Search

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.

Introduction: How can biotechnology and organic agriculture be fused and promoted simultaneously to overcome the main challenges in drug delivery systems. The role of organic agriculture in future human health treatment still represents a binary organic-conventional question. However, exosomes-like nanoparticles define a new organic path that plants and vegetables can release. In this review, we concisely propose plant-derived exosome-like nanovesicles and discuss their most important biological and pharmacological roles, representing a new tool for drug delivery., Areas Covered: Plant-derived exosomes-like nanovesicles; nature farming; green manufacturing practice; drug delivery; organic agriculture., Expert Opinion: There is growing interest in the potential use of plant-derived exosomes-like nanovesicles for various diagnostic and therapeutic applications that should translate into a supplement to current nano-pharmaceuticals. Despite their clinical potential, the lack of sensitive preparatory and analytical technologies for plant-derived exosomes-like nanovesicles poses a barrier to clinical translation. An increasing number of articles are recently published on new analytical platforms to address these challenges in cross-comparison with conventional assay methods. This review also mentions two patents from ExoLab-Italia on plant-derived exosome-like nanovesicles, respectively, on plant-derived exosome-like nanovesicles' ability to naturally deliver a series of potentially therapeutic molecules and a novel approach to upload them with therapeutic molecules.

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., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021. Published by Elsevier Ltd.)

In this paper we want to introduce a hot topic for clinical and translational research in oncology and all the related medical fields: the "exosomology", i.e., the science that looks at exosomes as nanovesicular tools for theranostics. Exosomes are extracellular vesicles (EVs) of nanometric sizes actively secreted by normal and, above all, tumor cells. Among the EVs, exosomes are surely the most investigated and with the most promising results, mainly for what concerns their potential as representing the future of the so-called "liquid biopsy". Unfortunately, the huge and increasing amount of data coming from preclinical studies was not followed by an adequate number of clinical investigations. However, those clinical studies published to date have provided encouraging but probably unexpected results, including the clinical relevance of the exosome plasmatic levels and the overexpression of well-known biomarkers on the circulating EVs. The clinical relevance of exosomes as a source of new tumor biomarkers (e.g., proteins and miRNA) has been sufficiently supported by clear data. We here want to provide our viewpoint about the existing clinical results based on the literature and our own experience to trigger discussion aimed at undertaking a new direction for future investigation on a role of exosomes in cancer diagnosis and treatment. We believe that a more strategic co-operation between the community of basic scientists and the clinical oncologists should be generated soon, in order to investigate the relevance of the impressive amount of data obtained in human tumor cell lines and animal models. For sure we need a more strategic behavior but also a far-sighted scientific investment in a field where nothing should be considered as granted; a field where we need a mutual collaboration between basic science, clinicians, governments and of course industry., Competing Interests: Declaration of Competing Interest There is not a competing interest for both SF and FC, (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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.

The immunocapture-based ELISA for extracellular vesicles (EVs)/exosomes, originally described in 2009 by Logozzi and colleagues, allows to capture, detect, characterize and quantify extracellular vesicles in both human body fluids and cell culture supernatants. It is based on the use of two antibodies directed one against a typical exosomal housekeeping protein and the second against either another exosomal housekeeping protein or a potential disease marker: the first antibody is used for the capture of exosomes, the second for the quantification and characterization of the captured vesicles. In fact, with this method it is possible both to characterize and count exosomes and to detect the presence of disease, including tumor, biomarkers. This needs of course to preliminary obtain an EVs purification from the clinical sample; the most agreed method to get to an EVs purification is the repeated rounds of ultracentrifugation, that, while far to be perfect, is the methodological approach allowing to not exclude EVs subpopulation from the separation procedure and to analyze a full range of EVs from both qualitative and quantitative point of view. The immunocapture-based approach has proven to be highly useful in screening, diagnosis and prognosis of tumors, in plasma samples. One amazing information provided by this method is that cancer patients have always significantly higher levels of EVs, in particular of exosomes, independently from the histological nature of the tumor. One microenvironmental factor that is fully involved in the increased exosome release by tumors is the extracellular acidity. However, few pre-clinical data suggest that plasmatic levels of exosomes may correlate with the tumor mass. Some recent clinical reports suggest also that circulating exosomes represent the real delivery system for some known tumor markers that are presently on trial (e.g., PSA). Here we review the pros and cons of the immunocapture-based technique in quantitative and qualitative evaluation of EVs in both health and disease., (© 2020 Elsevier Inc. All rights reserved.)

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.

The use of nanomaterials is increasing but the real risk associated with their use in humans has to be defined. In fact, nanomaterials tend to accumulate in organs over a long period of time and are slowly degraded or eliminated by the body. Exosomes are nanovesicles actively shuttle molecules, including chemical products and metals, through the body. Macrophages scavenge the body from both organic and inorganic substances, and they use to release high amounts of exosomes. We hypothesized that macrophages may have a role in eliminating nanomaterials through their exosomes. We treated human primary macrophages with 20 nm gold nanoparticles (AuNPs), analyzing the presence of AuNPs in both cells and the released exosomes by the implementation of different techniques, including SP-ICP-MS and NTA. We showed that macrophages endocytosed AuNPs and released them through exosomes. Our study on one hand provide the evidence for a new methodology in the early identification of the nanomaterials levels in exposed subjects. On the other hand we depict a way our body shuttle virtually intact nanoparticles through macrophage-released exosomes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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.

Specifically targeted drug delivery systems with low immunogenicity and toxicity are deemed to increase efficacy of cancer chemotherapy. Acridine Orange (AO) is an acidophilic dye with a strong tumoricidal action following excitation with a light source at 466 nm. However, to date the clinical use of AO is limited by the potential side effects elicited by systemic administration. The endogenous nanocarrier exosomes have been recently introduced as a natural delivery system for therapeutic molecules. In this article, we show the outcome of the administration to human melanoma cells of AO charged Exosomes (Exo-AO), in both monolayer and spheroid models. The results showed an extended drug delivery time of Exo-AO to melanoma cells as compared to the free AO, improving the cytotoxicity of AO. This study shows that Exo-AO have a great potential for a real exploitation as a new theranostic approach against tumors based on AO delivered through the exosomes.

Prostate specific antigen (PSA) test is the most common, clinically validated test for the diagnosis of prostate cancer (PCa). While neoplastic lesions of the prostate may cause aberrant levels of PSA in the blood, the quantitation of free or complexed PSA poorly discriminates cancer patients from those developing benign lesions, often leading to invasive and unnecessary surgical procedures. Microenvironmental acidity increases exosome release by cancer cells. In this study we evaluated whether acidity, a critical phenotype of malignancy, could influence exosome release and increase the PSA expression in nanovesicles released by PCa cells. To this aim, we exploited Nanoparticle Tracking Analysis (NTA), an immunocapture-based ELISA, and nanoscale flow-cytometry. The results show that microenvironmental acidity induces an increased release of nanovesicles expressing both PSA and the exosome marker CD81. In order to verify whether the changes induced by the local selective pressure of extracellular acidity may correspond to a clinical pathway we used the same approach to evaluate the levels of PSA-expressing exosomes in the plasma of PCa patients and controls, including subjects with benign prostatic hypertrophy (BPH). The results show that only PCa patients have high levels of nanovesicles expressing both CD81 and PSA. This study shows that tumor acidity exerts a selective pressure leading to the release of extracellular vesicles that express both PSA and exosome markers. A comparable scenario was shown in the plasma of prostate cancer patients as compared to both BPH and healthy controls. These results suggest that microenvironmental acidity may represent a key factor which determines qualitatively and quantitatively the release of extracellular vesicles by malignant tumors, including prostate cancer. This condition leads to the spill-over of nanovesicles into the peripheral blood of prostate cancer patients, where the levels of tumor biomarkers expressed by exosomes, such as PSA-exosomes, may represent a novel, non-invasive clinical tool for the screening and early diagnosis of prostate cancer., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: Cell-to-cell communication is imperative for life and it is mediated by sending and receiving information via the secretion and subsequent receptor-mediated detection of biological molecules. Exosomes (EXs) secreted from cells to the extracellular environment play an important role in intercellular communication in normal and pathological conditions. Areas covered: New evidence indicates that tumor cells-derived EXs contribute to cancer progression through the modulation of tumor microenvironment. The exosomal heat shock protein 60 (HSP60) is very likely a key player in intercellular cross-talk, particularly during the progress of diseases, such as cancer. Many studies have focused on the extracellular roles played by HSP60 that pertain to cancer development and immune system stimulation. Our experimental data in vitro and in vivo demonstrated that HSP60 occurs on the surface of EXs secreted by tumour cells. Expert commentary: Exosomal HSP60 has great potential for clinical applications, as a 'liquid biopsy', including its use as biomarker for diagnostics, assessing prognosis, and monitoring disease progression and response to treatment, particularly in cancer.

Despite considerable research efforts, the finding of reliable tumor biomarkers remains challenging and unresolved. In recent years a novel diagnostic biomedical tool with high potential has been identified in extracellular nanovesicles or exosomes. They are released by the majority of the cells and contain detailed molecular information on the cell of origin including tumor hallmarks. Exosomes can be isolated from easy accessible body fluids, and most importantly, they can provide several biomarkers, with different levels of specificity. Recent clinical evidence shows that the levels of exosomes released into body fluids may themselves represent a predictive/diagnostic of tumors, discriminating cancer patients from healthy subjects. The aim of this review is to highlight these latest challenging findings to provide novel and groundbreaking ideas for successful tumor early diagnosis and follow-up., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Cancer cells, including colorectal cancer ones (CRC), release high amounts of nanovesicles (exosomes), delivering biochemical messages for paracrine or systemic crosstalk. Mesenchymal stromal cells (MSCs) have been shown to play contradicting roles in tumor progression., Results: CRC exosomes induce in cMSCs: i) atypical morphology, higher proliferation, migration and invasion; ii) formation of spheroids; iii) an acidic extracellular environment associated with iv) a plasma membrane redistribution of vacuolar H+-ATPase and increased expression of CEA. Colon cancer derived MSCs, which were isolated from tumor masses, produce umbilicated spheroids, a future frequently observed in the inner core of rapidly growing tumors and recapitulate the changes observed in normal colonic MSCs exposed to CRC exosomes., Materials and Methods: Tissue specific colonic (c)MSCs were exposed to primary or metastatic CRC exosomes and analysed by light and electron microscopy, proliferation in 2D and 3D cultures, migration and invasion assays, Western blot and confocal microscopy for vacuolar H+-ATPase expression., Conclusions: CRC exosomes are able to induce morphological and functional changes in colonic MSCs, which may favour tumor growth and its malignant progression. Our results suggest that exosomes are actively involved in cancer progression and that inhibiting tumor exosome release may represent a way to interfere with cancer., Competing Interests: The authors state no conflicts of interest.

Background: We recently demonstrated that epicardial progenitor cells participate in the regenerative response to myocardial infarction (MI) and factors released in the pericardial fluid (PF) may play a key role in this process. Exosomes are secreted nanovesicles of endocytic origin, identified in most body fluids, which may contain molecules able to modulate a variety of cell functions. Here, we investigated whether exosomes are present in the PF and their potential role in cardiac repair., Methods and Results: Early gene expression studies in 3day-infarcted mouse hearts showed that PF induces epithelial-to-mesenchymal transition (EMT) in epicardial cells. Exosomes were identified in PFs from non-infarcted patients (PFC) and patients with acute MI (PFMI). A shotgun proteomics analysis identified clusterin in exosomes isolated from PFMI but not from PFC. Notably, clusterin has a protective effect on cardiomyocytes after acute MI in vivo and is an important mediator of TGFβ-induced. Clusterin addition to the pericardial sac determined an increase in epicardial cells expressing the EMT marker α-SMA and, interestingly, an increase in the number of epicardial cells ckit(+)/α-SMA(+), 7days following MI. Importantly, clusterin treatment enhanced arteriolar length density and lowered apoptotic rates in the peri-infarct area. Hemodynamic studies demonstrated an improvement in cardiac function in clusterin-treated compared to untreated infarcted hearts., Conclusions: Exosomes are present and detectable in the PFs. Clusterin was identified in PFMI-exosomes and might account for an improvement in myocardial performance following MI through a framework including EMT-mediated epicardial activation, arteriogenesis and reduced cardiomyocyte apoptosis., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Heat shock protein 60 (Hsp60) is a chaperonin involved in tumorigenesis, but its participation in tumor development and progression is not well understood and its value as a tumor biomarker has not been fully elucidated. In the current study, the authors presented evidence supporting the theory that Hsp60 has potential as a biomarker as well as a therapeutic target in patients with large bowel cancer., Methods: The authors studied a population of 97 subjects, including patients and controls. Immunomorphology, Western blot analysis, and quantitative real-time polymerase chain reaction were performed on tissue specimens. Exosomes were isolated from blood and characterized by electron microscopy, biochemical tests, and Western blot analysis., Results: Hsp60 was found to be increased in cancerous tissue, in which it was localized in the tumor cell plasma membrane, and in the interstitium associated with cells of the immune system, in which it was associated with exosomes liberated by tumor cells and, as such, circulated in the blood. An interesting finding was that these parameters returned to normal shortly after tumor removal., Conclusions: The data from the current study suggested that Hsp60 is a good candidate for theranostics applied to patients with large bowel carcinoma and encourage similar research among patients with other tumors in which Hsp60 has been implicated., (© 2015 American Cancer Society.)

In most forms of prion diseases, blood is infectious, but detection by immunochemistry techniques of the only available marker of infection (the misfolded prion protein, PrPTSE) in blood remains elusive. We developed a novel method for the detection of PrPTSE in blood of prion-infected rodents based on the finding that PrPTSE is associated with plasma exosomes. However, further purification of the exosomes on a sucrose gradient was necessary to remove plasma immunoglobulins, which interfere with PrPTSE, masking its detection by immunochemistry. Finally, we report that about 20% of plasma infectivity is associated with exosomes.

Mendelian laws provide the universal founding paradigm for the mechanism of genetic inheritance through which characters are segregated and assorted. In recent years, however, parallel with the rapid growth of epigenetic studies, cases of inheritance deviating from Mendelian patterns have emerged. Growing studies underscore phenotypic variations and increased risk of pathologies that are transgenerationally inherited in a non-Mendelian fashion in the absence of any classically identifiable mutation or predisposing genetic lesion in the genome of individuals who develop the disease. Non-Mendelian inheritance is most often transmitted through the germline in consequence of primary events occurring in somatic cells, implying soma-to-germline transmission of information. While studies of sperm cells suggest that epigenetic variations can potentially underlie phenotypic alterations across generations, no instance of transmission of DNA- or RNA-mediated information from somatic to germ cells has been reported as yet. To address these issues, we have now generated a mouse model xenografted with human melanoma cells stably expressing EGFP-encoding plasmid. We find that EGFP RNA is released from the xenografted human cells into the bloodstream and eventually in spermatozoa of the mice. Tumor-released EGFP RNA is associated with an extracellular fraction processed for exosome purification and expressing exosomal markers, in all steps of the process, from the xenografted cancer cells to the spermatozoa of the recipient animals, strongly suggesting that exosomes are the carriers of a flow of information from somatic cells to gametes. Together, these results indicate that somatic RNA is transferred to sperm cells, which can therefore act as the final recipients of somatic cell-derived information.

Intrinsic resistance to cytotoxic drugs has been a main issue in cancer therapy for decades. Microenvironmental acidity is a simple while highly efficient mechanism of chemoresistance, exploited through impairment of drug delivery. The latter is achieved by extracellular protonation and/or sequestration into acidic vesicles. This study investigates the importance of extracellular acidosis and nanovesicle (exosome) release in the resistance of human tumour cell to cisplatin (CisPt); in parallel to proton pump inhibitors (PPI) ability of interfering with these tumour cell features. The results showed that CisPt uptake by human tumour cells was markedly impaired by low pH conditions. Moreover, exosomes purified from supernatants of these cell cultures contained various amounts of CisPt, which correlated to the pH conditions of the culture medium. HPLC-Q-ICP-MS analysis revealed that exosome purified from tumour cell culture supernatants contained CisPt in its native form. PPI pre-treatment increased cellular uptake of CisPt, as compared to untreated cells, in an acidic-depend manner. Furthermore, it induced a clear inhibition of exosome release by tumour cells. Human tumours obtained from xenografts pretreated with PPI contained more CisPt as compared to tumours from xenografts treated with CisPt alone. Further analysis showed that in vivo PPI treatment induced a clear reduction in the plasmatic levels of tumour-derived exosomes which also contained lower level of CisPt. Altogether, these findings point to the identification of a double mechanism that human malignant melanoma use in resisting to a dreadful cellular poison such as cisplatin. This framework of resistance includes both low pH-dependent extracellular sequestration and an exosome-mediated elimination. Both mechanisms are markedly impaired by proton pump inhibition, leading to an increased CisPt-dependent cytotoxicity.

Exosomes are nanovesicles secreted into the extracellular environment upon internal vesicle fusion with the plasma membrane. The molecular content of exosomes is a fingerprint of the releasing cell type and of its status. For this reason, and because they are released in easily accessible body fluids such as blood and urine, they represent a precious biomedical tool. A growing body of evidence suggests that exosomes may be used as biomarkers for the diagnosis and prognosis of malignant tumors. This article focuses on the exploitation of exosomes as diagnostic tools for human tumors and discusses possible applications of the same strategies to other pathologies, such as neurodegenerative diseases.

EBV is a human herpesvirus associated with a number of malignancies. Both lymphoblastoid cell lines (LCLs), and EBV-infected nasopharyngeal carcinoma (NPC) cells have been demonstrated to release exosomes containing the EBV-encoded latent membrane protein 1 (LMP1), and mature micro-RNAs (EBV-miRNAs). Here we analyze the EBV protein and nucleic acid content of exosomes from different EBV-infected cells (LCL, 721 and Daudi) and we show for the first time that exosomes released from LCLs and 721 also contain EBV-encoded latent phase mRNAs. This confirms and strengthens exosomes pathogenetic potential, and might provide insights for development of novel diagnostic and therapeutic strategies., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Nanomedicine aims to exploit the improved and often novel physical, chemical, and biological properties of materials at the nanometric scale, possibly with the highest level of biomimetism, an approach that simulates what 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 in nanomedicine. 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, whereas those released by cells in pathological condition, such as tumor or infected cells, may induce undesired, dangerous, and mostly unknown effects, but we cannot exclude the possibility that exosomes may also be detrimental for the body in normal conditions. However, whether we consider extracellular vesicles as a whole, thus including MVs, it appears that even in normal conditions, extracellular vesicles may lead to unwanted effects, depending on gender and age. This review aims to critically emphasize existing data in the literature that support the possible roles of exosomes in both diagnostic and therapeutic scopes.

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.

Background: Metastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up., Methodology/principal Findings: We designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504+/-315) or caveolin-1 (619+/-310) were significantly increased in melanoma patients as compared to healthy donors (223+/-125 and 228+/-102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group., Conclusions/significance: We describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients.