Xiang Li1,2, Shaomin Wang1, Wei Mu3, Jennifer Barry1, Anna Han4, Richard L Carpenter5, Bing-Hua Jiang1, Stephen C Peiper1, Mỹ G Mahoney6, Andrew E Aplin4, Hong Ren7, Jun He8. 1. Department of Pathology, Anatomy & Cell Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, USA. 2. Department of Otorhinolaryngology-Head and Neck Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, P. R. China. 3. School of Public Health, Shanghai Jiaotong University School of Medicine, Shanghai, China. 4. Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, USA. 5. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Bloomington, IN, 47405, USA. 6. Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, USA. 7. Department of Thoracic Surgery and Oncology, Cancer Center, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, P. R. China. renhong@xjtu.edu.cn. 8. Department of Pathology, Anatomy & Cell Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, USA. Jun.he@jefferson.edu.
Abstract
BACKGROUND: Cancer cells have an imbalance in oxidation-reduction (redox) homeostasis. Understanding the precise mechanisms and the impact of the altered redox microenvironment on the immunologic reaction to tumors is limited. METHODS: We isolated exosomes from ovarian cancer cells through ultracentrifuge and characterized by Western-blots and Nanoparticle Tracking Analysis. 2D, 3D-coculture tumor model, and 3D live cell imaging were used to study the interactions between tumor cells, macrophages and CD3 T cells in vitro. The role of exosomal miR-155-5p in tumor growth was evaluated in xenograft nude mice models and immune-competent mice models. Flow cytometry and flow sorting were used to determine the expression levels of miR-155-5p and PD-L1 in ascites and splenic macrophages, and the percentages of CD3 T cells subpopulations. RESULTS: The elevation of reactive oxygen species (ROS) greatly downregulated exosomal miR-155-5p expression in tumor cells. Neutralization of ROS with N-acetyl-L-cysteine (NAC) increased the levels of miR-155-5p in tumor exosomes that were taken up by macrophages, leading to reduction of macrophage migration and tumor spheroid infiltration. We further found that programmed death ligand 1 (PD-L1) is a functional target of miR-155-5p. Co-culture of macrophages pre-treated with NAC-derived tumor exosomes or exosomal miR-155-5p with T-lymphocytes leading to an increased percentage of CD8+ T-lymphocyte and a decreased CD3+ T cell apoptosis through PD-L1 downregulation. Tumor growth in nude mice was delayed by treatment with NAC-derived tumor exosomes. Delivery of tumor exo-miR-155-5p in immune-intact mice suppressed ovarian cancer progression and macrophage infiltration, and activated CD8+ T cell function. It is of note that exo-miR-155-5p inhibited tumor growth more potently than the PD-L1 antibody, suggesting that in addition to PD-L1, other pathways may also be targeted by this approach. CONCLUSIONS: Our findings demonstrate a novel mechanism, ROS-induced down-regulation of miR-155-5p, by which tumors modulate the microenvironment that favors tumor growth. Understanding of the negative impact of ROS on the tumor immune response will improve current therapeutic strategies. Targeting miR-155-5p can be an alternative approach to prevent formation of an immunosuppressive TME through downregulation of PD-L1 and other immunosuppressive factors.
BACKGROUND: Cancer cells have an imbalance in oxidation-reduction (redox) homeostasis. Understanding the precise mechanisms and the impact of the altered redox microenvironment on the immunologic reaction to tumors is limited. METHODS: We isolated exosomes from ovarian cancer cells through ultracentrifuge and characterized by Western-blots and Nanoparticle Tracking Analysis. 2D, 3D-coculture tumor model, and 3D live cell imaging were used to study the interactions between tumor cells, macrophages and CD3 T cells in vitro. The role of exosomal miR-155-5p in tumor growth was evaluated in xenograft nude mice models and immune-competent mice models. Flow cytometry and flow sorting were used to determine the expression levels of miR-155-5p and PD-L1 in ascites and splenic macrophages, and the percentages of CD3 T cells subpopulations. RESULTS: The elevation of reactive oxygen species (ROS) greatly downregulated exosomal miR-155-5p expression in tumor cells. Neutralization of ROS with N-acetyl-L-cysteine (NAC) increased the levels of miR-155-5p in tumor exosomes that were taken up by macrophages, leading to reduction of macrophage migration and tumor spheroid infiltration. We further found that programmed death ligand 1 (PD-L1) is a functional target of miR-155-5p. Co-culture of macrophages pre-treated with NAC-derived tumor exosomes or exosomal miR-155-5p with T-lymphocytes leading to an increased percentage of CD8+ T-lymphocyte and a decreased CD3+ T cell apoptosis through PD-L1 downregulation. Tumor growth in nude mice was delayed by treatment with NAC-derived tumor exosomes. Delivery of tumor exo-miR-155-5p in immune-intact mice suppressed ovarian cancer progression and macrophage infiltration, and activated CD8+ T cell function. It is of note that exo-miR-155-5p inhibited tumor growth more potently than the PD-L1 antibody, suggesting that in addition to PD-L1, other pathways may also be targeted by this approach. CONCLUSIONS: Our findings demonstrate a novel mechanism, ROS-induced down-regulation of miR-155-5p, by which tumors modulate the microenvironment that favors tumor growth. Understanding of the negative impact of ROS on the tumor immune response will improve current therapeutic strategies. Targeting miR-155-5p can be an alternative approach to prevent formation of an immunosuppressive TME through downregulation of PD-L1 and other immunosuppressive factors.