Browsing by Subject "extracellular vesicles"

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.

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.

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.

Abstract Faculty: Faculty of Biological and Environmental Sciences Degree programme: Genetics and Molecular Biosciences Study track: Molecular and Analytical Health Biosciences Author: Annie Stendahl Title: Measurement repeatability of flow cytometry and nanoparticle tracking analysis for optimization of extracellular vesicle measurements Level: Master’s thesis Month and year: 11/2022 Number of pages: 92 Keywords: extracellular vesicles, repeatability measurements, metrology, traceability, flow cytometry, nanoparticle tracking analysis, reference material, METVES Supervisor or supervisors: Virpi Korpelainen, Katariina Maaninka and Pia Siljander Collaborative partner: VTT Technical Research Centre of Finland Ltd. Where deposited: E-thesis Extracellular vesicles (EVs) are lipid bilayer-enclosed vesicles secreted by all cells, containing variable cargo from nucleic acids and proteins to carbohydrates, metabolites, and lipids. EVs are considered to be involved in many physiological and pathological cell functions. Due to their presence in biofluids hence enabling semi-invasive liquid biopsies, EVs have indicated great promise for utilization as biomarkers in clinical settings. The innate properties of EVs and their cargo could also be harnessed into therapeutic use. However, the current methods and reference materials for determining EV concentration and size have not yet achieved the metrological level of repeatability and traceability, which is needed for EV measurements to be utilized in clinical settings. The aim of this thesis project was to evaluate repeatability of the methods typically used for EV quantification and size determination, flow cytometry (FCM) and nanoparticle tracking analysis (NTA). The repeatability was analyzed with reference material made of hollow organosilica beads and biological EV test samples, both developed in an ongoing EU metrology-project METVES II for EVs. A similar biological EV test sample was also prepared as part of the thesis project. Finally, the repeatability measurements were conducted with calibration beads recommended by the instrument manufacturers. The calibration beads gave repeatable results with FCM and one of the two NTA instruments tested, but neither the reference beads nor the biological EV test samples produced repeatable results to enable determination of repeatability. However, valuable understanding was gained on what can be optimized during the measurements and operation of the instruments to generate more repeatable results with FCM and NTA in EV analysis. Prior knowledge of both the sample type and method used for measuring would enable optimization of the measurement and instrument operation. Whether the aim is EV quantification or size determination, instrument errors and bias could then be minimized by adjusting the settings according to sample type. Furthermore, EV quantification and size determination would benefit from combining different methods to ensure more reliable and repeatable results. It is clear that more research needs to be done, for i.e., the tested reference beads need to be further developed to be established as EV reference material and enabling standardization of EV measurements. Standardizing EV quantification and size determination is required to achieve metrological repeatability and ultimately, traceability, and thus for EVs to be utilized in clinical settings as biomarkers or therapeutic use.

Background and objectives: Cells secrete extracellular vesicles (EV) and it has been found that cells communicate via EVs. EVs are liposome-like vesicles. Membrane is consisting of a lipid bilayer and hydrophilic moiety is inside the vesicle. It has been found that EVs carry e.g. nucleic acids, lipids and proteins. The aim of this master thesis was to determine whether EVs can transport non-coding RNA (siRNA) into the central nervous system through the blood-brain barrier. In the literature review, investigated methods which has been used to load siRNA into the EVs and how EVs are transported through the blood-brain barrier. The aim of the experimental part was to produce and isolate EVs and to load FAM-labeled dsDNA and siRNA into EVs by physical methods such as sonication and electroporation. Fluorescence measurements were taken to demonstrate FAM-labeled DNA loading into EVs and the functionality of the siRNA-loaded EVs was measured by measuring the expression level of the gapdh gene. Methods: Extracellular vesicles were produced in ARPE-19 and PC-3 cells. EVs were isolated from the cell culture medium by two-step differential centrifugation (DC) and further purified by gradient centrifugation (GC) by using the OptiPrep™-reagent. OptiPrep™-reagent was purified by Amicon 10kDa filtration tubes. The average particle size and size distribution of the isolated EVs were determined by NTA analysis, protein concentration was measured by colorimetric BCA method and EVs were characterized by Western blot method using HSP70 and CD9 antibodies. EVs were loaded with 21 bp length FAM-labeled dsDNA or siRNA by sonication or electroporation. Free nucleic acid and OptiPrep™-reagent were purified from EVs by the size-exclusion chromatography with Sephacryl (S-300) column. Loading efficient of the EVs were studied by measuring the fluorescence (ex 485 nm, em 520 nm) and qPCR method was used to demonstrate the functionality of the siRNA loaded EVs. In qPCR, the expression level of the gapdh gene was measured in dividing ARPE-19 cells. Results: DC and GC purified ARPE-19 and PC-3 EVs had an average particle size of about 140 nm and were successfully characterized by Western blot method. PC-3 EVs were produced in the bioreactor and the yields were enough for loading experiments. ARPE-19 cells produced only small amounts of EVs in culture flasks. The size-exclusion chromatography was a good method to purification free nucleic acids from EVs. The sonication method did not cause EVs to be degradation under the conditions used. Based on fluorescence measurement, FAM-labeled dsDNA could not be loaded into EVs. The functionality of siRNA-loaded EVs could not be demonstrated in ARPE-19 cell experiments. After electroporation large number of EVs were lost and this method of loading siRNA into EVs did not proved to be suitable. Conclusions: ARPE-19 EVs must be produced in the bioreactor to produce enough EVs for loading experiments. The EV purification protocol should be further optimized since the recovery-% of EVs were low after several purification steps. The size-exclusion chromatography is suitable for the purification of the free siRNA from EVs, but the chromatography method needs further optimization and miniaturization. Loaded EVs should be produced by aseptically or alternatively sterilized prior to ARPE-19 cell assay. Physical loading method, such as sonication, can be scaled to larger scale. Sonication method should be optimized e.g. by experimenting with higher temperatures and longer sonication times. The probe sonicator should be tested instead of the water bath sonicator. According to the literature review, the use of extracellular vesicles as carriers for biomolecule delivery into the central nervous system seems to be promising.