tumoral immune suppression by macrophages expressing ...€¦ · were used; ho-1 mm00516006_m1,...

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Tumoral Immune Suppression by Macrophages ExpressingFibroblast Activation Protein-a and Heme Oxygenase-1

James N. Arnold, Lukasz Magiera, Matthew Kraman, and Douglas T. Fearon

AbstractThe depletion of tumor stromal cells that are marked by their expression of the membrane protein fibroblast

activation protein-a (FAP) overcomes immune suppression and allows an anticancer cell immune response tocontrol tumor growth. In subcutaneous tumors established with immunogenic Lewis lung carcinoma cellsexpressing ovalbumin (LL2/OVA), the FAPþ population is comprised of CD45þ and CD45� cells. In the presentstudy, we further characterize the tumoral FAPþ/CD45þ population as a minor subpopulation of F4/80hi/CCR2þ/CD206þ M2 macrophages. Using bone marrow chimeric mice in which the primate diphtheria toxinreceptor is restricted either to the FAPþ/CD45þ or to the FAPþ/CD45� subset, we demonstrate by conditionallydepleting each subset that both independently contribute to the immune-suppressive tumor microenviron-ment. A basis for the function of the FAPþ/CD45þ subset is shown to be the immune inhibitory enzyme, hemeoxygenase-1 (HO-1). The FAPþ/CD45þ cells are the major tumoral source of HO-1, and an inhibitor of HO-1, Snmesoporphyrin, causes the same extent of immune-dependent arrest of LL2/OVA tumor growth as does thedepletion of these cells. Because this observation of immune suppression by HO-1 expressed by the FAPþ/CD45þ stromal cell is replicated in a transplanted model of pancreatic ductal adenocarcinoma, we concludethat pharmacologically targeting this enzyme may improve cancer immunotherapy. Cancer Immunol Res; 2(2);121–6. �2013 AACR.

IntroductionThe failure of the immune system to control the growth of

immunogenic cancers has been ascribed to two general pro-cesses: cancer immunoediting and immune suppression.Immunoediting has been demonstrated in models of autoch-thonous soft tissue sarcomas induced either by a mutagenicagent, methylcholanthrene (1), or by tissue-specific, Cre/LoxP-regulated expression of oncogenic K-rasG12D and deletion ofp53 (2). Tumoral immune suppression has been shown inmodels of transplanted, ectopic tumors (3), and recentlyin an autochthonous model of lung adenocarcinoma (4). Inrelation to immune suppression, progress has beenmade in theclinic with the introduction of therapeutic antibodies to CTLA-4, PD-1, and PD-L1 that antagonize immune checkpoints (5–7).However, as a high frequency of patients do not respond tothese therapeutic antibodies, it is appropriate to continue

studies of the tumoral stromal cells that have immune-sup-pressive function, including the cell that is identified by itsexpression of the membrane dipeptidyl dipeptidase, fibroblastactivation protein-a (FAP; ref. 8).

FAPþ stromal cells were first demonstrated in humanadenocarcinomas, and subsequently were found in variousnonneoplastic, chronic inflammatory lesions (9, 10). Recently,in a genetically modified mouse model in which FAPþ cellsexpress the primate diphtheria toxin receptor (DTR), theconditional depletion of these cells from an established, immu-nogenic, transplanted tumor caused its growth arrest. Thecontrol of tumor growth induced by depleting FAPþ cellsdepended on adaptive immunity, but did not involve enhancedpriming of the CD8þ T cells, leading to the conclusion thatFAPþ stromal cells suppressed the function of effector T cellsin the tumor microenvironment (8).

Understanding themeans of immune suppression by tumor-al FAPþ stromal cells is especially challenging because twosubtypes occur, a CD45� mesenchymal population and ahematopoietic subset that is CD45þ/CD11bþ/Gr-1� (8). Thepresent study focuses on the FAPþ/CD45þ tumoral cells,demonstrating that they are a subset of inflammatory macro-phages with an M2 phenotype that mediate immune suppres-sion by their expression of HO-1.

Materials and MethodsMice

FAP/enhanced GFP (EGFP) bacterial artificial chromosome(BAC) transgenic (Tg) and FAP/DTR BAC Tg mice havepreviously been described (8). C57BL/6-Ly5.1 (CD45.1) mice,

Authors' Affiliation: University of Cambridge, Cancer Research UK Cam-bridge Institute, Li Ka Shing Centre, Cambridge, United Kingdom

Note: Supplementary data for this article are available at Cancer Immu-nology Research Online (http://cancerimmunolres.aacrjournals.org/).

Current address for J.N. Arnold: King's College London, New Hunt'sHouse, Guy's Campus, London SE1 1UL, United Kingdom.

Corresponding Author: Douglas T. Fearon, Cancer Research UK Cam-bridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE,UK. Phone: 44-1223-769565; Fax: 44-1223-769881; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-13-0150

�2013 American Association for Cancer Research.

CancerImmunology

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C57BL/6 Rag2�/�, and C57BL/6 (CD45.2; The Jackson Labo-ratory) were used as indicated. The use of animals was app-roved by the Ethical Review Committee at the University ofCambridge and the Home Office, United Kingdom.

Subcutaneous tumor studies and HO inhibitionLewis lung carcinoma (LL2)/Thy1.1, LL2/Thy1.1-ovalbumin

(OVA; original line purchased from American Type CultureCollection), and PDA (TB 11381, D. Tuveson, CRUK CambridgeInstitute) were injected into mice, and the subsequent tumorsmeasured as previously described (8). Sn (IV) mesoporphyrinIX dichloride (SnMP; Frontier Scientific) was dissolved in 0.1mol/L NaOH and diluted using 0.1 mol/L NaHCO3, pH7. Forblocking IFN-g and TNF-a, mice were injected intraperitone-ally at day�1 and 0, relative to SnMP administration, with 12.5mg/g anti-IFN-g (XMG1.2) and 10 mg/g anti-TNF-a (MP6-XT3)or 22.5 mg/g nonimmune IgG (eBRG1; eBioscience). Tumortissue was enzyme-digested to release single cells as previouslydescribed (8).

Flow cytometryAntibodies were purchased from eBioscience unless other-

wise stated, the following antibodies were used: CCR2 (R&DSystems), CD3 (145-2C11), CD4 (RM4-5), CD8b (eBioH35-17.2),CD11b (M1/70), CD11c (N418), CD14 (Sa2-8), CD16/32 (93),CD31 (390), CD45 (30-F11), CD45.1 (Biolegend, A20), CD45.2(Biolegend, 104), CD69 (H1.2F3), F4/80 (BM8), Gr-1 (RB6-8C5),LAMP-1 (eBio1D4B), MHCII (M5/114.15), and Thy1.2 (53-2.1).Anti-mannose receptor (CD206; R&D Systems) was fluores-cently conjugated using the Alexa Fluor 488 antibody labelingkit (Invitrogen) before use. Fc receptors were blocked usinganti-CD16/32 (BD Bioscience, 2.4G2). Foxp3þ CD4 T cells werestained using the Mouse Regulatory T Cell Staining Kit(eBioscience) according to the manufacturer's protocol.FAPþ cells were stained as previously described (8). Tissuefactor-expressing cellswere stainedwith goat anti-tissue factor(R&D Systems) and detected as described for FAP staining (8).Dead cells were excluded using 7-amino actinomycin D (Cal-biochem). Datawere collected using a BDLSR II cytometer andanalyzed using FlowJo software (Treestar Inc.).

Bone marrow chimerasBone marrow cells were flushed from the femurs and tibias

of male FAP/DTR BAC Tg (CD45.2) or C57BL/6-Ly5.1 (CD45.1)mice and stained for FAP as previously described in PBS, 1%fetal calf serum (8). The streptavidin was phycoerythrin (PE)-conjugated (eBioscience). Bone marrow cells were depleted ofFAP-expressing cells using anti-PE Microbeads (Miltenyi Bio-tech) on LD columns according to manufacturer's protocol toprevent transfer of any donor FAPþ/CD45� cells present in thebonemarrow. FAP-depleted bonemarrow cells (10� 106) weretransferred via tail vein injection into recipient female mice,which had been lethally irradiated using a Cesium-137 sourceirradiator with 2 doses of 6 Gy, 3 hours apart. After 16 weeks,when the bonemarrow had reconstituted, LL2/OVA cells wereinjected. FAPþ cells expressing the FAP/DTR BAC transgenewere ablated through the administration of DTX (List Biolo-gicals) at 25 ng/g/24 hours via intraperitoneal injection.

ImmunofluorescenceFrozen optimum cutting temperature–embedded tumor

tissue, which were grown in FAP/EGFP BAC Tg mice, weresectioned and stained as previously described for the preser-vation of native GFP fluorescence (8). The following antibodieswere used; rat anti-F4/80 (AbD Serotec, CI:A3-1), rabbit anti-HO-1 (Insight Biotechnology, EP1391Y) and these weredetected using NL637 goat anti-rat IgG (R&D Systems) andAlexa Fluor-546 donkey anti-rabbit IgG (Invitrogen). Nucleiwere stained using 40,6-diamidino-2-phenylindole (DAPI; Invi-trogen). Images were acquired using a Leica SP2 confocalmicroscope and associated LCS analysis software.

qRT-PCR analysismRNA from tumor tissue and sorted cells was extracted

using the Absolutely RNAMiniprep Kit (Agilent Technologies).mRNA from sorted populations was amplified using the Trans-plex Complete whole transcriptome amplification kit (Sigma-Aldrich), and subsequent cDNA purified using a PCR Purifi-cation Kit (Qiagen) according to the manufacturer's protocols.qRT-PCR was performed as previously described using theSuperScript III Platinum One-step qRT-PCR system (Invitro-gen; ref. 8). The following primer/probes (Applied Biosystems)were used; HO-1 mm00516006_m1, HO-2 mm00468922_m1,IFN-g mm99999071_m1, Tbp mm01277045_m1, TNF-amm99999068_m1.

Statistical analysesStatistical analysis of tumor growth rates was performed

using the "CompareGrowthCurves" function of the statmodsoftware package (11). For comparing treatment groups, aMann–Whitney U test was used when n was <6 and a two-tailed Student t test when n was �6 and greater using Graph-Pad Prism 6 software.

Results and DiscussionThe present study was prompted by the recent finding that

stromal cells expressing the membrane protein, FAP, mediateimmune suppression in immunogenic transplanted tumorsestablished with the LL2/OVA cancer cell line (8). Because thisreport showed that the population of tumoral FAPþ cellscomprised CD45þ and CD45� subsets, this first follow-upstudy addresses the important aim of defining their respectiveimmune-suppressive functions. To characterize the hemato-poietic FAPþ stromal cell, which was equally prevalent in LL2and LL2/OVA tumors (Supplementary Fig. S1), single-cellsuspensions derived from subcutaneous LL2/OVA tumorswere subjected to flow cytometry analysis after staining withantibodies specific for CD45, F4/80, and FAP. All FAPþ/CD45þ

cells were present in the F4/80hi population (Fig. 1A), andcomprised a mean of 1.3 � 0.7% of all tumoral cells andapproximately 10% of all F4/80hi cells (Fig. 1B). The cell surfacephenotype of the FAPþ/F4/80hi subset did not differ from thatof FAP�/F4/80hi cells, in that all were CCR2þ, CD11bþ,CD11clo, CD14þ, CD16/32þ, Gr-1�, MHCIIlo, and CD206þ

(mannose receptor; Fig. 1C). The absence of Gr-1 excludedtheir identity as myeloid-derived suppressor cells (12), and theexpression of CD14 indicated that they were not fibrocytes

Arnold et al.

Cancer Immunol Res; 2(2) February 2014 Cancer Immunology Research122




(Fig. 1C; ref. 13). Thus, FAP is expressed by a minor proportionof tumoral macrophages that has an inflammatory M2 phe-notype, being CCR2þ, MHCIIlo, CD11clo, and CD206þ (Fig. 1C).To determine whether this FAPþ subset of tumoral F4/80hi

macrophages had immune-suppressive function, we generatedbone marrow chimeric mice by adoptively transferring male

bone marrow from C57BL/6-Ly5.1 or FAP/DTR BAC Tg micethat had been depleted of FAPþ cells into lethally irradiatedfemale recipients that were either C57BL/6-Ly5.1, C57BL/6(CD45.2), or FAP/DTR BAC Tg (Fig. 2A). In the resultingchimeric mice, peripheral blood leukocytes were always of atleast 95% donor origin (Supplementary Fig. S2). The recipient

Figure 2. The selective depletion ofDTR-expressing FAPþ/CD45þ andFAPþ/CD45� tumoral cells frombone marrow chimeric mice by theadministration of DTX. A, sketchdepicting the donor-recipientcombinations for generating thechimeric mice. B, the proportionsof each of the two subsets of FAPþ

cells in LL2/OVA tumors inindividual bone marrow chimericmice that had received DTX for 3consecutive days. C, the tumorvolumes (left) and the calculatedpercent change in growth (right) ofLL2/OVA tumors in the groups ofbone marrow chimeric mice thatwere given DTX for 3 consecutivedays. ns, not significant; �,P < 0.05;��, P < 0.01; ��, P < 0.001.

Figure 1. FAP expression bytumoral CD45þ cells marks asubpopulation of F4/80hi

macrophages. A, a representativeFACSdot plot of gatedCD45þcellsfrom a single-cell suspension of anLL2/OVA tumor stained with theadditional antibodies shown. B, theproportion of cells in five LL2/OVAtumors that were F4/80hi and eitherFAPþ or FAP�. C, FACShistograms of gated tumoral FAPþ

and FAP�F4/80hi cells stainedwiththe specific antibodies shown(open) and isotype controls(shaded).

FAPþ Macrophages Suppress Antitumor Immunity through HO-1

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origin of FAPþ/CD45� cells in tumors was verified by a lack ofthe Y chromosome (data not shown). The bone marrowchimeric mice were challenged by subcutaneous injection ofLL2/OVA cells. When tumors were established, mice weregiven DTX for 3 consecutive days, which led to the depletionof only the DTR-expressing subset of tumoral FAPþ cells (Fig.2B). At the initiation of DTX there was no statistically signif-icant difference in the tumor sizes between the non-Tg controland either DTR chimeric group (P > 0.05) (Supplementary Fig.S3). However, thereafter, there was slowing of tumor growth inboth types of DTR chimeric mice, but not in mice lacking theexpression of DTR in either FAPþ subset (Fig. 2C). Therefore,FAPþ cells of both hematopoietic and mesenchymal originhave immune-suppressive function, and, in this tumor model,both are required for tumor immune protection.

Having determined that the FAPþ/F4/80hi stromal cellswere capable of immune suppression in the LL2/OVA tumor,we sought a mechanism for this function. The findings of anassociation of the immunomodulatory enzyme, HO-1, with M2macrophages (14), and of the M2 phenotype of the FAPþ/F4/80hi tumoral cells (Fig. 1C) suggested the possibility that HO-1may be involved in this process. Indeed, FAPþ/F4/80þ cellscould be found expressing HO-1 in frozen sections from thetumors grown in the bone marrow chimeric mice lacking the

expression of DTR in either FAPþ subset (Supplementary Fig.S4). The FAPþ/F4/80þ cells, although distributed throughoutthe tumor, tended to be localized toward the tumor periphery.To evaluate the relative contribution of FAPþ F4/80þ cells tototal tumoral HO-1, LL2 cells were implanted in FAP/EGFPBAC Tg mice in which FAPþ cells expressed EGFP, and frozensections of the subsequent tumors were assessed by confocalmicroscopy for EGFP fluorescence, and expression of F4/80and HO-1 (Fig. 3A). Across three tumors, 75� 4.9% of the HO-1–expressing tumoral cells were double positive for EGFP andF4/80 (Fig. 3B). Moreover, HO-1 mRNA was chiefly detected inFACS sort-purified FAPþ/F4/80hi cells (Fig. 3C). Therefore, theFAPþ/F4/80þ stromal cell comprised the majority of HO-1–expressing cells in the LL2 tumor. It should be noted that therewas comparatively little expression of the closely relatedenzyme HO-2 in LL2/OVA tumors compared with HO-1 (Sup-plementary Fig. S5).

To determinewhether the expression ofHO-1 by the tumoralFAPþ/F4/80hi macrophages contributed to their immune sup-pressive function, C57BL/6 mice bearing established nonim-munogenic LL2 or immunogenic LL2/OVA tumors were trea-ted with SnMP, a specific inhibitor of HO that has no identifiedoff-target effects or toxicities. A response to a porphyrin-relatedinhibitor of HO-1, themost selective of which is SnMP, has been

Figure 3. Expression of HO-1 bythe FAPþ/F4/80þ tumoralmacrophages. A, frozen sectionsof LL2 tumors from FAP/EGFPBAC Tg mice in which FAPþ cellsare identified by native EGFPfluorescence (green) were alsostained with DAPI (blue) andantibodies specific for F4/80(white) and HO-1 (red). B, randomlyselected HO-1þ cells acrossmultiple sections and fields wereevaluated in three separate LL2tumors for coexpression of FAP/EGFP and F4/80 (>300 total HO-1þ

events were characterized). C,relative HO-1 mRNA expression insubpopulations of FACS-sortedtumoral populations from an LL2/OVA tumor.

Arnold et al.





considered to be indicative of the role of this enzyme in abiologic process (15–20). While inhibition of HO using SnMPdid not affect the growth rate of the LL2 tumors (Fig. 4A), itsignificantly slowed the growth of immunogenic LL2/OVAtumors, with this response occurring as early as 24 hours afterthe initiation of the treatment (Fig. 4A). The effect of SnMP onthe growth of the LL2/OVA tumor was confirmed to beimmune-mediated, as the inhibitor had no effect on the growthof LL2/OVA tumors in Rag2�/� mice (Fig. 4A).A subcutaneous tumor established with a cell line derived

from an autochthonous pancreatic ductal adenocarcinoma(PDA) also demonstrated the occurrence of FAPþ/F4/80þ/HO-1þ stromal cells (Supplementary Fig. S6A). Inhibition ofHO-1 by the administration of SnMP to C57BL/6 mice bearingestablished PDA tumors, which induce an immune response toan unknown antigen(s), slowed tumor growth by amechanismthat was immunemediated, as demonstrated by the absence oftumor control in SnMP-treated Rag2�/� mice bearing subcu-taneous PDA tumors (Supplementary Fig. S6B and S6C).The acute cessation of LL2/OVA growth induced by SnMP

(Fig. 4B) was not associated with a change in the immune cellcomposition of the tumors (Supplementary Fig. S7A) orwith an

increase in the activation state of the tumoral CD8 T cells, asassessed by CD69 and Lamp-1 expression (SupplementaryFig. S7B). Immune control induced by SnMP did correlatewith an increase in activated tumoral CD31þ tissue factorþ

(TF, coagulation factor III, CD142) endothelial cells (Fig. 4B).The T-cell cytokines, TNF-a and IFN-g , act synergistically to

activate endothelial cells (21). Because SnMP did not alter themRNA levels of IFN-g and TNF-a in the LL2/OVA tumors(Supplementary Fig. S8), inhibition of HO-1 may have sensi-tized endothelial cells to these cytokines. For example, one ofthe products of HO-1, carbon monoxide, suppresses the proa-poptotic effects of TNF-a on endothelial cells (22). To addressthis possibility, mice bearing LL2/OVA tumors were treatedwith neutralizing antibodies to IFN-g and TNF-a and thentreated with SnMP. In the presence of these anticytokineantibodies, SnMPdidnot arrest the growth of LL2/OVA tumors(Fig. 4D), consistent with the activity of HO-1 in the tumormicroenvironment being related to suppressing the responseof endothelial cells to these cytokines.

Of the many studies of HO-1 and inflammation, perhaps themost relevant to the current findings are those demonstratingthe ability of this enzyme and its product, carbonmonoxide, to

Figure 4. Effect of inhibiting HO-1 on adaptive immune- and cytokine-dependent regulation of tumor growth. A, C57BL/6 mice bearing established,nonimmunogenic LL2or immunogenic LL2/OVA tumors, andRag2�/�micewith LL2/OVA tumorswere treatedwith daily administration of SnMP (25mmol/kg)or vehicle control, with the arrowdenoting the initiation of treatment. B andC,mice bearing established LL2/OVA tumorswere treatedwith SnMP (25mmol/kg)or vehicle for 24 hours after which the change in tumor volumes (B) and the expression of TF by CD31þ endothelial cells were measured (C). D, micebearing LL2/OVA tumors that had been pretreated with neutralizing anti-TNF-a and anti-IFN-g antibodies or with isotype control antibody were given SnMP(25 mmol/kg) or vehicle, and tumor volumes were measured 24 hours later. The curves describing tumor growth in A were compared for differences using the"CompareGrowthCurves" permutation test. ns, not significant; �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

FAPþ Macrophages Suppress Antitumor Immunity through HO-1

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suppress rejection of allografts in rodent transplantation mod-els (23). Although this earlier study did not determine whichcells expressed HO-1, the present finding that HO-1 is pre-dominantly expressed by tumoral FAPþ/F4/80hi cells, whencoupled with the demonstration of their immune-suppressivefunction, is consistent with the theme that the host response tocancers involves tissue-protective processes that occur inother biologic settings. While the molecular basis for theimmune suppression that is mediated by the mesenchymalFAPþ/CD45� stromal cell remains to be determined, thepresent findings suggest that SnMP, which has been given tohumans to inhibit heme oxygenases (19), may have clinicalefficacy in overcoming tumoral immune suppression whenHO-1–expressing stromal cells are found to be present.

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

Authors' ContributionsConception and design: J.N. Arnold, M. Kraman, D.T. FearonDevelopment ofmethodology: J.N. Arnold, L.Magiera,M. Kraman, D.T. Fearon

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.N. Arnold, L. Magiera, M. KramanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.N. Arnold, L. Magiera, D.T. FearonWriting, review, and/or revision of the manuscript: J.N. Arnold, L. Magiera,D.T. FearonAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Magiera

AcknowledgmentsThe authors thank Dr. Alice Denton for tail vein injections of mice, Dr. Brian

Huntly for supplying the C57BL/6-Ly5.1mice (University of Cambridge), Mr. LoicTauzin for cell sorting, Dr. Stefanie Reichelt and Dr. Alexander Schreiner foradvice on confocal microscopy analyses (CRUK Cambridge Institute), andMr. John Cruise for the preparation of tumor tissue mRNA.

Grant SupportThis research was supported by the Wellcome Trust, the Ludwig Institute for

Cancer Research, and the Anthony Cerami and Anne Dunne Foundation forWorld Health (to D.T. Fearon). J.N. Arnold was supported by the IrvingtonInstitute Fellowship Program of the Cancer Research Institute.

Received September 13, 2013; revised November 11, 2013; accepted November12, 2013; published OnlineFirst November 27, 2013.

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2014;2:121-126. Published OnlineFirst November 18, 2013.Cancer Immunol Res James N. Arnold, Lukasz Magiera, Matthew Kraman, et al.

and Heme Oxygenase-1αFibroblast Activation Protein-Tumoral Immune Suppression by Macrophages Expressing

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