Isolation Strategies and Proteomic Characterization of Extracellular Vesicles

“Extracellular vesicles” is the collective term used to describe vesicular entities that are released from cells into the extracellular environment. These vesicles are composed of a delineating lipid membrane and its cargo which can comprise of bioactive molecules such as lipids, RNA, DNA and proteins which can be shuttled between cells and thus function as a means of cell-to-cell communication. The aims of this thesis were to address how discrepancies in isolation procedure effects the isolate, to distinguish vesicular proteins from co-isolated proteins, to determine the proteome of tissue resident EVs in tumors of colorectal cancer patients and finally to develop a method for high quality vesicle isolates from blood plasma. We demonstrate that different rotor types will influence not only the yield of isolated vesicles, but also the purity. Furthermore, prolonged ultracentrifugation can up to a point produce higher yields at no apparent cost to purity. Even after purification of vesicles with a density gradient, however, there are proteins in the isolate whose vesicular nature can be questioned as they are susceptible to membrane-impermeable proteolytic digestion. Interestingly, proteolysis of perceived luminal motifs of transmembrane proteins suggests the existence of proteins with unconventional topological orientation within the membrane. We further illustrate that vesicles isolated directly from colorectal tumor tissue greatly differ from vesicles from corresponding healthy tissue in their proteomic makeup. Lastly, we demonstrate the possibility of attaining a highly purified vesicle isolate from blood plasma that is of high enough quality for relevant proteomic evaluation. In conclusion, we demonstrate how both yield and purity can be optimized in cultured samples as well as in complex biological samples

Isolation Strategies and Proteomic Characterization of Extracellular Vesicles

“Extracellular vesicles” is the collective term used to describe vesicular entities that are released from cells into the extracellular environment. These vesicles are composed of a delineating lipid membrane and its cargo which can comprise of bioactive molecules such as lipids, RNA, DNA and proteins which can be shuttled between cells and thus function as a means of cell-to-cell communication. The aims of this thesis were to address how discrepancies in isolation procedure effects the isolate, to distinguish vesicular proteins from co-isolated proteins, to determine the proteome of tissue resident EVs in tumors of colorectal cancer patients and finally to develop a method for high quality vesicle isolates from blood plasma. We demonstrate that different rotor types will influence not only the yield of isolated vesicles, but also the purity. Furthermore, prolonged ultracentrifugation can up to a point produce higher yields at no apparent cost to purity. Even after purification of vesicles with a density gradient, however, there are proteins in the isolate whose vesicular nature can be questioned as they are susceptible to membrane-impermeable proteolytic digestion. Interestingly, proteolysis of perceived luminal motifs of transmembrane proteins suggests the existence of proteins with unconventional topological orientation within the membrane. We further illustrate that vesicles isolated directly from colorectal tumor tissue greatly differ from vesicles from corresponding healthy tissue in their proteomic makeup. Lastly, we demonstrate the possibility of attaining a highly purified vesicle isolate from blood plasma that is of high enough quality for relevant proteomic evaluation. In conclusion, we demonstrate how both yield and purity can be optimized in cultured samples as well as in complex biological samples

The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles

Background: Extracellular vesicles (EV), the collective term for vesicles released from cells, consist of vesicle species ranging in size from 30 nm to 5 µm in diameter. These vesicles are most commonly isolated by differential centrifugations, which pellets particles based on their differential movement through the liquid medium in which they are immersed. Multiple parameters, including the utilization of different rotor types, can influence the yield and purity of isolated vesicles; however, the understanding of how these factors affect is limited. Materials and methods: Here, we compare the influence of multiple centrifugation parameters, including the use of swinging bucket and fixed angle rotors, as well as different centrifugation times, for the isolation of the smallest EVs, “exosomes.” In particular, we determine the yields of exosomal RNA and protein, as well as the nature of the isolated vesicles and possible protein contamination with methods such as electron microscopy, western blot and flow cytometry. Results: Our results show that application of a specific g-force or rotation speed by itself does not predict the ability of pelleting exosomes, and that prolonged centrifugation times can achieve greater yields of exosomal RNA and protein, whereas very long centrifugation times result in excessive protein concentrations in the exosome pellet. Conclusion: In conclusion, rotor type, g-force and centrifugation times significantly influence exosome yield during centrifugation-based isolation procedures, and current commonly recommended isolation protocols may not be fully optimized for yield and purity of exosomes

Exosomes purified from a single cell type have diverse morphology

Extracellular vesicles (EVs) are produced by all known organisms and are important for cell communication and physiology. Great morphological diversity has been described regarding EVs found in body fluids such as blood plasma, breast milk, and ejaculate. However, a detailed morphological analysis has never been performed on exosomes when purified from a single cell type. In this study we analysed and quantified, via multiple electron microscopy techniques, the morphology of exosomes purified from the human mast cell line HMC-1. The results revealed a wide diversity in exosome morphology, suggesting that subpopulations of exosomes with different and specific functions may exist. Our findings imply that a new, more efficient way of defining exosome subpopulations is necessary. A system was proposed where exosomes were classified into nine different categories according to their size and shape. Three additional morphological features were also found in exosomes regardless of their morphological classification. These findings show that exosomes purified from a single cell line are also morphologically diverse, similar to previous observations for EVs in body fluids. This knowledge can help to improve the interpretation of experimental results and widen our general understanding of the biological functions of exosomes

T2 and T17 cytokines alter the cargo and function of airway epithelium-derived extracellular vesicles

BACKGROUND: Asthma is a common and heterogeneous disease that includes subgroups characterized by type 2 (T2) or type 17 (T17) immune responses for which there is a need to identify the underlying mechanisms and biomarkers in order to develop specific therapies. These subgroups can be defined by airway epithelium gene signatures and the airway epithelium has also been implicated to play a significant role in asthma pathology. Extracellular vesicles (EVs) carry functional biomolecules and participate in cell-to-cell communication in both health and disease, properties that are likely to be involved in airway diseases such as asthma. The aim of this study was to identify stimulus-specific proteins and functionality of bronchial epithelium-derived EVs following stimulation with T2 or T17 cytokines. METHODS: EVs from cytokine-stimulated (T2: IL-4 + IL-13 or T17: IL-17A + TNFα) human bronchial epithelial cells cultured at air-liquid interface (HBEC-ALI) were isolated by density cushion centrifugation and size exclusion chromatography and characterized with Western blotting and electron microscopy. Transcriptomic (cells) and proteomic (EVs) profiling was also performed. RESULTS: Our data shows that EVs are secreted and can be isolated from the apical side of HBEC-ALI and that cytokine stimulation increases EV release. Genes upregulated in cells stimulated with T2 or T17 cytokines were increased also on protein level in the EVs. Proteins found in T17-derived EVs were suggested to be involved in pathways related to neutrophil movement which was supported by assessing neutrophil chemotaxis ex vivo. CONCLUSIONS: Together, the results suggest that epithelial EVs are involved in airway inflammation and that the EV proteome may be used for discovery of disease-specific mechanisms and signatures which may enable a precision medicine approach to the treatment of asthma

Proteomic profiling of tumour tissue‐derived extracellular vesicles in colon cancer

Abstract Colon cancer is one of the most commonly occurring tumours among both women and men, and over the past decades the incidence has been on the rise. As such, the need for biomarker identification as well as an understanding of the underlying disease mechanism has never been greater. Extracellular vesicles are integral mediators of cell‐to‐cell communication and offer a unique opportunity to study the machinery that drives disease progression, and they also function as vectors for potential biomarkers. Tumour tissue and healthy mucosal tissue from the colons of ten patients were used to isolate tissue‐resident EVs that were subsequently subjected to global quantitative proteomic analysis through LC‐MS/MS. In total, more than 2000 proteins were identified, with most of the common EV markers being among them. Bioinformatics revealed a clear underrepresentation of proteins involved in energy production and cellular adhesion in tumour EVs, while proteins involved in protein biosynthesis were overrepresented. Additionally, 53 membrane proteins were found to be significantly upregulated in tumour EVs. Among them were several proteins with enzymatic functions that degrade the extracellular matrix, and three of these, Fibroblast activating factor (FAP), Cell surface hyaluronidase (CEMIP2), as well as Ephrin receptor B3 (EPHB3), were validated and found to be consistent with the global quantitative results. These stark differences in the proteomes between healthy and cancerous tissue emphasise the importance of the interstitial vesicle secretome as a major player of disease development

Extracellular vesicles in motion

By secreting extracellular vesicles (EVs), including exosomes and microvesicles, into the extracellular milieu, cells can convey complex biological messages between each other. These vesicles are generally thought to be static packages lacking the flexibility of their parental cells in terms of motility and the ability to change shape. However, cryo-electron micrographs reveal the presence of actin-like filaments in a subpopulation of EVs, raising the question if these vesicles could possess motile capabilities similar to that produced by actin in cells. We here show that fluorescently labeled EVs change their shape in a matter of minutes, regardless of whether they are isolated from human body fluids, mouse tissue or cell culture of human cells or yeast. Our findings therefore cast doubt on movement being confined to cells, suggesting that some EVs indeed have an intrinsic capacity to move. This novel observation showing morphological plasticity among EVs adds another level of complexity to the already multifaceted vesicular secretome, and may lead to new ways in which we perceive these nano-carriers of intercellular signals