Potential synergistic anti-tumor activity between lenalidomide and sorafenib in hepatocellular carcinoma.

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doi:10.1111/jgh.12708

H E PAT O L O G Y

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: [email protected]; [email protected] 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. Depletion of relevant T-cell subpopulations or cytokines was done by co-administration of relevant antibodies with study drug treatment. Tumor cell apoptosis and tumor angiogenesis were measured by transferase deoxytidyl uridine end labeling assay and immunohistochemical 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+ lymphocytes 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

Journal of Gastroenterology and Hepatology 29 (2014) 2021–2031 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

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Lenalidomide plus sorafenib in HCC

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Introduction

Methods and material

The multikinase inhibitor sorafenib is the current standard therapy for patients with advanced hepatocellular carcinoma (HCC).1 However, resistance to sorafenib treatment is a tremendous challenge, as evidenced by the low objective response rate (2–3%) and the modest survival benefit (prolongation of median survival time by 2 to 3 months, compared with placebo) with sorafenib treatment.2,3 Mechanisms of sorafenib resistance in HCC have been extensively studied to develop combination strategies for better treatment efficacy.4 Resistance of HCC to sorafenib treatment may involve aberrations in cellular signaling of cancer cells per se and in the tumor microenvironment.4 While most previous studies focused on aberrations of cancer cells per se, it is increasingly recognized that the immune modulatory effects of sorafenib and other molecular targeted agents may contribute to drug resistance.5 Sorafenib was found to have immunosuppressive effects by inhibiting T-cell activation, natural killer (NK) cell activity, and dendritic cell maturation.6–9 We hypothesized that these immunosuppressive effects of sorafenib may contribute to sorafenib resistance in HCC, and combination of sorafenib with immune modulatory agents that can enhance the host immune function may further improve the therapeutic efficacy of sorafenib. Lenalidomide, an immune modulatory derivative of thalidomide, has been approved for the treatment of multiple myeloma and myelodysplastic syndrome.10 Lenalidomide can stimulate T cells that have been partially activated through the T-cell receptor, increase interleukin (IL)-2 and interferon-γ (IFN-γ) production, and enhance the cytotoxic effects of NK cells and NK T cells. Lenalidomide can also inhibit tumor angiogenesis, which may help reverse the immune suppression induced by tumor angiogenesis.11,12 Preliminary data from a phase II trial of advanced HCC patients who had tumor progression on or intolerance with sorafenib treatment suggested promising efficacy of lenalidomide.13 In that study, 6 of the 37 patients had radiographic tumor response and 9 of the 32 patients who had elevated alphafetoprotein (AFP) levels at baseline had > 50% decrease in AFP levels after lenalidomide. In this study, we tested the hypothesis that combination of sorafenib with lenalidomide can improve the therapeutic efficacy of sorafenib. The efficacy and safety of the combination therapy were tested by using an orthotopic, immune-competent liver cancer model. The immune effectors contributing to the potential anti-tumor synergy between sorafenib and lenalidomide were explored.

Cell lines and animals. The cell lines used for in vitro experiments included human HCC cell lines PLC5, Hep3B (from American Type Culture Collection [ATCC]), and Huh-7 (from Health Science Research Resources Bank, Japan), the murine liver cancer cell line BNL-1MEA (from ATCC) and the murine breast cancer cell line 4T1 (from ATCC). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 μg/mL). Primary human umbilical venous endothelial cells (HUVEC) were cultured as previously described.14 The cells were maintained in a humidified incubator under 5% CO2 at 37°C. The orthotopic HCC model was established by injecting about 2 × 105 BNL-1MEA cells into the subcapsular area of the left liver lobe of male BALB/c or SCID mice at age of 6–7 weeks as previously described.15 The protocol for the animal experiments in this study was approved by the Institutional Animal Care and Use Committee of the College of Medicine, National Taiwan University, and conformed to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health.

Drug treatment and efficacy/safety evaluation. In in vitro studies, cell viability was measured by MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay and the extent of apoptosis was measured by flow cytometry (sub-G1 fraction analysis) after 72 h of drug treatment. In in vivo studies, mice were randomized to different treatment groups on day 6 after tumor implantation. Sorafenib (Bayer Schering Pharma, Berlin, Germany) was given daily by gavage and lenalidomide (Celgene, Summit, NJ, USA) was given daily by intra-peritoneal injection. Tumor volumes were measured after 22 days of treatment by using the following formula: volume (mm3) = (width)2 × length × 0.5. At the end of treatment, blood samples were collected to measure hemogram and biochemistry, and formalin-fixed, paraffinembedded tumor samples were collected for analysis of tumor apoptosis (transferase deoxytidyl uridine end labeling [TUNEL] assay) and tumor angiogenesis (immunohistochemical (IHC) staining). The change in tumor vasculature after drug treatment was also measured by the vascular normalization index (VNI) using the following formula:16

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Figure 1 Lenalidomide and sorafenib had synergistic anti-tumor efficacy in vivo but not in vitro. (a) Lenalidomide did not impair cell viability significantly of the hepatocellular carcinoma (HCC) cell lines (Hepa3B, BNL-1MEA, PLC5, and Huh-7) and human umbilical venous endothelial cells (HUVEC), as measured by MTT assay. Cells in 96-well plates were treated with drugs at the indicated concentrations for 72 h, and cell viability was assessed by MTT assay. Points, mean averages (n = 4); bars, SD. (b) Lenalidomide did not enhance the apoptosis-inducing effects of sorafenib, as measured by flow cytometry (sub-G1 fraction analysis). Columns, mean averages of three independent experiments; bars, SD. (c–e) Synergistic in vivo anti-tumor effects between lenalidomide and sorafenib in terms of tumor volume (c), tumor weight (d), and animal survival (e). Values presented in (c) and (d) were the means ± SD (n = 6 in each group). Statistical significance was set at *P < 0.05 by one-way analysis of variance (ANOVA). (e) The median survival time was 48 days in the vehicle group, 55 days in the sorafenib group, 52 days in the lenalidomide group, and 83 days in the , Hep3B; , BNL-MEA; , PLC5; , Huh-7; , HUVEC. , Control; combination group (P < 0.001, log-rank test, 8 or 9 mice/group). , Lenalidomide 30 μM; , Sorafenib 5 μM; , Sorafenib 5 μM + Lenalidomide 30 μM. , vehicle; , sorafenib; , lenalidomide; , sorafenib + lenalidomide.

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Figure 2 Effects of lenalidomide and sorafenib on tumor angiogenesis, tumor cell proliferation, and apoptosis. After 22 days of treatment, tumor tissues of mice were collected, formalin-fixed, and paraffin-embedded for analysis of tumor apoptosis (transferase deoxytidyl uridine end labeling [TUNEL] assay) and tumor angiogenesis (immunohistochemical staining). (a) Representative figures and quantification of tumor microvessel density (MVD, CD31 staining), apoptosis (TUNEL assay), and tumor cell proliferation (Ki-67 staining). (b) Combination of sorafenib and lenalidomide significantly increased vascular normalization, as measured by the Density α-SMA/Density Collagen IV ratio by IHC staining. *P < 0.05; **P < 0.01 by Student’s t-test. Columns, mean averages (n = 6); bars, SD.

VNI = MVD × ( Density α −SMA Density Collagen IV )

measured by an automated dry-chemistry analyzer system (Spotchem SP-4410; Kyoto Daiichi-kagaku, Kyoto, Japan).

MVD (microvessel density) was determined by immunohistochemical (IHC) staining with CD31 antibody. Density α-smooth muscle actin (SMA) and Density Collagen IV will be determined by IHC staining with respective antibodies. An increase in (Density α-SMA/Density Collagen IV) ratio indicated increase in the pericyte coverage and reduction in the thickness of the basement membrane in the tumor vessels and suggested improvement in vascular structure, i.e., vascular normalization.16 Parallel experiments were done to compare animal survival in different treatment groups. Blood samples were obtained via cardiac puncture after 22 days of drug treatment to check the hematology and blood biochemistry data. Hematology data were measured by an automated hematology analyzer (Medonic CA620 VET, Boule Medical AB, Sweden). Blood biochemistry data were 2024

Exploration of immune mediators. To determine activated T cells in tumors and activated BNL-specific CD8+ T cells in spleens, tumor-infiltrating lymphocytes (TILs) and splenocytes were harvested from mice after drug treatment as previously described.15,17 To collect TILs, tumor tissues were cut into small pieces, followed by incubation with HBSS containing collagenase type I (0.05 mg/mL), collagenase type IV (0.05 mg/mL), hyaluronidase (0.025 mg/mL), DNase (0.01 mg/mL), and soybean trypsin inhibitor (1 mg/mL) (all from Sigma-Aldrich, St. Louis, MO, USA) for 15 min. TILs were recovered by using Ficoll-Paque gradient and subjected to lymphocyte subpopulations analysis by flow cytometry (FACScan Caliber, Becton Dickinson, Franklin Lakes, NJ, USA; data processed by using the CELLQest Pro


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Figure 3 Safety of lenalidomide and sorafenib. Effects of drug treatment on animal body weight (a), white blood cells (b), lymphocytes (c), and granulocytes (d). Values presented are the means ± SD (n = 5 in each group). *, p < 0.05; **, p < 0.01. , Vehicle; , Lena; , Sor; , Sor + Lena.

Software, BD Biosciences Pharmingen, San Jose, CA, USA). CD4+ T cells, CD8+ T cells, NK cells, or NKT cells were determined by double staining with specific antibodies recognizing murine CD3 (clone 145-2C11), CD4 (cone GK1.5), CD8 (clone 53-6.7), or Pan-NK (clone DX5) (BD Biosciences Pharmingen). For staining of intracellular molecules, including IFN-γ, FoxP3, granzyme B, and perforin, cells were fixed after surface marker staining and permeabilized according to the manufacturer protocol (BD Biosciences Pharmingen), and then stained with allophycocyanin-conjugated anti-IFN-γ (clone XMG1.2), antiFoxP3 (clone MF23) (BD Biosciences Pharmingen), antigranzyme B (clone NGZB), or anti-perforin (eBioOMAK-D) antibodies, respectively. To determine the anti-tumor specificity of CD8+ splenocytes or TILs after drug treatment, in vitro stimulation was conducted by culturing 2 × 106 splenocytes or TILs with 1 × 105 irradiated BNL1MEA cells or 4T1 cells (as control) for 24 h in the presence of anti-mouse CD28 (1 ug/mL), IL-2 (20 ng/mL), and 2 μM

monensin. The activated tumor-specific T cells were then evaluated by staining with specific antibodies and flow cytometry. To analyze the NK cytolytic activities of splenocytes, the lactate dehydrogenase (LDH) assay (Promega) was used. Splenocytes isolated from mice after treatment with sorafenib, lenalidomide, or their combination were used as effector cells and YAC-1 cells as target cells, and the ratios of LDH release by effector cells/target cells were determined according to manufacturer’s instructions. Briefly, 2 × 104 YAC-1 cells were incubated with effector cells at effector cells/target cells ratios of 0.1, 1, and 10 for 195 min at 37°C, 5% CO2. LDH released by lysed target cells was quantified by colorimetry (absorbance at 490 nm). Target cells were incubated either in culture medium alone or in a mixture of 2% Triton X-100 to determine spontaneous and maximal LDH release, respectively. The percentage of specific lysis were calculated by the following formula: percent cytotoxicity = ([experimental LDH release − spontaneous LDH release by effector and target]/ [maximal LDH release − spontaneous LDH release]) × 100.


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Figure 4 Effects of lenalidomide and sorafenib on lymphocyte subpopulations in tumor microenvironment and in the spleen. (a) Subpopulations of interferon-γ (IFN-γ) + tumor-infiltrating lymphocytes (TILs); (b) IFN-γ + CD8+ lymphocytes in splenocytes; (c) IFN-γ + NKT cells in splenocytes; (d) CD25+FoxP3+ T regulatory cells of TILs after drug treatment. The bars represented mean cell number ± SD/mg tumor tissue of the double positive , Vehicle; , cells. Each group consisted of five mice. *P < 0.05; **P < 0.01, compared with vehicle (one-way analysis of variance [ANOVA]). , Sor; , Sor + Lena. Lena;

Depletion of pertinent cellular immune effectors and cytokines. To evaluate the roles of cellular immune effectors and cytokines in the anti-tumor efficacy of sorafenib and lenalidomide, CD8+ T cells were depleted by intraperitoneal injection of anti-CD8 (clone 53-6.72), or isotype controls (clone 2A3 or HRPN) (all from Bio × Cell, West Lebanon, NH, USA), respectively. The dosage of antibody treatment was 0.5 mg on day 5, and then 0.25 mg on days 8, 11, 14, and 17 after tumor implantation. Depletion of CD8+ cells was confirmed by flow cytometry as previously described.15 Statistical analysis. Animal survival was calculated by the Kaplan–Meier method and compared by log-rank test. Other laboratory results were expressed as means ± standard deviation (SD) and one-way analysis of variance were used to evaluate the statistical significance of the difference between treatment groups.

Results Lenalidomide and sorafenib had synergistic antitumor efficacy in vivo but not in vitro. Our in vitro studies showed that lenalidomide as single-agent treatment did not 2026

impair cell viability significantly in all the cell lines tested (HCC and HUVEC) (Fig. 1a and Supplementary Fig. S1a). No synergistic apoptosis-inducing effects were found when lenalidomide was combined with sorafenib (Fig. 1b and Supplementary Fig. S1b). By contrast, in vivo studies in the BALB/c orthotopic model showed significant anti-tumor synergy between lenalidomide and sorafenib in terms of tumor growth (Fig. 1c,d) and animal survival (Fig. 1e). Combination of lenalidomide with low- (5 mg/kg/day) or high- (30 mg/kg/day) dose sorafenib showed similar anti-tumor synergy (Supplementary Fig. S2). Therefore, we chose the low dosage of sorafenib in all subsequent experiments. By contrast, in vivo studies using the SCID model did not show synergistic antitumor activity (Supplementary Fig. S3). The above data indicated that lenalidomide can enhance the anti-tumor efficacy of sorafenib through modulation of tumor microenvironment. Combination of lenalidomide and sorafenib induced a significant decrease in tumor MVD, measured by CD31 staining, and increase in tumor cell apoptosis, measured by TUNEL assay, than treatment with either sorafenib or lenalidomide alone (Fig. 2a). A trend toward decrease in the proliferation marker Ki-67 was also noted with the lenalidomide–sorafenib combination (Fig. 2a). Treatment with sorafenib or lenalidomide increased α-smooth Journal of Gastroenterology and Hepatology 29 (2014) 2021–2031

© 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd

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Figure 5 Activation of CD8+ lymphocytes in TILs and splenocytes. (a) CD8+ TILs were stained with granzyme B, perforin, or interferon-γ. (b) CD8+ splenocytes were isolated in vitro stimulated with irradiated BNL liver cancer cells or 4T1 cells (irrelevant breast cancer cells) for 24 h, followed by intracellular granzyme B, perforin, or interferon-γ staining. Positive cells were counted by flow cytometry. (a) The bars represent mean number of the double positive cells/mg tumor tissue. (b) The bars represent mean number of double positive cells/105 splenocytes. Each group consisted of five mice. *P < 0.05; **P < 0.01, compared with vehicle (one-way analysis of variance [ANOVA]).

muscle actin (α-SMA) and decreased collagen IV staining (Fig. 2b). Therefore, VNI significantly increased in the combination treatment group, suggesting increasing vascular normalization (Fig. 2b). In this orthotopic liver cancer model, tumor implantation induced leukocytosis, lymphocytosis, and granulocytosis, while other hematology and biochemistry data were not significantly different from mice without tumor implantation (Fig. 3 and Supplementary Fig. S4). Lenalidomide, alone or in combination with sorafenib, significantly reduced the leukocytosis and lymphocytosis. The animal body weight and other hematology and biochemistry data were not significantly different in different treatment groups. The about data indicated that combination of lenalidomide and sorafenib is a well-tolerated regimen. Lenalidomide increased the IFN-γ+CD8+ subpopulation in TILs. We hypothesized that immune modulation of the tumor microenvironment by lenalidomide plays important roles in the anti-tumor synergy between sorafenib and lenalidomide. To explore the potential immune mediators, TILs from the liver tumors and splenocytes were measured. Combination of sorafenib and lenalidomide induced a significant increase of IFN-γ+CD8+ lymphocytes in TILs and splenocytes (Fig. 4a,b). Increase of NKT lymphocytes were found in TILs but not in splenocytes (Fig. 4a,c). In addition, combination of sorafenib and lenalidomide induced a significant decrease of

Treg lymphocytes in TILs than either treatment alone (Fig. 4d). The activation of CD8+ lymphocytes in TILs and splenocytes was further measured by staining with granzyme B, perforin, and IFN-γ. Combination of sorafenib and lenalidomide induced a significant increase in granzyme B, perforin, and IFN-γ-expressing CD8+ TILs than either treatment alone (Fig. 5a). In vitro studies using CD8+ T cells derived from TILs, after stimulation of BNL liver cancer cells or 4T1 breast cancer cells (as control), showed a significant increase in granzyme B, perforin, and IFN-γ expression after combination of sorafenib and lenalidomide treatment (Supplementary Fig. S5). CD8+ T cells derived from splenocytes after stimulation of BNL liver cancer cells or 4T1 breast cancer cells in vitro showed a similar trend of increase in granzyme B, perforin, and IFN-γ expression after combination of sorafenib and lenalidomide treatment (Fig. 5b). On the other hand, the cytotoxic activity of NK cells in splenocytes did not differ significantly between the treatment groups (Supplementary Fig. S6). The above data indicate that CD8+ T cells can be activated by sorafenib and lenalidomide combination treatment and may play a more important role than NK/NKT cells in mediating the anti-tumor synergy between sorafenib and lenalidomide. Depletion of CD8+ lymphocytes abolished the anti-tumor synergy between sorafenib and lenalidomide. To confirm the roles of CD8+ TILs, we performed depletion of CD8+ lymphocytes in the orthotopic liver


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Figure 6 Depletion of CD8+ lymphocytes abolished the anti-tumor synergy between sorafenib and lenalidomide. Tumor-bearing mice were treated by sorafenib, lenalidomide, or the combination along with treatment with anti-CD8 antibodies. (a) Comparison of tumor volumes with or without depletion. Values presented were the means ± SD (n = 3 in each group). (b–d) Effects of CD8+ lymphocyte depletion on tumor cell apoptosis (b), microvessel density (MVD) (c), and vascular normalization index (d) in mice treated by vehicle (control) and lenalidomide plus sorafenib. *P < 0.05; , control lg; , anti-CD8. **P < 0.01 by Student’s t-test. Values presented were the means ± SD (n = 6 in each group).

cancer model as previously described.15 The anti-tumor synergy between sorafenib and lenalidomide was almost completely abolished after depletion of CD8+ lymphocytes (Fig. 6a). Depletion of CD8+ lymphocytes significantly reduced the proportion of apoptotic tumor cells (Fig. 6b), but did not affect the extent of angiogenesis inhibition and vascular normalization induced by sorafenib plus lenalidomide (Fig. 6c,d). The above data indicate that lenalidomide-induced immune modulation plays a more important role than angiogenesis inhibition in the anti-tumor synergy between sorafenib and lenalidomide, and CD8+ TILs are the most important mediators of this synergy.

Discussion In this study, we found that the efficacy of sorafenib in HCC can be significantly improved by lenalidomide through immune modulation. Immune modulation has been extensively studied in HCC. The liver has distinctive subpopulations of lymphocytes that play important immune surveillance and immune modulatory functions.18 In addition, HCC-associated antigens can induce immunosuppressive effects.19,20 Therefore, immune modulation should be an important approach to improve treatment efficacy for HCC. 2028

Lenalidomide can affect the tumor microenvironment through both angiogenesis inhibition and immune modulation. Tumor angiogenesis can induce immune suppression because these two pathological processes share some common regulatory mechanisms. The tumor vasculature and the ambient pro-angiogenic factors can reduce the adhesion and trafficking of immune effector cells, promote infiltration of the immunosuppressive tumorassociated macrophages (TAMs) and Treg cells, and suppress T-cell activation.12 Vascular normalization induced by anti-angiogenic therapy can reduce hypoxia and production of pro-angiogenic factors in the tumor microenvironment, and improve trafficking of immune effector cells into the tumors.21 In the present study, both vascular normalization and IFN-γ+CD8+ immune effector cells in the tumors were increased by combination of lenalidomide and sorafenib, and the latter appears to be the more direct mediator of the anti-tumor synergy between lenalidomide and sorafenib. More studies are needed to dissect the complex interaction between the anti-angiogenic and the immune modulatory effects of lenalidomide and other anti-angiogenic agents in HCC. The immune modulatory effects of sorafenib and other molecular targeted agents have gained increasing attention in recent years.22–25 While earlier studies focused on the modulatory effects of molecular targeted agents on individual components of the


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immune system, more recent studies explored the complex interactions between target therapy and the immune regulatory mechanisms. Data from our study and other investigators indicate that sorafenib may enhance the anti-tumor immunity by inhibiting the immunosuppressive cell populations, including Treg and myeloidderived suppressor cells,23,24,26 and promote pro-inflammatory activities of TAMs.25 These findings support the combination of molecular targeted therapy and immunotherapy for cancer treatment.21,27 Future studies should focus on identification of immunerelated biomarkers for patient enrichment and monitoring of therapeutic response. Recently, agents targeting specific immune checkpoint proteins, including CTLA-4 and PD-1/PD-L1, demonstrated promising anti-cancer activities in clinical trials for advanced cancer patients.26,28 These checkpoint proteins function as co-receptors on the surfaces of T cells and help regulate T-cell responses following T-cell activation. Both CTLA-4 and PD-1/PD-L1 signaling were found to be associated with hepatocarcinogenesis and aggressive tumor behavior.29–32 A clinical trial of the anti-CTLA-4 antibody tremelimumab reported promising anti-tumor efficacy in patients with advanced HCC.33 An important concern of these immune checkpoint modulators is the immune-related adverse events, including hepatitis, and most of the clinical trials of these checkpoint modulators excluded patients with chronic viral hepatitis.34,35 However, emerging evidence suggested that these checkpoint modulators may also have anti-viral activities in patients with chronic hepatitis.33,36 Therefore, future clinical trials of these checkpoint modulators for the treatment of HCC are definitely warranted. There are several aspects that warrant further investigation. First, while CD8+ T cells were identified as the major mediator of the antitumor synergy between sorafenib and lenalidomide, further studies should be done to dissect the drug effects on different stages of T-cell priming/activation/cytotoxic action and clarify the mechanisms of interaction between immune effectors and cancer cells in the HCC microenvironment.37 Second, clarification of the mechanisms of interaction will facilitate the combination of other immune modulatory agents, such as antiCTLA4 and anti-PD1, to further enhance treatment efficacy. Third, lenalidomide can enhance the function of other immune effector cells, such as NK or NKT cells, which may play important roles in the liver immune system. The impact of NK/NKT cells in HCC microenvironment on treatment efficacy should be explored. In conclusion, our data demonstrate anti-tumor synergy between lenalidomide and sorafenib in HCC. Combination of molecular targeted agents and immunotherapy should be a promising approach for the treatment of HCC.

Acknowledgments The authors thank Professor Ping-Ning Hsu, Graduate Institute of Immunology, College of Medicine, National Taiwan University, and the Laboratory Animal Center, College of Medicine, National Taiwan University, for support of the animal studies. The authors thank Liver Disease Prevention and Treatment Foundation, Taiwan, for logistic support.


References 1 Shen YC, Hsu C, Cheng AL. Molecular targeted therapy for advanced hepatocellular carcinoma: current status and future perspectives. J. Gastroenterol. 2010; 45: 794–807. 2 Cheng AL, Kang YK, Chen Z et al. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol. 2009; 10: 25–34. 3 Llovet JM, Ricci S, Mazzaferro V et al. Sorafenib in advanced hepatocellular carcinoma. N. Engl. J. Med. 2008; 359: 378–90. 4 Berasain C. Hepatocellular carcinoma and sorafenib: too many resistance mechanisms? Gut 2013; 62: 1674–5. 5 Seliger B, Massa C, Rini B, Ko J, Finke J. Antitumour and immune-adjuvant activities of protein-tyrosine kinase inhibitors. Trends Mol. Med. 2010; 16: 184–92. 6 Hipp MM, Hilf N, Walter S et al. Sorafenib, but not sunitinib, affects function of dendritic cells and induction of primary immune responses. Blood 2008; 111: 5610–20. 7 Houben R, Voigt H, Noelke C, Hofmeister V, Becker JC, Schrama D. MAPK-independent impairment of T-cell responses by the multikinase inhibitor sorafenib. Mol. Cancer Ther. 2009; 8: 433–40. 8 Krusch M, Salih J, Schlicke M et al. The kinase inhibitors sunitinib and sorafenib differentially affect NK cell antitumor reactivity in vitro. J. Immunol. 2009; 183: 8286–94. 9 Zhao W, Gu YH, Song R, Qu BQ, Xu Q. Sorafenib inhibits activation of human peripheral blood T cells by targeting LCK phosphorylation. Leukemia 2008; 22: 1226–33. 10 Chanan-Khan AA, Cheson BD. Lenalidomide for the treatment of B-cell malignancies. J. Clin. Oncol. 2008; 26: 1544–52. 11 Davies F, Baz R. Lenalidomide mode of action: linking bench and clinical findings. Blood Rev. 2010; 24 (Suppl. 1): S13–9. 12 Motz GT, Coukos G. The parallel lives of angiogenesis and immunosuppression: cancer and other tales. Nat. Rev. Immunol. 2011; 11: 702–11. 13 Safran H, Charpentier KP, Kaubisch A et al. Lenalidomide for second-line treatment of advanced hepatocellular cancer (HCC): a Brown University Oncology Group phase II study. Am J Clin Oncol. 2013 Jul 15. [Epub ahead of print]. 14 Ou DL, Shen YC, Liang JD et al. Induction of Bim expression contributes to the antitumor synergy between sorafenib and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor CI-1040 in hepatocellular carcinoma. Clin. Cancer Res. 2009; 15: 5820–8. 15 Chang CJ, Chen YH, Huang KW et al. Combined GM-CSF and IL-12 gene therapy synergistically suppresses the growth of orthotopic liver tumors. Hepatology 2007; 45: 746–54. 16 Zhou Q, Guo P, Gallo JM. Impact of angiogenesis inhibition by sunitinib on tumor distribution of temozolomide. Clin. Cancer Res. 2008; 14: 1540–9. 17 Chang CJ, Yang YH, Liang YC et al. A novel phycobiliprotein alleviates allergic airway inflammation by modulating immune responses. Am. J. Respir. Crit. Care Med. 2011; 183: 15–25. 18 Nemeth E, Baird AW, O’Farrelly C. Microanatomy of the liver immune system. Semin. Immunopathol. 2009; 31: 333–43. 19 Alisa A, Boswell S, Pathan AA, Ayaru L, Williams R, Behboudi S. Human CD4(+) T cells recognize an epitope within alpha-fetoprotein sequence and develop into TGF-beta-producing CD4(+) T cells. J. Immunol. 2008; 180: 5109–17. 20 Cao M, Cabrera R, Xu Y et al. Hepatocellular carcinoma cell supernatants increase expansion and function of CD4(+)CD25(+) regulatory T cells. Lab. Invest. 2007; 87: 582–90. 21 Huang Y, Goel S, Duda DG, Fukumura D, Jain RK. Vascular normalization as an emerging strategy to enhance cancer immunotherapy. Cancer Res. 2013; 73: 2943–8.


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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 Lenalidomide and sorafenib did not have synergistic anti-tumor efficacy in multiple doses combination in vitro. (a) Lenalidomide did not impair cell viability significantly of the hepatocellular carcinoma (HCC) cell lines (BNL-1MEA, Hep3B) and HUVEC, as measured by MTT assay. Points, mean averages (n = 4); bars, SD. (b) Lenalidomide did not enhance the apoptosisinducing effects of sorafenib, as measured by flow cytometry 2030

(sub-G1 fraction analysis). Columns, means of three independent experiments; bars, SD. Figure S2 Lenalidomide could enhance anti-tumor efficacy of sorafenib at different doses in vivo. Male BALB/c mice were randomized to different treatment groups on day 5 after tumor implantation with BNL-1MEA cells and treated by gavage or intra-peritoneal injection as indicated: • • • • •

Vehicle; Sor-5, sorafenib 5 mg/kg/day; Sor-30, sorafenib 30 mg/kg/day; Lena-50, lenalidomide 50 mg/kg/day; Sor-5 + Lena-50, sorafenib 5 mg/kg/day + lenalidomide 50 mg/ kg/day; • Sor-30 + Lena-50, sorafenib 30 mg/kg/day + lenalidomide 50 mg/kg/day) daily. Body weight and tumor volume were measured as described in the Method section. (a) Effects of lenalidomide and sorafenib on mice body weight. (b–d) Synergistic in vivo anti-tumor effects between lenalidomide and low or high dose of sorafenib in terms of tumor volume (b and c) and tumor weight (d). Values presented are the means ± SD (n = 5 in each group). Statistical value was calculated by one-way analysis of variance (ANOVA). Figure S3 Lenalidomide and sorafenib had no synergistic antitumor efficacy in immune compromised mouse hepatocellular carcinoma (HCC) model. Male SCID mice were treated as indicated (Vehicle, control; Lena, lenalidomide 50 mg/kg/day; Sor, sorafenib 5 mg/kg/day; Sor + Lena, sorafenib 5 mg/kg/day + lenalidomide 50 mg/kg/day; healthy, tumor free) daily by gavage or intra-peritoneal injection after 6 days of orthotopic tumor implantation with BNL-1MEA cells. Mice were randomized to different treatment groups (n = 8 per group) on day 5 after tumor implantation. The median survival time was 27 days in the vehicle group, 34 days in the sorafenib group, 35 days in the lenalidomide group, and 30 days in the combination group (P > 0.05, log-rank test). Figure S4 Safety of lenalidomide and sorafenib in vivo. Effects of drug treatment on hematology (a) and biochemistry (b) values in blood samples from mice treatment by sorafenib, lenalidomide, or the combination. Hematology data were measured by an automated hematology analyzer (Medonic CA620 VET, Boule Medical AB, Sweden). Blood biochemistry data were measured by an automated dry-chemistry analyzer system (Spotchem SP-4410; Kyoto Daiichi-kagaku, Kyoto, Japan). Values presented are the means ± SD (n = 5 in each group). ALT, alanine aminotransferase; BUN, blood urea nitrogen; Cre, creatinine; Hb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume; PLT, platelets; RBC, red blood cells; T-Pro, total protein. Figure S5 Activation of CD8+ lymphocytes in tumor-infiltrating lymphocytes (TILs). CD8+ TILs were isolated in vitro stimulated with irradiated BNL cells or 4T1 cells (irrelevant tumor control) for 24 h, followed by intracellular granzyme B, perforin, or interferon-γ staining. Y-axis indicated ratio of cell count data from BNL- stimulation and 4T1-stimulation experiments. The bars represent mean cell number of the double positive cells/105 TILs. Each group consisted of 5 mice.


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Figure S6 Cytotoxic activity of natural killer (NK) cells in splenocytes isolated from mice treated with sorafenib, lenalidomide, or the combination. The lactate dehydrogenase (LDH) assay using YAC-1 cells as target cells was used and the ratios of LDH release by effector cells/target cells were determined according to manufacturer’s instructions. The percentage of


specific lysis were calculated by the following formula: percent cytotoxicity = ([experimental LDH release − spontaneous LDH release by effector and target]/[maximal LDH release − spontaneous LDH release]) × 100. Results shown in this figure were from experiments with effector/target cell of 0.1:1.


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Knockout of ADAM10 enhances sorafenib antitumor activity of hepatocellular carcinoma in vitro and in vivo.

Phase I Study of Lenalidomide and Sorafenib in Patients With Advanced Hepatocellular Carcinoma.

BRD4 Inhibitor SF1126 with Sorafenib in Hepatocellular Carcinoma.

Synergistic anti-tumor efficacy of sorafenib and fluvastatin in hepatocellular carcinoma.

Synergistic effects of ascorbate and sorafenib in hepatocellular carcinoma: New insights into ascorbate cytotoxicity.

Transarterial chemoembolization and sorafenib in hepatocellular carcinoma.

C-Myc-dependent pathways in hepatocellular carcinoma.

The Role of Sorafenib in Hepatocellular Carcinoma.

Sorafenib Effectiveness in Advanced Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC): beyond sorafenib-chemotherapy.

Pre-clinical activity of PR-104 as monotherapy and in combination with sorafenib in hepatocellular carcinoma.

Decade in review-hepatocellular carcinoma: HCC-subtypes, stratification and sorafenib.

The synergistic in vitro and in vivo antitumor effect of combination therapy with salinomycin and 5-fluorouracil against hepatocellular carcinoma.

2 inhibitor refametinib (BAY 86-9766) in combination with sorafenib exhibits antitumor activity in preclinical murine and rat models of hepatocellular carcinoma.

Potential efficacy of therapies targeting intrahepatic lesions after sorafenib treatment of patients with hepatocellular carcinoma.

Anti-tumor activity of sorafenib in a model of a pediatric hepatocellular carcinoma.

Sorafenib use in hepatocellular carcinoma: more questions than answers.

The Eltrombopag antitumor effect on hepatocellular carcinoma.

Sorafenib-based combined molecule targeting in treatment of hepatocellular carcinoma.

Sorafenib inhibits macrophage-mediated epithelial-mesenchymal transition in hepatocellular carcinoma.

Eupafolin Exhibits Potent Anti-Angiogenic and Antitumor Activity in Hepatocellular Carcinoma.

Chemotherapy for advanced hepatocellular carcinoma in the sorafenib age.

Sorafenib induced alteration of protein glycosylation in hepatocellular carcinoma cells.

The synergistic interaction between the calcineurin B subunit and IFN-γ enhances macrophage antitumor activity.