HEPATOLOGY Potential synergistic anti-tumor activity between lenalidomide and sorafenib in hepatocellular carcinoma Da-Liang Ou,* 1 Chun-Jung Chang,* 1 Yung-Ming Jeng, † Yi-Jang Lin, ‡ Zhong-Zhe Lin, ‡ Anita K Gandhi, § Sheng-Chieh Liao, ‡ Zi-Ming Huang, ‡ Chiun Hsu* ,‡,¶ and Ann-Lii Cheng* ,‡,¶ *Graduate Institute of Oncology, National Taiwan University College of Medicine, and Departments of † Pathology, ‡ Oncology and ¶ Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan; and § Department of Translational Development, Celgene Corporation, Summit, New Jersey, USA Key words immune modulatory, lenalidomide, sorafenib, tumor-infiltrating lymphocytes, vascular normalization. Accepted for publication 11 May 2014. Correspondence Professor Ann-Lii Cheng and Dr Chiun Hsu, Department of Oncology, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei 100, Taiwan. Email: alcheng@ntu.edu.tw; hsuchiun@gmail.com 1 These authors contribute equally to this work. Financial disclosure: Dr Ann-Lii Cheng is a consultant for Sanofi-Aventis Inc, Pfizer, Bayer Schering Pharma, Bristol-Myers Squibb (Taiwan) Ltd, Boehringer Ingelheim Taiwan Limited, and Novartis Inc. Dr Chiun Hsu received a research grant from Celgene Corporation. Dr Anita K Gandhi is an employee of Celgene Corporation. Other authors have no relevant financial interests related to this article. Financial support: This study was supported by grants NSC 100–2321-B-002 -053, NSC 101–2321-B-002 -014, NSC 102–2314-B-002 -142 -MY3, NSC 102–2325-B-002–038, and NSC 102–2321-B-002 -008 from National Science Council, Taiwan. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract Background and Aim: The immune modulatory drug lenalidomide has shown promising anti-tumor activity in a clinical trial of patients with advanced hepatocellular carcinoma (HCC). The present study explored whether lenalidomide can enhance the anti-tumor activity of sorafenib, the standard molecular targeted therapy for HCC. Methods: The anti-tumor efficacy of single-agent or combination treatment was measured by change in tumor volume and animal survival using an orthotopic liver cancer model. Distribution of T-cell subpopulations in tumor-infiltrating lymphocytes (TILs) and splenocytes derived from tumor-implanted mice was measured by flow cytometry. Deple- tion of relevant T-cell subpopulations or cytokines was done by co-administration of relevant antibodies with study drug treatment. Tumor cell apoptosis and tumor angiogen- esis were measured by transferase deoxytidyl uridine end labeling assay and immunohis- tochemical study, respectively. Results: Combination of sorafenib and lenalidomide produced significant synergistic anti-tumor efficacy in terms of tumor growth delay and animal survival. This synergistic effect was associated with a significant increase in interferon-γ expressing CD8 + lympho- cytes in TILs and a significantly higher number of granzyme- or perforin-expressing CD8 + T cells, compared with vehicle- or single-agent treatment groups. Combination treatment significantly increased apoptotic tumor cells and vascular normalization in tumor tissue. The synergistic anti-tumor effect was abolished after CD8 depletion. Conclusions: Lenalidomide can enhance the anti-tumor effects of sorafenib in HCC through its immune modulatory effects, and CD8 + TILs play an important role in the anti-tumor synergism. Authors’ Contributions Conception and design: D-L Ou, C-J Chang, C Hsu, A-L Cheng Development of methodology: D-L Ou, C-J Chang, Y-J Lin, S-C Liao, Z-M Huang Acquisition of data (participated in study discussion, provided facilities, etc.): D-L Ou, C-J Chang, Y-M Jeng, Z-Z Lin, AK Gandhi, C Hsu Writing, review, and/or revision of the manuscript: C Hsu, A-L Cheng Administrative, technical, or material support (i.e. reporting or organizing data, constructing databases): D-L Ou, C-J Chang, Y-M Jeng, Z-Z Lin, AK Gandhi Study supervision: C Hsu, A-L Cheng doi:10.1111/jgh.12708 2021 Journal of Gastroenterology and Hepatology 29 (2014) 2021–2031 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd