Extracellular vesicles (EVs) are phospholipid bilayer- enclosed particles, released from cells into body fluids. EVs’ morphology and molecular cargo reflect (pato)physiological state of their cell-of-origin, making them ideal candidates for minimally invasive biomarkers. However, they are largely unexplored due to lack of established isolation methods from complex fluids. Here, we established a method for effective isolation of pure EVs from blood and used it to evaluate EV-miRNAs as biomarkers of treatment outcome in malignant mesothelioma (MM). Methods: For method development, EVs were isolated from plasma of 10 healthy subjects, using density-based (ultracentrifugation on 20% sucrose cushion; sUC) and size- based (size exclusion chromatography; SEC) isolation methods. Size, concentration and purity of isolates were determined with NTA, AF4-MALS, TEM and miRNA levels with qPCR. Results: SEC generally led to a higher number but lower quality of isolated particles compared to sUC (P (0.001), due to contamination with lipoproteins and aggregates. sUC method was highly repeatable and resulted in purer EVs isolate with more miRNA cargo. Conclusion: sUC method led to higher yield and purity of isolated EVs and can be used used for biomarker studies in MM.
F.09 Development of a new technological process or technology
COBISS.SI-ID: 34540761Malignant mesothelioma (MM) is a rare cancer of the pleura or the peritoneum, mostly associated with asbestos exposure. It is usually diagnosed in the advanced stages and is characterized by poor prognosis and short survival. Extracellular vesicles (EVs) are membranebound particles released from many cell types into different body fluids. EVs can transport various molecules such as RNAs, proteins and metabolites and their molecular composition reflects the characteristics of the origin cell. EVs or their cargo could therefore serve as new minimally invasive biomarkers that would enable earlier detection of MM or better prediction of treatment response. Our aim was to evaluate miRNAs enriched in serum extracellular vesicles as potential biomarkers in patients with MM. We included 20 MM patients and 10 control subjects that were occupationally exposed to asbestos but did not develop any asbestos-related diseases. In MM, good response was defined as overall survival of more than 18 months, while poor response was defined as overall survival of less than 10 months. EVs were isolated from serum samples before and after treatment using ultracentrifugation on 20% sucrose cushion. Levels of miRNAs miR-103-3p, miR-126-3p and miR-625-3p from EVs were analysed using qPCR. MiR-425-5p and let-7i-5p were used for normalisation. Nonparametric tests and survival analysis were used to evaluate the biomarker potential of miRNAs from EVs. In the EVs isolated from serum of MM patients, relative expression of miR-103-3p was significantly lower compared to healthy asbestos-exposed controls (P=0.001), while relative expression of miR-126-3p was significantly higher (P=0.001). MiRNA expression before treatment was not associated with survival of MM patients. After treatment, relative expression of miR-625-3p and miR-126-3 increased only in MM patients with poor treatment response (P=0.012 and P=0.036, respectively), while no differences were observed in patients with good response (P=0.173 and P=0.374, respectively). Relative increase in miR-625-3p expression after treatment for more than 3.2% was associated with much shorter progression-free survival (7.5 vs 19.4 months, P=0.024) and overall survival (12.5 vs 49.1 months, P=0.043) of MM patients. In conclusion, miRNAs from EVs could serve as diagnostic or prognostic biomarkers in MM.
F.18 Transfer of new know-how to direct users (seminars, fora, conferences)
COBISS.SI-ID: 34608345Several pleural diseases have been associated with asbestos exposure. Asbestos exposure may lead to the development of benign pleural diseases, such as pleural plaques, diffuse pleural thickening, and pleural effusion, as well as to the development of malignant mesothelioma, a highly aggressive tumour of the pleura. Asbestos exposure related to pleural diseases may be occupational or environmental. Although the causal relationship between asbestos-related pleural diseases and asbestos exposure has been well confirmed, the role of genetic factors in the development of these diseases needs to be further investigated and elucidated. The results of the studies performed so far indicate that in addition to asbestos exposure, genetic factors as well as the interactions between genetic factors and asbestos exposure may have an important impact on the risk of asbestos-related pleural diseases, especially malignant mesothelioma. This book chapter presented how the risk of developing asbestos-related pleural diseases may be influenced by asbestos exposure, genetic factors, interactions between different genetic factors, as well as interactions between different genetic factors and asbestos exposure
F.30 Professional assessment of the situation
COBISS.SI-ID: 16466691Asbestos-related diseases, including asbestosis, benign pleural diseases, lung cancer, other types of cancer, and especially malignant mesothelioma (MM), still represent an enormous problem all over the world and are among the most investigated occupational diseases. Considering that MM is a highly aggressive and severe malignant cancer of pleura, peritoneum and other serosal surfaces, new blood biomarkers for earlier diagnosis, following response to treatment and disease progression, have been intensively investigated. Several studies suggested that soluble mesothelin-related peptides, fibulin-3, survivin, osteopontin, vimentin, calretinin, and many others could be helpful in diagnosis, detecting the progression of MM and evaluating tumour response to treatment; however, these biomarkers have not been validated in clinical practice. Therefore, search for novel better stand-alone or composite biomarkers is under way. The aim of this chapter is to present the importance of blood biomarkers in evaluating the risk of developing asbestos-related diseases, early diagnosis, following the response to treatment and progression of these diseases, with special emphasis on MM.
F.30 Professional assessment of the situation
COBISS.SI-ID: 17842179Within the project, we also pay a lot of attention not only to research, but also to education and dissemination of information on diseases related to asbestos exposure. Prof. Viljem Kovač was an invited speaker at a meeting organized by the Association of Patients with asbestos diseases (Društvo obolelih zaradi azbesta) onNovember 6th 2018 in Deskle and presented recent developments in the diagnosticis and treatment of malignant mesothelioma. Such lectures enable the rapid transfer of information on key scientific findings, available diagnostic methods and new treatment options to those who need it the most: people who have been exposed to asbestos, patients with asbestos diseases, their families and healthcare professionals caring for this population.
F.18 Transfer of new know-how to direct users (seminars, fora, conferences)
COBISS.SI-ID: 3180411