Recent findings have shown the significance of CD163-positive macrophages in tumor progression, yet there have been few studies on the function of CD163 in macrophages. Here, we uncover the role of CD163 in macrophage activation using CD163-deficient mice and human samples. We detected CD163 in 62 undifferentiated pleomorphic sarcoma samples, in which a high percentage of CD163-positive macrophages was associated with decreased overall survival and higher histologic grade. We observed macrophage-induced tumor cell proliferation in cocultures of human monocyte-derived macrophages and leiomyosarcoma (TYLMS-1) and myxofibrosarcoma (NMFH-1) cell lines, which was abrogated by silencing of CD163. Tumor development of sarcoma (MCA205 and LM8) cells in CD163-deficient mice was significantly abrogated in comparison with wild-type (WT) mice. Coculture with WT peritoneal macrophages significantly increased proliferation of MCA205 cells but decreased in the presence of CD163-deficient macrophages. Production of IL6 and CXCL2 in CD163-deficient macrophages was suppressed in comparison with WT macrophages, and overexpression of CD163 in CD163-deficient macrophages induced production of IL6 and CXCL2. Silencing of IL6 but not CXCL2 abrogated macrophage-induced proliferation of MCA205 cells. Taken together, our results show that CD163 is involved in protumoral activation of macrophages and subsequent development and progression of tumors in mice and humans.

Significance: Macrophage CD163-mediated induction of IL6 promotes tumor development and progression in murine and human malignant tumors. Cancer Res; 78(12); 3255–66. ©2018 AACR.

Host-derived immune cells, fibroblasts, and endothelial cells constitute the tumor microenvironment and are known to be related to tumor progression, and macrophages are a critical population of immune cells that induce tumor cell growth, angiogenesis, metastasis, and immune suppression (1–3). Macrophages are suggested to be broadly classified into classically activated macrophages (M1/kill macrophages) and alternatively activated macrophages (M2/repair macrophages) according to their functions and expression markers (4–6). Many studies using mouse models have indicated that M1-like macrophages produce proinflammatory cytokines that stimulate antitumor immune responses, whereas M2-like macrophages promote angiogenesis, immunosuppression, and tumor progression via secreting growth-promoting molecules. Macrophages infiltrating cancer tissues are referred to as tumor-associated macrophages (TAM), which are closely involved in the development of the tumor microenvironment, and heterogeneity of macrophage phenotypes is observed among TAMs in various malignant tumors, including sarcomas (7–9). Several clinicopathologic studies have recently demonstrated the significance of TAMs in the growth and progression of malignant tumors. Notably, a high density of CD163+ TAMs is found to be positively associated with a worse prognosis in many malignant tumors (9). However, it remains unclear how CD163 works in the protumoral activation of TAMs.

Undifferentiated pleomorphic sarcoma (UPS) is the most frequent soft-tissue sarcoma that shows no line of differentiation (10). It has long been known that large numbers of TAMs are detected in UPS, and UPS was previously referred to as malignant fibrous histiocytoma (10). Regarding sarcoma, a high density of TAMs has been found to be a prognostic factor in leiomyosarcoma (11, 12). However, little is known about the significance of TAMs in UPS. In the present study, we newly found a significant correlation between a high density of CD163+ TAMs and poor clinical course in patients with UPS. Because CD163 is known to be upregulated by Th2-type cytokines, CD163 is also considered a marker for M2/repair or protumor phenotype of macrophages (13, 14). Based on this background, CD163 is suggested to be critically involved in the protumor functions of TAMs; however, few research studies have investigated the functions of CD163 in the tumor microenvironment. Therefore, we studied the functions of CD163 in TAMs using an animal model and in vitro coculture study in the present study.

Patients and assessment of CD163-positive TAMs

We evaluated 62 tumors diagnosed as UPS that were registered in the Department of Anatomic Pathology, Kyushu University (Fukuoka, Japan), and in the Department of Cell Pathology, Kumamoto University (Kumamoto, Japan). All samples were primary cases, and radiation-induced sarcomas and secondary sarcomas after chemotherapy were excluded. The reassessed diagnosis of UPS was made according to the World Health Organization (WHO) 2013 classification (10). A study using this set of UPS cases was previously published (15). We evaluated the extent of necrosis and mitosis to define each tumor's French Federation of Cancer Centers (FNCLCC) grade. The seventh edition of the American Joint Committee on Cancer (AJCC) staging system was applied to every case. The Institutional Review Board at Kyushu University and Kumamoto University approved this retrospective study (#27-78 and #1175). Immunohistochemistry of Iba1, CD68, and CD163 was performed as described in a previous study (16). Iba1- and CD163-positive cells were counted in 10 randomly selected areas of high-power field of a microscope by two pathologists who were blinded to information about the patients' backgrounds or their prognosis.

Tumor cell lines

MCA205 (mouse fibrosarcoma of C57BL background), 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 Bank between 2014 and 2017. Cells were maintained in RPMI 1640 or DMEM/Ham's F-12 (WAKO) supplemented with 10% fetal bovine serum (FBS) and were regularly tested using a Mycoplasma test kit (TAKARA). These cells were cultured for less than 3 months before reinitiating the cultures and routinely inspecting microscopically for a stable phenotype.

Human macrophages

Peripheral blood mononuclear cells were acquired from healthy volunteer donors, and written informed consent was obtained from each participant. All the protocols using human macrophages were approved by the Kumamoto University Review Board (No. 486) and were conducted in accordance with the approved guidelines. CD14+ monocytes were purified from peripheral blood mononuclear cells by positive selection via magnetic-activated cell sorting technology (Miltenyi Biotec). The monocytes were cultured in RPMI supplemented with 2% FBS and 50 ng/mL macrophage colony-stimulating factor (M-CSF, WAKO) for 5 days to differentiate into macrophages.

Flow cytometry

Human macrophages were fixed with 2% paraformaldehyde and then reacted with anti-CD163 antibody or isotype matched control antibody (clone AM-3K and RM4; ref. 18) diluted in FACS buffer (BioLegend) with 0.5% Triton-X100. Then cells were reacted anti-mouse IgG antibody labeled with Alexa 488 (BioLegend). For flow cytometric analysis of mouse samples, antibodies for Gr1 (FITC, BioLegend), CD11b (Pacific Blue, BioLegend), CD3 (APC, BioLegend), EMR1 (PE, BioLegend), and CD163 (PE, Bioss Antibodies) were used. The stained cell samples were analyzed on a FACSverse (Becton Dickinson) flow cytometer with FACSuite (Becton Dickinson) software.

Coculture and 5-bromo-2′-deoxyuridine incorporation assay

Tumor 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 assayed using a BrdUrd Cell Proliferation Kit (Roche) according to the manufacturer's protocol.

Cell proliferation assay

Briefly, 10,000 tumor cells were cultured in a 96-well plate in quadruplicate before treatment. The cells were then cultured in the presence of IL6 or CXCL2. Cell viability was determined using a WST assay (WST-8 cell counting kit; Dojin Chemical) according to the manufacturer's protocol.

Cytokine array

Cytokine array analysis was performed using a mouse cytokine array kit, panel A (R&D Systems), according to the manufacturer's protocol.

Akt signaling array

Akt signaling array analysis was performed using a PathScan Akt Signaling Antibody Array Kit (Cell Signaling Technology), according to the manufacturer's protocol.

Animal studies

All animal experiments have been conducted in accordance with an Institutional Animal Care and Use Committee. CD163-deficient (CD163−/−) mice in the C57BL/6N background were obtained from the Knockout Mouse Project, and wild-type (WT) mice in the C57BL/6N or C3H background were obtained from CLEA Japan. CD163−/− mice were backcrossed to the C3H strain for more than seven generations. Mice were housed in a temperature-controlled room with a 12-hour light/dark cycle. During the course of the experiment, we observed no significant difference in body weights between CD163−/− and wild-type littermate mice. All animal experiments were approved by the Ethics Committee for Animal Experiments of Kumamoto University (#A23-154, #22-023).

Murine tumor-bearing model

Female mice (8–10 weeks) were subcutaneously injected with MCA205 or LM8 (subclone #5) cells (5 × 105) suspended in 50 μL of RPMI 1640. Mice were sacrificed on day 7 or 14 or 21, followed by the determination of subcutaneous tumor development and lung metastasis. The mice were monitored to assess survival. For cytokine measurement, blood samples were drawn 7 or 14 days after injection.

Murine macrophages

Resident peritoneal macrophages from mice (8–10 weeks) were obtained by peritoneal lavage using 6 mL of PBS. Cells were incubated in DMEM medium with 10% FBS. For the bone marrow–derived macrophage (BMDM) cultures, femurs and tibias were excised from mice, and the soft tissue was removed. Briefly, marrow was flushed with sterilized PBS, and erythrolysation was performed. Cells were seeded in polystyrene culture dishes with DMEM medium supplemented with 10% FBS. Cells were incubated in medium containing 20 ng/mL recombinant murine macrophage colony-stimulating factor (M-CSF; PeproTech).

Immunohistochemistry of murine samples

Subcutaneous tumor tissues were embedded in OCT compound (Sakura Finetech). After sectioning (5-μm thick), the tissues were fixed with cold acetone, and treated with the following primary antibodies: anti-CD4 (GK1.5; ATCC), anti-CD8 (53–6.72; ATCC), and anti-CD31 (MEC13.3; BD Pharmingen). After sectioning (3 μm thick), paraffin-embedded tumor tissues were used 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 visualized using diaminobenzidine including 0.1% NaN3.

Western blot analysis

Human and murine CD163 expression, STAT3 activation, and Akt signaling were evaluated by Western blot analysis as described previously (12). Briefly, macrophages were solubilized with Triton X-100, and the protein concentration was determined using the BCA protein assay reagent, followed by pretreatment by boiling for 5 minutes in 2% SDS and 2-mercaptoethanol. The protein (10 μg) was run on a 10% SDS-polyacrylamide gel and was transferred to a PVDF membrane (Millipore,). The membranes 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-pAkt antibody (D9E; Cell Signaling Technology), anti-Akt antibody (40D4; Cell Signaling Technology), anti-pPTEN antibody (Cell Signaling Technology), and anti-PTEN antibody (Cell Signaling Technology). These membranes were re-blotted with an anti–β-actin antibody as an internal calibration control.

Statistical analysis

Statistical analyses were carried out using JMP10 (SAS Institute) and StatMate III (ATOMS). The χ2test, the Kaplan–Meier method, and the Cox hazard test were used to analyze the clinical course associations. All data presented from animal studies and cell culture studies are representative of at least two or three independent experiments. The data are expressed as the mean ± standard deviation (SD, n = 3–6 each groups). Differences between the groups were examined for statistical significance using the Mann–Whitney U test and a nonrepeated measures ANOVA. A value of P < 0.05 was considered statistically significant. A P value of <0.05 was considered to denote the presence of a statistically significant difference.

A high percentage of CD163-positive cells in TAMs was related to a shortened overall survival

First, we examined whether Iba1-postive and CD163-positive TAMs were correlated with tumor progression using human resected UPS samples. CD68 is also useful as a pan-macrophage marker; however, we selected Iba1 instead of CD68 because CD68 positivity was also seen in sarcoma cells (Supplementary Fig. S1). Tumor cells are negative for Iba1 and CD163, and CD163-positive signals were overlapped in Iba1-positive TAMs (Fig. 1A and B). The median densities of Iba1-postive and CD163-positive TAMs were 683/mm2 and 406/mm2, respectively, and median percentage 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 of CD163-positive TAMs and a high percentage of CD163-positive TAMs were significantly associated with high AJCC stage and high FNCLCC grade respectively (Table 1). Notably, we found that a high percentage of CD163-positive TAMs was significantly associated with decreased overall survival (P = 0.043, Wilcoxon test; Fig. 1D). The high density of CD163-positive TAMs also seemed to be linked to a worse clinical course; however, the results did not reach statistical significance (P > 0.05; Fig. 1D; Supplementary Fig. S2; Supplementary Table S1). Whereas there was no significant association between the density of Iba1-positive TAMs and all clinical parameters (Table 1; Supplementary Fig. S2). These observations indicated the significance of CD163 in the protumor functions of TAMs.

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.

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

CD163+ TAMsIba1+ TAMs% of CD163+
Low (<400)High (≥400)Low (<700)High (≥700)Low (<80)High (≥80)
Variablen3032P3230P3527P
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 N.S. N.S. 12 N.S. 
 ≥5 cm 44 19 25  23 21  23 21  
Location 
 Superficial 22 13 N.S. 13 10 N.S. 13 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 
 3 24 11 13  13 11  15  
AJCC(ed 7) 
 II 22 15 0.006a 12 10 N.S. 15 N.S. 
 III 17  10   
 IV 23 16  10 13  11 12  
CD163+ TAMsIba1+ TAMs% of CD163+
Low (<400)High (≥400)Low (<700)High (≥700)Low (<80)High (≥80)
Variablen3032P3230P3527P
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 N.S. N.S. 12 N.S. 
 ≥5 cm 44 19 25  23 21  23 21  
Location 
 Superficial 22 13 N.S. 13 10 N.S. 13 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 
 3 24 11 13  13 11  15  
AJCC(ed 7) 
 II 22 15 0.006a 12 10 N.S. 15 N.S. 
 III 17  10   
 IV 23 16  10 13  11 12  

Abbreviation: N.S., not significant.

aStatistically significant.

Effect of CD163 on tumor proliferation in human sarcoma cell lines

Next, we tested whether CD163-positive macrophages support sarcoma cells in a coculture study using human macrophages and sarcoma cell lines. Human monocyte-derived macrophages and human sarcoma cell lines (TYLMS-1, HT-1080, NMFH-1) were cocultured, and the BrdUrd incorporation assay was performed as shown in Fig. 2A. Coculture with macrophages induced tumor cell proliferation when TYLMS-1 and NMFH-1 cells were used, whereas the cell proliferation of HT-1080 was not changed by coculture with macrophages (Fig. 2B). No effect was observed when the cell lines were cocultured with monocytes (Fig. 2B). Next, we found that the tumor culture supernatant (TCS) of TYLMS-1 and NMFH-1 cells more significantly upregulated CD163 expression on macrophages than those of HT-1080 cells (Fig. 2C and D), suggesting that CD163 might play important roles in the protumoral activation of macrophages. Therefore, following the silencing of CD163 expression in macrophages by siRNA (Fig. 2E), coculture and the BrdUrd incorporation assay were performed. The knockdown of CD163 in macrophages abrogated macrophage-induced tumor cell proliferation (Fig. 2F).

Figure 2.

Cell–cell interaction between macrophages and sarcoma cell lines. A and B, Human monocyte-derived macrophages (Mø) or monocytes (Mo) were incubated with human 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 CD163 expression was examined by Western blot analysis (C) and flow cytometry (D). E and F, Following the silencing of CD163 expression on human monocyte-derived macrophages by siRNA (E), coculture was performed, and tumor cell proliferation was assessed via BrdUrd incorporation assays (F). N.C., negative control.

Figure 2.

Cell–cell interaction between macrophages and sarcoma cell lines. A and B, Human monocyte-derived macrophages (Mø) or monocytes (Mo) were incubated with human 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 CD163 expression was examined by Western blot analysis (C) and flow cytometry (D). E and F, Following the silencing of CD163 expression on human monocyte-derived macrophages 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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Tumor development and metastasis were significantly impaired in CD163−/− mice

We next compared tumor development between WT mice and CD163−/− mice to study the significance of CD163 in the protumor activation of macrophages. First, we confirmed the deletion 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 background; Fig. 3A and B). Similar results were seen in the C3H background mice (Supplementary Fig. S3A). The numbers and expression of M2-related markers in resident macrophages did not differ between WT mice and CD163−/− mice (Supplementary Fig. S3B). There were no significant differences in the laboratory blood data of the two types of mice (Supplementary Table S2). During these procedures, we found that CD163 expression was strongly induced by IL4 in resident peritoneal macrophages, whereas weak or no expression of CD163 was observed in macrophages differentiated from bone marrow–derived cells (Fig. 3C and D). The MCA205 TCS also induced CD163 expression in peritoneal macrophages isolated from WT mice (Fig. 3D and E). Next, two murine sarcoma cell lines (MCA205 and LM8) were injected subcutaneously into WT mice and CD163−/− mice, and tumor development and metastasis were compared. Following the injection of MCA205 cell lines subcutaneously, tumor nodules were resected, and the tumor weight was examined at day 7. The results showed that tumor development was significantly inhibited in CD163−/− mice compared with that in WT mice (Fig. 3F). Lung metastasis of MCA205 cells was not observed, whereas LM8 cells are known to metastasize to the lung. As shown in Fig. 3G, both subcutaneous tumor development and lung metastasis at day 21 were significantly suppressed in CD163−/− mice compared with that in WT mice. The survival time of LM8-injected mice was extended in CD163−/− mice compared with that in WT mice (Fig. 3H). These observations indicated that CD163 is closely involved in tumor development and metastasis.

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.

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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CD163 is involved in the engraftment of tumor cells by supporting cell proliferation

Next, immunohistochemical studies were performed to uncover the mechanisms related to the suppression of tumor progression in CD163−/− mice. The results showed that the numbers of infiltrating EMR1-positive macrophages, CD4-positive lymphocytes, and CD8-positive lymphocytes did not differ between WT mice and CD163−/− mice in the developed MCA205 tumor nodules at day 7 (Supplementary Figs. S4 and S5). Angiogenesis and the Ki-67 expression in tumor cells did not differ between WT mice and CD163−/− mice (Supplementary Fig. S4). Similar results were also seen in LM8 tumor nodules at day 21 (Supplementary Fig. S4). These data suggested that the chemotaxis of macrophages, antitumor immune response, angiogenesis, or tumor cell proliferation were not associated with impaired tumor progression in CD163−/− mice. During the immunohistochemical analysis, we noted that no or low CD163 expression was observed in TAMs, whereas resident macrophages around the tumor nodule strongly expressed CD163 (Supplementary Fig. S6). Therefore, we concluded that CD163 expressed on resident macrophages was preferentially involved in tumor cell development. Because resident macrophages are associated with tumor engraftment at an early stage of tumor development, we next examined the difference in the rejection rate of tumor cells between WT mice and CD163−/− mice. As shown in Fig. 4A, the rejection rate of MCA205 cells was higher in CD163−/− mice than in WT mice. Next, a coculture experiment using peritoneal macrophages and MCA205 cells was performed to investigate whether WT macrophages could support tumor cell proliferation using the BrdUrd incorporation assay. As shown in Fig. 4B, the proliferation of MCA205 cells was significantly enhanced by coculture with WT macrophages; interestingly, this protumor function of the macrophages was decreased by coculture with CD163−/− macrophages (Fig. 4B). Furthermore, the proliferation of MCA205 cells was significantly enhanced by coculture with CD163 knock-in CD163−/− macrophages compared with coculture with CD163−/− macrophages (Fig. 4C). These results indicate that CD163 contributes to tumor engraftment and tumor proliferation in an early stage of tumor development.

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.

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 related to IL6 and CXCL2 production from macrophages

To examine the effect of CD163 on protumoral activation under coculture conditions, we investigated cytokine production using a cytokine array kit. As shown in Fig. 4D, the production of some cytokines was significantly reduced in CD163−/− macrophage monoculture or coculture with CD163−/− macrophages, and some cytokines were shown to be linked to CD163-related macrophage activation. We focused on IL6 and CXCL2 because these two cytokines are known to be associated with the growth of several tumor cells. The ELISA data showed that MCA205 cells did not secrete IL6 and CXCL2, and the concentration of IL6 and CXCL2 in the supernatant was significantly increased in the coculture condition (Fig. 4E). In addition, IL6 and CXCL2 production was significantly impaired in CD163−/− macrophages in cases with or without coculture (Fig. 4E). By contrast, both IL6 and CXCL2 production was significantly increased in CD163 knock-in CD163−/− macrophages under the coculture condition (Fig. 4F). These results suggest that CD163 is related to IL6 and CXCL2 production from macrophages.

Effect of IL6 and CXCL2 on tumor proliferation in MCA205 cells

We next examined the effect of IL6 and CXCL2 on tumor cell proliferation using MCA205 cells. As shown in Fig. 5A, IL6 and CXCL2 induced tumor proliferation, indicating that IL6 and CXCL2 play an important role in tumor proliferation under the coculture condition. Furthermore, the knockdown of the IL6 receptor in MCA205 cells suppressed tumor proliferation under the coculture condition, and the knockdown of IL6 or IL6R in macrophages also suppressed tumor proliferation under the coculture condition when WT macrophages were used for the culture study (Fig. 5B–D). Notably, the knockdown of IL6R or IL6 did not influence tumor cell proliferation when cells are cocultured with CD163−/− macrophages (Fig. 5C and D). However, tumor proliferation was not inhibited by the knockdown of CXCL2 in macrophages (Fig. 5E). Furthermore, the WT macrophage culture supernatant significantly induced the activation of STAT3, which plays an important role in tumor progression in tumor cells, compared with that in control cells, whereas the CD163−/− macrophage culture supernatant affected STAT3 activation slightly (Fig. 5F). IL6 also significantly induced STAT3 activation (Fig. 5F). These data indicate that IL6, rather than CXCL2, derived from macrophages is involved in tumor proliferation under the coculture condition.

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 β-actin levels by Western blot analysis, as described in Materials and Methods.

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 β-actin levels by Western blot analysis, as described in Materials and Methods.

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CD163 is a member of the scavenger receptor super family class B and is specifically expressed on myeloid lineages such as monocytes and macrophages (19–21). CD163 is a cell surface glycoprotein receptor with a molecular weight of 130 kDa and was first found to be a receptor of the hemoglobin (Hb) and haptoglobin (Hp) complex. The Hb-Hp complex was recognized by CD163 for clearance by receptor-mediated endocytosis (22). CD163 was also found to be related to the recognition of bacteria by macrophages (23). The soluble form of CD163 in human serum has been reported to be useful for evaluating macrophage activation in sepsis, rheumatic disease, and liver diseases (24–26); therefore, CD163 is now of interest as a marker of macrophage activation in several diseases.

In the present study, we revealed that CD163 was closely associated with the protumoral activation of macrophages, although the detailed signaling pathways via CD163 have never been uncovered. CD163 is related to the production of protumor cytokines such as IL6 and CXCL2 from macrophages, and IL6 was found to be more preferentially associated with macrophage-related tumor cell proliferation. IL6 plays a central role in tumor development and progression via supporting tumor cell proliferation, survival, and metastasis via Stat3 activation (27). Although tumor cell lines that do not secrete IL6 were used in this study, some tumor cells secrete IL6 and form paracrine and autocrine IL6 loops by inducing IL6 production from stromal cells (28). IL6 derived from TAMs was associated with the expansion of cancer stem-like cells in hepatocellular carcinoma (29) and was also involved in the niche for cancer stem-like cells and drug resistance in the MC38 murine sarcoma model (30). An increased density of CD163-positive TAMs is positively correlated with the expression of stem-cell markers, including CD44 and ALDH1, in some types of human cancers (31–33). These observations indicated that CD163-related macrophage activation is associated with the maintenance of cancer stem-like cells and induces tumor progression.

In the present study, we demonstrated the discrepancy of human and murine macrophages; CD163 is expressed on both resident and monocyte-derived macrophages in humans (34), whereas CD163 is detected on resident macrophages but not on BMDMs in mice. When the tumor is small, resident macrophages in the surrounding tissues accumulate intratumorally, but monocyte-derived macrophages become the main source of TAMs after the vascular network of the tumor tissue is formed (35). Different gene expression profiles between resident macrophages and BMDMs have been reported in mice, and EMR1 (F4/80 antigen), as well as CD163, is also expressed on resident macrophages at a higher level than that on BMDMs (36). In the present study, EMR1 was expressed on TAMs and resident macrophages around the tumor nodule, and the staining intensity seemed to be higher on resident macrophages than on TAMs. However, CD163 was expressed on resident macrophages but not on TAMs. Therefore, CD163 is considered useful to distinguish resident macrophages from BMDMs.

The detailed mechanisms of CD163-related signals have not been clarified; however, we found that IL6 and CXCL2 expression was significantly impaired in CD163−/− macrophages. It was reported approximately 10 years ago that antibody-dependent CD163 stimulation induced inflammatory cytokines such as IL1β and IL6 from macrophages (37) and activation of casein kinase II and protein kinase C (38); however, these observations were not reflected in our unpublished data. A recent study using CD163−/− mice demonstrated that soluble CD163 acts as a decoy receptor for TWEAK, and a deficiency of soluble CD163 increased the concentration of TWEAK in peripheral blood to promote tissue regeneration after ischemic injury (39); however, no description related to CD163-related signals has been seen. TWEAK has been reported to promote tumor cell invasion/progression rather than apoptosis (40, 41); therefore, the result of our present study that tumor development was impaired in CD163−/− mice is probably not due to an increased TWEAK. In our preliminary study, the PTEN-Akt signal in tumor cells was activated by coculture with macrophages, and this cell–cell interaction was impaired when CD163−/− macrophages were used for the coculture study. PTEN demonstrates phosphatase activity against the phospholipid products of PI3-kinase activity, leading to the inactivation of Akt signaling (42). PTEN activity is controlled by phosphorylation and phosphorylated 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 suppressing PTEN activity are thought to be secreted by macrophages, and CD163 is potentially related to tumor survival and growth indirectly via Akt signaling activation and PTEN inactivation in tumor cells.

In the present study, we used human cell lines and tissue samples to demonstrate the significance of CD163 in tumor cell proliferation, and an unknown CD163-related signal was suggested to be related to the protumoral activation of human macrophages as well as murine macrophages. Although a similar observation was observed in our previous study using malignant lymphoma samples, the detailed molecular mechanisms of CD163-related macrophage activation have never been clarified (43). CD163 is known to mediate cell–cell contacts between macrophages and erythroblasts (44), and CD163 might promote cell–cell adhesion between macrophages and tumor cells via unknown ligands.

In conclusion, we demonstrated the significance of CD163-related macrophage activation in the cell–cell interaction between tumor cells and TAMs using mouse and human cells. Although the 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 antitumor therapy.

No potential conflicts of interest were disclosed.

Conception and design: Y. Fujiwara, Y. Komohara

Development of methodology: D. Shiraishi, T. Iriki, Y. Komohara

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D. Shiraishi, Y. Fujiwara, H. Horlad, Y. Saito, 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, Junko Tsuboki, H. Bekki, Y. Komohara

Writing, review, and/or revision of the manuscript: Y. Fujiwara, Y. Komohara

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Komohara

Study supervision: H. Mizuta, M. Takeya, Y. Komohara

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. Komohara received JSPS KAKENHI grant No. 16H05162, Y. Fujiwara received JSPS KAKENHI grant No. 16K09247, and D. Shiraishi received JSPS KAKENHI grant No. 16K10865.

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

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Supplementary data