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Microenvironment and Immunology Chemotherapy Alters Monocyte Differentiation to Favor Generation of Cancer-Supporting M2 Macrophages in the Tumor Microenvironment Eveline M. Dijkgraaf, Moniek Heusinkveld, Bart Tummers, Lisa T.C. Vogelpoel, Renske Goedemans, Veena Jha, Johan W.R. Nortier, Marij J.P. Welters, Judith R. Kroep, and Sjoerd H. van der Burg Abstract Current therapy of gynecologic malignancies consists of platinum-containing chemotherapy. Resistance to therapy is associated with increased levels of interleukin (IL)-6 and prostaglandin E2 (PGE 2 ), 2 inammatory mediators known to skew differentiation of monocytes to tumor-promoting M2 macrophages. We investigated the impact of cisplatin and carboplatin on 10 different cervical and ovarian cancer cell lines as well as on the ability of the tumor cells to affect the differentiation and function of cocultured monocytes in vitro. Treatment with cisplatin or carboplatin increased the potency of tumor cell lines to induce IL-10producing M2 macrophages, which displayed increased levels of activated STAT3 due to tumor-produced IL-6 as well as decreased levels of activated STAT1 and STAT6 related to the PGE 2 production of tumor cells. Blockade of canonical NF-kB signaling showed that the effect of the chemotherapy was abrogated, preventing the subsequent increased production of PGE 2 and/or IL-6 by the tumor cell lines. Treatment with the COX-inhibitor indomethacin and/or the clinical monoclonal antibody against interleukin-6 receptor (IL-6R), tocilizumab, prevented M2-differentiation. Impor- tantly, no correlation existed between the production of PGE 2 or IL-6 by cancer cells and their resistance to chemotherapy-induced cell death, indicating that other mechanisms underlie the reported chemoresistance of tumors producing these factors. Our data suggest that a chemotherapy-mediated increase in tumor-promoting M2 macrophages may form an indirect mechanism for chemoresistance. Hence, concomitant therapy with COX inhibitors and/or IL-6R antibodies might increase the clinical effect of platinum-based chemotherapy in otherwise resistant tumors. Cancer Res; 73(8); 248092. Ó2013 AACR. Introduction Current treatment of advanced cervical and epithelial ovar- ian cancer includes platinum-based multimodality therapy (1, 2). Many patients with gynecologic cancer develop resistance to platinum drugs, thereby limiting further treatment options and decreasing overall survival rates (1, 3). Gynecologic cancers are generally known as immunogenic, and prominent correla- tions exist between the inltration of tumors by immune cells and clinical outcome (4, 5). Macrophages are the most abun- dant immune cells present in the tumor microenvironment. Macrophages originate from monocytic precursors in the blood and undergo specic differentiation depending on cues in the local tissue. Two extreme polarization states of macro- phages are known, M1 and M2, of which the latter has poor antigen-presenting capacity, prevents T-cell activation, con- tributes to suppressing dendritic cell (DC) functions, as well as enhances angiogenesis and metastasis (6). The presence of M2 macrophages in tumors is correlated to poor prognosis in several human cancers (7, 8). Previously, we and others showed the inuence of cervical (9) and ovarian cancer cells (10, 11) on differentiation of monocytes into DC or macrophages. The majority of cancer cells either hampered monocyte to DC differentiation or skewed their differentiation toward M2-like macrophages, depending on their ability to produce prosta- glandin E2 (PGE 2 ) and/or interleukin (IL)-6. Blocking these cytokines completely restored their differentiation toward DC (911). Interestingly, in gynecologic malignancies, upregula- tion of the COX enzymes has been associated with platinum drug resistance (12, 13). In addition, high levels of IL-6 in sera and ascites of patients with these gynecologic malignancies have also been related to chemoresistance and poor clinical outcome (14, 15). IL-6 is one of the major immunoregulatory cytokines present in the tumor microenvironment and induces several pathways leading to tumor proliferation, angiogenesis, and chemoresistance (16, 17). One important pathway stim- ulated by IL-6 is activation of STAT3 by phosphorylation. High levels of phosphorylated STAT3 are found in tumors (18) and tolerogenic antigen-presenting cells (APC; ref. 19). Authors' Afliation: Department of Clinical Oncology, Leiden University Medical Center, Albinusdreef, Leiden, the Netherlands Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Sjoerd H. van der Burg, Department of Clinical Oncology, K1-P, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, the Netherlands. Phone: 31-71-526-1180; Fax: 31-71-526- 6760; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-12-3542 Ó2013 American Association for Cancer Research. Cancer Research Cancer Res; 73(8) April 15, 2013 2480 on February 12, 2019. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst February 22, 2013; DOI: 10.1158/0008-5472.CAN-12-3542

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Page 1: Chemotherapy Alters Monocyte Differentiation to Favor …cancerres.aacrjournals.org/content/canres/73/8/2480.full.pdf · Microenvironment and Immunology Chemotherapy Alters Monocyte

Microenvironment and Immunology

Chemotherapy Alters Monocyte Differentiation to FavorGeneration of Cancer-Supporting M2 Macrophages in theTumor Microenvironment

EvelineM.Dijkgraaf,MoniekHeusinkveld, Bart Tummers, Lisa T.C. Vogelpoel, RenskeGoedemans, VeenaJha,Johan W.R. Nortier, Marij J.P. Welters, Judith R. Kroep, and Sjoerd H. van der Burg

AbstractCurrent therapy of gynecologic malignancies consists of platinum-containing chemotherapy. Resistance to

therapy is associated with increased levels of interleukin (IL)-6 and prostaglandin E2 (PGE2), 2 inflammatorymediators known to skew differentiation of monocytes to tumor-promoting M2 macrophages. We investigatedthe impact of cisplatin and carboplatin on 10 different cervical and ovarian cancer cell lines aswell as on the abilityof the tumor cells to affect the differentiation and function of cocultured monocytes in vitro. Treatment withcisplatin or carboplatin increased the potency of tumor cell lines to induce IL-10–producing M2 macrophages,which displayed increased levels of activated STAT3 due to tumor-produced IL-6 as well as decreased levels ofactivated STAT1 and STAT6 related to the PGE2 production of tumor cells. Blockade of canonical NF-kB signalingshowed that the effect of the chemotherapy was abrogated, preventing the subsequent increased production ofPGE2 and/or IL-6 by the tumor cell lines. Treatment with the COX-inhibitor indomethacin and/or the clinicalmonoclonal antibody against interleukin-6 receptor (IL-6R), tocilizumab, prevented M2-differentiation. Impor-tantly, no correlation existed between the production of PGE2 or IL-6 by cancer cells and their resistance tochemotherapy-induced cell death, indicating that other mechanisms underlie the reported chemoresistance oftumors producing these factors. Our data suggest that a chemotherapy-mediated increase in tumor-promotingM2 macrophages may form an indirect mechanism for chemoresistance. Hence, concomitant therapy with COXinhibitors and/or IL-6R antibodies might increase the clinical effect of platinum-based chemotherapy inotherwise resistant tumors. Cancer Res; 73(8); 2480–92. �2013 AACR.

IntroductionCurrent treatment of advanced cervical and epithelial ovar-

ian cancer includes platinum-based multimodality therapy (1,2). Many patients with gynecologic cancer develop resistanceto platinum drugs, thereby limiting further treatment optionsanddecreasing overall survival rates (1, 3). Gynecologic cancersare generally known as immunogenic, and prominent correla-tions exist between the infiltration of tumors by immune cellsand clinical outcome (4, 5). Macrophages are the most abun-dant immune cells present in the tumor microenvironment.Macrophages originate from monocytic precursors in theblood and undergo specific differentiation depending on cuesin the local tissue. Two extreme polarization states of macro-

phages are known, M1 and M2, of which the latter has poorantigen-presenting capacity, prevents T-cell activation, con-tributes to suppressing dendritic cell (DC) functions, as well asenhances angiogenesis and metastasis (6). The presence of M2macrophages in tumors is correlated to poor prognosis inseveral human cancers (7, 8). Previously, we and others showedthe influence of cervical (9) and ovarian cancer cells (10, 11) ondifferentiation of monocytes into DC or macrophages. Themajority of cancer cells either hampered monocyte to DCdifferentiation or skewed their differentiation toward M2-likemacrophages, depending on their ability to produce prosta-glandin E2 (PGE2) and/or interleukin (IL)-6. Blocking thesecytokines completely restored their differentiation toward DC(9–11). Interestingly, in gynecologic malignancies, upregula-tion of the COX enzymes has been associated with platinumdrug resistance (12, 13). In addition, high levels of IL-6 in seraand ascites of patients with these gynecologic malignancieshave also been related to chemoresistance and poor clinicaloutcome (14, 15). IL-6 is one of the major immunoregulatorycytokines present in the tumormicroenvironment and inducesseveral pathways leading to tumor proliferation, angiogenesis,and chemoresistance (16, 17). One important pathway stim-ulated by IL-6 is activation of STAT3 by phosphorylation.High levels of phosphorylated STAT3 are found in tumors(18) and tolerogenic antigen-presenting cells (APC; ref. 19).

Authors' Affiliation: Department of Clinical Oncology, Leiden UniversityMedical Center, Albinusdreef, Leiden, the Netherlands

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

Corresponding Author: Sjoerd H. van der Burg, Department of ClinicalOncology, K1-P, Leiden University Medical Center, Albinusdreef 2, 2333ZA, Leiden, the Netherlands. Phone: 31-71-526-1180; Fax: 31-71-526-6760; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-3542

�2013 American Association for Cancer Research.

CancerResearch

Cancer Res; 73(8) April 15, 20132480

on February 12, 2019. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 22, 2013; DOI: 10.1158/0008-5472.CAN-12-3542

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Consequently, STAT3 signaling in APC is linked to the induc-tion of T-cell tolerance (20).In view of the observations that PGE2 and IL-6 are associated

with chemoresistance and with the tumor-induced differenti-ation of tumor-promoting M2 macrophages as well as recentliterature indicating that chemotherapy may bear impact onthe function and thereby the efficacy of tumor-infiltratingimmune cells (21), we investigated the potential effects ofplatinum-based chemotherapeutics on the differentiation andfunction of APC under the influence of cervical and epithelialovarian cancer cells in vitro.We found that the treatment of tumor cells with cisplatin or

carboplatin increased the potency of some tumor cell lines toskew monocytes to M2-like macrophages. These M2-likemacrophages displayed IL-6–mediated increased levels of acti-vated STAT3 and PGE2-mediated decreased levels of activatedSTAT1 and STAT6, which are associated with immune poten-tiating pathways. The underlyingmechanismwas a cisplatin orcarboplatin-induced enhanced activation of the NF-kB path-way likely through the chemotherapy-induced DNA damageresponse (DDR). This resulted in an increased production ofPGE2 and IL-6 by cancer cells, but only if they already producedthese factors, and an enhanced skewing of monocytes towardM2-likemacrophages. Increased numbers of tumor-promotingM2-like macrophages may form an indirect mechanism forchemoresistance suggesting that concomitant therapy withCOX inhibitors and/or blocking of IL-6 receptor (IL-6R) mightincrease the antitumor effect of chemotherapy.

Materials and MethodsMedia and reagentsAPC and cancer cell lines were cultured as described earlier

(9) in RPMI-1640medium (Invitrogen) supplementedwith 10%fetal calf serum (FCS; Greiner Bio-one), 2 mmol/L L-glutamine(Cambrex), 100 U/mL penicillin, 100 mg/mL streptomycin, and50 mmol/L b-mercaptoethanol (Invitrogen), also referred to ascomplete or control medium. Adherent cells were harvestedusing trypsin/EDTA (Invitrogen). The following factors wereused to culture APC: 500 U/mL IL-4 (Invitrogen), 800 U/mLgranulocyte macrophage colony-stimulating factor (GM-CSF;Immunotools), 25 ng/mL macrophage colony-stimulating fac-tor (M-CSF; R&D), 1 to 10 ng/mL PGE2 (Sigma-Aldrich), and 1to 50 ng/mL IL-6 (Immunotools). The toll-like receptor (TLR)ligand 0.25 mg/mL lipopolysaccharide (LPS; Sigma-Aldrich)was used to activate APC; to mimic T-cell interaction, APCswere stimulated with irradiated CD40 ligand (CD40L)-expres-sing mouse fibroblasts. The following cytokines were used toinduce STAT phosphorylation: 500 U/mL IFN-g (Bender Med-systems), 10 ng/mL IL-10 (Peprotech), or 500 U/mL IL-4.

Chemotherapeutics, COX inhibitor, and monoclonalantibody against IL-6(R) and Bay 11-7082Cells were treated with 0.2 to 50 mg/mL cisplatin or 2 to 500

mg/mL carboplatin (Pharmachemie) and/or 25 mmol/L indo-methacin (Cayman Chemical) dissolved in dimethyl sulfoxide(DMSO) or as a control only with the corresponding concen-tration of DMSO. The monoclonal antibody (mAb) used to

block the (s)IL-6Rwas tocilizumab, 5 to 50 mg/mL (RoActemra,Roche BV).We used 0.02 to 200mg/mL of the selective inhibitorof kB-a (Ik-Ba) inhibitor Bay 11-7082 (Sigma-Aldrich).

Tumor cell line cultureThe human cervical cancer cell lines HELA, CASKI, CSCC1,

CSCC7, and CC8 were typed and cultured as described earlier(9). Ovarian cancer cell lines SKOV3 andA2780were purchasedfrom the European Collection of Animal Cell Cultures. CAOV3was obtained from the American Type Culture Collection(ATCC), and OVCAR3 and COV413B were kindly provided bythe department of Clinical Pathology of the Leiden UniversityMedical Center (Leiden, the Netherlands). All human ovariancancer cell lines were of epithelial origin. Cell lines wereauthenticated every half year by short tandem repeat (STR)DNA markers as described previously (22). In brief, PCRamplification of 8 highly polymorphic microsatellite STR lociand gender determination weremeasured, and the uniquenessof DNA profiles was compared for identity control within theSTR database of ATCC (23). Stock vials were thawed andcultured for 10 passages and routinely tested for the presenceof mycoplasma.

Cell lines were grown in culture flasks at 80% to 90%confluence and harvested with trypsin/EDTA and cultured in6- or 12-well plates (Corning) for 24 hours, treated withchemotherapeutics and/or COX inhibitors and/or tocilizumabas indicated. After 24 hours of treatment, cells were washedcarefully and medium was refreshed. Tumor supernatants(TSN) were harvested after an additional 24 hours of cultureand stored at �20�C. Cancer cell lines were cultured in thepresence of 2 mg/mL cisplatin or 20 mg/mL carboplatin for 24hours. These doses are estimated levels of chemotherapy in thetumor tissue because in patients, the maximum doses forcisplatin or carboplatin as measured in the blood are 5 to 6mg/mL and 40 to 80 mg/mL, respectively. Assuming that thelevels in (poorly vascularized) tumor tissue are somewhatlower, the doses used in vitro are representative for the in vivosituation (24–29).

APC cultureAPC were differentiated as described earlier (9). A brief

explanation is given in Supplementary Material and Methods.To address the direct cytotoxic effect of chemotherapy on APC,the cultures were supplemented with 20% TSN or controlmedium (monocyte-derived DC; mo-DC) and titrated dosesof chemotherapeutics, as indicated. To investigate the capacityof chemotherapy to alter the differentiation of APC by actingthrough tumor cell-mediated mechanisms, the cultures weresupplemented with 20% TSN of untreated and treated cancercell lines or control medium (mo-DC).

Transwell assayMonocytes were cultured in a 24-well plate in complete

medium with or without 2 mg/mL cisplatin or 20 mg/mLcarboplatin. Cancer cells were cultured in the top compart-ment of a Transwell 0.4 mm pore insert (Corning). After 3 days,complete medium with cytokines was added. At day 6, cellswere analyzed by flow cytometry.

Effect of Chemotherapy on Tumor Microenvironment

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Cell survivalTo determine the survival of the cancer cells upon chemo-

therapeutic treatment, an MTT assay (Trevigen) was con-ducted, according to the manufacturer's instructions. Cellsurvival was calculated as follows: (OD570–655 nm for treated/OD570–655 nm for untreated)� 100%. To determine APC survivalupon chemotherapeutic treatment, cells were analyzed by flowcytometry. Cell survival was calculated as follows: (% of cells inlive gate for treated/% of cells in live gate for untreated) �100%.

Phosphorylated STAT analysisPhosphorylated STAT (pSTAT) analysis was conducted

according to Krutzik and colleagues (30). Cells were fixed in1.5% paraformaldehyde (Sigma-Aldrich) for 10 minutes atroom temperature, harvested, and washed twice in PBS con-taining 2% FCS (PAA) and 0.02% sodium azide (AZL Pharma-cy). Then, cells were permeabilized in 90% methanol (Sigma-Aldrich) for 10minutes on ice, washed, and stained for pSTAT1(pY701), pSTAT3 (pY705), or pSTAT6 (pY641; all PE; all BDBiosciences). Expression was calculated as follows: geometricmean of fluorescence intensity of condition of interest �geometric mean of fluorescence intensity of the correspondingunstained control. Relative expression (ratio) was calculated asfollows: expression of condition of interest/expression of con-trol condition (mo-DC).

Cytokine analysisIL-12p70 and IL-10 were analyzed using ELISA Kits from BD

Biosciences or by inflammatory cytometric bead array (CBA)according to the manufacturer's instructions. To evaluate thecytokines present in supernatant of cancer cells, VEGF, IL-1b,IL-6, and IL-8 were determined by ELISA or CBA andM-CSF byBioplex (Bio-Rad). PGE2 levels were measured using the com-petitive PGE2 Immunoassay Kit (Enzo Life Sciences).

PRDX1 knockdown using shRNALentivirus expressing short hairpin RNA (shRNA) against

peroxiredoxin 1 (PRDX1) or TurboGFP (sh control) wereproduced and used to infect the CC8 cells at multiplicity ofinfection 5. Further details can be read in the SupplementaryMaterial and Methods.

ResultsDC differentiation is altered by cervical and ovariancarcinoma cell lines

Previously, we showed that when the TSN of cervical cancercells was added to monocyte cultures, this could skew thedifferentiation of monocytes to cells resembling M2 macro-phages (M2-like macrophages; ref. 9). We investigated whetheralso ovarian cancer cells could have this effect. Therefore, notonly 5 cervical cancer cell lines but also 5 ovarian cancer celllines were tested. First, the phenotype of in vitro differentiatedmonocytes cultured with TSN of cervical cancer and ovariancancer cell lines was assessed by the use of a panel of APCmarkers at day 6 of differentiation.We found that TSN from the2 ovarian cancer cell lines, COV413B and CAOV3, skewed

differentiation of monocytes toward a M2-like macrophagephenotype, and we confirmed the M2-like macrophage skew-ing capacity of HELA, CC8, and CSCC7.Wewill refer to these asM2-like macrophages and the TSN inducing these cells as M2-TSN (HELA, CC8, CSCC7, COV413B, and CAOV3). Monocytescultured in the presence of cancer cell lines that hamperedmonocyte differentiation, but did not induce M2-like macro-phages, are referred to as tumor APC and the supernatantinducing this phenotype as APC-TSN (CSCC1, CASKI, SKOV3,OVCAR3, and A2780; Supplementary Table S1).

Then, the functionality of these culturedAPCwas tested by asubsequent stimulation with LPS (a TLR4 agonist), most oftenused to stimulate tumor resident DC in vitro or with CD40L-expressing fibroblasts (CD40L) to mimic APC-T–cell interac-tion. The production of IL-12 and IL-10 was measured after 48hours. In accordance with our previous results, monocytesskewed by the TSN of ovarian cell lines COV413B and CAOV3toward an M2-like phenotype produced almost no IL-12, butinstead high levels of IL-10 when compared with mo-DC. APC-TSN displayed a similar balance in IL-12/IL-10 production asmo-DC.

To understand which soluble products in the TSN of ovariancancer cells drove the differentiation of M2 macrophages, wemeasured the production of TGFb, IL-1b, IL-6, IL-8, VEGF,PGE2, and M-CSF (Supplementary Table S2) and found thatCOV413B produced large amounts of IL-6 (25 ng/mL) but noPGE2, whereas CAOV3 produced both IL-6 (14 ng/mL) andPGE2 (2 ng/mL). Blocking of both these cytokines revealed thatthey are responsible for induction of phenotypically and func-tionally M2-like macrophages not only by cervical cancer celllines (9) but also by ovarian cancer cell lines (data not shown).

Cisplatin and carboplatin alter APC differentiation andfunction by affecting cancer cells

First, to address the cytotoxic effect of cisplatin and carbo-platin on our panel of cancer cell lines, we applied an MTTassay (Supplementary Fig. S1). We observed that the PGE2and/or IL-6–producing cell lines, HELA and CC8, were mostsensitive for chemotherapy, whereas COV413B, CAOV3, andCSCC7 producing PGE2 and/or IL-6, were most resistant tochemotherapy. In addition, SKOV3 was chemoresistant,whereas the cells did not produce PGE2 and/or IL-6 (Supple-mentary Fig. S1). This showed that there was no direct relationbetween the production of IL-6 and/or PGE2 and chemoresis-tance of cancer cells.

Furthermore, the direct cytotoxic effect of cisplatin andcarboplatin on the different types of APC (mo-DC, APC-TSN,and M2-TSN) was addressed. Monocytes were cultured in thepresence of TSN and increasing doses of chemotherapy. Fol-lowing treatment, cells were analyzed by flow cytometry.Interestingly, M2-like macrophages were more vulnerable tochemotherapy than tumor APC or mo-DC (Fig. 1A). Subse-quently, the sensitivity of classical M1 macrophages (mono-cytes cultured in the presence of GM-CSF) and classical M2macrophages (monocytes cultured in the presence of M-CSF)to cisplatin and carboplatin was tested (31). The classical M2macrophages were also more sensitive to chemotherapy thanM1 macrophages and DC (Fig. 1B).

Dijkgraaf et al.

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We previously showed that IL-6 and PGE2 promoted thedifferentiation of M2 macrophages (9), cells, which are asso-ciated with a worse response to therapy (7, 8). Therefore, westudied the effect of chemotherapy treatment on tumor cellsfocusing on the tumor-induced differentiation of APC. Tumorcells were incubated with a dose of chemotherapy represen-tative for the level within the tumor microenvironment. Sub-sequently, monocytes were cultured in the presence of TSNisolated from untreated, cisplatin, or carboplatin-treated can-cer cells. A clear increase in the percentage of CD1a-CD14þCD206þCD163þM2macrophages was observed whenM2-TSN from treated cancer cells was used compared withM2-TSN from untreated cells. This effect was not observed fortumor cells producing APC-TSN (Fig. 2), excluding the possi-bility that cell debris would have been the cause for the effectsobserved with the M2-TSN of chemotherapy-treated tumor

cells. To mimic the natural situation, we used a Transwellsystem to culture monocytes and cancer cells together in thepresence of cisplatin or carboplatin. This set-up confirmed thatplatinum-containing chemotherapeutics did not influence thedifferentiation of the APC directly as the phenotype of mo-DCremained the same upon treatment. However, addition ofplatinum chemotherapy to the culture system resulted in anincreased percentage of M2-like macrophages (Fig. 2A).

As expected (9), stimulation of differentiated monocyteswith LPS or CD40L resulted in a strong cell-surface expressionof the markers CD80, CD83, CD86, HLA-DR, and PD-L1 (datanot shown). Furthermore, chemotherapeutic treatmentresulted in a significant (P < 0.01) functional enhancement ofM2-like macrophage function (measured by indirect as well asTranswell assay). These M2-like macrophages produced sig-nificantly less IL-12 and more IL-10 upon activation with LPS

Figure 1. Survival of monocytesupon treatment with cisplatin andcarboplatin. CxCa, cervical cancercell line; OvCa, ovarian cancer cellline. A, example of survival of mo-DC, APC-TSN (TSN CASKI), andM2-TSN (TSN CAOV3) upontreatment with increasing doses ofcisplatin. B, survival of different APCupon chemotherapy treatment.Tumor APC/M2-like macrophagesreflect a mean of 3 experiments with5 different APC-TSN and M2-TSN.

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(data not shown) and CD40L after chemotherapy comparedwith their untreated controls (Fig. 2C).

Platinum-containing chemotherapy enhance PGE2 andIL-6 production by cancer cells

The capacity of cervical and ovarian cancer cells to skewmonocytes to M2-like macrophages depends mainly on theirability to produce PGE2 and/or IL-6 (9–11). Treatment of

tumor cells with cisplatin resulted in an increased productionof PGE2 and IL-6 in cell lines that already produced thesecytokines: CASKI and COV413B (IL-6), CSCC7 (PGE2), CC8,HELA, andCAOV3 (both PGE2 and IL-6). Carboplatin displayeda significant effect on PGE2 levels in HELA and CAOV3 as wellas enhanced the production of IL-6 in all IL-6–producing celllines (Fig. 3). As expected by the increased cytokine levels, theexpression of both COX-2 and/or IL-6, but not COX-1 mRNA

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Figure 2. Platinum-based chemotherapy treatment of cervical and ovarian cell lines alters APC differentiation. Mean (with SEM) of 3 experiments. Treatedtumor APC/M2-likemacrophages were compared with untreated tumor APC/M2-likemacrophages with a paired t test; �,P < 0.05; ��,P < 0.01; ���,P < 0.001.A, typical example of a flow cytometric analysis. Numbers in quadrants represent the percentage of cells within the live gate. Phenotype of mo-DC is notaffected by chemotherapy following direct incubation with chemotherapy. B, bar plots showing the percentage of CD1aþ, CD1a�CD14�, and CD14þ cellsinduced by TSN of untreated and treated tumor cells. C, the production of IL-12 and IL-10 of the different types of APC depicted in B is shown. M2-likemacrophages predominantly produce IL-10, whereas tumor APCs produce IL-12 and lower amounts of IL-10 compared with mo-DC. This effect issignificantly enhanced upon chemotherapy treatment.

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levelswere increased uponplatinum-based treatment in accor-dancewith the cytokine profile of the cell lines (SupplementaryFig. S2). Notably, cisplatin or carboplatin treatment of thecancer cells did not induce or enhance the production of TGFb,IL-1b, IL-8, VEGF, and M-CSF (Supplementary Table S2).

Altered levels of phosphorylated STAT1, 3, and 6 in TSN-differentiated macrophagesTo elucidate the mechanisms underlying the effect of

chemotherapy on APC differentiation and function, westudied the involvement of STAT. Therefore, the intracellu-lar pSTAT levels of APC were determined (Fig. 4A). Com-pared with mo-DC, APC cultured in the presence of all IL-6–producing cancer cell lines (CASKI, CC8, HELA, COV413B,and CAOV3), had significantly increased levels of pSTAT3(Fig. 4B and data not shown). A number of APC culturesdisplayed a decrease in the levels of pSTAT1 and pSTAT6.This was associated with the presence of PGE2 in the TSN(Fig. 4B and data not shown). Monocytes cultured in thepresence of cancer cell lines that did not produce PGE2 or IL-6 (CCSC1, SKOV3, OVCAR3, and A2780) did not showalteration in pSTAT levels. To test whether these factorswere responsible for the altered levels of phophorylatedSTATs, we differentiated monocytes in the presence ofincreasing doses of PGE2 and IL-6. Indeed, the levels of

pSTAT1 and pSTAT6 were dose dependently decreased inresponse to PGE2, whereas the levels of pSTAT3 increasedupon increasing amounts of IL-6 (Fig. 4C). We hypothesizedthat the observed increase in M2-skewing capacity of tumorcells treated with cisplatin or carboplatin would correlatewith further alteration of pSTAT levels. Indeed, the use ofM2-TSN isolated from cancer cell lines treated with cisplatinand carboplatin resulted in M2-like macrophages displayingsignificantly higher pSTAT3 and decreased pSTAT1 andpSTAT6 levels than M2-like macrophages cultured withM2-TSN from untreated cancer cells in almost all cases (Fig.4C). Notably, M2-like macrophages cultured with TSN ofCAOV3 already displayed low levels of pSTAT1 and pSTAT6compared with mo-DC, and when the TSN of CAOV3 treatedwith carboplatin was used, no significant further decrease inpSTAT1/6 levels was found, despite the increase in PGE2production.

Targeting PGE2 and (s)IL-6R prevent chemotherapy-enhanced M2-like macrophage skewing

We blocked the effect of IL-6 with tocilizumab, a humanmonocloncal antibody against (s)IL-6R that is clinically suc-cessful in the treatment of rheumatoid arthritis and Castlemandisease (32, 33). The effect of PGE2 was blocked by treating thecancer cells with the COX (1 and 2) inhibitor indomethacin to

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Figure 3. Alterations in phenotype and function of APC is associated with changes in PGE2 and/or IL-6 production by tumor cells. Mean (with SEM) of 3experiments. Treated tumor cells were compared with untreated tumor cells with a paired t test; �, P < 0.05; ��, P < 0.01; ���, P < 0.001.

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inhibit PGE2 production via COX-2 (Fig. 5A). Restoration to thefull phenotype ofmo-DC (Fig. 5B) aswell as function,measuredby the production of IL-12 and IL-10, could be obtained whenthe cancer cells were treated with indomethacin and/or toci-lizumab (Fig. 5C). Targeting COX-2 and (s)IL-6R also had a

clear effect on the levels of pSTAT. As expected from ourprevious experiments, treatment with tocilizumab decreasedthe levels of pSTAT3, whereas indomethacin treatmentrestored pSTAT1 and pSTAT6 levels similar to that observedin mo-DC (Fig. 5C).

mo-DC

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Figure4. Platinum-basedchemotherapy treatment of cancer cell lines influencesSTATphosphorylation inAPC.A, examples of pSTAT1, pSTAT3, andpSTAT6expression ofmonocytes cultured in the presence of (un)treated supernatant of cancer cells. B, overview of pSTAT levels inM2-likemacrophages.Mean (withSEM) of 3 experiments carried out on 3 different donors. Treated tumor APC/M2-like macrophages were compared with untreated tumor APC/M2-likemacrophages with a paired t test; �, P < 0.05; ��, P < 0.01; ���, P < 0.001. C, pSTAT1 and 6 are PGE2 dependent, whereas pSTAT3 is IL-6 dependent. Dottedlines indicate pSTAT levels in mo-DC.

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Activation of the NF-kB pathway via the DNA damageresponse is required for the chemotherapy-enhancedPGE2 and IL-6 productionTo find the underlying mechanism of the enhanced PGE2

and IL-6 production within the tumor cells upon treatment

with cisplatin and carboplatin, we investigated 2 hypotheses.Previously, Wang and colleagues showed that cisplatin canselectively crosslink a complex of the chaperone proteinPRDX1 and NF-kB at the NF-kB promotor site of the COX-2promotor region, thereby promoting COX-2 expression (34).

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Figure 5. Targeting COX-2 and IL-6 restores phenotype/function/STAT phosphorylation of APC. Mean of 3 experiments. Blocked conditions were comparedwith unblocked conditions with a paired t test; �,P < 0.05; ��,P < 0.01; ���,P < 0.001. A, PGE2 and IL-6 production is increased upon cisplatin treatment. PGE2

production is decreasedby inhibition by indomethacin. B,phenotypeof APC is restored by targetingCOX-2with indomethacin (HELA,CC8,CAOV3) and/or byblocking the (s)IL-6R with tocilizumab (COV413B, CAOV3). C, IL-10 production is decreased, whereas IL-12p70 production is restored upon targeting bothCOX-2 and the IL-6R. D, indomethacin treatment restores pSTAT1 and pSTAT6 levels to levels similar to mo-DC, whereas treatment with tocilizumabdecreases the levels of pSTAT3.

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Therefore, the expression of PRDX1 was blocked by shRNA, toprevent this protein to form a complex and hence, to preventthe increased production of PGE2. We successfully downregu-lated the expression of PRDX1, but this did not result indecreased COX-2 mRNA levels upon chemotherapy treatment(Supplementary Fig. S3).

The second mechanism that could play a role in theincreased production of PGE2 and IL-6 by cancer cells is theactivation of DDR pathway as this can lead toNF-kB activationand subsequently to the production of PGE2 and IL-6 (35, 36).To block the NF-kB pathway, Bay 11-7082, a selective inhibitorof I-kB phosphorylation and as such also of the canonical NF-kB pathway, was used. Both untreated and cisplatin-treatedcancer cells were incubated with increased doses of Bay 11-7082. Indeed, the cisplatin-induced increase in PGE2 and IL-6production was blocked by Bay 11-7082 in a dose-dependentfashion (Fig. 6).

DiscussionHere, we showed 2 potential effects of platinum-based

chemotherapy of gynecologic cancers on the immune sys-tem. First, we showed that M-CSF–induced M2 macrophagesand M2-like macrophages are most vulnerable for chemo-therapy. Monocyte-derived DC and M1 macrophages wereonly affected at higher doses, suggesting a selective survivalbenefit of these cells. This can be considered beneficial for

the patient. However, while it is clear that a sufficient doseof cisplatin or carboplatin to kill M2-like macrophages willbe reached within the blood stream, one may questionwhether this also occurs within the tumor microenviron-ment. The second effect concerns tumors in which theNF-kB pathway, leading to the production of PGE2 and/orIL-6, is already activated. Treatment of these tumors withcisplatin and carboplatin resulted in an increased produc-tion of these 2 inflammatory mediators and subsequentlyin a more pronounced skewing of monocyte differentia-tion toward the tumor promoting M2-like macrophages,reflected by their production of large amounts of IL-10,decreased production of IL-12, activation of tolerogenicSTAT3 pathway, and a decrease in the immune potentiatingSTAT1 and STAT6 pathways. This effect should be consid-ered detrimental to patients as it implies that, upon treat-ment with platinum-based regimens of PGE2 and/or IL-6–producing tumors, the number of local tumor-promotingM2 macrophages may increase, helping the tumor to defythe chemotherapeutic treatment. This would fit well withthe existing literature showing that chemoresistance ofcervical and ovarian cancer is associated with increasedlevels of PGE2 and IL-6 (12–15).

We studied 2 possible mechanisms responsible for thiseffect and showed that activation of the NF-kB pathway wasrequired, which occurred most likely via the DDR pathway.Indeed platinum-containing chemotherapeutics act by

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Figure 6. Enhanced PGE2 and IL-6 production in cancer cells in response to NF-kB activation. Increasing doses of the selective inhibitor of IkBphosphorylation and also of the canonical NF-kB pathway, Bay 11-7082, were added 1 hour before platinum treatment. Mean (with SEM) of 3 experiments.

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binding to and causing crosslinking of DNA and thus maytrigger the DDR pathway. Recently, several studies reportedthat upon DDR, NF-kB activationmay result in the productionof PGE2 and IL-6 (35, 36). More recently, it was shown thatexcessive DNA damage-induced NF-kB activation promotedexpression of IL-6 in HELA cells (37). Upon chemotherapy-induced DDR, the I-kB kinase (IKK) complex phosphorylatesIk-Ba for ubiquination and proteasomal degradation, thusallowing the NF-kB complex to translocate to the nucleus(18, 38). As the DDR pathway-activated NF-kB–mediatedproduction of PGE2 and IL-6 only occurred in tumor cell linesoriginally producing sufficient amounts of IL-6 and/or PGE2,an autocrine role for these cytokines can be envisaged ref.(39; Fig. 7).STATs represent central regulators of cancer-associated

inflammation and influence interactions between cancer cellsand their immune microenvironment that determine whetherthe inflammation promotes or inhibits cancer. The majority ofgynecologic tumors have high STAT3 activity and this wasassociated with poor survival and chemoresistance (40). Inter-estingly, it has been reported that COX-2 is a transcriptionaltarget of STAT3 signaling (41). Furthermore, high COX-2expression results in enhanced STAT3 phosphorylation in anIL-6–dependent manner (42). Together, this may form anautocrine mechanism. Notably, STAT3 and NF-kB interact atmultiple levels, thereby promoting protumorigenic inflamma-tory conditions in the tumor microenvironment (increasedPGE2 and IL-6 production), increasing tumor cell proliferationand survival as well as chemoresistance, tumor angiogenesis,and metastasis (18). Our data suggest that the autocrinesignaling loops of PGE2 and IL-6 in the tumor cells are

enhanced by the NF-kB–activating signals of the DDR pathwayinduced by platinum treatment.

Tumor-produced IL-6 induced the activation of the STAT3-signaling pathway in M2 macrophages, a signaling pathwaythat is known to be activated in tolerogenic APC (19, 20) Incontrast, STAT1 and STAT6 can support antitumor immunity(18). We found a reduction of pSTAT1 and pSTAT6 in M2-likemacrophages and showed that this was PGE2 dependent.These effects were enhanced when cancer cells were treatedwith cisplatin and carboplatin and sustain the notion thatplatinum-based chemotherapy may indirectly skew the localimmune environment to a more tolerogenic and tumor-pro-moting milieu in cancers actively producing PGE2 and/or IL-6.In a mouse tumor model, platinum-based therapy causedimmunogenic cell death, thereby activating local APC andenhancing antitumor T-cell responses. Cisplatin triggered therelease of the TLR4-stimulating protein HMGB-1 (43, 44). Wepreviously showed (9), and confirm here, that tumor-inducedmacrophages respond to the TLR4 agonist LPS as reflected bythe production of IL-10. Therefore, it is highly likely thatchemotherapy-mediated release of HMGB-1 may not onlyactivate DC, but activate local tumor-promoting macrophagesas well. Because tumor-associated macrophages can easilyoutnumber tumor-infiltrating DC, the overall effect may beless beneficial in case there is an excess of M2 macrophages.

Chemoresistance was thought for a long time to arise as aconsequence of cell intrinsic genetic changes, including upre-gulation of drug efflux pumps, activation of detoxifyingenzymes, or apoptotic defects. Recent evidence suggests thatresistance to chemotherapy can also result from cell extrinsicfactors such as cytokines and growth factors (45), implying an

Figure 7. Proposed mechanism ofenhanced PGE2 and IL-6 productionby cancer cells upon chemotherapytreatment. DNA damage caused bycisplatin and carboplatinphosphorylates Ik-Ba forubiquination and proteasomaldegradation, allowing the NF-kBcomplex to translocate to thenucleus. When STAT3 is active intumors, NF-kB prefers STAT3–p300interaction, thereby promoting theproduction of mediators, such as IL-6 and COX-2, which are associatedwith cancer-promotinginflammation. Figure adapted fromYu and colleagues (18).

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Effect of Chemotherapy on Tumor Microenvironment

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important role for the tumor microenvironment. Our datasupport this hypothesis and suggest an immunologic expla-nation for the correlation of high COX-2 expression and IL-6levels with poor response to treatment. We found no correla-tion of PGE2 and/or IL-6 production of the cancer cell lineswith chemoresistance of the cancer cell lines, whereas severalother studies show that high COX-2 expression and high levelsof IL-6 in serum and ascites identified patients with a poorresponse to cisplatin and unfavorable prognosis (13, 17, 46).This effect was explained by enhanced apoptosis resistance ofthe cancer cells but this was not reflected by our in vitro testswhere cancer cell lines were incubated with different doses ofcisplatin and carboplatin. Our observation that platinum-containing chemotherapy of cell lines with high COX-2and/or IL-6 expression promoted the differentiation of mono-cytes to tumor-promoting M2-like macrophages substantiatesthe notion that also cell extrinsic factors such as cytokinesplay a role in chemoresistance (48). In our opinion, chemore-sistance of a tumor may not only be mediated by the resis-tance of the cancer cells themselves but can occur through askewed tumor microenvironment that is geared to promotetumor cell growth for instance through the action of M2macrophages. This fits well with the finding that in glioma, theCOX-2 pathway promotes gliomagenesis by supporting thedevelopment of myeloid suppressor cells in the tumor micro-environment (47). If so, blocking the tumor-promoting effectsof PGE2 and IL-6 might even enhance the sensitivity ofotherwise more resistant tumors to chemotherapy, but thiswill require new studies.

Previously, we showed that monocytes differentiated in thepresence of HELA, CC8, and CSCC7 or its supernatant inducedan M2-like phenotype and that treatment with indomethacin(COX-inhibitor) and mAb against both IL-6 and IL-6R fullyrestored their phenotype and functionality toward that of DC.In recent clinical studies, where the effects of IL-6 weretargeted with the mAb siltuximab against IL-6, the resultswere discordant (48–50). One explanation given was the factthat if tumors produce high levels of IL-6, it will be difficult toneutralize all the soluble IL-6 present. In our experiments, weused a mAb against both the cell-surface bound form and thesoluble form of the IL-6R called tocilizumab. This antibody,which successfully blocks the detrimental effects of IL-6 in theclinic when treating rheumatoid arthritis and Castleman dis-ease (31, 32), was capable to fully block the effects of IL-6produced by untreated or chemotherapy-treated tumor cells.Pilot experiments revealed that this antibody to some extentalso blocked IL-6 signaling in the tumor cells themselves(Supplemental Fig. S4).

In summary, chemoresistance of tumors have long beenassociated with the activation of COX-2 and the production ofIL-6. Our data showed that tumor cells do not produce thesame levels of IL-6 and/or PGE2, and that there was no direct

correlation between the efficacy of cisplatin and carboplatinto kill tumor cells and the levels of these cytokines. Incontrast, we found that these chemotherapeutic compoundselicited the NF-kB pathway leading to an increased produc-tion of PGE2 and IL-6 in tumors that actively produce theseinflammatory mediators. As IL-6 or PGE2 by themselves or incombination can skew M2 macrophage differentiation, che-motherapeutic treatment of tumors will favor the differen-tiation of M2-like tolerogenic macrophages, despite thedifferences in levels produced by tumor cells. In the end,this will result in a stronger immune suppressive tumor-promoting tumor microenvironment known to be associat-ed with therapy resistance (8). Therefore, our data suggestthat a chemotherapy-induced increase in the number ofintratumoral tumor-promoting M2 macrophages forms anindirect mechanism underlying chemoresistance. Successfulblockers of PGE2 and the IL-6/IL-6R pathway are already inthe clinic. It will be of great interest to study the effects of acombined therapy of cytotoxic agents and these clinicallyavailable compounds in patients with apparently chemore-sistant tumors.

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

Authors' ContributionsConception and design: E.M. Dijkgraaf, J.W.R. Nortier, M.J.P. Welters, J.R.Kroep, S.H. van der BurgDevelopment ofmethodology: E.M. Dijkgraaf, M. Heusinkveld, B. Tummers, L.T.C. Vogelpoel, M.J.P. WeltersAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E.M. Dijkgraaf, M. Heusinkveld, B. Tummers, L.T.C.Vogelpoel, R. GoedemansAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E.M. Dijkgraaf, L.T.C. Vogelpoel, R. Goedemans, V.Jha, M.J.P. Welters, J.R. Kroep, S.H. van der BurgWriting, review, and/or revision of the manuscript: E.M. Dijkgraaf, M.Heusinkveld, L.T.C. Vogelpoel, J.W.R. Nortier, M.J.P. Welters, J.R. Kroep, S.H. vander BurgAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): B. Tummers, L.T.C. Vogelpoel, R.GoedemansStudy supervision: M.J.P. Welters, J.R. Kroep, S.H. van der Burg

AcknowledgmentsThe authors thank Drs. Thorbald van Hall and Bianca Querido for authen-

ticating all cell lines.

Grant SupportM.J.P. Welters was financially supported by a grant from the Dutch Cancer

Society 2009-4400 andB. Tummers and R. Goedemans are supported byNWO40-00812-98-09012.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 10, 2012; revised January 10, 2013; accepted January 17,2013; published OnlineFirst February 22, 2013.

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2013;73:2480-2492. Published OnlineFirst February 22, 2013.Cancer Res   Eveline M. Dijkgraaf, Moniek Heusinkveld, Bart Tummers, et al.   Microenvironmentof Cancer-Supporting M2 Macrophages in the Tumor Chemotherapy Alters Monocyte Differentiation to Favor Generation

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