ILGD INSTAND 2024 - A German extravaganza of tiny packages!

The intricate world of extracellular vesicles (EVs) continues to captivate researchers with its profound implications across various fields from basic research to clinical translation. In 2024, the German Society for Extracellular Vesicles (GSEV) once again took part in the annual IGLD INSTAND Symposium in Leipzig. As attendees delved into the latest findings, methodologies, and challenges surrounding EV research, the symposium emerged as a great platform for advancing our understanding of the plethora of EV functions. The ten EV-sessions were complemented by an industrial exhibition, enabling scientists to engage in productive discussions with business partners. This platform also facilitated the exploration of new devices and products within the EV and laboratory diagnostic market.
In this review article, we aim to comprehensively summarize the symposium, focusing particularly on the revelations and advancements pertaining to EVs, shedding light on their diverse functional roles, potential applications, and the evolving methodologies employed.

Between Kingdoms: EVs from Non-human Life Forms

The symposium was commenced by Claudia Günther from the University Hospital Erlangen exploring the communication between microorganisms
and hosts in gut and liver health by studying the transfer of microbial biomolecules through outer membrane vesicles (OMVs) [1]. Through advanced visualization techniques, she was able to reveal the transfer of bacterial OMVs to recipient cells in organs, including the intestine, liver and brain, revealing the interplay between gut microbiota and host cells.
After some last-minute schedule changes due to the German-wide train strike Janis Müller from the Institute of Virology at the University of Marburg stepped in to present his research journey from viruses to EVs and their interconnections. Having described the pro-viral enhancement of semen in HIV infections, his team investigated the effects of semen in Zika infections. He presented that viral attachment and cellular uptake of Zika virus employs apoptotic mimicry as mode of infection. This infection route is inhibited by physiological concentrations of EVs present in semen and saliva. Applying lipidomic analysis and flow cytometry, he demonstrated that phosphatidylserine (PS) is exposed on the EVs surface, blocking the PS receptors TIM and TAM, allowing for the infection by apoptotic mimicry [2]. Infection of viruses using other entry receptors, such as HIV, Hepatitis C virus and herpesviruses were not affected by EVs. His findings showcase the role of PS-rich EVs in body fluids as a defense against infection by viral apoptotic mimicries, explaining their preferential infection routes via PS-EV-deficient blood or blood ingesting arthropods.

Methods, Rigor and Standardization

Kicking off the next session, André Görgens from the Karolinska Institutet Huddinge, Sweden, underscored the significance of reporting experimental details when working with EVs. Emphasizing the importance of standardization and calibration as well as of continual development and enhancement of robust measures, he presented the inter-lab comparison of mesenchymal stem cell (MSC-)derived EV marker signatures. He referred to the recently published MISEV guidelines 2023 that were summarized by 60 drafting authors and 1045 authors in total. To name only few MISEV recommendations, EV researches should specifically report on the isolation specifics such as filter concentrations, precipitation, size-exclusion chromatography, differential ultracentrifugation, density gradients, asymmetric flow field-flow fractionation and immune-precipitation or affinity-precipitation. Furthermore, refinements in EV nomenclature should facilitate the discrimination of extracellular particles into non-vesicular extracellular particles (NVEPs) and vesicular particles with further separation of EVs, synthetic vesicles (SVs) and artificial cell-derived vesicles (ACDV) [3]. Applying the presented rigorous methods, André showed how the decoration of EVs with monoclonal antibodies targeting HER2 or PD-L1 could be loaded with drugs for specific tumor cell targeting.

Meike Saul from the University Hospital Hamburg Eppendorf shared her research on the isolation of EVs by anion exchange chromatography, a well-established method leveraging the negative surface charge of EVs. They suggested that the presented anion exchange resin facilitates the productive and scalable separation and purification of EVs with an overall loss of 20% during the isolation procedure [4]. In addition, DNA and chromatin could be isolated in a second distinct peak fraction.
Extending the scalability of EV production, Ferdinand Groten, engineer at the Fraunhofer Institute for Production Technology Aachen, introduced his newly developed fully automated cell production workflow [5].  In their setup, induced pluripotent stem cell, iPSCs, are cultured as mesenchymal stem cells, MSCs, and chondrocytes with the ultimate aim to use the cells or their EVs for treating osteoarthritis, a prevalent global health issue. The fully-automated setup can overcome current limitations of time-consuming and error prone manual manufacturing and facilitate standardization and quality control processes.  The presented workbench with liquid handlers, cell culture incubators, cell counters, freezing devices, might build the essential basis to scale up the production.

Dirk Strunk from the Medical University Graz, Austria, gave a comprehensive summary of the plethora of EV literature. His team compiled an extensive literature review of over 20.000 original EV research articles published to date. Employing an artificial-intelligence, AI, tool to extract information about EV source, isolation method as well as cargo and function, they could identify confounding factors of EV research [6]. They conclude that EV source and isolation methodology have the strongest impact on cargo and function based on published research. Making the condensed data available for the EV community, they launched the online platform EV-zone (EV-Zone.org), offering an extensive database with a search tool for EV literature. Through the interactive EV-checklist tool, they encourage authors of future EV publications to report their methodology with the ultimate aim to facilitate and promote transparency and reproducibility.

In the last talk of this session Stefan Holdenrieder, director of the Institute of Laboratory Medicine at the German Heart Centre Munich, gave an apprehensive overview of practical and regulatory requirements needed to translate research findings into diagnostic biomarker applications. He brought to the attention of the audience that biomarker research currently lacks essential standardization considerations, which will hamper clinical implementation. He stressed the importance of considering inter-laboratory reproducibility early on to establish highly replicable methodology for sensitive and specific biomarkers allowing clinical validation. From a clinical routine diagnostic point, a majority of biomarkers are not yet ready for laboratory diagnosis, which highlights that increased efforts should be taken towards harmonization to ensure reliable inter-laboratory results.

Translational aspects of stem cell and EV research

Halvard Bönig from the Institute for Transfusion Medicine and Immunology in Frankfurt a.M. opened the session on translational aspects of stem cell and EV research. In his talk about severe graft-versus-host disease, he highlighted the compelling treatment outcomes with Obnitix. Obnitix as a mesenchymal stromal cell (MSC)-based treatment has the ability to modulate the immune response and provide anti-inflammatory properties following hematopoietic stem cell-, umbilical cord blood-, or bone marrow transplantation. He pointed out that this positive immune modulation was primarily attributed to growth and anti-inflammatory factors as well as the release of EVs [7].
The following promising talk was given by Maurizio Muraca from the University of Padova, Italy, who presented the treatment potential of EVs for bronchopulmonary dysplasia (BPD) in premature neonates. He discussed data on the administration of MSC-conditioned media containing EVs, which has shown promising novel treatment effects in an BPD animal model, offering a safer alternative to direct cell administration by avoiding potential risks associated with engraftment or adverse effects [8].
Pieter Vader from the University Medical Center Utrecht, Netherlands, presented a talk titled EV-mediated gene delivery and editing. The talk was focused on his latest findings in EV-mediated delivery of CRISPR/Cas9 ribonucleoprotein complexes targeting the Pcsk9 gene in primary mouse hepatocytes. He and his team revealed that EV-mediated delivery effectively targets the Pcsk9 gene which led to an increase in low-density cholesterol receptor (LDLR) recycling in vitro [9].

Liquid Biopsy and EVs – Next Steps to Clinical Diagnostics

The session on liquid biopsy and EVs was kicked off by Vendula Hlaváčková Pospíchalová, from the Masaryk University in Brno, Czech Republic, and president of the newly launched Czech Society for Extracellular Vesicles. She presented her research on EVs in ascites of ovarian cancer patients. Isolated EVs were shown to have core ascitic EV-associated proteins which includes a subset specific for high-grade serous carcinoma (HGSC) of the ovary, fallopian tube and peritoneum [10]. Surprisingly the origin of HGSC-specific EVs could be mostly traced to non-malignant cells such as macrophages and fibroblasts. Nonetheless analysis of ascitic cells with simultaneous analysis of cell type-specific EV markers in HGSC had a higher prognostic potential than the analysis of ascitic cells alone.

Eva-Maria Krämer-Albers from the University of Mainz followed by exploring the potential of multiparametric phenotyping of central nervous system (CNS)-derived EVs as a diagnostic tool. She provided a comprehensive overview of how EVs interact with cellular barriers like the blood-brain barrier (BBB) and blood-CSF barrier (BCB). Elucidating the role of EVs in facilitating communication between the brain and periphery, with a particular focus on their involvement in modulating barrier properties and transferring cargo [11], she then showed how circulating CNS-derived EVs could be used as liquid biopsy markers for neurodegenerative diseases and brain tumors with the help of a multiplex bead-based flow-cytometry assay. In summary, the data demonstrated the potential to differentiate between glioblastoma patients and healthy controls1 [2].

Switching yet again to another biofluid, Smiths Sengkwawoh Lueong from the University Hospital Essen presented his work on the isolation and characterization of EV-associated DNA from blood samples for molecular diagnostics. Starting in lung cancer cell line models, he demonstrated a higher concentration of DNA, particularly of long DNA fragments, in large compared to small EVs. As the centrifugation-based isolation of EVs did not influence the yield or quality of cell-free DNA (cfDNA) in the sample, he was able to isolate and compare both compartments from plasma of non-small cell lung cancer (NSCLC) patients. His results suggested that, although the yield of EV-DNA from plasma was much lower compared to cfDNA, it allowed the detection of molecular alterations (e.g. KRAS mutations) at a much higher sensitivity.
On the topic of ctDNA, Verena Haselmann from the University Clinic Mannheim presented the necessary steps to fully realize the clinical value of liquid profiling of ctDNA [13]. She discussed the importance of standardizing protocols for sample collection, processing, and analysis to ensure reproducibility and reliability across clinical settings. Additionally, she emphasized the need for further validation studies to demonstrate the efficacy of ctDNA liquid profiling as a standard component in her research field, namely colorectal cancer care.

The first day was rounded off by two industry talks followed by an informal get-together which proved to be the perfect opportunity to continue discussions and network especially for young researchers that have recently joined the EV field.

EVs and LNPs in drug delivery: can we learn from each other?

Widely discussed in the field of EV-research is the potential of using EVs and lipid nanoparticles (LNPs) as potential drug delivery system. Therefore, the first session of the second day was focused on cellular uptake, trafficking and biodistribution of nanovesicular drug delivery vehicles such as LNPs, EVs and virus-like particles (VLPs). The opening talk was presented by Nicole Meisner-Kober from the Paris Lodron University of Salzburg, Austria. She discussed the role of nanovesicles as potential intercellular messengers and vehicles for drug delivery, highlighting their transfer of proteins, lipids and RNA. Focusing on the tracking of nanovesicles she found that EVs exhibited behavior akin to pathogenic particles, utilizing filopodia-surfing and grabbing motion to reach endocytic hotspots of the cell.  After EV internalization, they scan the ER and end up in lysosomes [14]. Next to that she discussed quantification and industrial upscaling of EV isolation for oral drug application. Besides the scientific input Nicole Meisner-Kober, announced the founding of the new Ludwig Boltzman Institute for Nanovesicular Precision Medicine.
The following talk was presented by Laura Dietz from the Max Plank Institute for Polymer Research in Mainz on the protein corona of EVs, LNPs and their role in cellular uptake. Her recently published work demonstrated that the presence of a protein corona enhances the uptake of EVs in human monocytes. In contrast to liposomes, for which uptake was triggered by the presence of immunoglobulins in the protein corona, blocking the Fc receptors on monocytes did not affect the uptake of EVs. This led them to suggest alternative docking mechanisms to cell membranes and uptake, highlighting distinct differences between EVs and liposomes as nanocarriers [15].
The presentation from Volker Mailänder from the Center for Translational Nanomedicine in Mainz was focused on uptake and intracellular trafficking as well as excretion of synthetic nanocarriers. The protein corona was the most defining factor to determine rate and route of endocytosis. He focused on the fate of the protein corona inside cells after uptake and investigated the separation of the protein corona from nanoparticles. They uncovered that within cells nanoparticles were directed towards recycling endosomes, whereas the corona ended up in multivesicular bodies. It is now assumed that nanoparticles are eliminated from the cell by exocytosis, while the protein corona is metabolized within cells [16].

Inside, Surface and Corona: Deciphering Functional EV Components

This session was opened by the ISEV president Edit Buzas from the Semmelweiss University in Budapest, Hungary.
Firstly, she described that dependent on the respective EV biogenesis pathway EV coronas can be extracellularly or intracellularly acquired. Intrigued by the complexity of EV pathways, they investigated whether the release of entire multivesicular large EVs was a common mechanism shared by cell types from various origins. Electron microscopy studies and fluorescent-tagging of membranes allowed them to identify a new so called ‘torn bag mechanism’ of EV release. As their data suggested a novel mechanism for small EVs distinct from ectosome release pathways and amphisome or multivesicular body exocytosis, they called the novel phenomenon amphiectosomes to describe the release of amphisomes through ectocytosis [17]. As potential markers of the released small EVs from amphiectosomes, CD63 or LC3 were found to be mutually exclusively present. The general mechanism of en bloc released membrane cloaked particles is well-described for a subset of viruses such as Hepatitis C, Noro or Zika virus [18] as well as in form of migrosomes [19] or spherosomes in cancer [20].

Maria Gomez-Serrano, from the Philipps University Marburg, took the audience to a more applied EV project focusing on the interplay of ovarian cancer with its tumor microenvironment. Displaying a unique anatomy of functionally distinct compartments, the microenvironment is characterized by tumor-associated macrophages, T-cells, mesothelial cells, fibroblasts and adipocytes. To decipher the communication routes, she investigates the cargo molecules of EVs isolated from patients’ ascites to explain observed tumor-promoting and immune suppressive properties. Whether metastasis of ovarian cancer is promoted by adipocyte-derived EVs from the omentum and the specific role of p53 and NF-kB signaling therein will be further studied.

Shifting from cancer to kidney diseases, Fabian Braun from the University Clinic Hamburg Eppendorf presented the role of small- and medium-sized EVs in podocytes and proximal tubular cells aiming to identify alterations in glomerular diseases and acute kidney injury. He showed that EV-mediated glomerular crosstalk was elevated in kidneys with glomerulonephritis and that those EVs altered motility and mitotic rates of parietal epithelial cells. While only minor proteomic changes were observed on medium-sized EVs isolated from supernatant of various podocyte stimulations, on the cellular level, they could observe an increased expression of DAB2. DAB2 is a regulator of LDL receptor recycling. Surprisingly, the isolated medium-sized EVs showed matryoshka-doll like membrane stacks when performing electron microscopy of EV cryosections.
The session was rounded off with latest updates on EV corona methodology by Martin Wolf from the Paracelsus University Salzburg, Austria. He presented how their scalable EV isolation protocol via tangential flow-filtration (TFF) recovered an imageable and functional protein corona [21]. Applying an artificial, functional EV corona by loading defined proangiogenic proteins, the so called VIEnA designer corona, revealed the importance of the EV corona on angiogenesis features such as relative network length. Facing the challenge of preserving the corona during storage, he could not just use the previously described HAT buffer [22] due to the overlapping human albumin from the buffer, which is also integral compound of the designer corona. Therefore, he studied the effect of coating experimental tubes before EV storage with various buffer formulation to discover the preservative value of HAT buffer coating on EV integrity and yield after storage. It can be hypothesized that this concept can be translated to other experimental scenarios in which HAT buffer substitution might need to be avoided.

EVs in the Tumor Microenvironment

Elke Pogge von Strandmann from the Philipps University Marburg could catch the attention of the audience in the last session. Within their project on pancreatic ductal adenocarcinoma, they studied the effect of BAG6 in tumor cells [23] and found a distinct mast cell population only present in BAG6 low expressing tumors. Inhibiting the release of BAG6-low EVs through the neutral sphingomyelinase inhibitor GW4896 could abrogate tumor growth. The better understanding of EVs in the tumor microenvironment allowed them to find further vulnerabilities of pancreatic ductal adenocarcinoma such as α-IL33 antibodies to inhibit mast cell activation by capturing IL33-positive EVs, as well as Imatinib inhibition of mast cell activity, and α-PDGF antibodies to inhibit mast cell tumor promoting activities. Currently patient stratification and treatment regimens are prepared for clinical translation.

Specific EV biogenesis pathways in breast cancer were the focus of GSEV president Kerstin Menck’s presentation from the University Hospital Münster. She investigated the proteome cargo of EVs originating from breast cancer cells after Syntenin, SDCBP, knockout. The intracellular adaptor protein SDCBP is known as central regulator of EV biogenesis and overexpression in breast cancer patients is associated with a poorer survival probability. They hypothesized that breast cancer tumor EVs can promote tumor cell proliferation, migration and invasion. In the proteome of SDCBP knock out cells, they found cell adhesion molecules significantly downregulated compared to wild type controls. Functional validation experiments revealed that SDCBP knock out EVs attached less efficiently to extracellular matrix in vitro, resulting in breast cancer tumor cells showing a reduced capacity for invasion and niche formation. Opportunities to translate these findings into breast cancer treatment will be further explored.
 
Daniel Hagey from the Karolinska Institute, Sweden, presented his work on the dynamic size and association profiles of tumor-derived DNA during pancreatic cancer progression. Therefore, EVs were separated according to size and small EV supernatant was included as soluble biomarker pool subsequently extracting DNA. Abundance of copy number variations and mutations of tumor origin were assessed in each of the isolated DNA fractions and tested for biomarker potential. Observed variability in tumor fractions is currently correlated to clinical data to leverage the full clinical biomarker potential of all tested EV populations.

The closing talk of the session and the conference was performed by Christian Pallasch from the University Clinic Cologne. They found that a loss of TP53 expression led to increased EV biogenesis and PD-L1 expression in B-cell lymphoma. Targeting PD-L1 by α-PDL1 antibodies and interfering with EV release by RAB27 knockout, had the potential to overcome TP53-mediated resistance to chemoimmunotherapy [24]. Increased macrophage activity such as elevated phagocytosis could synergistically inhibit tumor growth in combination with chemoimmunotherapy.

The IGLD Leipzig meeting 2024 was closed with an unforgettable evening at the social network event, set in the unique and historic atmosphere of an old-timer museum, where classic cars and elegant surroundings created the perfect backdrop for networking and camaraderie.