Malene Jørgensen

Extracellular Vesicle (EV) Array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping

Background Exosomes are one of the several types of cell-derived vesicles with a diameter of 30–100 nm. These extracellular vesicles are recognized as potential markers of human diseases such as cancer. However, their use in diagnostic tests requires an objective and high-throughput method to define their phenotype and determine their concentration in biological fluids. To identify circulating as well as cell culture-derived vesicles, the current standard is immunoblotting or a flow cytometrical analysis for specific proteins, both of which requires large amounts of purified vesicles. Methods Based on the technology of protein microarray, we hereby present a highly sensitive Extracellular Vesicle (EV) Array capable of detecting and phenotyping exosomes and other extracellular vesicles from unpurified starting material in a high-throughput manner. To only detect the exosomes captured on the EV Array, a cocktail of antibodies against the tetraspanins CD9, CD63 and CD81 was used. These antibodies were selected to ensure that all exosomes captured are detected, and concomitantly excluding the detection of other types of microvesicles. Results The limit of detection (LOD) was determined on exosomes derived from the colon cancer cell line LS180. It clarified that supernatant from only approximately 104 cells was needed to obtain signals or that only 2.5×104 exosomes were required for each microarray spot (~1 nL). Phenotyping was performed on plasma (1–10 µL) from 7 healthy donors, which were applied to the EV Array with a panel of antibodies against 21 different cellular surface antigens and cancer antigens. For each donor, there was considerable heterogeneity in the expression levels of individual markers. The protein profiles of the exosomes (defined as positive for CD9, CD63 and CD81) revealed that only the expression level of CD9 and CD81 was approximately equal in the 7 donors. This implies questioning the use of CD63 as a standard exosomal marker since the expression level of this tetraspanin was considerably lower.

Diagnostic and prognostic potential of extracellular vesicles in peripheral blood

PURPOSE Extracellular vesicles (EVs) are small, membrane-enclosed entities released from cells in many different biological systems. These vesicles play an important role in cellular communication by virtue of their protein, RNA, and lipid content, which can be transferred among cells. The complement of biomolecules reflects the parent cell, and their characterization may provide information about the presence of an aberrant process. Peripheral blood is a rich source of circulating EVs, which are easily accessible through a blood sample. An analysis of EVs in peripheral blood could provide access to unparalleled amounts of biomarkers of great diagnostic and prognostic value. The objectives of this review are to briefly present the current knowledge about EVs and to introduce a toolbox of selected techniques, which can be used to rapidly characterize clinically relevant properties of EVs from peripheral blood. METHODS Several techniques exist to characterize the different features of EVs, including size, enumeration, RNA cargo, and protein phenotype. Each technique has a number of advantages and pitfalls. However, with the techniques presented in this review, a possible platform for EV characterization in a clinical setting is outlined. FINDINGS Although EVs have great diagnostic and prognostic potential, a lack of standardization regarding EV analysis hampers the full use of this potential. Nevertheless, the analysis of EVs in peripheral blood has several advantages compared with traditional analyses of many soluble molecules in blood. IMPLICATIONS Overall, the use of EV analysis as a diagnostic and prognostic tool has prodigious clinical potential.

Exosomal proteins as potential diagnostic markers in advanced non-small cell lung carcinoma

Background Lung cancer is one of the leading causes of cancer-related death. At the time of diagnosis, more than half of the patients will have disseminated disease and, yet, diagnosing can be challenging. New methods are desired to improve the diagnostic work-up. Exosomes are cell-derived vesicles displaying various proteins on their membrane surfaces. In addition, they are readily available in blood samples where they constitute potential biomarkers of human diseases, such as cancer. Here, we examine the potential of distinguishing non-small cell lung carcinoma (NSCLC) patients from control subjects based on the differential display of exosomal protein markers. Methods Plasma was isolated from 109 NSCLC patients with advanced stage (IIIa–IV) disease and 110 matched control subjects initially suspected of having cancer, but diagnosed to be cancer free. The Extracellular Vesicle Array (EV Array) was used to phenotype exosomes directly from the plasma samples. The array contained 37 antibodies targeting lung cancer-related proteins and was used to capture exosomes, which were visualised with a cocktail of biotin-conjugated CD9, CD63 and CD81 antibodies. Results The EV Array analysis was capable of detecting and phenotyping exosomes in all samples from only 10 µL of unpurified plasma. Multivariate analysis using the Random Forests method produced a combined 30-marker model separating the two patient groups with an area under the curve of 0.83, CI: 0.77–0.90. The 30-marker model has a sensitivity of 0.75 and a specificity of 0.76, and it classifies patients with 75.3% accuracy. Conclusion The EV Array technique is a simple, minimal-invasive tool with potential to identify lung cancer patients.

Cloning and Characterization of Purple Acid Phosphatase Phytases from Wheat ( Triticum aestivum L.), Barley ( Hordeum vulgare L.), Maize ( Zea maize L.) and Rice ( Oryza sativa L.)

Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic analyses indicated that PAPhys possess four conserved domains unique to the PAPhys. In barley and wheat, the PAPhy genes can be grouped as PAPhy_a or PAPhy_b isogenes (barley, HvPAPhy_a, HvPAPhy_b1, and HvPAPhy_b2; wheat, TaPAPhy_a1, TaPAPhy_a2, TaPAPhy_b1, and TaPAPhy_b2). In rice and maize, only the b type (OsPAPhy_b and ZmPAPhy_b, respectively) were identified. HvPAPhy_a and HvPAPhy_b1/b2 share 86% and TaPAPhya1/a2 and TaPAPhyb1/b2 share up to 90% (TaPAPhy_a2 and TaPAPhy_b2) identical amino acid sequences. despite of this, PAPhy_a and PAPhy_b isogenes are differentially expressed during grain development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed that the PAPhy_a isogene set present in wheat/barley but not in rice/maize is the origin of high phytase activity in mature grains.

Exosomal Proteins as Diagnostic Biomarkers in Lung Cancer

Introduction: Exosomes have been suggested as promising biomarkers in NSCLC because they contain proteins from their originating cells and are readily available in plasma. In this study, we explored the potential of exosome protein profiling in diagnosing lung cancers of all stages and various histological subtypes in patients. Methods: Plasma was isolated from 581 patients (431 with lung cancer and 150 controls). The extracellular vesicle array was used to phenotype exosomes. The extracellular vesicle array contained 49 antibodies for capturing exosomes. Subsequently, a cocktail of biotin‐conjugated CD9, CD81, and CD63 antibodies was used to detect and visualize captured exosomes. Multimarker models were made by combining two or more markers. The optimal multimarker model was evaluated by area under the curve (AUC) and random forests analysis. Results: The markers CD151, CD171, and tetraspanin 8 were the strongest separators of patients with cancer of all histological subtypes versus patients without cancer (CD151: AUC = 0.68, p = 0.0002; CD171: AUC = 0.60, p = 0.0002; and TSPAN8: AUC = 0.60, p = 0.0002). The multimarker models with the largest AUC in the cohort of patients with all lung cancer histological subtypes and in the cohort of patients with adenocarcinoma only covered 10 markers (all cancer: AUC = 0.74 [95% confidence interval: 0.70–0.80]; adenocarcinoma only: AUC = 0.76 [95% confidence interval: 0.70–0.83]). In squamous cell cancer and SCLC, multimarker models did not exceed CD151 as an individual marker in separating patients with cancer from controls. Conclusion: We have demonstrated exosome protein profiling to be a promising diagnostic tool in lung cancer independently of stage and histological subtype. Multimarker models could make a fair separation of patients, demonstrating the perspectives of exosome protein profiling as a biomarker.

Potentials and capabilities of the Extracellular Vesicle (EV) Array

Extracellular vesicles (EVs) and exosomes are difficult to enrich or purify from biofluids, hence quantification and phenotyping of these are tedious and inaccurate. The multiplexed, highly sensitive and high-throughput platform of the EV Array presented by Jørgensen et al., (J Extracell Vesicles, 2013; 2: 10) has been refined regarding the capabilities of the method for characterization and molecular profiling of EV surface markers. Here, we present an extended microarray platform to detect and phenotype plasma-derived EVs (optimized for exosomes) for up to 60 antigens without any enrichment or purification prior to analysis.

Exosomal proteins as prognostic biomarkers in non-small cell lung cancer

Use of exosomes as biomarkers in non‐small cell lung cancer (NSCLC) is an intriguing approach in the liquid‐biopsy era. Exosomes are nano‐sized vesicles with membrane‐bound proteins that reflect their originating cell. Prognostic biomarkers are needed to improve patient selection for optimal treatment. We here evaluate exosomes by protein phenotyping as a prognostic biomarker in NSCLC.

Age-Related Changes in Plasma Extracellular Vesicle Characteristics and Internalization by Leukocytes

Cells release lipid-bound extracellular vesicles (EVs; exosomes, microvesicles and apoptotic bodies) containing proteins, lipids and RNAs into the circulation. Vesicles mediate intercellular communication between both neighboring and distant cells. There is substantial interest in using EVs as biomarkers for age-related diseases including cancer, and neurodegenerative, metabolic and cardiovascular diseases. The majority of research focuses on identifying differences in EVs when comparing disease states and matched controls. Here, we analyzed circulating plasma EVs in a cross-sectional and longitudinal study in order to address age-related changes in community-dwelling individuals. We found that EV concentration decreases with advancing age. Furthermore, EVs from older individuals were more readily internalized by B cells and increased MHC-II expression on monocytes compared with EVs from younger individuals, indicating that the decreased concentration of EVs with age may be due in part to increased internalization. EVs activated both monocytes and B cells, and activation of B cells by LPS enhanced EV internalization. We also report a relative stability of EV concentration and protein amount in individual subjects over time. Our data provide important information towards establishing a profile of EVs with human age, which will further aid in the development of EV-based diagnostics for aging and age-related diseases.

The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray

The research field of extracellular vesicles (EVs) is increasing immensely and the potential uses of EVs seem endless. They are found in large numbers in various body fluids, and blood samples may well serve as liquid biopsies. However, these small membrane-derived entities of cellular origin are not straightforward to work with in regard to isolation and characterization. A broad range of relevant preanalytical issues was tested, with a focus on the phenotypic impact of smaller EVs. The influences of the i) blood collection tube used, ii) incubation time before the initial centrifugation, iii) transportation/physical stress, iv) storage temperature and time (short term and long term), v) choice of centrifugation protocol, vi) freeze-thaw cycles, and vii) exosome isolation procedure (ExoQuick™) were examined. To identify the impact of the preanalytical treatments, the relative amounts (detected signal intensities of CD9-, CD63- and/or CD81-positive) and phenotypes of small EVs were analyzed using the multiplexed antibody-based microarray technology, termed the EV Array. The analysis encompassed 15 surface- or surface-related markers, including CD9, CD63, CD81, CD142, and Annexin V. This study revealed that samples collected in different blood collection tubes suffered to varying degrees from the preanalytical treatments tested here. There is no unequivocal answer to the questions asked. However, in general, the period of time and prospective transportation before the initial centrifugation, choice of centrifugation protocol, and storage temperature were observed to have major impacts on the samples. On the contrary, long-term storage and freeze-thawing seemed to not have a critical influence. Hence, there are pros and cons of any choice regarding sample collection and preparation and may very well be analysis dependent. However, to compare samples and results, it is important to ensure that all samples are of the same type and have been handled similarly.

Prospects and limitations of antibody-mediated clearing of lipoproteins from blood plasma prior to nanoparticle tracking analysis of extracellular vesicles

ABSTRACT Introduction: Nanoparticle tracking analysis (NTA) enables measurement of extracellular vesicles (EVs) but lacks the ability to distinct between EVs and lipoproteins which are abundantly present in blood plasma. Limitations in ultracentrifugation and size exclusion chromatography applied for EV isolation may result in inadequate EV purification and preservation. In this proof of concept study, we aimed to evaluate the potential of antibody-mediated removal of lipoproteins from plasma prior to extracellular vesicle (EV) analysis by nanoparticle tracking analysis (NTA). Methods: Ten platelet-free plasma (PFP) samples from healthy fasting subjects were incubated with magnetic beads coated with antibodies against apolipoprotein B-48 and B-100 (ApoB). Plasma samples were analysed with NTA before and after application of the bead procedure. Four fasting PFP samples were analysed with an ELISA specific for human ApoB to estimate the degree of removal of lipoproteins and EV array analysis was used for identification of possible EV loss. Results: The magnetic bead separation procedure resulted in a median reduction of the particle concentration in plasma by 62% (interquartile range 32–72%). The mean size of the remaining particles generally increased. ApoB concentration was reduced to a level close to the background signal, whereas a median reduction of the EV content by 21% (range 8–43%) was observed. Conclusion: Anti-ApoB antibody coated magnetic beads may hold potential for removal of lipoproteins from human PFP prior to EV measurement by NTA but some artefactual effect and EV loss may have to be endured.