9:30 - 10:00Registration
10:00 - 10:15
Opening words - Sulev Kõks
10:15 - 10:55

Bernd Giebel - Mesenchymal stem cell-derived extracellular vesicles: a potential new tool in regenerative medicine (Abstract)Human mesenchymal stem/stromal cells (MSCs) represent a promising tool in regenerative medicine. Up to now, more than 800 NIH-registered clinical trials investigated their immunomodulatory and pro-regenerative therapeutic potential in various diseases, including graft-versus-host disease (GvHD) and ischemic stroke. Despite controversial reports regarding the efficacy of MSC-treatments, MSCs seem to exert their beneficial effects rather in a paracrine manner than by cell replacement. In this context, extracellular vesicles (EVs), such as exosomes and microvesicles, are discussed to execute the MSCs’ therapeutic effects. Indeed, we observed beneficial therapeutic impacts of MSC-EVs in a patient, who suffered from steroid-refractory acute GvHD. Furthermore, beneficial effects were observed in animal models for several different diseases. Due to contradictory reports on the efficacy of MSC therapies, we currently compare functional properties of EVs harvested from supernatants of independent MSC preparations. In a first set of experiments, we compared T cell proliferation modulating activities of EV fractions harvested from cell culture-supernatants of MSCs derived from bone marrow probes of 20 different healthy donors. In addition, proteomic analyses of all applied EV-fractions were performed. Correlations between proteomic patterns and the MSC-EVs’ T cell proliferation modulating activities were observed. Aiming to dissect the EVs’ heterogeneity in giving samples we have set up and optimized an imaging flow cytometry based method to analyse EVs at the single vesicle level. By using sets of 3 different antibodies, including the EV markers CD9 and CD81, we are able to discriminate different EV-subpopulations, now. In our ongoing work we try to identify surrogate markers or marker combinations reflecting the immunomodulatory properties of given MSC-EV fractions. Furthermore, we optimize EV purification protocols to translate MSC-EVs into the clinics. 

10:55 - 11:15

Helena Sork - Small RNA profile of vesicular and non-vesicular extracellular RNA (Abstract)Extracellular vesicles (EVs) are known to contain a range of bioactive molecules, such as regulatory noncoding RNA, which can be transferred between organs and directly alter the functional state of the recipient cell. Owing to that, there is an increasing interest to understand in detail the composition of vesicular RNA, especially miRNAs, representing one of the most potent gene expression modulators. While previous studies on blood plasma provide evidence that the majority of extracellular miRNA is secreted independently of EVs, it is accepted that only EV-associated portion of miRNAs can exert biological effects. To shed light on the composition and sorting patterns of vesicular and non-vesicular pools of RNA, we conducted small RNA deep sequencing on vesicular-, non-vesicular- and cellular samples from two mouse- and two human cell-lines. As expected, the vesicular and non-vesicular portion of extracellular small RNA appears to be more similar in its content to each-other than to their parental cells. Nevertheless, certain miRNAs are still preferentially secreted either via- or independent of EVs. This study is important for understanding the basic biology of vesicular sorting patterns of small RNAs as well as for the development of EV-based carrier systems for therapeutic interventions. 

11:15 - 11:35

Artjom Stepanjuk - Proteolytic Modification of the Integrins in Microvesicles Secreted From the Human Trophoblast Cell Lines (Abstract)Artjom Stepanjuk, Andres Salumets, Sulev Ingerpuu Microvesicles released from the cell membrane have an important role in the cellular communication and transfer of information between different tissues. Microvesicles regulate various pathological processes, including tumor progression and preeclampsia. There is no general biomarker for microvesicles found till now due to their particular mechanism of biogenesis. Every cell type has its own surface proteome which means that the released microvesicles have specific membrane composition. The aim of our work was to find reliable surface biomarkers for microvesicles secreted by the human trophoblasts. For this purpose we studied the expression of different integrins on human trophoblast cell line JAR and JEG-3 cells and secreted from these cells microvesicles. Microvesicles were collected from the growth media of the cells by differential centrifugation. The expression of integrin α6β1 and α6β4 chains was established after western blotting with chain-specific antibodies. We used antibodies, which specifically recognize the integrin chains extracellular N-terminal or cytoplasmatic C-terminal epitopes. We detected by Western blot analysis that microvesicles secreted by the JAR and JEG-3 cells contain integrin α6, β1 and β4 chains N-terminal fragments, instead of intact molecules. To affirm these results, we performed integrin chain fragments mass-spectrometry. Appeared, that last peptides identified in the integrin β4 chain fragment localized close to proteolytic cleavage site for caspases 3 and 7. It has not been shown before that integrin chains may be proteolytically modified during microvesicle biogenesis. Our further task is to elucidate the role of caspases 3 and 7 in proteolytic degradation of the β4 integrin during microvesicles formation. We plan to study next whether integrin proteolytic modification is crucial for microvesicle formation in trophoblast cells. Our second task is to confirm that truncated integrin chains can be used as a microvesicles specific markers. 

11:35 - 11:55Coffee break
11:55 - 12:25Victoria James - Extracellular vesicles, cell-to-cell communication and the role of RNA (Abstract)The role of extracellular vesicles (EVs) as vehicles for cell-to-cell communication between a tumour and its environment is a relatively new concept. However, the hypothesis that EVs may play a critical role in co-opting tissues by tumours to generate a metastatic niche is generating significant interest. Many studies have focused on the protein component of tumour-derived EVs, but due to an absence of tools to track the movement of the RNA contained in EVs, the role of the RNA component cargo has been overlooked. This talk will present the novel use of RNA tracking technologies to demonstrate the delivery of prostate cancer cell EV-RNA cargos to osteoblasts, the proposed target cell type in osteoblastic prostate metastasis. Using a subset of genes involved in the regulation of osteoclast activation and bone remodelling mechanisms to demonstrate the complex relationship between the prostate cancer cell EV-RNA cargo and the recipient osteoblast transcriptome.  
12:25 - 12:55
Stoyan Tankov - Characterization of extracellular vesicles in early stages of embryo development (Abstract)Extracellular vesicles (EVs) are recognised as potent vehicles for intercellular communication. To date, there is little information available regarding the role of EVs during the early stages of embryonic development. The aim of this study was to develop techniques for the recovery of EVs secreted by bovine embryos in an in vitro culture system. The EVs were characterized according to size, concentration and electrical surface properties (zeta potential), in order to understand the role of EVs secreted by the bovine embryos during very early stages of development. Bovine embryos were created by in vitro fertilization (IVF) and cultured for 2, 5 and 8 days in modified synthetic oviductal fluid media. Embryos were cultured in groups of 0 (control), 1 (individual), 5 and 10 embryos over time. Conditioned media, at days 2, 5 or 8 post-IVF, was collected and EVs were isolated using a series of centrifugations and size-exclusion chromatography. The size, concentration and zeta potential of EVs was characterized using a Nanoparticle tracking analysis (Particlemetrix, Germany). Using this method of isolation, we were able to collect and characterise EVs produced by a single embryo at a very early stage of development (2-cell stage). Analysis confirmed the presence of EVs at all stages of development, with the concentration of EVs higher in blastocysts (~10^8 particles/ml), as compared to 2-cell stage embryos (~10^7 particles/ml) in embryos cultured in groups. These data were reflected in embryos cultured individually, where the size and concentration of EVs secreted at the 2-cell stage were notably different to EVs secreted by morula and blastocysts stage embryos. We have demonstrated that bovine embryos secrete EVs during the early stages of their development. We have developed a sensitive protocol for the isolation of EVs from embryos cultured individually or in groups. Furthermore, the concentration of EVs secreted varies depending on embryo developmental stage. 
12.55 - 14:10Lunch
14:10 - 14:50Stuart Hunt - Extracellular vesicles as a source of biomarkers for head and neck cancer diagnosis (Abstract)Worldwide, there are 560,000 new cases of head and neck cancer (HNC) diagnosed and 300,000 deaths annually. Due to their ability to spread they have a poor outcome, with only ~50% of patients surviving 5 years after diagnosis. HNC is strongly associated with lifestyle and environmental risk factors, which include tobacco and alcohol consumption. There is also strong evidence that some HNCs are caused by human papilloma virus (HPV). Approximately 75% of head and neck cancers start in the oral cavity. The incidence of oral cavity cancer has been increasing among younger people, possibly due to an increase in sexually transmitted HPV infection. Extracellular vesicles (EVs) are small particles that are released by a variety of cell types in the human body and can be found in many fluids such as saliva, blood plasma and urine. They are believed to facilitate cell-to-cell communication as they are vehicles carrying molecular messages between cells. The cargo of EVs is altered in cancer, which has a pro-tumorigenic impact on this cell-to-cell communication and supports the development and spread of tumours. EVs were isolated from a panel of HNC cell lines, with protein and RNA cargo being determined by mass spectrometry and small RNA sequencing analysis, respectively. The miRNA profile of EVs was found to be related to HPV status of the producing cell.  
14:50 - 15:10Reet Kurg - Cancer-testis antigens MAGE-A proteins are incorporated into extra-cellular vesicles released by cells (Abstract)Melanoma antigens (MAGE-A) represent a unique class of tumor antigens which are expressed in a wide variety of malignant tumors, while their expression in healthy normal tissues is restricted to germ cells of testis, fetal ovary and placenta. Their restricted expression and immunogenicity make them ideal targets for immunotherapy in human cancers. MAGE-A expression is observed mainly in cancers that have acquired malignant phenotypes, invasiveness or metastasis, and the expression of MAGE-A family proteins has been linked to a poor prognosis in cancer patients. Recently we showed that MAGEA4 and MAGEA10 proteins can be expressed on the surface of retrovirus Gag-protein induced virus-like particles (VLP-s). In the current study, we have analyzed the expression of MAGE-A proteins in naturally occurring extracellular vesicles (EVs) released by cells. We show that ectopically expressed MAGE-A proteins are incorporated into extracellular vesicles of different size. MAGE-A proteins are expressed on the surface of vesicles and are resistant to treatment with salt and non-ionic detergents. The possible role of EV-s carrying MAGE-A proteins in cell-cell signaling and stimulating of cell migration will be discussed. (Anneli Kuldkepp, Magda Karakai, Olavi Reinsalu, Kadri Õunap, Jasper August Tootsi, Mart Ustav and Reet Kurg) 
15:10 - 15:40

Imre Mäger - Small RNA transcriptome and RNA bidning proteins of extracellular vesicles (Abstract)Extracellular vesicles (EVs) mediate specific functions in cell-to-cell communication in health and disease by delivering or displaying biological macromolecules such as proteins and nucleic acids, such as miRNA, to their recipient cells. In order to understand miRNA content in EVs better, in relation to other small RNA species, we conducted next generation sequencing and proteomics experiments of EV samples derived from number of different cell types. Whereas EVs contain significant amount or miRNA, it is not the main EV-associated RNA species even among small RNAs. This is also reflected by the presence of respective RNA binding proteins of EVs. Furthermore, the small proportion of miRNAs that are enriched in EVs as compared to their parent cells, are not necessarily abundant miRNAs in cells. These findings are interesting in the context of EV biology, but also important for EV bioengineering for RNA delivery and for developing EVs for potential biomarkers. 

15:40 - 15:55Arina Lavrits - The role of vesicles originated from human endometrial cells in trophoblast invasion and formation of the structure of decidua (Abstract)Lavrits, A., Koel, A., Salumets, A., Ingerpuu, S. In our study, we investigated EVs (microvesicles) generated by human trophoblasts and endometrial cells that constitute the main part of placenta, an organ developing during pregnancy in woman´s organism. To supply fetus with nutrients and oxygen, trophoblast cells invade endometrium and create a complex system where both types of cells cooperate with each other guaranteeing normal development of embryo. Thus, the purpose of our investigation is to analyze whether receptive endometrium takes up more trophoblast EVs than non-receptive. In the present study, we used endometrial carcinoma cell lines RL95-2 and HEC-1A that serve as models of receptive and non-receptive endometrium, respectively. HTR-8/Svneo cell line was used as a model of trophoblast cells and H1299 as representative of non-endometrial cells. We isolated EVs by differential centrifugation using an ultracentrifuge. To investigate an internalization of trophoblast-derived EVs into endometrial cells we incubated receptive and non-receptive endometrial cells with previously labeled EVs for 1, 2 and 4 hours. The results were further analyzed by flow cytofluorimetry and confocal microscopy. We revealed that majority of the EVs were internalized after 4 hours of incubation and that H1299 cells internalized EVs more effectively than endometrial cell lines. While comparing the internalization of EVs between receptive and non-receptive endometrium, we found out that HEC-1A cells took up 1.6 times more EVs than RL95-2. Interestingly, endometrial cells were also able to internalize H1299-derived EVs. Our preliminary results showed that trophoblast-derived EVs enter non-endometrial cells as well, which means that the internalization of EVs for cancer originated cells most probably does not need specific communication between these cells. Hence, our next step is to investigate the cooperation of trophoblasts and primary endometrial cells. In addition to this, we are going to isolate two types of EVs (microvesicles and exosomes) derived from both endometrial and trophoblast cells and to analyze the difference between their structure and the mechanism of internalization into recipient cells.  
15:55 - 16:10Madli Tamm - Detecting cancer related mutations in cell-free DNA of lung cancer patients (Abstract)M. Tamm, K. Oselin, P. A. Kivistik, M. Kals, K. Lokk, K. Keerma, M. Saare, R. Roosipuu, T. Leismann, H. Roomere, A. Metspalu, N. Tõnisson Introduction: According to WHO, lung cancer is the most common cause of cancer deaths worldwide. Cancer burden can be reduced by early detection and response-adjusted treatment. It can be achieved by molecular diagnostic analysis of tumor cell-free DNA (cfDNA) from blood samples. This minimally invasive method can capture the whole tumor heterogeneity in almost real-time setting. Materials and Methods: The study cohort comprises of 106 lung adenocarcinoma patients who have donated cfDNA plasma samples. FFPE tumor samples were available for 70% of the study subjects. We have set up an amplicon-based multiplex next-generation sequencing (NGS) workflow for 9 lung adenocarcinoma-related genes. To initially verify our results, FFPE analysis results by certified diagnostic laboratories were used. In later analysis, NGS of FFPE samples served as an internal control for cfDNA. Results: Our results showed that cfDNA NGS can detect ~1% mutant allele content. The overall concordance between diagnostic laboratory and cfDNA NGS results was 89%. FFPE analysis provided higher detection sensitivity, whereas cfDNA analysis has higher specificity. We found a resistance mutation in cfDNA of a patient with previous history of long-term EGFR-TKI therapy whose treatment should be changed to third generation EGFR TKI-s. Perspectives: Our further aim is to collect longitudinal samples and perform a larger screen of drug susceptibility and resistance mutations in our study cohort, involving detection of copy number variations and DNA methylation changes. We believe that regular cfDNA analysis should become a clinical routine in various cancers. 
16:10 - 16:20Closing remarks
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