CD163 Is Required for Protumoral Activation of Macrophages ...Tumor Biology and Immunology CD163 Is...

Tumor Biology and Immunology

CD163 Is Required for Protumoral Activation ofMacrophages in Human and Murine SarcomaDaisuke Shiraishi1,2, Yukio Fujiwara1, Hasita Horlad1, Yoichi Saito1, Toyohisa Iriki1,Junko Tsuboki1, Pan Cheng1, Naomi Nakagata3, Hiroshi Mizuta2, Hirofumi Bekki4,5,Yasuharu Nakashima5, Yoshinao Oda5, Motohiro Takeya1, and Yoshihiro Komohara1

Abstract

Recent findings have shown the significance of CD163-positivemacrophages in tumor progression, yet there have been fewstudies on the function of CD163 in macrophages. Here, weuncover the role of CD163 in macrophage activation usingCD163-deficient mice and human samples. We detected CD163in 62 undifferentiated pleomorphic sarcoma samples, in which ahigh percentage of CD163-positive macrophages was associatedwith decreased overall survival and higher histologic grade. Weobserved macrophage-induced tumor cell proliferation in cocul-tures of human monocyte-derived macrophages and leiomyosar-coma (TYLMS-1) and myxofibrosarcoma (NMFH-1) cell lines,whichwas abrogated by silencing of CD163. Tumor developmentof sarcoma (MCA205andLM8) cells inCD163-deficientmicewassignificantly abrogated in comparison with wild-type (WT) mice.

Coculture with WT peritoneal macrophages significantlyincreased proliferation of MCA205 cells but decreased in thepresence of CD163-deficient macrophages. Production of IL6and CXCL2 in CD163-deficient macrophages was suppressed incomparisonwithWTmacrophages, and overexpression of CD163in CD163-deficient macrophages induced production of IL6 andCXCL2. Silencing of IL6 but not CXCL2 abrogated macrophage-inducedproliferation ofMCA205 cells. Taken together, our resultsshow that CD163 is involved in protumoral activation of macro-phages and subsequent development and progression of tumorsin mice and humans.

Significance: Macrophage CD163-mediated induction of IL6promotes tumor development and progression in murine andhumanmalignant tumors. Cancer Res; 78(12); 3255–66.�2018 AACR.

IntroductionHost-derived immune cells, fibroblasts, and endothelial cells

constitute the tumor microenvironment and are known to berelated to tumor progression, and macrophages are a criticalpopulation of immune cells that induce tumor cell growth,angiogenesis,metastasis, and immune suppression (1–3).Macro-phages are suggested to be broadly classified into classicallyactivated macrophages (M1/kill macrophages) and alternativelyactivated macrophages (M2/repair macrophages) according totheir functions and expressionmarkers (4–6).Many studies usingmousemodels have indicated that M1-likemacrophages produceproinflammatory cytokines that stimulate antitumor immune

responses, whereas M2-like macrophages promote angiogenesis,immunosuppression, and tumor progression via secretinggrowth-promoting molecules. Macrophages infiltrating cancertissues are referred to as tumor-associated macrophages (TAM),which are closely involved in the development of the tumormicroenvironment, and heterogeneity of macrophage pheno-types is observed among TAMs in various malignant tumors,including sarcomas (7–9). Several clinicopathologic studies haverecently demonstrated the significance of TAMs in the growth andprogression of malignant tumors. Notably, a high density ofCD163þ TAMs is found to be positively associated with a worseprognosis in many malignant tumors (9). However, it remainsunclear how CD163 works in the protumoral activation of TAMs.

Undifferentiated pleomorphic sarcoma (UPS) is the mostfrequent soft-tissue sarcoma that shows no line of differentiation(10). It has long been known that large numbers of TAMs aredetected in UPS, and UPS was previously referred to as malignantfibrous histiocytoma (10). Regarding sarcoma, a high density ofTAMshas been found to be aprognostic factor in leiomyosarcoma(11, 12). However, little is known about the significance of TAMsin UPS. In the present study, we newly found a significantcorrelation between a high density of CD163þ TAMs and poorclinical course in patients with UPS. Because CD163 is known tobe upregulated by Th2-type cytokines, CD163 is also considered amarker for M2/repair or protumor phenotype of macrophages(13, 14). Based on this background, CD163 is suggested to becritically involved in the protumor functions of TAMs; however,few research studies have investigated the functions of CD163 inthe tumormicroenvironment. Therefore,we studied the functionsof CD163 in TAMs using an animal model and in vitro coculturestudy in the present study.

1Department ofCell Pathology, Graduate School ofMedical Sciences, KumamotoUniversity, Honjo, Kumamoto, Japan. 2Department of Orthopaedic surgery,Graduate School of Medical Sciences, Kumamoto University, Honjo, Kumamoto,Japan. 3Division of Reproductive Engineering, Center for Animal Resources andDevelopment, Kumamoto University, Honjo, Kumamoto, Japan. 4Department ofOrthopaedic Surgery, Graduate School of Medical Science, Kyushu University,Maidashi, Higashi-ku, Fukuoka, Japan. 5Anatomic Pathology, Graduate School ofMedical Science, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan.

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

D. Shiraishi and Y. Fujiwara contributed equally to this article.

Corresponding Author: Yoshihiro Komohara, Graduate School of MedicalSciences, Faculty of Life Sciences, Kumamoto University, 1-1-1, Honjo, Chuo-ku,Kumamoto 860-8556, Japan. Phone: 81-96-373-5095; Fax: 81-96-373-5096;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-2011

�2018 American Association for Cancer Research.

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Materials and MethodsPatients and assessment of CD163-positive TAMs

We evaluated 62 tumors diagnosed as UPS that were registeredin the Department of Anatomic Pathology, Kyushu University(Fukuoka, Japan), and in the Department of Cell Pathology,Kumamoto University (Kumamoto, Japan). All samples wereprimary cases, and radiation-induced sarcomas and secondarysarcomas after chemotherapy were excluded. The reassessed diag-nosis of UPS was made according to the World Health Organi-zation (WHO) 2013 classification (10). A study using this set ofUPS cases was previously published (15).We evaluated the extentof necrosis and mitosis to define each tumor's French Federationof Cancer Centers (FNCLCC) grade. The seventh edition of theAmerican Joint Committee on Cancer (AJCC) staging system wasapplied to every case. The Institutional Review Board at KyushuUniversity and Kumamoto University approved this retrospectivestudy (#27-78 and #1175). Immunohistochemistry of Iba1,CD68, and CD163 was performed as described in a previousstudy (16). Iba1- and CD163-positive cells were counted in 10randomly selected areas of high-power field of a microscope bytwo pathologists who were blinded to information about thepatients' backgrounds or their prognosis.

Tumor cell linesMCA205 (mousefibrosarcomaofC57BLbackground),NMFH-

1 (human UPS), HT-1080 (human fibrosarcoma), TYLMS-1(human leiomyosarcoma), and LM8 (mouse osteosarcoma;ref. 17) were purchased from RIKEN Cell Bank or JCRB Cell Bankbetween 2014 and 2017. Cells were maintained in RPMI 1640 orDMEM/Ham's F-12 (WAKO) supplemented with 10% fetalbovine serum (FBS) and were regularly tested using aMycoplasmatest kit (TAKARA). These cells were cultured for less than 3monthsbefore reinitiating the cultures and routinely inspecting micro-scopically for a stable phenotype.

Human macrophagesPeripheral blood mononuclear cells were acquired from

healthy volunteer donors, and written informed consent wasobtained from each participant. All the protocols using humanmacrophageswere approvedby theKumamotoUniversity ReviewBoard (No. 486) and were conducted in accordance with theapproved guidelines. CD14þ monocytes were purified fromperipheral blood mononuclear cells by positive selection viamagnetic-activated cell sorting technology (Miltenyi Biotec). Themonocytes were cultured in RPMI supplemented with 2% FBSand 50 ng/mL macrophage colony-stimulating factor (M-CSF,WAKO) for 5 days to differentiate into macrophages.

Flow cytometryHuman macrophages were fixed with 2% paraformaldehyde

and then reacted with anti-CD163 antibody or isotype matchedcontrol antibody (clone AM-3K and RM4; ref. 18) diluted in FACSbuffer (BioLegend) with 0.5% Triton-X100. Then cells werereacted anti-mouse IgG antibody labeled with Alexa 488 (BioLe-gend). For flow cytometric analysis of mouse samples, antibodiesforGr1 (FITC, BioLegend),CD11b (PacificBlue, BioLegend),CD3(APC, BioLegend), EMR1 (PE, BioLegend), and CD163 (PE, BiossAntibodies) were used. The stained cell samples were analyzed ona FACSverse (Becton Dickinson) flow cytometer with FACSuite(Becton Dickinson) software.

Coculture and 5-bromo-20-deoxyuridine incorporation assayTumor cells (10,000 cells/well) and macrophages (10,000

cells/well) were directly cocultured in 96-well plates for 2 days.5-bromo-2'-deoxyuridine (BrdUrd) incorporation was assayedusing a BrdUrd Cell Proliferation Kit (Roche) according to themanufacturer's protocol.

Cell proliferation assayBriefly, 10,000 tumor cells were cultured in a 96-well plate in

quadruplicate before treatment. The cells were then cultured inthe presence of IL6 or CXCL2. Cell viability was determined usinga WST assay (WST-8 cell counting kit; Dojin Chemical) accordingto the manufacturer's protocol.

Cytokine arrayCytokine array analysis was performed using a mouse cytokine

array kit, panel A (R&D Systems), according to themanufacturer'sprotocol.

Akt signaling arrayAkt signaling array analysiswasperformedusing aPathScanAkt

Signaling Antibody Array Kit (Cell Signaling Technology), accord-ing to the manufacturer's protocol.

Animal studiesAll animal experiments have been conducted in accordance

with an Institutional Animal Care and Use Committee. CD163-deficient (CD163�/�) mice in the C57BL/6N background wereobtained from the Knockout Mouse Project, and wild-type (WT)mice in the C57BL/6N or C3H background were obtained fromCLEA Japan. CD163�/� mice were backcrossed to the C3H strainfor more than seven generations. Mice were housed in a temper-ature-controlled roomwith a 12-hour light/dark cycle. During thecourse of the experiment, we observed no significant difference inbody weights between CD163�/� and wild-type littermate mice.All animal experiments were approved by the Ethics Committeefor Animal Experiments of Kumamoto University (#A23-154,#22-023).

Murine tumor-bearing modelFemale mice (8–10 weeks) were subcutaneously injected with

MCA205or LM8 (subclone #5) cells (5� 105) suspended in50mLof RPMI 1640. Mice were sacrificed on day 7 or 14 or 21, followedby the determination of subcutaneous tumor development andlung metastasis. The mice were monitored to assess survival. Forcytokine measurement, blood samples were drawn 7 or 14 daysafter injection.

Murine macrophagesResident peritoneal macrophages from mice (8–10 weeks)

were obtained by peritoneal lavage using 6 mL of PBS. Cellswere incubated in DMEM medium with 10% FBS. For the bonemarrow–derived macrophage (BMDM) cultures, femurs andtibias were excised from mice, and the soft tissue was removed.Briefly, marrow was flushed with sterilized PBS, and erythro-lysation was performed. Cells were seeded in polystyreneculture dishes with DMEM medium supplemented with 10%FBS. Cells were incubated in medium containing 20 ng/mLrecombinant murine macrophage colony-stimulating factor(M-CSF; PeproTech).

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Cancer Res; 78(12) June 15, 2018 Cancer Research3256




Immunohistochemistry of murine samplesSubcutaneous tumor tissues were embedded in OCT com-

pound (Sakura Finetech). After sectioning (5-mm thick), thetissues were fixed with cold acetone, and treated with the follow-ing primary antibodies: anti-CD4 (GK1.5; ATCC), anti-CD8 (53–6.72; ATCC), and anti-CD31 (MEC13.3; BD Pharmingen). Aftersectioning (3 mm thick), paraffin-embedded tumor tissues wereused for the immunostaining with anti–Ki-67 antibody (DAKO),anti-EMR1 antibody (F4/80, DAKO), and anti-CD163 antibody(CosmoBio). The sections were subsequently treated with HRP-conjugated secondary antibody (Nichirei). Reactions were visu-alized using diaminobenzidine including 0.1% NaN3.

Western blot analysisHuman and murine CD163 expression, STAT3 activation, and

Akt signalingwere evaluated byWestern blot analysis as describedpreviously (12). Briefly, macrophages were solubilized with Tri-ton X-100, and the protein concentration was determined usingthe BCA protein assay reagent, followed by pretreatment byboiling for 5 minutes in 2% SDS and 2-mercaptoethanol. Theprotein (10 mg) was run on a 10% SDS-polyacrylamide gel andwas transferred to a PVDF membrane (Millipore,). The mem-branes were exposed to anti-human CD163 antibody (clone PM-2K, Transgenic), anti-murine CD163 antibody (Cosmo Bio.),anti-pSTAT3 antibody (D3A7; Cell Signaling Technology), anti-STAT3 antibody (sc-8019; Santa Cruz Biotechnology), anti-pAktantibody (D9E; Cell Signaling Technology), anti-Akt antibody(40D4; Cell Signaling Technology), anti-pPTEN antibody (CellSignaling Technology), and anti-PTEN antibody (Cell SignalingTechnology). These membranes were re-blotted with an anti–b-actin antibody as an internal calibration control.

Statistical analysisStatistical analyseswere carried out using JMP10 (SAS Institute)

and StatMate III (ATOMS). The c2test, the Kaplan–Meiermethod,and the Cox hazard test were used to analyze the clinical courseassociations. All data presented from animal studies and cellculture studies are representative of at least two or three inde-pendent experiments. The data are expressed as the mean �standard deviation (SD, n ¼ 3–6 each groups). Differencesbetween the groups were examined for statistical significanceusing the Mann–Whitney U test and a nonrepeated measuresANOVA. A value of P < 0.05 was considered statistically signif-icant. A P value of <0.05 was considered to denote the presence ofa statistically significant difference.

ResultsA high percentage of CD163-positive cells in TAMs was relatedto a shortened overall survival

First, we examined whether Iba1-postive and CD163-positiveTAMs were correlated with tumor progression using humanresected UPS samples. CD68 is also useful as a pan-macrophagemarker; however, we selected Iba1 instead of CD68becauseCD68positivity was also seen in sarcoma cells (Supplementary Fig. S1).Tumor cells are negative for Iba1 andCD163, andCD163-positivesignals were overlapped in Iba1-positive TAMs (Fig. 1A and B).The median densities of Iba1-postive and CD163-positive TAMswere 683/mm2 and 406/mm2, respectively, and median percent-age of CD163-positive cells in Iba1-postive TAMs was 78% (Fig.1C). Cases were classified into high and low groups depending on

each number or percentage by setting the threshold value to 700/mm2 (Iba1), 400/mm2 (CD163), and 80% (CD163/Iba1),respectively. Statistical analysis revealed that a high density ofCD163-positive TAMs and a high percentage of CD163-positiveTAMswere significantly associated with high AJCC stage and highFNCLCC grade respectively (Table 1). Notably, we found that ahigh percentage of CD163-positive TAMs was significantly asso-ciated with decreased overall survival (P ¼ 0.043, Wilcoxontest; Fig. 1D). The high density of CD163-positive TAMs alsoseemed tobe linked to aworse clinical course; however, the resultsdid not reach statistical significance (P > 0.05; Fig. 1D; Supple-mentary Fig. S2; Supplementary Table S1). Whereas there was nosignificant association between the density of Iba1-positive TAMsand all clinical parameters (Table 1; Supplementary Fig. S2).These observations indicated the significance of CD163 in theprotumor functions of TAMs.

Effect of CD163 on tumor proliferation in human sarcoma celllines

Next, we tested whether CD163-positive macrophages supportsarcoma cells in a coculture study using humanmacrophages andsarcoma cell lines. Human monocyte-derived macrophages andhuman sarcoma cell lines (TYLMS-1, HT-1080, NMFH-1) werecocultured, and the BrdUrd incorporation assay was performed asshown in Fig. 2A.Coculturewithmacrophages induced tumor cellproliferation when TYLMS-1 andNMFH-1 cells were used, where-as the cell proliferation of HT-1080 was not changed by coculturewithmacrophages (Fig. 2B). No effect was observed when the celllines were cocultured with monocytes (Fig. 2B). Next, we foundthat the tumor culture supernatant (TCS) of TYLMS-1 andNMFH-1 cells more significantly upregulated CD163 expression onmacrophages than those of HT-1080 cells (Fig. 2C and D),suggesting that CD163 might play important roles in the protu-moral activation of macrophages. Therefore, following the silenc-ing of CD163 expression in macrophages by siRNA (Fig. 2E),coculture and the BrdUrd incorporation assay were performed.The knockdown of CD163 in macrophages abrogated macro-phage-induced tumor cell proliferation (Fig. 2F).

Tumordevelopment andmetastasiswere significantly impairedin CD163�/� mice

We next compared tumor development between WT mice andCD163�/� mice to study the significance of CD163 in the pro-tumor activationofmacrophages. First, we confirmed the deletionof CD163 protein in peritoneal macrophages by Western blotanalysis, and no CD163 protein was detected in peritonealmacrophages derived from CD163�/� mice (C57BL/6background; Fig. 3A and B). Similar results were seen in theC3H background mice (Supplementary Fig. S3A). The numbersand expression of M2-related markers in resident macrophagesdid not differ between WT mice and CD163�/� mice (Supple-mentary Fig. S3B). There were no significant differences in thelaboratory blood data of the two types of mice (SupplementaryTable S2). During these procedures, we found that CD163 expres-sion was strongly induced by IL4 in resident peritoneal macro-phages, whereas weak or no expression of CD163was observed inmacrophages differentiated frombonemarrow–derived cells (Fig.3C and D). The MCA205 TCS also induced CD163 expression inperitoneal macrophages isolated from WT mice (Fig. 3D and E).Next, two murine sarcoma cell lines (MCA205 and LM8) wereinjected subcutaneously into WT mice and CD163�/� mice, and

CD163 Is Involved in Sarcoma Progression

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Iba1- and CD163-positive macrophages in undifferentiated pleomorphic sarcoma. A, Immunohistochemical observations of Iba1- and CD163-positive TAMs arepresented. B, Double-immunofluorescence of Iba1 (red) and CD163 (green) was performed. C, Dot blot data of the densities of Iba1- and CD163-positiveTAMs and the percentage of CD163-positive TAMs in Iba1-positive TAMs. D, Kaplan–Meier survival curves for the 62 patients as related to the number and thepercentage of CD163-positive TAMs and statistical analysis was done by the Wilcoxon test.

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tumor development and metastasis were compared. Followingthe injection of MCA205 cell lines subcutaneously, tumornodules were resected, and the tumor weight was examined atday 7. The results showed that tumor development was signifi-cantly inhibited in CD163�/� mice compared with that in WTmice (Fig. 3F). Lung metastasis of MCA205 cells was notobserved, whereas LM8 cells are known to metastasize to thelung. As shown in Fig. 3G, both subcutaneous tumor develop-ment and lung metastasis at day 21 were significantly sup-pressed in CD163�/� mice compared with that in WT mice. Thesurvival time of LM8-injected mice was extended in CD163�/�

mice compared with that in WT mice (Fig. 3H). These observa-tions indicated that CD163 is closely involved in tumor devel-opment and metastasis.

CD163 is involved in the engraftment of tumor cells bysupporting cell proliferation

Next, immunohistochemical studies were performed to uncov-er the mechanisms related to the suppression of tumor progres-sion in CD163�/� mice. The results showed that the numbers ofinfiltrating EMR1-positive macrophages, CD4-positive lympho-cytes, and CD8-positive lymphocytes did not differ between WTmice and CD163�/� mice in the developed MCA205 tumornodules at day 7 (Supplementary Figs. S4 and S5). Angiogenesisand the Ki-67 expression in tumor cells did not differ betweenWTmice andCD163�/�mice (Supplementary Fig. S4). Similar resultswere also seen in LM8 tumor nodules at day 21 (SupplementaryFig. S4). These data suggested that the chemotaxis of macro-phages, antitumor immune response, angiogenesis, or tumor cellproliferation were not associated with impaired tumor progres-sion in CD163�/� mice. During the immunohistochemical anal-ysis, we noted that no or low CD163 expression was observed inTAMs, whereas resident macrophages around the tumor nodulestrongly expressed CD163 (Supplementary Fig. S6). Therefore, weconcluded that CD163 expressed on resident macrophages waspreferentially involved in tumor cell development. Because res-ident macrophages are associated with tumor engraftment at anearly stage of tumor development, we next examined the differ-

ence in the rejection rate of tumor cells between WT mice andCD163�/� mice. As shown in Fig. 4A, the rejection rate ofMCA205 cells was higher in CD163�/� mice than in WT mice.Next, a coculture experiment using peritoneal macrophages andMCA205 cells was performed to investigate whether WT macro-phages could support tumor cell proliferation using the BrdUrdincorporation assay. As shown in Fig. 4B, the proliferation ofMCA205 cells was significantly enhanced by coculture with WTmacrophages; interestingly, this protumor function of the macro-phages was decreased by coculture with CD163�/� macrophages(Fig. 4B). Furthermore, the proliferation of MCA205 cells wassignificantly enhanced by coculture with CD163 knock-inCD163�/� macrophages compared with coculture withCD163�/� macrophages (Fig. 4C). These results indicate thatCD163 contributes to tumor engraftment and tumorproliferationin an early stage of tumor development.

CD163 is related to IL6 and CXCL2 production frommacrophages

To examine the effect of CD163 on protumoral activationunder coculture conditions, we investigated cytokine produc-tion using a cytokine array kit. As shown in Fig. 4D, theproduction of some cytokines was significantly reduced inCD163�/� macrophage monoculture or coculture withCD163�/� macrophages, and some cytokines were shown tobe linked to CD163-related macrophage activation. We focusedon IL6 and CXCL2 because these two cytokines are known to beassociated with the growth of several tumor cells. The ELISAdata showed that MCA205 cells did not secrete IL6 and CXCL2,and the concentration of IL6 and CXCL2 in the supernatant wassignificantly increased in the coculture condition (Fig. 4E). Inaddition, IL6 and CXCL2 production was significantly impairedin CD163�/� macrophages in cases with or without coculture(Fig. 4E). By contrast, both IL6 and CXCL2 production wassignificantly increased in CD163 knock-in CD163�/� macro-phages under the coculture condition (Fig. 4F). These resultssuggest that CD163 is related to IL6 and CXCL2 productionfrom macrophages.

Table 1. Clinicopathologic parameters and the TAMs

CD163þ TAMs Iba1þ TAMs % of CD163þ

Low (<400) High (�400) Low (<700) High (�700) Low (<80) High (�80)Variable n 30 32 P 32 30 P 35 27 P

GenderMale 31 15 16 N.S. 16 15 N.S. 20 11 N.S.Female 31 15 16 16 15 15 16

Age<65 25 12 13 N.S. 13 12 N.S. 15 10 N.S.�65 37 18 19 19 18 20 17

Size<5 cm 18 11 7 N.S. 9 9 N.S. 12 6 N.S.�5 cm 44 19 25 23 21 23 21

LocationSuperficial 22 13 9 N.S. 13 10 N.S. 13 9 N.S.Deep 40 17 23 20 20 22 18

FNCLCC2 38 19 19 N.S. 19 19 N.S. 26 12 0.017a

3 24 11 13 13 11 9 15AJCC(ed 7)II 22 15 7 0.006a 12 10 N.S. 15 7 N.S.III 17 8 9 10 7 9 8IV 23 7 16 10 13 11 12

Abbreviation: N.S., not significant.aStatistically significant.






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Cell–cell interaction between macrophages and sarcoma cell lines. A and B, Human monocyte-derived macrophages (Mø) or monocytes (Mo) were incubated withhuman sarcoma cells (TYLMS-1, HT1080, and NMFH-1) under direct coculture conditions for 2 days, followed by performance of the BrdUrd incorporation assay.N.S., not significant. C and D, Human monocyte-derived macrophages were incubated with the human sarcoma culture supernatant for 24 hours, and then CD163expression was examined by Western blot analysis (C) and flow cytometry (D). E and F, Following the silencing of CD163 expression on human monocyte-derivedmacrophages by siRNA (E), coculture was performed, and tumor cell proliferation was assessed via BrdUrd incorporation assays (F). N.C., negative control.

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In vivo sarcoma model in WT and CD163�/� mice. A and B, Loss of CD163 expression in peritoneal macrophages isolated from CD163�/� mice was confirmedby Western blot analysis (A) and flow cytometry (B). C, CD163 expression in peritoneal macrophages and BMDMs was tested by Western blot analysis.D and E, TCS-induced CD163 overexpression was evaluated by flow cytometry (D) and Western blot analysis (E). F, Following the injection of MCA205 cellsinto WT and CD163�/� mice subcutaneously, tumor weight was evaluated at day 7. G, Following the injection of LM8 cells into WT and CD163�/� micesubcutaneously, tumor weight and lung metastasis were evaluated at day 21. H, Survival time was observed in WT and CD163�/� mice transplanted withLM8. I, Immunohistochemistry for EMR1, CD31, CD4, CD8, and Ki67 were performed in tumor samples from WT and CD163�/� mice transplanted with MCA205,and representative data are presented.






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Figure 4.

Protumuor function and cytokine production of macrophages. A, Following the injection of MCA205 cells (50, 500, or 5000 cells per mice) into WT and CD163�/�

mice, tumor development was assessed after 1 month. B, MCA205 cells were cocultured with peritoneal macrophages isolated from WT and CD163�/� micefor 24 hours, and the BrdUrd incorporation assay was performed to evaluate tumor cell proliferation. C, MCA205 cells were cocultured with CD163�/� peritonealmacrophages and CD163 knock-in CD163�/� peritoneal macrophage for 24 hours, and the BrdUrd incorporation assay was performed to evaluate tumor cellproliferation. D, MCA205 cells were cocultured with peritoneal macrophages isolated from WT and CD163�/� mice for 24 hours, and cytokine production in theculture supernatant was tested by cytokine array. E,MCA205 cells were cocultured with peritoneal macrophages isolated fromWT and CD163�/�mice for 24 hours,and the concentration of IL6 andCXCL2 in the culture supernatantwas testedby ELISA.F,MCA205 cellswere coculturedwith CD163�/�peritonealmacrophages andCD163 knock-in CD163�/� peritoneal macrophage for 24 hours, and the concentration of IL6 and CXCL2 in the culture supernatant was tested by ELISA.


Shiraishi et al.




Effect of IL6 andCXCL2on tumor proliferation inMCA205 cellsWe next examined the effect of IL6 and CXCL2 on tumor cell

proliferation using MCA205 cells. As shown in Fig. 5A, IL6 andCXCL2 induced tumor proliferation, indicating that IL6 andCXCL2 play an important role in tumor proliferation under thecoculture condition. Furthermore, the knockdown of the IL6receptor in MCA205 cells suppressed tumor proliferation underthe coculture condition, and the knockdown of IL6 or IL6R inmacrophages also suppressed tumor proliferation under thecoculture condition when WT macrophages were used for theculture study (Fig. 5B–D). Notably, the knockdown of IL6R or IL6did not influence tumor cell proliferation when cells are cocul-tured with CD163�/� macrophages (Fig. 5C and D). However,tumor proliferation was not inhibited by the knockdown ofCXCL2 in macrophages (Fig. 5E). Furthermore, the WT macro-phage culture supernatant significantly induced the activation ofSTAT3, which plays an important role in tumor progression intumor cells, compared with that in control cells, whereas theCD163�/� macrophage culture supernatant affected STAT3 acti-vation slightly (Fig. 5F). IL6 also significantly induced STAT3activation (Fig. 5F). These data indicate that IL6, rather thanCXCL2, derived from macrophages is involved in tumor prolif-eration under the coculture condition.

DiscussionCD163 is amember of the scavenger receptor super family class

B and is specifically expressed on myeloid lineages such asmonocytes and macrophages (19–21). CD163 is a cell surfaceglycoprotein receptorwith amolecularweight of 130 kDaandwasfirst found to be a receptor of the hemoglobin (Hb) and hapto-globin (Hp) complex. The Hb-Hp complex was recognized byCD163 for clearance by receptor-mediated endocytosis (22).CD163 was also found to be related to the recognition of bacteriaby macrophages (23). The soluble form of CD163 in humanserum has been reported to be useful for evaluating macrophageactivation in sepsis, rheumatic disease, and liver diseases (24–26);therefore, CD163 is now of interest as a marker of macrophageactivation in several diseases.

In the present study, we revealed that CD163 was closelyassociated with the protumoral activation of macrophages,although the detailed signaling pathways via CD163 have neverbeen uncovered. CD163 is related to the production of protumorcytokines such as IL6 and CXCL2 frommacrophages, and IL6 wasfound to be more preferentially associated with macrophage-related tumor cell proliferation. IL6 plays a central role in tumordevelopment and progression via supporting tumor cell prolif-eration, survival, and metastasis via Stat3 activation (27).Although tumor cell lines that do not secrete IL6 were used inthis study, some tumor cells secrete IL6 and form paracrine andautocrine IL6 loops by inducing IL6 production from stromal cells(28). IL6 derived fromTAMswas associatedwith the expansion ofcancer stem-like cells in hepatocellular carcinoma (29) and wasalso involved in the niche for cancer stem-like cells and drugresistance in theMC38murine sarcomamodel (30). An increaseddensity of CD163-positive TAMs is positively correlated with theexpression of stem-cell markers, including CD44 and ALDH1, insome types of human cancers (31–33). These observations indi-cated that CD163-related macrophage activation is associatedwith themaintenance of cancer stem-like cells and induces tumorprogression.

In the present study, we demonstrated the discrepancy ofhuman and murine macrophages; CD163 is expressed on bothresident and monocyte-derived macrophages in humans (34),whereas CD163 is detected on resident macrophages but not onBMDMs in mice. When the tumor is small, resident macrophagesin the surrounding tissues accumulate intratumorally, but mono-cyte-derived macrophages become the main source of TAMs afterthe vascular network of the tumor tissue is formed (35). Differentgene expression profiles between resident macrophages andBMDMs have been reported in mice, and EMR1 (F4/80 antigen),as well as CD163, is also expressed on resident macrophages at ahigher level than that onBMDMs (36). In the present study, EMR1was expressed on TAMs and resident macrophages around thetumor nodule, and the staining intensity seemed to be higher onresident macrophages than on TAMs. However, CD163 wasexpressed on resident macrophages but not on TAMs. Therefore,CD163 is considered useful to distinguish resident macrophagesfrom BMDMs.

The detailed mechanisms of CD163-related signals have notbeen clarified; however, we found that IL6 and CXCL2 expres-sion was significantly impaired in CD163�/� macrophages. Itwas reported approximately 10 years ago that antibody-depen-dent CD163 stimulation induced inflammatory cytokines suchas IL1b and IL6 frommacrophages (37) and activation of caseinkinase II and protein kinase C (38); however, these observa-tions were not reflected in our unpublished data. A recent studyusing CD163�/� mice demonstrated that soluble CD163 acts asa decoy receptor for TWEAK, and a deficiency of soluble CD163increased the concentration of TWEAK in peripheral blood topromote tissue regeneration after ischemic injury (39); how-ever, no description related to CD163-related signals has beenseen. TWEAK has been reported to promote tumor cell inva-sion/progression rather than apoptosis (40, 41); therefore, theresult of our present study that tumor development wasimpaired in CD163�/�mice is probably not due to an increasedTWEAK. In our preliminary study, the PTEN-Akt signal in tumorcells was activated by coculture with macrophages, and thiscell–cell interaction was impaired when CD163�/� macro-phages were used for the coculture study. PTEN demonstratesphosphatase activity against the phospholipid products of PI3-kinase activity, leading to the inactivation of Akt signaling (42).PTEN activity is controlled by phosphorylation and phosphor-ylated PTEN attenuates its inhibitory function of Akt signaling,leading to the promotion of survival and growth in tumor cells(42). Therefore, it is suggested that unknown factors suppres-sing PTEN activity are thought to be secreted by macrophages,and CD163 is potentially related to tumor survival and growthindirectly via Akt signaling activation and PTEN inactivation intumor cells.

In the present study, we used human cell lines and tissuesamples to demonstrate the significance of CD163 in tumor cellproliferation, and an unknown CD163-related signal was sug-gested to be related to the protumoral activation of humanmacrophages as well as murine macrophages. Although a similarobservation was observed in our previous study using malignantlymphoma samples, the detailed molecular mechanisms ofCD163-related macrophage activation have never been clarified(43). CD163 is known to mediate cell–cell contacts betweenmacrophages and erythroblasts (44), and CD163might promotecell–cell adhesion between macrophages and tumor cells viaunknown ligands.






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Protumor effect of IL6 andCXCL2.A,MCA205 cellswere incubatedwith the indicated concentrations of IL6 or CXCL2 for 2 days, and then tumor cell proliferationwastested by the WST-8 assay. B and C, Following the silencing of IL6 expression on macrophages by siRNA (B), coculture study was performed, and tumor cellproliferation was assessed by the BrdUrd incorporation assay (C).D, Following the silencing of IL6R expression onMCA205 cells by siRNA, the same coculture studywas performed. E, Following the silencing of CXCL2 expression on macrophages by siRNA, coculture study and BrdUrd incorporation assays were performed.F,MCA205 cells were incubated withmacrophage culture supernatant (Mø CS) for 24 hours, followed by the determination of the pSTAT3, STAT3, and b-actin levelsby Western blot analysis, as described in Materials and Methods.


Shiraishi et al.




In conclusion, we demonstrated the significance of CD163-relatedmacrophage activation in the cell–cell interaction betweentumor cells andTAMsusingmouse andhuman cells. Although thedetailed signal activation pathways of CD163-related macro-phages activation remain to be uncovered, CD163 or CD163-related signals are considered a new therapeutic target for anti-tumor therapy.

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

Authors' ContributionsConception and design: Y. Fujiwara, Y. KomoharaDevelopment of methodology: D. Shiraishi, T. Iriki, Y. KomoharaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Shiraishi, Y. Fujiwara, H. Horlad, Y. Saito,N. Nakagata, Y. Oda, Y. KomoharaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):D. Shiraishi, Y. Fujiwara, H. Horlad, Y. Saito, T. Iriki,Junko Tsuboki, H. Bekki, Y. Komohara

Writing, review, and/or revision of the manuscript: Y. Fujiwara, Y. KomoharaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. KomoharaStudy supervision: H. Mizuta, M. Takeya, Y. Komohara

AcknowledgmentsWe thank Mrs. Emi Kiyota, Mr. Takenobu Nakagawa, and Ms. Ikuko

Miyakawa for their technical assistance. This work was supported by JSPSKAKENHI. M. Takeya received JSPS KAKENHI grant No. 25293089, Y.Komohara received JSPS KAKENHI grant No. 16H05162, Y. Fujiwarareceived JSPS KAKENHI grant No. 16K09247, and D. Shiraishi received JSPSKAKENHI grant No. 16K10865.

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

Received July 19, 2017; revisedNovember 30, 2017; acceptedMarch28, 2018;published first April 2, 2018.

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2018;78:3255-3266. Published OnlineFirst April 2, 2018.Cancer Res Daisuke Shiraishi, Yukio Fujiwara, Hasita Horlad, et al. Human and Murine SarcomaCD163 Is Required for Protumoral Activation of Macrophages in

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