Exosomal Markers CD63 Research Articles

Proteomic Signature of Extracellular Vesicles for Lung Cancer Recognition.

The proteins of extracellular vesicles (EVs) that originate from tumors reflect the producer cells’ proteomes and can be detected in biological fluids. Thus, EVs provide proteomic signatures that are of great interest for screening and predictive cancer diagnostics. By applying targeted mass spectrometry with stable isotope-labeled peptide standards, we assessed the levels of 28 EV-associated proteins, including the conventional exosome markers CD9, CD63, CD81, CD82, and HSPA8, in vesicles derived from the lung cancer cell lines NCI-H23 and A549. Furthermore, we evaluated the detectability of these proteins and their abundance in plasma samples from 34 lung cancer patients and 23 healthy volunteers. The abundance of TLN1, TUBA4A, HSPA8, ITGB3, TSG101, and PACSIN2 in the plasma of lung cancer patients was measured using targeted mass spectrometry and compared to that in plasma from healthy volunteers. The most diagnostically potent markers were TLN1 (AUC, 0.95), TUBA4A (AUC, 0.91), and HSPA8 (AUC, 0.88). The obtained EV proteomic signature allowed us to distinguish between the lung adenocarcinoma and squamous cell carcinoma histological types. The proteomic cargo of the extracellular vesicles represents a promising source of potential biomarkers.

MP39-13 FUNCTION OF EXOSOMAL ANTITUMOR MICRORNA-1 IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP39)1 Sep 2021MP39-13 FUNCTION OF EXOSOMAL ANTITUMOR MICRORNA-1 IN RENAL CELL CARCINOMA Issei Kawakami, Hirofumi Yosino, Hideki Enokida, and Masayuki Nakagawa Issei KawakamiIssei Kawakami More articles by this author , Hirofumi YosinoHirofumi Yosino More articles by this author , Hideki EnokidaHideki Enokida More articles by this author , and Masayuki NakagawaMasayuki Nakagawa More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002054.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: MicroRNAs, a class of small non-coding RNAs about 22 nucleotides in length, are involved in many cellular processes and aberrantly expressed in cancer tissues. For example, miR-1 functions as a tumor suppressor in renal cell carcinoma (RCC) as demonstrated in our previous study. Exosomes are 40–100 nm nano-sized extracellular vesicles, and are receiving increasing attention as novel structures participating in intracellular communication. We hypothesized that miR-1 functions as a tumor suppressor through exosomes in RCC, and investigated that in this study. METHODS: First, we performed statistical analyses of miR-1 based on the TCGA database of clear cell RCC (ccRCC) patients. We also checked miR-1 expression in serum exosomes between healthy controls and RCCs. Exosomes in cell lysate and human serum were isolated by a spin column-based method, and determined using Nanosight nanoparticle tracking analysis and western blot analysis with exosome marker CD63. Exosomes labeled with PKH26 were used to indicate that exosomes were fused into recipient cells, and miR-1 expressions were investigated by RT-PCR in recipient RCC cells with the addition of miR-1 transfected cell derived exosomes. By using the recipient RCC cells, we performed functional analyses such as proliferation, migration and invasion. We also performed functional analyses by using serum exosomes derived from RCC patients with IVC invasion or metastasis. In addition, we performed RNA sequence analyses to identify the mechanism in RCC cell with the addition of exosomal miR-1. RESULTS: MiR-1 expression was significantly downregulated in clinical ccRCC samples compared to that in normal kidney samples (p<0.001), and patients with low miR-1 expression (n=455) had poor overall survival (p=0.0447) in comparison to patients with high expression (n=50). Exosomes in cell lysate and human serum were detected by Nanosight and western blot analysis with CD63. Exosomes labeled with PKH26 were detected in the recipient cells, and miR-1 expressions were elevated nearly 40 and 10 times in exosomal miR-1 fused 786o and A498 cells respectively compared to in exosomal miR-control fused cells. Exosomal miR-1 also significantly inhibited cell proliferation, migration and invasion compared to exosomal miR-control. Furthermore, RNA sequence analyses showed that several genes and pathways might be regulated by exosomal miR-1. CONCLUSIONS: This study showed that exosomal miR-1 might exogenously function as a therapeutic target in the treatment of RCCs. In addition, exosomal miR-1 and/or other miRNAs could be a diagnostic tumor marker of RCCs. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e709-e709 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Issei Kawakami More articles by this author Hirofumi Yosino More articles by this author Hideki Enokida More articles by this author Masayuki Nakagawa More articles by this author Expand All Advertisement Loading ...

Abstract MP36: Human Urinary Exosomes Contain Functional ACE2

The Ang II convertase and SARS-COV-2 co-receptor ACE2 is highly expressed on proximal tubules within the kidney. ACE2 is also present in urine and reportedly correlates with various renal pathologies that may reflect enhanced shedding of the peptidase through activation of ADAMs. Indeed, 95 kDa ACE2 is typically detected in urine consistent with a shorter, soluble form of the peptidase; however, the full-length, membrane-bound form of ACE2 (120 kDa) is also evident in urine which is difficult to reconcile with ACE2 shedding. To account for these isoforms, we evaluated ACE2 expression in exosomes isolated from human urine. Morning collections from males [50 to 64 years of age, non-smokers] were immediately processed for exosome isolation by cibacron blue binding of albumin followed by 0.2 μmicron filtration to remove microvesicles and apoptotic bodies, Amicon 100 kDa concentration, and ultracentrifugation (UC) to pellet exosomes. Analysis of the UC pellet fraction revealed the exosomal markers ALIX, CD63 and HSP70, as well as the proximal tubule peptidases neprilysin (NEP) and ACE2. Exosomal ACE2 content was 45 ± 11 ng/mL (mean ± SEM; N=5) by ELISA and exosomal activity hydrolyzed Ang II to Ang-(1-7) that was abolished by the ACE2 inhibitor MLN4760. Fluorescent nanotracking analysis (f-NTA) with Alexa Fluor antibodies and CellMask Deep Red membrane stain (CMDR) demonstrate a similar density of ACE2+ and NEP+ exosomes that were ~50% of total urinary exosomes (*P&lt;0.05 vs. CD63+, N=3) while particle sizes were comparable and in the expected range of exosomes (100-150 nm). We conclude that human urinary exosomes express functional ACE2 which may originate from proximal tubule release.

Protective effects of intravitreal administration of mesenchymal stem cell-derived exosomes in an experimental model of optic nerve injury

Traumatic optic neuropathy results in the loss of retinal ganglion cells (RGCs), leading to unavoidable visual impairment. However, there is no effective therapy by far. Accumulated studies support the perception that mesenchymal stem cells (MSCs) secrete exosomes that serve as a protective paracrine factor. The study aimed to explore and evaluate the potential therapeutic effects of intravitreal transplantation of MSC-derived exosomes (MSC-exos) in an experimental model of optic nerve crush (ONC). Exosomes were isolated from rat MSCs and characterized by transmission electron microscope and western blotting. At the onset of ONC, a single intravitreal injection of exosomes or PBS was administered to the rats. At day 30, hematoxylin and eosin staining, immunohistochemistry, and βIII-tubulin staining were performed to evaluate the survival of RGCs. Moreover, TUNEL assay was used to examine the apoptosis of RGCs. Inflammation-relevant factors were identified via quantitative polymerase chain reaction. The expression levels of cell apoptosis-related molecules and key members of the PI3K/AKT signaling pathway were determined via western blot analysis. We found that MSC-exos exhibited typical characteristic morphologies (cup-shaped) and sizes (peak size of 93 nm). Furthermore, they exhibited substantial expression of the exosome markers CD63 and TSG101, but lacked the expression of the cellular marker GM130. Treatment with intravitreal MSC-exos notably promoted the survival of RGCs in ONC rats. The level of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-8, and MCP-1, were reduced, whereas those of the anti-inflammatory factor IL-10 were increased. Moreover, the apoptosis induced by ONC was decreased by the administration of MSC-exos via upregulation of the Bcl-2/Bax ratio and downregulation of caspase-3 activity. Furthermore, MSC-exos significantly stimulated AKT phosphorylation, whereas LY294002 restored the apoptosis-preventing effects of MSC-exos. The results of our results demonstrated that intravitreal administration of MSC-exos ameliorates ONC-induced injury in a rat model. These findings might aid in the development of effective exosome-based therapeutic strategies for the treatment of optic nerve degeneration.

Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function.

Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissues characterized by scant T cell infiltration and predominance of immunosuppressive myeloid cells. Given the important roles of extracellular vesicles (EVs) in cancer-host crosstalk, we hypothesized that EVs secreted by EwS tumors target myeloid cells and promote immunosuppressive phenotypes. Here, EVs were purified from EwS and fibroblast cell lines and exhibited characteristics of small EVs, including size (100–170 nm) and exosome markers CD63, CD81, and TSG101. Treatment of healthy donor-derived CD33+ and CD14+ myeloid cells with EwS EVs but not with fibroblast EVs induced pro-inflammatory cytokine release, including IL-6, IL-8, and TNF. Furthermore, EwS EVs impaired differentiation of these cells towards monocytic-derived dendritic cells (moDCs), as evidenced by reduced expression of co-stimulatory molecules CD80, CD86 and HLA-DR. Whole transcriptome analysis revealed activation of gene expression programs associated with immunosuppressive phenotypes and pro-inflammatory responses. Functionally, moDCs differentiated in the presence of EwS EVs inhibited CD4+ and CD8+ T cell proliferation as well as IFNγ release, while inducing secretion of IL-10 and IL-6. Therefore, EwS EVs may promote a local and systemic pro-inflammatory environment and weaken adaptive immunity by impairing the differentiation and function of antigen-presenting cells.

The Diagnostic Value of Serum Exosomal Has_circ_0000615 for Breast Cancer Patients.

BackgroundTo explore the expression level of has_circ_0000615 in peripheral blood samples and evaluate its diagnostic value for breast cancer patients.MethodsThe peripheral blood samples of 95 breast cancer patients who underwent curative surgical resection and 95 age-matched healthy volunteers in our institutions from September 2019 to November 2020 were systematically collected. The expression level of has_circ_0000615 in the plasma was amplified and detected by qRT-PCR, and its correlation to clinicopathological characteristics of breast cancer patients were analyzed.ResultsBreast cancer patients had a significantly higher expression level of has_circ_0000615 in the plasma than healthy controls (P < 0.01), and its high expression was closely associated with advanced tumor stage (P=0.010), lymph node metastasis (P = 0.001) and high grade of recurrence risk (P=0.012). The receiver operator characteristic (ROC) curves showed that the area under curve (AUC) value, sensitivity and specificity of has_circ_0000615 for the diagnosis of non-metastatic breast cancer was 0.904 (95% CI: 0.863–0.944), 76.8% and 88.4%, respectively. Serum has_circ_0000615 expression had a better diagnostic efficiency than routine tumor biomarkers such as CA153, CA125 and CEA for distinguishing breast cancer patients from healthy individuals. TEM revealed that isolated exosomes from the culture medium of breast cancer cells had a disk-like appearance with a diameter of 80–200 nm vesicles, and the expression of exosome markers CD9 and CD81 was markedly increased. More importantly, the expression of has_circ_0000615 was detected in the exosomes and its expression level was markedly upregulated in breast cancer cell lines compared with normal ductal epithelial cells. The stability assay showed that there was no difference between RNA extraction at 0 hour and 24 hours in terms of the expression of has_circ_0000615 (P =0.327). Has_circ_0000615 might as exosomes be secreted into the circulating blood of breast cancer patients, resulting in a high expression level in plasma samples.ConclusionThe detection of has_circ_0000615 might be a promising diagnostic method for breast cancer.

Protective effect of MSC-derived exosomes against cisplatin-induced apoptosis via heat shock protein 70 in auditory explant model

Therapeutics based on stem cell technology, including stem cell-derived exosomes, have emerged in recent years for the treatment of what were otherwise considered incurable diseases. In this study, we evaluated the efficacy of human MSC-derived exosomes for protection against cisplatin induced ototoxic hearing loss. Incubation of cochlear explants with MSC-derived exosomes prior to addition of cisplatin induced a reduction in cisplatin-induced drug toxicity in auditory hair cells but not when the exosomes were introduced simultaneously with or after cisplatin. The delivery of MSC-derived exosomes to cochlear explants was confirmed by the increasing protein levels of the exosome markers CD63 and HSP70 to reduce apoptosis. These results were consistent with those from a model in which MSC-derived exosomes protect auditory hair cells from cisplatin-induced drug toxicity in an ex vivo cochlear explant model and support future studies into the therapeutic benefits of stem cell-derived exosomes in clinical applications.

The Depolarization-Evoked, Ca2+-Dependent Release of Exosomes From Mouse Cortical Nerve Endings: New Insights Into Synaptic Transmission.

Whether exosomes can be actively released from presynaptic nerve terminals is a matter of debate. To address the point, mouse cortical synaptosomes were incubated under basal and depolarizing (25 mM KCl-enriched medium) conditions, and extracellular vesicles were isolated from the synaptosomal supernatants to be characterized by dynamic light scattering, transmission electron microscopy, Western blot, and flow cytometry analyses. The structural and biochemical analysis unveiled that supernatants contain vesicles that have the size and the shape of exosomes, which were immunopositive for the exosomal markers TSG101, flotillin-1, CD63, and CD9. The marker content increased upon the exposure of nerve terminals to the high-KCl stimulus, consistent with an active release of the exosomes from the depolarized synaptosomes. High KCl-induced depolarization elicits the Ca2+-dependent exocytosis of glutamate. Interestingly, the depolarization-evoked release of exosomes from cortical synaptosomes also occurred in a Ca2+-dependent fashion, since the TSG101, CD63, and CD9 contents in the exosomal fraction isolated from supernatants of depolarized synaptosomes were significantly reduced when omitting external Ca2+ ions. Differently, (±)-baclofen (10 µM), which significantly reduced the glutamate exocytosis, did not affect the amount of exosomal markers, suggesting that the GABAB-mediated mechanism does not control the exosome release. Our findings suggest that the exposure of synaptosomes to a depolarizing stimulus elicits a presynaptic release of exosomes that occurs in a Ca2+-dependent fashion. The insensitivity to the presynaptic GABAB receptors, however, leaves open the question on whether the release of exosomes could be a druggable target for new therapeutic intervention for the cure of synaptopathies.

Analysis of extracellular vesicles as a potential index for monitoring differentiation of neural lineage cells from induced pluripotent stem cells

To improve cell production efficacy, it is important to evaluate cell conditions during culture. Extracellular vesicles (EVs) secreted from various cells are involved in stem cell differentiation. As EVs carry information about their source cells, we hypothesized that they may serve as a noninvasive index of cell conditions. We evaluated changes in EV morphology, concentration, and microRNA (miRNA) and protein expression in culture supernatants during the differentiation of induced pluripotent stem cells (iPSCs) into neural lineage cells, for application in regenerative medicine for Parkinson's disease. We observed EVs (50-150nm) in culture supernatants of iPSCs and differentiated cells. The EVs expressed the exosome markers CD63, CD81, and CD9. Throughout differentiation, the EV concentration in the supernatants decreased, and EV miRNA and protein expression changed substantially. Especially, miR-106b, involved in neural stem cell differentiation and normal brain development, was considerably downregulated. CD63 expression correlated with the CORIN-positive cell rate, which is an index of differentiation. Thus, EV concentration and miRNA and protein expression may reflect the differentiation status of iPSCs. These findings pave the way for the development of novel and sensitive cell culture monitoring methods.

Abstract 2903: The exosome marker CD63 and Caveolin-1 in breast cancer patients

Abstract The tetraspanin CD63, a proposed exosome marker, has been reported to be expressed in solid tumors. For instance, CD63 status in carcinoma cells was reported to be a significant independent prognostic factor in gastric cancer patients, with its overexpression causing adversity in patients with gastrointestinal stromal tumors. However, the expression of CD63 has remained virtually unknown in breast cancer pathological specimens. In addition, the detection of high levels of exosomes expressing CD63 and Caveoloin-1 in the plasma of melanoma patients was reported as a novel tool for the clinical management of cancer patients. Caveolin-1 has also been reported to negatively regulate the endocytosis of exosomes. Tetraspanin proteins are shown to form complicated interaction networks in cells to exert their functions. In this study, we immunolocalized CD63 and Caveolin-1 in the surgical pathology specimens of 88 breast cancer patients. CD63 was immunolocalized in the cytoplasm of both the cancer and stromal cells. The number of CD63-positive cancer cells was significantly higher in HER2-positive patients and the levels of CD63-positive stromal cells were also significantly higher in those with high pathologic T factor (pT) and proliferation marker protein Ki-67 labeling index and low pathologic N factor (pN). The number of CD63-double-positive cancer cells and stromal cells was associated with high histological grade, high pT, and high Ki67 expressions. CD63-positive cancer cells and Caveolin-1-positive cancer cells were both significantly higher in Estrogen receptor-positive breast cancer patients with high histological grade and pT. To the best of our knowledge, this is the first study demonstrating the significance of CD63 in breast cancer. Our results indicated that CD63-double-positive cancer cells and stromal cells could be involved in clinically and biologically aggressive subtypes of breast cancer. Further studies are required to clarify the clinical significance of the associations between CD63 and Caveolin-1 in patients with breast cancer. Citation Format: Erina Iwabuchi, Yasuhiro Miki, Ayako Kanai, Takanori Ishida, Hironobu Sasano. The exosome marker CD63 and Caveolin-1 in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2903.

Urinary Exosomal CA9 mRNA as a Novel Liquid Biopsy for Molecular Diagnosis of Bladder Cancer.

ObjectiveThe objective of this study was to assess the possibility of using urinary exosomal CA9 mRNA as a novel liquid biopsy for the molecular diagnosis of bladder cancer.Patients and MethodsA total of 168 bladder cancer patients and 90 control subjects were included in the study. An isolation kit was used to isolate urinary exosomes. Transmission electron microscopy (TEM) was used to examine the presence of exosomes. Flow cytometry was used to examine the exosomal marker CD63. The expression level of exosomal CA9 mRNA was detected by RT-qPCR. The diagnostic performance of urinary urinary exosomal CA9 mRNA was evaluated.ResultsTEM confirmed the enriched exosomes from urinary bladder patients. Flow cytometry indicated a strong positive expression of exosome marker CD63. Successful extraction of RNA was performed from exosome samples. The level of urinary exosomal CA9 mRNA was significantly higher in bladder cancer group than in control group (p<0.001). The area under the ROC curve was 0.837 (95% CI: 0.743–0.859) with a sensitivity of 85.18% and a specificity of 83.15% for the diagnosis of bladder cancer.ConclusionWe found that the urinary exosomes were abundant in the urine of bladder cancer patients. CA9 mRNA could be detectable in urinary exosomes. The urinary exosomal CA9 mRNA may present a new liquid biopsy for the diagnosis of bladder cancer.

Isolation of extracellular vesicles from byproducts of cheesemaking by tangential flow filtration yields heterogeneous fractions of nanoparticles

Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, β-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV.

Abstract 561: ALK fusions can be clearly detected in extracellular vesicles from NSCLC cell lines: A become of hope for non-invasive ALK testing

Abstract Background: Identification of ALK rearrangements in NSCLC patients has important implications as it significantly expands their therapeutic opportunities. However, the availability of tumors biopsies is sometimes limited in the clinical setting. The analysis of liquid biopsies can potentially overcome such limitation. Nevertheless, the identification of ALK rearrangements using liquid biopsies remains challenging as fusion transcripts are rapidly degraded by RNAses present in the blood. Nonetheless, encapsulated RNA inside extracellular vesicles (EVs) is protected from RNAses. The main objective of this study is to explore the potential value of exosomes for non-invasive ALK testing. Methods: H3122 and H2228 cell lines, derived from NSCLC patients with EML4-ALK variant 1 (E13;A20) and variant 3 (E6a/b;A20) respectively, were used to isolate exosomes by the sequential centrifugation of cell culture supernatants. Briefly, supernatants were centrifuged twice for 10 min at 200xg, twice for 500xg for 10 min, once for 30 min at 10,000xg and a final ultracentrifugation at 100,000×g for 2 h at 4 °C. Purified exosomes were verified in terms of morphology and size by transmission electron microscopy (TEM). In addition, specific EVs markers, namely CD63, CD81 and CD9, were measured by flow cytometry and Western-Blot. EML4-ALK variants were analyzed at the protein level by Western blot using 5A4 antibody, and RNA level by digital PCR (dPCR), using a QuantStudio® 3D Digital PCR System and specific TaqMan® assays for EML4-ALK variant 1 and 3. Samples were also submitted to NGS analysis using the Oncomine™ Pan-Cancer Cell-Free Assay kit. Results: TEM showed spherical vesicles around 100 nm in both cell lines. Flow cytometry and Western blot confirmed expression of the exosomal specific markers CD63, CD81 and CD9 tetraspanins. Western blot also showed EML4-ALK variant 1 (120 kDa) and variant 3 (87 kDa) in H3122 and H2228 purified exosomes, respectively. Isolated exosome RNA profile was composed of small RNA (&amp;lt; 200 nt), although 18S and 28S rRNA peaks were also present. dPCR and NGS confirmed EML4-ALK rearrangements in exosome derived from both cell lines. Conclusions: EML4-ALK fusions can be detected at the RNA levels and at the protein levels analyzing EVs derived from H3122 and H2228 cell lines. These results set the stage for the development of EV-based non-invasive ALK testing in NSCLC patients. Citation Format: Estela Sánchez-Herrero, Carmen Campos-Silva, Yaiza Cáceres Martell, Sandra Sanz-Moreno, Roberto Serna-Blasco, Alejandro Rodríguez Festa, Miguel Ángel Molina-Vila, Mariano Provencio, Mar Valés-Gómez, Atocha Romero. ALK fusions can be clearly detected in extracellular vesicles from NSCLC cell lines: A become of hope for non-invasive ALK testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 561.

Marrow Stromal Cells Secrete FGF2 in Exosomes, Providing Paracrine Protection for Leukemia Cells from Kinase Inhibitors

Marrow Stromal Cells Secrete FGF2 in Exosomes, Providing Paracrine Protection for Leukemia Cells from Kinase Inhibitors

Abstract P-43: Effect of Glucocerebrosidase Dysfunction on the Pool of Plasma Exosomes of Patients with Gaucher Disease

Background: Extracellular vesicles (EVs) are small membrane vesicles released from different types of cells. EVs are found in many human biological fluids. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies with the plasma membrane. This type of vesicles is characterized by specific exosomal markers. Exosomes extracted from peripheral body liquids could have specific properties associated with different physiological conditions as well as human disorders, including neurodegenerative diseases. Gaucher disease (GD) – is the most common form of lysosomal storage disorders caused by mutations in the glucocerebrosidase (GBA) gene. Lysosome functionality is critical for the regulation of extracellular vesicle secretion and content. In model animals, the inhibition of glucocerebrosidase has been shown to increase the secretion of extracellular vesicles in brain tissues. Amount evaluation of EVs and their size in the biological fluids of patients with GD has not been early performed; therefore, it is unknown whether lysosomal dysfunction found in GD patients influences the plasma pool of EVs. The aim of this study was to evaluate the amount of blood plasma EVs in patients with GD and their characterization for morphology and size. Methods: EVs were isolated from the blood plasma of 8 GD patients and 8 controls by ultracentrifugation, and were characterized using cryo-electron microscopy (cryo-EM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS). Also, the presence of exosomal markers CD9, CD63, CD81, and HSP70 was analyzed by flow cytometry and western blot. Results: Here, it was first shown an increased proportion of exosome fraction in EVs from plasma of GD patients compared to controls by DLS and cryo-EM (p&lt;0.001) that was confirmed by mode size detected by NTA (p&lt;0.02). Moreover, an increased number of double and multilayer vesicles in plasma EVs from GD patients was demonstrated by cryo-EM. We also detected an increase in the expression of exosomal markers on the surface of vesicles from the blood plasma of patients with GD compared to controls. Conclusion: Here, we firstly report that the exosomes obtained from the blood plasma of GD patients have a larger size and altered morphology. Thus, we have shown that lysosomal dysfunction in GD patients leads to a striking alteration of blood plasma extracellular vesicle pool.

Bovine Mammary Alveolar Mac-T Cells Secrete Exosomes With Properties Similar to Bovine Milk Exosomes (BMEs)

ObjectivesExosomes are natural nanoparticles that play an important role in cell-to-cell communication. Communication is achieved through the binding of exosomes to the surface of recipient cells or delivery of regulatory cargos such as microRNAs (miRs) to recipient cells. Exosomes can be detected in most body fluids including milk. We reported that exosomes and miR cargos do not originate exclusively in endogenous synthesis but humans and other mammals absorb BMEs. The field of BME bioavailability is somewhat controversial and would benefit from the availability of a tool that facilitates BME tracking in organisms. The objective of this study was to compare the characteristics of BMEs and exosomes secreted by Mac-T cells as a step toward using Mac-T cell exosomes in future studies of BME bioavailability and distribution. MethodsExosomes were isolated from Mac-T conditioned culture media and skim milk by using ultracentrifugation. Exosome count, size, morphology, and purity were assessed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and immunoblot analysis, respectively. MiR cargos were assessed by qPCR. Levene’s test, Shapiro-Wilk test, and unpaired t-test were applied for statistical analysis; p < 0.05 was considered statistically significant. ResultsMorphology (not shown) and size were similar in BMEs (101 + 7.6 nm) and Mac-T derived exosomes (94 ± 10 nm; n = 3, p > 0.05). Exosome marker proteins, CD9, CD63 and TSG101, were detected in both BMEs and Mac-T cell-derived exosomes. Likewise, both preparations expressed miR-320a, miR-200c and let7a (p > 0.05). ConclusionsMac-T cell-derived exosomes have characteristic similar to BMEs and may be a useful tool in studies of BME bioavailability and distribution. Funding SourcesNIH 1P20GM104320, NIFA 2016-67,001-25,301 and 2020-67,017-30,834, USDA Hatch-1,011,996 and W4002 (all to J.Z.). J.Z is a consultant for PureTech Health, Inc.

Delivery of miR-224-5p by Exosomes from Cancer-Associated Fibroblasts Potentiates Progression of Clear Cell Renal Cell Carcinoma

Objectives Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma. Cancer-associated fibroblasts (CAFs) as the primary components of cancer stroma can affect tumor progression by secreting exosomes, while exosomes are carriers for proteins, nucleic acids, and other agents that responsible for delivery of biological information. Given this, exosomes derived from CAFs are emerging as promising biomarkers in clinical cancer diagnosis. Nevertheless, their role in clear cell renal cell carcinoma (ccRCC) remains poorly understood. Methods Here, we separated fibroblasts from ccRCC tissue, extracted exosomes, observed their morphology, and detected the expression of exosome marker proteins including Hsp70, CD9, and CD63. In the meantime, we labeled exosomes and performed coculture experiment to verify the delivery of miR-224-5p from CAFs to 769-P cells with exosomes as a carrier, so as to clarify the effect of CAF-derived exosomes on ccRCC cell malignant behaviors, as well as to discuss how miR-224-5p involves in above regulation. Results Transmission electron microscopy was firstly applied, and it was noted that the exosomes we isolated were in normal range. Besides, Western blot also confirmed the presence of exosome marker proteins Hsp70, CD9, and CD63. Furthermore, coculture experiments were performed and the CAF-derived exosomes were observed to be able to facilitate the malignant behaviors of ccRCC cells, and the exosomal miR-224-5p could be internalized by ccRCC cells to participate in regulation of cell proliferation, migration, invasion, and apoptosis. Conclusion To sum up, miR-224-5p can enter ccRCC cells via CAF-derived exosomes, in turn, promoting the malignant behaviors of ccRCC cells, which indicates that miR-224-5p has the potential severing as a therapeutic target for ccRCC.

Unraveling Secretory Mechanisms that Control Pentraxin 3 Secretion in Adipocytes During Inflammation

As a soluble pattern recognition receptor, Pentraxin 3 (PTX3) plays an important role in innate immunity and obesity-associated metabolic inflammation. PTX3 is abundantly expressed and secreted in adipocytes in response to lipopolysaccharide (LPS) stimulation. Appropriate regulation of PTX3 secretion is critical for maintaining inflammatory homeostasis. This study aims to unravel the mechanisms that control PTX3 secretion in adipocytes during LPS-induced inflammation. Upon 6h treatment of LPS, PTX3 expression and secretion were significantly induced in 3T3-L1 and stromal-vascular (SV) differentiated adipocytes, but to a lesser extent in SV cells or 3T3-L1 fibroblasts. However, LPSdoes not significantly stimulate PTX3 expression and secretion in macrophages. Using chemical inhibitors of conventional and unconventional protein secretion, we explored the mechanisms for controlling LPS-stimulated PTX3 secretion. 3T3-L1 adipocytes were treated with LPS for 6h in the presence or absence of various inhibitors blocking protein secretion from the Golgi complex (Monensin and Brefeldin A), mitochondrial oxidation (carbonyl cyanide 3-chlorophenylhydrazone [CCCP]), autophagy-lysosome (chloroquine and 3-methyladenine) and inflammasome (Bay 11–7082 and wedelolactone) activation, or exosome synthesis and trafficking (GW4869, manumycin A, calpeptin, and Y-27632). There were no significant effects of all inhibitors except for Monensin, Brefeldin A, and CCCP on intracellular and secreted levels of PTX3 in adipocytes. We found that Monensin and Brefeldin A significantly blocked LPS-stimulated PTX3 secretion, resulting in cellular PTX3 accumulation in adipocytes. Disrupting mitochondrial membrane potential by CCCP caused the reduction in PTX3 secretion from adipocytes. Additionally, we detected PTX3 in exosomes isolated from LPS-treated adipocytes. Inhibiting exosome synthesis by Manumycin A attenuated LPS-stimulated PTX3 secretion in both adipocyte culture media and isolated exosomes but not in the non-exosomal fraction of media, suggesting the involvement of the exosomal pathway in PTX3 secretion. However, the levels of exosomal PTX3 were significantly lower than that of the non-exosomal PTX3, and only 4.3% of secreted PTX3 was detected in the exosomal fraction of cultural media. Inhibiting the Golgi complex pathway blocked both the exosomal and non-exosomal secretion of PTX3 in adipocytes. After further fractionation of isolated crude exosomes by the iodixanol density gradient centrifugation, we showed that the majority of PTX3 was found in the non-extracellular vesicular (EV) fractions; only a small portion of secreted PTX3 overlapped with the exosomal marker CD63 in the small EV fractions. We conclude that PTX3 is secreted mainly through the conventional protein secretion pathway and minimally through the exosomal or EV pathway in response to LPS stimulation.

Comparing Approaches to Normalize, Quantify, and Characterize Urinary Extracellular Vesicles.

Urinary extracellular vesicles (uEVs) are a promising source for biomarker discovery, but optimal approaches for normalization, quantification, and characterization in spot urines are unclear. Urine samples were analyzed in a water-loading study, from healthy subjects and patients with kidney disease. Urine particles were quantified in whole urine using nanoparticle tracking analysis (NTA), time-resolved fluorescence immunoassay (TR-FIA), and EVQuant, a novel method quantifying particles via gel immobilization. Urine particle and creatinine concentrations were highly correlated in the water-loading study (R2 0.96) and in random spot urines from healthy subjects (R2 0.47-0.95) and patients (R2 0.41-0.81). Water loading reduced aquaporin-2 but increased Tamm-Horsfall protein (THP) and particle detection by NTA. This finding was attributed to hypotonicity increasing uEV size (more EVs reach the NTA size detection limit) and reducing THP polymerization. Adding THP to urine also significantly increased particle count by NTA. In both fluorescence NTA and EVQuant, adding 0.01% SDS maintained uEV integrity and increased aquaporin-2 detection. Comparison of intracellular- and extracellular-epitope antibodies suggested the presence of reverse topology uEVs. The exosome markers CD9 and CD63 colocalized and immunoprecipitated selectively with distal nephron markers. Conclusions uEV concentration is highly correlated with urine creatinine, potentially replacing the need for uEV quantification to normalize spot urines. Additional findings relevant for future uEV studies in whole urine include the interference of THP with NTA, excretion of larger uEVs in dilute urine, the ability to use detergent to increase intracellular-epitope recognition in uEVs, and CD9 or CD63 capture of nephron segment-specific EVs.

Subendothelial Accumulation of Exosomes and Coronary Microvascular Dysfunction in Mice Lacking Acid Ceramidase

Coronary microvascular dysfunction (CMD) has been demonstrated to be one of the major factors contributing to ischemic heart disease. However, the mechanisms initiating CMD remain poorly understood. The present study tested the hypothesis that lysosomal acid ceramidase finely controls subendothelial accumulation of exosomes leading CMD and subsequent myocardial ischemia. Using an endothelial-specific acid ceramidase (AC) gene (mouse gene code: Asah1) knockout (Asah1fl/fl/ECcre) mouse model fed with the Western diet (WD), we found that Asah1fl/fl/ECcre mice on the WD have more thickening of mid-layer in their small coronary arteries as shown significantly increased media/lumen ratio compared to the WD-fed wild type littermates (WT/WT). By nanoparticle tracking analysis (NTA) using NanoSight NS300 nanoparticle analyzer, we also observed that exosomes (~80-130 nm) in the blood stream were markedly increased in WD-fed Asah1fl/fl/ECcre mice compared to WT/WT mice. There were also more exosomes detected in the subendothelial space of coronary arterial wall, as shown by remarkable increase in colocalization of exosome marker CD63 or Annexin II with proteoglycans, bioglycan or decorin (subendothelial matrix markers) in Asah1fl/fl/ECcre mice. By electron microscopy (EM), we further confirmed that Asah1fl/fl/ECcre mice on the WD also significantly augmented the accumulation of exosomes under the endothelium small coronary arteries. Functionally, electrocardiogram (ECG) recording showed that in response to ergonovine, a coronary artery constrictor, the elevation of S-T segment was more remarkable in WD-fed Asah1fl/fl/ECcre than WT/WT mice. Using HypoxyProbe-1, the myocardial ischemic response to ergonovine was also detected more in the heart of WD-fed Asah1fl/fl/ECcre than WT/WT mice. Together, these results indicate that subendothelial exosome accumulation due to defect of lysosomal AC gene expression and activity may be a crucial pathogenic factor for CMD.