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Microenvironmental pH is a key factor for exosome traffic in tumor cells.

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.

High Levels of Exosomes Expressing CD63 and Caveolin-1 in Plasma of Melanoma Patients

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.

CD95 (APO‐1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway

CD95 (APO‐1/Fas) is a member of the tumor necrosis factor receptor family, which can trigger apoptosis in a variety of cell types. However, little is known of the mechanisms underlying cell susceptibility to CD95‐mediated apoptosis. Here we show that human T cells that are susceptible to CD95‐mediated apoptosis, exhibit a constitutive polarized morphology, and that CD95 colocalizes with ezrin at the site of cellular polarization. In fact, CD95 co‐immunoprecipitates with ezrin exclusively in lymphoblastoid CD4+ T cells and primary long‐term activated T lymphocytes, which are prone to CD95‐mediated apoptosis, but not in short‐term activated T lymphocytes, which are refractory to the same stimuli, even expressing equal levels of CD95 on the cell membrane. Pre‐treatment with ezrin antisense oligonucleotides specifically protected from the CD95‐mediated apoptosis. Moreover, we show that the actin cytoskeleton integrity is essential for this function. These findings strongly suggest that the CD95 cell membrane polarization, through an ezrin‐mediated association with the actin cytoskeleton, is a key intracellular mechanism in rendering human T lymphocytes susceptible to the CD95‐mediated apoptosis.

Exosomes: the future of biomarkers in medicine

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.

pH-dependent antitumor activity of proton pump inhibitors against human melanoma is mediated by inhibition of tumor acidity

Metastatic melanoma is associated with poor prognosis and still limited therapeutic options. An innovative treatment approach for this disease is represented by targeting acidosis, a feature characterizing tumor microenvironment and playing an important role in cancer malignancy. Proton pump inhibitors (PPI), such as esomeprazole (ESOM) are prodrugs functionally activated by acidic environment, fostering pH neutralization by inhibiting proton extrusion. We used human melanoma cell lines and xeno‐transplated SCID mice to provide preclinical evidence of ESOM antineoplastic activity. Human melanoma cell lines, characterized by different mutation and signaling profiles, were treated with ESOM in different pH conditions and evaluated for proliferation, viability and cell death. SCID mice engrafted with human melanoma were used to study ESOM administration effects on tumor growth and tumor pH by magnetic resonance spectroscopy (MRS). ESOM inhibited proliferation of melanoma cells in vitro and induced a cytotoxicity strongly boosted by low pH culture conditions. ESOM‐induced tumor cell death occurred via rapid intracellular acidification and activation of several caspases. Inhibition of caspases activity by pan‐caspase inhibitor z‐vad‐fmk completely abrogated the ESOM‐induced cell death. ESOM administration (2.5 mg kg−1) to SCID mice engrafted with human melanoma reduced tumor growth, consistent with decrease of proliferating cells and clear reduction of pH gradients in tumor tissue. Moreover, systemic ESOM administration dramatically increased survival of human melanoma‐bearing animals, in absence of any relevant toxicity. These data show preclinical evidence supporting the use of PPI as novel therapeutic strategy for melanoma, providing the proof of concept that PPI target human melanoma modifying tumor pH gradients.

Potent Immune Response against HIV-1 and Protection from Virus Challenge in hu-PBL-SCID Mice Immunized with Inactivated Virus-pulsed Dendritic Cells Generated in the Presence of IFN-α

A major challenge of AIDS research is the development of therapeutic vaccine strategies capable of inducing the humoral and cellular arms of the immune responses against HIV-1. In this work, we evaluated the capability of DCs pulsed with aldrithiol-2–inactivated HIV-1 in inducing a protective antiviral human immune response in SCID mice reconstituted with human PBL (hu-PBL-SCID mice). Immunization of hu-PBL-SCID mice with DCs generated after exposure of monocytes to GM-CSF/IFN-α (IFN-DCs) and pulsed with inactivated HIV-1 resulted in a marked induction of human anti–HIV-1 antibodies, which was associated with the detection of anti-HIV neutralizing activity in the serum. This vaccination schedule also promoted the generation of a human CD8+ T cell response against HIV-1, as measured by IFN-γ Elispot analysis. Notably, when the hu-PBL-SCID mice immunized with antigen-pulsed IFN-DCs were infected with HIV-1, inhibition of virus infection was observed as compared with control animals. These results suggest that IFN-DCs pulsed with inactivated HIV-1 can represent a valuable approach of immune intervention in HIV-1–infected patients.

Exosome Release and Low pH Belong to a Framework of Resistance of Human Melanoma Cells to Cisplatin

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.

Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine

Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.

Human intestinal lamina propria lymphocytes are naturally permissive to HIV-1 infection.

The presence of HIV‐1 in the intestinal mucosa of AIDS patients has been reported and human intestinal lamina propria lymphocytes (LPL) have been proposed as important targets for HIV‐1 infection. However, little information is available concerning the permissiveness of human intestinal CD4+ T lymphocytes to HIV‐1 infection. Here, we show that human LPL, in contrast to autologous peripheral blood lymphocytes (PBL), are permissive to both X4 T‐tropic and R5 M‐tropic strains of HIV‐1, as well as to clinical isolates, in the absence of exogenous stimuli. Flow cytometry showed that the vast majority of T LPL were CD45RO+ and CD69+, and that CD4+ T LPL highly expressed CC chemokine receptor 5 (CCR5) as compared to PBL, while CX chemokine receptor 4 was equally expressed on LPL and PBL. Exogenous RANTES and macrophage inflammatory protein‐1α (natural CCR5 ligands) virtually abolished the entry of the R5 M‐tropic strain HIV‐1 into human LPL. Thus, we infer that human intestinal CD4+ T lymphocytes are naturally susceptible to HIV‐1 infection, due to their physiological state of activation and to marked expression of HIV‐1 coreceptors, independently of the route of primary (either mucosal or parental) infection and the shifts of the virus phenotype occurring during the course of AIDS.

The natural alliance between type I interferon and dendritic cells and its role in linking innate and adaptive immunity.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) and thus play a pivotal role in induction of the immune response. Recent studies in both human and mouse models have shown that type I IFN, cytokines originally characterized for their antiviral activity and exerting multiple biologic effects, efficiently promote the differentiation and activation of DCs. These observations, together with the findings that DCs can express biologically relevant levels of type I interferon (IFN) and, in particular, that high amounts of these cytokines are released by specialized DC precursors (i.e., plasmacytoid DCs) in response to viral infections, strongly suggest the existence of a natural alliance between type I IFN and DCs, which is instrumental in ensuring an efficient immune response to both infectious agents and tumors. Further recent knowledge on the interactions between type I IFN and DCs emphasizes the importance of these cytokines in linking innate and adaptive immunity and may lead to new perspectives in their use as vaccine adjuvants as well as in strategies for the development of DC-based vaccines.