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      Targeting and Therapy of Glioblastoma in a Mouse Model Using Exosomes Derived From Natural Killer Cells

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          Abstract

          Objective

          Glioblastoma is a highly aggressive primary brain tumor that is resistant to radiotherapy and chemotherapy. Natural killer (NK) cells have been used to treat incurable cancers. Recent studies have investigated the effectiveness of NK-cell-derived exosomes (NK-Exo) for treating incurable cancers such as melanoma, leukemia, and neuroblastoma; however, NK-Exo have not been used to treat glioblastoma. In the present study, we investigated the antitumor effects of NK-Exo against aggressive glioblastoma both in vitro and in vivo and determined the tumor-targeting ability of NK-Exo by performing fluorescence imaging.

          Methods

          U87/MG cells were transfected with the enhanced firefly luciferase (effluc) and thy1.1 genes; thy1.1-positive cells were selected using microbeads. U87/MG/F cells were assessed by reverse transcription polymerase chain reaction (RT-PCR), western blotting, and luciferase-activity assays. NK-Exo were isolated by ultracentrifugation, purified by density gradient centrifugation, and characterized by transmission electron microscopy, dynamic light scattering (DLS), nanoparticle-tracking analysis (NTA), and western blotting. Cytokine levels in NK-Exo were compared to those in NK cells and NK-cell medium by performing an enzyme-linked immunosorbent assay (ELISA). NK-Exo-induced apoptosis of cancer cells was confirmed by flow cytometry and western blotting. In vivo therapeutic effects and specificity of NK-Exo against glioblastoma were assessed in a xenograft mouse model by fluorescence imaging. Xenograft mice were treated with NK-Exo, which was administered seven times through the tail vein. Tumor growth was monitored by bioluminescence imaging (BLI), and tumor volume was measured by ultrasound imaging. The mice were intraperitoneally injected with dextran sulfate 2 h before NK-Exo injection to decrease the liver uptake and increase the tumor specificity of NK-Exo.

          Results

          RT-PCR and western blotting confirmed the gene and protein expression of effluc in U87/MG/F cells, with the bioluminescence activity of U87/MG/F cells increasing with an increase in cell number. NTA and DLS results indicated that the size of NK-Exo was ~100 nm, and the western blot results confirmed that NK-Exo expressed exosome markers CD63 and Alix. We confirmed the in vitro cytotoxic effects of NK-Exo on U87/MG/F cells by performing BLI, and the killing effect on U87/MG and U87MG/F cells was measured by CCK-8 and MTT assays ( p < 0.001). ELISA results indicated that NK-Exo contained tumor necrosis factor-α and granzyme B. In vivo NK-Exo treatment inhibited tumor growth compared to in control mice ( p < 0.001), and pretreatment of xenograft mice with dextran sulfate 2 h before NK-Exo treatment increased the antitumor effect of NK-Exo ( p < 0.01) compared to in control and NK-Exo-alone-treated mice.

          Conclusion

          NK-Exo targeted and exerted antitumor effects on glioblastoma cells both in vitro and in vivo, suggesting their utility in treating incurable glioblastoma.

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          Most cited references 52

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          Exosome-mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth.

          Multipotent mesenchymal stromal cells (MSCs) have potential therapeutic benefit for the treatment of neurological diseases and injury. MSCs interact with and alter brain parenchymal cells by direct cell-cell communication and/or by indirect secretion of factors and thereby promote functional recovery. In this study, we found that MSC treatment of rats subjected to middle cerebral artery occlusion (MCAo) significantly increased microRNA 133b (miR-133b) level in the ipsilateral hemisphere. In vitro, miR-133b levels in MSCs and in their exosomes increased after MSCs were exposed to ipsilateral ischemic tissue extracts from rats subjected to MCAo. miR-133b levels were also increased in primary cultured neurons and astrocytes treated with the exosome-enriched fractions released from these MSCs. Knockdown of miR-133b in MSCs confirmed that the increased miR-133b level in astrocytes is attributed to their transfer from MSCs. Further verification of this exosome-mediated intercellular communication was performed using a cel-miR-67 luciferase reporter system and an MSC-astrocyte coculture model. Cel-miR-67 in MSCs was transferred to astrocytes via exosomes between 50 and 100 nm in diameter. Our data suggest that the cel-miR-67 released from MSCs was primarily contained in exosomes. A gap junction intercellular communication inhibitor arrested the exosomal microRNA communication by inhibiting exosome release. Cultured neurons treated with exosome-enriched fractions from MSCs exposed to 72 hours post-MCAo brain extracts significantly increased the neurite branch number and total neurite length. This study provides the first demonstration that MSCs communicate with brain parenchymal cells and may regulate neurite outgrowth by transfer of miR-133b to neural cells via exosomes. Copyright © 2012 AlphaMed Press.
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            Functional significance of the perforin/granzyme cell death pathway.

            Perforin/granzyme-induced apoptosis is the main pathway used by cytotoxic lymphocytes to eliminate virus-infected or transformed cells. Studies in gene-disrupted mice indicate that perforin is vital for cytotoxic effector function; it has an indispensable, but undefined, role in granzyme-mediated apoptosis. Despite its vital importance, the molecular and cellular functions of perforin and the basis of perforin and granzyme synergy remain poorly understood. The purpose of this review is to evaluate critically recent findings on cytotoxic granule-mediated cell death and to assess the functional significance of postulated cell-death pathways in appropriate pathophysiological contexts, including virus infection and susceptibility to experimental or spontaneous tumorigenesis.
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              Bovine milk-derived exosomes for drug delivery.

              Exosomes are biological nanovesicles that are involved in cell-cell communication via the functionally-active cargo (such as miRNA, mRNA, DNA and proteins). Because of their nanosize, exosomes are explored as nanodevices for the development of new therapeutic applications. However, bulk, safe and cost-effective production of exosomes is not available. Here, we show that bovine milk can serve as a scalable source of exosomes that can act as a carrier for chemotherapeutic/chemopreventive agents. Drug-loaded exosomes showed significantly higher efficacy compared to free drug in cell culture studies and against lung tumor xenografts in vivo. Moreover, tumor targeting ligands such as folate increased cancer-cell targeting of the exosomes resulting in enhanced tumor reduction. Milk exosomes exhibited cross-species tolerance with no adverse immune and inflammatory response. Thus, we show the versatility of milk exosomes with respect to the cargo it can carry and ability to achieve tumor targetability. This is the first report to identify a biocompatible and cost-effective means of exosomes to enhance oral bioavailability, improve efficacy and safety of drugs.
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                Author and article information

                Contributors
                Journal
                Front Immunol
                Front Immunol
                Front. Immunol.
                Frontiers in Immunology
                Frontiers Media S.A.
                1664-3224
                23 April 2018
                2018
                : 9
                Affiliations
                Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital , Daegu, South Korea
                Author notes

                Edited by: Anahid Jewett, University of California, Los Angeles, United States

                Reviewed by: Eliana Ruggiero, San Raffaele Hospital (IRCCS), Italy; Emily R. Levy, National Institutes of Health (NIH), United States

                *Correspondence: Byeong-Cheol Ahn, abc2000@ 123456knu.ac.kr

                Specialty section: This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology

                Article
                10.3389/fimmu.2018.00824
                5924812
                Copyright © 2018 Zhu, Oh, Gangadaran, Kalimuthu, Baek, Jeong, Lee, Lee and Ahn.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                Page count
                Figures: 13, Tables: 0, Equations: 0, References: 79, Pages: 18, Words: 10522
                Categories
                Immunology
                Original Research

                Immunology

                exosomes, natural killer cells, tumor targeting, immunotherapy, glioblastoma

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