Extracellular Vesicles Studies Research Articles

Understanding the Conundrum of Pancreatic Cancer in the Omics Sciences Era.

Pancreatic cancer (PC) is an increasing cause of cancer-related death, with a dismal prognosis caused by its aggressive biology, the lack of clinical symptoms in the early phases of the disease, and the inefficacy of treatments. PC is characterized by a complex tumor microenvironment. The interaction of its cellular components plays a crucial role in tumor development and progression, contributing to the alteration of metabolism and cellular hyperproliferation, as well as to metastatic evolution and abnormal tumor-associated immunity. Furthermore, in response to intrinsic oncogenic alterations and the influence of the tumor microenvironment, cancer cells undergo a complex oncogene-directed metabolic reprogramming that includes changes in glucose utilization, lipid and amino acid metabolism, redox balance, and activation of recycling and scavenging pathways. The advent of omics sciences is revolutionizing the comprehension of the pathogenetic conundrum of pancreatic carcinogenesis. In particular, metabolomics and genomics has led to a more precise classification of PC into subtypes that show different biological behaviors and responses to treatments. The identification of molecular targets through the pharmacogenomic approach may help to personalize treatments. Novel specific biomarkers have been discovered using proteomics and metabolomics analyses. Radiomics allows for an earlier diagnosis through the computational analysis of imaging. However, the complexity, high expertise required, and costs of the omics approach are the main limitations for its use in clinical practice at present. In addition, the studies of extracellular vesicles (EVs), the use of organoids, the understanding of host-microbiota interactions, and more recently the advent of artificial intelligence are helping to make further steps towards precision and personalized medicine. This present review summarizes the main evidence for the application of omics sciences to the study of PC and the identification of future perspectives.

A comparative analysis of extracellular vesicles (EVs) from human and feline plasma

Extracellular vesicles (EVs) are nanoparticles found in all biological fluids, capable of transporting biological material around the body. Extensive research into the physiological role of EVs has led to the development of the Minimal Information for Studies of Extracellular Vesicles (MISEV) framework in 2018. This framework guides the standardisation of protocols in the EV field. To date, the focus has been on EVs of human origin. As comparative medicine progresses, there has been a drive to study similarities between diseases in humans and animals. To successfully research EVs in felines, we must validate the application of the MISEV guidelines in this group. EVs were isolated from the plasma of healthy humans and felines. EV characterisation was carried out according to the MISEV guidelines. Human and feline plasma showed a similar concentration of EVs, comparable expression of known EV markers and analogous particle to protein ratios. Mass spectrometry analyses showed that the proteomic signature of EVs from humans and felines were similar. Asymmetrical flow field flow fractionation, showed two distinct subpopulations of EVs isolated from human plasma, whereas only one subpopulation was isolated from feline plasma. Metabolomic profiling showed similar profiles for humans and felines. In conclusion, isolation, and characterisation of EVs from humans and felines show that MISEV2018 guidelines may also be applied to felines. Potential comparative medicine studies of EVs may provide a model for studying naturally occurring diseases in both humans and felines.

Addressing challenges in the removal of unbound dye from passively labelled extracellular vesicles.

Studies of extracellular vesicles (EVs), their trafficking and characterization often employ fluorescent labelling. Unfortunately, little attention has been paid thus far to a thorough evaluation of the purification of EVs after labelling, although the presence of an unbound dye may severely compromise the results or even lead to wrong conclusions on EV functionality. Here, we systematically studied five dyes for passive EV labelling and meticulously compared five typical purification methods: ultracentrifugation (UC), ultracentrifugation with discontinuous density gradient (UCG), ultrafiltration (UF), size exclusion chromatography (SEC), and anion exchange chromatography (AEC). A general methodology for evaluation of EV purification efficiency after the labelling was developed and tested to select the purification methods for the chosen dyes. Firstly, we found that some methods initially lead to high EV losses even in the absence of the dye. Secondly, the suitable purification method needs to be found for each particular dye and depends on the physical and chemical properties of the dye. Thirdly, we demonstrated that the developed parameter Erp (relative purification efficiency) is a useful tool for the pre-screening of the suitable dye-purification method combinations. Additionally, it was also shown that the labelled EVs properly purified from the unbound dye may show significantly reduced contrast and visibility in the target application, e.g. in the live cell fluorescence lifetime imaging.

The Role of Extracellular Vesicles of Various Origins in the Development of Preeclampsia

Pre-eclampsia is a clinically unfavorable pregnancy outcome that determines the main indicators of maternal and/or perinatal morbidity and mortality. According to modern concepts, the placenta plays a central role in the development of PE, while intercellular and intervesicular communications involving extracellular vesicles (EVs, extracellular vesicles) initiate a cascade of various biological effects, determining the mechanisms of ontogenesis of the gestational process in normal and pathological conditions. Achievements in studies of extracellular vesicles (EVs, extracellular vesicles) are of particular interest both to clinicians and to researchers studying the pathophysiology of gestational complications. Extracellular vesicles (EVs) in preeclampsia are produced both by scintiotrophoblast and the maternal body - blood cells (platelets, red blood cells, white blood cells) and the cardiovascular (vascular endothelium, smooth muscle) system. Changes in the concentration of these EVs can contribute to the implementation of preeclampsia, enhancing the pro-inflammatory and procoagulant states inherent in the gestation process. This review focuses on freely available information on the possible interactions between placental and maternal EVs. Understanding the contribution of EVs to the development of preeclampsia can help to deepen knowledge about the pathogenesis of this pathology and determine the diagnostic and prognostic significance of extracellular vesicles as biomarkers.

Palmitoylated mNeonGreen Protein as a Tool for Visualization and Uptake Studies of Extracellular Vesicles.

Extracellular vesicles (EVs) are membranous nanoparticles released by cells as vital mediators of intercellular communication. As such, EVs have become an attractive target for pathogens and cancer cells, which can take control over their cargo composition, as well as their trafficking, shaping the pathogenesis. Despite almost four decades of research on EVs, the number of specific and efficient EV labeling methods is limited, and there is still no universal method for the visualization of their transport in living cells. Lipophilic dyes that non-specifically intercalate into the EVs membranes may diffuse to other membranes, leading to the misinterpretation of the results. Here, we propose a palmitoylated fluorescent mNeonGreen (palmNG) protein as an alternative to chemical dyes for EVs visualization. The Branchiostoma lanceolatum-derived mNeonGreen is a brighter, more stable, and less sensitive to laser-induced bleaching alternative to green fluorescent protein (GFP), which makes it a more potent tag in a variety of fluorescence-based techniques. A palmNG-expressing stable human melanoma cell line was generated using retrovirus gene transfer and cell sorting. This protein partially localizes to cellular membranes, and can be detected inside size-exclusion (SEC)-purified EVs. With the use of flow cytometry and fluorescent confocal microscopy, we performed qualitative and quantitative analyses of palmNG-EVs uptake in recipient human hepatoma cells, in comparison to PKH67-labeled vesicles. Our findings confirm that membrane-embedded mNeonGreen can be successfully applied as a tool in EVs transfer and uptake studies.

Abstract 6493: Optimized methods for studies of extracellular vesicles in kidney cancer

Abstract Background: Extracellular vesicles (EVs) are small membranous vesicles. All cells can secrete EVs into ECM and biofluids. The EV membrane helps to protect protein or nucleotide cargos, that can be transferred from the parental to recipient cells in an autocrine or paracrine manner. Thus, EVs are a mode of intercellular communication and valuable source of (cancer) biomarkers. The majority of human cancer EV research studies EVs from cell culture, blood or urine. Cell culture EVs are reproducible but lack sensitivity. Benign cells contribute to the total EV population found in blood and urine and thus lack tumor-specificity. We optimized EV isolation directly from renal cell carcinoma (RCC) tissue, which can aid RCC biomarker discovery. We isolated from human RCC and healthy kidney tissue. The isolated EV-product was evaluated in correspondence with the latest guidelines MISEV-guidelines (Minimal Information for Studies of Extracellular Vesicles). Methods: Following radical nephrectomy, three technical replicates and three biological replicates were obtained for both normal and clear cell RCC. The tissues were processed and used to incubate media. EVs were isolated using a combination of differential centrifugation, filtration, and ultracentrifugation. Vesicle yield and size distribution of each sample were measured using two methods; the conventional Nanoparticle Tracking Analysis (NanoSight), and the novel method Nano Flowcytometry (NanoFCM). Transmission Electron Microscopy (TEM) was used to determine presence of intact vesicles in the EV samples. Western blot was used to detect presence of EV protein markers (CD81, CD63, Flotillin-1), and absence of cellular debris (calnexin). Results: The normalized vesicle counts for the technical replicates of normal kidney EVs were 1.44 × 1010 ± 2.51 × 109 p/mL (mean ± SD) measured by NanoSight, and 1.67 × 1010 ± 6.89 × 109 p/mL (mean ± SD) measured by NanoFCM. Vesicle counts for replicates RCC EVs measured by NanoSight were 1.80 × 1010 ± 3.59 × 109 p/mL (mean ± SD), and they were 1.68 × 1010 ± 4.18 × 109 p/mL (mean ± SD) measured by NanoFCM. We observed an acceptable biological variance in EV counts of less than 10-fold among different patients. Comparing NanoSight with NanoFCM, the differences in measured vesicle counts of identical samples were less than 5-fold. TEM confirmed presence of small EVs in all technical and biological replicates. Protein analyses demonstrate presence of CD81, CD63, and Flotillin-1 in all samples. Furthermore, calnexin was absent in all samples. Conclusions: We optimized a method for EV isolation from human kidney cancer and normal kidney tissue. Our protocol consistently results in high vesicle counts, as we confirmed using a standard method and a new method. Particle concentrations of both methods were similar, but the NanoFCM performs better in assessing particle size distribution. We confirmed the presence of intact vesicles and EV proteins, and the absence of cellular contamination. This optimized protocol can contribute to RCC-biomarker discovery and aid biological kidney cancer research. Citation Format: Richard C. Zieren, Liang Dong, Phillip M. Pierorazio, Kenneth J. Pienta, Theo M. de Reijke, Sarah R. Amend. Optimized methods for studies of extracellular vesicles in kidney cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6493.

THE ROLE OF INTERACTIONS BETWEEN TROPHOBLASTDERIVED EXTRACELLULAR MICROVESICLES, IMMUNE CELLS AND ENDOTHELIUM IN PATHOGENESIS OF PRE-ECLAMPSIA

Pre-eclampsia is a multisystemic disease that occurs in the second half of pregnancy, being characterized by the development of hypertension and proteinuria. Pre-eclampsia is still one of the main causes of maternal and neonatal morbidity and mortality. Pre-eclampsia is believed to be a result of complex interactions between maternal and placental factors. However, the exact pathophysiology of this syndrome remains unclear. Intercellular interactions are the basis of fetoplacental development in physiological pregnancy. A special mechanism of intercellular interactions is associated with the release of membranebound extracellular microvesicles by cells. Concentration and molecular composition of extracellular vesicles in biological fluids depend on the producer cell types, as well as the stimuli that trigger their formation. The studies of extracellular vesicles in pre-eclampsia focus on the particles produced by the cells of maternal cardiovascular system (endothelium, smooth muscle cells of blood vessels), and blood (erythrocytes, leukocytes, and platelets), like as by the cells of syncytiotrophoblast. Changed concentrations and molecular composition of these extracellular vesicles can contribute to the pathophysiology of pre-eclampsia, due to increased proinflammatory and procoagulant state occurring during pregnancy. This review focuses, firstly, on characteristics of the extracellular vesicles produced by syncytiotrophoblast, and possible role of their interaction with maternal immune cells, endothelial cells and platelets in the course of developing pre-eclampsia. Understanding the role of extracellular vesicles of syncytiotrophoblast in pathogenesis of pre-eclampsia could suggest an opportunity of providing these results for early and non-invasive diagnostics of placental disorders, as well as for predicting development of this disease.

Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA.

Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here, using RNA sequencing, we demonstrate that FBS contains a diverse repertoire of protein-coding and regulatory RNA species, including mRNA, miRNA, rRNA, and snoRNA. The majority of them (>70%) are retained even after extended ultracentrifugation in the preparations of vesicle-depleted FBS (vdFBS) commonly utilized in the studies of extracellular vesicles (EV) and intercellular communication. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. Many evolutionally conserved FBS-derived RNA species can be falsely annotated as human or mouse transcripts. Notably, specific miRNAs abundant in FBS, such as miR-122, miR-451a and miR-1246, have been previously reported as enriched in cell-culture derived EVs, possibly due to the confounding effect of the FBS. Analysis of publically available exRNA datasets supports the notion of FBS contamination. Furthermore, FBS transcripts can be taken up by cultured cells and affect the results of highly sensitive gene expression profiling technologies. Therefore, precautions for experimental design are warranted to minimize the interference and misinterpretations caused by FBS-derived RNA.