Effects of an acute exercise bout in hypoxia on extracellular vesicle release in healthy and prediabetic subjects.

The purpose of this study is to investigate exosome-like vesicle (ELV) plasma concentrations and markers of multivesicular body (MVB) biogenesis in skeletal muscle in response to acute exercise. Seventeen healthy [body mass index (BMI): 23.5 ± 0.5 kg·m-2] and 15 prediabetic (BMI: 27.3 ± 1.2 kg·m-2) men were randomly assigned to two groups performing an acute cycling bout in normoxia or hypoxia ([Formula: see text] 14.0%). Venous blood samples were taken before (T0), during (T30), and after (T60) exercise, and biopsies from m. vastus lateralis were collected before and after exercise. Plasma ELVs were isolated by size exclusion chromatography, counted by nanoparticle tracking analysis (NTA), and characterized according to international standards, followed by expression analyses of canonical ELV markers in skeletal muscle. In the healthy normoxic group, the total number of particles in the plasma increased during exercise from T0 to T30 (+313%) followed by a decrease from T30 to T60 (-53%). In the same group, an increase in TSG101, CD81, and HSP60 protein expression was measured after exercise in plasma ELVs; however, in the prediabetic group, the total number of particles in the plasma was not affected by exercise. The mRNA content of TSG101, ALIX, and CD9 was upregulated in skeletal muscle after exercise in normoxia, whereas CD9 and CD81 were downregulated in hypoxia. ELV plasma abundance increased in response to acute aerobic exercise in healthy subjects in normoxia, but not in prediabetic subjects, nor in hypoxia. Skeletal muscle analyses suggested that this tissue did not likely play a major role of the exercise-induced increase in circulating ELVs.

Report of a new six-panel flow cytometry marker for early differential diagnosis of APL from HLA-DR negative Non-APL leukemia.

Although AML-M3 (APL) and HLA-DR negative non-APL are characterized by negative HLA-DR antigen, they are different entities with similar morphology in some cases. The aim of this study is the precise, differential diagnosis of APL from HLA-DR negative non-APL by flow cytometry to narrow the diagnosis window. Bone marrow or blood samples of 580 AML patients were analyzed, and flow cytometry and molecular analysis were performed for the diagnosis of blood disorders. In 105 HLA-DR negative AML patients, expression of HLA-DR, CD33, CD117, CD11b, CD64, CD34, CD9 and myeloperoxidase staining pattern were evaluated. Fifty-six patients were diagnosed with APL, and 49 patients were diagnosed with HLA-DR negative non-APL. The APL blasts expressed CD33, CD117, CD64, and CD9 in 100%, 80.3%, 94.6%, and 100% of the cases, respectively. HLA-DR negative non-APL blasts expressed CD33, CD117, CD64 and CD9 in 75.5%, 59.1%, 32.6%, and 73.4% of the cases, respectively. APL cells were negative for HLA-DR, CD11b, and CD34 in 96.4%, 94.6%, and 91.0%, respectively. Blasts in HLA-DR negative non M3-AML were negative for CD11b, CD117, and CD34 in 77.5%, 40.9%, and 22.4%, respectively. We also investigated myeloperoxidase (MPO) staining pattern and found strong diffuse reaction in APL cells while HLA-DR negative non-APL cells showed focal positive reaction. In all of the APL patients, except for one, PML/RARA translocation was positive, and in another case with HLA-DR negative non-APL, PML/RARA and other translocations were not detected. The six-panel combination profile rapidly and specifically identifies APL from other HLA-DR negative AML.

High serum extracellular vesicle miR-10b expression predicts poor prognosis in patients with acute myeloid leukemia.

BACKGROUND: Increasing evidence have demonstrated that serum extracellular vesicle microRNAs (EV-miRNAs) are promising noninvasive biomarkers for various cancer types. OBJECTIVE: In this study, we aimed to investigate and evaluate the potential clinical significance of serum EV-miR-10b for acute myeloid leukemia (AML). METHODS: Blood samples were collected from a cohort of 95 de novo AML patients and 80 healthy individuals. Of all AML patients, 51 patients were considered as cytogenetic normal AML (CN-AML). Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to measure the expression levels of serum EV-miR-10b. RESULTS: The extracellular vesicles we extracted from the serum samples were positive for EV/exosome markers including TSG101, CD63, Flotillin-1 and CD9. The expression levels of serum EV-miR-10b were significantly higher in AML/CN-AML patients than healthy controls. In addition, serum EV-miR-10b expression was strongly correlated with aggressive clinical characteristics. Moreover, receiver operating characteristic analysis showed serum EV-miR-10b yielded an area under the curve of 0.875, with 77.89% specificity and 82.50% sensitivity in identifying AML patients from normal controls. Furthermore, AML patients with higher serum EV-miR-10b expression had significantly shorter survival and serum EV-miR-10b was demonstrated to be an independent prognostic marker for overall survival in AML. CONCLUSIONS: Taken together, serum EV-miR-10b might serve as a promising biomarker for predicting the prognosis of AML.

Multiplex isolation and profiling of extracellular vesicles using a microfluidic DICE device.

Profiling of extracellular vesicles (EVs) is an emerging area in the field of liquid biopsies because of their innate significance in diseases and abundant information reflecting disease status. However, unbiased enrichment of EVs and thorough profiling of EVs is challenging. In this paper, we present a simple strategy to immobilize and analyze EVs for multiple markers on a single microfluidic device and perform differentiated immunostaining-based characterization of extracellular vesicles (DICE). This device, composed of four quadrants with a single inlet, captures biotinylated EVs efficiently and facilitates multiplexed immunostaining to profile their extracellular proteins, allowing for a multiplexed approach for non-invasive cancer diagnostics in the future. From controlled sample experiments using cancer cell line derived EVs and specific fluorescence staining with lipophilic dyes, we identified that the DICE device is capable of isolating biotinylated EVs with 84.4% immobilization efficiency. We extended our study to profile EVs of 9 clinical samples from non-small cell lung cancer (NSCLC) patients and healthy donors and found that the DICE device successfully facilitates immunofluorescent staining for both the NSCLC patients and the healthy control. This versatile and simple method to profile EVs could be extended to EVs of any biological origin, promoting discoveries of the role of EVs in disease diagnostics and monitoring.

Prostate cancer sheds the αvß3 integrin in vivo through exosomes.

The αvß3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvß3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvß3 travels through exosomes and tested its levels in pools of vesicles, which we designate extracellular vesicles highly enriched in exosomes (ExVs), and in exosomes isolated from the plasma of prostate cancer patients. Here, we show that the αvß3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvß3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to ß3-negative recipient cells. We also demonstrate the intracellular localization of ß3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvß3 and CD9 as compared to plasma ExVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the αvß3 integrin is expressed in a subset of exosomes characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2. Finally, we present evidence that the levels of αvß3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvß3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvß3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvß3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target.

Extracellular vesicles do not contribute to higher circulating levels of soluble LRP1 in idiopathic dilated cardiomyopathy.

Idiopathic dilated cardiomyopathy (IDCM) is a frequent cause of heart transplantation. Potentially valuable blood markers are being sought, and low-density lipoprotein receptor-related protein 1 (LRP1) has been linked to the underlying molecular basis of the disease. This study compared circulating levels of soluble LRP1 (sLRP1) in IDCM patients and healthy controls and elucidated whether sLRP1 is exported out of the myocardium through extracellular vesicles (EVs) to gain a better understanding of the pathogenesis of the disease. LRP1 α chain expression was analysed in samples collected from the left ventricles of explanted hearts using immunohistochemistry. sLRP1 concentrations were determined in platelet-free plasma by enzyme-linked immunosorbent assay. Plasma-derived EVs were extracted by size-exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis and cryo-transmission electron microscopy. The distributions of vesicular (CD9, CD81) and myocardial (caveolin-3) proteins and LRP1 α chain were assessed in SEC fractions by flow cytometry. LRP1 α chain was preferably localized to blood vessels in IDCM compared to control myocardium. Circulating sLRP1 was increased in IDCM patients. CD9- and CD81-positive fractions enriched with membrane vesicles with the expected size and morphology were isolated from both groups. The LRP1 α chain was not present in these SEC fractions, which were also positive for caveolin-3. The increase in circulating sLRP1 in IDCM patients may be clinically valuable. Although EVs do not contribute to higher sLRP1 levels in IDCM, a comprehensive analysis of EV content would provide further insights into the search for novel blood markers.

[Isolation and characterization of exosomes from blood plasma of breast cancer and colorectal cancer patients]. / Vydelenie i kharakterizatsiia ékzosom plazmy krovi bol'nykh rakom molochnoi zhelezy i kolorektal'nym rakom.

A simple approach for isolation of exosomes from the blood plasma, which allows to obtain highly purified preparations of microvesicles no larger than 100 nm has been proposed. The presence of different subpopulations of exosomes in the blood plasma of healthy donors and cancer patients has been recognized. We found the presence of the universal markers CD9, CD24 and CD81 on exosomes isolated from blood plasma that can be used to their routine typing.

Reproducibility and efficiency of serum-derived exosome extraction methods.

OBJECTIVES: Exosomes are emerging as a source of biomarkers with putative prognostic and diagnostic value. However, little is known about the efficiency, reproducibility and reliability of the protocols routinely used to quantify exosomes in the human serum. DESIGN AND METHODS: We used increasing amounts of the same serum sample to isolate exosomes using two different methods: ultracentrifugation onto a sucrose cushion and ExoQuick™. Quantitative analysis of serum-derived exosomes was performed by determining protein concentration (BCA assay) and the number of nanoparticles (Nanosight™ technology). Exosome quality was assessed by Coomassie staining and Western blotting for CD9, LAMP2 exosomal markers and a negative marker Grp94. RESULTS: Correlation between serum volume and the number of isolated exosomes is significant for both methods when exosomes are quantified using protein concentration. However, when the number of nanoparticles is used to quantify exosomes, ExoQuick™ is the only reproducible and efficient method. CD9, LAMP2 and Grp94 exosomal markers are equivalently expressed in both methods. However, exosomes isolated using ultracentrifuge method are strongly contaminated with albumin and IgG. CONCLUSION: ExoQuick™ is an efficient and reproducible method to isolate exosomes for quantitative studies, whereas ultracentrifugation is not. Moreover, high albumin contamination of ultracentrifuged-derived exosomes impairs the use of protein concentration as a mean to quantify serum-derived exosomes.