Quality Of Exosomes Research Articles

Preparation of a CaTiO3/Al3+/Pr3+/Sm3+ nanocomposite for enrichment of exosomes in human serum

Exosomes (30–200 nm) play important roles in intercellular communication. Because their contents differ between healthy individuals and subjects diagnosed with various diseases, exosomes have been regarded as potential sources of biomarkers for clinical diagnosis. However, the accuracy of diagnosis by exosomal biomarkers is highly dependent on the extraction efficiency, yield, and the quality of exosomes. Hence, inexpensive, convenient, and fast exosome separation methods are required. In the present study, the CaTiO3/Al3+/Pr3+/Sm3+ nanocomposite was synthesized and applied in highly selective and efficient separation of exosomes. Notably, the developed material exhibited higher specificity and efficiency than commercially available TiO2. Moreover, CaTiO3/Al3+/Pr3+/Sm3+ could be reused at least three times without any significant decrease in efficiency. The synthesized material was also used for the extraction of exosomes from the serums of patients with Alzheimer's disease (AD) and healthy controls. The exosomes were subjected to two-dimensional gel electrophoresis (2-DE) separation and matrix-assisted laser desorption/ionization-time of flight (MALDI TOF/TOF) mass spectrometry analysis. It was found that five proteins in the exosomes were evidently upregulated, while one protein was downregulated. Among the detected proteins, serum amyloid P-component (SAP) has been reported to be closely related to pathogenesis of AD. The obtained results indicated that the developed method involving separation and analysis of serum exosomes could be used for disease diagnosis or postoperative clinical monitoring.

Comparison and Investigation of Exosomes Derived from Platelet-Rich Plasma Activated by Different Agonists

PRP-Exos are nanoscale cup-shaped vesicles that carry a variety of proteins, mRNAs, microRNAs, and other bioactive substances. PRP-Exos can be formed through several induction pathways, which determine their molecular profiles and facilitate their tailormade participation in intercellular communication. Currently, little is known on how the PRP-Exos activation method influences the quality and quantity of PRP-Exos. The present study aims to observe and analyze the number, profile, and growth factors of PRP-Exos through TEM, Nanoflow, and WB after PRP activation and compare the difference in function of PRP-Exos on HUVECs, with different stimuli (calcium gluconate, thrombin, or both). We found that PRP activated with both thrombin and calcium gluconate harvested the highest concentration of exosomes [(7.16 ± 0.46) × 1010 particles/ml], compared to thrombin group [(4.87 ± 0.15) × 1010 particles/ml], calcium gluconate group [(5.85 ± 0.43) × 1010 particles/ml], or saline group [(7.52 ± 0.19) × 109 particles/ml], respectively (P < 0.05) via Nanoflow analysis. The WB analysis showed that cytokines (VEGF, PDGFBB, bFGF, TGF-β) are differentially encapsulated in PRP-Exos, depending on the PRP stimulus, in which the mixture-PRP-Exos yielded the highest concentration of cytokines. In the function assay of PRP-Exos on HUVECs, the mixture-PRP-Exos promoted HUVECs proliferation, increased HUVECs migration, promoted the formation of vessel-like by HUVECs via the AKT ERK signal pathway more dramatically, compared with other groups. In summary, our studies showed that PRP activated by the mixture of calcium gluconate and thrombin harvested the best quality of exosomes which had the top biological functions. This study provides a protocol for selecting appropriate PRP activators to obtain high-quality exosomes for future applications.

How hypoxia regulate exosomes in ischemic diseases and cancer microenvironment?

Exosomes, as natural occurring vesicles, play highly important roles in the behavior and fate of ischemic diseases and different tumors. Secretion, composition, and function of exosomes are remarkably influenced by hypoxia in ischemic diseases and tumor microenvironment. Exosomes secreted from hypoxic cells affect development, growth, angiogenesis, and progression in ischemic diseases and tumors through a variety of signaling pathways. In this review article, we discuss how hypoxia affects the quantity and quality of exosomes, and review the mechanisms by which hypoxic cell-derived exosomes regulate ischemic cell behaviors in both cancerous and noncancerous cells.

Exosomes Derived from Cancer Associated Fibroblasts Elicit Survival and Drug Resistance of Primary Lymphoma Cells

BackgroundCancer associated fibroblasts (CAFs) are one of the major components constituting the tumor microenvironment and known to be deeply involved in the growth and metastasis of solid tumors. We previously reported that CAFs supported survival of primary lymphoma cells via the increased glycolytic metabolism (Aoki et al. Oncotarget 2017). Recent findings indicate that exosomes secreted from tumor cells play an important role of the survival and metastasis. Although the information about exosomes secreted from tumor cells has been accumulated, the role of them secreted from cells constituting the tumor microenvironment has been largely unexplored.PurposeTo uncover the role of exosomes in lymphoma microenvironment, we investigated the function of them derived from CAFs those were isolated from primary lymphoma samples.MethodsCAFs were successfully isolated from primary lymph node samples of various types of non-Hodgkin lymphoma including follicular lymphoma, diffuse large B-cell lymphoma, angioimmunoblastic T-cell lymphoma, mantle cell lymphoma, and T lymphoblastic lymphoma (N=20), and subsequently exosomes secreted from the representative 4 CAFs were obtained by a standard procedure using ultracentrifugation. Exosomes were confirmed by immunoblotting, electron microscope, and the nano tracking analysis. The functional role of CAFs and exosomes in an interaction between lymphoma cells and its microenvironment were investigated. Written informed consent for the experimental use of patient lymph node samples was obtained, and all experimental procedures were approved by the institutional review board of Nagoya University Hospital.ResultsWe isolated CAFs from different types of non-Hodgkin lymphoma samples, and studied whether the survival of patient lymphoma cells could be supported in co-culture with CAFs. The survival of cells differed depending on CAFs indicating the diversity of the ability of CAFs to support lymphoma cells. Then we analyzed cellular glycolysis and ATP production of lymphoma cells in co-culture with CAFs. As expected, the increase of glycolysis and production of ATP differed among 4 CAFs probably due to the extent of the Warburg effect. Next, we investigated whether CAFs secreted extracellular vesicles into culture supernatant. We found that vesicles with CD9 positive and matched exosomes in size were accumulated in culture media (Figure A, B), and the amount of released exosomes differed among CAFs in line with the ability to support the survival of lymphoma cells. Exosomes displayed survival support of lymphoma cells in a dose-dependent manner(Figure C), and cellular glycolysis and ATP production were increased in the presence of exosomes as well as CAFs, which indicated that the ability of CAFs to support lymphoma cells was, at least in part, elicited by exosomes. While exosomes secreted from CAFs with the strong ability to support lymphoma cells displayed the support of the survival, the survival of lymphoma cells was not observed in the presence of exosomes derived from CAFs with the weak support of lymphoma cells even in higher dose of exosomes, indicating that not only the quantity but also the quality of exosomes could be a determinant of the survival effect. Next, we focused on exosomes secretion-related proteins of nSMase2 and Rab27b to uncover the underlying mechanism of secretion of exosomes. NSMase2 required for exosomes formation and Rab27b involved in the migration of multivesicular body from the pericule to the membrane were strongly expressed in CAFs with higher ability to support lymphoma cells. Using nSMase2- and Rab27b- specific siRNA, the amount of exosomes secretion was reduced resulting in the decreased survival support. Finally, we studied the presence of CAFs or exosomes associated with the drug resistance. As expected, primary lymphoma cells demonstrated resistance to gemcitabine and cytarabine in the presence of CAFs or exosomes, and the resistance was restored in the presence of CAFs transduced with Rab27b-specific siRNA.ConclusionOur results suggest that CAFs and exosomes secreted from them are involved in the survival and drug resistance of patient lymphoma cells and play a pivotal role in the microenvironment of non-Hodgkin lymphoma. Exosomes would be a novel attractive therapeutic target. [Display omitted] DisclosuresKiyoi:Sanofi K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Novartis Pharma K.K.: Research Funding; Astellas Pharma Inc.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria.

Abstract 3162: Blood-based exosomal miRNAs as biomarkers for diagnosis and prognosis of clear cell renal cell cancer

Abstract Introduction &amp; objectives: In previous studies we identified specific miRNA alterations in tumor tissues of clear cell renal cell cancer (ccRCC) with diagnostic and prognostic value relating to the presence of metastasis. For few years it has been known, that miRNAs are actively packed in exosomes which can be released into body fluids. Therefore, we hypothesize that in a simple blood based test we are able to use specific miRNA signatures as minimal invasive biomarkers for confirmation of the diagnosis and evaluation of the metastatic risk in ccRCC. Materials &amp; methods: Initially, exosomes were isolated from 1 ml serum of 10 ccRCC patients (metastatic and non metastatic tumors) and 10 healthy volunteers using appropriated exosome isolation kit protocol (Thermo Fisher Scientific). Quality of exosomes was proven using exosomal markers (CD63, CD81, Alix, Synthenin) and Nano Particle Tracking Analysis. Exosomal totalRNA was isolated using miRNeasy Mini Kit (Qiagen). For miRNA analyses total RNA was reverse transcribed using TaqMan Reverse Transcription Kit (Thermo Fisher Scientific), cDNA was preamplified using TaqMan® PreAmp Master Mix (Thermo Fisher Scientific) and real time PCR was performed using Gene Expression master mix (Thermo Fisher Scientific). Expression differences were calculated using REST Software and SPSS. Results: We have shown that 1 ml serum is sufficient to analyze miRNA expression in exosomes. Exosomes isolated from serum exhibit high amount of exosomal markers and possess the typical exosomal size (30-120 nm). CcRCC serum samples are characterized by downregulation of several miRNAs (including miR-10b and miR451). Furthermore, specific miRNAs (including miR-30c) differentiate patients with or without metastases as well as healthy volunteers. Conclusion: These initial data confirm our hypothesis that the tissue based miRNA signature could be used as biomarkers for detection of aggressive ccRCC analyzing exosomes from liquid biopsy. This minimal invasive blood based test is easy to handle in clinical practice and could be a powerful tool for early detection and monitoring of metastatic disease. To validate these results the expansion of the sample set is ongoing. Citation Format: Joana Heinzelmann, Sophie Baumgart, Sebastian Hoelters, Martin Janssen, Michael Stöckle, Kerstin Junker. Blood-based exosomal miRNAs as biomarkers for diagnosis and prognosis of clear cell renal cell cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3162.

MP78-07 BLOOD BASED EXOSOMAL MIRNAS AS BIOMARKERS FOR DIAGNOSIS AND PROGNOSIS OF CLEAR CELL RENAL CELL CANCER

You have accessJournal of UrologyKidney Cancer: Epidemiology & Evaluation/Staging II1 Apr 2016MP78-07 BLOOD BASED EXOSOMAL MIRNAS AS BIOMARKERS FOR DIAGNOSIS AND PROGNOSIS OF CLEAR CELL RENAL CELL CANCER Joana Heinzelmann, Sophie Baumgart, Seastian Hölters, Martin Janssen, Michael Stöckle, and Kerstin Junker Joana HeinzelmannJoana Heinzelmann More articles by this author , Sophie BaumgartSophie Baumgart More articles by this author , Seastian HöltersSeastian Hölters More articles by this author , Martin JanssenMartin Janssen More articles by this author , Michael StöckleMichael Stöckle More articles by this author , and Kerstin JunkerKerstin Junker More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1963AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In previous studies we identified specific miRNA alterations in tumor tissues of clear cell renal cell cancer (ccRCC) with diagnostic and prognostic value relating to the presence of metastasis. For few years it has been known, that miRNAs are actively packed in exosomes which can be released into body fluids. Therefore, we hypothesize that in a simple blood based test we are able to use specific miRNA signatures as minimal invasive biomarkers for confirmation of the diagnosis and evaluation of the metastatic risk in ccRCC. METHODS Initially, exosomes were isolated from 1 ml serum of 10 ccRCC patients (metastatic and non metastatic tumors) and 10 healthy volunteers using appropriated exosome isolation kit protocol (Thermo Fisher Scientific). Quality of exosomes was proven using exosomal markers (CD63, CD81, Alix, Synthenin) and Nano Particle Tracking Analysis. Exosomal totalRNA was isolated using miRNeasy Mini Kit (Qiagen). For miRNA analyses total RNA was reverse transcribed using TaqMan Reverse Transcription Kit (Thermo Fisher Scientific), cDNA was preamplified using TaqMan® PreAmp Master Mix (Thermo Fisher Scientific) and real time PCR was performed using Gene Expression master mix (Thermo Fisher Scientific). Expression differences were calculated using REST Software and SPSS. RESULTS We have shown that 1 ml serum is sufficient to analyze miRNA expression in exosomes. Exosomes isolated from serum exhibit high amount of exosomal markers and possess the typical exosomal size (30-120 nm). CcRCC serum samples are characterized by downregulation of several miRNAs (including miR-10b and miR451). Furthermore, specific miRNAs (including miR-30c) differentiate patients with or without metastases as well as healthy volunteers. CONCLUSIONS These initial data confirm our hypothesis that the tissue based miRNA signature could be used as biomarkers for detection of aggressive ccRCC analyzing exosomes from liquid biopsy. This minimal invasive blood based test is easy to handle in clinical practice and could be a powerful tool for early detection and monitoring of metastatic disease. To validate these results the expansion of the sample set is ongoing. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1026-e1027 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Joana Heinzelmann More articles by this author Sophie Baumgart More articles by this author Seastian Hölters More articles by this author Martin Janssen More articles by this author Michael Stöckle More articles by this author Kerstin Junker More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Exosomes Secreted By Multiple Myeloma Cell Lines (MMCLs) and Bone Marrow Stromal Cells (BMSCs) Reciprocally Modulate Cell Adhesion and Impact on MM Cell Migration and Bortezomib-Induced Apoptosis

Introduction: In MM pathogenesis, the interaction of plasma cells and the bone marrow microenvironment plays a crucial role. Exosomes are secreted by nearly all cell types, including cancer cells, and have been implicated in intercellular communication, drug resistance and tumor progression. We therefore purified, characterized and studied the influence of exosomes secreted by MMCLs and BMSCs on cell adhesion, bortezomib-induced apoptosis and cell migration.Methods: Exosomes were purified from the supernatants of MMCLs (IM-9, MM.1S, U266, RPMI8226) and M2-10B4 BMSCs after 72h of culture with RPMI1640+10% exosome-depleted FBS by differential centrifugation. Purity and quality of exosomes was confirmed by electron microscopy and western blot for CD63. Exosomal protein concentration per 5x106 cells was quantified by Bradford. CD44 and ICAM1 were evaluated by flow cytometry. Apoptosis was assessed via annexin V/7-AAD-staining. Chemotaxis to M2-10B4 exosomes (100-500μg/ml) was studied using 24-well chemotaxis chambers. Migrated cells were counted by flow cytometry.Results: Exosomes from IM-9 cells were identified by electron microscopy, thereby showing a characteristic saucer-like morphology, ranging in diameter between 30 and 120nm. Both MM cell and M2-10B4 exosomes expressed CD63. Mean exosomal protein concentration was 1.4μg/μl for M2-10B4 and varied for MMCLs between 1.2 and 1.7μg/μl. M2-10B4 cells expressed higher levels of both adhesion molecules CD44 and ICAM-1 when incubated with exosomes from MMCLs compared to basal conditions, however, not reaching statistical significance. Conversely, M2-10B4 exosomes induced a significant increase in CD44 expression on IM-9 cells (p=0.03), reflecting an increased adhesion to components of the extracellular matrix that might contribute to cell-adhesion mediated drug resistance. IM-9 apoptotic cells after 24h with 10nM bortezomib decreased from 72% to 61% in the presence of M2-10B4 exosomes and from 74% to 62% with IM-9 exosomes, suggesting a lower sensitivity to bortezomib. Migration of IM-9 cells slightly increased with 200μg/ml M2-10B4 exosomes and decreased with higher exosome concentrations.Conclusions: Our findings underline the role of M2-10B4 exosomes on adhesion and migration of one representative MMCL (IM-9), and the impact of M2-10B4 and IM-9 exosomes on bortezomib cytotoxicity in this MMCL, suggesting their involvement in a paracrine/autocrine loop modulating treatment response and MM cell survival. DisclosuresNo relevant conflicts of interest to declare.