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Human Cancer Biology Inammatory Monocyte Mobilization Decreases Patient Survival in Pancreatic Cancer: A Role for Targeting the CCL2/CCR2 Axis Dominic E. Sanford 1 , Brian A. Belt 1 , Roheena Z. Panni 1 , Allese Mayer 4 , Anjali D. Deshpande 4 , Danielle Carpenter 3 , Jonathan B. Mitchem 1 , Stacey M. Plambeck-Suess 1 , Lori A. Worley 1 , Brian D. Goetz 1 , Andrea Wang-Gillam 5,6 , Timothy J. Eberlein 1,6 , David G. Denardo 2,3,6 , Simon Peter Goedegebuure 1,6 , and David C. Linehan 1,6 Abstract Purpose: To determine the role of the CCL2/CCR2 axis and inflammatory monocytes (CCR2 þ /CD14 þ ) as immunotherapeutic targets in the treatment of pancreatic cancer. Experimental Design: Survival analysis was conducted to determine if the prevalence of preoperative blood monocytes correlates with survival in patients with pancreatic cancer following tumor resection. Inflammatory monocyte prevalence in the blood and bone marrow of patients with pancreatic cancer and controls was compared. The immunosuppressive properties of inflammatory monocytes and macrophages in the blood and tumors, respectively, of patients with pancreatic cancer were assessed. CCL2 expression by human pancreatic cancer tumors was compared with normal pancreas. A novel CCR2 inhibitor (PF-04136309) was tested in an orthotopic model of murine pancreatic cancer. Results: Monocyte prevalence in the peripheral blood correlates inversely with survival, and low monocyte prevalence is an independent predictor of increased survival in patients with pancreatic cancer with resected tumors. Inflammatory monocytes are increased in the blood and decreased in the bone marrow of patients with pancreatic cancer compared with controls. An increased ratio of inflammatory monocytes in the blood versus the bone marrow is a novel predictor of decreased patient survival following tumor resection. Human pancreatic cancer produces CCL2, and immunosuppressive CCR2 þ macrophages infiltrate these tumors. Patients with tumors that exhibit high CCL2 expression/low CD8 T-cell infiltrate have significantly decreased survival. In mice, CCR2 blockade depletes inflammatory monocytes and macrophages from the primary tumor and premetastatic liver resulting in enhanced antitumor immunity, decreased tumor growth, and reduced metastasis. Conclusions: Inflammatory monocyte recruitment is critical to pancreatic cancer progression, and targeting CCR2 may be an effective immunotherapeutic strategy in this disease. Clin Cancer Res; 19(13); 3404–15. Ó2013 AACR. Introduction Pancreatic ductal adenocarcinoma, often called pancre- atic cancer, is an aggressive malignancy with a death rate nearly equal to its incidence and a 5-year survival of less than 5% (1). Pancreatic cancer is characterized by a unique- ly dense stroma that confers resistance to therapy (2–5). Within this stroma are abundant immunosuppressive mye- loid cells, which include monocytes/macrophages (2). Although it is appreciated that tumor-associated macro- phages (TAM) possess important tumor-promoting prop- erties in several malignancies (6), the contribution of monocyte mobilization from the bone marrow to the tumor has not been well studied. Here, we investigate the key role of monocyte migration from the bone marrow to the primary tumor and premetastatic site in pancreatic cancer, and show the efficacy of CCR2 blockade in the treatment of this disease. Monocytes are produced and stored in the bone marrow, and are CD45 þ /CD11b þ /CD115 þ /HLA-DR þ in humans and CD45 þ /CD11b þ /CD115 þ /F4/80 þ /MHCII in mice (7). However, monocytes are composed of heterogeneous subsets, which include resident monocytes and inflam- matory monocytes (8). Resident monocytes are CD16 þ / Authors' Afliations: Departments of 1 Surgery, 2 Medicine, and 3 Pathol- ogy and Immunology, Divisions of 4 Health Behavior Research and 5 Oncol- ogy, Department of Medicine, Washington University School of Medicine; and 6 Alvin J. Siteman Cancer Center, St. Louis, Missouri Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: David C. Linehan, Washington University School of Medicine, 660 South Euclid Ave. Box 8109, St. Louis, MO 63110. Phone: 314-747-2938; Fax: 314-222-6255; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-13-0525 Ó2013 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 19(13) July 1, 2013 3404 on June 21, 2018. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst May 7, 2013; DOI: 10.1158/1078-0432.CCR-13-0525

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Page 1: flammatory Monocyte Mobilization Decreases …clincancerres.aacrjournals.org/content/clincanres/19/13/3404.full.pdfInflammatory Monocyte Mobilization Decreases Patient Survival in

Human Cancer Biology

Inflammatory Monocyte Mobilization Decreases PatientSurvival in Pancreatic Cancer: A Role for Targeting theCCL2/CCR2 Axis

Dominic E. Sanford1, Brian A. Belt1, Roheena Z. Panni1, Allese Mayer4, Anjali D. Deshpande4,Danielle Carpenter3, Jonathan B. Mitchem1, Stacey M. Plambeck-Suess1, Lori A. Worley1, Brian D. Goetz1,Andrea Wang-Gillam5,6, Timothy J. Eberlein1,6, David G. Denardo2,3,6, Simon Peter Goedegebuure1,6, andDavid C. Linehan1,6

AbstractPurpose: To determine the role of the CCL2/CCR2 axis and inflammatory monocytes (CCR2þ/CD14þ)

as immunotherapeutic targets in the treatment of pancreatic cancer.

Experimental Design: Survival analysis was conducted to determine if the prevalence of preoperative

blood monocytes correlates with survival in patients with pancreatic cancer following tumor resection.

Inflammatory monocyte prevalence in the blood and bone marrow of patients with pancreatic cancer and

controls was compared. The immunosuppressive properties of inflammatory monocytes and macrophages

in the blood and tumors, respectively, of patients with pancreatic cancer were assessed. CCL2 expression

by human pancreatic cancer tumors was compared with normal pancreas. A novel CCR2 inhibitor

(PF-04136309) was tested in an orthotopic model of murine pancreatic cancer.

Results: Monocyte prevalence in the peripheral blood correlates inversely with survival, and low

monocyte prevalence is an independent predictor of increased survival in patients with pancreatic cancer

with resected tumors. Inflammatory monocytes are increased in the blood and decreased in the bone

marrow of patients with pancreatic cancer compared with controls. An increased ratio of inflammatory

monocytes in the blood versus the bonemarrow is a novel predictor of decreased patient survival following

tumor resection. Human pancreatic cancer produces CCL2, and immunosuppressive CCR2þ macrophages

infiltrate these tumors. Patients with tumors that exhibit high CCL2 expression/low CD8 T-cell infiltrate

have significantly decreased survival. In mice, CCR2 blockade depletes inflammatory monocytes and

macrophages from the primary tumor and premetastatic liver resulting in enhanced antitumor immunity,

decreased tumor growth, and reduced metastasis.

Conclusions: Inflammatory monocyte recruitment is critical to pancreatic cancer progression, and

targeting CCR2 may be an effective immunotherapeutic strategy in this disease. Clin Cancer Res; 19(13);

3404–15. �2013 AACR.

IntroductionPancreatic ductal adenocarcinoma, often called pancre-

atic cancer, is an aggressive malignancy with a death ratenearly equal to its incidence and a 5-year survival of lessthan 5% (1). Pancreatic cancer is characterized by a unique-

ly dense stroma that confers resistance to therapy (2–5).Within this stroma are abundant immunosuppressive mye-loid cells, which include monocytes/macrophages (2).Although it is appreciated that tumor-associated macro-phages (TAM) possess important tumor-promoting prop-erties in several malignancies (6), the contribution ofmonocyte mobilization from the bone marrow to thetumor has not been well studied. Here, we investigate thekey role of monocyte migration from the bone marrow tothe primary tumor and premetastatic site in pancreaticcancer, and show the efficacy of CCR2 blockade in thetreatment of this disease.

Monocytes are produced and stored in the bonemarrow,and are CD45þ/CD11bþ/CD115þ/HLA-DRþ in humansand CD45þ/CD11bþ/CD115þ/F4/80þ/MHCII� in mice(7). However, monocytes are composed of heterogeneoussubsets, which include resident monocytes and inflam-matory monocytes (8). Resident monocytes are CD16þ/

Authors' Affiliations: Departments of 1Surgery, 2Medicine, and 3Pathol-ogy and Immunology, Divisions of 4Health Behavior Research and 5Oncol-ogy, Department of Medicine, Washington University School of Medicine;and 6Alvin J. Siteman Cancer Center, St. Louis, Missouri

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: David C. Linehan, Washington University Schoolof Medicine, 660 South Euclid Ave. Box 8109, St. Louis, MO63110. Phone:314-747-2938; Fax: 314-222-6255; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-0525

�2013 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 19(13) July 1, 20133404

on June 21, 2018. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 7, 2013; DOI: 10.1158/1078-0432.CCR-13-0525

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CX3CR1high/CD14�/CCR2� in humans and mice (7). Res-

ident monocytes represent approximately 15% of circulat-ingmonocytes in normal healthy humans and 40% to 50%in mice (9, 10). These cells arise from the differentiation ofinflammatorymonocytes in the peripherywhere they play arole in steady-state immunosurveillance and inflammationresolution (7, 9). In contrast, inflammatory monocytes areCD14þ/CCR2þ/CD16�/CX3CR1

low in humans and Ly6Chi

/CCR2þ/CD16�/CX3CR1low in mice. These cells are the

predominant monocytes in circulation representingapproximately 85% of circulating monocytes in normalhealthy humans and 50% to 60% in mice (8, 9). Underphysiologic conditions, the CCL2/CCR2 chemokine axis iscritical to the mobilization of inflammatory monocytesfrom the bone marrow to the blood as well as their recruit-ment to sites of inflammation where they extravasate intotissues and differentiate intomacrophages or dendritic cells(7). Macrophages can be critical regulators of tumor pro-gression (6); however, these cells are not highly prevalent innormal pancreas and must be selectively recruited duringmalignant progression (2). As such,monocytemobilizationfrom thebonemarrow is a vital pathway formacrophages toinfiltrate tumors in the periphery. Once assimilated, macro-phages acquire an immunosuppressive, trophic (alterna-tively activated or M2) phenotype in the tumor microen-vironment and at the premetastatic site (6).In these studies, we show that both the prevalence of

monocytes in the peripheral blood and the mobilization ofinflammatory monocytes from the bone marrow are pre-dictive of survival in patients with pancreatic cancer. Fur-thermore, we show that pancreatic cancer uses the CCL2/CCR2 axis to favor the mobilization and recruitment ofinflammatory monocytes from the bone marrow to the

primary tumor and premetastatic liver where these cellsfacilitate tumor growth and metastasis. We identify CCR2inhibition (CCR2i) using a novel agent (PF-04136309) asan adjunct to standard chemotherapy in pancreatic cancer,which blocks inflammatory monocyte recruitment result-ing in the reduction of tumor growth and metastasis.

Materials and MethodsAnalysis of peripheral blood monocyte prevalence andsurvival with multivariate analysis

All patients (n¼ 483) with pancreatic cancer undergoingpancreaticoduodenectomy at Barnes-Jewish Hospital (St.Louis, MO) between 1997 and 2011 were followed forsurvival in a prospectively maintained database under anInternal Review Board (IRB)–approved protocol. Weexcluded patients with elevated preoperative leukocytecounts (>11,000 cells/dL; n ¼ 50), patients who did nothave preoperative complete blood counts (CBC) obtainedat our institution (n¼ 49), and patients who died within 30days of surgery (n¼ 7). Patients were stratified into 3 groupsbased on the percentage of blood leukocytes, which weremonocytes (monocyte prevalence) on preoperative CBC:low (<6%), normal (�6% to <11%), and high (�11%)monocyte groups. Ranges were established such that allpatients in themid group fell within 1 SDof themean; thus,patients in the low and high monocyte groups were greaterthan 1 SD below and above the mean, respectively. Multi-variate analysis was conducted using pancreatic cancerpatient demographic and pathologic data. Further analyticdetails are described in the Supplementary Methods.

Isolation of blood, bone marrow, and tumor frompancreatic cancer patients

Informed consent was obtained from all patients inaccordance with institutional Human Studies CommitteeProtocol. Peripheral blood and bonemarrowmononuclearcells (BMMC) were isolated from healthy volunteers andpatients with pancreatic cancer before chemotherapy,radiotherapy, or surgery, as has been previously described(11). Human pancreatic adenocarcinomas and normalpancreas were snap-frozen in liquid nitrogen or minced,mechanically dissociated, digested in enzyme buffer for 30minutes, and filtered.

Mice, cell lines, and murine pancreatic cancer modelC57BL/6andCCR2�/�mice (B6.129S4-Ccr2tm1Ifc/J)were

purchased from The Jackson Laboratory. The murine pan-creatic adenocarcinoma cell line KCKO, a metastatic tumorline, was the kind gift of Dr. PinkuMukherjee (University ofNorth Carolina at Charlotte, Charlotte, NC; ref. 12). Eight-to 10-week-old mice were anesthetized and injected in thetail of the pancreas with 1 � 105 KCKO cells suspended ina 1:1 PBS:Matrigel mixture. After mice were sacrificed atindicated times, bone marrow was extracted from thefemurs and blood collected in heparinized capillary tubes.Blood and bone marrow cells were subjected to red bloodcell (RBC) lysis (BioLegend) as permanufacturer’s protocol.Orthotopic tumor burden was measured by the gross wet

Translational RelevancePancreatic cancer is an aggressive malignancy with a

dismal prognosis, due in part to a high rate of metastaticdissemination and chemoresistance. Macrophages arepredominant in the uniquely dense pancreatic cancerstroma, and these cells enhance tumor growth andmetastasis. Inflammatory monocytes (CD14þ/CCR2þ)depend on CCL2/CCR2 signaling for mobilization fromthe bone marrow to the blood as well as recruitment tosites of inflammation where they extravasate into tissuesand become macrophages. The role of inflammatorymonocytes in pancreatic cancer as well as the effect oftargeting these cells through CCR2 blockade has notbeen studied. These studies show that inflammatorymonocyte recruitment from the bone marrow is prog-nostically and therapeutically important in pancreaticcancer. This study has laid the foundation for anongoing phase Ib trial using CCR2 blockade in com-bination with chemotherapy in patients with pancre-atic cancer with locally advanced, nonmetastaticdisease (NCT01413022).

Role of Inflammatory Monocytes in Pancreatic Cancer

www.aacrjournals.org Clin Cancer Res; 19(13) July 1, 2013 3405

on June 21, 2018. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 7, 2013; DOI: 10.1158/1078-0432.CCR-13-0525

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weight of the pancreas. Metastatic and disseminatedtumors were scored by serial sectioning and gross evalu-ation, which was validated by tissue pathology.

Chemotherapy and CCR2 inhibitorPF-04136309 (Pfizer) is a CCR2 kinase antagonist and

the details have been published previously (13). Mice wereinjected subcutaneously with 100 mg/kg of PF-04136309twice-daily beginning 2 days after tumor implantation.Mice were also injected intravenously with 50 mg/kg ofgemcitabine (GEM; Hospira) into the retro-orbital sinusevery 4 days. When indicated gemcitabine and PF-04136309were given in combination without altering doseor schedule of either agent separately.

Flow cytometryHuman and mouse single-cell suspensions were blocked

with TruStain FcX or anti-CD16/32 antibody, respectively(BioLegend) and stained with fluorescent antibodies usingstandard protocols for flow cytometry. Cells undergoingintracellular staining were permeabilized with eBiosciencePermeabilization Buffer according to the manufacturer’sprotocol. Antibodies used for human staining are listed inSupplementary Methods.

RNA isolation and real-time PCRTotal RNA was isolated by TRIzol extraction and reverse

transcribed into cDNA. Quantitative real-time PCR (qRT-PCR) was conducted using predesigned TaqMan GeneExpression Assays (Life Technologies) on a 7500 Fast Ther-mal Cycler (Applied Biosystems). Target gene expressionwas normalized to glyceraldehyde-3-phosphate dehydro-genase (GAPDH), HPRT1, or b-actin. The normalizedexpression levels of genes were analyzed using the 7500software for 7500 RT-PCR system V2.0.6.

T-cell proliferation assaysCD14þ cells were isolated from peripheral blood mono-

nuclear cells (PBMC) of patients with pancreatic cancerbefore tumor resection and single cell tumor suspensionswith the EasySep Human CD14 Positive Selection Kit as permanufacturer’s instructions (STEMCELL Technologies).CD14-depleted PBMCs were labeled with carboxyfluores-cein succinimidyl ester (CFSE; Life Technologies) and cocul-tured in 96-well round-bottomplates (Corning) coatedwithLEAF purified anti-human CD3 (BioLegend, clone OKT3)with varying concentrations of autologous CD14þ cells incomplete media supplemented with human interleukin(IL)-2 (NIH, Bethesda, MD). Cell cultures were harvestedafter incubating for 72 hours at 37�C and the CFSE dilutionof theCD4þ andCD8þT-cell fractionswere analyzedbyflowcytometry. The division index (defined as the average num-ber of divisions that a cell present in the starting populationhas undergone) was calculated using FlowJo software.

Immunofluorescence and immunohistochemistryTissue sections from formalin-fixed paraffin-embedded

tissue blocks of human pancreatic cancer and normal pan-

creas were stained as previously described (11). Confocalimages were acquired on an Axiovert 100M microscopeequipped with a LSM 510 META Confocal Laser ScanningMicroscope system (Zeiss). Immunohistochemical imageswere acquired at �10 magnification on an Olympus BX51microscope with a SPOT RT Slider digital camera andsoftware (Diagnostic Instrument, Inc.). See SupplementaryMethods for further details.

Pancreatic cancer tissue microarray survival analysisAfter obtaining IRB approval, tissue microarray (TMA)

studies were conducted on a cohort of 60 previouslyuntreated patients with pancreatic cancer who underwentpancreaticoduodenectomy at Barnes-Jewish Hospital.Patients did not receive neo-adjuvant therapy and weretypically treated with adjuvant chemotherapy. To constructthe TMA, well-defined areas of tumor were demarcated andpunched (1-mm diameter) from paraffin-embedded tumorblocks. An Aperio Scan-Scope XT Slide Scanner (AperioTechnologies) system was used to acquire digital imagesusing a �20 objective. A tumor-specific nuclear algorithm(IHC-MARK) developed in-house (14, 15) was modified toquantify CCL2 and CD8 expression.

Statistical analysisAll data (other thanmultivariate analyses) were analyzed

using GraphPad Prism version 5.01 (GraphPad SoftwareInc.). Calculating differences in numerical values was con-ducted using Mann–Whitney test for nonparametric data.Fisher exact test was used to compare categorical data. P <0.05 defined statistically significant differences.

See Supplementary Methods for additional methodolo-gic data.

ResultsDecreased monocytes in the peripheral blood isassociated with better survival in pancreatic cancerpatients

To investigate the importance ofmonocytes in pancreaticcancer, we examined whether there was a correlationbetween survival and the prevalence of monocytes in theblood of 377 patients with pancreatic cancer. All patientswere chemotherapy na€�ve, diagnosed with local, surgicallyresectable disease, and underwent pancreaticoduodenect-omy. Patients were stratified into low (<6% of leukocytes,>1 SD below mean), mid (�6% to <11% of leukocytes,within 1 SD of mean), or high (�11% of leukocytes, 1 SDabove mean) monocyte groups based on the prevalence ofmonocytes in their preoperative CBC. There was a signifi-cant correlation between the prevalence of preoperativemonocytes in the peripheral blood of patients with pan-creatic cancer and overall survival (Supplementary TableS1). Patients with high blood monocytes had significantlydecreased overall survival compared with the rest of thecohort with a 5-year survival of 11.0% versus 22.1% (P ¼0.03; Fig. 1A). In addition, low blood monocyte count wasfound to be a prognostic factor for survival in patients with

Sanford et al.

Clin Cancer Res; 19(13) July 1, 2013 Clinical Cancer Research3406

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pancreatic cancer compared with the rest of the cohort witha 5-year survival of 28.8% versus 20.2% (P¼ 0.03; Fig. 1B).When comparing patients in the low, mid, and highmonocyte groups separately, there was an incrementaldecrease in survival as blood monocytes increased; 5-yearsurvival was 28.8% versus 20.9% versus 11.0% (meansurvival ¼ 35.6 months vs. 27.6 months vs. 21.1 months;P ¼ 0.02) in the low, mid, and high groups, respectively(Fig. 1C).Asmonocyte prevalence in the blood correlated inversely

with survival on univariate analysis, we conducted a mul-tivariate analysis using patient demographic and pathologicdata to determine if monocyte count was an independentpredictor of survival in patients with pancreatic cancer.Indeed, lowbloodmonocyte prevalencewas independentlyassociated with increased survival on multivariate analysis[HR, 0.58; 95% confidence interval (CI), 0.40–0.86]. How-ever, the association between high monocyte prevalenceand survival was not independent of other pathologicfactors. Compared with the rest of the cohort, patients inthe high monocyte group had a strong trend toward havinga higher incidence of lymph node–positive tumors (81.5%vs. 68.1%; P ¼ 0.066), which is a strong predictor ofdecreased survival after tumor resection (16). Thesefindingsindicate that monocyte prevalence in the peripheral bloodis a prognostic indicator for patient survival, and thustargetingmonocytesmight represent an attractive therapeu-tic strategy.

Inflammatory monocyte mobilization from the bonemarrow is prognostic in pancreatic cancer patientsAs with other chronic inflammatory conditions, mono-

cytes are elevated in the peripheral blood of patients withsolid organ malignancy (17), including pancreatic cancer(Supplementary Fig. S1A). Given the fact that the bonemarrow acts as a storage reservoir for monocytes, wehypothesized that inflammatory monocytes are beingmobilized from the bone marrow to the peripheral bloodin human pancreatic cancer (7). Therefore, we analyzed the

prevalence of inflammatorymonocytes and residentmono-cytes in PBMCs and BMMCs from patients with nonmeta-static pancreatic cancer before any treatment (i.e., chemo-therapy, radiotherapy, or surgical resection) and comparedthese with normal controls (Fig. 2A).We found that inflam-matory monocytes were significantly more prevalent in theblood of patients with pancreatic cancer (Fig. 2B), however,the prevalence of resident monocytes was unchanged (Sup-plementary Fig. S1B). Consistent with previous reports,inflammatory monocytes made up 85% of blood mono-cytes in healthy individuals (8), and were increased to 92%in patients with pancreatic cancer (Supplementary Fig.S1C). Analysis of the bone marrow revealed that the prev-alence of inflammatory monocytes were significantlydecreased in patients with pancreatic cancer compared withcontrols, whereas resident monocytes were unchanged,suggesting that there is a shift in inflammatory monocyteequilibrium from the bonemarrow to the peripheral bloodduring pancreatic cancer (Fig. 2C).

Given the finding that patients with elevated peripheralblood monocytes have worse overall survival, we hypoth-esized that the ratio of inflammatory monocytes in theblood versus the bone marrow (blood:bone marrowinflammatory monocyte ratio) may serve as a surrogatefor inflammatory monocyte mobilization which couldcontribute to pancreatic cancer progression and ultimatelydecrease survival. This ratio was increased in patients withpancreatic cancer compared with normal controls (Supple-mentary Fig. S1D). Importantly, patients with pancreaticcancer who experienced rapid treatment refractory recur-rence and death within the first year following tumorresection had significantly higher blood:bone marrowinflammatory monocyte ratios compared with 1-year pan-creatic cancer survivors (Fig. 2D). Similarly, patients withpancreatic cancer with blood:bone marrow inflammatorymonocyte ratios �1.5 had significantly decreased survival(Supplementary Fig. S1D). This suggests that inflammatorymonocytemobilization plays a key role in pancreatic cancerpatient outcome.

Figure 1. Theprevalenceof peripheral bloodmonocytes is prognostic in patientswith pancreatic cancer. A, patientswere stratified into low (<6%of leukocytes,>1 SD belowmean), mid (�6% to < 11% of leukocytes, within 1 SD of mean), and high (� 11% of leukocytes, >1 SD above mean) peripheral blood monocytegroups. Kaplan–Meier survival curves compare patients with pancreatic cancer in high (A) and low (B) bloodmonocyte groups to the rest of the cohort as wellas patients in low, mid, and high blood monocyte groups separately (C). P values are by log-rank (Mantel–Cox) test.

Role of Inflammatory Monocytes in Pancreatic Cancer

www.aacrjournals.org Clin Cancer Res; 19(13) July 1, 2013 3407

on June 21, 2018. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 7, 2013; DOI: 10.1158/1078-0432.CCR-13-0525

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Human pancreatic tumors express CCL2 and areinfiltrated by immunosuppressive CCR2þ

macrophagesThe recruitment of inflammatory monocytes in various

human inflammatory diseases, such as rheumatoid arthritis(18), type I diabetes (19), and atherosclerosis (20) is medi-ated by the chemokine CCL2 and its receptor CCR2. Geneexpression analysis found that human pancreatic cancertissue has elevated CCL2 mRNA compared with normalpancreas by both qRT-PCR analysis of surgical specimensand by retrospective analysis of published datasets(ref. 21; Fig. 3A; Supplementary Fig. S2A). Upon furtheranalysis of tumors, CCL2 protein was highly expressed bymalignant ducts as well as cells apparently within thestroma, which could represent stromal cells and/or cancercells in epithelial–mesenchymal transition (Fig. 3A andSupplementary Fig. S2B).

Pancreatic tumors possess a highly immunosuppressivemicroenvironment in which myeloid cells are crucial (2,3, 11, 22). There is evidence that inflammatory monocytesare recruited to tumors where they differentiate into immu-nosuppressive TAM (23–25). CCR2þ TAM (CD45þ/CD11bþ/CD115þ/HLA-DRþ/CD14þ/CCR2þ) reside inhuman pancreatic tumors, and make up roughly 28% oftumor-infiltrating leukocytes (Fig. 3B and SupplementaryFig. S2C). However, effector T cells are significantly out-numberedby immunosuppressive TAMasCD8T cellsmakeup only around 7%of tumor-infiltrating leukocytes (Fig. 3Band Supplementary Fig. S2C). We sought to determine ifinflammatory monocytes are immunosuppressive in theperipheral blood of patients with pancreatic cancer orwhether tumor infiltration is a prerequisite. We isolated

both inflammatory monocytes and TAM from the freshblood and tumors, respectively, of individual patients withpancreatic cancer (Supplementary Fig. S2D), and comparedtheir abilities to suppress autologous effector T-cell prolif-eration. We observed that while TAMs were markedlyimmunosuppressive, inflammatory monocytes were not(Fig. 3C). This suggests that the tumor microenvironmentchanges the phenotype of inflammatorymonocytes follow-ing infiltration.

Patients with pancreatic cancer having tumors with highTAM:CD8 T-cell ratios have a poor prognosis (15). Simi-larly, patients having tumors which express high CCL2witha low CD8 T-cell infiltrate (upon univariate analysis of aTMA) also have significantly reduced survival comparedwith patients with tumors expressing low CCL2 and havinghigh CD8 T cells (Fig. 3D). This further shows the criticaltumor cell–stroma interplay that is characteristic of pancre-atic cancer (5).

CCR2 mediates inflammatory monocyte mobilizationin murine pancreatic cancer

Todetermine if targeting inflammatorymonocyte recruit-ment through CCR2 signaling inhibition could preventinflammatory monocyte mobilization from the bonemarrow in pancreatic cancer, we used a murine model.KCKO is a metastatic cell line derived from a geneticallyengineered, spontaneous murine pancreatic cancer model(LSL-KRASG12D � p48-Cre; ref. 12). Like human pancreaticcancer, KCKO tumors express increased CCL2 comparedwith normal murine pancreas (Supplementary Fig. S3A).Also, KCKO does not express CCR2 in vivo. We injectedKCKO orthotopically into the pancreas of wild-type (WT)

A Human control blood

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Figure 2. Inflammatory monocyte(IM) equilibrium in the peripheralblood and bone marrow isprognostic in patients withpancreatic cancer (PC). A, flowcytometrygating strategies used todefine inflammatory monocytes(CD115þ/CD16�/CX3CR1

low/CD14þ/CCR2þ) and residentmonocytes (RM) (CD115þ/CD16þ/CX3CR1

hi/CD14�/CCR2low) inperipheral blood and bone marrowof patients with pancreatic cancer(n ¼ 21) and healthy controls(n ¼ 11). Graphs compare theprevalence of inflammatorymonocytes in the blood (B) andbone marrow (C) of patients withpancreatic cancer and controls.D, graph compares the blood:bonemarrow inflammatory monocyteratios of patients with pancreaticcancer with <1 year survival to� 1 year survivors following tumorresection. All graphs show means� SEM, and P values are byMann–Whitney test.

Sanford et al.

Clin Cancer Res; 19(13) July 1, 2013 Clinical Cancer Research3408

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mice to study the effects of pancreatic cancer on inflamma-tory monocyte mobilization from the bone marrow. Mim-icking our observations in patients with pancreatic cancer,tumor-bearing mice displayed significant increases in cir-culating inflammatory monocytes, whereas these cells weredecreased in the bone marrow (Fig. 4A). Signaling throughCCR2 is crucial to monocyte egress from the bone marrow(26, 27). We observed that tumor-bearing WT mice treatedwith a CCR2 antagonist (PF-04136309) and tumor-bearingCCR2�/� mice exhibit a significant decrease in circulatinginflammatory monocytes (Fig. 4A and B and Supplemen-tary Fig. S3B). In addition, these mice had an increasedprevalence of inflammatorymonocytes in the bonemarrowcompared with vehicle-treated tumor-bearing mice, sug-

gesting that these cells are retained in the bone marrow(Fig. 4A and C and Supplementary Fig. S3C). Furthermore,CCR2 blockade reverses the blood:bone marrow inflam-matory monocyte ratio in tumor-bearing mice, which wehave shown to be prognostically important in patients withpancreatic cancer (Fig. 4D).

CCR2 inhibition promotes antitumor immunity inmurine pancreatic cancer

Like human pancreatic cancer, KCKO implanted in thepancreas of mice is characterized by a dense stromal infil-trate with a predominance of immunosuppressive myeloidcells (Supplementary Fig. S4A). We found that tumor-bear-ing WT mice treated with CCR2i and tumor-bearing

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Figure 3. Human pancreatic cancers express CCL2 and are infiltrated by immunosuppressive CCR2þ macrophages. A, bar graph compares CCL2 geneexpression in human pancreatic cancer (PC; n ¼ 11) to normal pancreas (n ¼ 10) and representative immunofluorescent confocal images (�63) of humanpancreatic cancer stained for cytokeratin 7 (green) and CCL2 (red) with nuclear (Topro) counterstain (blue). B, representative flow cytometry images(gated on CD45þ cells) and immunofluorescent confocal images [�63; CD14, green; CCR2, red; and nuclear (Topro) counterstain, blue] of human pancreatictumors reveal tumor-infiltrating CCR2þ TAM, whereas bar graph compares tumor-infiltrating TAM to CD8 T cells using flow cytometry. C, representativeflow cytometry histogram from T-cell suppression assay depicts stimulated, CFSE-labeled CD8 T cells alone (red) or cocultured at a 1:1 inflammatorymonocyte (IM; blue) or TAM (green) to lymphocyte ratio after 72 hours, and bar graph compares effect of inflammatory monocytes and TAMon T-cell divisionindex at various ratios. n ¼ 6 experiments. All graphs depict means � SEM and �, P < 0.05 by Mann–Whitney test. D, Kaplan-Meier survival curvescompare overall survival in patients with pancreatic cancer with high CD8 T-cell tumor infiltrate/low tumor CCL2 expression to patients with low CD8 T-cellinfiltrate/high tumor CCL2 expression by TMA (n ¼ 60 tumors). P value is by log-rank (Mantel–Cox) test.

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CCR2�/� mice displayed marked decreases in tumor-infil-trating inflammatory monocytes and macrophages(CD45þ/CD11bþ/F4/80þ/Ly6Clow/MHCIIþ; Fig. 5A). Incontrast, tumor-infiltrating effector T cells were increasedwith a concomitant decrease in regulatory T cells (CD45þ/CD4þ/CD25þ/FoxP3þ; Fig. 5B) suggesting an enhancedantitumor immune response. Tumors fromWTmice treatedwithCCR2i and fromCCR2�/�mice exhibited a shift fromaT-helper cell (TH)2 to a TH1 gene expression profile, char-acterized by decreased Arg1, TGF-b, IL-1b, IL-6, and IL-10with an increase in IFN-g (Fig. 5C; ref. 28). In addition,mRNA expression of the monocyte/macrophage recruit-ment mediators, CCL2, and macrophage colony-stimulat-ing factor (M-CSF), were both increased in tumors from

CCR2i-treated mice and from CCR2�/� mice by qRT-PCR(Supplementary Figs. S4B and S4C). This may suggest aregulatory feedback mechanism between tumors and infil-trating macrophages; specifically, tumors deprived of TAMmay attempt to recruit additional macrophages by bothupregulating CCL2 production and adapting to use alter-native mechanisms, such as M-CSF production.

During tumorigenesis, granulocytes acquire immunosup-pressive properties and promote tumor growth (22, 29, 30).These granulocytes are often referred to as granulocyticmyeloid-derived suppressor cells (G-MDSC), and theirimportance has previously been shown in human andmurine pancreatic cancer (11, 31). Gemcitabine is a stan-dard chemotherapeutic agent used in human pancreatic

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Figure 4. CCR2 mediatesinflammatory monocytemobilization from the bone marrowto the blood in murine pancreaticcancer. A, representative flowcytometry images showchanges ininflammatory monocyte (IM)prevalence in the blood and bonemarrow of control, vehicle-treated,and CCR2i-treated tumor-bearingWT mice after 28 days. Bar graphsshow the prevalence ofinflammatory monocytes in theblood (B) and bone marrow (C) aswell as the blood:bone marrowinflammatory monocyte ratios(D) of control, vehicle-treated,CCR2�/�, and CCR2i-treatedtumor-bearingWTmice. All graphsdepict means � SEM andhorizontal bars denote statisticallysignificant differences betweengroups defined as P < 0.05 byMann–Whitney test.

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cancer (32). Gemcitabine targets rapidly dividing tumorcells, but also selectively depletes granulocytes (CD45þ/CD11bþ/Gr1hi/Ly6Gþ; ref. 33). In contrast, monocytes/macrophages are more resistant to the effects of gemci-tabine, and we have found that inflammatory monocytespersist in the blood of patients with pancreatic cancertreated with chemotherapy (Supplementary Fig. S5). Weobserved a complementary effect of gemcitabine on gran-ulocyte depletion in the tumors of CCR2i-treated mice, asCCR2i alone resulted in a slight increase in granulocytes(Fig. 5A). Alternatively, gemcitabine alone led to anincrease in TAM, which CCR2i effectively depleted in thetumors of mice treated with the combination of these 2agents (Fig. 5A). CCR2�/� mice and CCR2i-treated WTmice displayed significantly reduced tumor growth ofboth subcutaneous and orthotopic tumors (Fig. 5D). Anadditive decrease in tumor growth was also observedwhen gemcitabine was given in combination with CCR2i(Fig. 5D).

CCR2 inhibition prevents liver metastasis in murinepancreatic cancerInflammatory monocytes and macrophages are believed

to play a crucial role in the establishment of metastasis (6).CCL2 has been shown to play an important role in therecruitment of metastasis-associated macrophages (MAM;CD11bþ/F4/80þ/Ly6Clow/CCR2þ) to the premetastatic

lung in an experimental model of breast cancer (34). Inpancreatic cancer, the liver is the most common site ofdistant metastasis (16). We observed a marked increase inCCL2 mRNA expression by qRT-PCR with a concomitantincrease in inflammatory monocytes and macrophages inthe premetastatic livers of WT mice bearing orthotopicpancreatic tumors (Fig. 6A–C and Supplementary Fig.S6). However, CCR2i efficiently blocked the recruitmentof inflammatory monocytes and macrophages to premeta-static livers, whereas gemcitabine did not (Fig. 6B–D andSupplementary Fig. S6). Twenty-eight days after implanta-tion, this was associated with a significant decrease in livermetastasis compared with vehicle- or gemcitabine only–treated mice (Fig. 6D). Strikingly, none of the 15 micetreated with the combination of gemcitabine and CCR2iacquiredhepaticmetastasis comparedwith 15of 20 vehicle-treated mice that developed liver metastasis (Fig. 6D). Thissuggests that targeting CCR2 can prevent liver metastasis inpancreatic cancer.

DiscussionMonocyte mobilization from the bone marrow has been

poorly characterized in human solid organmalignancy. Wehave identified an important pathophysiologic process inpatientswithpancreatic cancer that has bothprognostic andtherapeutic implications. Current pancreatic cancer staging

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Figure 5. CCR2blockade promotes antitumor immunity and impairs tumor growth. Bar graphs compare tumormyeloid infiltrate (A) aswell as the prevalence oftumor infiltrating T lymphocytes (B) and immune gene expression (C) in WT tumor-bearing mice treated with vehicle, gemcitabine, CCR2i, and CCR2iþGEMcombination after 28 days. PMN, granulocytes. D, tumor growth curves and graph compare effect of CCR2 blockade on subcutaneous and orthotopic(at 28 days) KCKO tumor growth. Note: CCR2�/� tumors were significantly smaller than CCR2i-treated tumors at days 25 and 28 on growth curve, P < 0.05.All graphs depict means � SEM. Horizontal bars or �, P < 0.05, whereas ��, P < 0.01 by Mann–Whitney test.

Role of Inflammatory Monocytes in Pancreatic Cancer

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systems, such as the American Joint Committee on Cancertumor–node–metastasis (TNM) staging, fail to identifythose patients who quickly recur and die from systemicprogression of their disease following removal of theirtumors (pancreatectomy; ref. 16). We found that survivaldecreases as the prevalence of bloodmonocytes increases inpatients with pancreatic cancer, and that a lowprevalence ofblood monocytes is an independent predictor of improvedsurvival. In addition, an increased blood:bone marrowinflammatory monocyte ratio predicts which patients withpancreatic cancer experience rapid recurrence and deathfollowing pancreatectomy. Perhaps, usingmonocytes in theblood and bone marrow as a biomarker can assist intherapeutic decision-making by indicating which patientswould benefit from early aggressive systemic treatment,such as chemotherapy or immunotherapy, rather thaninitial local treatment, such as surgery. Furthermore,patients with pancreatic cancer with evidence of increased

monocyte mobilization from the bone marrow may be theideal candidates inwhich to use antimonocyte/macrophagetherapies.

Tumors are sites of chronic inflammation and mono-cytes/macrophages influence outcome (35). Immunosup-pressive myeloid cells are the predominant tumor-infil-trating leukocytes in pancreatic cancer—not T cells(3, 11, 22). Tumors depend on the stroma for survivaland spread, and monocyte/macrophages are critical. TAMarise from monocytes, and these cells suppress antitumorimmunity (36) as well as directly promote tumor growth(15), invasiveness (37), angiogenesis (38), and chemore-sistance (14). TAMs within the tumor microenvironmenthave been shown to correlate with decreased patientsurvival in several human malignancies, including humanpancreatic cancer (39–41). However, we introduce thenovel concept that there is a shift in inflammatory mono-cyte equilibrium from the bone marrow to the blood,

ALiver CCL2 Expression

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Figure 6. CCR2 inhibition reduces inflammatory monocytes andMAMs in premetastatic livers and impairs hepatic metastasis in murine pancreatic cancer. A,graph compares CCL2 expression using qRT-PCR in baseline (day 0), premetastatic (day 6) and metastatic (day 28) livers of WT mice. B, representativeimmunofluorescence images (�20) of livers 9 days postinjection from control (Matrigel only, MG) mice, vehicle-treated tumor-bearing mice, andCCR2i-treated tumor-bearingmice for F4/80 (green) expressionwith a nuclear (Topro) stain (blue). C, graphs depict macrophage and inflammatorymonocyteprevalence in the livers of MG control or tumor-bearing mice treated with vehicle, CCR2i, gemcitabine, or CCR2iþGEM combination by flow cytometry.All flow cytometry and qRT-PCR conducted on grossly normal liver (i.e., excluded metastatic liver deposits). All graphs depict means � SEM andhorizontal bars denote statistically significant differences between groups defined as P < 0.05 by Mann–Whitney test. D, graph shows the incidence of livermetastasis after 28 days in tumor-bearingmice treatedwith vehicle, gemcitabine, CCR2i, andCCR2iþGEMcombination aswell as CCR2�/�mice. n¼ 13–20mice per group. Horizontal bars denote P < 0.05 by Fisher exact test.

Sanford et al.

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which is also predictive of survival in patients with pan-creatic cancer.CCR2þ monocytes have been shown to mediate immu-

nosuppression (42) and metastasis (34) in murine cancermodels. The CCL2/CCR2 chemokine axis plays an essentialrole in the recruitment of inflammatory monocytes fromthe bonemarrow to peripheral sites of inflammation. Oncerecruited, the phenotype of monocytes/macrophagesdepends on the local immune environment (43). Tumorsuse this same pathway to recruit monocytes to the primarytumor where these cells acquire an alternatively activated(M2) phenotype (34, 44). CCL2 expression is also upregu-lated in the premetastatic liver of mice bearing orthotopicpancreatic cancer tumors. We hypothesize that inflamma-tory monocyte mobilization in pancreatic cancer is a sur-rogate for macrophage infiltration in the tumor andpremetastatic liver. Our experiments in mice support thistheory as the ratio of blood:bone marrow inflammatorymonocytes correlated with the prevalence of macrophagesin theprimary tumor andpremetastatic liver. Targeting TAMin pancreatic cancer is a relatively new strategy (45). Wehave chosen the approach of depleting macrophages in thetumor and liver by targeting inflammatory monocytes withCCR2 blockade, which acts at the level of the bone marrow(26, 27). Unlike many immunotherapeutics, CCR2i doesnot depend on the delivery of drug to the stroma-dense,poorly vascular tumormicroenvironment. On the contrary,CCR2i depends on delivery to the highly vascular bonemarrow (26).In the present study, we revealed that circulating inflam-

matory monocytes were not immunosuppressive in thepatients with pancreatic cancer examined. However, amonocytic subset of MDSC (Mo-MDSC; CD14þ/HLA-DRlow/�) with immunosuppressive properties has beendescribed in the blood of patients with advanced malig-nancy (46, 47). Although we did not observe Mo-MDSC inthe blood of the patients with pancreatic cancer examined,this could be due to differences in the patient populationsstudied. We evaluated the blood and bone marrow ofsurgically resectable patients with nonmetastatic pancreaticcancer without evidence of active infection (i.e., preopera-tive blood leukocytes < 11,000cells/dL) before any treat-ment to limit confounding factors which often plaguepatients with advanced pancreatic cancer, such as infection,biliary obstruction,multiple courses of chemotherapy/radi-ation, tumor necrosis, and bowel obstruction (16). Admit-tedly, there is significant overlap between cells defined asMo-MDSC and inflammatory monocytes. All CD14þ

/CCR2þ cells in the peripheral blood of patients withpancreatic cancer in the current study were HLA-DRþ anddid not suppress T-cell proliferation ex vivo—therebymeeting the definition of inflammatory monocytes asopposed to Mo-MDSC (29). It is likely that inflammatorymonocytes and Mo-MDSC are closely related, and Mo-MDSC may in fact represent a subset of inflammatorymonocytes as has been suggested (48). There is evidencein patients with gastrointestinal malignancy that theextent of cell-mediated immune responses (mediated by

monocytes/macrophages) is inversely proportional to thestage of disease (49, 50). Perhaps inflammatory mono-cytes in the blood of patients with more advanced malig-nancy may downregulate HLA-DR expression and acquireimmunosuppressive properties—thereby meeting the def-inition of Mo-MDSC.

In summary, inflammatory monocyte mobilizationfrom the bone marrow predicts survival in human pan-creatic cancer, and the CCL2/CCR2 axis plays a criticalrole in the recruitment of inflammatory monocytes to thetumor microenvironment and premetastatic liver. CCR2imay be an ideal compliment to standard chemothera-peutics as this therapy had additive effect on the tumorwhile dramatically reducing metastasis. On the basis ofthe data presented here, we are proceeding with a phaseIb/II clinical trial using PF-04136309 combined withstandard chemotherapy in patients with pancreaticcancer with locally advanced, nonmetastatic disease[NCT01413022; ClinicalTrials.gov]. Tumor, blood, andbone marrow are being collected pre- and posttreatmentwith CCR2i to determine if changing the blood:bonemarrow inflammatory monocyte ratio can reduce TAMin the tumor, decrease metastasis, and improve patientsurvival. We believe the work presented here along withour clinical trial will make substantial contributions tothe fields of cancer immunotherapy and tumor mono-cyte/macrophage biology.

Disclosure of Potential Conflicts of InterestD.C. Linehan has a commercial research grant from Pfizer. No potential

conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: D.E. Sanford, B.A. Belt, D.G. Denardo, S.P.Goedegebuure, D.C. LinehanDevelopment of methodology:D.E. Sanford, B.A. Belt, J.B. Mitchem, D.G.DenardoAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.):D.E. Sanford, B.A. Belt, R.Z. Panni, D. Carpenter,S.M. Plambeck-Suess, D.C. LinehanAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): D.E. Sanford, B.A. Belt, R.Z. Panni,A. Mayer, A.D. DeshpandeWriting, review, and/or revision of the manuscript: D.E. Sanford, B.A.Belt, R.Z. Panni, A.D. Deshpande, J.B. Mitchem, A. Wang-Gillam, T.J.Eberlein, D.G. Denardo, S.P. Goedegebuure, D.C. LinehanAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases):D.E. Sanford, B.A. Belt, L.A.Worley,B.D. Goetz, A. Wang-Gillam, D.G. Denardo, D.C. LinehanStudy supervision: D.G. Denardo, S.P. Goedegebuure, D.C. Linehan

Grant SupportThis study was supported by NCI cancer center grant P30 CA091842 (to

A.D. Deshpande), the Siteman Cancer Center Frontier Fund Team ScienceAward (to A. Wang-Gillam, D.C. Linehan, and D.G. Denardo), WU/PfizerBiomedical Research Grant PW0457 (to D.C. Linehan), Lustgarten Foun-dation, V Foundation, Edward Mallinckrodt Jr. Award, the Cancer ResearchFoundation, and Siteman Cancer Center Career Development Award (toD.G. Denardo), and NCI grant T32 CA 009621 (to J.B. Mitchem and D.E.Sanford).

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 February 25, 2013; revised April 1, 2013; accepted April 19, 2013;published OnlineFirst May 7, 2013.

Role of Inflammatory Monocytes in Pancreatic Cancer

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Role of Inflammatory Monocytes in Pancreatic Cancer

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