D, natural cytotoxicity receptors, perforin, granzyme B, transforming growth factor beta (TGF-β), killer-cell immunoglobulin-like receptors, and PD-1. NK cell-derived exosomes were co-incubated with target cells, AML cell lines and primary leukemia cells, at different exosome:target (E:T) ratios using escalating doses of exosomes (10-70 µg). NK cell-derived exosomes mediated strong anti-leukemic activity against AML cell lines and primary leukemic blasts. Importantly, with higher doses of exosomes, higher levels of cytotoxicity were observed, suggesting that exosome-mediated lysis is concentration dependent. NK cell-derived exosomes mediated leukemia killing via different cell death pathways including apoptosis and necroptosis. Conclusion: NK cell-derived exosomes mediating cytotoxicity against leukemic targets represents a novel therapeutic modality for patients with AML. Disclosures No relevant conflicts of interest to declare.

Syntaxin 6-mediated exosome secretion regulates enzalutamide resistance in prostate cancer.

Prostate cancer (PCa) deaths are typically the result of metastatic castration-resistant PCa (mCRPC). Recently, enzalutamide (Enz), an oral androgen receptor inhibitor, was approved for treating patients with mCRPC. Invariably, all PCa patients eventually develop resistance against Enz. Therefore, novel strategies aimed at overcoming Enz resistance are needed to improve the survival of PCa patients. The role of exosomes in drug resistance has not been fully elucidated in PCa. Therefore, we set out to better understand the exosome's role in the mechanism underlying Enz-resistant PCa. Results showed that Enz-resistant PCa cells (C4-2B, CWR-R1, and LNCaP) secreted significantly higher amounts of exosomes (2-4 folds) compared to Enz-sensitive counterparts. Inhibition of exosome biogenesis in resistant cells by GW4869 and dimethyl amiloride strongly decreased their cell viability. Mechanistic studies revealed upregulation of syntaxin 6 as well as its increased colocalization with CD63 in Enz-resistant PCa cells compared to Enz-sensitive cells. Syntaxin 6 knockdown by specific small interfering RNAs in Enz-resistant PCa cells (C4-2B and CWR-R1) resulted in reduced cell number and increased cell death in the presence of Enz. Furthermore, syntaxin 6 knockdown significantly reduced the exosome secretion in both Enz-resistant C4-2B and CWR-R1 cells. The Cancer Genome Atlas analysis showed increased syntaxin 6 expressions associated with higher Gleason score and decreased progression-free survival in PCa patients. Importantly, IHC analysis showed higher syntaxin 6 expression in cancer tissues from Enz-treated patients compared to Enz naïve patients. Overall, syntaxin 6 plays an important role in the secretion of exosomes and increased survival of Enz-resistant PCa cells.

Environmental pollutant induced cellular injury is reflected in exosomes from placental explants

IntroductionExosomes are intercellular signaling vesicles whose cargo reflects the physiological status of the cell of their origin and can regulate gene expression in other tissues. Polybrominated diphenyl ethers (PBDEs) and bisphenols (A [BPA], Tetrabromobisphenol A [TBBPA], and 2,4,6-Tribromophenol [TBP]) are common environmental pollutants known to increase the risk for spontaneous preterm birth (PTB). We hypothesized that placental exposure to these environmental pollutants causes exosome cargo changes that reflect exposure associated placental response. MethodsFull-term, C-section placenta explants were treated with PBDE congeners (47, 100, 153, 209), TBBPA, TBP or BPA for 24 h. Exosomes were isolated from media by sequential ultracentrifugation and purified by size exclusion chromatography. Exosomes were characterized by electron microscopy, nanoparticle tracking analysis and Western blot. Proteomics identified differentially expressed exosomal proteins and Ingenuity pathway analysis (IPA) determined biological functions and pathways represented by identified proteins. ResultsRegardless of treatment, placental expressed exosomes markers (PLAP, CD9, CD63, 81 and ALIX), had a size distribution between 50 and 175 nm and were present in the conditioned medium at 5–8 x 1011 exosomes/mL. Proteomic analysis identified 2598 proteins which demonstrated that specific pollutants caused differential expression of specific proteins, including alarmin, High Mobility Group Box 1 (HMGB1), MAPK14 (p38 MAPK) and GSK3β. IPA revealed an inhibition of pathways associated with cell survival, tissue repair and proliferation, as well as activation of cell death pathways (e.g. necrosis). ConclusionEnvironmental exposure of placental explants did not change the quantity of exosomes or their characteristics. However, exosome cargo composition exposed to some environment pollutants may be involved in placental nuclear and cellular injury and inflammation.

P2581Unique molecular profile of exosomes from epicardial fat of patients with atrial fibrillation

Abstract Background and aim Exosome, the smallest vesicles that originate from intracellular compartments, contains and delivers specific bioactive proteins, lipids, mRNA, and miRNA to facilitate intercellular signaling. However, the role and mechanism of exosomes in atrial remodeling and fibrillation (AF) has not been determined. While epicardial fat (eFat) and inflammation have been linked to atrial remodeling and fibrillation (AF), the role of eFat exosomes in the pathogenesis of AF remains unknown. Thus, we aimed to determine whether eFat exosomes play a role in the initiation and progression of AF. Methods and results We collected eFat specimens from patients with and without chronic or paroxysmal AF undergoing heart surgery. Isolated fat specimens were cut into small pieces and incubated as organ cultures. We isolated exosomes from the medium of the explant by differential ultra-centrifugation and characterized the vesicle size distributions, morphology, specific markers, and molecular cargo. Immunoblotting of CD63, CD81 and TSG101 confirmed the presence of exosomes (Fig.1 B). Significantly, eFat from patients with AF secreted higher amounts of exosomes compared with controls (p<0.001), which confirmed by Nanoparticle Tracking Analysis (NTA) (Fig.1 A). The levels of exosomal pro-inflammatory and pro-fibrotic cytokines were higher in exosomes from patients with, compared with those without, AF (Fig.1 C, D). Measurements of micro RNA (miRNA) by real-time PCR showed lower levels of anti-fibrotic miRNA-133a (p=0.2), and higher levels of pro-fibrotic miRNA-146b (p=0.2) in exosomes from patients with, compared with those without, AF (Fig.1 E, F) Conclusion We show, for the first time, that exosomes from eFat of patients with AF have a unique pro-inflammatory and pro-fibrotic profile. We suggest a novel mechanism that links eFat to AF and eFat exosomes as novel biomarkers and potential therapeutic targets for AF.

The Role of Exo-miRNAs in Cancer: A Focus on Therapeutic and Diagnostic Applications.

Exosomes are extracellular vesicles released into biological fluids where they act as carriers of various molecules, including proteins, lipids, and RNAs, between cells, modulating or perturbing specific physiological processes. Recently, it has been suggested that tumoral cells release excessive amounts of exosomes that, through their cargo, promote tumor progression, stimulating growth, angiogenesis, metastasis, insensitivity to chemotherapy, and immune evasion. Increasing evidence highlights exosomal microRNAs (exo-miRNAs) as important players in tumorigenesis. MicroRNA (miRNA) are a class of small non-coding RNA able to regulate gene expression, targeting multiple mRNAs and inducing translational repression and/or mRNA degradation. Exo-miRNAs are highly stable and easily detectable in biological fluids, and for these reasons, miRNAs are potential cancer biomarkers useful diagnostically and prognostically. Furthermore, since exosomes are natural delivery systems between cells, they can be appropriately modified to carry therapeutic miRNAs to specific recipient cells. Here we summarize the main functions of exo-miRNAs and their possible role for diagnostic and therapeutic applications.

Abstract 130: Development of Bioinspired Synthetic Exosomes With Proangiogenic Potential

Exosomes with variable microRNA cargos are released from different cell types and stimulate angiogenesis in animal models. Therefore, exosomes are currently considered for their potential to represent a safer and easier biological-based alternative to stem cell therapies. However, the limited amount of exosomes that can be reproducibly prepared from cultured stem cells and the complexity of the cargo of endogenous exosomes make them difficult to develop as off-the-shelf “pharmaceutical products” and limit their clinical potential. These issues could be surpassed by the use of artificial (i.e. nanoparticles mimicking the natural product) exosomes carrying a therapeutic cargo. In consideration of the possibility that not all cargo components of exosomes are required for their therapeutic functions, we reasoned that bioinspired artificial exosomes (AEs) incorporating key therapeutically relevant molecules and devoid of potentially negative or indifferent factors could further improve their therapeutic potential. Therefore, we developed and tested AEs containing exosomal proangiogenic miRNAs as a “proof-of-concept” for therapeutic angiogenesis. In order to achieve this, we: (1) characterized the common microRNA cargo of endogenous proangiogenic exosomes (from stem cells, pericytes and pericardial fluid) using bioinformatics, (2) exploited this knowledge to develop off-the-shelf artificial exosomes (AEs) with proangiogenic capacities, (3) validated the angiogenic potential of the bioinspired AEs. Bioinformatics analyses integrating data of miRNA arrays and panels of proangiogenic exosomes confirmed the enrichment of let-7 family in these exosomes. After testing the angiogenic potential of different members of let-7 family, we produced AEs containing let-7b (the most potent in the family) by microfluidic micromixing. The AEs were uptaken by human ECs cultured under hypoxic conditions, without causing toxicity. let-7b-AEs transferred functional let-7b, thus decreased the expression of validated targets of let-7b in recipient cells. let-7b-AEs improved EC survival, proliferation and angiogenesis in vitro and in vivo . These data suggest the therapeutic potential of bioinspired AEs containing let-7b.

Leptin Modulates Exosome Biogenesis in Breast Cancer Cells: An Additional Mechanism in Cell-to-Cell Communication.

Exosomes—small membrane vesicles secreted by both normal and malignant cells upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane—play an important role in cell-to-cell communication. During the last decade, several reports have highlighted the involvement of these nanovesicles in many aspects of breast cancer development and progression, but the extracellular signals governing their generation in breast cancer cells have not been completely unraveled. Here, we investigated the role of the obesity hormone leptin, a well-known adipokine implicated in mammary tumorigenesis, on the mechanisms regulating exosome biogenesis and release in both estrogen receptor α (ERα)—positive MCF-7 and triple-negative MDA-MB-231 breast cancer cells. We found that leptin treatment enhanced the number of MVBs in the cytoplasm of breast cancer cells and increased the amount of exosomes released in cell conditioned media. At molecular level, leptin increased the protein expression of Tsg101—a key component of the endosomal sorting complex required for transport I (ESCRT-I)—by a post-transcriptional mechanism involving its direct interaction with the chaperone protein Hsp90. Targeting leptin signaling, by a selective leptin receptor antagonist the peptide LDFI (Leu-Asp-Phe-Ile), abrogated leptin effects on Tsg101 expression and on exosome secretion in breast cancer cells. In conclusion, our findings, identifying for the first time leptin/leptin receptor/Hsp90 axis as an important regulator of exosome generation in mammary carcinoma cells, suggest that targeting this signaling pathway might represent a novel therapeutic strategy to impair exosome secretion and interrupt the dangerous cell-to-cell communication in breast cancer.

Excessive exosome release is the pathogenic pathway linking a lysosomal deficiency to generalized fibrosis.

Lysosomal exocytosis is a ubiquitous process negatively regulated by neuraminidase 1 (NEU1), a sialidase mutated in the glycoprotein storage disease sialidosis. In Neu1-/- mice, excessive lysosomal exocytosis is at the basis of disease pathogenesis. Yet, the tissue-specific molecular consequences of this deregulated pathway are still unfolding. We now report that in muscle connective tissue, Neu1-/- fibroblasts have features of myofibroblasts and are proliferative, migratory, and exocytose large amounts of exosomes. These nanocarriers loaded with activated transforming growth factor-β and wingless-related integration site (WNT)/β-catenin signaling molecules propagate fibrotic signals to other cells, maintaining the tissue in a prolonged transitional status. Myofibroblast-derived exosomes fed to normal fibroblasts convert them into myofibroblasts, changing the recipient cells' proliferative and migratory properties. These findings reveal an unexpected exosome-mediated signaling pathway downstream of NEU1 deficiency that propagates a fibrotic disease and could be implicated in idiopathic forms of fibrosis in humans.

Abstract 1991: Exploring the potential of exosomes for cancer diagnosis and management: Comparative analysis of isolation methods for optimum yield purity and downstream applications

Abstract Exosomes have received significant attention due to their involvement in several diseases, including cancer, and are being explored as a tool for disease diagnosis and management. As a result, several kits based on different principles are now available in the market for exosome isolation. In this study, we have compared the relative efficacy of four commercial kits from Invitrogen, 101Bio, Wako and iZON along with conventional ultracentrifugation-based method for exosome yield, purity and quality for downstream applications. Cancer cell culture supernatant was used as an abundant and uniform source of exosomes for all methods and exosome quantity, size distribution, zeta potential, marker expression, RNA (qRT-PCR) and protein (mass spectrometry) quality was determined. The Invitrogen method showed the highest yield but the preparation showed broader size distribution likely due to co-precipitation of microvesicles and had the least magnitude of zeta potential (dispersion stability). Exosome preparations from other methods showed accepted size range (<150 nm) with a variable zeta potential (-12mV to -29mV) with good purity. The gel filtration method; however, showed a broader size distribution in the lower size range, which could be due contamination of aggregates of proteins and vitamins molecules. Quality of RNA isolated from all preparations was comparable; however, proteins isolated from Invitrogen method-based exosomal preparation showed contamination of polyethylene glycol (PEG) in mass spectrometry analyses. Similarly, other chemical impurities coming from the precipitant in Invitrogen kit could also be the reason for toxicity of resultant exosomal preparation in biological growth measurement assay. Together, these findings from comparative analyses should serve as a guide for choice and further optimization of exosome isolation methods for their desired downstream applications in cancer biology, therapy and/or biomarker discovery. Citation Format: Girijesh K. Patel, Mohammad Aslam Khan, Haseeb Zubair, Sanjeev Kumar Srivastava, Moh’d Khushman, Seema Singh, Ajay Pratap Singh. Exploring the potential of exosomes for cancer diagnosis and management: Comparative analysis of isolation methods for optimum yield purity and downstream applications [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 1991.

Targeted delivery of doxorubicin to HER2 positive tumor models.

BackgroundExosomes are natural nanovesicles with unique characteristics, such as long circulating half-life, the intrinsic ability to target tissues, biocompatibility, and minimal or no inherent systemic toxicity. Mesenchymal stem cells produce large amounts of exosomes with regenerative properties and more stability in human plasma. TUBO breast cancer cell lines overexpress rat HER2/neu protein.MethodsTargeted exosomes were isolated from transduced bone marrow mesenchymal stem cells. Doxorubicin was encapsulated into exosomes by electroporation. Flow cytometry was used to assess the attachment of exosomes to the target cells. The in vitro cytotoxicity effect of targeted doxorubicin-loaded exosomes on TUBO cells was determined using MTT assay. Selective delivery of doxorubicin to tumor tissues was analyzed by measuring the auto-fluorescence of doxorubicin by in vivo imaging system. Moreover, tumor growth inhibition and body weight were monitored following injection of free doxorubicin, and targeted and untargeted doxorubicin-loaded exosomes in a TUBO breast cancer model. Finally, mouse tissues were examined for the presence of intrinsic fluorescence of doxorubicin.ResultsFlow cytometry results revealed significant differences in binding of targeted exosomes to HER2-positive (46.05%) and HER2-negative (13.9%) cells. The results of MTT assay showed that cytotoxicity of targeted doxorubicin-loaded exosomes was higher than free doxorubicin at 72 hours. Selective distribution of targeted doxorubicin-loaded exosomes in the target tissues of the murine breast cancer model suggested specific delivery of doxorubicin by targeted exosomes, rather than untargeted exosomes. Free doxorubicin and untargeted doxorubicin-loaded exosomes showed insignificant effects, whereas targeted doxorubicin-loaded exosomes reduced the tumor growth rate.ConclusionHerein, we report efficient delivery of targeted doxorubicin-loaded exosomes in vitro, corroborated with a significant reduction of murine breast cancer model tumor growth rate.

Safety of nivolumab and ipilimumab in combination with radiotherapy in patients with locally advanced squamous cell carcinoma of the head and neck (LA SCCHN).

6070 Background: Standard cisplatin-based chemoradiotherapy for LA SCCHN is associated with toxicity and less than desirable survival in high-risk patients (pts). The combination of the anti-PD1 antibody nivolumab (nivo) and the anti-CTLA-4 antibody ipilimumab (ipi) has been effective in melanoma and renal cell carcinoma and is under investigation in other solid tumors including SCCHN. Combining RT and immunotherapy (IO) has a strong preclinical rationale and is being evaluated in the clinic. This pilot trial combines IO and RT to build upon these observations. Methods: We enrolled previously untreated high-risk pts with AJCC 7th edition stage IVA-IVB SCCHN of the oral cavity, oropharynx (OP), hypopharynx, larynx. HPV+ OP tumors were T4, or N2c or N3 by AJCC 7th edition criteria. Nivo 3 mg/kg was administered every 2 weeks IV x 17 doses and ipi 1 mg/kg every 6 weeks x 6 doses beginning 2 weeks before IMRT 2 Gy/fraction/day given to total dose 70 Gy. The primary safety endpoint was acute in-field toxicity. Exploratory correlative studies include tumor PD-L1 expression, tumor immune bias, and exosome quantity and composition. The total sample size is 24 pts with 12 enrolled in the first stage and 12 in the expansion cohort. Results: A planned safety analysis was performed in the first 12 pts (8 OP; 1 hypopharynx; 3 larynx). No acute grade (G) 4 or dose-limiting toxicities were seen. Acute G 3 in-field toxicity occurred during RT in 50% of pts: dysphagia (4 pts), mucositis (3), odynophagia (2), hoarseness (1), dermatitis (1). 50% of pts had immune-related toxicity at any time. 3 pts discontinued treatment at > 3 months post RT: 1 for an immune-related cause (G 3 colitis that resolved with steroids), and 2 for non-immune-related causes (1 G 5 bleeding due to carotid rupture secondary to an in-field ulcer at 4 months post RT in a pt with complete response and 1 G 3 OP ulcer resolving with hyperbaric oxygen). 1 pt with a laryngeal primary developed a solitary lung metastasis vs new primary 6 months post RT. 10 of 12 pts are alive with no evidence of disease (follow up 7.2-18.4 months). Conclusions: Preliminary results with this non-platinum-containing RT plus IO regimen are encouraging. Longer follow-up is needed for assessment of late effects and efficacy. Enrollment is ongoing in the expansion cohort. This study is supported by BMS. Clinical trial information: NCT03162731.