Metformin Inducing the Change of Functional and Exhausted Phenotypic Tumor-Infiltrated Lymphocytes and the Correlation with JNK Signal Pathway in Triple-Negative Breast Cancer

Ruibin Wang,1 Yuchen Li,2 Yanjie Zhao,3 Feng Shi,4 Quan Zhou,5 Jiangping Wu,6 Shuzhen Lyu,7 Qingkun Song8,9 1Department of Emergency, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of China; 2Cell and Molecular Biology, Sid Faithfull Brain Cancer Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia; 3Department of Medical Oncology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of China; 4Department of Pathology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of China; 5Department of Pathology, Cancer Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, People’s Republic of China; 6Department of Cancer Research, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of China; 7Department of Breast Surgery, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of China; 8Department of Clinical Epidemiology, Beijing Youan Hospital, Capital Medical University, Beijing, People’s Republic of China; 9Department of Clinical Epidemiology, Beijing Shijitan Hospital, Capital Medical University, Beijing, People’s Republic of ChinaCorrespondence: Qingkun Song, Department of Clinical Epidemiology, Beijing Youan Hospital, Capital Medical University, Xitoutiao 8, Fengtai Dist, Beijing, People’s Republic of China, Email songqingkun@aliyun.comBackground: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Metformin has been shown to have the potential to inhibit the proliferation of malignant cells. This study aimed to investigate the regulatory effect of metformin on phenotypic tumor-infiltrated lymphocytes (TILs) and mechanisms in TNBC.Methods: Microarray analysis was performed on 4T1 cells post metformin treatment. BALB/c mice were inoculated with 4T1 cells with knockdown/overexpression of C-Jun N-terminal kinase (JNK), and administered with metformin. Phenotypic TILs in the tumor microenvironment (TME) were visualized by immunofluorescence staining.Results: Metformin inhibited 4T1 cell proliferation and increased expression of JNK by 21% in vitro. In vivo, Metformin increased cell counts of CD4+ and CD8+TILs by 100% and 85%, respectively, and the increase of TILs was associated with JNK pathway. Cell counts of CD4+/PD-1+ and CD8+/PD-1+TILs were reduced by 64% and 58%, respectively, post metformin treatment, but the reduction of exhausted TILs was not associated with JNK pathway. Metformin induced a 11% and 20% reduction of IL-6 and TNF-α level in the TNBC model.Conclusion: Our study demonstrated that metformin increased the functional phenotype of TILs and associated with JNK pathway, and suppressed the exhausted phenotype of TILs independently to JNK pathway in TNBC microenvironment. Further studies are needed to explore the basic mechanism of action of the drug. Metformin has potentially enhanced efficacy when used in combination with immunotherapy against TNBC.Keywords: triple-negative breast cancer, metformin, phenotypic tumor-infiltrated lymphocytes, C-Jun N-terminal kinase