Browsing by Subject "extracellular vesicles"

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  • Kyykallio, Heikki; Faria, Alessandra V. S.; Hartmann, Rosabella; Capra, Janne; Rilla, Kirsi; Siljander, Pia R-M (2022)
    Recent advances in cell biology research regarding extracellular vesicles have highlighted an increasing demand to obtain 3D cell culture-derived EVs, because they are considered to more accurately represent EVs obtained in vivo. However, there is still a grave need for efficient and tunable methodologies to isolate EVs from 3D cell cultures. Using nanofibrillar cellulose (NFC) scaffold as a 3D cell culture matrix, we developed a pipeline of two different approaches for EV isolation from cancer spheroids. A batch method was created for delivering high EV yield at the end of the culture period, and a harvesting method was created to enable time-dependent collection of EVs to combine EV profiling with spheroid development. Both these methods were easy to set up, quick to perform, and they provided a high EV yield. When compared to scaffold-free 3D spheroid cultures on ultra-low affinity plates, the NFC method resulted in similar EV production/cell, but the NFC method was scalable and easier to perform resulting in high EV yields. In summary, we introduce here an NFC-based, innovative pipeline for acquiring EVs from 3D cancer spheroids, which can be tailored to support the needs of variable EV research objectives.
  • Syrjä, Pernilla; Palviainen, Mari; Jokinen, Tarja; Kyöstilä, Kaisa; Lohi, Hannes; Roosje, Petra; Anderegg, Linda; Leeb, Tosso; Sukura, Antti; Eskelinen, Eeva-Liisa (2020)
    Lagotto Romagnolo breed dogs develop a progressive neurological disease with intracellular vacuolar storage when homozygous for a variant in the autophagy-related gene 4D (ATG4D). A lysosomal enzyme deficiency has not been proven in this disease, despite its overlapping morphology with lysosomal storage diseases. Instead, basal autophagy was altered in fibroblasts from affected dogs. The aim of this study was to clarify the origin of the limiting membrane of the accumulating vacuoles and determine whether altered basal autophagy affects the extracellular release of vesicles in cells from diseased dogs. When assessed by immunoelectron microscopy, the membrane of the cytoplasmic vacuoles in affected tissues contained ATG4D, markers for autolysosomes (microtubule-associated protein 1A/B light chain 3 and lysosome-associated membrane protein 2) and for recycling endosomes (transferrin receptor 2), indicating that the vacuoles are hybrid organelles between endocytic and autophagic pathways. Ultracentrifugation, nanoparticle tracking analysis, and mass spectrometry were used to analyze the vesicles released from cultured fibroblasts of affected and control dogs. The amount of extracellular vesicles (EVs) released from affected fibroblasts was significantly increased during basal conditions in comparison to controls. This difference disappeared during starvation. The basal EV proteome of affected cells was enriched with cytosolic, endoplasmic reticulum, and mitochondrial proteins. Heat shock proteins and chaperones, some of which are known substrates of basal autophagy, were identified among the proteins unique to EVs of affected cells. An increased release of extracellular vesicles may serve as a compensatory mechanism in disposal of intracellular proteins during dysfunctional basal autophagy in this spontaneous disease.
  • Fu, Shu Yi Vicky (Helsingin yliopisto, 2022)
    Biomacromolecules are large particles found in biological fluids. The upregulations and downregulation of some biomacromolecules, such as extracellular vesicles (EVs) have been linked to cancer and infectious diseases. The study of these biological particles can help us in understanding the progression of those conditions better. Furthermore, studying naturally occurring biological molecules, e.g., immunoglobulin G (IgG), DNA, nucleic acids and glycoproteins can help us to gain more insight to important biological processes in the human body. The first part of this thesis is a literature review of monolithic columns in the separation of large biological molecules in liquid chromatographic and capillary electrochromatographic applications. Columns, including novel monolithic stationary phases, also known as monoliths, have been developed to counter some of the problems associated with the traditionally used packed beds in separation science. Monoliths have a unique structure of interconnecting porous channels, which allows faster separation with better resolution, reproducibility and mass transfer characteristics compared to packed beds. Organic-based polymer monoliths are the most widely used monolithic materials in biological applications, but the use of inorganic-based silica monoliths and hybrid monoliths have grown in the last couple of decades. Monolithic columns are versatile and they can be utilized in several chromatographic techniques, such as reversed-phase chromatography, affinity chromatography, ion-exchange chromatography, capillary electrochromatography and mixed-mode chromatography. Due to the growing interest, miniaturized monoliths e.g. in microfluidic devices, small capillaries and microarrays have been exploited to allow faster separation using sample volumes even as low as a few femtolitres. For higher sample throughput, monoliths in the format of 96-well plates, tips, sheets and disks have been introduced, especially for sample pre-treatment purposes. In the experimental part, affinity monolithic chromatography was employed for the isolation of lipoproteins and EVs in both exomere and exosome size range. The main function of EVs is transporting signal molecules from cell-to-cell to maintain homeostasis of the body. Low-density lipoprotein (LDL), very-low-density lipoprotein and chylomicrons are lipoproteins that transport different lipids in the human blood stream. The study of these particles is important because lipoproteins and especially LDL have been associated with atherosclerotic cardiovascular diseases. The experimental part of this thesis is focused on studying the feasibility of Convective Interaction Media (CIM) monoliths in disk (1.3 µm pores, 0.34 ml) and 96-well plate (2.1 µm pores, 0.1 ml) formats in purifying nanosized biomacromolecules from human plasma. The preparation of the affinity monoliths and the isolation of particles in the disk format was conducted following existing protocols and methods, which were modified for the monolithic 96-well plate. Six different monoclonal antibodies (mAbs), anti-CD9, anti-CD34, anti-CD61, anti-CD63, anti-CD81 and anti-CD82 were immobilized on the monolithic supports to target EVs. Anti-apoB100 mAb was used in targeting apolipoprotein B100 present on the surface of apoB100-containing lipoproteins. The isolation in the disk format was done using an on-line immunoaffinity chromatography – asymmetric flow field-flow fractionation method connected to ultraviolet, dynamic light scattering and diode array detectors. To compare the two different formats with different pore sizes in lipoprotein and EV isolation, the immobilization protocol and isolation conditions were optimized for the monolithic well plate. The isolation on the monolithic 96-well plate was done within 20 minutes, and the operation consumed three times less sample and buffer than in the disk format. Both monolithic formats were suitable for LDL isolation and the disks could also be used in EV isolation and separation. However, due to the larger pore size, EVs were found to be unstable in the monolithic wells.
  • Amatya, Sajeen Bahadur; Salmi, Sonja; Kainulainen, Veera; Karihtala, Peeter; Reunanen, Justus (2021)
    Simple Summary:& nbsp;Microbial dysbiosis has been credited as one of the contributing factors to the development and progression of gastrointestinal tract cancer. The altered microbiota influences carcinogenesis through the induction of instability and damage to genetic material, modulation of host metabolic and inflammatory pathways, production of carcinogenic metabolites, and suppression of host antitumor response. These microbes secrete extracellular vesicles that are possibly carrying carcinogenic bioactive metabolites within their cargo. Studies have illustrated the ability of bacterial extracellular vesicles to cross the intestinal epithelial barrier and selectively accumulate near intestinal tumor cells. The purpose of this systemic review was to highlight the possible role of gut bacterial vesicles in the development, progression, and pathogenesis of gastrointestinal tract cancer and their possible involvement in the modulation of the tumor microenvironment. An infinitesimal amount of research has been carried out on the impact of bacterial extracellular vesicles on oncogenesis and tumor progression. This review aimed to encourage more investigations on this subject.Bacterial extracellular vesicles are membrane-enclosed, lipid bi-layer nanostructures that carry different classes of biomolecules, such as nucleic acids, lipids, proteins, and diverse types of small molecular metabolites, as their cargo. Almost all of the bacteria in the gut secrete extracellular vesicles to assist them in competition, survival, material exchange, host immune modulation, infection, and invasion. The role of gut microbiota in the development, progression, and pathogenesis of gastrointestinal tract (GIT) cancer has been well documented. However, the possible involvement of bacterial extracellular vesicles (bEVs) in GIT cancer pathophysiology has not been given due attention. Studies have illustrated the ability of bEVs to cross physiological barriers, selectively accumulate near tumor cells, and possibly alter the tumor microenvironment (TME). A systematic search of original published works related to bacterial extracellular vesicles on gastrointestinal cancer was performed for this review. The current systemic review outlines the possible impact of gut microbiota derived bEVs in GIT cancer in light of present-day understanding. The necessity of using advanced sequencing technologies, such as genetic, proteomic, and metabolomic investigation methodologies, to facilitate an understanding of the interrelationship between cancer-associated bacterial vesicles and gastrointestinal cancer is also emphasized. We further discuss the clinical and pharmaceutical potential of bEVs, along with future efforts needed to understand the mechanism of interaction of bEVs in GIT cancer pathogenesis.
  • Zhao, Yue (Helsingfors universitet, 2017)
    Background: 5-year survival rate of oral tongue squamous cell carcinoma (OTSCC) has been low (less than 60%) despite developing treatment modalities. A previous research revealed that different populations of inflammatory cells infiltration in OTSCC were associated with different clinical outcomes. On the other hand, extracellular vesicles (EVs) secreted by OTSCC cells suggested crosstalk between OTSCC cells and tumor infiltrating inflammatory cells. Study aims: This study aims to investigate the interaction between OTSCC cells and inflammatory cells and answer 3 questions: (1) Can human peripheral blood mononuclear cells (MNCs) affect activities of OTSCC cells such as proliferation, migration and invasion? (2) Can EVs of OTSCC cells affect polarization of macrophages? (3) Can EVs of OTSCC cells affect cytotoxic activity of CD8+ T cells and NK cells? Materials and methods: Two OTSCC cell lines (HSC-3 and SCC-25) were used. OTSCC cells and human peripheral blood MNCs were co-cultured using a 3D organotypic myoma model. Proliferation and invasion into myoma tissue of OTSCC cells were detected by Immunohistochemical staining of pan-cytokeratin and Ki67. Invasion area and depth of OTSCC cells were measured using ImageJ software. Migration of OTSCC cells in the presence of MNCs was monitored using a scratch wound healing assay with IncuCyte™ system. OTSCC EVs were isolated with ultracentrifugation and characterized with NTA and Immuno-EM. Human primary monocytes, CD8+ T cells and NK cells were isolated using MACS, and their purity was checked using FACS. Expression of macrophage phenotypic markers was checked with qPCR. Cytotoxic activity was evaluated using an IncyCyte™ cell killing assay. Results: Activated human peripheral blood MNCs significantly reduced proliferation of both OTSCC cell lines, and invasion area of only HSC-3. None of the inflammatory cells in the experiment had any effect on invasion depth and migration of OTSCC cells. On the other hand, OTSCC cell-derived EVs didn't influence macrophage polarization, but had heterogeneous modulating effects on cytotoxic activity of CD8+ T cells and NK cells. Conclusion: We detected effects of OTSCC cells and inflammatory cells on each other by secreted molecule mediators or EVs, but the results were not uniform and varied in different OTSCC cell lines or inflammatory cell populations and sources. The outcome of the study emphasizes the importance of a personalized design of cancer treatment, which takes other components in tumor microenvironment such as inflammatory cells and EVs into consideration.
  • Palviainen, Mari; Laukkanen, Kirsi; Tavukcuoglu, Zeynep; Velagapudi, Vidya; Kärkkäinen, Olli; Hanhineva, Kati; Auriola, Seppo; Ranki, Annamari; Siljander, Pia (2020)
    Cancer alters cell metabolism. How these changes are manifested in the metabolite cargo of cancer-derived extracellular vesicles (EVs) remains poorly understood. To explore these changes, EVs from prostate, cutaneous T-cell lymphoma (CTCL), colon cancer cell lines, and control EVs from their noncancerous counterparts were isolated by differential ultracentrifugation and analyzed by nanoparticle tracking analysis (NTA), electron microscopy (EM), Western blotting, and liquid chromatography-mass spectrometry (LC-MS). Although minor differences between the cancerous and non-cancerous cell-derived EVs were observed by NTA and Western blotting, the largest differences were detected in their metabolite cargo. Compared to EVs from noncancerous cells, cancer EVs contained elevated levels of soluble metabolites, e.g., amino acids and B vitamins. Two metabolites, proline and succinate, were elevated in the EV samples of all three cancer types. In addition, folate and creatinine were elevated in the EVs from prostate and CTCL cancer cell lines. In conclusion, we present the first evidence in vitro that the altered metabolism of different cancer cells is reflected in common metabolite changes in their EVs. These results warrant further studies on the significance and usability of this metabolic fingerprint in cancer.
  • Puutio, Johanna (Helsingin yliopisto, 2020)
    Extracellular vesicles (EVs) are phospholipid bilayer-enclosed nanoparticles that are secreted by eukaryotic and prokaryotic cells. EVs carry macromolecules and signalling molecules to adjacent cells and play an important role in intercellular communication under both pathologic and homeostatic conditions. Therefore, they have become of significant interest for their therapeutic, diagnostic and prognostic potential. EVs are small and highly heterogeneous in size, shape, cargo and membrane composition, posing several challenges for establishing analytical and clinical guidelines. Therefore, EV research requires standardized and robust methods for their separation and characterization. In this study physical and immunochemical methods were employed to characterize human platelet-derived EVs (pEVs) generated from platelets activated with different external biochemical stimuli. The platelet-activating effect of the pro-inflammatory S100A8/A9 protein complex and a combination of thrombin and collagen were studied with nano flow cytometry. The size distribution of pEVs was studied with nanoparticle tracking analysis (NTA) and asymmetrical flow field-flow fractionation (AF4), which represents a newly emerging method on the EV field. Finally, fluorescent labelling and co-localization analysis were employed to characterize membrane marker composition of pEVs and assess its usefulness as an analytic tool for EV research. We succeeded in providing new hints towards meaningful discoveries in platelet biology by characterizing the way platelets respond to inflammatory and hemostatic signals by shedding pEVs. When platelet activation markers are characterized with flow cytometry, the S100A8/A9 protein appeared to cause a shift in membrane activation markers when compared to the thrombin- collagen mix and the baseline control. Increased TLT-1 translocation and decreased integrin αIIbβ3 expression on pEV surfaces suggests that S100A8/A9 induced pEV secretion through differently packed platelet α-granules, rather than from the plasma membrane. An increase in TLT-1 expression compared to decreased P-selectin and αIIbβ3 suggests that S100A8/A9 stimulation shifts platelet phenotype towards secretion rather than aggregation. A protocol for small pEV separation with AF4-MALS was set up. With this method, subtle differences between small pEV populations were seen that were not distinguishable with NTA or flow cytometry. When investigated with AF4-MALS, S100A8/A9 induced pEVs appeared larger than those produced with thrombin- collagen activation. The mean particle sizes of the pEV populations obtained from activated platelets were generally also larger than those produced without an activator. We tested novel methods to detect subtle differences in small EV population sizes that are easily missed with conventional methods due to their technical limitations. A well-optimised AF4 protocol can detect different pEV subpopulations and is a promising tool for EV. In the future, when AF4 is combined with a MALS detector and a fraction collector, nanoimaging of fluorescently labelled EVs could be combined with it as a downstream application to obtain information on their versatile biological functions.
  • Barreiro, Karina; Dwivedi, Om Prakash; Leparc, German; Rolser, Marcel; Delic, Denis; Forsblom, Carol; Groop, Per-Henrik; Groop, Leif; Huber, Tobias B.; Puhka, Maija; Holthofer, Harry (2020)
    Urinary Extracellular Vesicles (uEV) have emerged as a source for biomarkers of kidney damage, holding potential to replace the conventional invasive techniques including kidney biopsy. However, comprehensive studies characterizing uEV isolation methods with patient samples are rare. Here we compared performance of three established uEV isolation workflows for their subsequent use in transcriptomics analysis for biomarker discovery in diabetic kidney disease. We collected urine samples from individuals with type 1 diabetes with macroalbuminuria and healthy controls. We isolated uEV by Hydrostatic Filtration Dialysis (HFD), ultracentrifugation (UC), and a commercial kit- based isolation method (NG), each with different established urine clearing steps. Purified EVs were analysed by electron microscopy, nanoparticle tracking analysis, and Western blotting. Isolated RNAs were subjected to miRNA and RNA sequencing. HFD and UC samples showed close similarities based on mRNA sequencing data. NG samples had a lower number of reads and different mRNA content compared to HFD or UC. For miRNA sequencing data, satisfactory miRNA counts were obtained by all methods, but miRNA contents differed slightly. This suggests that the isolation workflows enrich specific subpopulations of miRNA-rich uEV preparation components. Our data shows that HFD,UC and the kit-based method are suitable methods to isolate uEV for miRNA-seq. However, only HFD and UC were suitable for mRNA-seq in our settings.
  • Al-Samadi, Ahmed; Awad, Shady Adnan; Tuomainen, Katja; Zhao, Yue; Salem, Abdelhakim; Parikka, Mataleena; Salo, Tuula (2017)
    The crosstalk between immune cells, cancer cells, and extracellular vesicles (EVs) secreted by cancer cells remains poorly understood. We created three-dimensional (3D) cell culture models using human leiomyoma discs and Myogel to study the effects of immune cells on highly (HSC-3) and less (SCC-25) invasive oral tongue squamous cell carcinoma (OTSCC) cell lines. Additionally, we studied the effects of EVs isolated from these cell lines on the cytotoxicity of CD8(+) T and NK cells isolated from three healthy donors. Our analysis included the effects of these EVs on innate immunity in zebrafish larvae. Activated immune cells significantly decreased the proliferation of both OTSCC cell lines and associated with a diminished invasion area of HSC-3 cells. In general, EVs from SCC-25 increased the cytotoxic activity of CD8(+) T and NK cells more than those from HSC-3 cells. However, this effect varied depending on the source and the immune and cancer cell subgroups. In zebrafish, the amount of IL-13 mRNA was decreased by SCC-25 EVs. This study describes promising in vitro and in vivo models to investigate interactions between immune cells, cancer cells, and EVs.
  • Aksela, Laura (Helsingfors universitet, 2016)
    Even though cancer treatment modalities have improved during last decades, there is still lack of specific, efficient and curative treatments especially in case of advanced and metastatic cancers. One relatively new approach is to use oncolytic adenoviruses, which selectively infect and kill cancerous cells leaving healthy cells unharmed. These viruses have shown to be effective especially when administered intratumorally and in combination with chemotherapeutics. However this approach has multiple challenges like rapid clearance by antibody neutralization in systemic administration. Another challenge is the cell entry of oncolytic adenovirus, which is mainly mediated by the Coxsackie-Adenovirus receptor and this receptor is downregulated in various cancer cells. Rapid clearance and reduced cell entry thus lead to decreased amount of oncolytic adenovirus in target cells and decreased efficacy. In order to overcome these limitations, this study explored the possibility to use cancer cell derived extracellular vesicles (EVs) as drug delivery system for oncolytic adenovirus. Since oncolytic adenoviruses have shown to be effective especially in combination with chemotherapeutics, the ability of EVs to deliver both oncolytic adenoviruses and chemotherapeutic drug paclitaxel was studied. The aims of this study were to i) study whether oncolytic adenoviruses could be encapsulated inside EVs (EV-virus complex) and load this complex with paclitaxel (EV-virus-PTX complex), ii) discover whether the surface charge or size distribution of EV-virus and EV-virus-PTX complexes differs from the control EVs and iii) study the infectivity/efficacy of EV-virus and EV-virus-PTX complex in comparison to noncapsulated adenovirus in vitro. Since this is a novel approach, the literature review focused on the characteristics, advantages and challenges of oncolytic adenoviruses and EVs. In order to determine whether cancerous cell are able to encapsulate oncolytic adenoviruses inside EVs, A549 lung cancer and PC-3 prostate cancer cells were infected with oncolytic adenovirus and the formed EVs were isolated form conditioned media using differential centrifugation. Paclitaxel was loaded into these EV-virus complexes with incubation. EV-virus complexes were imaged using transmission electron microscopy (TEM) (i). The characteristics of these EV-virus and EV-virus-paclitaxel complexes were studied by determining the surface charge by electrophoretic light scattering and the size distribution by nanoparticle tracking analysis (ii). In order to determine the infectivity/efficacy of these complexes in autologous use, three in vitro level assays were performed (cell viability, immunocytochemistry and transduction assay) (iii). In addition confocal microscopy was used to observe the localization of EV-virus complexes inside the cell. These studies pointed out that both cell lines were able to encapsulate oncolytic adenovirus inside EVs, which was observed by TEM. The size distribution of these EV-virus and EV-virus-PTX complexes may support this observation and the size was in range 50-500 nm. In addition the determined surface charge was shown to be similar in EV-virus and EV-virus-PTX- complexes when compared to control EVs derived from noninfected cells - however more specific assays in order to characterize the surface properties of EV-virus complexes are needed. As a main finding, these EV-virus and EV-virus-PTX complexes were shown to significantly increase the efficacy of oncolytic adenovirus in comparison to free oncolytic adenovirus, paclitaxel and paclitaxel+virus combination in all three in vitro assays. In addition localization of the EV-virus complex was seen with confocal microscopy imaging. These results indicate that EVs may enhance the delivery of oncolytic adenovirus into cancerous cells. Using EVs as a drug delivery system for both oncolytic adenovirus and chemotherapeutic drug paclitaxel was shown to increase the efficacy of oncolytic adenovirus in comparison to free virus. This characteristic could potentially enhance the targeting ability to cancerous cells and thus lead to decreased amount of side-effects of healthy tissues especially in case of chemotherapeutics. These promising results of this novel approach are however preliminary due to relatively low number of repetitions (n~3) and more research is needed especially in order to characterize, purify and concentrate the EV-virus complexes.
  • Perut, Francesca; Tasso, Roberta; Mannerström, Bettina (2022)
  • Kornilov, Roman; Puhka, Maija; Mannerström, Bettina; Hiidenmaa, Hanna; Peltoniemi, Hilkka; Siljander, Pia Riitta-Maria; Seppänen-Kaijansinkko, Riitta; Kaur, Sippy (2018)
    Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EVdepleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories.
  • Niemi, Liisa (Helsingfors universitet, 2016)
    Extracellular vesicles are cell-derived vesicles which consist of two lipid layers. Extracellular vesicles involve in intercellular communication, maintaining of homeostase and development of pathophysiological states in human body. Extracellular vesicles are promising biomarkers and drug carriers in future. The aim of this study was to develop a method based on time resolved fluorescence microscopy and autologous extracellular vesicles labelled with environmentally sensitive fluorescent probes for studying the distribution of mitose-inhibitor paclitaxel in prostate cancer cells (PC-3) carried by extracellular vesicles. The efficacy of paclitaxel loaded extracellular vesicles was compared to synthetic liposomes. The two subpopulations of extracellular vesicles, exosome -and microvesicle-enriched, were isolated from the PC-3 cell media by differential ultracentrifugation. The size distribution and particle concentration of extracellular vesicles was determined by nanoparticle tracking analysis. DSPC-Cholesterol liposomes were prepared by reverse-phase evaporation method and the size distribution of the liposomes was determined by dynamic laser diffraction and nanoparticle tracking analysis. Paclitaxel was loaded into the liposomes in hydration phase and into the extracellular vesicles by incubating vesicles and paclitaxel. Unbound paclitaxel was removed from samples by ultracentrifugation. The the dose-dependent sytotoxicity of paclitaxel loaded extracellular vesicles and liposomes was evaluated with Alamar Blue viability assay. The release and distribution of paclitaxel from extracellular vesicles in living PC-3 cells was investigated by confocal microscopy and time-resolved fluorescence microscopy. The exosomes had approximately 50 nm smaller diameter than microvesicles and exosome particle concentrations were significantly higher compared to microvesicles. According to viability assays conducted with wide range of concentrations, paclitaxel loaded in microvesicles were slightly more effective than paclitaxel loaded in exosomes. The time-resolved fluorescence microscopy was useful method for investigating the release and distribution of extracellular vesicle bound paclitaxel, since we succesfully detected changes in Paclitaxel-OregonGreen fluorescence lifetime in different phases of the drug delivery process. With confocal microscopy we detected that paclitaxel loaded extracellular vesicles were already uptaken inside the cells after two hours of incubation and after few hours, paclitaxel was detected in microtubules of PC-3 cells and killed PC-3 cells. Extracellular vesicles may improve the accumulation of paclitaxel into tumor cells thus preventing the side-effects of paclitaxel. Nevertheless, PC-3 cell derived extracellular vesicles have ability to increase the PC-3 cell viability, which limits their potential use as drug carrier due to safety issues. In addition, extracellular vesicles characterization and isolation methods lack standardization and the isolation of exosomes and microvesicles is impossible due to this fact. Extracellular vesicles involvement in physiological and pathophysiological states should be investigated throughoutly and their safety as drug carriers should be examined both in animal and human.
  • Zhang, Xu; Zhang, Hongbo; Gu, Jianmei; Zhang, Jiayin; Shi, Hui; Qian, Hui; Wang, Dongqing; Xu, Wenrong; Pan, Jianming; Santos, Hélder A. (2021)
    Extracellular vesicles (EVs) have emerged as a novel cell-free strategy for the treatment of many diseases including cancer. As a result of their natural properties to mediate cell-to-cell communication and their high physiochemical stability and biocompatibility, EVs are considered as excellent delivery vehicles for a variety of therapeutic agents such as nucleic acids and proteins, drugs, and nanomaterials. Increasing studies have shown that EVs can be modified, engineered, or designed to improve their efficiency, specificity, and safety for cancer therapy. Herein, a comprehensive overview of the recent advances in the strategies and methodologies of engineering EVs for scalable production and improved cargo-loading and tumor-targeting is provided. Additionally, the potential applications of engineered EVs in cancer therapy are discussed by presenting prominent examples, and the opportunities and challenges for translating engineered EVs into clinical practice are evaluated.
  • Mannerström, Bettina; Kornilov, Roman; Abu-Shahba, Ahmed G.; Chowdhury, Iftekhar M.; Sinha, Snehadri; Seppänen-Kaijansinkko, Riitta; Kaur, Sippy (2019)
    Extracellular vesicles (EVs) are central to intercellular communication and play an important role in cancer progression and development. Osteosarcoma (OS) is an aggressive bone tumour, characterized by the presence of malignant mesenchymal cells. The specific tumour-driving genetic alterations that are associated with OS development are currently poorly understood. Mesenchymal stem cells (MSCs) of osteogenic lineage have been postulated as likely candidates as the cells of origin for OS, thus indicating that MSCs and OS stroma cells may be related cell types. Therefore, this study set out to examine the EV-mediated intercellular crosstalk of MSCs and OS. MSCs and pre-osteoblasts were treated with OS-EVs at different time points, and the epigenetic signature of OS-EVs was assessed by methylation analysis of LINE-1 (long interspersed element) and tumour suppressor genes. In addition, surface markers and expression of specific genes were also evaluated. Our data indicated that OS-EVs mediated LINE-1 hypomethylation in MSCs, whereas an opposite effect was seen in pre-osteoblasts, indicating that MSCs but not pre-osteoblasts were susceptible to epigenetic transformation. Thus, OS-EVs modulated the fate of MSCs by modulating the epigenetic status, and also influenced the expression of genes related to bone microenvironment remodelling. Overall, this study provided evidence that epigenetic regulation appears to be an early event in the transformation of MSCs during the development of OS. Elucidating the mechanisms of EV-mediated communication may lead to new avenues for therapeutic exploitation.
  • Puhka, Maija; Thierens, Lisse; Nicorici, Daniel; Forsman, Tarja; Mirtti, Tuomas; af Hällström, Taija; Serkkola, Elina; Rannikko, Antti (2022)
    Simple Summary Prostate cancer lacks non-invasive specific biomarkers for aggressive disease. Urinary extracellular vesicles (uEV) could provide such markers; however, due to technical challenges, little is known regarding the pathogenesis pathways reflected in uEV. We performed a miRNA, target mRNA and pathway study focused on uEV, exploring the differences between cancer (1) status groups (Gleason score) and (2) progression groups. The uEV provided a surprisingly comprehensive presentation of differentially expressed miRNAs, target mRNAs and pathogenesis pathways. The miRNAs associated with prostate cancer status or progression were mostly unique, but still targeted overlapping sets of signalling, resistance, hormonal and immune pathways. Interestingly, mRNA targets of the key miRNAs (miR-892a, miR-223-3p, miR-146a-5p) were widely expressed in both uEV and plasma EV from PCa patients. The study thus suggests that uEV carry a vast presentation of PCa status and progression-linked RNAs that are worth further exploration in large personalized medicine trials. Background: Prostate cancer (PCa) lacks non-invasive specific biomarkers for aggressive disease. We studied the potential of urinary extracellular vesicles (uEV) as a liquid PCa biopsy by focusing on the micro RNA (miRNA) cargo, target messenger RNA (mRNA) and pathway analysis. Methods: We subjected uEV samples from 31 PCa patients (pre-prostatectomy) to miRNA sequencing and matched uEV and plasma EV (pEV) from three PCa patients to mRNA sequencing. EV quality control was performed by electron microscopy, Western blotting and particle and RNA analysis. We compared miRNA expression based on PCa status (Gleason Score) and progression (post-prostatectomy follow-up) and confirmed selected miRNAs by quantitative PCR. Expression of target mRNAs was mapped in matched EV. Results: Quality control showed typical small uEV, pEV, RNA and EV-protein marker enriched samples. Comparisons between PCa groups revealed mostly unique differentially expressed miRNAs. However, they targeted comprehensive and largely overlapping sets of cancer and progression-associated signalling, resistance, hormonal and immune pathways. Quantitative PCR confirmed changes in miR-892a (Gleason Score 7 vs. >= 8), miR-223-3p (progression vs. no progression) and miR-146a-5p (both comparisons). Their target mRNAs were expressed widely in PCa EV. Conclusions: PCa status and progression-linked RNAs in uEV are worth exploration in large personalized medicine trials.
  • de Andrade, A. L. D. L.; de Oliveira, C. E.; Dourado, M. R.; Macedo, C. C. S.; Winck, F. V.; Paes Leme, A. F.; Salo, T.; Coletta, R. D.; de Almeida Freitas, R.; Galvao, H. C. (2018)
    BackgroundA new intercellular communication mode established by neoplastic cells and tumor microenvironment components is based on extracellular vesicles (EVs). However, the biological effects of the EVs released by tumor cells on angiogenesis are not completely understood. Here, we aimed to understand the biological effects of EVs isolated from two cell lines of oral squamous cell carcinoma (OSCC) (SCC15 and HSC3) on endothelial cell tubulogenesis. MethodsOSCC-derived EVs were isolated with a polymer-based precipitation method, quantified using nanoparticle tracking analysis and verified for EV markers by dot blot. Functional assays were performed to assess the angiogenic potential of the OSCC-derived EVs. ResultsThe results showed that EVs derived from both cell lines displayed typical spherical-shaped morphology and expressed the EV markers CD63 and Annexin II. Although the average particle concentration and size were quite similar, SCC15-derived EVs promoted a pronounced tubular formation associated with significant migration and apoptosis rates of the endothelial cells, whereas EVs derived from HSC3 cells inhibited significantly endothelial cell tubulogenesis and proliferation. ConclusionThe findings of this study reveal that EVs derived from different OSCC cell lines by a polymer-based precipitation method promote pro- or anti-angiogenic effects.
  • Somersalo, Petter (Helsingfors universitet, 2017)
    Cells release different types of phospholipid bilayer-limited vesicles into the extracellular space. These are commonly referred to as extracellular vesicles (EVs). Exosomes (EXOs), ca 50-100 nm in diameter and microvesicles (MVs), ca 100-1000 nm in diameter, having different intracellular origin, are the two main subpopulations of EVs. EVs have been demonstrated to carry a range of proteins and nucleic acids subsequently delivered to recipient cells, making them attractive as drug delivery vehicles. Several mechanisms for the cellular uptake of EVs have been established. When a nanoparticle is introduced into blood plasma, plasma proteins are adsorbed to its surface, forming a protein corona. The formation of the corona is a dynamic process, governed by individual protein concentrations as well as their respective affinities for the surface. Proteins of the corona interact with surrounding cells, thus being able to influence the cellular uptake of the nanoparticle. In the current study, the uptake of PC-3-derived EVs into PC-3 cells was investigated. Moreover, the impact of a human blood plasma-derived protein corona on said uptake was assessed. EVs were isolated from collected PC-3 cell culture medium using differential centrifugation. Experiments were performed separately for MVs (20000xg EV-fraction) and EXOs (110000xg EVfraction). SDS-PAGE analysis revealed adsorption of plasma proteins to EVs, following their exposure to plasma. Prior to uptake experiments DiO-labelled EVs were either incubated or not incubated in plasma. Plasma incubation lasted overnight. PC-3 cells were then treated with either of the two EV-preparations. Following incubation, EV uptake was assessed using confocal microscopy by determining the percentage of positive fluorescent cells in cell cultures. Pre-study plasma incubation resulted in a reduced or unchanged uptake of MVs and in a reduced uptake of EXOs, when compared to their native counterparts. In conclusion, the plasma-derived protein corona was shown not to improve EV uptake. It is worth noting that the current study limits itself to the use of PC-3-derived EVs and PC-3 cells as recipient cells in uptake experiments.
  • Yates, Abi G.; Pink, Ryan C.; Erdbrugger, Uta; Siljander, Pia R-M.; Dellar, Elizabeth R.; Pantazi, Paschalia; Akbar, Naveed; Cooke, William R.; Vatish, Manu; Dias-Neto, Emmanuel; Anthony, Daniel C.; Couch, Yvonne (2022)
    It is clear from Part I of this series that extracellular vesicles (EVs) play a critical role in maintaining the homeostasis of most, if not all, normal physiological systems. However, the majority of our knowledge about EV signalling has come from studying them in disease. Indeed, EVs have consistently been associated with propagating disease pathophysiology. The analysis of EVs in biofluids, obtained in the clinic, has been an essential of the work to improve our understanding of their role in disease. However, to interfere with EV signalling for therapeutic gain, a more fundamental understanding of the mechanisms by which they contribute to pathogenic processes is required. Only by discovering how the EV populations in different biofluids change-size, number, and physicochemical composition-in clinical samples, may we then begin to unravel their functional roles in translational models in vitro and in vivo, which can then feedback to the clinic. In Part II of this review series, the functional role of EVs in pathology and disease will be discussed, with a focus on in vivo evidence and their potential to be used as both biomarkers and points of therapeutic intervention.
  • Hepo-oja, Pilvi (Helsingin yliopisto, 2020)
    Fecal microbiota transplantation (FMT) is used to treat recurrent Clostridioides difficile infection (rCDI), and its potential as a treatment for other inflammatory conditions, like inflammatory bowel diseases (IBD), or irritable bowel syndrome (IBS), has been extensively studied lately. It has been noticed that some bacteria in fecal transplants do not require physical contact with intestinal epithelium to alleviate inflammation, and extracellular vesicles (EVs) have been proposed to carry the anti-inflammatory properties of those beneficial bacteria. In this thesis project, an isolation protocol was set up to isolate EVs from two fecal-originated Bacteroides isolates, Bacteroides ovatus and Bacteroides vulgatus, which had shown anti-inflammatory potential in previous studies. Isolation of EVs succeeded, and both isolates were confirmed to produce EVs. To study the anti-inflammatory potential, human colon epithelial cells (HT-29) were treated with several dilutions of isolated EVs, and then challenged with lipopolysaccharide (LPS) to induce inflammation. Amount of produced interleukin (IL-) 8 was measured as a marker of inflammation. EVs of both Bacteroides isolates continuously showed anti-inflammatory potential, but statistically significant conclusions could not be made. EVs have a potential to be used as a treatment in different inflammatory conditions and as adjuvant factors in synthetic FMT. To study the immunomodulatory potential of EVs of Bacteroides species more, proteomic analysis of contents of EVs, as well as potential to improve intestinal barrier are suggested. Also, testing the ability to alleviate production of other inflammatory markers could reveal more anti-inflammatory potential.