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  • Research Article

    Tumor-Associated Monocytes/Macrophages Impair NK-Cell Function via TGFb1 in Human Gastric Cancer Liu-sheng Peng1, Jin-yu Zhang1, Yong-sheng Teng1, Yong-liang Zhao2, Ting-ting Wang1, Fang-yuan Mao1, Yi-pin Lv1, Ping Cheng1,Wen-hua Li1, Na Chen1, Mubing Duan3, Weisan Chen3, Gang Guo1, Quan-ming Zou1, and Yuan Zhuang1


    Natural killer (NK) cells are a major component of the host antitumor immune response in human cancer. However, the nature, functional regulation, and clinical relevance of NK cells in gastric cancer remain largely unknown. In this study, we showed that the percentages of NK cells in tumors were significantly decreased, and low percentages of tumor-infil- trating NK cells were positively correlated with poor survival and disease progression. Although the expression of activating and inhibitory receptors on NK cells was shown to be not different between tumor and nontumor tissues, NK cells in tumors had impaired effector functions, characterized by decreased IFNg , TNFa, and Ki-67 expression. We found that

    tumor-infiltrating monocytes/macrophages were physically close to NK cells, and their percentages negatively correlated with IFNgþ and TNFaþ NK-cell percentages. Ex vivo study showed that isolated tumor-associated monocytes/macro- phages could impair NK-cell expression of IFNg , TNFa, and Ki-67. Blockade of TGFb1 attenuated such monocytes/macro- phages-mediated impairment of NK-cell function. Our data suggest that human NK-cell function was impaired by tumor- associated monocytes/macrophages, and that restoring NK- cell function may be an important therapeutic strategy to prevent tumor immune escape in gastric cancer. Cancer Immunol Res; 5(3); 248–56. �2017 AACR.

    Introduction Gastric cancer is one of themost commonhumanmalignancies

    and accounts for approximately 900,000 total cases and 700,000 deaths globally per annum (1). The prognosis of gastric cancer patients with early-stage disease is generally good, but the control of advanced-stage gastric cancer remains challenging. It is gener- ally accepted that the development and progression of gastric cancer is influenced by tumor and host immune system cross-talk, with many studies supporting a central role for adaptive immu- nity in determining the clinical outcomes of gastric cancer patients (2–4). However, less is known about the role of innate immunity

    and innate immune cell cross-regulation during gastric cancer progression.

    Natural killer (NK) cells are a group of innate lymphoid cells that confer antitumor as well as antiviral host protection. This process is mediated by cell surface recognition of altered or cancerous cells compared with normal host cells, through their differential engagement of NK-inhibitory or -activating receptors (5). Once activated, NK cells secrete cytotoxic enzymes and cytokines to kill their target cells. However, many studies focusing on the role of NK cells in cancer have shown that the function of NK cells is impaired within the tumor microenvironment (6–8). In gastric cancer, impairment of the cytotoxic function of circu- lating NK cells has also been reported to be associated with advanced disease progression (9), suggesting that the progression of gastric cancer may be linked to defects in antitumor NK-cell effector function. However, the phenotype, functional regulation, and clinical relevance of NK cells within the human gastric cancer microenvironment remain largely unknown.

    Monocytes/macrophages are another important group of innate immune cells that reside within the tumor milieu and modulate local antitumor immunity (10). Monocytes/macro- phages can be reprogrammed by tumor-derived signals to express inhibitory molecules and/or release soluble inhibitory factors, which in turn suppress cytotoxic T-cell activation and effector function (11, 12). Monocytes/macrophages isolated from tumors can also induce NK-cell dysfunction during hepatocellular carci- noma (13), suggesting a role for tumor-associated monocytes/ macrophages in impairing antitumor NK-cell function during tumor progression. However, the presence of interactions between tumor-associatedmonocytes/macrophages andNKcells,

    1National Engineering Research Center of Immunological Products, Department of Microbiology and Biochemical Pharmacy, College of Pharmacy, Third Military Medical University, Chongqing, PR China. 2Department of General Surgery and Center of Minimal Invasive Gastrointestinal Surgery, Southwest Hospital, Third Military Medical University, Chongqing, PR China. 3La Trobe Institute for Molec- ular Science, La Trobe University, Bundoora, Victoria, Australia.

    L.-s. Peng and J.-y. Zhang contributed equally to this article.

    Note: Supplementary data for this article are available at Cancer Immunology Research Online (http://cancerimmunolres.aacrjournals.org/).

    Corresponding Authors: Yuan Zhuang, Third Military Medical University, No.30 Gaotanyan stree, Chongqing 400038, PR China. Phone: 086-023-68752377; Fax: 086-023- 68752377; E-mail: yuanzhuang1983@yahoo.com; or Quan-ming Zou, qmzou@tmmu.edu.cn

    doi: 10.1158/2326-6066.CIR-16-0152

    �2017 American Association for Cancer Research.

    Cancer Immunology Research

    Cancer Immunol Res; 5(3) March 2017248

    on April 24, 2020. © 2017 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

    Published OnlineFirst February 1, 2017; DOI: 10.1158/2326-6066.CIR-16-0152


  • as well as their precise mechanisms of communication in gastric cancer, remains unknown.

    In the present study, we showed that the percentages of tumor- infiltrating NK cells were significantly decreased, and low percen- tages of these cells were positively correlated with tumor progres- sion and poor overall survival of gastric cancer patients. Mean- while, tumor-infiltrating NK cells displayed functional impairment, and such impaired functions were mediated by tumor-associated monocytes/macrophages-derived TGFb1 pro- duction. Restoring NK-cell function may be an important thera- peutic strategy to prevent tumor immune escape in gastric cancer.

    Materials and Methods Patients and tissue samples

    Fresh peripheral blood, autologous nontumor, and tumor gastric tissues were obtained from gastric cancer patients during surgery at the Southwest Hospital of the Third Military Medical University. None of the patients had received chemotherapy or radiotherapy before sampling, and individuals with autoimmune disease, infectious diseases, or multi-primary cancer were exclud- ed. The clinical stages of tumors were determined according to the tumor–node–metastasis (TNM) classification systemby the Inter- national Union against Cancer (Edition 7). The study was approved by the Ethics Committee of the Third Military Medical University, andpriorwritten informed consentwasobtained from each patient. The clinical characteristics of gastric cancer patients were presented in Supplementary Table S1.

    Cell isolation Fresh nontumor and tumor tissues were used for the isola-

    tion of tissue-infiltrating lymphocytes as previously described (14). In brief, paired nontumor and tumor tissues were cut into small pieces, then suspended in RPMI 1640 containing 1 mg/mL collagenase IV (Sigma-Aldrich) and 10 mg/mL DNase I (Roche), and gently mechanically dissociated using the MACS Dissociator (Miltenyi Biotec). Dissociated cell suspensions were further incubated for 1 hour at 37�C under continuous rotation and then filtered through 70 mm cell strainers to obtain single-cell suspensions. Cell suspensions were then used for flow cytometry analysis.

    Flow cytometric analysis Cell suspensions were stained with appropriate surface anti-

    bodies, and then fixed, permeabilized for 20 minutes using Cytofix/Cytoperm (BD Biosciences). Permeabilized cells were subsequently stained with antibodies against intracellular mole- cules. For intracellular staining of IFNg and TNFa, cells were stimulated for 4 hours with phorbol myristate acetate (PMA, 50 ng/mL) and ionomycin (1 mg/mL) in the presence of Golgistop (BD Biosciences) before staining. The fluorochrome-labeled anti- bodies are listed in Supplementary Table S2.

    Immunohistochemistry Paraffin-embedded samples were cut into 5-mm sections. After

    being deparaffinized and hydrated, sections immersed in Tris- EDTA buffer (pH 9.0) were subjected to heat-induced antigen retrieval in a microwave oven and then treated with 3% hydrogen peroxide for endogenous peroxide quenching. Following incu- bation with mouse monoclonal anti-human CD57 (NK-1) anti- body (Zhongshan Golden Bridge Biotech.) or TGFb1 antibody

    (Abcam) overnight at 4�C, slides were incubatedwith horseradish peroxidase–conjugated secondary anti-mouse antibody (Zhong- shan Golden Bridge Biotech.) and then developed with 3,3'- diaminobenzidine tetrahydrochloride. For colocalization analy- sis, after CD57 staining, slides were incubated with a mouse anti- human CD68 antibody (DAKO) and stained using EnVision G2 System/AP Rabbit/Mouse (Permanent Red; DAKO). Slides were examined using a microscope (Nikon Eclipse 80i; Nikon).

    In vitro coculture of tissue-infiltratingmonocytes/macrophages and NK cells

    Cell suspensions derived from paired nontumor and tumor tissues were stained by PE-Cy7 anti-human CD45 and PerCP- Cy5.5 anti-human CD14 antibodies, and CD45þCD14þ mono- cytes/macrophages were then purified by fluorescence-activated cell sorting (FACSAria II; BD Biosciences). Peripheral blood NK cells were purified using the humanNK-cell enrichment Kit (Stem Cell Technologies, Inc.). The purity of monocytes/macrophages and NK cells was >90%. Purified tissue-associated monocytes/ macr


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