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. Author manuscript; available in PMC: 2017 Jul 15.
Published in final edited form as: Cancer Res. 2016 May 17;76(14):4283–4292. doi: 10.1158/0008-5472.CAN-15-2812

MFG-E8 drives melanoma growth by stimulating mesenchymal stromal cell-induced angiogenesis and M2 polarization of tumor-associated macrophages

Kazuya Yamada 1,*, Akihiko Uchiyama 1,*, Akihito Uehara 1, Buddhini Perera 1, Sachiko Ogino 1, Yoko Yokoyama 1, Yuko Takeuchi 1, Mark C Udey 2, Osamu Ishikawa 1, Sei-ichiro Motegi 1
PMCID: PMC5033700  NIHMSID: NIHMS791644  PMID: 27197197

Abstract

Secretion of the powerful angiogenic factor MFG-E8 by pericytes can bypass the therapeutic effects of anti-VEGF therapy, but the mechanisms by which MGF-E8 acts are not fully understood. In this study, we investigated how this factor acts to promote the growth of melanomas which express it. We found that mouse bone marrow-derived mesenchymal stromal cells (MSC) expressed a substantial amount of MFG-E8. To assess its expression from this cell type we implanted melanoma cells and MSC derived from wild-type (WT) or MFG-E8-deficient (KO) into mice and monitored tumor growth. Tumor growth and M2 macrophages were each attenuated in subjects co-implanted with KO-MSC compared to WT-MSC. In both xenograft tumors and clinical specimens of melanoma, we found that MFG-E8 expression was heightened near blood vessels where MSC could be found. Through in vitro assays we confirmed that WT-MSC-conditioned medium was more potent at inducing M2 macrophage polarization, compared to KO-MSC-conditioned medium. VEGF and ET-1 expression in KO-MSC was significantly lower than in WT-MSC, correlating in vivo with reduced tumor growth and numbers of pericytes and M2 macrophages within tumors. Overall, our results suggested that MFG-E8 acts at two levels, by increasing VEGF and ET-1 expression in MSC and by enhancing M2 polarization of macrophages, to increase tumor angiogenesis.

Keywords: MFG-E8, melanoma, mesenchymal stromal cell, angiogenesis, tumor-associated macrophages

Introduction

Mesenchymal stromal cells (MSC) are bone marrow (BM)-derived nonhematopoietic pluripotent progenitor cells with the capacity to differentiate into various cell types, including chondrocytes, adipocytes and osteocytes (1-3). Recent evidence indicates that pericytes and MSC are similar cells that are located external to the vasculature and that are involved in angiogenesis, repair and tissue maintenance (4-6). It has been reported that malignant tumor cells, such as malignant glioma and melanoma, can recruit MSC from surrounding tissue or the circulation and stimulate the growth of MSC by the secretion of soluble factors, including platelet-derived growth factor (PDGF) (7-9). These tumor-associated MSC secrete growth factors or cytokines, resulting in the promotion of angiogenesis (7-9). In addition, several studies have reported that MSC can differentiate into fibroblasts, myofibroblasts or pericyte-like cells and enhance angiogenesis, resulting in tumor progression and metastasis in vivo (10-14). MSC also have an immunosuppressive function and may help tumor escape from immune surveillance (11). Tumor-resident and injected MSC have been demonstrated to promote the recruitment of tumor associated macrophages (TAM) (15, 16). These findings have led to an increased interest in understanding the role of MSC in tumor growth and the potential of MSC to serve as therapeutic targets in melanoma.

Milk fat globule-epidermal growth factor (EGF) factor 8 (MFG-E8) is a secreted glycoprotein, and consists of two EGF-like domains and two discoidin-like domains with sequence homology to the blood coagulation factors V and VIII (17, 18). The second EGF-like domain contains an RGD motif that binds to integrin αvβ3/5 (18, 19). The carboxy-terminal domains of MFG-E8 can bind to negatively charged phospholipids (20), resulting in the opsonization of apoptotic cells for uptake by phagocytes (19, 21). In addition, interactions between MFG-E8 and integrin αv have been implicated in the enhancement of angiogenesis in mice (22-24).

Many studies have indicated that MFG-E8 enhances tumor cell survival, invasion and angiogenesis and contributes to local immune suppression (23, 25-29). In a murine melanoma model, MFG-E8 enhanced the tumorigenicity and metastatic capacity through Akt and Twist-dependent pathways (26). In addition, MFG-E8 produced by TAM in melanomas activated signal transducer and activator of transcription-3 (STAT3) and Sonic Hedgehog pathways in cancer stem cells (27). Furthermore, systemic MFG-E8 blockade using an anti-MFG-E8 antibody cooperates with cytotoxic chemotherapy, molecular targeted therapy and radiation to induce the destruction of murine tumors, including melanoma (28).

In prior studies, we demonstrated that pericytes and/or pericyte precursors were important sources of MFG-E8 in melanoma tumors, and pericytes/pericyte precursor-derived MFG-E8 enhanced angiogenesis in melanoma tumors (29). MFG-E8 associated with integrin αv and PDGF receptor β (PDGFRβ) on the surface of pericytes/pericyte precursors after PDGF treatment, and the formation of this complex inhibited degradation of PDGFRβ, resulting in the enhancement of PDGFRβ signaling (30). These findings suggest that MFG-E8 expressing perivascular cells regulate angiogenesis and tumor growth in melanoma by potentiating PDGFR signaling in perivascular cells. Considering that pericytes and MSC are similar perivascular cells, and that tumor-recruited MSC secrete proangiogenic factors, we hypothesized that MSC might express a substantial amount of MFG-E8, and that MFG-E8 might regulate the functions of MSC, including angiogenesis in melanoma tumors. To test this hypothesis, we analyzed the expression of MFG-E8 in BM-derived MSC (BM-MSC) and compared the effects of MSC from MFG-E8 wild type (WT) and knockout (KO) mice on melanoma tumor growth and vascularity.

Materials and Methods

Cell culture

The mouse melanoma cell line (B16-F10) and the mouse monocyte/macrophage cell line (RAW 264.7) were obtained directly from the ATCC between 2011 and 2015, and frozen aliquots of cells were prepared upon receipt. Cells were used within 6 months after receipt, and a link to the method of authentication is provided here: http://www.atcc.org/support/faqs/eae27/Authenticating%20cell%20lines-249.aspx.

Mice

MFG-E8 KO C57BL/6 mice were generated, and genotyped as described previously (24, 29, 31). MFG-E8 KO mice were generated by interbreeding homozygous animals carrying the targeted MFG-E8 allele. Interbreeding homozygous C57BL/6 mice and C57BL/6-Tg (CAG-EGFP) mice were purchased from Japan SLC. Eight- to twelve-week-old mice were used for all experiments.

Isolation and characterization of bone marrow-derived MSC

BM cell suspensions were obtained from MFG-E8 WT/KO C57BL/6 female mice and cultured. Magnetic-activated cell sorting (MACS) (Miltenyi Biotec) was performed to remove CD11b+ cells. For examination of surface expression of MSC markers, BM-MSC were incubated consecutively with Alexa 488-conjugated anti-human Sca-1, CD105, CD44, CD45, CD11b Ab or isotype control Ab (BioLegend) before flow cytometric analysis with a FACS Calibur instrument (BD Biosciences).

MSC differentiation assay

To analyze the differentiation potential of MSC, we used Mouse Mesenchymal Stem Cell Functional Identification Kit (R&D systems) according to the manufacture's instructions.

RNA extraction and real-time RT-PCR

Total RNA was isolated using RNeasy Mini Kits (Qiagen) and was subjected to reverse transcription with a SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). Quantitative RT-PCR was performed via the TaqMan system (Applied Biosystems) using a 7300 Real-Time PCR machine (Applied Biosystems).

To inhibit MFG-E8 production, 5 × 105 MSC were transfected with 10 nmol/L small interfering RNA (siRNA) using HiPerFect transfection reagent (Qiagen). At 24 hours after siRNA transfection, cells were incubated under hypoxic condition (1% O2, 5% CO2 and 94% N2) for 24 hours, and then, RNA was extracted.

Melanoma xenograft model

B16F10 melanoma cells (2×105 cells) and MFG-E8 WT/KO-MSC (2×105 cells) were implanted subcutaneously into the flanks of MFG-E8 WT/KO mice. MSC was incubated under hypoxic condition for 24 hours prior to implantation. Tumor sizes (width × length; mm2) were determined with calipers every 2 or 3 days. To analyze the localization of implanted MSC in melanomas, MSC derived from BM of green fluorescent protein (GFP) transgenic mice were incubated under hypoxic condition for 24 hours, and then GFP-MSC were implanted with B16F10 cells into MFG-E8 KO mice. MSC was labelled with CM-DiI (Molecular Probe) as previously described (32). CM-DiI-labelled MSC were implanted with B16F10 cells into mice. To analyze the effect of macrophages depletion on tumor growth, 200 μl clodronate liposomes (Clophosome-A, FormuMax) or placebo control liposomes (FormuMax) was injected intraperitoneally at day 0 and then 100 μl clodronate liposomes or control was injected twice per week.

Immunofluorescence staining

Tumors (100 mm2) were excised from flank skin and fixed in 4% paraformaldehyde and 30% Sucrose/H2O. After blocking, frozen sections (4 μm thick) were stained with the antibody of interest, followed by the secondary antibody conjugated with Alexa Fluor 488 or 568. Human melanoma tissues were used after surgical resection in the patients treated at the Department of Dermatology, of Gunma University hospital. The study was approved by the institutional review board of Gunma University.

Macrophage differentiation assay

MFG-E8 WT/KO-MSC (5×105 cells) were incubated for 24 hours under hypoxic conditions, and then conditioned media was collected. RAW 264.7 cells (1×106 cells) were incubated in 1 ml of MFG-E8 WT/KO-MSC-conditioned medium. After 48 hours incubation, RNA was extracted. To assess the effect of recombinant MFG-E8 (rMFG-E8) on macrophage differentiation, RAW 264.7 cells were incubated with or without rMFG-E8 (500 ng/ml) (R&D Systems) for 48 hours, then RNA was extracted.

BM chimeric mice and tumor implantation

C57BL/6 MFG-E8 WT/KO-BM cells were collected from the femurs of mice by aspiration and flushing. Recipient C57BL/6 mice were irradiated with 12 Gy and then 5 × 106 MFG-E8 WT/KO-BM were injected intravenously. Eight weeks after BM cell injection, B16F10 melanoma cells were implanted subcutaneously into the flanks of mice.

Statistical analysis

P values were calculated using the Student's t-test (two-sided) or by analysis of one-way ANOVA followed by Bonferroni's post test as appropriate. Error bars represent standard errors of the mean, and numbers of experiments (n) are as indicated.

Results

Mice bone marrow-derived MSC produced a substantial amount of MFG-E8

To examine the expression of MFG-E8 in MSC, mRNA and protein levels of MFG-E8 in mouse BM cells, BM-MSC, 10T1/2 pericyte-like cells and B16 melanoma cells were compared. We found that BM-MSC produced much more MFG-E8 mRNA and protein as compared to BM cells, 10T1/2 cells and B16 melanoma cells (Fig. 1A). Next, the surface markers of MFG-E8 WT- and KO-MSC were compared. Typical MSC markers, such as Sca-1, CD105, and CD44, were expressed in more than 80% of MSC, and negative markers, CD45 and CD11b were not expressed (Fig. 1B). There were no differences between the surface marker profiles of WT- and KO-MSC (Fig. 1B). Next, the differentiation potential of MFG-E8 WT- and KO-MSC was compared. Adipogenic differentiation assessed by staining with Oil-red-O in MFG-E8 KO-MSC was significantly enhanced (Fig. 1C). The adipogenic marker FABP4 mRNA expression in KO-MSC was also markedly enhanced compared to that in WT-MSC (Fig. 1C). In contrast, osteogenic differentiation in KO-MSC was significantly suppressed (Fig. 1D). There was no obvious difference in the chondrogenic differentiation capacity of WT- and KO-MSC (Fig. 1E). These results suggest that MSC expressed a substantial amount of MFG-E8, and MFG-E8 might suppress adipogenic differentiation, but promote osteogenic differentiation of MSC.

Figure 1.

Figure 1

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Mice BM-MSC produced a substantial amount of MFG-E8. A. mRNA and protein levels of MFG-E8 in BM, BM-MSC, 10T1/2 pericyte-like cells and B16 melanoma cells. mRNA and protein level in BM was assigned value of 1. Values were determined in 3 independent experiments. B. Flow cytometry analyses of the surface expression of Sca-1, CD105, CD44, CD45, CD11b in MFG-E8 WT- and KO-MSC. Data are representative of n=3 experiments. C. Adipogenic differentiation was assessed by Oil-red-O staining. MSC were incubated in adipogenic differentiation medium (AM) or normal medium (NM) for 2 weeks. FABP4 mRNA levels in WT- and KO-MSC were measured 2 weeks after incubation with NM or AM. mRNA level in WT-MSC incubated with NM was assigned value of 1. Values were determined in 3 independent experiments. D. Osteogenic differentiation was assessed by alizarin red S staining. MSC were incubated in osteogenic differentiation medium (OM) or normal medium (NM) for 2 weeks. Concentration of Alizarin red S was determined by absorbance measurement. The absorbance amount in WT-MSC incubated with NM was assigned value of 1. Values were determined in 4 independent experiments. E. Chondrogenic differentiation was assessed by collagen II staining. MSC were incubated in chodrogenic differentiation medium for 3 weeks. Scare bar = 20 μm. Data are representative of n=3 experiments. **P<0.01, *P<0.05.

MSC were localized around CD31+ EC and expressed pericytes marker NG2 and MFG-E8 in melanoma tumors

Next, we examined the localization of MSC and the expression of MFG-E8 in MSC in melanoma tumors co-implanted with MSC. B16 melanoma cells and GFP-MSC were co-injected into MFG-E8 KO mice. Recent studies have shown that hypoxia preconditioning improved the viability and tissue repair capacity of MSC after transplantation, leading to increased angiogenesis and the improvement of myocardial and brain ischemic model (33, 34). We found that hypoxic treatment significantly enhanced cell proliferation and MFG-E8 mRNA expression in MSC (Supplementary Fig. S1A, B). Therefore, MSC were incubated under hypoxic conditions for 24 hours prior to implantation in this study. GFP-MSC were localized around CD31+ endothelial cells (EC) (Fig. 2A) and several GFP-MSC expressed pericytes marker NG2 (Fig. 2B). MFG-E8 staining was weakly positive in melanoma cells and strongly positive in GFP-MSC and around the vessel-like structures in melanoma tumors (Fig. 2C). These results suggest that MSC might localize to the perivascular area, and MFG-E8 derived from MSC might be involved in MSC-induced tumor angiogenesis and tumor growth.

Figure 2.

Figure 2

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Perivascular MSC expressed MFG-E8, and MFG-E8 in MSC accelerated the growth of melanoma tumors. A. Localization of GFP-MSC and CD31+ EC in melanoma tumors. Arrowheads indicate perivascular GFP-MSC. B. Localization of GFP-MSC and NG2 expression in melanoma tumors. Arrowheads indicate NG2 expressing GFP-MSC. C. Localization of GFP-MSC and MFG-E8 expression in melanoma tumors. Arrowheads indicate MFG-E8 expressing GFP-MSC. D. Localization of CM-DiI-labelled WT/KO-MSC (red) and CD31+ EC (green) in melanoma tumors. Arrowheads indicate perivascular CM-DiI-labelled MSC. E. Localization of CM-DiI-labelled WT/KO-MSC (red) and NG2 expression (green) in melanoma tumors. Arrowheads indicate NG2 expressing CM-DiI-labelled MSC. Both melanoma cells and MSC were implanted subcutaneously, and tumors were analyzed 10 days after inoculation. Scare bar = 20 μm. F. Tumor sizes of malanomas co-implanted with/without MFG-E8 WT/KO-MSC. G. Survival in mice implanted with melanoma cells and MFG-E8 WT/KO-MSC. Representative data from 3 independent experiments (6 mice / group). **P<0.01 in WT-MSC vs. KO-MSC.

To examine the distribution of MFG-E8 WT- and KO-MSC in B16 tumor, MFG-E8 WT- and KO-MSC were labelled with CM-DiI, and co-implanted with B16 melanoma cells into mice. CM-DiI-labelled WT- and KO-MSC were localized around CD31+ EC (Fig. 2D). There was no obvious differences of the distribution of WT- and KO-MSC in B16 tumors. In addition, several CM-DiI labelled WT- and KO-MSC expressed NG2 (Fig. 2E). These results suggest that MFG-E8 in MSC may not be associated with the distribution of MSC in melanoma tumors.

MFG-E8 in MSC accelerated the growth of melanoma tumors

It has been reported that MSC promote tumor growth through enhanced angiogenesis and immune modulation (10-14). In contrast, few studies have reported that MSC inhibit tumor growth (35). Therefore, we initially examined the effect of MSC on melanoma tumor growth, and found that MSC enhanced the growth of melanoma tumors compared to that of melanoma cells alone (Fig. 2F; B16 alone vs B16+WT- or KO-MSC). Next, both melanoma cells and MFG-E8 WT/KO-MSC were implanted subcutaneously into mice. We found that the enhancement of tumor growth by co-implantation of KO-MSC was less than that caused by co-implantation of WT-MSC (Fig. 2F; B16+WT-MSC vs B16+KO-MSC). The enhancement of survival in mice co-implanted with melanoma cells and KO-MSC was also significantly less than that in mice co-implanted with WT-MSC (Fig. 2G). These results indicate that MFG-E8 in MSC might contribute to MSC-induced melanoma tumor growth.

MFG-E8 in MSC accelerated the number of pericytes in melanoma tumors

Next, we examined the vascularity in tumors and found that the numbers of NG2+ pericytes in melanoma tumors co-implanted with MFG-E8 WT-MSC were significantly higher than those in tumors co-implanted with KO-MSC (Fig. 3A, B). The numbers of CD31+ EC in melanoma tumors co-implanted with WT-MSC tended to be higher than those in tumors co-implanted with KO-MSC, however, this difference was not significant (Fig. 3A, B). The level of pericyte coverage, which was assessed by the pericytes/EC ratio in tumors co-implanted with WT-MSC, tended to be higher than those in tumors co-implanted with KO-MSC (Fig. 3B). These results suggest that MFG-E8 in MSC might accelerate angiogenesis, especially the number of pericytes in melanoma tumors.

Figure 3.

Figure 3

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MFG-E8 in MSC accelerated the number of pericytes in melanoma tumors. A. The amount of EC (CD31+) and pericytes (NG2+) in melanoma co-injected with/without MFG-E8 WT/KO-MSC. Scare bar = 100 μm. B. Quantification of the extent of vascularization (EC and pericytes (PC) area) and PC coverage (PC/EC ratio) in melanoma tumors. Values were determined in 5 random fields in n=3 tumors per group. **P<0.01, *P<0.05.

MFG-E8 in MSC induced tumor-associated M2 macrophages

We next assessed the numbers of infiltrated TAM in melanoma tumors co-implanted with MFG-E8 WT- or KO-MSC. The numbers of CD68+, CD206+ M2 macrophages in melanoma tumors co-implanted with KO-MSC were significantly reduced compared with those in tumors co-implanted with WT-MSC (Fig. 4A), suggesting that MFG-E8 in MSC might be involved in the increased infiltration of TAM into melanoma tumors.

Figure 4.

Figure 4

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MFG-E8 WT-MSC induced tumor-associated M2 macrophages. A. Quantification of the amount of CD68+ macrophages and CD68+, CD206+ M2 macrophages in melanoma co-injected with MFG-E8 WT/KO-MSC. Values were determined in 5 random fields in n=3 tumors per group. *P<0.05. B. Tumor sizes of malanomas co-implanted with/without MFG-E8 WT/KO-MSC in mice treated with clodronate liposomes or control liposomes (n=10 for each time points and groups). **P<0.01, *P<0.05 in Ctl+B16+WT-MSC vs. Ctl+B16+KO-MSC. C. Tumor sizes of melanoma co-implanted with/without MFG-E8 WT/KO-MSC in mice treated with clodronate liposomes or control at 15 days after inoculation. Values were determined in n=10 mice per groups. **P<0.01, *P<0.05. D. mRNA levels of iNOS, arginase-1, CD206 and Ym1 in RAW 264.7 cultured with MFG-E8 WT/KO-MSC-conditioned medium for 48 hours. mRNA level in macrophages cultured with normal medium as a control was assigned value of 1. Values were determined in 3 independent experiments. **P<0.01, *P<0.05. E. Arginase-1, CD206 and Ym1 stainings in RAW 264.7 cultured with MFG-E8 WT/KO-MSC-conditioned medium for 48 hours. Scare bar = 20 μm. F. mRNA levels of iNOS and arginase-1 in RAW 264.7 cultured with or without rMFG-E8 for 48 hours. mRNA level in macrophages cultured with normal medium as a control was assigned value of 1. Values were determined in 3 independent experiments.

We next examined the effect of the depletion of macrophages on MFG-E8 WT-MSC-induced melanoma tumor growth. The number of CD68+ macrophages in melanoma tumor in mice treated with clodronate liposomes were significantly inhibited compared to that in mice treated with control liposomes (Supplementary Fig. S2A). We found that clodronate liposomes treatment inhibited tumor growth of B16 melanoma alone and co-implanted with MFG-E8 WT/KO-MSC (Fig. 4B, C; Ctl+B16 alone, Ctl+B16+WT-MSC or KO-MSC vs Clodronate+B16 alone, Clodronate+ B16+WT-MSC or KO-MSC). In addition, the enhancement of tumor growth and survival in mice by co-implantation of WT-MSC was partially reduced by clodoronate liposomes treatment, but not completely attenuated (Fig. 4B, C, Supplementary Fig. S2B). These results suggest that TAM might partially contribute to the WT-MSC-induced tumor growth.

It has been reported that tumor resident and injected MSC increased the infiltration of TAM and tumor growth (15, 16). In addition, M2 markers, arginase-1, CD206 and Ym1 in macrophages, including RAW264.7 cells were increased by culturing with MSC-conditioned medium (36-39). Therefore, we examined whether MFG-E8 plays a role in MSC-induced M2 macrophage polarization. The expression of iNOS, a M1 macrophage marker, in macrophages cultured with WT- or KO-MSC-conditioned medium were unchanged. Culturing RAW 264.7 cells with WT-MSC-conditioned medium increased mRNA and protein levels of arginase-1, CD206 and Ym1 expressions in macrophages, however, KO-MSC-conditioned medium did not (Fig. 4D, E). Next, to assess the direct effect of MFG-E8 on M2 macrophage polarization, rMFG-E8 was added to the medium of cultured macrophages. The addition of rMFG-E8 in the culture medium of RAW 264.7 macrophages did not affect the expression of M1 and M2 markers in macrophages (Fig. 4F). These results suggest that MFG-E8 in MSC might regulate MSC-induced M2 macrophage polarization, in conjunction with factors other than MFG-E8 that are also secreted from MSC.

Depletion of MFG-E8 decreased the production of ET-1 and VEGF in MSC

The effect of MFG-E8 on the cell proliferation of MSC was analyzed by an in vitro assay. The proliferation of MFG-E8 KO-MSC tended to be decreased in comparison with that in WT-MSC, however, the difference was not significant (Fig. 5A). We next examined the role of MFG-E8 in the production of angiogenic factors in MSC in vitro. The levels of VEGF and ET-1 mRNA in KO-MSC were significantly suppressed (Fig. 5B, C). The mRNA levels of angiopoietin in MFG-E8 WT/KO-MSC were unchanged (Fig. 5D). We also examined the role of MFG-E8 in MSC using MFG-E8 siRNA. MFG-E8 siRNA inhibited mRNA levels of MFG-E8 by 60% (Fig. 5E). VEGF and ET-1 mRNA levels in MFG-E8 siRNA-treated MSC were reduced compared with those in control siRNA-treated MSC (Fig. 5F, G). The mRNA levels of angiopoietin in control or MFG-E8 siRNA-treated MSC were unchanged (Fig. 5H). These results suggest that MFG-E8 might regulate the production of angiogenic factors, such as VEGF and ET-1, in MSC.

Figure 5.

Figure 5

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Depletion of MFG-E8 decreased the production of ET-1 and VEGF in MSC. A. Proliferation of MFG-E8 WT/KO-MSC cultured under hypoxia condition for 48 hours. Values were determined in 3 independent experiments and normalized to the extent of proliferation in WT-MSC. B-D. Quantification of mRNA levels of VEGF (B), ET-1 (C) and angiopoietin (D) in MFG-E8 WT/KO-MSC cultured under hypoxia condition for 24 hours. mRNA levels in WT-MSC was assigned a value of 1. E-H. Quantification of mRNA levels of MFG-E8 (E), VEGF (F), ET-1 (G) and angiopoietin (H) in MSC transfected with control or MFG-E8 siRNA cultured under hypoxia condition for 24 hours. mRNA levels in MSC transfected with control siRNA was assigned a value of 1. Values were determined in 3-4 independent experiments. **P<0.01, *P<0.05.

Contribution of MFG-E8+ BM-derived cells to melanoma tumor growth, the number of pericytes and tumor-associated M2 macrophages

We examined the contribution of BM-derived MFG-E8+ cells to melanoma tumor growth using BM chimeric mice generated from MFG-E8 WT and KO mice. Melanoma tumor growth in MFG-E8 KO-BM-transplanted mice was modestly inhibited beginning 12 days after inoculation compared with that in WT-BM-transplanted mice (Fig. 6A). The number of NG2+ pericytes in tumors in MFG-E8 KO-BM-transplanted mice was significantly decreased (Fig. 6B). In addition, the number of CD68+, CD206+ M2 macrophages in tumors was significantly reduced in KO-BM-transplanted mice (Fig. 6C). These results suggest that BM-derived MFG-E8+ cells might contribute to the number of pericytes and TAM and melanoma tumor growth.

Figure 6.

Figure 6

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Contribution of MFG-E8+ BM-derived cells to melanoma tumor growth, the number of pericytes and TAM. A. Tumor sizes of malanomas in MFG-E8 WT- or KO-BM-transplanted WT mice. WT-BM in WT mice; n= 8, KO-BM in WT mice; n= 11, for each time points and group. B. The amount EC (CD31+) and pericytes (NG2+) in melanoma in WT- or KO-BM in WT mice. Values were determined in 10 random fields in n=3 tumors per group. **P<0.01. C. Quantification of the amount of CD68+ macrophages and CD68+, CD206+ M2 macrophages in melanoma in WT- or KO-BM in WT mice. Values were determined in 10 random fields in n=3 tumors per group. *P<0.05.

Perivascular expression of MFG-E8 in human melanoma tumors

Finally, we examined the distribution of MFG-E8 in human melanoma tumors. We found that MFG-E8 staining in human melanoma was primarily observed around blood vessels, especially in αSMA+ pericytes (Fig. 7A, B). MFG-E8 staining around the vessels was more frequently observed inside of melanoma tumors compared to outside of the tumors (Fig. 7A, lower panels). These results suggest that the perivascular expression of MFG-E8 in human melanoma tumors might be associated with tumor angiogenesis and tumor growth.

Figure 7.

Figure 7

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Perivascular expressions of MFG-E8 in human melanoma tumors. A, B. Expression of MFG-E8 in relationship to EC (CD31+) or pericytes (PC) (αSMA+) in human melanoma tumor. Scare bar = 20 μm. Dotted line is the border of tumor, and right side of border is inside tumor. Data are representative of n=5 tumors. C. Model for the role of MFG-E8 in MSC-induced angiogenesis, M2 macrophage polarization and melanoma tumor growth. MFG-E8 in MSC might increase the expression of VEGF and ET-1 in MSC and enhance M2 macrophage polarization, leading to the enhancement of angiogenesis and tumor growth.

Discussion

We previously demonstrated that PDGFRβ+ pericytes/pericyte precursors are a predominant source of MFG-E8 in melanoma tumors (29). In this study, we demonstrated that BM-MSC produce a substantial amount of MFG-E8. Since several reports have proposed that the perivascular zone is the MSC niche in vivo, and perivascular MSC contribute to angiogenesis (4-6), suggesting that MSC-derived MFG-E8 might regulate the functions of MSC and contribute to tumor angiogenesis and growth.

We demonstrated that adipogenic differentiation in MFG-E8 KO-MSC was markedly enhanced, and osteogenic differentiation in KO-MSC was significantly suppressed. With respect to MFG-E8 and osteogenesis, Abe et al. reported that chronic periodontal bone loss occurred more frequently in MFG-E8 KO mice compared with that in WT mice (40). However, the mechanisms regulating MSC differentiation by MFG-E8 remain unknown.

We determined that GFP-MSC localized in the perivascular area and expressed NG2 and MFG-E8 in melanoma tumors. We also found that the number of pericytes and the level of pericyte coverage in melanomas co-implanted with MSC were higher than those in melanomas without MSC co-implantation. Interestingly, the increased number of pericytes by co-implantation with MSC was reduced in melanomas co-implanted with MFG-E8 KO-MSC as compared with MFG-E8 WT-MSC. These results suggest that MSC might localize around the vessels and act as pericytes in melanoma tumors, and MFG-E8 in MSC might be involved in these mechanisms.

Regarding TAM and MFG-E8, MFG-E8 induced efferocytosis of apoptotic prostate cancer cells and promoted M2 phenotype polarization in macrophages (41), suggesting that MFG-E8 might regulate the functions of TAM. We demonstrated that the enhancement of melanoma tumor growth by co-implantation of WT-MSC was partially suppressed by macrophages depletion in vivo, suggesting that MFG-E8 in MSC might enhance tumor growth via the regulation of TAM. Furthermore, we determined that MFG-E8 KO-MSC reduced M2 macrophage infiltration in vivo and that MFG-E8 KO-MSC reduced MSC-induced M2 macrophage polarization in vitro. These results suggest that MFG-E8 in MSC might positively regulate TAM in melanoma tumors. MFG-E8 WT MSC-conditioned medium induced M2 macrophage polarization, however, rMFG-E8 did not, suggesting that secreted factors other than MFG-E8 might also be required for M2 macrophage polarization. We further examined the protein levels of IL-10 and IL-4 in the conditioned medium of WT- and KO-MSC incubated under hypoxic condition for 24, 48 and 72 hours by ELISA. However, IL-10 and IL-4 secretions were not detected in the supernatant of WT- and KO-MSC (data not shown), suggesting that IL-10 and IL-4 might not be associated with WT-MSC conditioned medium-induced M2 polarization of macrophages.

ET-1/ET receptor signaling has been shown to play a role in the growth and progression of melanoma (42, 43). In addition, ET-1 contributes to angiogenesis, extracellular matrix degeneration and macrophage chemoattraction (44). According to these results, MFG-E8 might increase the expression of ET-1 and VEGF in MSC, resulting in enhancement of angiogenesis, macrophage infiltration and tumor growth in melanoma.

Using BM chimeric mice, we identified that BM-derived MFG-E8+ cells contribute to increased numbers of pericytes, M2 macrophage infiltration and melanoma tumor growth. Although BM-derived MFG-E8+ cells include various types of cells, such as leukocytes, macrophages and endothelial progenitor cells, BM-MSC express a greater amount of MFG-E8 than other BM-derived cells; therefore, we speculate that MFG-E8 in MSC may regulate the migration of MSC from BM to tumors. Our previous results that MFG-E8 enhanced PDGF:PDGFRβ signaling and the migration of 10T1/2 MSC cells (29, 30) are consistent with this speculation. However, the present results using a tumor model in BM chimeric mice do not prove that MFG-E8 regulates MSC migration in vivo, and further investigation is required.

Collectively, we propose the following model to clarify the role of MFG-E8 in MSC-induced angiogenesis, M2 macrophage polarization and melanoma tumor growth (Fig. 7C). BM-MSC may migrate and localize around blood vessels in melanoma tumors. MSC produce substantial amounts of MFG-E8, and MFG-E8 production is additionally induced by hypoxic conditions in tumors. MFG-E8 in MSC might increase the expression of VEGF and ET-1 in MSC and enhance M2 macrophage polarization, leading to the enhancement of angiogenesis and tumor growth. As previously reported, MFG-E8 also acts on pericytes and EC by potentiating the stimulatory effects of PDGF and VEGF, respectively, leading to enhanced angiogenesis (22, 30). This regulation of MSC by MFG-E8 might provide new insight into the pathogenesis of tumor growth in melanoma.

In human melanoma, MFG-E8 was predominantly expressed around blood vessels, especially in pericytes, suggesting that our concept may be involved in the pathogenesis of human melanoma progression. However, additional studies regarding the role of MSCs in human melanoma are warranted.

Supplementary Material

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Acknowledgments

Grant Support: This work was financially supported by Japanese Dermatological Association research grant (Shiseido donation), Takeda Science Foundation, Research Grant 2013, and JSPS KAKENHI, Grant Number 24791135 and 26461654 (to S. Motegi), and the Intramural Program of NIH, Center for Cancer Research, National Cancer Institute (to M.C. Udey).

Footnotes

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

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