OCT4 Expression in Gliomas Is Dependent on Cell Metabolism.

The OCT4 transcription factor is necessary to maintain cell stemness in the early stages of embryogenesis and is involved in the formation of induced pluripotent stem cells, but its role in oncogenesis is not yet entirely clear. In this work, OCT4 expression was investigated in malignant gliomas. Twenty glioma cell lines and a sample of normal adult brain tissue were used. OCT4 expression was found in all studied glioma cell lines but was not detected in normal adult brain tissue. For one of these lines, OCT4 knockdown caused tumor cell death. By varying the culture conditions of these cells, we unexpectedly found that OCT4 expression increased when cells were incubated in serum-free medium, and this effect was significantly enhanced in serum-free and L-glutamine-free medium. L-glutamine and the Krebs cycle, which is slowed down in serum-free medium according to our NMR data, are sources of α-KG. Thus, our data indicate that OCT4 expression in gliomas may be regulated by the α-KG-dependent metabolic reprogramming of cells.

Extracellular Vesicles Secreted by Adipose Tissue during Obesity and Type 2 Diabetes Mellitus Influence Reverse Cholesterol Transport-Related Gene Expression in Human Macrophages.

Obesity is a risk factor for type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). Adipose tissue (AT) extracellular vesicles (EVs) could play a role in obesity and T2DM associated CVD progression via the influence of their specific cargo on gene expression in recipient cells. The aim of this work was to evaluate the effects of AT EVs of patients with obesity with/without T2DM on reverse cholesterol transport (RCT)-related gene expression in human monocyte-derived macrophages (MDMs) from healthy donors. AT EVs were obtained after ex vivo cultivation of visceral and subcutaneous AT (VAT and SAT, respectively). ABCA1, ABCG1, PPARG, LXRß (NR1H2), and LXRα (NR1H3) mRNA levels in MDMs as well as in origine AT were determined by a real-time PCR. T2DM VAT and SAT EVs induced ABCG1 gene expression whereas LXRα and PPARG mRNA levels were simultaneously downregulated. PPARG mRNA levels also decreased in the presence of VAT EVs of obese patients without T2DM. In contrast ABCA1 and LXRß mRNA levels tended to increase with the addition of obese AT EVs. Thus, AT EVs can influence RCT gene expression in MDMs during obesity, and the effects are dependent on T2DM status.

Potential of Plant Exosome Vesicles from Grapefruit (Citrus × paradisi) and Tomato (Solanum lycopersicum) Juices as Functional Ingredients and Targeted Drug Delivery Vehicles.

Plant-derived extracellular vesicles (PEVs) have gained attention as promising bioactive nutraceutical molecules; their presence in common fruit juices has increased their significance because human interaction is inevitable. The goal of this study was to evaluate the potential of PEVs derived from grapefruit and tomato juices as functional ingredients, antioxidant compounds, and delivery vehicles. PEVs were isolated using differential ultracentrifugation and were found to be similar in size and morphology to mammalian exosomes. The yield of grapefruit exosome-like vesicles (GEVs) was higher than that of tomato exosome-like vesicles (TEVs), despite the latter having larger vesicle sizes. Furthermore, the antioxidant activity of GEVs and TEVs was found to be low in comparison to their juice sources, indicating a limited contribution of PEVs to the juice. GEVs showed a higher efficiency in being loaded with the heat shock protein 70 (HSP70) than TEVs, as well as a higher efficiency than TEV and PEV-free HSP70 in delivering HSP70 to glioma cells. Overall, our results revealed that GEVs present a higher potential as functional ingredients present in juice and that they exert the potential to deliver functional molecules to human cells. Although PEVs showed low antioxidant activity, their role in oxidative response in cells should be further addressed.

Experimental validation of proton boron capture therapy for glioma cells.

Proton boron capture therapy (PBCT) has emerged from particle acceleration research for enhancing the biological effectiveness of proton therapy. The mechanism responsible for the dose increase was supposed to be related to proton-boron fusion reactions (11B + p → 3α + 8.7 MeV). There has been some experimental evidence that the biological efficiency of protons is significantly higher for boron-11-containing prostate or breast cancer cells. The aim of this study was to evaluate the sensitizing potential of sodium borocaptate (BSH) under proton irradiation at the Bragg peak of cultured glioma cells. To address this problem, cells of two glioma lines were preincubated with 80 or 160 ppm boron-11, irradiated both at the middle of 200 MeV beam Spread-Out Bragg Peak (SOBP) and at the distal end of the 89.7 MeV beam SOBP and assessed for the viability, as well as their ability to form colonies. Our results clearly show that BSH provides for only a slight, if any, enhancement of the effect of proton radiation on the glioma cells in vitro. In addition, we repeated the experiments using the Du145 prostate cancer cell line, for which an increase in the biological efficiency of proton irradiation in the presence of sodium borocaptate was demonstrated previously. The data presented add new argument against the efficiency of proton boron capture therapy when based solely on direct dose-enhancement effect by the proton capture nuclear reaction, underlining the need to investigate the indirect effects of the secondary alpha irradiation depending on the state and treatment conditions of the irradiated tissue.

Cryo-electron microscopy of adipose tissue extracellular vesicles in obesity and type 2 diabetes mellitus.

Extracellular vesicles (EVs) are cell-derived membrane vesicles which play an important role in cell-to-cell communication and physiology. EVs deliver biological information from producing to recipient cells by transport of different cargo such as proteins, mRNAs, microRNAs, non-coding RNAs and lipids. Adipose tissue EVs could regulate metabolic and inflammatory interactions inside adipose tissue depots as well as distal tissues. Thus, adipose tissue EVs are assumed to be implicated in obesity-associated pathologies, notably in insulin resistance and type 2 diabetes mellitus (T2DM). In this study we for the first time characterize EVs secreted by visceral (VAT) and subcutaneous adipose tissue (SAT) of patients with obesity and T2DM with standard methods as well as analyze their morphology with cryo-electron microscopy. Cryo-electron microscopy allowed us to visualize heterogeneous population of EVs of various size and morphology including single EVs and EVs with internal membrane structures in samples from obese patients as well from the control group. Single vesicles prevailed (up to 85% for SAT, up to 75% for VAT) and higher proportion of EVs with internal membrane structures compared to SAT was typical for VAT. Decreased size of single and double SAT EVs compared to VAT EVs, large proportion of multilayered EVs and all EVs with internal membrane structures secreted by VAT distinguished obese patients with/without T2DM from the control group. These findings could support the idea of modified biogenesis of EVs during obesity and T2DM.

Delivery of functional exogenous proteins by plant-derived vesicles to human cells in vitro.

Plant-derived extracellular vesicles (EVs) gain more and more attention as promising carriers of exogenous bioactive molecules to the human cells. Derived from various edible sources, these EVs are remarkably biocompatible, biodegradable and highly abundant from plants. In this work, EVs from grapefruit juice were isolated by differential centrifugation followed by characterization of their size, quantity and morphology by nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy and cryo-electron microscopy (Cryo-EM). In Cryo-EM experiments, we visualized grapefruit EVs with the average size of 41 ± 13 nm, confirmed their round-shaped morphology and estimated the thickness of their lipid bilayer as 5.3 ± 0.8 nm. Further, using cell culture models, we have successfully demonstrated that native grapefruit-derived extracellular vesicles (GF-EVs) are highly efficient carriers for the delivery of the exogenous Alexa Fluor 647 labeled bovine serum albumin (BSA) and heat shock protein 70 (HSP70) into both human peripheral blood mononuclear cells and colon cancer cells. Interestingly, loading to plant EVs significantly ameliorated the uptake of exogenous proteins by human cells compared to the same proteins without EVs. Most importantly, we have confirmed the functional activity of human recombinant HSP70 in the colon cancer cell culture upon delivery by GF-EVs. Analysis of the biodistribution of GF-EVs loaded with 125I-labeled BSA in mice demonstrated a significant uptake of the grapefruit-derived extracellular vesicles by the majority of organs. The results of our study indicate that native plant EVs might be safe and effective carriers of exogenous proteins into human cells.

Hsp70-containing extracellular vesicles are capable of activating of adaptive immunity in models of mouse melanoma and colon carcinoma.

The release of Hsp70 chaperone from tumor cells is found to trigger the full-scale anti-cancer immune response. Such release and the proper immune reaction can be induced by the delivery of recombinant Hsp70 to a tumor and we sought to explore how the endogenous Hsp70 can be transported to extracellular space leading to the burst of anti-cancer activity. Hsp70 transport mechanisms were studied by analyzing its intracellular tracks with Rab proteins as well as by using specific inhibitors of membrane domains. To study Hsp70 forms released from cells we employed the assay consisting of two affinity chromatography methods. Hsp70 content in culture medium and extracellular vesicles (EVs) was measured with the aid of ELISA. The properties and composition of EVs were assessed using nanoparticle tracking analysis and immunoblotting. The activity of immune cells was studied using an assay of cytotoxic lymphocytes, and for in vivo studies we employed methods of affinity separation of lymphocyte fractions. Analyzing B16 melanoma cells treated with recombinant Hsp70 we found that the chaperone triggered extracellular transport of its endogenous analog in soluble and enclosed in EVs forms; both species efficiently penetrated adjacent cells and this secondary transport was corroborated with the strong increase of Natural Killer (NK) cell toxicity towards melanoma. When B16 and CT-26 colon cancer cells before their injection in animals were treated with Hsp70-enriched EVs, a powerful anti-cancer effect was observed as shown by a two-fold reduction in tumor growth rate and elevation of life span. We found that the immunomodulatory effect was due to the enhancement of the CD8-positive response and anti-tumor cytokine accumulation; supporting this there was no delay in CT-26 tumor growth when Hsp70-enriched EVs were grafted in nude mice. Importantly, pre-treatment of B16 cells with Hsp70-bearing EVs resulted in a decline of arginase-1-positive macrophages, showing no generation of tumor-associated macrophages. In conclusion, Hsp70-containing EVs generated by specifically treated cancer cells give a full-scale and effective pattern of anti-tumor immune responses.

Proteome of Glioblastoma-Derived Exosomes as a Source of Biomarkers.

Extracellular vesicles (EV) are involved in important processes of glioblastoma multiforme (GBM), including malignancy and invasion. EV secreted by glioblastoma cells may cross the hematoencephalic barrier and carry molecular cargo derived from the tumor into the peripheral circulation. Therefore, the determination of the molecular composition of exosomes released by glioblastoma cells seems to be a promising approach for the development of non-invasive methods of the detection of the specific exosomal protein markers in the peripheral blood. The present study aimed to determine the common exosomal proteins presented in preparations from different cell lines and search potential glioblastoma biomarkers in exosomes. We have performed proteomics analysis of exosomes obtained from the conditioned culture medium of five glioblastoma cell lines. A list of 133 proteins common for all these samples was generated. Based on the data obtained, virtual two-dimensional electrophoresis (2DE) maps of proteins presented in exosomes of glioblastoma cells were constructed and the gene ontology (GO) analysis of exosome proteins was performed. A correlation between overexpressed in glial cell proteins and their presence in exosomes have been found. Thus, the existence of many potential glioblastoma biomarkers in exosomes was confirmed.

Evaluation of immune and chemical precipitation methods for plasma exosome isolation.

Exosomes are a type of extracellular vesicles (EVs) secreted by multiple mammalian cell types and involved in intercellular communication. Numerous studies have explored the diagnostic and therapeutic potential of exosomes. The key challenge is the lack of efficient and standard techniques for isolation and downstream analysis of nanovesicles. Conventional isolation methods, such as ultracentrifugation, precipitation, filtration, chromatography, and immune-affinity-based approaches, rely on specific physical properties or on surface biomarkers. However, any of the existing methods has its limitations. Various parameters, such as efficacy, specificity, labor input, cost and scalability, and standardization options, must be considered for the correct choice of appropriate approach. The isolation of exosomes from biological fluids is especially challenged by the complex nature and variability of these liquids. Here, we present a comparison of five protocols for exosome isolation from human plasma: two chemical affinity precipitation methods (lectin-based purification and SubX™ technology), immunoaffinity precipitation, and reference ultracentrifugation-based exosome isolation method in two modifications. An approach for the isolation of exosomes based on the phenomenon of binding and aggregation of these particles via clusters of outer membrane phosphate groups in the presence of SubX™ molecules has been put forward in the present study. The isolated EVs were characterized based upon size, quantity, and protein content.

Cryo-electron microscopy of extracellular vesicles from cerebrospinal fluid.

Extracellular vesicles (EVs) are membrane-enclosed vesicles which play important role for cell communication and physiology. EVs are found in many human biological fluids, including blood, breast milk, urine, cerebrospinal fluid (CSF), ejaculate, saliva etc. These nano-sized vesicles contain proteins, mRNAs, microRNAs, non-coding RNAs and lipids that are derived from producing cells. EVs deliver complex sets of biological information to recipient cells thereby modulating their behaviors by their molecular cargo. In this way EVs are involved in the pathological development and progression of many human disorders, including neurodegenerative diseases. In this study EVs purified by ultracentrifugation from CSF of patients with Parkinson's disease (PD) and individuals of the comparison group were characterized using nanoparticle tracking analysis, flow cytometry and cryo-electron microscopy. Vesicular size and the presence of exosomal marker CD9 on the surface provided evidence that most of the EVs were exosome-like vesicles. Cryo-electron microscopy allowed us to visualize a large spectrum of extracellular vesicles of various size and morphology with lipid bilayers and vesicular internal structures. Thus, we described the diversity and new characteristics of the vesicles from CSF suggesting that subpopulations of EVs with different and specific functions may exist.