Targeting Tumor-Inﬁltrating Macrophages Decreases Tumor … · Microenvironment and Immunology...

Microenvironment and Immunology

Targeting Tumor-Infiltrating Macrophages DecreasesTumor-Initiating Cells, Relieves Immunosuppression, andImproves Chemotherapeutic Responses

Jonathan B. Mitchem1, Donal J. Brennan10, Brett L. Knolhoff2, Brian A. Belt1, Yu Zhu2, Dominic E. Sanford1,Larisa Belaygorod2, Danielle Carpenter3, Lynne Collins4,5, David Piwnica-Worms4,5,6, Stephen Hewitt8,Girish Mallya Udupi11, William M. Gallagher11, Craig Wegner7, Brian L. West9, Andrea Wang-Gillam2,Peter Goedegebuure1,6, David C. Linehan1,6, and David G. DeNardo2,3,6

AbstractTumor-infiltrating immune cells can promote chemoresistance and metastatic spread in aggressive tumors.

Consequently, the type and quality of immune responses present in the neoplastic stroma are highly predictive ofpatient outcome in several cancer types. In addition to host immune responses, intrinsic tumor cell activities thatmimic stem cell properties have been linked to chemoresistance, metastatic dissemination, and the induction ofimmune suppression. Cancer stem cells are far from a static cell population; rather, their presence seems to becontrolled by highly dynamic processes that are dependent on cues from the tumor stroma. However, the impactimmune responses have on tumor stem cell differentiation or expansion is not well understood. In this study, weshow that targeting tumor-infiltratingmacrophages (TAM) and inflammatorymonocytes by inhibiting either themyeloid cell receptors colony-stimulating factor-1 receptor (CSF1R) or chemokine (C–Cmotif) receptor 2 (CCR2)decreases the number of tumor-initiating cells (TIC) in pancreatic tumors. Targeting CCR2 or CSF1R improveschemotherapeutic efficacy, inhibits metastasis, and increases antitumor T-cell responses. Tumor-educatedmacrophages also directly enhanced the tumor-initiating capacity of pancreatic tumor cells by activating thetranscription factor STAT3, thereby facilitating macrophage-mediated suppression of CD8þ T lymphocytes.Together, our findings show how targeting TAMs can effectively overcome therapeutic resistance mediated byTICs. Cancer Res; 73(3); 1128–41. �2012 AACR.

IntroductionTumor-infiltrating immune cells are a hallmark ofmost solid

tumors, and the presence of varied immune populations sig-nificantly affects clinical outcomes for patients with cancer (1,2). Historically, tumor-infiltrating immune cells have beenviewed as restraining tumor progression (3), but in recent years,it has become more widely appreciated that chronic immuneresponses play critical roles in promoting tumor progression,

metastasis, and resistance to cytotoxic therapies (1). Therefore,understanding the molecular mechanisms by which malignantcells derail antitumor immune responses to favor diseaseprogression is critical to identify potential therapeutic targets.Recently, we reported that selective depletion of tumor-infil-tratingmacrophages (TAM) by neutralizing colony-stimulatingfactor-1 (CSF1) or inhibiting CSF1 receptor (CSF1R) activityimproves the efficacy of chemotherapy inmammary tumors, inpart by instigating antitumor responses by CD8þ T cells (4).Similarly, deficiency in the CSF1 gene in op/op mice leads todecreased mammary tumor metastasis and slows pancreaticneuroendocrine tumor development (5, 6). Although the potentcapacity ofmacrophages to induce tumor progression has beenwell established, the mechanisms by which macrophage affectchemoresistance are not well defined.

In addition to immune regulation of cancer progression andchemoresistance, tumor cells that acquire stem-like or tumor-initiating properties (often called "cancer stem cells") exhibitenhanced resistance to cytotoxic therapy and increasedpropensity for metastatic dissemination (7, 8). Several linesof evidence suggest that the tumor-initiating capacity ofmalignant cells is rooted in inflammatory signals (9). How-ever, the mechanisms by which different populations ofleukocytes might support the expansion of tumor-initiatingcells (TIC) are unknown. One possibility is that reciprocal

Authors' Affiliations: Departments of 1Surgery, 2Medicine, and 3Pathol-ogy and Immunology; 4Molecular Imaging Center, Mallinckrodt Institute ofRadiology; 5BRIGHT Institute; 6Siteman Cancer Center, Washington Uni-versity School of Medicine; 7Pfizer Inc., St Louis, Missouri; 8NationalCancer InstituteAdvancedTechnologyCenter, Bethesda,Maryland; 9Plex-xikon Inc., Berkeley, California; 10Central Clinical Division, School of Med-icine, Queensland Centre for Gynaecological Cancer, University ofQueensland, Brisbane, Australia; and 11UCD School of Biomolecular andBiomedical Science, UCD Conway Institute, University College Dublin,Belfield, Dublin, Ireland

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: David G. DeNardo, Department of Medicine,Washington University School of Medicine, 660 South Euclid Ave, Box8069, St Louis, MO 63110. Phone: 314-362-9524; Fax: 314-747-2797;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-2731

�2012 American Association for Cancer Research.

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cross-talk between tumor-infiltrating leukocytes and malig-nant cells regulates the development of cells with stem-likeproperties, which in turn facilitates resistance to therapeuticinterventions. A recent study showed that macrophagescan induce tumor stem-like properties in vitro in murinelung and colon cancer cell lines (10). However, it isunclear whether this interaction can be exploited pharma-cologically, and if so, whether it also affects tumor-derivedimmunosuppression.In this study, we investigated the mechanisms by which

macrophages and TICs collaborate to regulate pancreaticductal adenocarcinoma (PDAC) progression, immunosup-pression, and responses to chemotherapy. We show that

targeting TAMs by inhibiting either CSF1R or chemokine (C–C motif) receptor 2 (CCR2) decreases the numbers of pan-creatic TICs and improves chemotherapeutic efficacy in vivo.We also found that TAMs directly induce TIC properties inpancreatic cancer cells by activating STAT3. In turn, TICsinduce immunosuppressive behavior in TAMs, and thusblock antitumor CD8þ T-lymphocyte responses during che-motherapeutic treatment.

Materials and MethodsPancreatic cancer tissuemicroarray cohort and analysis

Tissue microarray (TMA) studies were conducted on apatient cohort constructed from 60 cases of invasive PDAC

Figure 1. ALDH1þ PDAC cells have high tumor-initiating potential. A, FACS analysis of ALDHBright cells from Kras-INK tumors. Plots of cells gated onCD45�mCherryþ and stained with Aldefluor� a DEAB inhibitor. ALDHBright (blue) and ALDHDim (red) cells are depicted. B, analysis tumor spheroid formationfrom ALDHBright and ALDHDim Kras-INK and KCM cells. The mean number of tumor spheroids formed after 2 weeks is depicted. C, paired aliquots ofFACS-sorted ALDHBright and ALDHDim Kras-INK cells were injected into subcutaneous in nude mice, and tumor formation was accessed. D, automatedquantitation of cytoplasmic ALDH1A1þ cells in normal human pancreas and PDAC tissues (n¼ 52 PDAC and 5 normal). E, flow-cytometry analysis of humanPDAC tissue gated on CD45� and stained for EpCAM and Aldefluor positivity. All error bars are SEM, and �, P < 0.05 by Mann–Whitney.

Macrophages Regulate Tumor-Initiating Cells

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Figure 2. Depletion of TAMs results in reduced ALDHBright TICs. A, analysis of leukocyte and ALDHBright TIC frequency in KCM tumors frommice treated for 21days with vehicle, CSF1Ri1, CCR2i, or in CCR2�/� hosts. (i) The presence of CD11bþLy6G�Ly6CLoF4/80HiMHCIIþ macrophage, CD11bþLy6GHiLy6Cþ

(G-MDSC/neutrophil), CD11bþLy6G�Ly6CHiF4/80þMHCIIþSSCLo monocyte (mono), CD11bþLy6G�Ly6CLoCD11cHiMHCIIHi (M-DC), CD11bþ

Ly6GþLy6C�MHCIIþ (basophil), and CD11bLoLy6G�Ly6C�CD11cHiMHCIIHi (Lymph-DC) subsets is depicted as the mean% of total live cells. (ii) ALDHBright

TICs are depicted as themean%of total live CD45�mCherryþ cells. B, analysis of macrophage subsets following CCR2 or CSF1R inhibition. (i) CD11bþCD3/19/49b�Ly6G�Ly6CLoF4/80þmacrophages were subdivided byMHCII and CD11c expression, and (ii) the mean frequency of each subset is displayed for alltreatment groups. (iii) Relative expression of F4/80, CD206, and mCherry (indicator of phagocytosis) is depicted. C, flow-cytometry analysis of TAMsandM-MDSCs infiltrating PAN02 tumors inmice treated for 4 or 8 dayswith vehicle, anti-CSF1, CCR2i, and/or CSF1Ri2 is depicted. D, themean frequency ofmacrophages and CD45�mCherryþALDHBright TICs in Kras-INK tumors following 8 days of CSF1Ri treatment is depicted. Representative flow-cytometryplots ofmCherryþALDHBright tumor cells (bluegate) are shown.E, quantitative real-timePCR (qRT-PCR) analysis in orthotopicKras-INK tumor tissue followingtreatment with CSF1Ri for 14 days. Graph depicts themean fold change compared vehicle. Flow cytometry and qRT-PCR data depict themean values from 5to 10 mice � SEM. �, Statistically significant differences at P < 0.05 (Mann–Whitney U test).

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diagnosed at the Department of Pathology at WashingtonUniversity (St Louis, MO). Patients had not received neoadju-vant therapy and underwent pancreaticoduodenectomy, typ-ically followed by adjuvant chemotherapy. To assemble TMAs,clearly defined areas of tumor tissue were demarked and 2biopsies (1.0-mm diameter) taken from each donor block. Weused 4.0-mmparaffin sections for immunohistochemical (IHC)analyses. The Washington University School of Medicine (StLouis, MO) ethical committee approved this study. Fullyautomated image acquisition was used using Aperio Scan-Scope XT Slide Scanner (Aperio Technologies) system tocapture whole-slide digital images with a �20 objective. Atumor-specific nuclear algorithm (IHC-MARK) developed in-house was modified to quantify CD8 and CD68 expression aspreviously published (4, 11).

CCR2 kinase, CSF1R kinase, and STAT3 inhibitorsCCR2 inhibitor PF-04136309 was provided by Pfizer and

administered tomice at a concentration of 100mg/kg in twice-daily subcutaneous injections. PF-04136309 details have beenpreviously published (12). Inhibitors of CSF1R1 and CSF1R2were provided by Plexxikon Inc. CSF1Ri1 is PLX6134 thatcontains the GW2580 compound, which was described indetail elsewhere (13). CSF1Ri2 is PLX3397, a selective biospe-cific inhibitor for c-Fms and the c-Kit receptor tyrosine kinases,with biochemical IC50 values of 0.02 and 0.01 mmol/L, respec-tively. PLX3397 was used as a confirmatory compound forPLX6134/GW2580with better specificity for CSF1R, anddetailswere presented elsewhere (4, 14, 15). Both CSF1R inhibitorswere administered to mice in a formulated diet at a concen-tration of 800 mg/kg chow. STATIC was obtained fromCalbiochem/EMD, used at doses less than the reported IC50

(<10 mmol/L) in vitro, and handled according to manufacturerinstructions.

Cell lines and constructsPAN02 cells were obtained from Dr. David C. Linehan

(Washington University School of Medicine, St. Louis, MO),KCM cells from Dr. Pinku Mukherjee (University of NorthCarolina, Charlotte, NC), and Kras-INK from Dr. DouglasHanahan's laboratory (Ecole Polytechnique Federale de Lau-sanne, Lausanne, Switzerland) and have been published else-where (16–18). Briefly, KCM and Kras-INK were derived frompancreatic adenocarcinomas from p48-CRE/LSL-KrasG12D/Muc1.Tg (17, 18) and p48-CRE/LSL-KrasG12D/INK4aflox (18,19) mice, respectively. All cell lines were Mycoplasma testedand labeled with a polycistronic click beetle red luciferase-mCherry reporter by lentiviral infection, and positive cells wereselected by fluorescence-activated cell sorting (FACS) sorting.These constructs were supplied by Dr. David Piwnica-Wormsand the BRIGHT Institute (St Louis, MO).

Orthotopic model and preclinical animal cohortsSyngeneic orthotopic PDAC tumors were established by

surgical implantation as previously described (20). Briefly, weinjected 50,000 to 200,000 cells with 50 mL Matrigel (BD-Bio-sciences) and cohorts of mice were randomized to treatmentgroups by bioluminescence imaging of click beetle red luciferase

at day 21 (21) or gross palpation of the pancreas. Gemcitabine(GEM; Hospira) was obtained from the Washington UniversitySchool of Medicine pharmacy and diluted in PBS. Mice weretreatedwith 50mg/kg gemcitabineby intravenous injection intothe right retro-orbital sinus every 4 to 5 days. Preclinical studieswere conducted with 10 to 15 age-matched 10-week-old femalemice/group. In survival studies, a death event was classified as aloss of 15% of body weight or poor body conditioning score.Disease and tumor burden were measured by the gross wetweight of the pancreas. Metastatic and disseminated tumorswere scored by gross evaluation, which was validated by eitherbioluminescence or tissue pathology.

Additional methodologic detail is in the SupplementaryData.

ResultsALDH� PDAC cells have high tumor-initiating capacity

To investigate how macrophages impact the presence ofTICs, we used 3 distinct murine pancreatic tumor cell lines,denoted as Kras-INK, KCM, and PAN02, derived fromPDACs arising from genetic models (p48-CRE/LSL-Kras/INK4Aflox/wt or Pdx-CRE/LSL-Kras/Tg-Muc1) or tumors aris-ing from 3-methylcholanthrene carcinogenesis (PAN02;refs. 16, 17, 22). To identify potential TIC cellular subsets, thesecell lines were labeled with mCherry and Click-beetle Red(CBR) luciferase and analyzed for EpCAM, CD24, CD44,CD29, CD49f, CD133, and cMet expression and Aldefluoractivity (a measure of aldehyde dehydrogenase activity)using mCherry to identify implanted tumor cells in vivo.Our analysis revealed a distinct population of tumor cellswith high Aldefluor activity (ALDHBright; Fig. 1A). No distinctpopulations of CD133þ or cMetþ cells were observed. Anal-ysis of cell sorted from orthotopic Kras-INK and KCM PDACtumors illustrated that ALDHBright cells express higher levelsof CD29, CD44, and CD49f, display increased tumor spheroidformation in vitro, and have increased tumorigenic potentialin nude mice (Fig. 1A–C, data not shown). Analysis of freshhuman PDAC tissue also revealed an identifiable populationof ALDHBright tumor cells with a frequency ranging from 2%to 15% of the total CD45� EpCAMþ cells (Fig. 1E). IHCanalysis of ALDH1A1 revealed a significant increase inpositive cells in PDAC tissue compared with the normalpancreas (Fig. 1D). These results are consistent with previ-ous reports showing the tumorigenic potential of ALDH1þ

cells in human tumors (23, 24).

Depletion of TAMs decreased the presence of ALDHBright

TICsTodeterminewhether targeting TAMs alters the frequency of

TICs, we treated mice bearing orthotopic KCM tumors with 2CSF1R tyrosine kinase antagonists and a CCR2 antagonistand used CCR2 knockout mice (CCR2�/�). The CSF1R kinaseinhibitors used were PLX6134, a preparation of the GW2580compound (CSF1Ri1), and PLX3397 (CSF1Ri2). To test CCR2antagonism, we used PF04136309 (CCR2i). Additional detailsand structures for these compounds are in the Materials andMethods and published elsewhere (12–15, 25). Analysis of tumortissue after 21 days of treatment revealed significant reductions






in numbers of tumor-infiltrating CD11bþLy6G�Ly6CLoF4/80HiMHCIIþ macrophages, CD11bþLy6G�Ly6CHiF4/80Mid-

MHCIIþ inflammatory monocytes, and CD11bþ

Ly6G�Ly6CloCD11cHiMHCIIHi dendritic cells (DC; presumablymyeloid-derived dendritic cells) in response to blockade ofCCR2 or CSF1R signaling. In contrast, we observed no alterationin the number of CD11bLoLy6G�Ly6C�CD11cHiMHCIIHi cells(presumably lymphoid-deriveddendritic cells), and thenumbersof CD11bþLy6GHiLy6Cþ immature granulocytes/neutrophilswere modestly increased (�30%; Fig. 2A). Despite possiblecellular diversity, we will use the terms granulocytic myeloid-derived suppressor cells (G-MDSC) for CD11bþLy6GHiLy6Cþ

cells and monocytic MDSCs (M-MDSC) for CD11bþ

Ly6G�Ly6CHiF4/80MidMHCIIþ cells.Analysis of the impact of CCR2 or CSF1R inhibition on TAM

subsets found that these inhibitors significantly depletemacrophages expressing high levels of MHCII, but notMHCIILo or Tie2þ TAMs (Fig. 2B and Supplementary Fig.S1A). Analysis of the MHCIIHi macrophages showed that thesecells express the highest levels of F4/80, display high tumorphagocytosis as measured by mCherry fluorescence, andexpress modest levels of extracellular MRC1 (features consis-tent with mature macrophages).

To determine whether CSF1R and CCR2 blockade havedifferential effects on tumor-infiltrating myeloid cells, weanalyzed the impact of CSF1R and CCR2 inhibitors as singleagents or in combination (Fig. 2C). Mice bearing established(�1 cm) PDAC tumors were treated for 4 or 8 days with CCR2i,anti-CSF1 immunoglobulin G (IgG; 5A1), CSF1Ri2, or CCR2iplus CSF1Ri2. Analysis of tumor-infiltrating M-MDSCs andTAMs revealed no additive effects of combined inhibition.Individually, anti-CSF1, CSF1Ri, and CCR2i all effectivelydepleted monocytes within 4 days. However, blockade ofCSF1/CSF1R depletedmature TAMs in thefirst 4 days, whereasCCR2 inhibition did not effectively deplete TAMs until after 8days of treatment. These results suggest that CSF1R and CCR2have redundant rather than divergent activities on depletingTAMs and M-MDSCs.

To assess the effects of these inhibitors on TICs, weconducted a parallel analysis of the KCM tumor cells,identified by CD45�mCherryþ, and found a 40% to 55%reduction in CD44HiALDHBright TICs following CCR2 orCSF1R blockade (Fig. 2A(i)). To determine whether macro-phage depletion can rapidly alter the presence of ALDHBright

TICs, we treated mice bearing approximately 1-cm Kras-INKtumors with CSF1R inhibitors for 8 days and found a 40% to70% decrease in CD44HiALDHBright TICs (Fig. 2C). Weobserved similar results in orthotopic PAN02 tumors (Sup-plementary Fig. S1B). Correlating with these observeddecreases in ALDHBright cells, we found that OCT4, Nanog,and SOX2 mRNA expression was decreased following treat-ment with CSF1R or CCR2 inhibitors (Fig. 2E and Supple-mentary Fig. S1C). Notably, IHC and mRNA analysis of CCR2or CSF1R expression in these PDAC tumors revealed they donot express significant levels of CCR2 or CSF1R in vivo and invitro (Supplementary Fig. S1D and S1E). Taken together,these results suggest that targeting TAMs can rapidly reducethe numbers of ALDHBright TICs.

TAMs can directly enhance the tumor-initiatingproperties of PDAC cells

To determine whether macrophages can directly enhancethe tumor-initiating properties of pancreatic cancer cells, wecocultured macrophages with PDAC cells. Coculture withmacrophages increased the frequency of ALDHBright cells inmurine and human PDAC cell lines (Fig. 3A). To determinewhether soluble factors derived from tumor-educated macro-phages enhanced TIC properties, we first cultured bone-mar-row–derived macrophages (BM-MAC) in PDAC-conditionedmedium (CM) and then used the resultant "tumor-educated"BM-MACs to create conditioned medium for tumor spheroidassays. Conditioned medium from tumor-activated BM-MACsenhanced the formation of tumor spheres in PAN02, Kras-INK,and KCM cells (Fig. 3B). Similar results were also seen in Kras-INK cells cultured in Transwells with BM-MACs (Supplemen-tary Fig. S2A). Consistent with enhanced tumor-initiatingproperties, we observed that BM-MAC coculture increasedCD29 and CD49f protein and OCT4, Nanog, and SOX2 mRNAexpression in PDAC cells (Fig. 3C and Supplementary Fig. S2B).

Another feature commonly associated with TICs isincreased resistance to chemotherapy. Fitting this,ALDHBright cells isolated from Kras-INK tumors displayeddecreased response to gemcitabine (Supplementary Fig.S2C). To elucidate whether macrophages can enhance theresistance to chemotherapy, we treated PDAC cells withgemcitabine and found reduced numbers of Annexin Vþ cellswhen BM-MACs were present in coculture (Fig. 3D and Sup-plementary Fig. S2D). Similar results were observed using aTranswell system or BM-MAC-CM (Supplementary Fig. S2Eand S2F). Together, these results suggest that tumor-educatedmacrophages produce soluble factors that can regulate bothtumor-initiating capacity and chemoresistance in PDAC cells.

Gemcitabine treatment increases macrophageinfiltration into PDAC tumors

Common chemotherapeutics have been reported to inducethe recruitment ofmyeloid cells to regressing tumors (4, 26, 27).To assess whether gemcitabine treatment alters myeloid cellrecruitment in PDAC tumors, we analyzed pancreatic tissuefrom normal mice or mice bearing Kras-INK tumors � GEMtreatment. The numbers of TAMs, G-MDSCs, inflammatorymonocytes, and CD4þFOXP3þ regulatory T cells (Treg) wereincreased by the presence of PDAC tumors, but only TAMsincreased in number following gemcitabine treatment (Fig.3E). Corresponding with increased TAM infiltration, CSF1 andCCL2 but not CCL5 were upregulated by gemcitabine treat-ment (Fig. 3F). Taken together, these results suggest thatblockade of CCR2 and/or CSF1R would improve the responseto gemcitabine in PDAC tumors.

Targeting TAMs enhances the response to chemotherapyand reduces metastasis

CSF1R and CCR2 inhibitors as single agents modestlyslow PDAC tumor growth, similar to gemcitabine therapy(Figs. 4A–C and Supplementary Fig. S3A–S3C). Similar resultswere also observed in CCR2�/�mice (Supplementary Fig. S3B),suggesting that these effects are due to alterations in the tumor

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stroma. To determinewhether inhibiting CSF1R or CCR2 couldimprove responses to chemotherapy, we treated mice bearingestablished orthotopic Kras-INK or Pan02 tumors with gem-citabine alone or in combination with CSF1R or CCR2 inhibi-tors. We found that CSF1Ri plus gemcitabine dramaticallyslowed tumor progression in both Pan02 and Kras-INK ortho-topic tumors (Fig. 4A and Supplementary Fig. S3A and S3C).

For example, in Kras-INK tumors, gemcitabine reduced tumorgrowth (compared with parallel mice sacrificed at the start oftreatment, Rx Start) by 32% as compared with vehicle-treatedtumors, whereas CSF1Ri plus gemcitabine reduced tumorgrowth by 81%. Similar but somewhat less dramatic resultswere obtained with gemcitabine in combination with CCR2i(Fig. 4B). Similar results were observed with paclitaxel plus

Figure 3. Macrophages promote TICproperties in vitro. A, analysis of ALDHBright cells frequency in humanandmousePDACcells coculturedwith humanbloodmonocyte-derived macrophages or murine BM-MACs for 36 hours. Mean fold changes in CD45�ALDHBright cells is depicted. B, quantitation of PDAC tumorspheroids following 14 days of culture in vehicle or BM-MACconditionedmedium.C, qRT-PCR results fromKras-INK tumor cells cocultured for 24 hourswithBM-MACs in a Transwell chamber. Normalized mean fold changes is depicted. D, Kras-INK or KCM cells in coculture with BM-MACs were treated withgemcitabine for 36 hours. The mean% of CD45�mCherryþAnnnexin Vþ tumor cells is depicted (n ¼ 3). E, analysis of immune cell infiltration followinggemcitabine treatment of mice bearing Kras-INK tumors. The mean frequency of macrophages, G-MDSC/neutrophil, monocytes (mono), and CD3þ

CD4þFOXP3þ Tregs is depicted (n ¼ 5 mice/group). F, analysis of mRNA expression from Kras-INK tumor tissue from mice treated with either vehicle orgemcitabine. Normalized mean fold change is depicted (n ¼ 5 mice/group). All graphs displayed as means � SEM; and �, P < 0.05 (Mann–Whitney U test).






CSF1Ri2 (Supplementary Fig. S3C). Detailed analysis of prima-ry tumor pathology revealed a high level of necrosis in tumorsfrommice treatedwith gemcitabine plus CSF1Ri, but not eitheragent alone. No alterations in stromal desmoplasia wereobserved (Fig. 4F).

Similar to human PDAC, orthotopic Kras-INK and Pan02tumors develop hepatic and peritoneal metastatic disease.In mice bearing Kras-INK tumors, gemcitabine or CSF1Rinhibition alone reduced peritoneal metastases, and com-bined treatment regimens had an additive effect (Fig. 4D).

Figure 4. Inhibition of CSF1RorCCR2overcomes chemoresistance. A andB,mice bearing establishedKras-INK tumorswere treatedwith vehicle orCSF1Ri1,CSF1Ri2, or CCR2i (B) � GEM. The tumor burden was accessed by wet tumor weight, and it is displayed as the mean% increase compared with 5 micesacrificed at the start of treatment (RX Start). C, the growth of subcutaneous PAN02 tumors in mice treated with � CCR2i is depicted (n ¼ 5/group). D andE, the frequency and/or severity of disseminated tumors in the perennial cavity or liver is depicted as the proportion of mice with gross tumors at necropsy(n ¼ 10–15 mice/group). F, (i) images of hematoxylin and eosin staining depicts necrotic tissue (Nec) following CSF1Ri and gemcitabine treatment, and (ii)TriChrome staining depicts collagen deposition (blue). G andH, flow-cytometry analysis of TAM and ALDHBright TIC frequency in Kras-INK tumors is depictedas themean%of the total number of live cells or CD45�mCherryþ cells (n¼ 5–8mice/group). �,P < 0.05 (Mann–WhitneyU test or unpaired t test) in all panels.

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We observed similar results in the Pan02 model (Fig. 4E).Gemcitabine or CSF1R inhibition alone also decreased thefrequency of hepatic metastases; however, an additive

reduction in the liver metastatic frequency was onlyobserved in the Pan02 model (Fig. 4D and E). Consistentwith these results, CSF1Ri plus gemcitabine also increased

Figure 5. TAM and TIC cross-talk to suppress CTLs. A–C, analysis of CD3þCD4þ and CD3þCD8þ T-cell (A and C) and FOXP3þ CD4þ Treg (B) infiltration intoKras-INK tumors frommice is depicted as themean%of total live cells (n¼5–6mice/group). D, cytokinemRNAexpression assessed in Kras-INK tumors frommice treated with GEM � CSF1Ri is depicted as mean normalized fold change compared with the gemcitabine alone treatment group (5 animals/group).E, mice bearing orthotopic Kras-INK tumors were treated with GEM � CSF1Ri2 � anti-CD8 IgG. Tumor burden is depicted as the mean% increasecompared with the start of treatment (RX Start). n ¼ 10 to 14 mice/group. F, CD8þ CTL suppression by tumor-infiltrating leukocytes. TAMs or G-MDSCsisolated by flow sorting were assayed for their ability to repress splenic CD8þ proliferation following anti-CD3/CD28 stimulation. The mean number ofproliferation cycles is measured by CFSE dilution after 70 hours of activation. A representative plot of CFSE signal intensity in CD8þ cells is depicted forunactivated, activated, and TAM cocultures (right). G, CD8þCTL suppression by TICs. Isolated ALDHBright and ALDHDim KCMcells assayed for their ability torepress CD8þ proliferation following anti-CD3/CD28 stimulation. H, suppression of CD8þ proliferation was assayed in cocultures with BM-MACs and/orisolated ALDHBright and ALDHDim KCM cells. The ratio of all cells to CD8þ CTLs is depicted. � or ��, P < 0.05 by Mann–Whitney in all panels.






Figure 6. Macrophage-induced chemoresistance and stem-like properties require STAT3 signaling. A, quantitation and �40 images of pSTAT3þ tumor cellsare depicted. The mean frequency of positive cells in PAN02 and KCM tumors treated with either vehicle or CSF1R or CCR2 inhibitors is shown (n > 5/group).B, ELISA analysis of STAT3 phosphorylation in Kras-INK cells following treatment with macrophage conditioned medium (MCM). C, normalized mean foldchanges in gene expression are depicted from Kras-INK tumor cells and BM-MACs alone or in coculture for 24 hours using a Transwell chamber.D, MCM was added to adherence-free Kras-INK tumor spheroid cultures� the STAT3 inhibitor STATIC, and the number of tumor spheroids after 14 days isdepicted. E, MCM was added to adherence-free KCM-shLacZ, KCM-shSTAT3#3, or KCM-shSTAT3#5 tumor spheroid cultures � the STAT3 inhibitorsand the number of tumor spheroids after 14 days is depicted. F, analysis of Annexin VþKras-INK cells in direct coculturewith BM-MACs�STATIC and treatedwith gemcitabine for 36 hours is depicted as percentage of CD45�mCherryþ tumor cells (n ¼ 3/group). G, analysis of Annexin Vþ KCM, KCM-shLacZ,KCM-shSTAT3#3, or KCM-shSTAT3#5 cells cocultured with BM-MACs � GEM is depicted (n ¼ 3/group).

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the survival of Pan02 tumor-bearing mice (SupplementaryFig. S3D).Paralleling the observed efficacy of CSF1Ri plus gemcitabine,

although gemcitabine treatment significantly increased thefrequency of both TAMs andALDHBright TICs, this increasewasabrogated by CSF1R inhibition (Fig. 4G and H). Similar but lessdramatic effects were observed with CCR2 inhibition (Supple-mentary Fig. S3E). Taken together, these results suggest thatblocking macrophage infiltration into tumors during therapyreduces the number of TICs and improves the efficacy ofchemotherapy.

Depletion of TAMs results in increased CTL responseduring chemotherapyPrevious studies showed that TAMs have significant immu-

nosuppressive capacity (4, 28, 29). To determine whethermacrophage depletion would restore antitumor T-cell activityin PDAC tumors, we analyzed tumor-infiltrating T lympho-cytes in mice treated with CCR2i or CSF1Ri � GEM. Analysisof tumor-infiltrating lymphocytes revealed significantlyincreased CD4þ and CD8þ T cell and reduced FOXP3þ Treg

infiltration when CSF1Ri or CCR2i was given in combinationwith gemcitabine (Fig. 5A–C). Thus, despite eliciting decreasedtumor growth, single-agent gemcitabine, CCR2i, or CFS1Ri didnot alter T-cell infiltration, suggesting that both tumor celldestruction by chemotherapy and macrophage depletion arenecessary to sustain CTL infiltration. Consistent with elevatedCTL responses, we also observed increased IFN-g , IFN-b1,granzyme B, perforin, and interleukin (IL)-12 p35 anddecreased TGF-b and arginase-1 mRNA expression in tumors(Fig. 5D). To determine the role of CD8þ T lymphocytes in theefficacy of combined therapy, we used CD8-depleting antibo-dies (clone 2.43) in the context of CSF1Ri plus gemcitabinetreatment. While treatment with CSF1Ri1 plus GEM signifi-cantly blunted tumor growth compared with the effects ofgemcitabine alone (�80%), this therapeutic efficacywas largelydependent on CD8þ T lymphocytes (Fig. 5E).

TAMs and TICs cross-talk to repress CD8� T lymphocytesImmunosuppression in the tumormicroenvironment can be

mediated by TAMs, M-MDSCs, G-MDSCs, Tregs, and immaturedendritic cells. Although TAMs are the most prevalent of thesecells, 2 recent reports showed that G-MDSCs mobilized bytumor-derived granulocyte macrophage colony-stimulatingfactor (GM-CSF) can also suppress CTL activation in PDACtumors (30, 31). To assess the immunosuppressive capacity ofthese leukocytes, we isolated tumor-infiltrating TAMs and G-MDSCs fromKras-INK tumors and compared their ability withsuppress CD8þ T lymphocyte proliferation (by Carboxyfluor-escein succinimidyl ester (CFSE) dilution) following polyclonalanti-CD3/CD28 activation. AlthoughbothTAMs andG-MDSCsare capable of suppressing CD8þ T-cell proliferation, TAMswere slightly more suppressive at lower dilutions (Fig. 5F).Enhanced T-cell–suppressive activity in TICs has been

reported in glioblastoma (32). To test if PDAC TICs are highlyimmunosuppressive, we analyzed the suppressive capacity ofALDHBright and ALDHDim KCM cells and found that althoughboth subsets inhibit CD8þ T-cell proliferation at high concen-

trations, ALDHBright cells exhibited modestly greater suppres-sive activity (Fig. 5G).

Activation of the immunosuppressive properties in innateimmune cells by tumor cells is a common feature of aggressivetumors. To determine whether PDAC TICs increase the immu-nosuppressive capacity of macrophages, we cocultured smallnumbers of ALDHBright and ALDHDim KCMcells with BM-MACs(Fig. 5H). Although tumor-na€�ve BM-MACs exhibited decreasedCD8-suppressive capacity compared with that of TAMs, CD8suppression by BM-MACs was significantly elevated by thepresence of ALDHBright but not ALDHDim KCM cells. Takentogether, thesedata suggest that inhibitingCSF1RorCCR2 in thecontext of chemotherapy allows productive CD8þ T-cellresponses via the combined action of the (i) reduced presenceof immunosuppressive ALDHBright TICs, (ii) reduced presence ofimmunosuppressive TAMs (as well as M-MDSC), and (iii) dis-ruption of TIC-induced macrophage immunosuppression.

STAT3 activation is necessary for macrophage-dependent increases in TIC numbers andchemoresistance

STAT3 is a key mediator of proinflammatory cytokinesand immune suppression in both leukocytes and neoplasticcells, and has also been linked to TIC survival and chemore-sistance in several cancer types (33–36). IHC analysis oftumor tissue from mice treated with CSF1R or CCR2 inhi-bitors revealed significantly reduced levels of phospho-STAT3 (pSTAT3, Ser205) in malignant cells, but not adjacentnormal or leukocytes (Fig. 6A). Correlating with these resultsmacrophage conditioned medium increased pSTAT3 levelsin Kras-INK cells in vitro (Fig. 6B). Corresponding with theactivation of STAT3-mediated transcription, we observedincreased IL-6, GP130, and STAT3 mRNA expression inPDAC cells and increased IL-1b, IL-6, and ARG1 mRNAexpression in macrophages when cocultured in a Transwell(Fig. 6C). To determine if STAT3 was necessary for TAM-mediated regulation of PDAC TICs, we used small-moleculeinhibitors of STAT3 signaling [STATIC (37) and WP1066]and STAT3 short hairpin RNA (shRNA) constructs. Treat-ment with STATIC or WP1066 abrogated the formation oftumor spheroids in the presence or absence of BM-MAC-CM(Fig. 6D and E). In addition, partial suppression of STAT3expression (�50%–60%) using shRNA reduced the inductionof tumor spheroid formation by BM-MAC-CM (Fig. 6E).Inhibition of STAT3 by either STATIC or shRNA was suffi-cient to overcome the chemoprotective effects of macro-phage coculture (Fig. 6F and G). These data suggest thatTAMs induce TIC properties and chemoresistance throughthe activation of STAT3.

STAT3 signaling is necessary for TIC-inducedimmunosuppression

Wenext sought to understand howSTAT3 activation in TICsregulated the immunosuppressive capacity of TAMs. Toaccomplish this, we analyzed the effects of shRNA againstSTAT3 on PDAC-induced immunosuppression. Correspond-ing to our previous results, ALDHBright KCM cells, but not KCMALDHDim cells, stably expressing shRNA against LacZ robustly






induced BM-MAC–mediated suppression of CD8þ T-cell pro-liferation. In contrast, ALDHBright KCM cells expressing STAT3shRNA were unable to enhance BM-MAC–mediated CD8þ T-cell suppression (Fig. 7A).

To understand the clinical implications of these interac-tions, we analyzed a TMA containing specimens from 59patients with PDAC. Tumors were scored for the presence ofCD68þ and CD8þ leukocytes and stratified into 2 groups(CD68Hi/CD8Lo and CD68Lo/CD8Hi). We observed that inpatients in whom CD68þ macrophages were the dominanttumor-infiltrating leukocyte (CD68Hi/CD8Lo), overall survivalwas significantly reduced compared with all other groups(denoted as CD68Lo/CD8Hi; Fig. 7B). Similarly, stratificationof patients by epithelial pSTAT3 revealed that high pSTAT3indicated reduced survival (Fig. 7C). Combined analysis foundthat CD68þ leukocytes, but not CD8þ T cells, correlated withepithelial pSTAT3 intensity (Spearman r¼ 0.32, P¼ 0.024) andtumors classified as CD68Hi/CD8Lo had increased epithelialpSTAT3 (Fig. 7D). Taken together, these results suggest thatepithelial STAT3 signaling is high in tumors in which macro-phages likely play an immunosuppressive role (e.g., CD68Hi

/CD8Lo tumors) and targeting TAMS in these patients couldresult in improved survival.

DiscussionOur results show that inhibiting CSF1R or CCR2 signaling

can increase chemotherapeutic efficacy and block metastasisby the combined action of reducing TIC numbers, and over-come macrophage-induced CD8þ CTL suppression. We illus-trate that macrophages can directly induce TIC properties inPDAC cells by enhancing STAT3 activation and that STAT3þ

TICs enhance TAM-mediated immunosuppression. Thus,cross-talk between TAMs and TICs through STAT3 regulatesthe chemotherapeutic response by repressing antitumor CTLactivity (Fig. 7E).

CCR2 and CSF1R as regulators of myeloid responsesTumor-infiltrating innate immune cells are composed of

diverse cellular populations including G-MDSCs, M-MDSCs,tie-2þ angiogenic monocytes and myeloid-derived dendriticcells. We found that blockade of CSF1R or CCR2 result in a verysimilar spectrum of alterations in tumor-infiltrating myeloidcells both reducing mature CD11bþLy6G�L6CMid-Lo

MHCIIHiF480þ macrophages and CD11bþLy6G�L6CHi

MHCIIþF480þ monocytes. Although the effects of CCR2 andCSF1R blockade on tumor-infiltrating leukocytes are similar,their mechanisms of action are likely divergent. Studies have

Figure 7. Immune status andSTAT3 activation predict patient survival. A, STAT3þ TICs augmentmacrophage-mediated immunosuppression. Suppression ofCD8 proliferation was assayed in cocultures with BM-MACs and/or isolated ALDHBright and ALDHDim KCM-shLacZ or KCM-shSTAT3#5 cells. The ratioof all cells to CD8þCTLs and themean number of proliferation cycles is depicted. B, the CD8 to CD68 ratio predicts survival of patient with PDAC. Automatedanalysis of CD68þ and CD8þ immunohistochemistry reveals the relationship between leukocyte density and overall survival. The Kaplan–Meierestimate of overall survival comparing CD68Hi/CD8Low with all other groups, denoted as CD68Low/CD8High, is shown. C, STAT3 phosphorylation predictssurvival of patient with PDAC. The Kaplan–Meier estimate of overall survival comparing patients divided into pSTAT3Hi and pSTAT3Lo subgroups by themeanintensity score is shown.D,meanepithelial pSTAT3score is depicted for tumors classifiedaseitherCD68Hi/CD8LoorCD68Hi/CD8Lo. E, schematic depictionofthe coordinated suppression of CD8þ CTLs by TAMs and TICs. �, P < 0.05 by Mann–Whitney, and survival P values are log-rank (Mantel–Cox).

Mitchem et al.





suggested that CCR2 mediates the trafficking of circulatingLy6CHiCCR2þCX3CR1Lo inflammatory monocytes to targettissues (38, 39), whereas CSF1R is more likely involved inmaturation and/or survival of these cells at inflamed sites.Both receptors can affect bone marrow mobilization. In thisand previous studies, significantly reduced macrophage infil-tration was observed as early as 2 to 4 days after CSF1Rinhibition (4). In contrast, CCR2 inhibition reduced monocytenumbers in 4 days but only depleted TAMs after 8 days oftreatment. Neither CSF1R nor CCR2 blockade resulted indramatic alterations in circulating monocyte numbers overthese periods. Thus, these results suggest that CCR2 inhibitionmay affect recruitment of inflammatory monocytes from thecirculation, whereas CSF1R inhibition may affect survival ofmonocytes/macrophages at the tumor site.Tumor-infiltrating immature granulocytes delineated as

CD11bþLy6GHi are often composed of highly heterogeneouspopulations, which can include tumor-activated neutrophilsand G-MDSCs (40). GM-CSF–dependent mobilization andactivation of these cells have been shown to mediate suppres-sion of CTL responses in PDAC tumors (30, 31). However, inthis study, targeting either CCR2 or CSF1R did not reduce thepresence of CD11bþLy6GHi cells (Fig. 2). Thesefindings suggestthat in the context of chemotherapy, CD11bþLy6GHi cellscannot overcome the loss of TAMs and M-MDSC. While notobserved in thismodel, gemcitabine treatment has been shownto reduce the numbers of G-MDSCs in mammary tumors (41)and may affect the immunosuppressive capacity of G-MDSCsin PDAC. Alternatively, the depletion of TAMs may alter thecellular activity CD11bþLy6GHi cells, pushing them towardmore mature, less immunosuppressive phenotypes. Thesedifferences in immune responses following CCR2 or CSF1/CSF1R inhibition are likely important considerations for theirclinical application.

Inflammation and TICsSTAT3 activity has been shown to be required for the

expansion and maintenance of cancer stem cells in severalcancers (33, 34). In pancreatic cancer, epithelial deletion ofSTAT3 results in significantly reduced Kras-induced tumorformation in part through alterations in matrix metallopro-teinase 7 (MMP7) expression and decreased immune infiltra-tion (35). These data are consistent with the idea that recip-rocal cross-talk between leukocytes and cancer cells sustaintumor progression. Work by Jinushi and colleagues showedthat macrophage-derived MGF-E8 and IL-6 enhance stem-likeproperties in lung and colon cancer cells by activating STAT3(10). Similarly, we revealed that depletion of TAMs by CSF1R orCCR2 inhibition leads to reduced STAT3 phosphorylation (Fig.6) and decreased numbers of ALDHBright TICs. However,regulation of STAT3 activationmay be one of several pathwaysregulating this process. TAMs are potent producers of WNTsand Sonic hedgehog ligands as well as growth factors, such asEGF, basic fibroblast growth factor, platelet-derived growthfactor, and hepatocyte growth factor, many of which have beenimplicated in stimulating cancer stem-like properties (42, 43).In addition, factors such as VEGF, MMP9, and Bv8 emanatingfrom tumor-infiltratingmyeloid cells can significantly alter the

quality of tumor vasculature, and endothelial cell/tumor cellinteractions may be a critical component of the cancer stemcell niche (44–46). Thus, multiple pathways are likely regulatedby tumor inflammation to influence the prevalence of TICs.

Macrophages and chemosensitivitySeveral recent studies revealed that macrophages can

directly regulate tumor cell chemoresistance. Previous workby Shree and colleagues (27) and Gocheva and colleagues(47) has illustrated that macrophage-derived cathepsinsregulate pancreatic neuroendocrine tumor progression andmammary tumor response to paclitaxel. Intriguingly,cathepsin activity has also been revealed as necessary forthe secretion and processing of proinflammatory cytokines.However, the manner in which these activities affect mam-mary TICs or STAT3 activation is not known. In addition,targeting CCR2 in the tumor microenvironment has beenshown to improve the delivery of chemotherapeutic regi-mens by regulating the tumor vasculature (48). These obser-vations further stress the importance of TAMs and thecomplexity of their roles in the tumor microenvironment.

Clinical prospectiveTherapeutic resistance and metastatic spread define the

lethality of aggressive cancers. Thus, understanding and tar-geting the mechanisms responsible is critical to improvingtherapeutic outcomes. Several studies have indicated thattargeting key pathways regulating TIC survival and/or differ-entiation can overcome therapeutic resistance. However, thedurability of such therapies remains unproven, as such target-ing the tumor stromal responses that support tumor "stem-ness" is an attractive alternative.

Disclosure of Potential Conflicts of InterestD.J. Brennan has ownership interest (including patents) in patent for CD8:

CD68 signature. D.C. Linehan has other commercial research support fromPfizerOncology and Novartis Oncology. No potential conflicts of interest were dis-closed by the other authors.

Authors' ContributionsConception and design: J.B. Mitchem, D.J. Brennan, B.A. Belt, C. Wegner,P. Goedegebuure, D.C. Linehan, D.G. DeNardoDevelopmentofmethodology: J.B.Mitchem, B.A. Belt, L. Belaygorod, S. Hewitt,W.M. Gallagher, B.L. West, P. Goedegebuure, D.C. Linehan, D.G. DeNardoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.B. Mitchem, D.J. Brennan, B.L. Knolhoff, B.A. Belt, D.E. Sanford, L. Collins, D. Piwnica-Worms, S. Hewitt, W.M. Gallagher, A. Wang-Gillam, D.C. Linehan, D.G. DeNardoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.B. Mitchem, D.J. Brennan, B.A. Belt, L. Collins, D.Piwnica-Worms, G.M. Udupi, D.C. Linehan, D.G. DeNardoWriting, review, and/or revision of the manuscript: J.B. Mitchem,D.J. Brennan, B.A. Belt, Y. Zhu, D. Piwnica-Worms, C. Wegner, B.L. West,P. Goedegebuure, D.C. Linehan, D.G. DeNardoAdministrative, technical, or material support (i.e., reporting or orga-nizingdata, constructingdatabases): J.B.Mitchem,D. Carpenter, G.M.Udupi,A. Wang-Gillam, D.C. Linehan, D.G. DeNardoStudy supervision: D.G. DeNardo

AcknowledgmentsThe authors thank the Siteman Frontier Funds Team Science Award, Drs.

Mukherjee and Hanahan for providing cell lines, and the efforts of the BrightImaging Center. The authors also thank Drs. Hawkins, Stewart, and Webber fordiscussions and input. The authors also thank generous support from theLustgarten Foundation, V Foundation, Edward Mallinckrodt Jr. Award, the






Cancer Research Foundation, and Siteman Cancer Center Career DevelopmentAward.

Grant SupportThis study was supported by WU/Pfizer Biomedical Research Grant PW0457

(D.C. Linehan), NIH P50 CA 94056 (D. Piwnica-Worms), and NCI grant T32 CA009621 (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 indicate thisfact.

Received July 16, 2012; revised November 16, 2012; accepted November 16,2012; published OnlineFirst December 5, 2012.

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2013;73:1128-1141. Published OnlineFirst December 5, 2012.Cancer Res Jonathan B. Mitchem, Donal J. Brennan, Brett L. Knolhoff, et al. Chemotherapeutic Responses

ImprovesTumor-Initiating Cells, Relieves Immunosuppression, and Targeting Tumor-Infiltrating Macrophages Decreases

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