Increased Plasmatic Levels of Exosomes Are Significantly Related to Relapse Rate in Patients with Oral Squamous Cell Carcinoma: A Cohort Study.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is characterized by an immunosuppressive tumor microenvironment. Their plasma-derived exosomes deliver immunomodulatory molecules and cargo that correlate significantly with clinical parameters. This study aims to assess the exosomal profile as a potential tool for early detection of relapse and long-term outcomes in OSCC patients undergoing conventional therapy. METHODS: 27 OSCC patients with a median 38-month follow-up were included in this study. The relationship between NTA-derived parameters and clinical pathological parameters was examined, and receiver operating characteristic (ROC) curves were utilized to evaluate the diagnostic efficacy of these values in detecting cancer relapse. RESULTS: Plasmatic levels of exosomes prior to surgery showed a drastic reduction after surgical intervention (8.08E vs. 1.41 × 109 particles/mL, p = 0.006). Postsurgical concentrations of exosomes were higher in patients who experienced relapse compared to those who remained disease-free (2.97 × 109 vs. 1.11 × 109 particles/mL, p = 0.046). Additionally, patients who relapsed exhibited larger exosome sizes after surgery (141.47 vs. 132.31 nm, p = 0.03). Patients with lower concentrations of exosomes prior to surgery demonstrated better disease-free survival compared to those with higher levels (p = 0.012). ROC analysis revealed an area under the curve of 0.82 for presurgical exosome concentration in identifying relapse. CONCLUSIONS: Presurgical exosomal plasmatic levels serve as independent predictors of early recurrence and survival in OSCC. All in all, our findings indicate that the detection of peripheral exosomes represents a novel tool for the clinical management of OSCC, with potential implications for prognosis assessment.

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 Importance of Detecting, Quantifying, and Characterizing Exosomes as a New Diagnostic/Prognostic Approach for Tumor Patients.

Exosomes are extracellular vesicles (EVs) of nanometric size studied for their role in tumor pathogenesis and progression and as a new source of tumor biomarkers. The clinical studies have provided encouraging but probably unexpected results, including the exosome plasmatic levels' clinical relevance and well-known biomarkers' overexpression on the circulating EVs. The technical approach to obtaining EVs includes methods to physically purify EVs and characterize EVs, such as Nanosight Tracking Analysis (NTA), immunocapture-based ELISA, and nano-scale flow cytometry. Based on the above approaches, some clinical investigations have been performed on patients with different tumors, providing exciting and promising results. Here we emphasize data showing that exosome plasmatic levels are consistently higher in tumor patients than in controls and that plasmatic exosomes express well-known tumor markers (e.g., PSA and CEA), proteins with enzymatic activity, and nucleic acids. However, we also know that tumor microenvironment acidity is a key factor in influencing both the amount and the characteristics of the exosome released by tumor cells. In fact, acidity significantly increases exosome release by tumor cells, which correlates with the number of exosomes that circulate through the body of a tumor patient.

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.

Plasmatic Exosome Number and Size Distinguish Prostate Cancer Patients From Healthy Individuals: A Prospective Clinical Study.

There is a urgent need for valuable strategy in early and less invasive diagnosis for cancer. Preliminary data have shown that the plasmatic levels of exosomes increase in cancer condition. This study investigates the relevance of plasmatic levels and size distribution of exosomes in 42 individuals with no signs of urological disease (CTR) as compared to 65 prostate cancer patients (PCa). It was used Nanoparticle Tracking Analysis (NTA), a highly reliable and sensitive method for exosomes characterization and quantification. The relation structure among the NTA-derived parameters was assessed by means of Principal Component Analysis, which allowed detecting the global discriminant power of NTA test in terms of Receiver Operating Characteristic (ROC) curve and the selection of cut-off thresholds. The results showed that PCa had significantly higher plasmatic levels of exosomes and that the exosomes were smaller in size as compared to the CTR; the values reached 89% sensitivity and 71% specificity, in distinguishing PCa from CTR. These results propose a new exosome-based non-invasive clinical approach for the clinical follow-up of prostate cancer undergoing surgical treatment; in addition this method may be developed as a new screening test for prostate cancer's early diagnosis. While this clinical study was performed in prostate cancer, it may represent a proof of concept extendable to virtually all cancers, as it is suggested by both pre-clinical evidence and clinical data obtained with different technical approaches.

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.

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.

Exosomes: A Source for New and Old Biomarkers in Cancer.

Clinical oncology needs reliable tumor biomarkers to allow a follow-up of tumor patients who do not necessarily need invasive approaches. To date, the existing biomarkers are not sufficiently reliable, and many of them have generated more problems than facilitating the commitment of clinical oncologists. Over the last decades, a broad family of extracellular vesicles, with size ranging between micro to nano, has been raised as a new hope for potential sources of new tumor biomarkers. However, while knowledge in the field is increasing, we do not currently have definitive information allowing a clinical use of extracellular vesicles in cancer clinics. Recent evidence provides new perspective in clinical oncology, based on data showing that circulating nanovesicles called exosomes may represent a valuable source of tumor biomarkers. In this review, we discuss the existing clinical data supporting a key role of exosomes as a source of tumor biomarkers, including proteins and miRNAs, but also discuss the importance of the expression of known tumor biomarkers when expressed on exosomes.

Anti-aging and anti-tumor effect of FPP® supplementation.

The beneficial effect of FPP® as antioxidant is known. Here we summarize recent data supporting future implementation of FPP® in tumor treatment and in controlling aging at the molecular level. We first showed that oral FPP® is able to control tumor growth and with inducing a potent and systemic anti-oxidant reaction (i.e. reduced ROS and increased GSH and SOD-1). Then we showed that FPP® is able to markedly increase the body anti-oxidant reaction together with increasing both telomerase activity in the blood and the telomeres length in bone marrow and ovary of treated mice as compared to the untreated mice.

Immunocapture-based ELISA to characterize and quantify exosomes in both cell culture supernatants and body fluids.

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.

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.

Increased Plasmatic Levels of PSA-Expressing Exosomes Distinguish Prostate Cancer Patients from Benign Prostatic Hyperplasia: A Prospective Study.

Prostate Specific Antigen (PSA) fails to discriminate between benign prostatic hyperplasia (BPH) and Prostate Cancer (PCa), resulting in large numbers of unnecessary biopsies and missed cancer diagnoses. Nanovesicles called exosomes are directly detectable in patient plasma and here we explore the potential use of plasmatic exosomes expressing PSA (Exo-PSA) in distinguishing healthy individuals, BPH, and PCa. Exosomes were obtained from plasma samples of 80 PCa, 80 BPH, and 80 healthy donors (CTR). Nanoparticle Tracking Analysis (NTA), immunocapture-based ELISA (IC-ELISA), and nanoscale flow-cytometry (NSFC), were exploited to detect and characterize plasmatic exosomes. Statistical analysis showed that plasmatic exosomes expressing both CD81 and PSA were significantly higher in PCa as compared to both BPH and CTR, reaching 100% specificity and sensitivity in distinguishing PCa patients from healthy individuals. IC-ELISA, NSFC, and Exo-PSA consensus score (EXOMIX) showed 98% to 100% specificity and sensitivity for BPH-PCa discrimination. This study outperforms the conventional PSA test with a minimally invasive widely exploitable approach.

Human primary macrophages scavenge AuNPs and eliminate it through exosomes. A natural shuttling for nanomaterials.

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.

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.

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.

Oral Administration of Fermented Papaya (FPP®) Controls the Growth of a Murine Melanoma through the In Vivo Induction of a Natural Antioxidant Response.

Prolonged oxidative stress may play a key role in tumor development. Antioxidant molecules are contained in many foods and seem to have a potential role in future anti-tumor strategies. Among the natural antioxidants the beneficial effect of Fermented Papaya (FPP®) is well known. The aim of this study was to investigate the effects of orally administered FPP® in either the prevention or treatment of a murine model of melanoma. The tumor growth was analyzed together with the blood levels of both oxidants (ROS) and anti-oxidants (SOD-1 and GSH). The results showed that FPP® controlled tumor growth, reducing the tumor mass of about three to seven times vs. untreated mice. The most significant effect was obtained with sublingual administration of FPP® close to the inoculation of melanoma. At the time of the sacrifice none of mice treated with FPP® had metastases and the subcutaneous tumors were significantly smaller and amelanotic, compared to untreated mice. Moreover, the FPP® anti-tumor effect was consistent with the decrease of total ROS levels and the increase in the blood levels of GSH and SOD-1. This study shows that a potent anti-oxidant treatment through FPP® may contribute to both preventing and inhibiting tumors growth.

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.

Microenvironmental pH and Exosome Levels Interplay in Human Cancer Cell Lines of Different Histotypes.

Exosomes are extracellular nanovesicles primarily involved in the pathogenesis of many diseases including cancer. This study was set out from recent evidence that extracellular acidity may increase the exosome release by cancer cells. However, this preliminary evidence did not provide solid information on whether the pH-dependent exosome over-release represents a common feature of all cancers. To the purpose of demonstrating that cancer acidity is a major determinant in inducing an increased exosome release by human cancer cells, we evaluated human tumor cell lines deriving from either colon, breast, prostate cancers, melanoma, or osteosarcoma. All cell lines were cultured in either the current 7.4 pH or the typical pH of cancer that is 6.5. The levels of released extracellular vesicles were measured by protein counts, nanoparticle tracking analysis (NTA), and nanoscale flow cytometry. The results showed that pH 6.5 induced a remarkable increase in exosome release, and buffering the medium significantly reduced the exosome release in all cancers. With these results, we provide, for the first time, evidence that tumor acidity and exosome levels represent common cancer phenotypes.

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.