Blocking TLR2 Activity Attenuates Pulmonary Metastases of Tumor

Blocking TLR2 Activity Attenuates Pulmonary Metastases of Tumor Hong-Zhen Yang1., Bing Cui1., Han-Zhi Liu1, Su Mi1, Jun Yan1, Hui-Min Yan1, Fang Hua1, Heng Lin1, Wen-Feng Cai1, Wen-Jie Xie1, Xiao-Xi Lv1, Xiao-Xing Wang1, Bing-Mu Xin1, Qi-Min Zhan2, Zhuo-Wei Hu1,2* 1 Molecular Immunology and Pharmacology Laboratory, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China, 2 State Key Laboratory of Molecular Oncology, Cancer Institute and Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Abstract Background: Metastasis is the most pivotal cause of mortality in cancer patients. Immune tolerance plays a crucial role in tumor progression and metastasis. Methods and Findings: In this study, we investigated the potential roles and mechanisms of TLR2 signaling on tumor metastasis in a mouse model of intravenously injected B16 melanoma cells. Multiple subtypes of TLRs were expressed on B16 cells and several human cancer cell lines; TLR2 mediated the invasive activity of these cells. High metastatic B16 cells released more heat shock protein 60 than poor metastatic B16-F1 cells. Importantly, heat shock protein 60 released by tumor cells caused a persistent activation of TLR2 and was critical in the constitutive activation of transcription factor Stat3, leading to the release of immunosuppressive cytokines and chemokines. Moreover, targeting TLR2 markedly reduced pulmonary metastases and increased the survival of B16-bearing mice by reversing B16 cells induced immunosuppressive microenvironment and restoring tumor-killing cells such as CD8+ T cells and M1 macrophages. Combining an anti-TLR2 antibody and a cytotoxic agent, gemcitabine, provided a further improvement in the survival of tumor-bearing mice. Conclusions and Significance: Our results demonstrate that TLR2 is an attractive target against metastasis and that targeting immunosuppressive microenvironment using anti-TLR2 antibody is a novel therapeutic strategy for combating a life-threatening metastasis. Citation: Yang H-Z, Cui B, Liu H-Z, Mi S, Yan J, et al. (2009) Blocking TLR2 Activity Attenuates Pulmonary Metastases of Tumor. PLoS ONE 4(8): e6520. doi:10.1371/ journal.pone.0006520 Editor: Jacques Zimmer, Centre de Recherche Public de la Santé (CRP-Santé), Luxembourg Received June 19, 2009; Accepted July 8, 2009; Published August 5, 2009 Copyright: ß 2009 Yang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants from the National Major Basic Research Program of China (2006CB503808) and from National Natural Science Foundation (30672468). Dr. ZW Hu is also supported by Cheung Kong Scholars Programme of the Ministry of Education and by a Foundation of the Ministry of Personnel of China for Returned Oversea Chinese Senior Scholars. Dr. HZ Yang is supported by a Grant from Basic Research Program of Institute of Materia Medica (2006QN32). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: . These authors contributed equally to this work. microenvironment are responsible for the fail of tumor immunosurveillance, leading to tumor progression and metastasis [5]. Although the mechanism is still to be elucidated, transcription factor Stat3 is constitutively activated at a very high level in cancer cell lines and tumors [6]. The constitutively-activated Stat3 (cStat3) mediates a crosstalk between tumor cells and host immune cells to direct immunosuppressive response in tumor tissues. Blocking Stat3 in highly metastatic melanoma cells suppresses the invasion of the cells and lung metastases; enforcing the expression of cStat3 converts poorly metastatic melanoma cells into highly metastatic cells [7]. Thus, Stat3 provides a link between oncogenesis and immunosuppression and plays a central role in the establishment of immunosuppressive environment and the progression of tumors [8]. TLRs have recently emerged as critical components of the immune system to sense danger signals, initiate innate immune response, and direct the polarization of adaptive immune Introduction Tumor metastases are the most common cause of death in cancer patients but there are no effective therapies to target the development and progression of metastases [1]. Recent evidence highlights that tumor metastasis requires collaborative interactions between tumor cells and immune cells at both primary and secondary tumor locations [2]. Actually, immune response functions as a double-edged sword: it is recognized as the surveillance component for the host to eliminate tumor cells and attenuate the development of metastases on one hand; it is also accepted as the mediator promoting primary tumor metastasis on the other hand [3]. A large number of infiltrating immune cells, such as regulatory T cells (Tregs), tumor-related macrophages, and mast cells, contribute to cancer growth, angiogenesis, metastasis, and tumor immune tolerance [4]. Indeed, an accumulation of suppressive factors and a reduction of tumoricidal factors in the tumor PLoS ONE | www.plosone.org 1 August 2009 | Volume 4 | Issue 8 | e6520 TLR2 and Tumor Metastasis receptor CCR8 (Fig. S2, Method S2). TLR2 activity mediated HSP60-stimulated release of the suppressive cytokines, chemokines, and the expression of CCR8 in B16 cells (Fig. S3). responses [9]. TLRs induce an acute inflammation against various pathogens by recognizing highly conserved pathogen-associated molecular patterns (PAMPs) in the pathogens. Also, TLRs induce chronic inflammation or immune tolerance after being activated by damage-associated molecular patterns (DAMPs) such as HMGB1, HSPs, and S100 proteins released from injured tissue or tumor tissue [10,11]. In general, stimulation of TLR3, 4, 7, 8, or 9 induces a TH1 or TH17 type of immune response; whereas stimulation of TLR2 induces a TH2, Treg, or TH17 type of immune response [12,13]. Obviously, TLR2 has an unique position in the regulation of tumor tolerance, cancer progression and metastasis [3]. Indeed, we recently find that targeting TLR2 reverses pulmonary fibrosis through regulating immunosuppressive microenvironment [14]. We therefore wonder if TLR2 signaling has a role in the regulation of tumor metastasis. We found that the basal activity of TLR2 determines the evasive capacity and the level of immunosuppressive factors in tumor cells. Targeting TLR2 significantly attenuated pulmonary metastases of B16 melanoma and animal death. Our study provides strong evidence to directly demonstrate that targeting TLR2 to overcome tumor cell-induced immunosuppressive microenviron (...truncated)