The Diagnostic and Prognostic Value of Plasmatic Exosome Count in Cancer Patients and in Patients with Other Pathologies.

The extent of both scientific articles and reviews on extracellular vesicles (EVs) has grown impressively over the last few decades [...].

Hydrogen-Rich Alkaline Water Supplementation Restores a Healthy State and Redox Balance in H2O2-Treated Mice.

Water is a major requirement for our bodies, and alkaline water has induced an antioxidant response in a model of natural aging. A series of recent reports have shown that aging is related to reduced water intake. Hydrogen-rich water has been suggested to exert a general antioxidant effect in relation to both improving lifestyle and preventing a series of diseases. Here, we wanted to investigate the effect of the daily intake of hydrogen-rich alkaline water (HAW) in counteracting the redox imbalance induced in a model of H2O2-treated mice. Mice were treated with H2O2 for two weeks and either left untreated or supplied with HAW. The results show that HAW induced a reduction in the ROS plasmatic levels that was consistent with the increase in the circulating glutathione. At the same time, the reduction in plasmatic 8-hydroxy-2'-deoxyguanosine was associated with reduced DNA damage in the whole body. Further analysis of the spleen and bone marrow cells showed a reduced ROS content consistent with a significantly reduced mitochondrial membrane potential and superoxide accumulation and an increase in spontaneous proliferation. This study provides evidence for a clear preventive and curative effect of HAW in a condition of systemic toxic condition and redox imbalance.

What we know on the potential use of exosomes for nanodelivery.

Antitumor therapy is taking into consideration the possibility to use natural nanovesicles, called exosomes, as an ideal delivery for both old and new anti-cancer molecules. This with the attempt to improve the efficacy, at the same time reducing the systemic toxicity of physical, chemical, and biological molecules. Exosomes may in fact increase the level of biomimetism, through simulating what really occurs in nature. Although extracellularly released vesicles include both microvesicles (MVs) and exosomes, only exosomes have the size that may be considered suitable for potential use to this purpose, also by analogy with the diffusely used artificial nanoparticles, such as lyposomes. In fact, recent reports have shown that exosomes are able to interact with target cells within an organ or at a distance using different mechanisms. Much is yet to be understood about exosomes, and currently, we are looking at the visible top of an iceberg, with most of what we have to understand on these nanovesicles still under the sea. In fact, we know that exosomes released by normal cells always trigger positive effects, while those released by cells in pathological condition, such as tumors may induce undesired, dangerous, and mostly unknown effects. To date we have many pre-clinical data available and possibly useful to think about a strategic use of exosomes as a delivery nanodevice in cancer treatment. However, this review wants to critically emphasize two important points actually hampering further discussion in the field : (i) the clinical data are virtually absent at the moment ; (ii) the best cellular source of exosomes to be used to deliver drugs is really far to be defined. Facing off these two points may well facilitate the attempt to figure out this very important issue for improving at the best future anti-cancer treatments.

Exosome Analysis in Prostate Cancer: How They Can Improve Biomarkers' Performance.

Exosomes are extracellular nanovesicles (EV), that is, carriers of different biomolecules such as lipids, proteins, nucleic acids. Their composition and the fact that their release dramatically increases in cases of tumorigenesis open up different scenarios on their possible application to research into new biomarkers. The first purpose of the present review was to specifically analyze and compare different methodologies available for the use of exosomes in prostate cancer (PC). The most widely applied methodologies include ultracentrifugation techniques, size-based techniques, immunoaffinity capture-based techniques (mainly ELISA), and precipitation. To optimize the acquisition of exosomes from the reference sample, more techniques can be applied in sequence for a single extraction, thereby determining an increase in labor time and costs. The second purpose was to describe clinical results obtained with the analysis of PSA-expressing exosomes in PC; this provides an incredibly accurate method of discriminating between healthy patients and those with prostate disease. Specifically, the IC-ELISA alone method achieved 98.57% sensitivity and 80.28% specificity in discriminating prostate cancer (PC) from benign prostatic hyperplasia (BPH). An immunocapture-based ELISA assay was performed to quantify and characterize carbonic anhydrase (CA) IX expression in exosomes. The results revealed that CA IX positive exosomes were 25-fold higher in plasma samples from PC patients than in those from healthy controls. The analysis of PC-linked exosomes represents a promising diagnostic model that can effectively distinguish patients with PC from those with non-malignant prostatic disease. However, the use of exosome analysis in clinical practice is currently limited by several issues, including a lack of standardization in the analytical process and high costs, which are still too high for large-scale use.

Ex Vivo Anti-Leukemic Effect of Exosome-like Grapefruit-Derived Nanovesicles from Organic Farming-The Potential Role of Ascorbic Acid.

Citrus fruits are a natural source of ascorbic acid, and exosome-like nanovesicles obtained from these fruits contain measurable levels of ascorbic acid. We tested the ability of grapefruit-derived extracellular vesicles (EVs) to inhibit the growth of human leukemic cells and leukemic patient-derived bone marrow blasts. Transmission electron microscopy and nanoparticle tracking analysis (NTA) showed that the obtained EVs were homogeneous exosomes, defined as exosome-like plant-derived nanovesicles (ELPDNVs). The analysis of their content has shown measurable amounts of several molecules with potent antioxidant activity. ELPDNVs showed a time-dependent antiproliferative effect in both U937 and K562 leukemic cell lines, comparable with the effect of high-dosage ascorbic acid (2 mM). This result was confirmed by a clear decrease in the number of AML blasts induced by ELPDNVs, which did not affect the number of normal cells. ELPDNVs increased the ROS levels in both AML blast cells and U937 without affecting ROS storage in normal cells, and this effect was comparable to ascorbic acid (2 mM). With our study, we propose ELPDNVs from grapefruits as a combination/supporting therapy for human leukemias with the aim to improve the effectiveness of the current therapies.

The Potentiality of Plant-Derived Nanovesicles in Human Health-A Comparison with Human Exosomes and Artificial Nanoparticles.

Research in science and medicine is witnessing a massive increases in literature concerning extracellular vesicles (EVs). From a morphological point of view, EVs include extracellular vesicles of a micro and nano sizes. However, this simplistic classification does not consider both the source of EVs, including the cells and the species from which Evs are obtained, and the microenvironmental condition during EV production. These two factors are of crucial importance for the potential use of Evs as therapeutic agents. In fact, the choice of the most suitable Evs for drug delivery remains an open debate, inasmuch as the use of Evs of human origin may have at least two major problems: (i) autologous Evs from a patient may deliver dangerous molecules; and (ii) the production of EVs is also limited to cell factory conditions for large-scale industrial use. Recent literature, while limited to only a few papers, when compared to the papers on the use of human EVs, suggests that plant-derived nanovesicles (PDNV) may represent a valuable tool for extensive use in health care.

The key role of extracellular vesicles in the metastatic process.

Extracellular vesicles (EVs), including exosomes, have a key role in the paracrine communication between organs and compartments. EVs shuttle virtually all types of biomolecules such as proteins, lipids, nucleic acids, metabolites and even pharmacological compounds. Their ability to transfer their biomolecular cargo into target cells enables EVs to play a key role in intercellular communication that can regulate cellular functions such as proliferation, apoptosis and migration. This has led to the emergence of EVs as a key player in tumor growth and metastasis through the formation of "tumor niches" in target organs. Recent data have also been shown that EVs may transform the microenvironment of primary tumors thus favoring the selection of cancer cells with a metastatic behavior. The release of EVs from resident non-malignant cells may contribute to the metastatic processes as well. However, cancer EVs may induce malignant transformation in resident mesenchymal stem cells, suggesting that the metastatic process is not exclusively due to circulating tumor cells. In this review, we outline and discuss evidence-based roles of EVs in actively regulating multiple steps of the metastatic process and how we can leverage EVs to impair metastasis.

Nanovesicles released by OKT3 hybridoma express fully active antibodies.

Recent findings have shown that nanovesicles preparations from either primary immune cells culture supernatants or plasma contain immunoglobulins, suggesting that a natural way of antibody production may be through exosome release. To verify this hypothesis, we used the OKT3 hybridoma clone, which produces a murine IgG2a monoclonal antibody used to reduce rejection in patients undergoing organ transplantation. We showed exosome-associated immunoglobulins in hybridoma supernatants, by Western blot, nanoscale flow cytometry and immunocapture-based ELISA. The OKT3-exo was also being able to trigger cytokines production in both CD4 and CD8 T cells. These results show that nanovesicles contain immunoglobulin and could be used for immunotherapy. These data could lead to a new approach to improve the effectiveness of therapeutic antibodies by exploiting their natural property to be expressed on nanovesicle membrane, that probably render them more stable and as a consequence more capable to interact with their specific ligand in the best way.

Nanovesicles from Organic Agriculture-Derived Fruits and Vegetables: Characterization and Functional Antioxidant Content.

Dietary consumption of fruits and vegetables is related to a risk reduction in a series of leading human diseases, probably due to the plants' antioxidant content. Plant-derived nanovesicles (PDNVs) have been recently receiving great attention regarding their natural ability to deliver several active biomolecules and antioxidants. To investigate the presence of active antioxidants in fruits, we preliminarily analyzed the differences between nanovesicles from either organic or conventional agriculture-derived fruits, at equal volumes, showing a higher yield of nanovesicles with a smaller size from organic agriculture-derived fruits as compared to conventional ones. PDNVs from organic agriculture also showed a higher antioxidant level compared to nanovesicles from conventional agriculture. Using the PDNVs from fruit mixes, we found comparable levels of Total Antioxidant Capacity, Ascorbic Acid, Catalase, Glutathione and Superoxide Dismutase 1. Finally, we exposed the nanovesicle mixes to either chemical or physical lytic treatments, with no evidence of effects on the number, size and antioxidant capacity of the treated nanovesicles, thus showing a marked resistance of PDNVs to external stimuli and a high capability to preserve their content. Our study provides for the first time a series of data supporting the use of plant-derived nanovesicles in human beings' daily supplementation, for both prevention and treatment of human diseases.

Biomarkers in Prostate Cancer Diagnosis: From Current Knowledge to the Role of Metabolomics and Exosomes.

Early detection of prostate cancer (PC) is largely carried out using assessment of prostate-specific antigen (PSA) level; yet it cannot reliably discriminate between benign pathologies and clinically significant forms of PC. To overcome the current limitations of PSA, new urinary and serum biomarkers have been developed in recent years. Although several biomarkers have been explored in various scenarios and patient settings, to date, specific guidelines with a high level of evidence on the use of these markers are lacking. Recent advances in metabolomic, genomics, and proteomics have made new potential biomarkers available. A number of studies focused on the characterization of the specific PC metabolic phenotype using different experimental approaches has been recently reported; yet, to date, research on metabolomic application for PC has focused on a small group of metabolites that have been known to be related to the prostate gland. Exosomes are extracellular vesicles that are secreted from all mammalian cells and virtually detected in all bio-fluids, thus allowing their use as tumor biomarkers. Thanks to a general improvement of the technical equipment to analyze exosomes, we are able to obtain reliable quantitative and qualitative information useful for clinical application. Although some pilot clinical investigations have proposed potential PC biomarkers, data are still preliminary and non-conclusive.

Extracellular acidity and increased exosome release as key phenotypes of malignant tumors.

The tumor milieu is characteristically acidic as a consequence of the fermentative metabolism of glucose that results in massive accumulation of lactic acid within the cytoplasm. Tumor cells get rid of excessive protons through exchangers that are responsible for the extracellular acidification that selects cellular clones that are more apt at surviving in this challenging and culling environment. Extracellular vesicles (EVs) are vesicles with diameters ranging from nm to µm that are released from the cells to deliver nucleic acids, proteins, and lipids to adjacent or distant cells. EVs are involved in a plethora of biological events that promote tumor progression including unrestricted proliferation, angiogenesis, migration, local invasion, preparation of the metastatic niche, metastasis, downregulation or hijacking of the immune system, and drug resistance. There is evidence that the release of specific exosomes is increased many folds in cancer patients, as shown by many techniques aimed at evaluating "liquid biopsies". The quality of the exosomal contents has been shown to vary at the different moments of tumor life such as local invasion or metastasis. In vitro studies have recently pointed out that cancer acidity is a major determinant in inducing increased exosome release by human cancer cells, by showing that exosomal release was increased as the pH moved from 7.4 pH to the typical pH of cancer that is 6.5. In this review, we emphasize the recent evidence that tumor acidity and exosomes levels are strictly related and strongly contribute to the malignant tumor phenotypes.

In vivo antiaging effects of alkaline water supplementation.

Telomeres length and telomerase activity are currently considered aging molecular stigmata. Water is a major requirement for our body and water should be alkaline. Recent reports have shown that aging is related to a reduced water intake. We wanted to investigate the effect of the daily intake of alkaline water on the molecular hallmark of aging and the anti-oxidant response. We watered a mouse model of aging with or without alkaline supplementation. After 10 months, we obtained the blood, the bone marrow and the ovaries from both groups. In the blood, we measured the levels of ROS, SOD-1, GSH, and the telomerase activity and analysed the bone marrow and the ovaries for the telomeres length. We found reduced ROS levels and increased SOD-1, GSH, telomerase activity and telomeres length in alkaline supplemented mice. We show here that watering by using alkaline water supplementation highly improves aging at the molecular level.

Lipidomic analysis of cancer cells cultivated at acidic pH reveals phospholipid fatty acids remodelling associated with transcriptional reprogramming.

Cancer cells need to modulate the biosynthesis of membrane lipids and fatty acids to adapt themselves to an accelerated rate of cell division and survive into an extracellular environment characterised by a low pH. To gain insight this crucial survival process, we investigated the lipid composition of Mel 501 melanoma cells cultured at either physiological or acidic pH and observed the remodelling of phospholipids towards longer and more unsaturated acyl chains at low pH. This modification was related to changes in gene expression profile, as we observed an up-regulation of genes involved in acyl chain desaturation, elongation and transfer to phospholipids. PC3 prostate and MCF7 breast cancer cells adapted at acidic pH also demonstrated phospholipid fatty acid remodelling related to gene expression changes. Overall findings clearly indicate that low extracellular pH impresses a specific lipid signature to cells, associated with transcriptional reprogramming.

Plasmatic exosomes from prostate cancer patients show increased carbonic anhydrase IX expression and activity and low pH.

Acidity, hypoxia and increased release of exosomes are severe phenotypes of tumours. The regulation of pH in tumours involves the interaction of several proteins, including the carbonic anhydrases which catalyze the formation of bicarbonate and protons from carbon dioxide and water. Among CA isoforms, CA IX is over-expressed in a large number of solid tumours, conferring to cancer cells a survival advantage in hypoxic and acidic microenvironment, but there isn't evidence that CA IX expression could have a real clinical impact. Therefore, in this study for the first time the expression and activity of CA IX have been investigated in the plasmatic exosomes obtained from patients with prostate carcinoma (PCa). For this purpose, the study was performed through different methodological approaches, such as NTA, western blot analysis, enzyme activity assay, Nanoscale flow cytometry, ELISA, confocal microscopy. The results showed that PCa exosomes significantly overexpressed CA IX levels and related activity as compared to healthy donors. Furthermore, CA IX expression and activity were correlated to the exosome intraluminal pH, demonstrating for the first time that PCa exosomes are acidic. Our data suggest the possible use of the exosomal CA IX expression and activity as a biomarker of cancer progression in PCa.

Extracellular Vesicles-Based Drug Delivery Systems: A New Challenge and the Exemplum of Malignant Pleural Mesothelioma.

Research for the most selective drug delivery to tumors represents a fascinating key target in science. Alongside the artificial delivery systems identified in the last decades (e.g., liposomes), a family of natural extracellular vesicles (EVs) has gained increasing focus for their potential use in delivering anticancer compounds. EVs are released by all cell types to mediate cell-to-cell communication both at the paracrine and the systemic levels, suggesting a role for them as an ideal nano-delivery system. Malignant pleural mesothelioma (MPM) stands out among currently untreatable tumors, also due to the difficulties in achieving an early diagnosis. Thus, early diagnosis and treatment of MPM are both unmet clinical needs. This review looks at indirect and direct evidence that EVs may represent both a new tool for allowing an early diagnosis of MPM and a potential new delivery system for more efficient therapeutic strategies. Since MPM is a relatively rare malignant tumor and preclinical MPM models developed to date are very few and not reliable, this review will report data obtained in other tumor types, suggesting the potential use of EVs in mesothelioma patients as well.

Prostate cancer cells and exosomes in acidic condition show increased carbonic anhydrase IX expression and activity.

Acidity and hypoxia are crucial phenotypes of tumour microenvironment both contributing to the selection of malignant cells under a micro evolutionistic pressure. During the tumour progression, nanovesicles, called exosomes and the metalloenzyme carbonic anhydrase IX (CA IX) affect the tumour growth and proliferation. Exosomes are released into the tumour microenvironment and spilt all over the body, while CA IX is a tumour-associated protein overexpressed in many different solid tumours. In the present study, to better understand the relationships between exosomes and CA IX, it has been used an in vitro cellular model of cells cultured in different pH conditions. The results showed that the acidic microenvironment induced upregulation of both expression and activity of CA IX in cancer cells and their exosomes, together with increasing the number of released exosomes. These data strongly support the importance of CA IX as a cancer biomarker and as a valuable target of new anticancer therapies.

On the Choice of the Extracellular Vesicles for Therapeutic Purposes.

Extracellular vesicles (EVs) are lipid membrane vesicles released by all human cells and are widely recognized to be involved in many cellular processes, both in physiological and pathological conditions. They are mediators of cell-cell communication, at both paracrine and systemic levels, and therefore they are active players in cell differentiation, tissue homeostasis, and organ remodeling. Due to their ability to serve as a cargo for proteins, lipids, and nucleic acids, which often reflects the cellular source, they should be considered the future of the natural nanodelivery of bio-compounds. To date, natural nanovesicles, such as exosomes, have been shown to represent a source of disease biomarkers and have high potential benefits in regenerative medicine. Indeed, they deliver both chemical and bio-molecules in a way that within exosomes drugs are more effective that in their exosome-free form. Thus, to date, we know that exosomes are shuttle disease biomarkers and probably the most effective way to deliver therapeutic molecules within target cells. However, we do not know exactly which exosomes may be used in therapy in avoiding side effects as well. In regenerative medicine, it will be ideal to use autologous exosomes, but it seems not ideal to use plasma-derived exosomes, as they may contain potentially dangerous molecules. Here, we want to present and discuss a contradictory relatively unmet issue that is the lack of a general agreement on the choice for the source of extracellular vesicles for therapeutic use.

A Role of Tumor-Released Exosomes in Paracrine Dissemination and Metastasis.

Metastatic diffusion is thought to be a multi-step phenomenon involving the release of cells from the primary tumor and their diffusion through the body. Currently, several hypotheses have been put forward in order to explain the origin of cancer metastasis, including epithelial⁻mesenchymal transition, mutagenesis of stem cells, and a facilitating role of macrophages, involving, for example, transformation or fusion hybridization with neoplastic cells. In this paradigm, tumor-secreted extracellular vesicles (EVs), such as exosomes, play a pivotal role in cell communications, delivering a plethora of biomolecules including proteins, lipids, and nucleic acids. For their natural role in shuttling molecules, EVs have been newly considered a part of the metastatic cascade. They have a prominent role in preparing the so-called "tumor niches" in target organs. However, recent evidence has pointed out an even more interesting role of tumor EVs, consisting in their ability to induce malignant transformation in resident mesenchymal stem cells. All in all, in this review, we discuss the multiple involvements of EVs in the metastatic cascade, and how we can exploit and manipulate EVs in order to reduce the metastatic spread of malignant tumors.

Exosomal Chaperones and miRNAs in Gliomagenesis: State-of-Art and Theranostics Perspectives.

Gliomas have poor prognosis no matter the treatment applied, remaining an unmet clinical need. As background for a substantial change in this situation, this review will focus on the following points: (i) the steady progress in establishing the role of molecular chaperones in carcinogenesis; (ii) the recent advances in the knowledge of miRNAs in regulating gene expression, including genes involved in carcinogenesis and genes encoding chaperones; and (iii) the findings about exosomes and their cargo released by tumor cells. We would like to trigger a discussion about the involvement of exosomal chaperones and miRNAs in gliomagenesis. Chaperones may be either targets for therapy, due to their tumor-promoting activity, or therapeutic agents, due to their antitumor growth activity. Thus, chaperones may well represent a Janus-faced approach against tumors. This review focuses on extracellular chaperones as part of exosomes' cargo, because of their potential as a new tool for the diagnosis and management of gliomas. Moreover, since exosomes transport chaperones and miRNAs (the latter possibly related to chaperone gene expression in the recipient cell), and probably deliver their cargo in the recipient cells, a new area of investigation is now open, which is bound to generate significant advances in the understanding and treatment of gliomas.

Natural extracellular nanovesicles and photodynamic molecules: is there a future for drug delivery?

Photodynamic molecules represent an alternative approach for cancer therapy for their property (i) to be photo-reactive; (ii) to be not-toxic for target cells in absence of light; (iii) to accumulate specifically into tumour tissues; (iv) to be activable by a light beam only at the tumour site and (v) to exert cytotoxic activity against tumour cells. However, to date their clinical use is limited by the side effects elicited by systemic administration. Extracellular vesicles are endogenous nanosized-carriers that have been recently introduced as a natural delivery system for therapeutic molecules. We have recently shown the ability of human exosomes to deliver photodynamic molecules. Therefore, this review focussed on extracellular vesicles as a novel strategy for the delivery of photodynamic molecules at cancer sites. This completely new approach may enhance the delivery and decrease the toxicity of photodynamic molecules, therefore, represent the future for photodynamic therapy for cancer treatment.