The Hamburg EV community: A present reality with a promising future

The participants presented projects focusing on diverse EV-related topics (neuroscience, nephrology, cancer research, infectious diseases, immunology, obstetrics, and materno-fetal medicine), with descriptions of the various separation methods and protocols, to prepare EVs from different sources, such as blood, urine, cerebral spinal fluid, tissue, and cell culture. Among the discussed topics, technical talks covering EV-proteomics, EV-DNA, and cryogenic electron microscopy (cryo-EM) were held. Moreover, we had presentations explaining the main aims of the European Liquid Biopsy Society and the first academic EV Core Facility established at the Philipps University Marburg.

Finally, all participants joined in a fruitful round table discussion on strategies to improve our collaborations on the topic and how to strengthen EV research in Hamburg, connected with the German and the international EV community. Here, we present the meeting’s details, the topics discussed, and the general conclusions concerning the anticipated future EV research network in Hamburg. Creating collaborative and stable relationships is crucial to developing EV research, and we are thrilled about this very positive start.

Retreat content

The UKE-EV Retreat consisted of one-and-a-half-day oral presentations distributed in six sessions. We had fifteen research talks, five technically focused presentations, one plenary session, a European Liquid Biopsy Society presentation, and two interactive round tables. Instead of having thematic blocks organized by classical disciplines and specific topics, the talks were randomly distributed during the two days. Our goal was to make EVs per se the sole core of the meeting, to stimulate interdisciplinary discussions and scientific interactions among researchers from distinct areas. Students, as well as full professors internationally recognized in their fields, had the opportunity to gather together and present their work.

Short summary of topics presented

Infectious diseases

The retreat comprised five presentations focusing on infectious diseases (LFF-FV74): i) Lidia Bosurgi (I. Department of Medicine, UKE, and Bernhard Nocht Institute for Tropical Medicine) introduced the Schistosoma mansoni infection and showed how EV released by dying cells could regulate the disease severity by modulating the macrophage function; ii) Linda Niemetz (Bernhard Nocht Institute for Tropical Medicine) presented her study to define the putative role of EVs as an antigen source for cross-presentation between dendritic and T cells in Ebola virus infections; iii) Maura Dandri (I. Department of Medicine, UKE) talked about the role and variety of EV-like viral and subviral components that are produced from human hepatitis viruses. The presentation mainly focused on the characterization of circulating pseudo-enveloped hepatitis E Virus particles detected in patients and human liver chimeric mice and on distinct EV-associated particles detected in the course of chronic infection with hepatitis B and D viruses; iv) Timo Trenkner (Leibniz Institute of Virology) showed how EVs released from B cells transformed by Epstein-Barr Virus infection inhibit innate immune receptors; v) Ute Westerkamp (Institute for Medical Microbiology, Virology and Hygiene, UKE) discussed the characteristics and functions of EVs in Merkel cell carcinoma (MCC) with an emphasis on the role of viral oncoproteins in virus-positive MCCs.

Neuroscience and brain cancer

We had three talks about neuroscience and brain cancer: i) Matthias Kneussel (Institute of Molecular Neurogenetics, Center for Molecular Neurobiology Hamburg (ZMNH)) presented his group’s work on neuronal proteins regulating EV release and EV interaction with target cells, pointing out the relevance of the interaction between cellular prion protein (PrP^C) and the trafficking adaptor muskelin in neuronal EV release; ii) Amanda Salviano-Silva (Department of Neurosurgery, UKE) talked about tumour-derived EVs as circulating biomarkers in glioblastoma, primarily focusing on their DNA content and using Imaging Flow Cytometry analysis for EV-surface markers characterization; iii) Berta Puig (Department of Neurology, UKE) presented the work of her group on EVs in stroke, pointing out a role for PrP^C on brain-derived EVs (BDEVs) in EV uptake, and analyzing the mRNA BDEV content in inflammation and recovery processes after ischemic damage.

Kidney-related pathologies

Two presentations were focused on the role of EVs in kidney-related pathologies: i) Fabian Braun (III. Department of Medicine, UKE) showed the relevance of EVs in intercellular communication upon glomerular damage and their signature in urine, working on kidney tissue and urine-derived EVs (uEVs); and ii) Karen Lahme (Institute of Cellular and Integrative Physiology, UKE) focused her talk on the role of uEVs on glomerular damage, showing an interesting model to discriminate EVs originated from different cells inside the kidney.

Obstetrics and materno-fetal medicine

Dennis Irfan Yüzen (Department of Obstetrics and Fetal Medicine, UKE) described blood-derived EVs as an indicator of maternal immune activation during SARS-CoV-2 infection.

Immunology

Moreover, two Immunology-focussed talks were included: i) Johannes Hartl (I. Department of Medicine, UKE), who described microparticles as a source of antigenic DNA in lupus, which could potentiate the autoantibodies in the pathology; and ii) Riekje Winzer (Department of Immunology, UKE), who reported that CD8+ T cells contribute to adenosine production, a potent immune suppressor, by releasing CD73+ EVs with AMPase activity.

Cancer progression and related biomarkers

Finally, two presentations focused on cancer progression assessment and biomarkers: i) Johann von Felden (I. Department of Medicine, UKE) presented blood-derived EVs as carriers of biomarkers for liver cancer, and ii) Julian Kött together with Glenn Geidel (Department of Dermatology, UKE) showed the potential of blood-derived EVs as biomarkers for metastatic melanoma.

Technical presentations

One of the main goals of this retreat was to share detailed technical knowledge, which is highly valuable regardless of the specific field of study. We also aimed to inform researchers of the technical resources and infrastructures available in Hamburg, distributed among different groups and institutions. For that, we invited some colleagues to describe in detail their research, focusing mainly on technical approaches.

Hannah Voß (Institute for Clinical Chemistry and Laboratory Medicine, Section Mass Spectrometry and Proteomics, UKE) presented her experience in proteomic analysis of EV samples and the new tools they developed to improve peptide detection. She emphasized the relevance of high-quality sample preparation and thoughtful protocol election to achieve an optimal analysis. Tim Benecke (Center of Structural Systems Biology, University of Hamburg) talked about Cryo-EM analysis of glycoprotein structure to target viral membrane fusion and how this microscopy technique could as well be applied to visualize EV shape, determine their size and, potentially, the structure of specific proteins present on/in the vesicles. Franz Ricklefs (Department of Neurosurgery, UKE) presented tools for improving the methylation and mutation analysis of EV-DNA extracted from the blood of patients with glioblastoma. DNA from EVs represent suitable biomarkers, as they can reflect the epigenetic characteristics of the tumour more precisely than analyses based on bulk cell-free DNA. Mohsin Shafiq, together with Andreu Matamoros-Angles (both from the Institute of Neuropathology, UKE), discussed the role of brain-derived EVs in Alzheimer’s disease, especially the function of PrP^C on the aggregation of amyloid-beta (Aβ) peptides. They presented the use of super-resolution microscopy, aggregation assays (analysed by small angle X-ray scattering (SAXS) at the DESY campus), and a new non-enzymatically-based BDEV isolation protocol to protect EV membrane proteins from ‘artificial’ cleavage during tissue extraction. Lastly, Jochen Behrends, the Head of the Core Facility Fluorescence Cytometry (Research Center Borstel), talked about the usage of flow cytometers for the detection and characterization of EVs, giving an overview of the development of the flow cytometers and their possibilities for practical applications in different EV-related contexts. The talk of Jochen Behrends, due to personal reasons, was postponed and therefore held on the 1^st of June 2023.

The above-mentioned presentations were also strengthened by an overview given by Berta Puig about the International Society of Extracellular Vesicles (ISEV), the MISEV guidelines and their relevance for reporting standards and reproducibility in the EV field, and publication opportunities within the ISEV journals “Journal of Extracellular Vesicles” and “Journal of Extracellular Biology”. This summary provided a valuable overview of the international EV community, which was especially useful for newcomers to the field.

As one of the plenary talks, we had an exciting presentation on the European Liquid Biopsy Society (ELBS, www.elbs.eu) by its chairman, Klaus Pantel (Department of Tumour Biology, UKE), that emphasized the necessity of performing EV analyses in the frame of the already widespread liquid biopsy field. The ELBS is a network that incorporates partners from academia, industry, clinics, and governmental agencies to address the challenges of clinical implementation of liquid biopsy assays, primarily, but not exclusively, on cancer patients. Liquid biopsies, including EVs, are crucial to find suitable biomarkers to improve disease treatments, diagnosis, and prognosis determination; therefore, standardized clinical protocols and analyses are needed.

Plenary talk by ‘external’ speaker: the experience of the first German EV Core Facility

We were delighted to have Christian Preußer, head of the EV Core Facility at the Philipps University Marburg, as a keynote speaker to share his experience establishing and running this type of unique* facility (*at least thus far in Germany). He presented the equipment and techniques they offer for EV isolation and characterization, especially the Free Flow Electrophoresis (FFE) as a new, reproducible EV separation technique. He also pointed out the relevance of intense communication and co-organization with other Core Facilities, such as Proteomics or Electron Microscopy, to address the complexity that a proper EV analysis requires. Finally, he answered and discussed concrete technical details faced daily in EV research, such as the inconsistency in the EV size determination depending on the method used and the advantages and disadvantages of different EV isolation methods. He also talked about the German Society for Extracellular Vesicles (www.gsev.org) and made the attendees aware of some interesting EV-related meetings and activities in the near future.

Round table discussion

At the end of both days, we held round table sessions to discuss how to improve EV research in Hamburg. On the first day, the discussion was institutionally-oriented, mainly focused on equipment, facilities, and resources we may need to strengthen EV-related projects. We also discussed the need to clearly define common strategies and well-defined scientific topics for future collaborative funding. On the second day, the debate was dedicated to implementing useful tools for our day-to-day work on/with EVs to share our expertise and enhance our organization and efficiency regarding the use of standardized protocols, reagents, equipment, and expertise already available in Hamburg. The group’s immediate consensus was to create: i) a website to list the EV groups at UKE and other institutions in Hamburg; ii) some EV task forces, i.e., working groups based on topic- and/or technique-related expertise (i.e., cytometry-based EV analysis; EV isolation from tissue or blood, etc.), and iii) a group responsible for generating a shared list of antibodies against EV-related proteins available in individual groups to enable rapid mutual support.

Summary and conclusions

The response to the first EV retreat in Hamburg was highly positive, showing that EV research in Hamburg has a solid present but likely an even more promising future. In Hamburg, but also in the EV community in Germany and worldwide, it is clearly manifested that a collaborative attitude in relation to sharing protocols, knowledge, and reagents at every level is essential. This retreat showed a genuinely collaborative spirit among basic and clinician scientists at different career stages, all interested in strengthening the EV field in the Hamburg area. By joining our expertise locally and further connecting with the national and international EV community, we intend to create a collaborative scientific spirit fostering medical research in the fascinating and complex field of EV research. Initiatives like the one presented here may be a tiny seed to accomplish another way to do science and create a more friendly environment and, ultimately, improve our daily lives as scientists to increase the value, reproducibility, impact, and social output of our research.

This EV meeting highlighted one of the most vital aspects of the EV Hamburg community: its interdisciplinarity regarding topics and approaches. The research ranges from basic molecular studies in vitro to long-term studies with patient-derived samples and disease-related animal models. Moreover, we can count on experts on: i) EV isolation from different biological sources (cell culture, biofluids, and tissue), ii) EV biochemical analysis (i.e., proteomics, transcriptomics), iii) EV functional studies (i.e., live imaging and uptake experiments), and iv) EV morphology and size characterization (i.e., electron microscopy and flow cytometry). This network provides a solid technical and conceptual baseline for the development of several EV projects.