Ming Wang1, Zhongyu Qin2, Jiajia Wan3, Yan Yan3, Xixi Duan3, Xiaohan Yao3, Ziming Jiang4, Wenqing Li3, Zhihai Qin5,6. 1. Medical Research Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou, 450052, Henan, China. wangmingheda@163.com. 2. Changzhi Medical College, Changzhi, 046000, Shanxi, China. 3. Medical Research Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou, 450052, Henan, China. 4. Department of Urology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China. 5. Medical Research Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou, 450052, Henan, China. zhihai@ibp.ac.cn. 6. Academy of Medical Sciences, Zhengzhou University, Zhengzhou, 450052, Henan, China. zhihai@ibp.ac.cn.
Abstract
BACKGROUND: Since the lung is one of the most common sites for cancer metastasis, it could provide a suitable microenvironment for pre-metastatic niche (PMN) formation to facilitate tumor cell colonization. Regulatory T cells (Tregs) are an immunosuppressive cell type found ubiquitously in tumors and may play a crucial role in PNM formation. In this study, we investigated tumor-derived exosome (TDE)-induced Treg differentiation in the lung PMN as well as the underlying mechanisms. METHODS: TDEs were isolated from the Lewis lung carcinoma cell line (LLC-exo) and their effects on mouse pulmonary fibroblasts was investigated in vitro as well as on lung tumor formation and metastasis in a pre-injected mouse model. Immune cell populations in the lung were analyzed by flow cytometry. Expression of CCL1 and CCR8 was evaluated by immunofluorescence staining, qRT-PCR and Western blot analyses. Cytokine expression was measured using mouse cytokine arrays and ELISA. RESULTS: The number of CD4+ FoxP3+ Tregs was significantly increased in lungs in a LLC-exo pre-injected mouse model. Lung fibroblasts secreted increased amounts of CCL1 after co-culture with LLC-exo, which induced Treg differentiation by activating its specific receptor CCR8, ultimately contributing to the establishment of an immunologically tolerant PMN. Moreover, inhibiting the release of LLC-exo by GW4869, or blocking the CCL1-CCR8 axis using AZ084, suppressed Tregs differentiation and tumor metastasis in the lung. CONCLUSIONS: Collectively, our study provides a novel mechanism by which Tregs are activated to form an immunologically tolerant PMN and demonstrates a critical link among lung fibroblasts, Tregs and metastatic tumor cells.
BACKGROUND: Since the lung is one of the most common sites for cancer metastasis, it could provide a suitable microenvironment for pre-metastatic niche (PMN) formation to facilitate tumor cell colonization. Regulatory T cells (Tregs) are an immunosuppressive cell type found ubiquitously in tumors and may play a crucial role in PNM formation. In this study, we investigated tumor-derived exosome (TDE)-induced Treg differentiation in the lung PMN as well as the underlying mechanisms. METHODS: TDEs were isolated from the Lewis lung carcinoma cell line (LLC-exo) and their effects on mouse pulmonary fibroblasts was investigated in vitro as well as on lung tumor formation and metastasis in a pre-injected mouse model. Immune cell populations in the lung were analyzed by flow cytometry. Expression of CCL1 and CCR8 was evaluated by immunofluorescence staining, qRT-PCR and Western blot analyses. Cytokine expression was measured using mouse cytokine arrays and ELISA. RESULTS: The number of CD4+ FoxP3+ Tregs was significantly increased in lungs in a LLC-exo pre-injected mouse model. Lung fibroblasts secreted increased amounts of CCL1 after co-culture with LLC-exo, which induced Treg differentiation by activating its specific receptor CCR8, ultimately contributing to the establishment of an immunologically tolerant PMN. Moreover, inhibiting the release of LLC-exo by GW4869, or blocking the CCL1-CCR8 axis using AZ084, suppressed Tregs differentiation and tumor metastasis in the lung. CONCLUSIONS: Collectively, our study provides a novel mechanism by which Tregs are activated to form an immunologically tolerant PMN and demonstrates a critical link among lung fibroblasts, Tregs and metastatic tumor cells.
Authors: Dillon S Richardson; Jonathan M Spehar; David T Han; Prathik A Chakravarthy; Steven T Sizemore Journal: Cells Date: 2022-05-14 Impact factor: 7.666