胆管癌外泌体通过抑制肿瘤坏死因子α与穿孔素分泌使细胞因子诱导的杀伤细胞抗肿瘤活性下降

Cholangiocarcinoma-derived exosomes inhibit the antitumor activity of cytokine-induced killer cells by down-regulating the secretion of tumor necrosis factor-α and perforin 胆管癌外泌体通过抑制肿瘤坏死因子α与穿孔素分泌使细胞因子诱导的杀伤细胞抗肿瘤活性下降 first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:caolipingzju@126.com †E-mail:caolipingzju@126.com caolipingzju@126.com Department of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China Department of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China Objective: 2 The aim of our study is to observe the impact of cholangiocarcinoma-derived exosomes on the antitumor activities of cytokine-induced killer (CIK) cells and then demonstrate the appropriate mechanism. Methods: 2 Tumor-derived exosomes (TEXs), which are derived from RBE cells (human cholangiocarcinoma line), were collected by ultracentrifugation. CIK cells induced from peripheral blood were stimulated by TEXs. Fluorescence-activated cell sorting (FACS) was performed to determine the phenotypes of TEX-CIK and N-CIK (normal CIK) cells. The concentrations of tumor necrosis factor-α (TNF-α) and perforin in the culture medium supernatant were examined by using an enzyme-linked immunosorbent assay (ELISA) kit. A CCK-8 kit was used to evaluate the cytotoxic activity of the CIK cells to the RBE cell line. Results: 2 The concentrations of TNF-α and perforin of the group TEX-CIK were 138.61 pg/ml and 2.41 ng/ml, respectively, lower than those of the group N-CIK 194.08 pg/ml ( P < 0.01) and 3.39 ng/ml ( P < 0.05). The killing rate of the group TEX-CIK was 33.35%, lower than that of the group N-CIK (47.35% ( P < 0.01)). The population of CD3 + , CD8 + , NK (CD56 + ), and CD3 + CD56 + cells decreased in the TEX-CIK group ((63.2±6.8)%, (2.5±1.0)%, (0.53±0.49)%, (0.45±0.42)%) compared with the N-CIK group ((90.3±7.3)%, (65.7±3.3)%, (4.2±1.2)%, (15.2±2.7)%), P < 0.01. Conclusions: 2 Our results suggest that RBE cells-derived exosomes inhibit the antitumor activity of CIK cells by down-regulating the population of CD3 + , CD8 + , NK (CD56 + ), and CD3 + CD56 + cells and the secretion of TNF-α and perforin. TEX may play an important role in cholangiocarcinoma immune escape. 目的 2 探索胆管癌来源外泌体（TEX）对细胞因子诱导的杀伤细胞（CIK）抗肿瘤活性的影响，并初步探讨其作用机制。 创新点 2 首次通过体外实验证明TEX可引起CIK抗肿瘤活性下降，且此作用与肿瘤坏死因子α（TNF-α）和穿孔素表达抑制相关。 方法 2 采用超速离心法提取人胆管癌细胞（RBE）来源的外泌体，同时CIK通过人外周血培养获得。将TEX负载到CIK培养体系中作为TEX-CIK组，不加TEX的CIK作为N-CIK组。流式细胞仪检测两组CIK细胞表型变化，酶联免疫吸附法（ELISA）检测两组培养基上清液中TNF-α和穿孔素的浓度，CCK-8法检测CIK对RBE细胞的杀伤活性。 结论 2 TEX能降低CIK细胞CD3 + 、CD8 + 、NK（CD56 + ）以及CD3 + CD56 + 比例，并且抑制TNF-α和穿孔素表达，从而降低CIK细胞的抗肿瘤效应。 Cholangiocarcinoma Tumor-derived exosomes Cytokine-induced killer cells Immune escape 胆管癌 肿瘤来源外泌体 细胞因子诱导的杀伤细胞 免疫逃逸 Introduction 1 Cholangiocarcinoma (CCA) is a devastating malignant disease that is proverbially difficult to diagnose and is associated with a high mortality. Although CCA accounts for only 3% of all gastrointestinal cancers, CCA represents the second most frequent primary hepatic malignant tumor after hepatocellular carcinoma, and a progressively increasing incidence rate was observed worldwide during the last decades (Khan et al., 2005 2005 ; Welzel et al., 2006 2006 ; Ewald et al., 2013 2013 ). Adoptive cellular immunotherapy is becoming an important application for cancer therapy. In recent decades, the application of cytokine-induced killer (CIK) cells has evolved from experimental observations into early clinical studies (Morse et al., 2005 2005 ; Wang et al., 2011 2011 ). CIK cells are a population of cells derived from human peripheral blood mononuclear cells (PBMCs) after ex vivo expansion with interferon-γ (IFN-γ), monoclonal antibody against cluster of differentiation 3 (CD3), and interleukin-2 (IL-2) (Lu and Negrin, 1994 1994 ). CIK cells have a higher proliferation rate, increased cytolytic activities, and a non-major histocompatibility complex (MHC)-restricted killing of tumor cells by secreting tumor necrosis factor-α (TNF-α) and perforin. CIK cells-based immunotherapy has proved to be effective in patients with a variety of malignancies, including CCA. Perforin and TNF-α, which are secreted by CIK cells, play a critical role in killing tumor cells and in immunotherapy against tumors. CIK cells have a potent cytotoxicity against tumor cells in vitro (Hongeng et al., 2003 2003 ), but the curative effect of immunotherapy based on CIK cells is not that satisfying (Morse et al., 2005 2005 ). Thus, we suspect that some substances secreted by tumor cells inhibit the activities of CIK cells. Exosomes are small vesicles, about 40–100 nm in size, that are secreted by a variety of cell types, such as B-cells, T-cells, dendritic cells, and tumor cells (Raposo et al., 1996 1996 ; Zitvogel et al., 1998 1998 ; Wolfers et al., 2001 2001 ). Tumor-derived exosomes (TEXs) have been shown to play an important role in the immune system, by mediating antigen and cytokine secretions (Bretz et al., 2013 2013 ; Thompson et al., 2013 2013 ). In this study, in order to confirm the impact of TEX to CIK cells, we used the RBE cell line (human CCA cell line) to generate TEX. CIK cells were stimulated by TEX to evaluate the antitumor activities to the RBE cell line and to probe into their mechanisms, which caused CIK therapy to be dissatisfactory. Materials and methods 1 Cell lines and culture 2 The RBE cell line, which is one of the human CCA cell lines, was obtained from the Committee of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). RBE cells were grown in the Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, USA) and antibiotics (50 μg/ml each of penicillin, streptomycin, and gentamicin; Gibco, USA) at 37 °C in a humidified 5% CO 2 and 95% of air atmosphere. Preparation of CIK cells 2 We collected 15 ml of peripheral blood in evacuated tubes that contained heparin. PBMCs were isolated by using Ficoll density-gradient centrifugation and incubated in tissue culture flasks at 37 °C for 2 h, supplemented with an RPMI-1640 medium. On Day 0, for the non-adherent PBMCs we added 1000 U/ml recombinant human IFN-γ (Genentech, USA). After 24 h, 500 U/ml IL-2 (Chiron, USA), and 50 μg/ml CD3 monoclonal antibody (OKT3; Ortho Biotech, USA) were added and then these cells were cultured for another 7 d. Half of the old medium was replaced every 2 d with fresh CIK medium and 500 U/ml IL-2, respectively. Preparation and identification of TEX 2 Cell culture 3 The RBE cells were cultured in an RPMI-1640 medium, supplemented with 10% FBS and antibiotics, as described previously, until the culture flask was almost full. Then, the culture media were discarded, and the cells were washed three times with phosphate-buffered saline (PBS; Gibco, USA). Next, new media supplemented with RPMI-1640 medium, without FBS, and the antibiotics mentioned above were added to the media, and the cells were cultured for another 72 h. Isolation of TEX 3 After that, the cell culture media were collected and sequential centrifugations were performed. The cell culture media were centrifuged at 300 g for 3 min at 4 °C to remove floating cells. These supernatants were then centrifuged at 2000 g for 15 min at 4 °C and at 12 000 g for 35 min at 4 °C to remove all cell debris. These supernatants were then passed through a 0.22-μm filter (Millipore, USA). The filtrates were ultracentrifuged at 120 000 g for 3 h at 4 °C to collect exosomes using an Optima TLX Ultracentrifuge (Beckman Coulter, CA, USA). Exosome pellets were resuspended in PBS and were further ultracentrifuged at 120 000 g for 3 h at 4 °C. Exosome protein quantification was performed using a BCA protein assay kit (KeyGen Biotechnologies, China). The final exosome pellets were stored at −80 °C until use (Chiba et al., 2012 2012 ). Nanoparticle tracking analysis 3 Isolated TEX from RBE cell lines were analyzed using the nanoparticle tracking analysis (NTA) Version 2.3 Build 0034. Analysis of the data was done using the software supplied with the machine. Graphical analysis shows the size distribution of the TEX. Western blot analysis 3 TEX was isolated by the procedure mentioned above from the RBE cell lines. A total of 20 μg TEX and RBE cell samples were solubilized in NP40 buffer (Boston BioProducts, MA, USA) and were run on a Western blot according to standard protocols. The following antibodies were used: mouse anti-CD63 (1:500, Abcam, UK), mouse anti-tumor susceptibility gene 101 (TSG101) (1:500, Abcam, UK), mouse anti-ALIX (1:500, Abcam, UK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, UK) as the loading control. Identification of CIK cells 2 An amount of 1×10 6 CIK cells was harvested and washed twice with PBS. These cells were resuspended in 150 μl of PBS, labeled with 20 μl of antibodies against CD3/4/8 (PerCP-conjugated anti-CD3, FITC-conjugated anti-CD4, PE-conjugated anti-CD8; BD Biosciences, USA) and 10 μl of anti-CD56 antibody (APC-conjugated anti-CD56; BD Biosciences, USA), and placed in the dark for 30 min at 4 °C, and then washed twice with PBS. Fluorescence-activated cell sorting (FACS) was applied by using a FACSCalibur flow cytometer (BD Biosciences, USA) and CellQuest software (BD Biosciences, USA). Detection of TNF-α and perforin 2 An amount of 1×10 6 CIK cells was added to a new 6-well flat-bottomed plate. These CIK cells, labeled as group TEX-CIK, were then treated with TEX at a ratio of 1×10 6 CIK cells:20 μg of TEX for 24 h. Another batch of 1×10 6 CIK cells, labeled as group N-CIK, was added to a new 6-well flat-bottomed plate for 24 h. After 24 h, the concentrations of TNF-α and perforin in the culture medium supernatant, with or without TEX, were examined by using an enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, USA). Each group was tested in triplicate. Cytotoxic examination of CIK cells 2 The RBE cells in the logarithmic growth phase were added to a 96-well plate (10 000 cells/well) as a target group. On the second day, TEX-CIK and N-CIK cells were added as an effector to the target ratio of 10:1 and tested in triplicate. Mixed incubation was done for 24 h. The supernatant was removed and all cells were washed twice with PBS. Then, CCK-8 was added, followed by incubation for another 3 h. The blank control was set with only PBS and CCK-8. The absorbance (optical density (OD)) was measured at 450 nm using a microplate reader. The killing rate was calculated as follows: killing rate=(OD experiment −OD blank )/(OD negative −OD blank )×100%. Statistical analysis 2 Data were expressed as the mean±standard deviation (SD). A t -test was used for analysis of comparisons, correlation, and variance using SPSS 19.0 software (IBM, USA). P < 0.05 was regarded as statistically significant. Results 1 Characterization of TEX 2 A Philips 208 transmission electron microscope (FEI Company, the Netherlands) was utilized to analyze our TEX preparation for the RBE cell supernatants from a morphological point of view. The electron microscopy indicated that TEX was accorded with the characteristics of the exosomes (Fig. 1a 1a ). The size distribution of TEX was confirmed by using the Nanoparticle tracking analysis (Version 2.3 Build 0034), which was consistent with the exosomes (Fig. 1b 1b ). The exosome identity markers ALIX, CD63, and TSG101 were confirmed by using a Western blot analysis (Fig. 1c 1c ). (a) Electron microscopy of TEX (bar=100 nm). The culture supernatants yielded pellets consisting of a similar population of vesicles 30–120 nm in diameter. (b) The size distribution of TEX. (c) Western plot showing the expressions of exosomal markers ALIX, TSG101, and CD63 in TEX, with GAPDH as the loading control Phenotypic analysis of CIK cells 2 To determine the immunological effects of TEX-CIK and N-CIK cells, the phenotypes were characterized. The population of CD3 + , CD8 + , NK (CD56 + ) and CD3 + CD56 + cells decreased in the TEX-CIK group ((63.2±6.8)%, (2.5±1.0)%, (0.53±0.49)%, (0.45±0.42)%) compared with the N-CIK group ((90.3±7.3)%, (65.7±3.3)%, (4.2±1.2)%, (15.2±2.7)%) ( P < 0.01; Fig. 2 2 ). The population of CD4 + cells in the TEX-CIK group was (10.1±2.5)%, not statistically significant when compared with that (12.3±2.7)% in the N-CIK group ( P > 0.05). The populations of CD3 + , CD8 + , NK (CD56 + ), and CD3 + CD56 + cells decreased in the TEX-CIK group compared with the N-CIK group ( P < 0.01). However, the population change of CD4 + cells was not statistically significant ( P > 0.05) TEX down-regulates the secretion of TNF-α and perforin 2 The concentrations of TNF-α and perforin of the group TEX-CIK were (138.61±46.01) pg/ml and (2.41±1.10) ng/ml, respectively, lower than those of the group N-CIK (194.08±35.14) pg/ml and (3.39±1.39) ng/ml ( P < 0.05; Fig. 3 3 ). (a) The concentration of TNF-α of the groups TEX-CIK and N-CIK were (138.61±46.01) pg/ml, (194.08±35.14) pg/ml, respectively. (b) The concentration of perforin of the groups TEX-CIK and N-CIK were (2.41±1.10) ng/ml, (3.39±1.39) ng/ml, respectively. The secretion of TNF-α ( P < 0.01) and perforin ( P < 0.05) was down-regulated by TEX in the CIK cells TEX inhibits the antitumor activity of the CIK cells 2 The killing rate of the group TEX-CIK was (33.35±10.05)%, significantly lower than that of the group N-CIK (47.35±12.74)% ( P < 0.01; Fig. 4 4 ). The killing rate of the group TEX-CIK was (33.35±10.05)%, significantly lower than that of the group N-CIK (47.35±12.74)% ( P < 0.01) Discussion 1 CCA was first reported by Durand-Fardel in 1840 (Olnes and Erlich, 2004 2004 ). The disease is notoriously difficult to diagnose and is usually fatal because of its late clinical identification and the lack of effective non-surgical therapeutic modalities (Khan et al., 2005 2005 ). Over the past two decades, the development of CIK cells for the treatment of carcinoma has received considerable attention. CIK cells have a major histocompatibility complex (MHC)-independent antitumor ability in both solid and hematologic malignancies. The antitumor activity is primarily related to a high percentage of the CD3 + CD56 + subset (Ma et al., 2012 2012 ; Nakano et al., 2012 2012 ). The exact mechanism is still unclear, but primarily relates to the secretion of cytokines to inhibit the growth of tumor cells (Pievani et al., 2011 2011 ; Wang et al., 2011 2011 ; Yang et al., 2012 2012 ). A large number of in vitro studies had confirmed the ability of CIK cells to kill various types of solid tumors. The CD3 + CD56 + fraction of CIK cells is considered the crucial effector for the antitumor activities. Proofs of antitumor activities were clearly provided but the clinical responses were much lower than expected (Mesiano et al., 2012 2012 ). Exosomes are microvesicles released from various cells into the extracellular space, including tumor cell lines (Thompson et al., 2013 2013 ). TEXs have been shown to play an important role in the immune system (Sun et al., 2012 2012 ) by mediating apoptosis of the T lymphocytes and down-regulating the cytokine’s presentation (Wolfers et al., 2001 2001 ). CD3 as a T-cell co-receptor is an important part of the T-cell receptor (TCR) complex. CD8 also serves as a co-receptor for the TCR and binds it to an MHC class I molecule (Garcia et al., 1996 1996 ). The importance of the MHC class and TCR in cancer immunology is greater than expected when compared with previous studies (Slansky et al., 2000 2000 ; Xue et al., 2013 2013 ). The results in this study showed the imporatance of TEX in down-regulating the population of CD3 + , CD8 + , NK (CD56 + ), and CD3 + CD56 + cells in the TEX-CIK group when compared with the N-CIK group ( P < 0.01). The concentrations of TNF-α and perforin in the group TEX-CIK were lower than those in the group N-CIK ( P < 0.05). The down-regulation of CD3 and CD8 may attenuate the ability of antigen in presenting, thus meditating the tumor immune escape. Taylor et al. ( 2003 2003 ) suggested that FasL associated with TEXs was responsible for apoptosis of the T lymphocytes and a decrease of CD3 + cells in patients with ovarian carcinoma. Wieckowski et al. ( 2009 2009 ) showed that tumor-derived microvesicles induce apoptosis in CD8 + T lymphocytes. The subset of the CD3 + CD56 + cells, which expresses both the T-cell marker CD3 and the natural killer cell marker CD56, is termed non-MHC-restricted T cells. These cells are capable of killing both autologous and allogeneic tumor targets, and show the greatest cytotoxicity (Schmidt-Wolf et al., 1993 1993 ). CD3 + CD56 + lymphocytes play a critical role in the antitumor process by secreting TNF-α and perforin. TNF-α is a cytokine able to induce hemorrhagic necrosis of tumors and is regarded as a promising approach for cancer therapeutics (Carswell et al., 1975 1975 ). TNF-α is able to induce fever, apoptotic cell death, cachexia, and inflammation, and to inhibit tumorigenesis and viral replication. Perforin is a cytolytic mediator produced by killer lymphocytes. The protein is partially homologous to the terminal components of the membrane attack complex, which complements and produces pores of up to 20 nm in diameter in the target membranes (Liu et al., 1995 1995 ). Upon degranulation, perforin inserts itself into the target cell’s plasma membrane, forming pores (Tschopp et al., 1986 1986 ). This protein creates transmembrane tubules and is capable of lysing non-specifically a variety of target cells. This protein is one of the main cytolytic proteins of cytolytic granules, and it is also known to be a key effector molecule for T-cell- and natural killer-cell-mediated cytolysis. The data above showed that TEX down-regulated the population of CD3 + , CD8 + , NK (CD56 + ), and CD3 + CD56 + cells, especially the population of CD3 + CD56 + cells, resulting in the secretion decreases of TNF-α and perforin, thus the antitumor ability of the TEX-CIK group was lower than that of the N-CIK group. An increasing number of literature show that transforming growth factor-β (TGF-β), Wnt signaling components, phosphatase and tensin homolog (PTEN), epidermal growth factor receptor (EGFR), and Fas ligand may play important roles in exosome-meditated tumor immunosuppression (Apte et al., 2013 2013 ). Thus, the further mechanism underlying the depletion of CD3 + CD56 + CIK cells upon exosome treatment needs additional research. 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Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes Compliance with ethics guidelines Jiong-huang CHEN, Jian-yang XIANG, Guo-ping DING, and Li-ping CAO declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. All participants or their guardians gave written consent for their blood samples to be used for scientific research. The research protocol was reviewed and approved by the Research Ethics Committee of Zhejiang University, Hangzhou, China. 10.1631/jzus.B1500266 Project supported by the National Natural Science Foundation of China (No. 81272671) and the Foundation of Health and Family Planning Commission of Zhejiang Province (No. 2015KYB218), China 2015-11-04 2016-06-18 2016-01-22 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2016 2016 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 07 17