blocking the ccl2 ccr2 axis using ccl2- neutralizing ...neutralizing antibody is an effective...

Large Molecule Therapeutics

Blocking the CCL2–CCR2 Axis Using CCL2-Neutralizing Antibody Is an Effective Therapy forHepatocellular Cancer in a Mouse ModelKun-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

Hepatocellular carcinoma, a deadly disease, commonlyarises in the setting of chronic inflammation. C-C motif chemo-kine ligand 2 (CCL2/MCP1), a chemokine that recruits CCR2-positive immune cells to promote inflammation, is highly upre-gulated in hepatocellular carcinoma patients. Here, we exam-ined the therapeutic efficacy of CCL2–CCR2 axis inhibitorsagainst hepatitis and hepatocellular carcinoma in the miR-122 knockout (a.k.a. KO) mouse model. This mouse modeldisplays upregulation of hepatic CCL2 expression, which corre-lates with hepatitis that progress to hepatocellular carcinomawith age. Therapeutic potential of CCL2–CCR2 axis blockadewas determined by treating KO mice with a CCL2-neutralizingantibody (nAb). This immunotherapy suppressed chronic liverinflammation in these mice by reducing the population of

CD11highGr1þ inflammatory myeloid cells and inhibitingexpression of IL6 and TNFa in KO livers. Furthermore, treatmentof tumor-bearing KO mice with CCL2 nAb for 8 weeks signif-icantly reduced liver damage, hepatocellular carcinoma inci-dence, and tumor burden. Phospho-STAT3 (Y705) andc-MYC, the downstream targets of IL6, as well as NF-kB, thedownstream target of TNFa, were downregulated upon CCL2inhibition, which correlated with suppression of tumor growth.In addition, CCL2 nAb enhanced hepatic NK-cell cytotoxicityand IFNg production, which is likely to contribute to the inhi-bition of tumorigenesis. Collectively, these results demonstratethat CCL2 immunotherapy could be an effective therapeuticapproach against inflammatory liver disease and hepatocellularcarcinoma. Mol Cancer Ther; 16(2); 312–22. �2016 AACR.

IntroductionHepatocellular carcinoma is the most common liver cancer

and the second leading cause of cancer-related deaths world-wide (1, 2). The incidence of hepatocellular carcinoma hastripled in the United States because of the precipitous increasein nonalcoholic fatty liver disease and hepatitis C virus (HCV)

infection in the past 20 years (3, 4). Furthermore, sorafenib, theonly FDA-approved drug for advanced hepatocellular carcino-ma extends overall survival by only 2.8 months (5). Recentclinical trials have shown that it is ineffective as an adjuvanttherapy after resection or ablation of the tumor (6). Thus, thereis an urgent need to develop novel therapeutic strategies for thetreatment of hepatocellular carcinoma.

miR-122 is the most abundant liver-specific miRNA in verte-brates, and loss of miR-122 is associated with metastasis andpoor prognosis in hepatocellular carcinoma patients (7). Wepreviously found that development of spontaneous hepatocel-lular carcinoma with age mimicked different stages of tumorprogression (e.g., steatohepatitis, fibrosis, primary, and meta-static hepatocellular carcinoma) in miR-122 knockout (KO)mice (8, 9). We also observed that miR-122 depletion in themouse liver leads to upregulation of chemokine (C-C motif)ligand 2 (CCL2), which recruits CCR2þCD11bhighGr1þ

immune cells to the liver (8). These cells, in turn, produceproinflammatory cytokines, including IL6 and TNFa in the liver,resulting in hepatitis and eventually hepatocellular carcinoma.

CCL2 is known to be involved in the pathogenesis of severaldiseases characterized by monocytic infiltrates, such as psori-asis, rheumatoid arthritis, and atherosclerosis (10, 11). CCL2also promotes local inflammation and macrophage infiltrationin the chronically injured liver (12). Very recently, Li andcolleagues demonstrated that a chemical inhibitor of CCR2,the receptor of CCL2, inhibited hepatocellular carcinomadevelopment by reducing monocytes/macrophage infiltrationand M2 macrophage polarization as well as CD8þ T-cell

1Molecular, Cellular and Developmental Biology Program, The Ohio State Uni-versity, Columbus, Ohio. 2Department of Pathology, The Ohio State University,Columbus, Ohio. 3Comprehensive Cancer Center, Wexner Medical Center, TheOhio State University, Columbus, Ohio. 4Center for Biostatistics, The Ohio StateUniversity, Columbus, Ohio. 5Virology, Immunology and Medical Genetics, TheOhio State University, Columbus, Ohio. 6Center for Systems Biology, Massachu-setts General Hospital, Harvard Medical School, Boston, Massachusetts. 7Jans-sen Research and Development, LLC, Spring House, Pennsylvania. 8Departmentof Molecular and Cellular Biochemistry, The Ohio State University, Columbus,Ohio.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

K.-Y. Teng and J. Han contributed equally to this article.

Current address for Shun He: Massachusetts General Hospital (MGH), HarvardMedical School, Center for Systems Biology, Boston, MA.

Corresponding Authors: Kalpana Ghoshal, Department of Pathology, The OhioState University, 420 West 12th Avenue, 646C TMRF Building, Columbus, OH43210. Phone: 614-292-5688; Fax: 614-688-4245; E-mail:[email protected]; and Jianhua Yu, [email protected]

doi: 10.1158/1535-7163.MCT-16-0124

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

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activation in the murine xenograft model (13). However,whether targeting the CCL2–CCR2 axis by immunotherapycould be an effective therapeutic approach against chronicinflammation and hepatocellular carcinoma has not beenaddressed. In this study, we addressed this important questionusing a novel preclinical model, miR-122 KOmice that developchronic inflammation–driven hepatocellular carcinoma (8).Our results clearly showed that targeting CCL2–CCR2 signalingby CCL2 immunotherapy could be an alternative approach insuppressing hepatitis and hepatocellular carcinoma develop-ment. Furthermore, our studies revealed that CCL2-neutraliz-ing antibody (nAb) immunotherapy in miR-122 KO mousemodel involves suppression of CD11bhighGr1þ inflammatorymyeloid cell recruitment and enhancement of natural killer(NK) cell cytotoxicity. These observations underscore theimportance of targeting CCL2–CCR2 axis as a potential therapyin a subset of human hepatocellular carcinoma patients withchronic hepatic inflammation and high CCL2.

Materials and MethodsTreatment of miR-122 KOmice with CCL2 antibody and CCR2inhibitor

miR-122 KO mice were generated as described previously (8).Animals were housed inHelicobacter-free facility under a 12-hourlight/dark cycle. Animals were handled following the guidelinesof theOhio State University Institutional Laboratory Animal CareCommittee.

CCL2 nAb (anti-mouse CCL2; clone C1142) was gener-ously provided by Janssen (14). miR-122 KO mice wereinjected intraperitoneally with CCL2 nAb (2 mg/kg). Thecontrol group was injected with vehicle (PBS). To study thefunction of CCL2 in hepatitis, 4-month-old KO mice weretreated with CCL2 nAb twice a week for 4 weeks. To studythe role of CCL2 in hepatocellular carcinoma development,12-month-old KO mice were injected with CCL2 nAb twice aweek for 8 weeks.

CCR2 inhibitor (Tocris Bioscience, cat# 2089) was givento KO mice in the drinking water daily (10 mg/kg) for 4 weeks.The control group for CCR2 inhibitor was fed with regularwater.

Flow cytometric analysisMurine mononuclear cells from livers were isolated as

described previously (8, 15). Erythrocytes were lysed usingRBC lysis buffer (BioLegend). Cells isolated from livers weretreated with Fc Block antibody (anti CD16/32, BD Biosciences).Cells were stained with mouse-specific immune cell surfacemarkers for 30 minutes at 4�C. The following anti-mouseantibodies were used at a dilution of 1:200: CD3-APC, CD3-PerCP-Cy5.5, NK1.1-APC, NK1.1-PE, CD19-FITC, CD69-FITC,CD27-PE-Cy7, CD11b-PE, CD11b-PerCP-Cy5.5, Gr1 V450,and IFNg FITC (BioLegend). For staining of IFNg , cells weretreated with Cytofix/Cytoperm (BD Biosciences) followinginitial cell surface staining, and then, intracellular staining wasperformed.

NK cytotoxicity assayTumor-derived NK cells were enriched by NK Cell Isolation

Kit II (Miltenyi Biotec) and then sorted using a FACSAria II CellSorter (BD Biosciences). Hepa1-6, a mouse hepatoma cell line,was generously provided by Dr. Gretchen Darlington (Baylor

College of Medicine, Houston, TX). Although we did notauthenticate these cells, they exhibited characteristics and geneexpression profile of mouse hepatoma cells. Hepa cells werelabeled with Chromium-51 (Cr-51) and then preincubatedwith C1142 antibody or isotype antibody at a concentrationof 10 mg/mL for 1 hour. Then, a standard 4-hour Cr-51 releaseassay (16) was performed to access cytotoxicity of mouse NKcells against Hepa cells.

ELISAFor a-fetoprotein (AFP), mouse serum was collected by man-

dibular punch (before treatment) or cardiac punch (after treat-ment). KOmouse older than 10months would be monitored fortheir body weight and serumAFP level by every 2weeks. AFP levelwas quantified by DRG AFP (Alpha Fetoprotein) Kit (DRG, cat#EIA-1468).

For IFNg , mouse liver mononuclear cells were isolated asdescribed previously (8, 15). Hepa cells cultured in DMEMcontaining 10% FBS, were preincubated with C1142 (CCL2nAb) at a concentration of 10 mg/mL for 1 hour. Then, 106

mononuclear cells were incubated with the same number ofHepa cells per well in a 96-well V-bottom plate at 37�C for 24hours. Culture supernatants were collected for IFNg estimationusing mouse IFNg ELISA Ready-SET-Go Kit (eBioscience, cat#88-7314-88).

MRIMRI of liver tumors in miR-122 KO mice was performed as

described previously (17).

Serologic, histologic, and immunohistochemical analysisSerum was isolated from mice by cardiac puncture after CO2

asphyxiation and cervical dislocation and stored at �80�C. Bio-chemical analysis of serum enzymeswas performed using VetACE(Alfa Wassermann system) as described previously (8). Macro-scopic tumors were counted, dissected, and weighed along withbenign liver tissues. A fraction of tissues was fixed in 4% para-formaldehyde as described previously (8), and the rest was snapfrozen for RNA and protein analysis.

For histology, paraffin-embedded tissue sections (4 mm) onglass slides were stained with hematoxylin and eosin (H&E) orimmunohistochemical analysis with Ki-67, AFP, and glypican3 (GPC3)-specific antibodies as described previously (8).Inflammation score was generated through blinded evaluationof H&E-stained slides (�100 magnification) as described pre-viously (8). Immunohistochemical analysis was done asdescribed previously (8) and quantified by ImageJ (imagej.nih.gov/ij/). Microscopic images were taken by phase-contrastmicroscope (BX41TF, OLYMPUS) and camera (DP71, OLYM-PUS). cellSens Standard 1.13 (OLYMPUS) software was usedto capture images. Antibody information is provided in theSupplementary Material.

qRT-PCRTotal RNA was extracted from liver tissue using TRIzol (Life

Technologies, cat#15596018) followed by DNase I treatment.DNase-treated RNA was reverse transcribed into cDNA usingHigh-Capacity cDNA Reverse Transcription Kit (Applied Biosys-tems, cat# 4368813). qRT-PCR analysis of each sample performedin triplicate was performed using SYBR Green chemistry. Gene

CCL2 Immunotherapy Suppresses Hepatitis and HCC

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expression was normalized to Gapdh. Relative expression wascalculated by DDCt method. Primer sequences are provided inthe Supplementary Table S1.

Immunoblot analysisProteins were extracted from whole liver and tumor tissues by

SDS lysis buffer, followed by immunoblotting with primaryantibodies (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 analysisAll bar diagrams are presented as mean � SD. Two sample t

tests or ANOVA were used for analysis for comparison of two ormore groups. For the GEO data analysis, CCL2 expression fromthe quantile-normalized data was compared among tumortissue types with ANOVA. Holm procedure was used to adjustfor multiple comparisons. Fisher exact test was used to test thedifference of tumor incidence and tumor size between the CCL2nAb–treated and vehicle-treated groups. P values <0.05 wereconsidered significant and represented as asterisks (�, P value�0.05; ��, P value �0.01; ��, P value �0.001).

ResultsCCL2 is upregulated in primary human hepatocellularcarcinoma

To verify whether CCL2 plays a role in human hepatocellularcarcinoma development, we first examined its expression acrossindependent microarray datasets (hepatocellular carcinoma vs.normal liver) downloaded from Oncomine (18). To validateCCL2 expression, we chose The Cancer Genome Atlas, Masliver, and Guichard liver that contain large patient cohorts(18–20). CCL2 DNA copy number or mRNA expressionshowed a significant increase in tumor tissues compared withthe adjacent benign liver across four datasets (Table 1; refs.18–20). To further evaluate CCL2 expression and disease prog-nosis, we analyzed hepatocellular carcinoma RNA microarraydata (n ¼ 115, mainly HCV positive) from GEO database(GSE14323; ref. 20). The results showed that CCL2 RNAexpression was significantly elevated in hepatocellular carcino-mas, cirrhotic livers, and cirrhotic hepatocellular carcinomascompared with normal livers (Fig. 1). These data imply that

CCL2 might be critical for progression to cirrhosis and hepa-tocellular carcinoma. Thus, blocking the CCL2–CCR2 axisseems to be a reasonable therapeutic approach for hepatocel-lular carcinoma or even early stages of hepatocellular carcino-ma development, such as hepatitis and cirrhosis.

CCL2–CCR2 blockade reduced chronic inflammation and liverinjuries in miR-122 KO mice

CCL2 is a major chemokine that is known to cause variousinflammatory diseases in humans (10, 21). Similarly, upregula-tion of CCL2 inmiR-122 KO liver correlated with chronic inflam-mation (8). To determine whether CCL2 is a key player inhepatitis, and blocking CCL2 could inhibit liver inflammation,CCL2nAbwas administered to4-month-oldmiR-122KOmicebyan intraperitoneal injection for 4 weeks (Fig. 2A). CCL2 nAb(C1142) displayed certain specificity toward murine CCL2 (14).In addition, CCL2 nAb was well tolerated in mice (14). Further-more, its antitumor efficacy has been demonstrated in murinebreast cancer metastasis (22), lung (23), brain (24), and prostatecancer (25, 26) models, and no adverse effects were reported.

To evaluate the effects of CCL2 nAb in suppressing hepatitis,we treated 4-month-old miR-122 KO mice with CCL2 nAb,followed by histopathologic and serologic analyses. As reportedin other mouse models (14, 22–26), CCL2 nAb is well toleratedin miR-122 KO mice. There were no obvious changes in theappearance, activity, or body weight (Supplementary Fig. S1).Liver histology showed reduced immune cell infiltration aftertreating with CCL2 nAb (Fig. 2B). Similarly, CD45 immuno-histochemical analysis revealed that CCL2 nAb treatmentdecreased leukocyte accumulation in the liver (Fig. 2C). Nota-bly, a significant reduction in serum alanine aminotransferase

Table 1. CCL2 expression across four independentmicroarrays in hepatocellularcarcinoma patients

Cancer vs. normal Array type P Fold change Sample size

Mas Liver RNA 0.012 3.324 n ¼ 115Guichard Liver cohort 2 DNA 0.036 2.025 n ¼ 52TCGA Liver DNA 0.003 2.063 n ¼ 212Guichard Liver cohort 1 DNA 0.026 2.153 n ¼ 185

NOTE: CCL2 expression in human clinical samples (cancer vs. normal). Dataprovided by Oncomine (www.oncomine.com, January 2016, Thermo FisherScientific). TCGA database (TCGA Research Network: http://cancergenome.nih.gov) contains >200 hepatocellular carcinoma specimens of different etiol-ogy (HBV, HCV, alcohol, and NAFLD). Mas liver is an RNA array, which contains>100 hepatocellular carcinoma samples (mostly HCV positive; ref. 20). TheGuichard liver DNA array consisting of 237 hepatocellular carcinomas of variousrisk factors, including HBV, HCV, alcohol, and NAFLD, was analyzed by exosomesequencing and SNP array (19).Abbreviation: TCGA, The Cancer Genome Atlas.

Figure 1.

CCL2 is overexpressed in human cirrhotic livers, as well as cirrhotic and non-cirrhotic hepatocellular carcinomas (HCC). CCL2 RNA expression retrieved fromGEO data (www.ncbi.nlm.nih.gov/geo/) with accession_GSE14323 (20) wascompared among the four types of liver tissues, and statistical analysiswas doneusing ANOVA.

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(ALT) and AST levels corroborated that the liver damage due tohepatitis in KO mice could be reversed by inhibition of CCL2(Fig. 2D). Alkaline phosphatase (ALP), a marker of biliarydysfunction, was also reduced by approximately 50%; however,it was not statistically significant (Fig. 2D). This result impliesthat blocking CCL2 may not be able to fully rescue hepato-biliary damage because ALP is a target of miR-122, which isderepressed in miR-122–depleted livers (8, 9). Serum gamma-glutamyl transferase and bilirubin (total and direct) levels werenot significantly altered in CCL2 nAb–injected mice (Supple-mentary Table S2).

We have shown that activation of the CCL2–CCR2 axis inmiR-122 KO liver correlates with the recruitment ofCD11bhighGr1þ cells and hepatic inflammation and injuries(8). In addition, CCR2 inhibitors showed anti-inflammatoryeffects in the animal models with different diseases, such asdiabetic nephropathy (27), kidney hypertension (28), steato-hepatitis (29), and renal atrophy (30) models, and no adverseeffects were reported. We decided to test whether treatment ofmice with a CCR2-specific inhibitor could reduce liver inflam-mation in KO mice. Indeed, KO mice fed water containing aCCR2 inhibitor exhibited reduced hepatic inflammation, as

Figure 2.

CCL2 nAb therapy reduces chronic liverinflammation and liver damage in adultmiR-122 KO mice. A, The schematicpresentation of the CCL2 nAbtreatment. HCC, hepatocellularcarcinoma. B, Liver histology of4-month-old male KO mice injectedintraperitoneally with CCL2 nAb(2 mg/kg, n¼ 5) or PBS (vehicle, n¼ 5).Representative images of H&E-stainedliver sections [scale bars, 100 (top) and25 mm (bottom)]. Right, quantitationof the inflammation scores generatedthrough blinded evaluation of H&E-stained liver sections (�100magnification, 100 mm). Theinflammatory area was quantified byImageJ of three randomly chosen fields(�100 magnification, 100 mm) peranimal (n¼ 5).C,Representative imagesof CD45-stained liver sections [scalebars, 100 (top) and 25 mm (bottom)].Right, CD45þ areas quantified byImageJ of three randomly chosen fields(�100 magnification, 100 mm) peranimal (n¼ 5).D,Analysis of serumALT,AST, and ALP levels (n ¼ 5). n.s., notsignificant.






revealed by the dramatic reduction in bridging inflammation(Supplementary Fig. S2). Collectively, these data showed thatblocking the CCL2–CCR2 axis could effectively inhibit hepa-titis in miR-122 KO mice.

CCL2 nAb therapy inhibited hepatitis by reducingCD11bhighGr1þ inflammatory myeloid cell accumulation

It is well established that immune cells play a critical rolein liver inflammation (31). Our previous study showedthat CD11bhighGr1þ inflammatory myeloid cells excessivelyaccumulated in miR-122–depleted livers (8). In addition,CD11bhighGr1þ cells, which express CCR2 (8), are known tobe recruited to the liver via CCL2–CCR2 interaction (32).Therefore, we quantitated CD11bhighGr1þ cells in KO liversdepleted of CCL2 or CCR2. Flow cytometric analysis of theimmune cells isolated from livers and spleen showed reducedCD11bhighGr1þ cell population in the KO mice treated with

CCL2 nAb without significant change in their numbers inperipheral blood (Fig. 3A and B; Supplementary Fig. S3).Consistent with other studies (12, 13), we also found decreasednumber of hepatic macrophages in liver by IHC upon blockingthe CCL2–CCR2 axis (Supplementary Fig. S4). However, nosignificant alterations in the number of hepatic B, T, and NKcells were observed (Supplementary Fig. S5A–S5D). In addi-tion, Immune cells isolated from CCR2 inhibitor–treated KOmice showed reduced hepatic CD11bhighGr1þ cells (Supple-mentary Fig. S6), further supporting the idea that blocking theCCL2–CCR2 axis reduces CD11bhighGr1þ inflammatory mye-loid cells in liver.

IL6 and TNFa are two proinflammatory cytokines that drivehepatitis and hepatocellular carcinoma in the liver (33).Previously, we have shown that CD11bhighGr1þ inflammatorymyeloid cells produce IL6 and TNFa in the miR-122 KO liver(8). Therefore, it is expected that hepatic IL6 and TNFa levels

Figure 3.

CCL2 nAb treatment decreases liverCD11bhighGr1þ cell population and Il6and Tnfa expression in adult KOmice.A,Injection of 4-month-old KO mice CCL2nAb reduced the hepatic CD11bhighGr1þ

population. Right, quantitation ofhepatic CD11bhighGr1þ inflammatorymyeloid cells (n ¼ 5). B, ReducedCD11bhighGr1þ population in spleen.Right, quantitation of splenicCD11bhighGr1þ inflammatory myeloidcells (n ¼ 5). C, qRT-PCR analysis ofinflammatory cytokines andchemokines.

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would be suppressed if the population of CD11bhighGr1þ

cells is decreased in the CCL2 nAb–treated mice. Indeed, theexpressions of both cytokines were reduced in the livers ofCCL2 nAb–treated group (Fig. 3C). In addition, we alsoobserved a decrease in Il1b expression upon CCL2 nAbtreatment (Fig. 3C). Notably, CCL2 nAb treatment did notalter the expressions of other inflammatory cytokines orchemokines, such as Ccl5, Ccl8, Cxcl9, Il10, Ccl2, or Ccr2(Fig. 3C). Taken together, these results suggest that inhibitionof the CCL2–CCR2 axis reduces liver inflammation by sup-pressing CD11bhighGr1þ inflammatory myeloid cells, whichleads suppression of the two major proinflammatory cyto-kines, IL6 and TNFa.

Attenuation of hepatocarcinogenesis in miR-122 KO micetreated with CCL2 nAb

As aforementioned, CCL2 is highly expressed in the tumortissues of hepatocellular carcinoma patients (Fig. 1; Table 1).Besides, blocking CCL2 suppresses chronic liver inflammationat early stages of hepatocellular carcinoma development in theKO mice (Fig. 2). Therefore, we speculated that impedingCCL2 function would suppress development of spontaneousliver tumors in KO mice. To test this, male KO mice (�12months old) bearing liver tumors confirmed by the serumAFP levels were randomly assigned to two treatment groups.Comparison of the serum AFP level with the tumor size in alarge number of KO mice revealed that mice with serum AFP

Figure 4.

Blocking CCL2 function suppresseshepatocellular carcinoma (HCC)development in KO mice. A, Theschematic depiction of theimmunotherapy protocol and therepresentative MRI of liver tumors (axialview, T1 weighted) for 12-month-oldmiR-122 KOmice. B, Five representativeimages of the livers harvested from thevehicle (n ¼ 9) and CCL2 nAb–treatedmice (n ¼ 11). C, Flow cytometricanalysis of hepatic CD11bhighGr1þ

immune cells in the vehicle- and CCL2nAb–treated mice. D, Representativeimages of liver histology, AFP, andGPC3 immunohistochemical images ofthe vehicle- and nAb-treated groups[scale bars: 250 (top), 100 (middle), and25 mm (bottom)].






levels >12 IU/mL usually developed tumors (SupplementaryFig. S7A). These ELISA data were further confirmed by MRI(Fig. 4A). KO mice were injected intraperitoneally with CCL2nAb (2 mg/kg) or vehicle twice a week for 8 weeks (Fig. 4A),and the tumor burden was analyzed one week after the lastinjection. We chose male KO mice for this study as these mice,like men, have higher hepatocellular carcinoma penetranceand tumor burden (8, 9).

Significant phenotypic differences were observed in the liverbetween the vehicle- and the CCL2 nAb–treated groups after 8weeks of CCL2 immunotherapy; however, there were no obvi-ous changes in the appearance, activity, or body weightbetween the two groups (Supplementary Fig. S7B). Althoughthe majority of the PBS-injected mice developed large macro-scopic liver tumors, CCL2 nAb–treated mice mostly developedsmaller and fewer tumors (Fig. 4B; Table 2). Consistent with theprevious data (Fig. 3A), we found hepatic CD11bhighGr1þ cellpopulation was reduced in the CCL2 nAb–treated tumor-bear-ing mice compared with vehicle-treated group (Fig. 4C). Inaddition, serum AFP and ALT levels were lower in theCCL2 nAb–treated mice (Table 2). Furthermore, the majorityof the hepatocellular carcinomas in the vehicle-treated groupwere AFP and GPC3 positive and grade 2–3 (intermediate topoorly differentiated) hepatocellular carcinomas, whereasthose in CCL2-inactivated mice were mostly grade 1 (welldifferentiated) hepatocellular carcinomas, AFP and GPC3 neg-ative (Fig. 4D). These results suggest that CCL2 blockade is notonly anti-inflammatory but also antitumorigenic in the liver.

Oncogenic signaling downstreamof IL6 and TNFawas blockedin miR-122 KO tumors upon CCL2 nAb therapy

Because CCL2 nAb treatment suppressed the level of IL6 andTNFa in the liver (Fig. 3C), we hypothesized that the downstreamoncogenic signaling pathways of IL6 and TNFa, namely STAT3andNF-kB, respectively, would be inhibited in theCCL2-depletedmice. Indeed, immunoblot analyses showed that phospho-STAT3(Y705) level was dramatically reduced, whereas total STAT3 leveldecreased only slightly in the tumors of CCL2 nAb–treated mice(Fig. 5A and C). Reduced nuclear STAT3 levels in the CCL2 nAb–treated mice confirmed that CCL2 inhibition suppresses STAT3functions in liver tumors (Fig. 5B and C). Similarly, the level oftwo major NF-kB subunits, p65 and p50, was reduced in bothwhole liver lysates andnuclear extracts (Fig. 5B andC). The level ofc-MYC, a downstream target of STAT3, was also reduced in theCCL2 nAb–treated mice (Fig. 5A and C). Histologically, p65 andc-MYC both showed reduced expression in the transformedhepatocytes, whereas pSTAT3 was decreased in immune cells aswell as tumor cells (Supplementary Fig. S8).

c-MYC is a well-known oncogene that promotes hepatocel-lular carcinoma proliferation (34, 35). Therefore, Ki-67 expres-sion, a marker of cell proliferation, was evaluated in tumor cellsby IHC. As expected, the number of Ki-67–positive cells wasmuch less in both CCL2-depleted tumor and nontumor tissues(Fig. 5D; Supplementary Fig. S9). PCNA, another cellularproliferation marker, was also reduced in the CCL2 nAb–treated tumor extracts (Fig. 5E). Of note, we found cell apo-ptosis is increased in the CCL2 nAb–treated group as demon-strated by slight increase in cleaved PARP and cleaved caspase-7levels (Supplementary Fig. S10). These data suggest that inhi-bition of hepatocellular carcinoma growth by targeting CCL2 is,at least in part, through the inhibition of STAT3 and NF-kBsignaling and c-MYC expression.

CCL2 nAb activatedNK cells in the tumormicroenvironment tosuppress liver cancer

Cancer immunotherapy has been widely conducted in theclinic over the past several years (36–38). As antibody-basedimmunotherapy is applied in this study, and NK cell is knownto be activated by the Fc domain of antibodies (37), we investi-gatedwhether enhancedNK cytotoxicity could be amechanismoftumor suppression in CCL2 nAb–treatedmice. Although the totalnumber of NK cells did not change significantly after CCL2 nAbimmunotherapy (Supplementary Fig. S4C and S4D), the activityof NK cells increased, as demonstrated by the upregulation ofhepatic Ifng expression (Fig. 6A). To verify the source of theelevated IFNg , primary hepatic immune cells isolated fromtumor-bearing KO mice were cocultured with mouse hepatoma(Hepa1-6) cells in the presence of CCL2 nAb. The culture super-natants were subjected to IFNg ELISA, and cells were analyzed byflowcytometry. BothELISA and intracellular stainingdata showedthat only NK cells (Fig. 6B and C) were the major producers ofIFNg . In addition, the cell surface expressionofCD69, amarker foractivated NK cells, increased when NK cells were cocultured withCCL2 nAb–treated mouse hepatoma (Hepa) cells (Fig. 6D),suggesting that NK cells could be activated upon exposure ofhepatoma cells to CCL2 nAb. To confirm the activated NK cellscould suppress cancer cell growth, NK-cell cytotoxicity wasassessed by Cr-51 release assay (16, 39). To this end, Hepa cellswere first labeled with Cr-51, followed by incubation with CCL2nAb and subsequently, with tumor-derived NK cells. Enhancedrelease of Cr-51 fromHepa cells is indicative of higher cytotoxicityof NK cells. Indeed, tumor-derived NK cells cocultured with Hepacells and CCL2 nAb exhibited higher cytotoxicity (Fig. 6E). Thisresult also suggested that the increased cytotoxicity of NK cellscould be triggered independent of other immune cells. Collec-tively, these results demonstrate that CCL2 nAb impedes tumorgrowth, at least in part, by activating NK cells.

DiscussionCCL2 is a chemotactic factor to tumor cells and inflammatory

macrophage/monocytes. Blocking the CCL2–CCR2 axis by CCR2inhibitor decreases the intrahepatic macrophage infiltration in adiet-induced steatohepatitis model (29). CCL2 Spiegelmer, aCCL2 RNA antagonist, suppresses the macrophage infiltration inthe carbon tetrachloride (CCl4) and methionine–choline-defi-cient diet-induced liver injury models (12). Recently, Li andcolleagues also demonstrated the importance of CCL2–CCR2axis in different hepatocellular carcinoma models (13). In line

Table 2. Histopathologic and serologic analysis of tumor-bearing miR-122 KOmice treated with CCL2 nAb for 8 weeks

Vehicle CCL2 nAb P

HCC incidence 8/9 5/11 0.07Tumor number 2.67 � 1.22 0.73 � 0.79 0.0004Sum of tumor diameter (cm) 1.71 � 1.24 0.57 � 0.65 0.01Serum ALT (U/L) 151.89 � 68.06 73.57 � 28.5 0.027Serum AFP (IU/mL) 14.07 � 3.51 9.95 � 0.61 0.001

NOTE: All measurements are presented as mean� SD. P values were calculatedusing Student two-tailed t test (for tumor number, sumof tumor diameter, tumorweight, ALT, and AFP) or Fisher exact test (for hepatocellular carcinomaincidence).Abbreviation: HCC, hepatocellular carcinoma.

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with the animal models mentioned above, it is clear that CCL2 iscrucial for regulating the inflammatory milieu in liver. In thisstudy, we aimed at elucidating the efficacy of CCL2 immunother-apy against chronic hepatitis and hepatocellular carcinoma in amurine model. This is a logical extension of our previous studythat demonstrated a causal role of CCL2 in the inflamed liver thatprogressively leads to hepatocellular carcinoma in miR-122 KOmice (8). Analysis of GEO, Oncomine, and Protein Atlas data-bases showed increased CCL2 expression in patients sufferingfrom hepatocellular carcinoma (18, 20, 40). These data suggestCCL2 may play a role in liver cancer. To further clarify the role ofthe CCL2–CCR2 axis in chronic liver inflammation and tumor

development, we used CCL2 nAb to block CCL2 signaling. Ourdata suggest that CCL2 nAb inhibits tumor development in miR-122 KO mice through at least two distinct mechanisms: suppres-sing IL6 and TNFa production by reducing the infiltration ofCD11bhighGr1þ inflammatory myeloid cells to the liver andenhancing NK-cell cytotoxicity (Supplementary Fig. S11).

An elegant study demonstrating the correlation of CCL2 pro-tein level with poor survival of hepatocellular carcinoma patientswas published (13) during the preparation of our manuscript. Liand colleagues showed that a clinically relevant CCR2 antagonistinhibitedmurine hepatocellular carcinoma progression by reduc-ing M2-type tumor-associated macrophage population and

Figure 5.

CCL2 nAb therapy suppresseshepatocellular carcinoma developmentin KO mice by inhibiting tumor cellproliferation. A, Immunoblotting of thekey downstream proteins of IL6 andTNFa in KO tumors. B, Immunoblottingof NF-kB subunit and STAT3 in thetumor nuclear extracts. C, Respectiveprotein levels were normalized to thoseof GAPDH or KU86. pSTAT3 level wasnormalized to tSTAT3, which isseparated 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 ofthree randomly chosen fields (�200magnification, 50 mm) per animal(n ¼ 5). E, Immunoblotting of PCNAin liver tumors.






increasing CD8þ cytotoxic T cells in orthotopic and subcutaneoushepatocellular carcinoma models (13). Their study supportsthe notion that blocking the CCL2–CCR2 axis has profoundeffects in modulating liver immune system to suppress hepato-cellular carcinoma progression. While Li and colleagues foundmacrophage and CD8þ T cells are regulated by a CCR2 inhibitorin xenograft models, we found CD11bhighGr1þ inflammatorymyeloid cells and NK cells could be modulated by CCL2 nAbin miR-122 KO mouse livers. Thus, these studies (ours and Liand colleagues') demonstrated that targeting CCL2–CCR2 axisby blocking the receptor or the ligand could be an effectivehepatocellular carcinoma therapy. In the present study, we used

our unique animal model to study the role of CCL2 in liverinflammation and hepatocellular carcinoma progression inlight of our previous work (8). We demonstrated two differenttypes of immune cells, including CD11bhighGr1þ inflammatorymyeloid cells and NK cells, that play key roles in regulating liverinflammation and tumor development after blocking CCL2function. In addition, CD11bhighGr1þ inflammatory myeloidcells promote tumorigenesis by increasing tumor cell prolifer-ation via IL6 and TNFa. Both IL6 and TNFa are well-knownproinflammatory cytokines that activate oncogenic transcrip-tion factor STAT3 to promote hepatocellular carcinoma devel-opment (33, 41). Consistent with the previous findings, our

Figure 6.

CCL2 nAb activates NK cells in KOmouse livers. A, qRT-PCR analysis ofhepatic Ifng in CCL2 nAb–treatedmice. B, IFNg quantification by ELISAin the supernatants of cultured Hepaand hepatic immune cells. C,Intracellular flow cytometric analysisof IFNg in the cocultured immunecells. D, Flow cytometric analysis ofthe activated NK-cell surface marker,CD69. E, Cytotoxicity of hepatic NKcells toward CCL2 nAb–treated Hepacells.

Teng et al.





current data reveal that CCL2 nAb reduces accumulation ofCD11bhighGr1þ cells, which decreases IL6 and TNFa levels inthe tumor and surrounding liver tissues, and reduces theactivation of STAT3 and the expression of c-MYC. Ours andLi and colleagues' data demonstrated that targeting the CCL2–CCR2 axis by blocking the receptor or the ligand could be aneffective hepatocellular carcinoma therapy. In future, it wouldbe interesting to investigate whether the combination of bothwould be a more effective therapeutic approach.

NK cells are keymodulators of liver diseases. Activated NK cellssuppress liver disease progression from chronic hepatitis to hepa-tocellular carcinoma (42). On the basis of ours and Li andcolleagues' data (13), CCL2 is expressed in liver tumors of bothmouse and human origin. The CCL2 nAb binds to Fc receptor(CD16) onNK cells (37), and the CCL2 secreted from tumor cellsbinds to the CCL2 nAb. This can trigger NK-cell activation (i.e.,enhancement of IFNg secretion and cytotoxicity), causingenhanced killing of liver tumor cells. In this study, we observedenhanced cytotoxic capability of NK cells against CCL2 nAb–treated hepatoma cells. We also noticed that the level of IFNg washigher in theNKcells isolated from the livers of tumor-bearingKOmice treated with CCL2 nAb, reinforcing the notion that NK cellswere activated by the CCL2 nAb. Consistent with other literaturesthat cytotoxic NK cells activate caspase-3, -7, and PARP (43, 44),we found increased cleaved caspase-7 and cleaved PARP in theCCL2 nAb–treated mice (Supplementary Fig. S10). Inhibitionof STAT3 activity has been shown to sensitize hepatocellularcarcinoma cells to NK cell–mediated cytotoxicity (45). Interest-ingly, our data show that STAT3 activity is reduced in the CCL2nAb–treated mice tumors (Fig. 5A–C), suggesting a reciprocalregulation.

Novel therapeutic strategies for hepatocellular carcinoma areurgently needed, as existing therapies could not cure or prolongpatients' survival even by 6months (5). In this study, we highlightthe importance of CCL2 in contributing to chronic liver inflam-mation and tumorigenesis. Furthermore, the efficacy of a CCL2-specific nAb in the miR-122 mouse model underscores the fea-sibility of immune-based therapy in inflammatory liver diseaseand cancer. Our study shows that CCL2 nAb immunotherapyelicits two distinct mechanisms that act in concert to inhibit

hepatitis and hepatocellular carcinoma development, and pro-vides rationales for future clinical trials in hepatocellular carci-noma patients with chronic inflammation.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K.-Y. Teng, L.A. Snyder, J. Yu, K. GhoshalDevelopment of methodology: K.-Y. Teng, J. Han, S. He, K. GhoshalAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.-Y. Teng, J. Han, S.-H. Hsu, S. He, J. Barajas,K. GhoshalAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.-Y. Teng, J. Han, X. Zhang, J. Yu, K. GhoshalWriting, review, and/or revision of the manuscript: K.-Y. Teng, J. Han,X. Zhang, S.-H. Hsu, L.A. Snyder, W.L. Frankel, M.A. Caligiuri, J. Yu, K. GhoshalAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K.-Y. Teng, N. Wani, J. Barajas, K. GhoshalStudy supervision: S.T. Jacob, J. Yu, K. GhoshalOther (review of slides): W.L. FrankelOther (provided valuable suggestions throughout this study, participated inmonthly discussions, and critically reviewed the manuscript): S.T. Jacob

AcknowledgmentsWe thank The Ohio State University Comprehensive Cancer Center Com-

parative Pathology and Mouse Phenotyping core for pathologic analysis. Wethank Dr. Gretchen Darlington (Baylor College of Medicine) for providingHepa1-6 cells, Dr. Huban Kutay for breeding miR-122 KO mice, and VivekChowdhary, Ryan Reyes, and Dr. Hui-Lung Sun for valuable discussions.

Grant SupportThis work was supported, in part, by NIH grants R01CA193244 (to

K. Ghoshal) and R01CA086978 (to S.T. Jacob and K. Ghoshal) as wellas by Pelotonia IDEA grants (to J. Yu and K. Ghoshal) and PelotoniaGraduate Fellowship (to K.-Y. Teng).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 3, 2016; revised October 24, 2016; accepted November 16,2016; published OnlineFirst December 15, 2016.

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2017;16:312-322. Published OnlineFirst December 15, 2016.Mol Cancer Ther Kun-Yu Teng, Jianfeng Han, Xiaoli Zhang, et al. an Effective Therapy for Hepatocellular Cancer in a Mouse Model

CCR2 Axis Using CCL2-Neutralizing Antibody Is−Blocking the CCL2

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