Blocking the CCL2–CCR2 Axis Using CCL2

Published OnlineFirst December 15, 2016; DOI: 10.1158/1535-7163.MCT-16-0124

Large Molecule Therapeutics Molecular Cancer Therapeutics Blocking the CCL2–CCR2 Axis Using CCL2- Neutralizing Antibody Is an Effective Therapy for Hepatocellular Cancer in a Mouse Model Kun-Yu Teng1,2,3, Jianfeng Han3, Xiaoli Zhang4, Shu-Hao Hsu2,3, Shun He3,5,6, Nissar A. Wani2,3,8, Juan M. Barajas2,3, Linda A. Snyder7, Wendy L. Frankel2,3, Michael A. Caligiuri3,5, Samson T. Jacob3,5,8, Jianhua Yu3,5, and Kalpana Ghoshal2,3,8

Abstract

þ Hepatocellular carcinoma, a deadly disease, commonly CD11highGr1 inflammatory myeloid cells and inhibiting arises in the setting of chronic inflammation. C-C motif chemo- expression of IL6 and TNFa in KO livers. Furthermore, treatment kine ligand 2 (CCL2/MCP1), a chemokine that recruits CCR2- of tumor-bearing KO mice with CCL2 nAb for 8 weeks signif- positive immune cells to promote inflammation, is highly upre- icantly reduced liver damage, hepatocellular carcinoma inci- gulated in hepatocellular carcinoma patients. Here, we exam- dence, and tumor burden. Phospho-STAT3 (Y705) and ined the therapeutic efficacy of CCL2–CCR2 axis inhibitors c-MYC, the downstream targets of IL6, as well as NF-kB, the against hepatitis and hepatocellular carcinoma in the miR- downstream target of TNFa, were downregulated upon CCL2 122 knockout (a.k.a. KO) mouse model. This mouse model inhibition, which correlated with suppression of tumor growth. displays upregulation of hepatic CCL2 expression, which corre- In addition, CCL2 nAb enhanced hepatic NK-cell cytotoxicity lates with hepatitis that progress to hepatocellular carcinoma and IFNg production, which is likely to contribute to the inhi- with age. Therapeutic potential of CCL2–CCR2 axis blockade bition of tumorigenesis. Collectively, these results demonstrate was determined by treating KO mice with a CCL2-neutralizing that CCL2 immunotherapy could be an effective therapeutic antibody (nAb). This immunotherapy suppressed chronic liver approach against inflammatory liver disease and hepatocellular inflammation in these mice by reducing the population of carcinoma. Mol Cancer Ther; 16(2); 312–22. 2016 AACR.

Introduction infection in the past 20 years (3, 4). Furthermore, sorafenib, the only FDA-approved drug for advanced hepatocellular carcino- Hepatocellular carcinoma is the most common liver cancer ma extends overall survival by only 2.8 months (5). Recent and the second leading cause of cancer-related deaths world- clinical trials have shown that it is ineffective as an adjuvant wide (1, 2). The incidence of hepatocellular carcinoma has therapy after resection or ablation of the tumor (6). Thus, there tripled in the United States because of the precipitous increase is an urgent need to develop novel therapeutic strategies for the in nonalcoholic fatty liver disease and hepatitis C virus (HCV) treatment of hepatocellular carcinoma. miR-122 is the most abundant liver-specificmiRNAinverte- brates, and loss of miR-122 is associated with metastasis and 1Molecular, Cellular and Developmental Biology Program, The Ohio State Uni- poor prognosis in hepatocellular carcinoma patients (7). We versity, Columbus, Ohio. 2Department of Pathology, The Ohio State University, Columbus, Ohio. 3Comprehensive Cancer Center, Wexner Medical Center, The previously found that development of spontaneous hepatocel- Ohio State University, Columbus, Ohio. 4Center for Biostatistics, The Ohio State lular carcinoma with age mimicked different stages of tumor University, Columbus, Ohio. 5Virology, Immunology and Medical Genetics, The progression (e.g., steatohepatitis, fibrosis, primary, and meta- Ohio State University, Columbus, Ohio. 6Center for Systems Biology, Massachu- static hepatocellular carcinoma) in miR-122 knockout (KO) 7 setts General Hospital, Harvard Medical School, Boston, Massachusetts. Jans- mice (8, 9). We also observed that miR-122 depletion in the sen Research and Development, LLC, Spring House, Pennsylvania. 8Department mouse liver leads to upregulation of chemokine (C-C motif) of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, þ high þ Ohio. ligand 2 (CCL2), which recruits CCR2 CD11b Gr1 immune cells to the liver (8). These cells, in turn, produce Note: Supplementary data for this article are available at Molecular Cancer fl Therapeutics Online (http://mct.aacrjournals.org/). proin ammatory cytokines, including IL6 and TNFa in the liver, resulting in hepatitis and eventually hepatocellular carcinoma. K.-Y. Teng and J. Han contributed equally to this article. CCL2 is known to be involved in the pathogenesis of several Current address for Shun He: Massachusetts General Hospital (MGH), Harvard diseases characterized by monocytic infiltrates, such as psori- Medical School, Center for Systems Biology, Boston, MA. asis, rheumatoid arthritis, and atherosclerosis (10, 11). CCL2 Corresponding Authors: Kalpana Ghoshal, Department of Pathology, The Ohio also promotes local inflammation and macrophage infiltration State University, 420 West 12th Avenue, 646C TMRF Building, Columbus, OH in the chronically injured liver (12). Very recently, Li and 43210. Phone: 614-292-5688; Fax: 614-688-4245; E-mail: colleagues demonstrated that a chemical inhibitor of CCR2, [email protected]; and Jianhua Yu, [email protected] the receptor of CCL2, inhibited hepatocellular carcinoma doi: 10.1158/1535-7163.MCT-16-0124 development by reducing monocytes/macrophage infiltration þ 2016 American Association for Cancer Research. and M2 macrophage polarization as well as CD8 T-cell

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activation in the murine xenograft model (13). However, College of Medicine, Houston, TX). Although we did not whether targeting the CCL2–CCR2 axis by immunotherapy authenticate these cells, they exhibited characteristics and gene could be an effective therapeutic approach against chronic expression profile of mouse hepatoma cells. Hepa cells were inflammation and hepatocellular carcinoma has not been labeled with Chromium-51 (Cr-51) and then preincubated addressed. In this study, we addressed this important question with C1142 antibody or isotype antibody at a concentration using a novel preclinical model, miR-122 KO mice that develop of 10 mg/mL for 1 hour. Then, a standard 4-hour Cr-51 release chronic inflammation–driven hepatocellular carcinoma (8). assay (16) was performed to access cytotoxicity of mouse NK Our results clearly showed that targeting CCL2–CCR2 signaling cells against Hepa cells. by CCL2 immunotherapy could be an alternative approach in suppressing hepatitis and hepatocellular carcinoma develop- ELISA ment. Furthermore, our studies revealed that CCL2-neutraliz- For a-fetoprotein (AFP), mouse serum was collected by man- ing antibody (nAb) immunotherapy in miR-122 KO mouse dibular punch (before treatment) or cardiac punch (after treat- high þ fl model involves suppression of CD11b Gr1 in ammatory ment). KO mouse older than 10 months would be monitored for myeloid cell recruitment and enhancement of natural killer their body weight and serum AFP level by every 2 weeks. AFP level (NK) cell cytotoxicity. These observations underscore the was quantified by DRG AFP (Alpha Fetoprotein) Kit (DRG, cat# – importance of targeting CCL2 CCR2axisasapotentialtherapy EIA-1468). in a subset of human hepatocellular carcinoma patients with For IFNg, mouse liver mononuclear cells were isolated as fl chronic hepatic in ammation and high CCL2. described previously (8, 15). Hepa cells cultured in DMEM containing 10% FBS, were preincubated with C1142 (CCL2 Materials and Methods nAb) at a concentration of 10 mg/mL for 1 hour. Then, 106 Treatment of miR-122 KO mice with CCL2 antibody and CCR2 mononuclear cells were incubated with the same number of inhibitor Hepa cells per well in a 96-well V-bottom plate at 37 Cfor24 miR-122 KO mice were generated as described previously (8). hours. Culture supernatants were collected for IFNg estimation Animals were housed in Helicobacter-free facility under a 12-hour using mouse IFNg ELISA Ready-SET-Go Kit (eBioscience, cat# light/dark cycle. Animals were handled following the guidelines 88-7314-88). of the Ohio State University Institutional Laboratory Animal Care Committee. MRI CCL2 nAb (anti-mouse CCL2; clone C1142) was gener- MRI of liver tumors in miR-122 KO mice was performed as ously provided by Janssen (14). miR-122 KO mice were described previously (17). injected intraperitoneally with CCL2 nAb (2 mg/kg). The control group was injected with vehicle (PBS). To study the Serologic, histologic, and immunohistochemical analysis function of CCL2 in hepatitis, 4-month-old KO mice were Serum was isolated from mice by cardiac puncture after CO2 treated with CCL2 nAb twice a week for 4 weeks. To study asphyxiation and cervical dislocation and stored at 80C. Bio- the role of CCL2 in hepatocellular carcinoma development, chemical analysis of serum enzymes was performed using VetACE 12-month-oldKOmicewereinjectedwithCCL2nAbtwicea (Alfa Wassermann system) as described previously (8). Macro- week for 8 weeks. scopic tumors were counted, dissected, and weighed along with CCR2 inhibitor (Tocris Bioscience, cat# 2089) was given benign liver tissues. A fraction of tissues was fixed in 4% para- to KO mice in the drinking water daily (10 mg/kg) for 4 weeks. formaldehyde as described previously (8), and the rest was snap The control group for CCR2 inhibitor was fed with regular frozen for RNA and protein analysis. water. For histology, paraffin-embedded tissue sections (4 mm) on glass slides were stained with hematoxylin and eosin (H&E) or Flow cytometric analysis immunohistochemical analysis with Ki-67, AFP, and glypican Murine mononuclear cells from livers were isolated as 3(GPC3)-specific antibodies as described previously (8). described previously (8, 15). Erythrocytes were lysed using Inflammation score was generated through blinded evaluation RBC lysis buffer (BioLegend). Cells isolated from livers were of H&E-stained slides (100 magnification) as described pre- treated with Fc Block antibody (anti CD16/32, BD Biosciences). viously (8). Immunohistochemical analysis was done as Cells were stained with mouse-specific immune cell surface described previously (8) and quantified by ImageJ (imagej. markers for 30 minutes at 4C. The following anti-mouse nih.gov/ij/). Microscopic images were taken by phase-contrast antibodies were used at a dilution of 1:200: CD3-APC, CD3- microscope (BX41TF, OLYMPUS) and camera (DP71, OLYM- PerCP-Cy5.5, NK1.1-APC, NK1.1-PE, CD19-FITC, CD69-FITC, PUS). cellSens Standard 1.13 (OLYMPUS) software was used CD27-PE-Cy7, CD11b-PE, CD11b-PerCP-Cy5.5, Gr1 V450, to capture images. Antibody information is provided in the and IFNg FITC (BioLegend). For staining of IFNg,cellswere Supplementary Material. treated with Cytofix/Cytoperm (BD Biosciences) following initial cell surface staining, and then, intracellular staining was performed. qRT-PCR Total RNA was extracted from liver tissue using TRIzol (Life NK cytotoxicity assay Technologies, cat#15596018) followed by DNase I treatment. Tumor-derived NK cells were enriched by NK Cell Isolation DNase-treated RNA was reverse transcribed into cDNA using Kit II (Miltenyi Biotec) and then sorted using a FACSAria II Cell High-Capacity cDNA Reverse Transcription Kit (Applied Biosys- Sorter (BD Biosciences). Hepa1-6, a mouse hepatoma cell line, tems, cat# 4368813). qRT-PCR analysis of each sample performed was generously provided by Dr. Gretchen Darlington (Baylor in triplicate was performed using SYBR Green chemistry. Gene

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expression was normalized to Gapdh. Relative expression was calculated by DDCt method. Primer sequences are provided in the Supplementary Table S1.

Immunoblot analysis Proteins were extracted from whole liver and tumor tissues by SDS lysis buffer, followed by immunoblotting with primary antibodies (8). Signals were developed using Pierce ECL reagent (ThermoFisher, cat# 32106) and quantiﬁed by ImageJ (imagej. nih.gov/ij/). Antibody information is provided in the Supple- mentary Material.

Statistical analysis All bar diagrams are presented as mean SD. Two sample t tests or ANOVA were used for analysis for comparison of two or more groups. For the GEO data analysis, CCL2 expression from the quantile-normalized data was compared among tumor tissue types with ANOVA. Holm procedure was used to adjust for multiple comparisons. Fisher exact test was used to test the difference of tumor incidence and tumor size between the CCL2 nAb–treated and vehicle-treated groups. P values <0.05 were considered significant and represented as asterisks (, P value 0.05; , P value 0.01; , P value 0.001). Figure 1. CCL2 is overexpressed in human cirrhotic livers, as well as cirrhotic and non- Results cirrhotic hepatocellular carcinomas (HCC). CCL2 RNA expression retrieved from CCL2 is upregulated in primary human hepatocellular GEO data (www.ncbi.nlm.nih.gov/geo/) with accession_GSE14323 (20) was carcinoma compared among the four types of liver tissues, and statistical analysis was done using ANOVA. To verify whether CCL2 plays a role in human hepatocellular carcinoma development, we first examined its expression across independent microarray datasets (hepatocellular carcinoma vs. CCL2 might be critical for progression to cirrhosis and hepa- normal liver) downloaded from Oncomine (18). To validate tocellular carcinoma. Thus, blocking the CCL2–CCR2 axis CCL2 expression, we chose The Cancer Genome Atlas, Mas seems to be a reasonable therapeutic approach for hepatocel- liver, and Guichard liver that contain large patient cohorts lular carcinoma or even early stages of hepatocellular carcino- – CCL2 (18 20). DNA copy number or mRNA expression ma development, such as hepatitis and cirrhosis. showed a significant increase in tumor tissues compared with the adjacent benign liver across four datasets (Table 1; refs. CCL2–CCR2 blockade reduced chronic inflammation and liver – CCL2 18 20). To further evaluate expression and disease prog- injuries in miR-122 KO mice nosis, we analyzed hepatocellular carcinoma RNA microarray CCL2 is a major chemokine that is known to cause various n ¼ data ( 115, mainly HCV positive) from GEO database inflammatory diseases in humans (10, 21). Similarly, upregula- CCL2 (GSE14323; ref. 20). The results showed that RNA tion of CCL2 in miR-122 KO liver correlated with chronic inflam- fi expression was signi cantly elevated in hepatocellular carcino- mation (8). To determine whether CCL2 is a key player in mas, cirrhotic livers, and cirrhotic hepatocellular carcinomas hepatitis, and blocking CCL2 could inhibit liver inflammation, compared with normal livers (Fig. 1). These data imply that CCL2 nAb was administered to 4-month-old miR-122 KO mice by an intraperitoneal injection for 4 weeks (Fig. 2A). CCL2 nAb (C1142) displayed certain specificity toward murine CCL2 (14). Table 1. CCL2 expression across four independent microarrays in hepatocellular In addition, CCL2 nAb was well tolerated in mice (14). Further- carcinoma patients more, its antitumor efficacy has been demonstrated in murine P Cancer vs. normal Array type Fold change Sample size breast cancer metastasis (22), lung (23), brain (24), and prostate n ¼ Mas Liver RNA 0.012 3.324 115 cancer (25, 26) models, and no adverse effects were reported. Guichard Liver cohort 2 DNA 0.036 2.025 n ¼ 52 TCGA Liver DNA 0.003 2.063 n ¼ 212 To evaluate the effects of CCL2 nAb in suppressing hepatitis, Guichard Liver cohort 1 DNA 0.026 2.153 n ¼ 185 we treated 4-month-old miR-122 KO mice with CCL2 nAb, NOTE: CCL2 expression in human clinical samples (cancer vs. normal). Data followed by histopathologic and serologic analyses. As reported provided by Oncomine (www.oncomine.com, January 2016, Thermo Fisher in other mouse models (14, 22–26), CCL2 nAb is well tolerated Scientific). TCGA database (TCGA Research Network: http://cancergenome. in miR-122 KO mice. There were no obvious changes in the nih.gov) contains >200 hepatocellular carcinoma specimens of different etiol- appearance, activity, or body weight (Supplementary Fig. S1). ogy (HBV, HCV, alcohol, and NAFLD). Mas liver is an RNA array, which contains Liver histology showed reduced immune cell infiltration after >100 hepatocellular carcinoma samples (mostly HCV positive; ref. 20). The Guichard liver DNA array consisting of 237 hepatocellular carcinomas of various treating with CCL2 nAb (Fig. 2B). Similarly, CD45 immuno- risk factors, including HBV, HCV, alcohol, and NAFLD, was analyzed by exosome histochemical analysis revealed that CCL2 nAb treatment sequencing and SNP array (19). decreased leukocyte accumulation in the liver (Fig. 2C). Nota- Abbreviation: TCGA, The Cancer Genome Atlas. bly, a significant reduction in serum alanine aminotransferase

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Figure 2. CCL2 nAb therapy reduces chronic liver inflammation and liver damage in adult miR-122 KO mice. A, The schematic presentation of the CCL2 nAb treatment. HCC, hepatocellular carcinoma. B, Liver histology of 4-month-old male KO mice injected intraperitoneally with CCL2 nAb (2 mg/kg, n ¼ 5) or PBS (vehicle, n ¼ 5). Representative images of H&E-stained liver sections [scale bars, 100 (top) and 25 mm (bottom)]. Right, quantitation of the inflammation scores generated through blinded evaluation of H&E- stained liver sections (100 magnification, 100 mm). The inflammatory area was quantified by ImageJ of three randomly chosen fields (100 magnification, 100 mm) per animal (n ¼ 5). C, Representative images of CD45-stained liver sections [scale bars, 100 (top) and 25 mm (bottom)]. Right, CD45þ areas quantified by ImageJ of three randomly chosen fields (100 magnification, 100 mm) per animal (n ¼ 5). D, Analysis of serum ALT, AST, and ALP levels (n ¼ 5). n.s., not significant.

(ALT) and AST levels corroborated that the liver damage due to We have shown that activation of the CCL2–CCR2 axis in hepatitis in KO mice could be reversed by inhibition of CCL2 miR-122 KO liver correlates with the recruitment of þ (Fig. 2D). Alkaline phosphatase (ALP), a marker of biliary CD11bhighGr1 cells and hepatic inflammation and injuries dysfunction, was also reduced by approximately 50%; however, (8). In addition, CCR2 inhibitors showed anti-inflammatory it was not statistically significant(Fig.2D).Thisresultimplies effects in the animal models with different diseases, such as that blocking CCL2 may not be able to fully rescue hepato- diabetic nephropathy (27), kidney hypertension (28), steato- biliary damage because ALP is a target of miR-122, which is hepatitis (29), and renal atrophy (30) models, and no adverse derepressed in miR-122–depleted livers (8, 9). Serum gamma- effects were reported. We decided to test whether treatment of glutamyl transferase and bilirubin (total and direct) levels were mice with a CCR2-specific inhibitor could reduce liver inflam- not significantlyalteredinCCL2nAb–injected mice (Supple- mation in KO mice. Indeed, KO mice fed water containing a mentary Table S2). CCR2 inhibitor exhibited reduced hepatic inflammation, as

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revealed by the dramatic reduction in bridging inflammation CCL2 nAb without significant change in their numbers in (Supplementary Fig. S2). Collectively, these data showed that peripheral blood (Fig. 3A and B; Supplementary Fig. S3). blocking the CCL2–CCR2axiscouldeffectivelyinhibithepa- Consistent with other studies (12, 13), we also found decreased titis in miR-122 KO mice. number of hepatic macrophages in liver by IHC upon blocking the CCL2–CCR2 axis (Supplementary Fig. S4). However, no CCL2 nAb therapy inhibited hepatitis by reducing significant alterations in the number of hepatic B, T, and NK þ CD11bhighGr1 inflammatory myeloid cell accumulation cells were observed (Supplementary Fig. S5A–S5D). In addi- It is well established that immune cells play a critical role tion, Immune cells isolated from CCR2 inhibitor–treated KO þ in liver inflammation (31). Our previous study showed mice showed reduced hepatic CD11bhighGr1 cells (Supple- þ that CD11bhighGr1 inflammatory myeloid cells excessively mentaryFig.S6),furthersupportingtheideathatblockingthe þ accumulated in miR-122–depleted livers (8). In addition, CCL2–CCR2 axis reduces CD11bhighGr1 inflammatory mye- þ CD11bhighGr1 cells, which express CCR2 (8), are known to loid cells in liver. berecruitedtotheliverviaCCL2–CCR2 interaction (32). IL6 and TNFa are two proinflammatory cytokines that drive þ Therefore, we quantitated CD11bhighGr1 cells in KO livers hepatitis and hepatocellular carcinoma in the liver (33). þ depleted of CCL2 or CCR2. Flow cytometric analysis of the Previously, we have shown that CD11bhighGr1 inflammatory immune cells isolated from livers and spleen showed reduced myeloid cells produce IL6 and TNFa in the miR-122 KO liver þ CD11bhighGr1 cell population in the KO mice treated with (8). Therefore, it is expected that hepatic IL6 and TNFa levels

Figure 3. CCL2 nAb treatment decreases liver CD11bhighGr1þ cell population and Il6 and Tnfa expression in adult KO mice. A, Injection of 4-month-old KO mice CCL2 nAb reduced the hepatic CD11bhighGr1þ population. Right, quantitation of hepatic CD11bhighGr1þ inflammatory myeloid cells (n ¼ 5). B, Reduced CD11bhighGr1þ population in spleen. Right, quantitation of splenic CD11bhighGr1þ inflammatory myeloid cells (n ¼ 5). C, qRT-PCR analysis of inflammatory cytokines and chemokines.

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Figure 4. Blocking CCL2 function suppresses hepatocellular carcinoma (HCC) development in KO mice. A, The schematic depiction of the immunotherapy protocol and the representative MRI of liver tumors (axial view, T1 weighted) for 12-month-old miR-122 KO mice. B, Five representative images of the livers harvested from the vehicle (n ¼ 9) and CCL2 nAb–treated mice (n ¼ 11). C, Flow cytometric analysis of hepatic CD11bhighGr1þ immune cells in the vehicle- and CCL2 nAb–treated mice. D, Representative images of liver histology, AFP, and GPC3 immunohistochemical images of the vehicle- and nAb-treated groups [scale bars: 250 (top), 100 (middle), and 25 mm (bottom)].

þ would be suppressed if the population of CD11bhighGr1 Attenuation of hepatocarcinogenesis in miR-122 KO mice cellsisdecreasedintheCCL2nAb–treated mice. Indeed, the treated with CCL2 nAb expressions of both cytokines were reduced in the livers of As aforementioned, CCL2 is highly expressed in the tumor CCL2 nAb–treated group (Fig. 3C). In addition, we also tissues of hepatocellular carcinoma patients (Fig. 1; Table 1). observed a decrease in Il1b expression upon CCL2 nAb Besides, blocking CCL2 suppresses chronic liver inflammation treatment (Fig. 3C). Notably, CCL2 nAb treatment did not at early stages of hepatocellular carcinoma development in the alter the expressions of other inflammatory cytokines or KO mice (Fig. 2). Therefore, we speculated that impeding chemokines, such as Ccl5, Ccl8, Cxcl9, Il10, Ccl2,orCcr2 CCL2 function would suppress development of spontaneous (Fig. 3C). Taken together, these results suggest that inhibition liver tumors in KO mice. To test this, male KO mice (12 of the CCL2–CCR2 axis reduces liver inflammation by sup- months old) bearing liver tumors confirmed by the serum þ pressing CD11bhighGr1 inflammatory myeloid cells, which AFP levels were randomly assigned to two treatment groups. leads suppression of the two major proinflammatory cyto- Comparison of the serum AFP level with the tumor size in a kines, IL6 and TNFa. largenumberofKOmicerevealedthatmicewithserumAFP

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levels >12 IU/mL usually developed tumors (Supplementary c-MYC is a well-known oncogene that promotes hepatocel- Fig. S7A). These ELISA data were further confirmed by MRI lular carcinoma proliferation (34, 35). Therefore, Ki-67 expres- (Fig. 4A). KO mice were injected intraperitoneally with CCL2 sion, a marker of cell proliferation, was evaluated in tumor cells nAb (2 mg/kg) or vehicle twice a week for 8 weeks (Fig. 4A), by IHC. As expected, the number of Ki-67–positive cells was and the tumor burden was analyzed one week after the last much less in both CCL2-depleted tumor and nontumor tissues injection. We chose male KO mice for this study as these mice, (Fig. 5D; Supplementary Fig. S9). PCNA, another cellular like men, have higher hepatocellular carcinoma penetrance proliferation marker, was also reduced in the CCL2 nAb– and tumor burden (8, 9). treated tumor extracts (Fig. 5E). Of note, we found cell apo- Significant phenotypic differences were observed in the liver ptosis is increased in the CCL2 nAb–treated group as demon- between the vehicle- and the CCL2 nAb–treated groups after 8 stratedbyslightincreaseincleavedPARPandcleavedcaspase-7 weeks of CCL2 immunotherapy; however, there were no obvi- levels (Supplementary Fig. S10). These data suggest that inhi- ous changes in the appearance, activity, or body weight bition of hepatocellular carcinoma growth by targeting CCL2 is, between the two groups (Supplementary Fig. S7B). Although at least in part, through the inhibition of STAT3 and NF-kB the majority of the PBS-injected mice developed large macro- signaling and c-MYC expression. scopic liver tumors, CCL2 nAb–treated mice mostly developed smaller and fewer tumors (Fig. 4B; Table 2). Consistent with the þ CCL2 nAb activated NK cells in the tumor microenvironment to previous data (Fig. 3A), we found hepatic CD11bhighGr1 cell suppress liver cancer population was reduced in the CCL2 nAb–treated tumor-bear- Cancer immunotherapy has been widely conducted in the ing mice compared with vehicle-treated group (Fig. 4C). In clinic over the past several years (36–38). As antibody-based addition, serum AFP and ALT levels were lower in the immunotherapy is applied in this study, and NK cell is known CCL2 nAb–treated mice (Table 2). Furthermore, the majority to be activated by the Fc domain of antibodies (37), we investi- of the hepatocellular carcinomas in the vehicle-treated group gated whether enhanced NK cytotoxicity could be a mechanism of were AFP and GPC3 positive and grade 2–3 (intermediate to tumor suppression in CCL2 nAb–treated mice. Although the total poorly differentiated) hepatocellular carcinomas, whereas number of NK cells did not change significantly after CCL2 nAb thoseinCCL2-inactivatedmiceweremostlygrade1(well immunotherapy (Supplementary Fig. S4C and S4D), the activity differentiated) hepatocellular carcinomas, AFP and GPC3 neg- of NK cells increased, as demonstrated by the upregulation of ative (Fig. 4D). These results suggest that CCL2 blockade is not hepatic Ifng expression (Fig. 6A). To verify the source of the only anti-inflammatory but also antitumorigenic in the liver. elevated IFNg, primary hepatic immune cells isolated from tumor-bearing KO mice were cocultured with mouse hepatoma Oncogenic signaling downstream of IL6 and TNFa was blocked (Hepa1-6) cells in the presence of CCL2 nAb. The culture super- in miR-122 KO tumors upon CCL2 nAb therapy natants were subjected to IFNg ELISA, and cells were analyzed by Because CCL2 nAb treatment suppressed the level of IL6 and flow cytometry. Both ELISA and intracellular staining data showed TNFa in the liver (Fig. 3C), we hypothesized that the downstream that only NK cells (Fig. 6B and C) were the major producers of oncogenic signaling pathways of IL6 and TNFa, namely STAT3 IFNg. In addition, the cell surface expression of CD69, a marker for and NF-kB, respectively, would be inhibited in the CCL2-depleted activated NK cells, increased when NK cells were cocultured with mice. Indeed, immunoblot analyses showed that phospho-STAT3 CCL2 nAb–treated mouse hepatoma (Hepa) cells (Fig. 6D), (Y705) level was dramatically reduced, whereas total STAT3 level suggesting that NK cells could be activated upon exposure of decreased only slightly in the tumors of CCL2 nAb–treated mice hepatoma cells to CCL2 nAb. To confirm the activated NK cells (Fig. 5A and C). Reduced nuclear STAT3 levels in the CCL2 nAb– could suppress cancer cell growth, NK-cell cytotoxicity was treated mice confirmed that CCL2 inhibition suppresses STAT3 assessed by Cr-51 release assay (16, 39). To this end, Hepa cells functions in liver tumors (Fig. 5B and C). Similarly, the level of were first labeled with Cr-51, followed by incubation with CCL2 two major NF-kB subunits, p65 and p50, was reduced in both nAb and subsequently, with tumor-derived NK cells. Enhanced whole liver lysates and nuclear extracts (Fig. 5B and C). The level of release of Cr-51 from Hepa cells is indicative of higher cytotoxicity c-MYC, a downstream target of STAT3, was also reduced in the of NK cells. Indeed, tumor-derived NK cells cocultured with Hepa CCL2 nAb–treated mice (Fig. 5A and C). Histologically, p65 and cells and CCL2 nAb exhibited higher cytotoxicity (Fig. 6E). This c-MYC both showed reduced expression in the transformed result also suggested that the increased cytotoxicity of NK cells hepatocytes, whereas pSTAT3 was decreased in immune cells as could be triggered independent of other immune cells. Collec- well as tumor cells (Supplementary Fig. S8). tively, these results demonstrate that CCL2 nAb impedes tumor growth, at least in part, by activating NK cells. Table 2. Histopathologic and serologic analysis of tumor-bearing miR-122 KO mice treated with CCL2 nAb for 8 weeks Discussion Vehicle CCL2 nAb P HCC incidence 8/9 5/11 0.07 CCL2 is a chemotactic factor to tumor cells and inflammatory Tumor number 2.67 1.22 0.73 0.79 0.0004 macrophage/monocytes. Blocking the CCL2–CCR2 axis by CCR2 Sum of tumor diameter (cm) 1.71 1.24 0.57 0.65 0.01 inhibitor decreases the intrahepatic macrophage infiltration in a Serum ALT (U/L) 151.89 68.06 73.57 28.5 0.027 diet-induced steatohepatitis model (29). CCL2 Spiegelmer, a Serum AFP (IU/mL) 14.07 3.51 9.95 0.61 0.001 CCL2 RNA antagonist, suppresses the macrophage infiltration in NOTE: All measurements are presented as mean SD. P values were calculated the carbon tetrachloride (CCl ) and methionine–choline-defi- using Student two-tailed t test (for tumor number, sum of tumor diameter, tumor 4 weight, ALT, and AFP) or Fisher exact test (for hepatocellular carcinoma cient diet-induced liver injury models (12). Recently, Li and incidence). colleagues also demonstrated the importance of CCL2–CCR2 Abbreviation: HCC, hepatocellular carcinoma. axis in different hepatocellular carcinoma models (13). In line

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Figure 5. CCL2 nAb therapy suppresses hepatocellular carcinoma development in KO mice by inhibiting tumor cell proliferation. A, Immunoblotting of the key downstream proteins of IL6 and TNFa in KO tumors. B, Immunoblotting of NF-kB subunit and STAT3 in the tumor nuclear extracts. C, Respective protein levels were normalized to those of GAPDH or KU86. pSTAT3 level was normalized to tSTAT3, which is separated from others by a dash line. Quantitation was done by ImageJ. D, Representative images of Ki-67 staining (scale bar, 100 mm; inset, 25 mm). Quantitation was done by ImageJ of three randomly chosen ﬁelds (200 magniﬁcation, 50 mm) per animal (n ¼ 5). E, Immunoblotting of PCNA in liver tumors.

with the animal models mentioned above, it is clear that CCL2 is development, we used CCL2 nAb to block CCL2 signaling. Our crucial for regulating the inflammatory milieu in liver. In this data suggest that CCL2 nAb inhibits tumor development in miR- study, we aimed at elucidating the efficacy of CCL2 immunother- 122 KO mice through at least two distinct mechanisms: suppres- apy against chronic hepatitis and hepatocellular carcinoma in a sing IL6 and TNFa production by reducing the infiltration of þ murine model. This is a logical extension of our previous study CD11bhighGr1 inflammatory myeloid cells to the liver and that demonstrated a causal role of CCL2 in the inflamed liver that enhancing NK-cell cytotoxicity (Supplementary Fig. S11). progressively leads to hepatocellular carcinoma in miR-122 KO An elegant study demonstrating the correlation of CCL2 pro- mice (8). Analysis of GEO, Oncomine, and Protein Atlas data- tein level with poor survival of hepatocellular carcinoma patients bases showed increased CCL2 expression in patients suffering was published (13) during the preparation of our manuscript. Li from hepatocellular carcinoma (18, 20, 40). These data suggest and colleagues showed that a clinically relevant CCR2 antagonist CCL2 may play a role in liver cancer. To further clarify the role of inhibited murine hepatocellular carcinoma progression by reduc- the CCL2–CCR2 axis in chronic liver inflammation and tumor ing M2-type tumor-associated macrophage population and

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Figure 6. CCL2 nAb activates NK cells in KO mouse livers. A, qRT-PCR analysis of hepatic Ifng in CCL2 nAb–treated mice. B, IFNg quantiﬁcation by ELISA in the supernatants of cultured Hepa and hepatic immune cells. C, Intracellular ﬂow cytometric analysis of IFNg in the cocultured immune cells. D, Flow cytometric analysis of the activated NK-cell surface marker, CD69. E, Cytotoxicity of hepatic NK cells toward CCL2 nAb–treated Hepa cells.

þ increasing CD8 cytotoxic T cells in orthotopic and subcutaneous our unique animal model to study the role of CCL2 in liver hepatocellular carcinoma models (13). Their study supports inflammation and hepatocellularcarcinomaprogressionin the notion that blocking the CCL2–CCR2 axis has profound light of our previous work (8). We demonstrated two different þ effects in modulating liver immune system to suppress hepato- types of immune cells, including CD11bhighGr1 inflammatory cellular carcinoma progression. While Li and colleagues found myeloid cells and NK cells, that play key roles in regulating liver þ macrophage and CD8 T cells are regulated by a CCR2 inhibitor inflammation and tumor development after blocking CCL2 þ þ in xenograft models, we found CD11bhighGr1 inflammatory function. In addition, CD11bhighGr1 inflammatory myeloid myeloid cells and NK cells could be modulated by CCL2 nAb cells promote tumorigenesis by increasing tumor cell prolifer- in miR-122 KO mouse livers. Thus, these studies (ours and Li ation via IL6 and TNFa.BothIL6andTNFa are well-known and colleagues') demonstrated that targeting CCL2–CCR2 axis proinflammatory cytokines that activate oncogenic transcrip- by blocking the receptor or the ligand could be an effective tion factor STAT3 to promote hepatocellular carcinoma devel- hepatocellular carcinoma therapy. In the present study, we used opment (33, 41). Consistent with the previous findings, our

320 Mol Cancer Ther; 16(2) February 2017 Molecular Cancer Therapeutics

CCL2 Immunotherapy Suppresses Hepatitis and HCC

current data reveal that CCL2 nAb reduces accumulation of hepatitis and hepatocellular carcinoma development, and pro- þ CD11bhighGr1 cells, which decreases IL6 and TNFa levels in vides rationales for future clinical trials in hepatocellular carci- the tumor and surrounding liver tissues, and reduces the noma patients with chronic inflammation. activation of STAT3 and the expression of c-MYC. Ours and – Li and colleagues' data demonstrated that targeting the CCL2 Disclosure of Potential Conflicts of Interest CCR2 axis by blocking the receptor or the ligand could be an No potential conflicts of interest were disclosed. effective hepatocellular carcinoma therapy. In future, it would be interesting to investigate whether the combination of both Authors' Contributions would be a more effective therapeutic approach. Conception and design: K.-Y. Teng, L.A. Snyder, J. Yu, K. Ghoshal NK cells are key modulators of liver diseases. Activated NK cells Development of methodology: K.-Y. Teng, J. Han, S. He, K. Ghoshal suppress liver disease progression from chronic hepatitis to hepa- Acquisition of data (provided animals, acquired and managed patients, tocellular carcinoma (42). On the basis of ours and Li and provided facilities, etc.): K.-Y. Teng, J. Han, S.-H. Hsu, S. He, J. Barajas, colleagues' data (13), CCL2 is expressed in liver tumors of both K. Ghoshal mouse and human origin. The CCL2 nAb binds to Fc receptor Analysis and interpretation of data (e.g., statistical analysis, biostatistics, (CD16) on NK cells (37), and the CCL2 secreted from tumor cells computational analysis): K.-Y. Teng, J. Han, X. Zhang, J. Yu, K. Ghoshal Writing, review, and/or revision of the manuscript: K.-Y. Teng, J. Han, binds to the CCL2 nAb. This can trigger NK-cell activation (i.e., X. Zhang, S.-H. Hsu, L.A. Snyder, W.L. Frankel, M.A. Caligiuri, J. Yu, K. Ghoshal enhancement of IFNg secretion and cytotoxicity), causing Administrative, technical, or material support (i.e., reporting or organizing enhanced killing of liver tumor cells. In this study, we observed data, constructing databases): K.-Y. Teng, N. Wani, J. Barajas, K. Ghoshal enhanced cytotoxic capability of NK cells against CCL2 nAb– Study supervision: S.T. Jacob, J. Yu, K. Ghoshal treated hepatoma cells. We also noticed that the level of IFNg was Other (review of slides): W.L. Frankel higher in the NK cells isolated from the livers of tumor-bearing KO Other (provided valuable suggestions throughout this study, participated in monthly discussions, and critically reviewed the manuscript): S.T. Jacob mice treated with CCL2 nAb, reinforcing the notion that NK cells were activated by the CCL2 nAb. Consistent with other literatures that cytotoxic NK cells activate caspase-3, -7, and PARP (43, 44), Acknowledgments we found increased cleaved caspase-7 and cleaved PARP in the We thank The Ohio State University Comprehensive Cancer Center Com- – parative Pathology and Mouse Phenotyping core for pathologic analysis. We CCL2 nAb treated mice (Supplementary Fig. S10). Inhibition thank Dr. Gretchen Darlington (Baylor College of Medicine) for providing of STAT3 activity has been shown to sensitize hepatocellular Hepa1-6 cells, Dr. Huban Kutay for breeding miR-122 KO mice, and Vivek carcinoma cells to NK cell–mediated cytotoxicity (45). Interest- Chowdhary, Ryan Reyes, and Dr. Hui-Lung Sun for valuable discussions. ingly, our data show that STAT3 activity is reduced in the CCL2 – – nAb treated mice tumors (Fig. 5A C), suggesting a reciprocal Grant Support regulation. This work was supported, in part, by NIH grants R01CA193244 (to Novel therapeutic strategies for hepatocellular carcinoma are K. Ghoshal) and R01CA086978 (to S.T. Jacob and K. Ghoshal) as well urgently needed, as existing therapies could not cure or prolong as by Pelotonia IDEA grants (to J. Yu and K. Ghoshal) and Pelotonia patients' survival even by 6 months (5). In this study, we highlight Graduate Fellowship (to K.-Y. Teng). the importance of CCL2 in contributing to chronic liver inflam- The costs of publication of this article were defrayed in part by the fi payment of page charges. This article must therefore be hereby marked mation and tumorigenesis. Furthermore, the ef cacy of a CCL2- advertisement fi in accordance with 18 U.S.C. Section 1734 solely to indicate speci c nAb in the miR-122 mouse model underscores the fea- this fact. sibility of immune-based therapy in inflammatory liver disease and cancer. Our study shows that CCL2 nAb immunotherapy Received March 3, 2016; revised October 24, 2016; accepted November 16, elicits two distinct mechanisms that act in concert to inhibit 2016; published OnlineFirst December 15, 2016.

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322 Mol Cancer Ther; 16(2) February 2017 Molecular Cancer Therapeutics

Blocking the CCL2−CCR2 Axis Using CCL2-Neutralizing Antibody Is an Effective Therapy for Hepatocellular Cancer in a Mouse Model

Kun-Yu Teng, Jianfeng Han, Xiaoli Zhang, et al.

Mol Cancer Ther 2017;16:312-322. Published OnlineFirst December 15, 2016.

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