CD163 Is Required for Protumoral Activation of Macrophages In

Published OnlineFirst April 2, 2018; DOI: 10.1158/0008-5472.CAN-17-2011

Cancer Tumor Biology and Immunology Research

CD163 Is Required for Protumoral Activation of Macrophages in Human and Murine Sarcoma Daisuke 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-positive Coculture with WT peritoneal macrophages significantly macrophages in tumor progression, yet there have been few increased proliferation of MCA205 cells but decreased in the studies on the function of CD163 in macrophages. Here, we presence of CD163-deficient macrophages. Production of IL6 uncover the role of CD163 in macrophage activation using and CXCL2 in CD163-deficient macrophages was suppressed in CD163-deficient mice and human samples. We detected CD163 comparison with WT macrophages, and overexpression of CD163 in 62 undifferentiated pleomorphic sarcoma samples, in which a in CD163-deficient macrophages induced production of IL6 and high percentage of CD163-positive macrophages was associated CXCL2. Silencing of IL6 but not CXCL2 abrogated macrophage- with decreased overall survival and higher histologic grade. We induced proliferation of MCA205 cells. Taken together, our results observed macrophage-induced tumor cell proliferation in cocul- show that CD163 is involved in protumoral activation of macro- tures of human monocyte-derived macrophages and leiomyosar- phages and subsequent development and progression of tumors coma (TYLMS-1) and myxofibrosarcoma (NMFH-1) cell lines, in mice and humans. which was abrogated by silencing of CD163. Tumor development Significance: Macrophage CD163-mediated induction of IL6 of sarcoma (MCA205 and LM8) cells in CD163-deficient mice was promotes tumor development and progression in murine and significantly abrogated in comparison with wild-type (WT) mice. human malignant tumors. Cancer Res; 78(12); 3255–66. 2018 AACR.

Introduction responses, whereas M2-like macrophages promote angiogenesis, immunosuppression, and tumor progression via secreting Host-derived immune cells, fibroblasts, and endothelial cells growth-promoting molecules. Macrophages infiltrating cancer constitute the tumor microenvironment and are known to be tissues are referred to as tumor-associated macrophages (TAM), related to tumor progression, and macrophages are a critical which are closely involved in the development of the tumor population of immune cells that induce tumor cell growth, microenvironment, and heterogeneity of macrophage pheno- angiogenesis, metastasis, and immune suppression (1–3). Macro- types is observed among TAMs in various malignant tumors, phages are suggested to be broadly classified into classically including sarcomas (7–9). Several clinicopathologic studies have activated macrophages (M1/kill macrophages) and alternatively recently demonstrated the significance of TAMs in the growth and activated macrophages (M2/repair macrophages) according to progression of malignant tumors. Notably, a high density of their functions and expression markers (4–6). Many studies using þ CD163 TAMs is found to be positively associated with a worse mouse models have indicated that M1-like macrophages produce prognosis in many malignant tumors (9). However, it remains proinflammatory cytokines that stimulate antitumor immune unclear how CD163 works in the protumoral activation of TAMs. Undifferentiated pleomorphic sarcoma (UPS) is the most 1Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto frequent soft-tissue sarcoma that shows no line of differentiation University, Honjo, Kumamoto, Japan. 2Department of Orthopaedic surgery, (10). It has long been known that large numbers of TAMs are Graduate School of Medical Sciences, Kumamoto University, Honjo, Kumamoto, detected in UPS, and UPS was previously referred to as malignant Japan. 3Division of Reproductive Engineering, Center for Animal Resources and fibrous histiocytoma (10). Regarding sarcoma, a high density of 4 Development, Kumamoto University, Honjo, Kumamoto, Japan. Department of TAMs has been found to be a prognostic factor in leiomyosarcoma Orthopaedic Surgery, Graduate School of Medical Science, Kyushu University, (11, 12). However, little is known about the significance of TAMs Maidashi, Higashi-ku, Fukuoka, Japan. 5Anatomic Pathology, Graduate School of in UPS. In the present study, we newly found a significant Medical Science, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan. þ correlation between a high density of CD163 TAMs and poor Note: Supplementary data for this article are available at Cancer Research clinical course in patients with UPS. Because CD163 is known to Online (http://cancerres.aacrjournals.org/). be upregulated by Th2-type cytokines, CD163 is also considered a D. Shiraishi and Y. Fujiwara contributed equally to this article. marker for M2/repair or protumor phenotype of macrophages Corresponding Author: Yoshihiro Komohara, Graduate School of Medical (13, 14). Based on this background, CD163 is suggested to be Sciences, Faculty of Life Sciences, Kumamoto University, 1-1-1, Honjo, Chuo-ku, critically involved in the protumor functions of TAMs; however, Kumamoto 860-8556, Japan. Phone: 81-96-373-5095; Fax: 81-96-373-5096; few research studies have investigated the functions of CD163 in E-mail: [email protected] the tumor microenvironment. Therefore, we studied the functions doi: 10.1158/0008-5472.CAN-17-2011 of CD163 in TAMs using an animal model and in vitro coculture 2018 American Association for Cancer Research. study in the present study.

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Materials and Methods Coculture and 5-bromo-20-deoxyuridine incorporation assay Tumor cells (10,000 cells/well) and macrophages (10,000 Patients and assessment of CD163-positive TAMs cells/well) were directly cocultured in 96-well plates for 2 days. We evaluated 62 tumors diagnosed as UPS that were registered 5-bromo-2'-deoxyuridine (BrdUrd) incorporation was assayed in the Department of Anatomic Pathology, Kyushu University using a BrdUrd Cell Proliferation Kit (Roche) according to the (Fukuoka, Japan), and in the Department of Cell Pathology, manufacturer's protocol. Kumamoto University (Kumamoto, Japan). All samples were primary cases, and radiation-induced sarcomas and secondary sarcomas after chemotherapy were excluded. The reassessed diag- Cell proliferation assay nosis of UPS was made according to the World Health Organi- Briefly, 10,000 tumor cells were cultured in a 96-well plate in zation (WHO) 2013 classification (10). A study using this set of quadruplicate before treatment. The cells were then cultured in UPS cases was previously published (15). We evaluated the extent the presence of IL6 or CXCL2. Cell viability was determined using of necrosis and mitosis to define each tumor's French Federation a WST assay (WST-8 cell counting kit; Dojin Chemical) according of Cancer Centers (FNCLCC) grade. The seventh edition of the to the manufacturer's protocol. American Joint Committee on Cancer (AJCC) staging system was applied to every case. The Institutional Review Board at Kyushu Cytokine array University and Kumamoto University approved this retrospective Cytokine array analysis was performed using a mouse cytokine study (#27-78 and #1175). Immunohistochemistry of Iba1, array kit, panel A (R&D Systems), according to the manufacturer's CD68, and CD163 was performed as described in a previous protocol. study (16). Iba1- and CD163-positive cells were counted in 10 fi randomly selected areas of high-power eld of a microscope by Akt signaling array two pathologists who were blinded to information about the Akt signaling array analysis was performed using a PathScan Akt patients' backgrounds or their prognosis. Signaling Antibody Array Kit (Cell Signaling Technology), according to the manufacturer's protocol. Tumor cell lines MCA205 (mouse fibrosarcoma of C57BL background), NMFH- Animal studies 1 (human UPS), HT-1080 (human fibrosarcoma), TYLMS-1 All animal experiments have been conducted in accordance (human leiomyosarcoma), and LM8 (mouse osteosarcoma; with an Institutional Animal Care and Use Committee. CD163- ref. 17) were purchased from RIKEN Cell Bank or JCRB Cell Bank deficient (CD163 / ) mice in the C57BL/6N background were between 2014 and 2017. Cells were maintained in RPMI 1640 or obtained from the Knockout Mouse Project, and wild-type (WT) DMEM/Ham's F-12 (WAKO) supplemented with 10% fetal mice in the C57BL/6N or C3H background were obtained from bovine serum (FBS) and were regularly tested using a Mycoplasma CLEA Japan. CD163 / mice were backcrossed to the C3H strain test kit (TAKARA). These cells were cultured for less than 3 months for more than seven generations. Mice were housed in a temper- before reinitiating the cultures and routinely inspecting micro- ature-controlled room with a 12-hour light/dark cycle. During the scopically for a stable phenotype. course of the experiment, we observed no significant difference in body weights between CD163 / and wild-type littermate mice. Human macrophages All animal experiments were approved by the Ethics Committee Peripheral blood mononuclear cells were acquired from for Animal Experiments of Kumamoto University (#A23-154, healthy volunteer donors, and written informed consent was #22-023). obtained from each participant. All the protocols using human macrophages were approved by the Kumamoto University Review Murine tumor-bearing model Board (No. 486) and were conducted in accordance with the Female mice (8–10 weeks) were subcutaneously injected with þ approved guidelines. CD14 monocytes were purified from MCA205 or LM8 (subclone #5) cells (5 105) suspended in 50 mL peripheral blood mononuclear cells by positive selection via of RPMI 1640. Mice were sacrificed on day 7 or 14 or 21, followed magnetic-activated cell sorting technology (Miltenyi Biotec). The by the determination of subcutaneous tumor development and monocytes were cultured in RPMI supplemented with 2% FBS lung metastasis. The mice were monitored to assess survival. For and 50 ng/mL macrophage colony-stimulating factor (M-CSF, cytokine measurement, blood samples were drawn 7 or 14 days WAKO) for 5 days to differentiate into macrophages. after injection.

Flow cytometry Murine macrophages Human macrophages were fixed with 2% paraformaldehyde Resident peritoneal macrophages from mice (8–10 weeks) and then reacted with anti-CD163 antibody or isotype matched were obtained by peritoneal lavage using 6 mL of PBS. Cells control antibody (clone AM-3K and RM4; ref. 18) diluted in FACS were incubated in DMEM medium with 10% FBS. For the bone buffer (BioLegend) with 0.5% Triton-X100. Then cells were marrow–derived macrophage (BMDM) cultures, femurs and reacted anti-mouse IgG antibody labeled with Alexa 488 (BioLe- tibias were excised from mice, and the soft tissue was removed. gend). For flow cytometric analysis of mouse samples, antibodies Briefly, marrow was flushed with sterilized PBS, and erythro- for Gr1 (FITC, BioLegend), CD11b (Pacific Blue, BioLegend), CD3 lysation was performed. Cells were seeded in polystyrene (APC, BioLegend), EMR1 (PE, BioLegend), and CD163 (PE, Bioss culture dishes with DMEM medium supplemented with 10% Antibodies) were used. The stained cell samples were analyzed on FBS. Cells were incubated in medium containing 20 ng/mL a FACSverse (Becton Dickinson) flow cytometer with FACSuite recombinant murine macrophage colony-stimulating factor (Becton Dickinson) software. (M-CSF; PeproTech).

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CD163 Is Involved in Sarcoma Progression

Immunohistochemistry of murine samples each number or percentage by setting the threshold value to 700/ Subcutaneous tumor tissues were embedded in OCT com- mm2 (Iba1), 400/mm2 (CD163), and 80% (CD163/Iba1), pound (Sakura Finetech). After sectioning (5-mm thick), the respectively. Statistical analysis revealed that a high density of tissues were fixed with cold acetone, and treated with the follow- CD163-positive TAMs and a high percentage of CD163-positive ing primary antibodies: anti-CD4 (GK1.5; ATCC), anti-CD8 (53– TAMs were significantly associated with high AJCC stage and high 6.72; ATCC), and anti-CD31 (MEC13.3; BD Pharmingen). After FNCLCC grade respectively (Table 1). Notably, we found that a sectioning (3 mm thick), paraffin-embedded tumor tissues were high percentage of CD163-positive TAMs was significantly asso- used for the immunostaining with anti–Ki-67 antibody (DAKO), ciated with decreased overall survival (P ¼ 0.043, Wilcoxon anti-EMR1 antibody (F4/80, DAKO), and anti-CD163 antibody test; Fig. 1D). The high density of CD163-positive TAMs also (CosmoBio). The sections were subsequently treated with HRP- seemed to be linked to a worse clinical course; however, the results conjugated secondary antibody (Nichirei). Reactions were visu- did not reach statistical significance (P > 0.05; Fig. 1D; Supple- alized using diaminobenzidine including 0.1% NaN3. mentary Fig. S2; Supplementary Table S1). Whereas there was no significant association between the density of Iba1-positive TAMs Western blot analysis and all clinical parameters (Table 1; Supplementary Fig. S2). Human and murine CD163 expression, STAT3 activation, and These observations indicated the significance of CD163 in the Akt signaling were evaluated by Western blot analysis as described protumor functions of TAMs. previously (12). Briefly, macrophages were solubilized with Tri- ton X-100, and the protein concentration was determined using Effect of CD163 on tumor proliferation in human sarcoma cell the BCA protein assay reagent, followed by pretreatment by lines boiling for 5 minutes in 2% SDS and 2-mercaptoethanol. The Next, we tested whether CD163-positive macrophages support protein (10 mg) was run on a 10% SDS-polyacrylamide gel and sarcoma cells in a coculture study using human macrophages and was transferred to a PVDF membrane (Millipore,). The mem- sarcoma cell lines. Human monocyte-derived macrophages and branes were exposed to anti-human CD163 antibody (clone PM- human sarcoma cell lines (TYLMS-1, HT-1080, NMFH-1) were 2K, Transgenic), anti-murine CD163 antibody (Cosmo Bio.), cocultured, and the BrdUrd incorporation assay was performed as anti-pSTAT3 antibody (D3A7; Cell Signaling Technology), anti- shown in Fig. 2A. Coculture with macrophages induced tumor cell STAT3 antibody (sc-8019; Santa Cruz Biotechnology), anti-pAkt proliferation when TYLMS-1 and NMFH-1 cells were used, where- antibody (D9E; Cell Signaling Technology), anti-Akt antibody as the cell proliferation of HT-1080 was not changed by coculture (40D4; Cell Signaling Technology), anti-pPTEN antibody (Cell with macrophages (Fig. 2B). No effect was observed when the cell Signaling Technology), and anti-PTEN antibody (Cell Signaling lines were cocultured with monocytes (Fig. 2B). Next, we found Technology). These membranes were re-blotted with an anti– that the tumor culture supernatant (TCS) of TYLMS-1 and NMFH- b-actin antibody as an internal calibration control. 1 cells more significantly upregulated CD163 expression on macrophages than those of HT-1080 cells (Fig. 2C and D), Statistical analysis suggesting that CD163 might play important roles in the protu- Statistical analyses were carried out using JMP10 (SAS Institute) moral activation of macrophages. Therefore, following the silenc- and StatMate III (ATOMS). The c2test, the Kaplan–Meier method, ing of CD163 expression in macrophages by siRNA (Fig. 2E), and the Cox hazard test were used to analyze the clinical course coculture and the BrdUrd incorporation assay were performed. associations. All data presented from animal studies and cell The knockdown of CD163 in macrophages abrogated macro- culture studies are representative of at least two or three inde- phage-induced tumor cell proliferation (Fig. 2F). pendent experiments. The data are expressed as the mean fi standard deviation (SD, n ¼ 3–6 each groups). Differences Tumor development and metastasis were signi cantly impaired / between the groups were examined for statistical significance in CD163 mice using the Mann–Whitney U test and a nonrepeated measures We next compared tumor development between WT mice and / fi ANOVA. A value of P < 0.05 was considered statistically signif- CD163 mice to study the signi cance of CD163 in the pro- fi icant. A P value of <0.05 was considered to denote the presence of tumor activation of macrophages. First, we con rmed the deletion a statistically significant difference. of CD163 protein in peritoneal macrophages by Western blot analysis, and no CD163 protein was detected in peritoneal macrophages derived from CD163 / mice (C57BL/6 Results background; Fig. 3A and B). Similar results were seen in the A high percentage of CD163-positive cells in TAMs was related C3H background mice (Supplementary Fig. S3A). The numbers to a shortened overall survival and expression of M2-related markers in resident macrophages First, we examined whether Iba1-postive and CD163-positive did not differ between WT mice and CD163 / mice (Supple- TAMs were correlated with tumor progression using human mentary Fig. S3B). There were no significant differences in the resected UPS samples. CD68 is also useful as a pan-macrophage laboratory blood data of the two types of mice (Supplementary marker; however, we selected Iba1 instead of CD68 because CD68 Table S2). During these procedures, we found that CD163 expres- positivity was also seen in sarcoma cells (Supplementary Fig. S1). sion was strongly induced by IL4 in resident peritoneal macro- Tumor cells are negative for Iba1 and CD163, and CD163-positive phages, whereas weak or no expression of CD163 was observed in signals were overlapped in Iba1-positive TAMs (Fig. 1A and B). macrophages differentiated from bone marrow–derived cells (Fig. The median densities of Iba1-postive and CD163-positive TAMs 3C and D). The MCA205 TCS also induced CD163 expression in were 683/mm2 and 406/mm2, respectively, and median percent- peritoneal macrophages isolated from WT mice (Fig. 3D and E). age of CD163-positive cells in Iba1-postive TAMs was 78% (Fig. Next, two murine sarcoma cell lines (MCA205 and LM8) were 1C). Cases were classified into high and low groups depending on injected subcutaneously into WT mice and CD163 / mice, and

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A Case 1 (TAM low) Case 2 (TAM high) C 2,000 ) 2 1,500

Iba1 1,000

50 μm 50 μm 500 Cell density Cell density (/mm

0 Iba1 CD163 CD163 150

B 100 Iba1 CD163 DAPI Merge

50 Relative population (%) 50 μm 0 CD163/Iba1

D 1.0 1.0

0.8 2 0.8 CD163 (/mm ) : Low CD163 (%) : Low 0.6 0.6 PFS 0.4 2 PFS 0.4 CD163 (/mm ) : High CD163 (%) : High 0.2 P = 0.16 0.2 P = 0.11 0.0 0.0 0 20 806040 100 120 140 20 806040 100 120 140 Months Months

1.0 1.0 CD163 (/mm2) : Low 0.8 0.8 CD163 (%) : Low

0.6 0.6 OS OS 0.4 CD163 (/mm2) : High 0.4 CD163 (%) : High 0.2 P 0.2 = 0.15 P = 0.043 0.0 0.0 0 20 806040 100 120 140 160 0 20 806040 100 120 140 160 Months Months

Figure 1. Iba1- and CD163-positive macrophages in undifferentiated pleomorphic sarcoma. A, Immunohistochemical observations of Iba1- and CD163-positive TAMs are presented. B, Double-immunoﬂuorescence of Iba1 (red) and CD163 (green) was performed. C, Dot blot data of the densities of Iba1- and CD163-positive TAMs 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 the percentage of CD163-positive TAMs and statistical analysis was done by the Wilcoxon test.

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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 Gender Male 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. 5cm 44 19 25 23 21 23 21 Location Superficial 22 13 9 N.S. 13 10 N.S. 13 9 N.S. Deep 40 17 23 20 20 22 18 FNCLCC 2 38 19 19 N.S. 19 19 N.S. 26 12 0.017a 32411 13 13 11 9 15 AJCC(ed 7) II 22 15 7 0.006a 12 10 N.S. 15 7 N.S. III 17 8 9 10 7 9 8 IV 23 7 16 10 13 11 12 Abbreviation: N.S., not significant. aStatistically significant.

tumor development and metastasis were compared. Following ence in the rejection rate of tumor cells between WT mice and the injection of MCA205 cell lines subcutaneously, tumor CD163 / mice. As shown in Fig. 4A, the rejection rate of nodules were resected, and the tumor weight was examined at MCA205 cells was higher in CD163 / mice than in WT mice. day 7. The results showed that tumor development was signifi- Next, a coculture experiment using peritoneal macrophages and cantly inhibited in CD163 / mice compared with that in WT MCA205 cells was performed to investigate whether WT macro- mice (Fig. 3F). Lung metastasis of MCA205 cells was not phages could support tumor cell proliferation using the BrdUrd observed, whereas LM8 cells are known to metastasize to the incorporation assay. As shown in Fig. 4B, the proliferation of lung. As shown in Fig. 3G, both subcutaneous tumor develop- MCA205 cells was significantly enhanced by coculture with WT ment and lung metastasis at day 21 were significantly sup- macrophages; interestingly, this protumor function of the macro- pressed in CD163 / mice compared with that in WT mice. The phages was decreased by coculture with CD163 / macrophages survival time of LM8-injected mice was extended in CD163 / (Fig. 4B). Furthermore, the proliferation of MCA205 cells was mice compared with that in WT mice (Fig. 3H). These observa- significantly enhanced by coculture with CD163 knock-in tions indicated that CD163 is closely involved in tumor devel- CD163 / macrophages compared with coculture with opment and metastasis. CD163 / macrophages (Fig. 4C). These results indicate that CD163 contributes to tumor engraftment and tumor proliferation CD163 is involved in the engraftment of tumor cells by in an early stage of tumor development. supporting cell proliferation Next, immunohistochemical studies were performed to uncov- CD163 is related to IL6 and CXCL2 production from er the mechanisms related to the suppression of tumor progres- macrophages sion in CD163 / mice. The results showed that the numbers of To examine the effect of CD163 on protumoral activation infiltrating EMR1-positive macrophages, CD4-positive lympho- under coculture conditions, we investigated cytokine produc- cytes, and CD8-positive lymphocytes did not differ between WT tion using a cytokine array kit. As shown in Fig. 4D, the mice and CD163 / mice in the developed MCA205 tumor production of some cytokines was significantly reduced in nodules at day 7 (Supplementary Figs. S4 and S5). Angiogenesis CD163 / macrophage monoculture or coculture with and the Ki-67 expression in tumor cells did not differ between WT CD163 / macrophages, and some cytokines were shown to mice and CD163 / mice (Supplementary Fig. S4). Similar results be linked to CD163-related macrophage activation. We focused were also seen in LM8 tumor nodules at day 21 (Supplementary on IL6 and CXCL2 because these two cytokines are known to be Fig. S4). These data suggested that the chemotaxis of macro- associated with the growth of several tumor cells. The ELISA phages, antitumor immune response, angiogenesis, or tumor cell data showed that MCA205 cells did not secrete IL6 and CXCL2, proliferation were not associated with impaired tumor progres- and the concentration of IL6 and CXCL2 in the supernatant was sion in CD163 / mice. During the immunohistochemical anal- significantly increased in the coculture condition (Fig. 4E). In ysis, we noted that no or low CD163 expression was observed in addition, IL6 and CXCL2 production was significantly impaired TAMs, whereas resident macrophages around the tumor nodule in CD163 / macrophages in cases with or without coculture strongly expressed CD163 (Supplementary Fig. S6). Therefore, we (Fig.4E).Bycontrast,bothIL6andCXCL2productionwas concluded that CD163 expressed on resident macrophages was significantly increased in CD163 knock-in CD163 / macro- preferentially involved in tumor cell development. Because res- phages under the coculture condition (Fig. 4F). These results ident macrophages are associated with tumor engraftment at an suggest that CD163 is related to IL6 and CXCL2 production early stage of tumor development, we next examined the differ- from macrophages.

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NMFH-1 Cell line A Cell proliferation (%) TYLMS-1 and 100 150 50 TYLMS-1 0 B, ua ooyedrvdmcohgs(ø rmncts(o eeicbtdwith incubated were (Mo) monocytes or (Mø) macrophages monocyte-derived Human D ). E 2day N.S. P <0.001 P<0.001 and D F, olwn h iecn fC13epeso nhmnmonocyte-derived human on expression CD163 of silencing the Following P <0.01 BrdUrd N.C.

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CD163 Is Involved in Sarcoma Progression

A B WT KO CD163 Isotype Isotype control control CD204

β-Actin Count % WT CD163-/- Count % C Resident PM

Cont. IL4 IL6 IL10 CD163 CD163 CD163 D Isotype WT KO Isotype control control TCS β-Actin TCS IL4 IL4 BMDM Count % Cont. IL4 IL6 IL10 Count %

CD163

β-Actin CD163 CD163 F MCA205 P᧸0.001 E Control TCS 250 CD163 200 150 β-Actin 100

Control LM8 TCS 50 CD163 0 MCA205 tumor weight tumor (mg) MCA205 β-Actin WT CD163-/-

GHWT CD163-/- 1.0 P WT Logrank = 0.003 0.8 Wilcoxon P = 0.004 0.6 -/- -/- WT CD163 CD163 1 cm 100 μm 0.4 Survival rate P᧸0.01 2 P᧸0.001 0.2 6,000 100 0 80 7065605550454035 4,000 60 Survival time (days) 40 2,000 20 0 LM8 tumor weight (mg) LM8 tumor weight 0 WT CD163-/- metastasis LM8 lung (mm ) WT CD163-/-

Figure 3. In vivo sarcoma model in WT and CD163/ mice. A and B, Loss of CD163 expression in peritoneal macrophages isolated from CD163/ mice was confirmed by 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 cells into WT and CD163/ mice subcutaneously, tumor weight was evaluated at day 7. G, Following the injection of LM8 cells into WT and CD163/ mice subcutaneously, tumor weight and lung metastasis were evaluated at day 21. H, Survival time was observed in WT and CD163/ mice transplanted with LM8. 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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A 100 WT C CD163-/- Mø CD163-/- Mø -/- + 80 CD163 + CD163-/- Mø Control adeno V mCD163 adeno V 60 40 20 0 Tumor rejection (%) Tumor 5,000 500 50 Injected cell numbers P < 0.05 P P B 300 < 0.001 < 0.05 180 160 P < 0.05 250 140 200 120 150 100 80 100 60 50 40 0 20 0 BrdU incorporation (%) BrdUrd incorporation (%) BrdUrd incorporation Mø CT Mø CT Mø CT MCA205 -/- Mø Mø -/- Mø mCD163 -/- Mø mCD163 D (WT) (CD163 ) + MCA205 -/- Mø Mø -/- CD163 + + + MCA205 -/- MCA205 CD163 (WT) (CD163 ) MCA205 MCA205 CD163 IL6 CD163

CXCL2 F P < 0.001 350 CXCL1 300 250 P < 0.001 IFN 200 150 CXCL10 100 IL6 (pg/mL) 50 0 CXCL13

I309

IL1Ra

M-CSF

CCL2 P < 0.05 120 TIMP-1 100 P < 0.05 E P < 0.001 80 P < 0.001 600 P < 0.001 1,000 P < 0.001 60 500 40 800 20

400 CXCL2 (pg/mL) 600 0 300 200 400

IL6 (pg/mL) 100 200 0 CXCL2 (pg/mL) 0

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/ mice for 24 hours, and the BrdUrd incorporation assay was performed to evaluate tumor cell proliferation. C, MCA205 cells were cocultured with CD163/ peritoneal macrophages and CD163 knock-in CD163/ peritoneal macrophage for 24 hours, and the BrdUrd incorporation assay was performed to evaluate tumor cell proliferation. D, MCA205 cells were cocultured with peritoneal macrophages isolated from WT and CD163/ mice for 24 hours, and cytokine production in the culture supernatant was tested by cytokine array. E, MCA205 cells were cocultured with peritoneal macrophages isolated from WT and CD163/ mice for 24 hours, and the concentration of IL6 and CXCL2 in the culture supernatant was tested by ELISA. F, MCA205 cells were cocultured with CD163/ peritoneal macrophages and CD163 knock-in CD163/ peritoneal macrophage for 24 hours, and the concentration of IL6 and CXCL2 in the culture supernatant was tested by ELISA.

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CD163 Is Involved in Sarcoma Progression

Effect of IL6 and CXCL2 on tumor proliferation in MCA205 cells In the present study, we demonstrated the discrepancy of We next examined the effect of IL6 and CXCL2 on tumor cell human and murine macrophages; CD163 is expressed on both proliferation using MCA205 cells. As shown in Fig. 5A, IL6 and resident and monocyte-derived macrophages in humans (34), CXCL2 induced tumor proliferation, indicating that IL6 and whereas CD163 is detected on resident macrophages but not on CXCL2 play an important role in tumor proliferation under the BMDMs in mice. When the tumor is small, resident macrophages coculture condition. Furthermore, the knockdown of the IL6 in the surrounding tissues accumulate intratumorally, but mono- receptor in MCA205 cells suppressed tumor proliferation under cyte-derived macrophages become the main source of TAMs after the coculture condition, and the knockdown of IL6 or IL6R in the vascular network of the tumor tissue is formed (35). Different macrophages also suppressed tumor proliferation under the gene expression profiles between resident macrophages and coculture condition when WT macrophages were used for the BMDMs have been reported in mice, and EMR1 (F4/80 antigen), culture study (Fig. 5B–D). Notably, the knockdown of IL6R or IL6 as well as CD163, is also expressed on resident macrophages at a did not influence tumor cell proliferation when cells are cocul- higher level than that on BMDMs (36). In the present study, EMR1 tured with CD163 / macrophages (Fig. 5C and D). However, was expressed on TAMs and resident macrophages around the tumor proliferation was not inhibited by the knockdown of tumor nodule, and the staining intensity seemed to be higher on CXCL2 in macrophages (Fig. 5E). Furthermore, the WT macro- resident macrophages than on TAMs. However, CD163 was phage culture supernatant significantly induced the activation of expressed on resident macrophages but not on TAMs. Therefore, STAT3, which plays an important role in tumor progression in CD163 is considered useful to distinguish resident macrophages tumor cells, compared with that in control cells, whereas the from BMDMs. CD163 / macrophage culture supernatant affected STAT3 acti- The detailed mechanisms of CD163-related signals have not vation slightly (Fig. 5F). IL6 also significantly induced STAT3 been clarified; however, we found that IL6 and CXCL2 expres- activation (Fig. 5F). These data indicate that IL6, rather than sion was significantly impaired in CD163 / macrophages. It CXCL2, derived from macrophages is involved in tumor prolif- was reported approximately 10 years ago that antibody-depen- eration under the coculture condition. dent CD163 stimulation induced inflammatory cytokines such as IL1b and IL6 from macrophages (37) and activation of casein Discussion kinase II and protein kinase C (38); however, these observations were not reflected in our unpublished data. A recent study CD163 is a member of the scavenger receptor super family class using CD163 / mice demonstrated that soluble CD163 acts as B and is specifically expressed on myeloid lineages such as a decoy receptor for TWEAK, and a deficiency of soluble CD163 monocytes and macrophages (19–21). CD163 is a cell surface increased the concentration of TWEAK in peripheral blood to glycoprotein receptor with a molecular weight of 130 kDa and was promote tissue regeneration after ischemic injury (39); how- first found to be a receptor of the hemoglobin (Hb) and hapto- ever, no description related to CD163-related signals has been globin (Hp) complex. The Hb-Hp complex was recognized by seen. TWEAK has been reported to promote tumor cell inva- CD163 for clearance by receptor-mediated endocytosis (22). sion/progression rather than apoptosis (40, 41); therefore, the CD163 was also found to be related to the recognition of bacteria result of our present study that tumor development was by macrophages (23). The soluble form of CD163 in human impaired in CD163 / mice is probably not due to an increased serum has been reported to be useful for evaluating macrophage TWEAK. In our preliminary study, the PTEN-Akt signal in tumor activation in sepsis, rheumatic disease, and liver diseases (24–26); cells was activated by coculture with macrophages, and this therefore, CD163 is now of interest as a marker of macrophage cell–cell interaction was impaired when CD163 / macro- activation in several diseases. phages were used for the coculture study. PTEN demonstrates In the present study, we revealed that CD163 was closely phosphatase activity against the phospholipid products of PI3- associated with the protumoral activation of macrophages, kinase activity, leading to the inactivation of Akt signaling (42). although the detailed signaling pathways via CD163 have never PTEN activity is controlled by phosphorylation and phosphor- been uncovered. CD163 is related to the production of protumor ylated PTEN attenuates its inhibitory function of Akt signaling, cytokines such as IL6 and CXCL2 from macrophages, and IL6 was leading to the promotion of survival and growth in tumor cells found to be more preferentially associated with macrophage- (42). Therefore, it is suggested that unknown factors suppres- related tumor cell proliferation. IL6 plays a central role in tumor sing PTEN activity are thought to be secreted by macrophages, development and progression via supporting tumor cell prolif- and CD163 is potentially related to tumor survival and growth eration, survival, and metastasis via Stat3 activation (27). indirectly via Akt signaling activation and PTEN inactivation in Although tumor cell lines that do not secrete IL6 were used in tumor cells. this study, some tumor cells secrete IL6 and form paracrine and In the present study, we used human cell lines and tissue autocrine IL6 loops by inducing IL6 production from stromal cells samples to demonstrate the significance of CD163 in tumor cell (28). IL6 derived from TAMs was associated with the expansion of proliferation, and an unknown CD163-related signal was sug- cancer stem-like cells in hepatocellular carcinoma (29) and was gested to be related to the protumoral activation of human also involved in the niche for cancer stem-like cells and drug macrophages as well as murine macrophages. Although a similar resistance in the MC38 murine sarcoma model (30). An increased observation was observed in our previous study using malignant density of CD163-positive TAMs is positively correlated with the lymphoma samples, the detailed molecular mechanisms of expression of stem-cell markers, including CD44 and ALDH1, in CD163-related macrophage activation have never been clarified some types of human cancers (31–33). These observations indi- (43). CD163 is known to mediate cell–cell contacts between cated that CD163-related macrophage activation is associated macrophages and erythroblasts (44), and CD163 might promote with the maintenance of cancer stem-like cells and induces tumor cell–cell adhesion between macrophages and tumor cells via progression. unknown ligands.

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P A P < 0.01 < 0.01 P < 0.01 P < 0.01 200 200

150 150

100 100 MCA205 MCA205 50 50 Proliferation (%) Proliferation (%) 0 0 0.1 1 0.1 1

IL6 (ng/mL) CXCL2 (ng/mL) Control B Control

CXCL2 IL6 IL6R

β-Actin β-Actin β-Actin Nonload Nonload Nonload IL6 siRNA IL6R siRNA Control siRNA CXCL2 siRNA Control siRNA Control siRNA

P < 0.001 P < 0.05 D C P < 0.001 200 P < 0.001 250 P < 0.05 P < 0.001 150 200 150 100 100 MCA205 50 MCA205 50 Proliferation (%) 0 Proliferation (%) 0 MCA MCA MCA MCA MCA MCA MCA MCA MCA MCA + + + + + + + + WT CD163-/- WT CD163-/- WT CD163-/- WT CD163-/-

Control IL6 Control IL6R siRNA siRNA siRNA siRNA E F P < 0.001 P < 0.001 250 200 S67$7 150 100 67$7 MCA205 50

Proliferation (%) 0 ˟-Actin MCA MCA MCA MCA MCA + + + + WT KO -/- -/- WT CD163 WT CD163 IL-6 Mø CS Control

Control CXCL2 Control siRNA siRNA

Figure 5. Protumor effect of IL6 and CXCL2. A, MCA205 cells were incubated with the indicated concentrations of IL6 or CXCL2 for 2 days, and then tumor cell proliferation was tested 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 cell proliferation was assessed by the BrdUrd incorporation assay (C). D, Following the silencing of IL6R expression on MCA205 cells by siRNA, the same coculture study was 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 with macrophage culture supernatant (Mø CS) for 24 hours, followed by the determination of the pSTAT3, STAT3, and b-actin levels by Western blot analysis, as described in Materials and Methods.

3264 Cancer Res; 78(12) June 15, 2018 Cancer Research

CD163 Is Involved in Sarcoma Progression

In conclusion, we demonstrated the significance of CD163- Writing, review, and/or revision of the manuscript: Y. Fujiwara, Y. Komohara related macrophage activation in the cell–cell interaction between Administrative, technical, or material support (i.e., reporting or organizing tumor cells and TAMs using mouse and human cells. Although the data, constructing databases): Y. Komohara Study supervision: H. Mizuta, M. Takeya, Y. Komohara detailed signal activation pathways of CD163-related macrophages activation remain to be uncovered, CD163 or CD163- related signals are considered a new therapeutic target for anti- Acknowledgments tumor therapy. We thank Mrs. Emi Kiyota, Mr. Takenobu Nakagawa, and Ms. Ikuko Miyakawa for their technical assistance. This work was supported by JSPS KAKENHI. M. Takeya received JSPS KAKENHI grant No. 25293089, Y. Disclosure of Potential Conflicts of Interest Komohara received JSPS KAKENHI grant No. 16H05162, Y. Fujiwara No potential conflicts of interest were disclosed. received JSPS KAKENHI grant No. 16K09247, and D. Shiraishi received JSPS KAKENHI grant No. 16K10865. Authors' Contributions Conception and design: Y. Fujiwara, Y. Komohara The costs of publication of this article were defrayed in part by the Development of methodology: D. Shiraishi, T. Iriki, Y. Komohara payment of page charges. This article must therefore be hereby marked Acquisition of data (provided animals, acquired and managed patients, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate provided facilities, etc.): D. Shiraishi, Y. Fujiwara, H. Horlad, Y. Saito, this fact. N. Nakagata, Y. Oda, Y. Komohara Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D. Shiraishi, Y. Fujiwara, H. Horlad, Y. Saito, T. Iriki, Received July 19, 2017; revised November 30, 2017; accepted March 28, 2018; Junko Tsuboki, H. Bekki, Y. Komohara published first April 2, 2018.

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3266 Cancer Res; 78(12) June 15, 2018 Cancer Research

CD163 Is Required for Protumoral Activation of Macrophages in Human and Murine Sarcoma

Daisuke Shiraishi, Yukio Fujiwara, Hasita Horlad, et al.

Cancer Res 2018;78:3255-3266. Published OnlineFirst April 2, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-2011

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