Evo Kristina Lindersson Søndergaard

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.

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.

Phenotyping of Leukocytes and Leukocyte-Derived Extracellular Vesicles

Extracellular vesicles (EVs) have a demonstrated involvement in modulating the immune system. It has been proposed that EVs could be used as biomarkers for detection of inflammatory and immunological disorders. Consequently, it is of great interest to investigate EVs in more detail with focus on immunological markers. In this study, five major leukocyte subpopulations and the corresponding leukocyte-derived EVs were phenotyped with focus on selected immunological lineage-specific markers and selected vesicle-related markers. The leukocyte-derived EVs displayed phenotypic differences in the 34 markers investigated. The majority of the lineage-specific markers used for identification of the parent cell types could not be detected on EVs released from monocultures of the associated cell types. In contrast, the vesicular presentation of CD9, CD63, and CD81 correlated to the cell surface expression of these markers, however, with few exceptions. Furthermore, the cellular expression of CD9, CD63, and CD81 varied between leukocytes present in whole blood and cultured leukocytes. In summary, these data demonstrate that the cellular and vesicular presentation of selected lineage-specific and vesicle-related markers may differ, supporting the accumulating observations that sorting of molecular cargo into EVs is tightly controlled.

Antibody-Based Assays for Phenotyping of Extracellular Vesicles

Extracellular vesicles (EVs) are a heterogeneous population of membrane-enclosed vesicles. EVs are recognized as important players in cell-to-cell communication and are described to be involved in numerous biological and pathological processes. The fact that EVs are involved in the development and progression of several diseases has formed the basis for the use of EV analysis in a clinical setting. As the interest in EVs has increased immensely, multiple techniques have been developed aiming at characterizing these vesicles. These techniques characterize different features of EVs, like the size distribution, enumeration, protein composition, and the intravesicular cargo (e.g., RNA). This review focuses on techniques that exploit the specificity and sensitivity associated with antibody-based assays to characterize the protein phenotype of EVs. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers. Thus, protein profiling of EVs holds great diagnostic and prognostic potential.

Enhanced Humoral Responses Induced by Targeting of Antigen to Murine Dendritic Cells

Dendritic cells (DCs) are superior in their ability to induce and control adaptive immune responses. These qualities have motivated the hypothesis that targeted delivery of antigen to DCs in vivo may be an effective way of enhancing immunization. Recent results show that antigen targeted to certain DC surface molecules may indeed induce robust immune responses. Targeting of antigen to DCs can be accomplished by the means of monoclonal antibodies. This study compared the humoral responses induced in mice by in vivo targeting of DCs using monoclonal antibodies specific for CD11c, CD36, CD205, Clec6A, Clec7A, Clec9A, Siglec‐H and PDC‐TREM. The results demonstrate that antigen delivery to different targets on DCs in vivo gives rise to humoral responses that differ in strength. Targeting of antigen to CD11c, CD36, CD205, Clec6A, Clec7A and PDC‐TREM induced significantly stronger antibody responses compared to non‐targeted isotype‐matched controls. Targeting of Clec9A and Siglec‐H did not lead to efficient antibody responses, which may be due to unfavourable properties of the targeting antibody, in which case, other antibodies with the same specificity might elicit a different outcome. Anti‐CD11c was additionally used for elucidating the impact of the route of vaccination, and the results showed only minor differences between the antibody responses induced after immunization either s.c., i.v. or i.p. Altogether, these data show that targeting of different surface molecules on DCs result in very different antibody responses and that, even in the absence of adjuvants, strong humoral responses was induced.

Characterization of a Cell-culturing System for the Study of Contact-independent Extracellular Vesicle Communication

Appropriate and well-documented in vitro cell-culturing systems are necessary to study the activity and biological function of extracellular vesicles (EVs). The aim of this study was to describe an experimental system, in which dynamic, vesicle-based cell communication can be investigated. A commercially available cell-culturing system was applied to study contact-independent cell communication, which separated two cell populations using a membrane with a pore size of 0.4 μm. The EV exchange characteristics between the two compartments in the culture set-up was preliminarily investigated in a cell-free set-up, and analysed using the Extracellular Vesicle (EV) Array and Nanoparticle Tracking Analysis. The application of the cell-culturing set-up was demonstrated using co-cultures of human primary cells. The effects of the relative placement of the two cell populations on the phenotype of EVs found in the cell supernatant were investigated. The results indicate that this placement can be important for the biological hypothesis that is being investigated. These observations are relevant for short (<24h) as well as long (several days) studies of vesicle-based cell communication. Moreover, the introduced cell-culturing set-up and analytical strategy can be used to study contact-independent vesicle communication in a reproducible manner.

Oxygen-Related Differences in Cellular and Vesicular Phenotypes Observed for Ovarian Cell Cancer Lines

Extracellular vesicles (EVs) are one of several tools that cells use to communicate with each other. This communication is facilitated by a number of surface-associated proteins and the cargo of the vesicles. For several cancer types, the amount of EVs is observed to be up-regulated in patients compared to healthy individuals, possibly signifying the presence of an aberrant process. The hypoxia-induced release of EVs from cancer cells has been hypothesized to cause the malignant transformation of healthy recipient cells. In this study, the phenotype of cells and EVs from the ovarian cancer cell lines, COV504, SKOV3, and Pt4, were quantified and analysed under normoxic and hypoxic conditions. It was shown that both cells and EVs express common markers and that the EV phenotype varies more than the cellular phenotype. Additionally, cells subjected to 24 hours of hypoxia compared to normoxia produced more EVs. The phenotyping of EVs from cancer cell lines provides information about their molecular composition. This information may be translated to knowledge regarding the functionality of EVs and lead to a better understanding of their role in cancer.

The phenotypical changes of plasma EVs over time in healthy donors

Book: ISEV2017 To cite this article: (2017) Abstract Book: ISEV2017, Journal of Extracellular Vesicles, 6:sup1, 1310414, DOI: 10.1080/20013078.2017.1310414 To link to this article: https://doi.org/10.1080/20013078.2017.1310414

Phenotyping and quantification of cascade-primed immune cells (CAPRI) and their EVs

Book: ISEV2017 To cite this article: (2017) Abstract Book: ISEV2017, Journal of Extracellular Vesicles, 6:sup1, 1310414, DOI: 10.1080/20013078.2017.1310414 To link to this article: https://doi.org/10.1080/20013078.2017.1310414

Phenotyping of leukocytes and leukocyte-derived extracellular vesicles

Extracellular vesicles (EVs) have a demonstrated involvement in modulating the immune system. It has been proposed that EVs could be used as biomarkers for detection of inflammatory and immunological disorders. Consequently, it is of great interest to investigate EVs in more detail with focus on immunological markers. In this study, five major leukocyte subpopulations and the corresponding leukocyte-derived EVs were phenotyped with focus on selected immunological lineage-specific markers and selected vesicle-related markers. The leukocyte-derived EVs displayed phenotypic differences in the 34 markers investigated. The majority of the lineage-specific markers used for identification of the parent cell types could not be detected on EVs released from monocultures of the associated cell types. In contrast, the vesicular presentation of CD9, CD63, and CD81 correlated to the cell surface expression of these markers, however, with few exceptions. Furthermore, the cellular expression of CD9, CD63, and CD81 varied between leukocytes present inwhole blood and cultured leukocytes. In summary, these data demonstrate that the cellular and vesicular presentation of selected lineage-specific and vesicle-relatedmarkersmay differ, supporting the accumulating observations that sorting of molecular cargo into EVs is tightly controlled.ISEV2016 is organized by The Local Organizing Committee Chair Edit I Buzás (Hungary), Aled Clayton (United Kingdom), Dolores Di Vizio (USA), Juan Manuel Falcon-Perez (Spain), Guido Jenster (The Netherlands), Lorraine O’Driscoll (Ireland), Yong Song Gho (South Korea), Marjolein van Driel (The Netherlands), Hans van Leeuwen (The Netherlands), Guillaume van Niel (France), Marca HM Wauben (The Netherlands), Kenneth W Witwer (USA), María Yáñez-Mó (Spain) Together with the Executive ISEV Board (2014 – 2016) President: Jan Lötvall Secretary General: Clotilde Théry Interim Treasurer: Kenneth W Witwer Executive Chair Science / Meetings: Marca Wauben Executive Chair Education: Yong Song Gho Executive Chair Communication: Andrew Hill Members at Large: Peter Quesenberry, Kenneth W Witwer, Susmita Sahoo, Dolores Di Vizio, Chris Gardiner, Edit I Buzás, Hidetoshi Tahara, Suresh Mathivanan, Igor Kurochkin