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DOI: 10.1002/eji.201343867

Serum amyloid A3 exacerbates cancer by enhancing the suppressive capacity of myeloid-derived suppressor cells via TLR2-dependent STAT3 activation Jung-Mi Lee1 , Eun-Kyung Kim1 , Hyungseok Seo2 , Insu Jeon2 , Min-Ji Chae1 , Young-Jun Park1 , Boyeong Song2 , Yun-Sun Kim1 , Yeon-Jeong Kim3 , Hyun-Jeong Ko4 and Chang-Yuil Kang1,2 1

Laboratory of Immunology, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, Korea 2 Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, and College of Medicine or College of Pharmacy, Seoul National University, Seoul, Korea 3 Laboratory of Microbiology and Immunology, College of Pharmacy, Inje University, Gyungnam, Korea 4 Laboratory of Microbiology and Immunology, College of Pharmacy, Kangwon National University, Chuncheon, Korea Myeloid-derived suppressor cells (MDSCs), which suppress diverse innate and adaptive immune responses and thereby provide an evasion mechanism for tumors, are emerging as a key population linking inflammation to cancer. Although many inflammatory factors that induce MDSCs in the tumor microenvironment are known, the crucial components and the underlying mechanisms remain elusive. In this study, we proposed a novel mechanism by which serum amyloid A3 (SAA3), a well-known inflammatory factor, connects MDSCs with cancer progression. We found that SAA3 expression in BALB/c mice increased in monocytic MDSCs (Mo MDSCs) with tumor growth. The induction of SAA3 by apo-SAA treatment in Mo MDSCs enhanced their survival and suppressive activity, while it inhibited GM-CSF-induced differentiation. Endogenous SAA3 itself contributed to the increase in the survival and suppressive activity of Mo MDSCs. We demonstrated that SAA3 induced TLR2 signaling, in turn increasing the autocrine secretion of TNF-α, that led to STAT3 activation. In addition, activated STAT3 enhanced the suppressive activity of Mo MDSCs. Furthermore, SAA3 induction in Mo MDSCs contributed to accelerating tumor progression in vivo. Collectively, these data suggest a novel mechanism by which Mo MDSCs mediate inflammation through SAA3-TLR2 signaling and thus exacerbate cancer progression by a STAT3-dependent mechanism.

Keywords: Myeloid-derived suppressor cell microenvironment



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Serum amyloid A3

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STAT3

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TLR2

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Tumor

Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction Correspondence: Dr. Chang-Yuil Kang e-mail: [email protected]  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Cancer and inflammation have been shown to be strongly correlated. These processes are connected by converging intrinsic and extrinsic pathways, resulting in the activation of transcription www.eji-journal.eu

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factors, specifically NF-κB, STAT3, and hypoxia-inducible factor 1α in tumor cells [1–5]. These transcription factors induce the production of inflammatory mediators that recruit and activate various leukocytes and in turn induce the same key transcription factors in inflammatory cells, stromal cells, and tumor cells. All of these cell types and transcription factors cooperate to generate a cancer-related inflammatory microenvironment. Moreover, recent studies have shown that another mechanism by which inflammation promotes tumor progression is through the induction of myeloid-derived suppressor cells (MDSCs). MDSCs are potent suppressors of immune surveillance and antitumor immunity, thereby facilitating tumor growth [6]. Although many factors linking cancer to inflammation are known, the crucial components remain to be elucidated. Serum amyloid A (SAA), a family of apolipoproteins that associate with high-density lipoprotein in plasma, is a well-known marker of inflammation. SAA levels in plasma are increased in patients with a broad spectrum of inflammatory diseases, such as atherosclerosis, rheumatoid arthritis, Crohn’s disease, diabetes, and ankylosing spondylitis [7–9]. In mice, three acutephase isoforms, SAA1, SAA2, and SAA3, have been identified. During the inflammatory response, SAA3 is expressed in various organs, while SAA1 and SAA2 are induced mainly in the liver. Bacterial antigens (such as LPS), proinflammatory cytokines (such as IL-1β, IL-6, and TNF-α), and glucocorticoids, alone or in combination, have been shown to induce acute phase SAA (A-SAA) synthesis in hepatocytes, macrophages, and synoviocytes [10–12]. A-SAA gene expression is induced through the activation of NF-κB, CCAAT-binding protein (C/EBP), STAT3, and SAA-activating factor, while it is repressed by the transcription factors YY1 and AP2 [8]. SAA is involved in several processes, including the transport of cholesterol to the liver for secretion into the bile, the recruitment of immune cells to inflammatory sites, and the induction of enzymes that degrade the ECM. SAA is significantly elevated in the blood of cancer patients [13–15] and tumor-bearing mice [16–18], and it has a positive correlation with cancer stage [19, 20], providing further evidence that SAA is involved in tumor progression. In recent studies, it has been reported that SAA regulates tumor microenvironment via its effects on myeloid cells. For example, SAA1 controls the plasticity of neutrophil differentiation by not only inducing the differentiation of anti-inflammatory IL-10-secreting neutrophils, but also by promoting the interaction of invariant NKT cells with these neutrophils [21]. SAA can act as an endogenous factor that stimulates G-CSF expression in isolated macrophages and induces neutrophilia in mice in a TLR2-dependent manner [22]. SAA also contributes to the polarization of macrophages toward an M2 profile, especially M2b- or M2d-like polarization, which exacerbates hepatocellular carcinoma cell invasion [23]. These data suggest that SAA could induce immunosuppressive MDSCs, a heterogeneous cell population of myeloid origin, to decrease tumor immunity and facilitate tumor growth. Furthermore, in our previous study, to identify the factors that induce changes in MDSCs related to tumor growth, we analyzed the gene expres C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Cellular immune response

sion patterns in the total RNA of monocytic MDSCs (Mo MDSCs) and PMN MDSCs isolated from na¨ıve, early time point or late time point tumor-bearing mice [24]. Interestingly, SAA3 mRNA levels were specifically increased in Mo MDSCs as the tumor grew. In the current study, we investigated whether SAA3 played a role in the induction of the suppressive capacity of MDSCs. To investigate this hypothesis, the effects of SAA3 on the survival, differentiation, and suppressive activity of MDSCs were analyzed. We found that SAA3 upregulation in Mo MDSCs significantly enhanced their viability. SAA3 also inhibited GM-CSF-induced Mo MDSC differentiation and augmented the suppressive activity of Mo MDSCs on Ag-specific T-cell proliferation. These effects were dependent on TLR2. SAA3-TLR2 signaling increased the autocrine secretion of IL-6 and TNF-α and induced the activation of STAT3 that fortified the suppressive capacity of Mo MDSCs thereby accelerating tumor growth in vivo. Collectively, we demonstrate here that SAA3 augments the suppressive capacity of MDSCs by specifically enhancing the survival and suppressive activity of Mo MDSCs, and by blocking their differentiation, suggesting that Mo MDSC mediated cancer exacerbation is induced by STAT3 activation via SAA3-TLR2 signaling.

Results Gene expression analysis of SAA isoforms in the subsets of MDSCs In a previous study, we analyzed the gene expression patterns in MDSC subsets, Mo MDSCs and PMN MDSCs, isolated from na¨ıve, early time point or late time point tumor-bearing mice [24]. The expression levels of inflammation-related factors were increased in both Mo MDSCs and PMN MDSCs. Among the SAA isoforms, the level of SAA3 was dramatically increased in Mo MDSCs as the tumor grew, while that of PMN MDSCs was unchanged. On the basis of this finding, we aimed to investigate the hypothesis that SAA3 could affect the biological characteristics of Mo MDSCs in tumor microenvironment. First, the mRNA levels of SAA isoforms were measured in MDSC subsets, Mo MDSCs and PMN MDSCs, isolated from na¨ıve or Her-2/CT26 tumor-bearing mice (Fig. 1A). We found that SAA3 mRNA was dramatically increased only in Mo MDSCs. To investigate the effect of SAA3 induction in MDSCs, Mo MDSCs (Ly-6Chigh Ly-6Glow CD11b+ ) and PMN MDSCs (Ly-6Clow Ly-6Ghigh CD11b+ ) isolated from the splenocytes of Her-2/CT26 tumorbearing mice (Fig. 1B) were treated with recombinant apo-SAA, which is known to have functional similarity to mouse SAA3 [25]. Four hours later, Saa3 gene expression was significantly increased only in Mo MDSCs by apo-SAA treatment but not by apo-SAA1 treatment, which was used as a control (Fig. 1C). The expression increased until 4–16 hours and then decreased afterwards (Supporting Information Fig. 1). Collectively, these data indicate that SAA3 is induced specifically in the Mo subset of MDSCs by either tumor progression or apo-SAA treatment. www.eji-journal.eu

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dependent (Fig. 2C and D). To confirm whether apo-SAA treatment also enhances proliferation, we analyzed the expression of proliferation marker Ki67 in MDSCs after apo-SAA treatment. We found that Ki67 expression was not increased in PMN MDSCs or Mo MDSCs 24 hour after incubation with apo-SAA (Supporting Information Fig. 2A). Furthermore, the percentage of PI− cells increased in apo-SAA-treated Mo MDSCs, suggesting that apoSAA elevates the proportion of Mo MDSCs by enhancing survival rather than by promoting proliferation (Supporting Information Fig. 2B). Taken together, these data demonstrate that SAA3 specifically affects Mo MDSCs and dramatically enhances the viability of this subpopulation, resulting in an increase in the proportion of Mo MDSCs among total splenocytes.

TLR2-mediated enhancement of Mo MDSC viability

Figure 1. Induction of SAA3 expression in Mo MDSCs with tumor growth and by apo-SAA treatment. (A) Mo MDSCs (Ly-6Chigh Ly6Glow CD11b+ ) and PMN MDSCs (Ly-6Clow Ly-6Ghigh CD11b+ ) were isolated from na¨ıve or Her-2/CT26 tumor-bearing mice. The gene expression pattern of Saa isoforms was analyzed in total RNA. Data are shown as relative expression compared to HPRT and represent mean + SEM of three samples representative of two independent experiments. (B) Ly6C/Ly6G expression in Mo MDSCs (Ly-6Chigh Ly-6Glow CD11b+ ) and PMN MDSCs (Ly-6Clow Ly-6Ghigh CD11b+ ) after gating CD11b+ cells was assessed by flow cytometry. Plot is representative of three independent experiments. (C) The gene expression pattern of Saa isoforms was analyzed by quantitative real-time PCR in Mo MDSCs and PMN MDSCs from Her-2/CT26 tumor-bearing mice treated with apo-SAA or apo-SAA1 (500 ng /mL) for 4 hour. Data are shown as relative expression compared to HPRT and represent mean + SEM of three samples representative of two independent experiments. **p < 0.01 (Student’s t-test).

Enhancement of Mo MDSC viability following apo-SAA treatment To analyze the effect of SAA3 induction in diverse splenic cell populations, total splenocytes from Her-2/CT26 tumor-bearing mice were treated with apo-SAA or apo-SAA1 for 48 hour, and changes in the cell populations were examined. While the ratio of MDSCs (CD11b+ Gr-1+ ), B cells (B220+ CD3ε− ), and T cells (CD3ε+ B220− ) in total splenocytes was unchanged, that of Mo MDSCs (Ly-6Chigh Ly-6Glow CD11b+ ) was highly increased in apoSAA-treated MDSCs (Fig. 2A). To elucidate the mechanism by which apo-SAA treatment leads to an increase in Mo MDSCs, we analyzed the changes in the expression of surface molecules and in the survival of MDSC subsets, Mo MDSCs and PMN MDSCs. Forty-eight hours after incubation of the isolated Mo and PMN MDSCs with apo-SAA, no changes in Ly6C/Ly6G expression were detected in Mo and PMN MDSCs (data not shown). However, apo-SAA treatment significantly enhanced the survival of Mo MDSCs, but not PMN MDSCs (Fig. 2B). We further confirmed that this effect was dose C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

TLR2, TLR4, and formyl peptide receptor 2 (FPR2) function as SAA receptors. Therefore, we tested whether these receptors mediated the SAA3-induced increase in Mo MDSC survival. We first examined the expression of these molecules in MDSC subsets. TLR2 and FPR2 were expressed on both Mo and PMN MDSCs, while the expression of TLR4 was relatively low on both types of MDSC (Fig. 3A). To examine the role of each receptor, Mo MDSCs were treated with apo-SAA for 24 hour, including a 1-hour preincubation with α-TLR2 Ab, α-TLR4 Ab, and WRW4, which blocked FPR2 [22, 23]. Although each blocking agent by itself did not alter the viability of Mo MDSCs, the apo-SAA-induced enhancement of viability was significantly decreased only when TLR2 was blocked (Fig. 3B). In addition, the apo-SAA-induced increase in survival was significantly lowered in TLR2−/− Mo MDSCs compared with WT Mo MDSCs (Fig. 3C). We also confirmed that the TLR2-dependent enhanced survival of splenic Mo MDSCs derived from diverse tumor types, such as 4T1, EL4, and MC38 (Fig. 3D). Next, we analyzed whether endogenous SAA3 expression in Mo MDSCs was also involved in increased viability. To examine this, SAA3 mRNA in freshly isolated Mo MDSCs was knocked down using an siRNA system. Treatment with siSAA3 downregulated Saa3 expression and significantly decreased the survival of Mo MDSCs compared with siCtrl treatment, indicating that endogenous Saa3 expression was required for increased viability of Mo MDSCs (Fig. 3E). Collectively, these data suggest that TLR2 mediates the SAA3-induced enhancement of viability in Mo MDSCs and that endogenous Saa3 expression also contributes to increasing the viability of Mo MDSCs.

Inhibition of Mo MDSC differentiation following apo-SAA treatment To investigate the effect of SAA3 on the differentiation of MDSCs, Mo MDSCs and PMN MDSCs were cultured with or without apoSAA for 3 days in the presence of GM-CSF as a differentiating agent. Before the incubation, Mo MDSCs had a light blue www.eji-journal.eu

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Figure 2. Enhancement of Mo MDSC viability by SAA3. (A) Total splenocytes from Her-2/CT26 tumor-bearing mice were treated with apo-SAA or apo-SAA1 (500 ng/mL) for 48 hour, and the changes in the cell populations were analyzed by flow cytometry. Numbers indicate the percentage of MDSCs (CD11b+ Gr-1+ ), B cells (B220+ CD3ε− ), and T cells (CD3ε+ B220− ) in total splenocytes and Mo MDSCs (Ly-6Chigh Ly-6Glow CD11b+ , rectangle) and PMN MDSCs (Ly-6Clow Ly-6Ghigh CD11b+ ) in gated CD11b+ cells. The data are representative of two independent experiments. (B) Mo MDSCs and PMN MDSCs isolated from Her-2/CT26 tumor-bearing mice were treated with apo-SAA or apo-SAA1 (500 ng/mL) for 24 hour. Cell viability was measured by CCK-8 assay at 24 hour after treatment, and the results are expressed as a percentage relative to vehicle-treated controls, which are plotted as 100% viability. The data show mean ± SEM of three samples representative of three independent experiments. Dose-dependent cell viability using (C) CCK-8 assay and (D) 7-AAD staining was analyzed in Mo MDSCs of Her-2/CT26 tumor-bearing mice treated with apo-SAA (100, 300, 1000 ng/mL) for 24hour. Data are shown as mean ± SEM of three samples representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

cytoplasm with a one-lobed nucleus, while PMN MDSCs had a faint cytoplasm with lobulated nucleus (Fig. 4A). After the incubation with GM-CSF, PMN MDSCs did not show any changes in Ly6C/Ly6G expression and morphology, while Mo MDSCs differentiated into activated macrophage-like cells characterized by larger size, larger granules, and decreased Ly6C expression. ApoSAA treatment, in the presence of GM-CSF, inhibited the differentiation of Mo MDSCs into activated macrophages, as determined based on their morphology (Fig. 4A) and lowered MHC class II expression (Fig. 4B). Decreased F4/80 expression, which is characteristic of activated macrophages, was also reversed by apo-SAA treatment (Fig. 4C). Furthermore, the effect of apo-SAA treatment on F4/80 expression was significantly reversed by TLR2 blockade (Fig. 4C). The enhanced expression of F4/80 by apo-SAA treatment was not observed in TLR2−/− Mo MDSC (Fig. 4D). Collectively, these data demonstrate that SAA3 inhibits the GM-CSFinduced differentiation of Mo MDSCs into activated macrophages in a TLR2-dependent manner.

Increased suppressive activity of Mo MDSCs following apo-SAA treatment Among the MDSC subsets, Mo MDSCs are known to be potent suppressor cells; therefore, we sought to determine whether SAA3 would augment the suppressive activity of Mo MDSCs. To investigate this possibility, gene expression patterns related to the suppressive mechanism of MDSCs were analyzed after a 16-hour incu C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

bation with apo-SAA (Fig. 5A) or a 72-hour incubation with apoSAA in the presence of GM-CSF (Fig. 5B). The expression levels of NOS 2, arginase 1, and Nox2, which are the main enzymes that mediate the suppressive activity of MDSCs, were increased by apo-SAA treatment with or without GM-CSF (Fig. 5A and B). The expression level of cEBPβ, which is a transcription factor involved in this suppressive activity, was increased by apo-SAA treatment; however, this change was not significant (Fig. 5A). As hypothesized, apo-SAA treatment increased the suppressive activity of Mo MDSCs in Ag-specific T-cell proliferation assays (Fig. 5C). In addition, while the TLR2 blockade alone did not statistically change the suppressive activity, the TLR2 blockade along with apo-SAA treatment significantly reversed the apo-SAA-induced increase in the suppressive activity of Mo MDSCs (Fig. 5D). To exclude the possibility of direct, SAA3-independent, TLR2-mediated effects of apo-SAA on Mo MDSCs, we used a knockdown approach. Mo MDSCs were nucleofected with siSAA3 or siCtrl, then apo-SAA was added. As a result, the increase in the suppressive activity by apo-SAA treatment was abrogated in the SAA3 knockdown Mo MDSCs, suggesting that these effects were mediated by SAA3 (Fig. 5E). Next, to examine the effect of endogenous SAA3 expression on the suppressive activity of Mo MDSCs, freshly isolated Mo MDSCs were treated with siSAA3. Transfection with siSAA3 downregulated Saa3 expression and significantly decreased the suppressive activity of Mo MDSCs compared with siCtrl (Fig. 5F). Taken together, these data demonstrate that SAA3 augments the suppressive activity of Mo MDSCs through TLR2-dependent signaling and that endogenous Saa3 expression is involved in increasing the suppressive activity of Mo MDSCs. www.eji-journal.eu

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Figure 3. TLR2 mediates the enhancement of Mo MDSC viability by SAA3. (A) TLR2, TLR4, and FPR2 expression was assessed in gated Mo (Ly-6Chigh Ly-6Glow CD11b+ ) and PMN MDSCs (Ly-6Clow Ly-6Ghigh CD11b+ ) by flow cytometry. Plots are representative of three independent experiments. (B) Mo MDSCs of Her-2/CT26 tumor-bearing mice were treated with apo-SAA (500 ng/mL) for 24 hour, including a 1-hour preincubation with α-TLR2 Ab (10 μg/mL), α-TLR4 Ab (10 μg/mL), and WRW4 (2 μM). The percent of 7-AAD-negative was assessed by flow cytometry. The data show mean + SEM of three samples representative of three independent experiments. (C) Mo and PMN MDSCs isolated from splenocytes of EL4 tumor-bearing WT or TLR2−/− mice were treated or not with apo-SAA (500 ng/mL) for 24 hour. Cell viability was determined using the CCK-8 assay and data are shown as mean + SEM of three samples representative of three independent experiments. (D) Cell viability in Mo MDSCs of 4T1, EL4, and MC38 tumor-bearing mice was determined using the CCK-8 assay and is shown as mean + SEM of three samples representative of two independent experiments. (E) Mo MDSCs of Her-2/CT26 tumor-bearing mice were nucleofected with 500 nM of siSAA3 or siCtrl according to the Amaxa nucleofection system protocol as detailed in the Materials and methods. mRNA levels of SAA3 were assessed 4 hour after transfection. Cell viability was determined 24 hour after transfection. Data are shown as mean + SEM of three samples representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

Mechanism of increased suppressive activity of Mo MDSCs following apo-SAA treatment To elucidate the mechanism of increased suppressive function of Mo MDSCs by SAA3, we examined the cytokine profiles that are known to mediate these properties in myeloid cells by TLR2 signaling [26, 27]. The secretion levels of IL-6 and TNF-α, but not GM-CSF and IL-1β, were significantly increased in the supernatant of Mo MDSCs treated by apo-SAA (Fig. 6A); this increase was dependent on TLR2 signaling (Fig. 6B). Since IL-6 and TNF-α are known to mediate the activation of STAT3, which could increase the immunosuppressive function of MDSCs, we next examined whether SAA3 induces STAT3 phosphoryla C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

tion. As expected, STAT3 activation was strongly induced in Mo MDSCs, but not in PMN MDSCs, by apo-SAA treatment (Fig. 6C), and this also was mediated by TLR2-dependent manner (Fig. 6D). To investigate which cytokine attributes to the activation of STAT3 induced by SAA treatment, α-IL-6- and α-TNF-α-neutralizing Abs, alone or in combination, were used. We found that, while the α-IL-6 Ab marginally decreased STAT3 activation, the α-TNF-α Ab completely blocked STAT3 activation in Mo MDSCs induced after SAA treatment, suggesting that STAT3 activation is mainly dependent upon TNF-α secretion (Fig. 6E). Finally, to confirm that STAT3 could mediate the increase of the suppressive function of Mo MDSCs induced by SAA, we examined whether STAT3 inhibition reversed these effects. www.eji-journal.eu

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Figure 4. Inhibitory effect of SAA3 on the differentiation of Mo MDSCs is mediated by TLR2. (A–C) Mo and PMN MDSCs of Her-2/CT26 tumor-bearing mice were cultured with apo-SAA (500 ng/mL) in the presence of GM-CSF (20 ng/mL) for 72 hour, including a 1 hour preincubation with α-TLR2 Ab (10 μg/mL), α-TLR4 Ab (10 μg/mL), and WRW4 (2 μM). Ly6C/Ly6G expression gated in CD11b+ cells was assessed by flow cytometry and nuclei and cytoplasm were visualized using Diff-Quick staining (original magnification, ×400). Data are representative of three independent experiments. (B) I-A/I-E expression was assessed by flow cytometry of cells treated as in (A). GM-CSF + vehicle (gray filled), GM-CSF + SAA (solid line) plots are representative of two independent experiments. (C) F4/80 expression in gated Mo (Ly-6Chigh Ly-6Glow CD11b+ ) and PMN MDSCs (Ly-6Clow Ly6Ghigh CD11b+ ) was assessed by flow cytometry using cells treated as in (A). The data are shown as mean + SEM of three samples representative of three independent experiments. **p < 0.01 (Student’s t-test). (D) Mo and PMN MDSCs isolated from splenocytes of B16F10 tumor-bearing WT or TLR2−/− mice were treated with apo-SAA (500 ng/mL) in the presence of GM-CSF (20 ng/mL) for 72 hour. F4/80 in gated Mo (Ly-6Chigh Ly-6Glow CD11b+ ) and PMN MDSCs (Ly-6Clow Ly-6Ghigh CD11b+ ) was assessed by flow cytometry. GM-CSF + vehicle (gray filled), GM-CSF + SAA (solid line). Data are representative of two independent experiments.

STAT3-specific inhibitors, JSI124 and STA21, significantly abrogated the increase of the suppressive function of Mo MDSCs by apo-SAA treatment (Fig. 6F). Especially, STA21 completely abrogated the suppressive function of Mo MDSCs, indicating that the suppressive function of MDSCs is mainly mediated by STAT3 activation regardless of the function of SAA3. Altogether, these data suggest that SAA3-TLR2 signaling increases the autocrine secretion of TNF-α, that triggers the STAT3-dependent suppressive function of Mo MDSCs.

Roles of SAA3 in Mo MDSCs of tumor-bearing mice To identify the roles of SAA3 in Mo MDSCs in vivo, we compared the tumor growth capacity between vehicle- and apo-SAAtreated Mo MDSCs in Her-2/CT26 model. Tumor + SAA-treated Mo MDSCs significantly accelerated tumor growth compared with tumor + vehicle-treated Mo MDSCs, although there was no difference between the tumor alone and tumor + vehicle-treated Mo MDSCs groups, except on day 20 (Fig. 7A). We confirmed this effect in another tumor model, MC38. SAA-treated Mo MDSCs coinjected with MC38 significantly increased tumor growth compared with vehicle-treated Mo MDSCs coinjected with MC38

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(Fig. 7B). These data suggest that SAA3 induction in Mo MDSCs may facilitate tumor growth due to the increased survival and suppressive activity of Mo MDSCs. Next, to elucidate the role of SAA3-TLR2 signaling in vivo, we analyzed the effect of SAA-treated Mo MDSCs from WT or TLR2−/− tumor-bearing mice during MC38 tumor growth. As a result, tumor growth was significantly decreased in mice injected with SAA-treated TLR2−/− Mo MDSC compared with those injected with SAA-treated WT Mo MDSC, suggesting that TLR2 signaling in SAA-treated Mo MDSCs mediate the enhancement of tumor growth (Fig. 7C). To further investigate the effect of endogenous SAA3-TLR2 signaling in tumor-bearing mice, we utilized TLR2−/− mice, albeit indirectly, because SAA3−/− mice were not available. Tumor was s.c. injected into WT or TLR2−/− mice, and Mo MDSCs and PMN MDSCs were examined. Tumor growth was significantly decreased in TLR2−/− mice compared with WT mice (Fig. 7D). Mo MDSCs were significantly decreased among tumor-infiltrating leukocytes in TLR2−/− mice compared with WT mice (Fig. 7E). PMN MDSCs were slightly increased among tumor-infiltrating leukocytes in TLR2−/− mice, but this difference was not significant. In addition, we determined that TLR2−/− Mo MDSCs had a lower suppressive activity compared with WT Mo MDSCs (Fig. 7F). Collectively, these data suggest that TLR2 signaling in vivo would

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Figure 5. SAA3 increases the suppressive activity of Mo MDSCs (A, B). mRNA expression levels of Nos2, Arg1, Nox2, c/EBPα, cEBPβ, and PU.1 were analyzed in Mo MDSCs of Her-2/CT26 tumor-bearing mice treated with (A) apo-SAA (500 ng/mL) for 16 hour and (B) 72 hour after coincubation with GM-CSF (20 ng/mL). *p < 0.05 (Student’s t-test). (C) Total splenocytes (3 × 105 ) from DO11.10 mice were stimulated with 250 μg/mL OVA protein; and serially diluted Mo MDSCs, which were preincubated with vehicle or apo-SAA (500 ng/mL) for 24 hour, were added. After 3 days of culture, T-cell proliferation was determined using an 18 hour pulse with 4μCi/mL 3 H-thymidine. The fractional numbers of the x-axis indicate the relative numbers of diluted MDSCs with regard to the total DO11.10 splenocytes (3 × 105 ). (D) Mo MDSCs from MC38 tumor-bearing mice were incubated with apo-SAA (500 ng/mL) for 24 hour, including a 1-hour preincubation with α-TLR2 Ab (10 μg/mL). Ag-specific T-cell proliferation was examined as described in (C). (E) Mo MDSCs purified from Her-2/CT26 tumor-bearing mice were nucleofected with 500 nM of siSAA3 or siCtrl R nucleofection system. Four hours after siRNA nucleofection, apo-SAA (500 ng/mL) was added for 24 hour. Top panel shows using the Amaxa the mRNA levels of SAA3 after incubation with apo-SAA. After washing, these cells were cocultured with antigen-stimulated DO11.10 cells for 3 days, and 3 H-thymidine incorporation was measured. (F) Mo MDSCs nucleofected with 500 nM of siSAA3 or siCtrl for 24 hour were cocultured with antigen-stimulated DO11.10 cells for 3 days, and 3 H-thymidine incorporation was measured. Top panel shows the mRNA levels of SAA3 after nucleofection. Data in (A-F) are shown as mean + SEM of three samples representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

contribute to increasing the survival and suppressive function of Mo MDSCs.

Discussion The study of myeloid cells could provide new clues about the mechanism by which chronic inflammation influences cancer onset and progression. MDSCs accumulate during cancer, inflammation, and infection and are known to be key suppressors of the immune response. They inhibit both innate and adaptive immunity by producing iNOS, arginase, ROS and IL-10, TCR nitration, cysteine deprivation, and induction of Treg cells [6, 28]. Multiple proinflammatory mediators, such as PGE2 [29], IL-1β [30–32],  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

IL-6 [27, 33], VEGF [34], S100A8/A9 proteins [35, 36], and the complement component C5a [37], secreted by tumor cells or host cells could induce and activate MDSCs. Although a pathological increase in the levels of these factors may be sufficient to induce neoplasia and provide a link between inflammation, MDSCs, and cancer progression, the main factors that regulate the suppressive capacity of MDSCs remain elusive. This study demonstrates a novel role of SAA3, a critical inflammatory mediator, in augmenting the suppressive capacity of Mo MDSCs. We showed that SAA3 enhanced the survival and suppressive activity of Mo MDSCs, while inhibiting their differentiation. In particular, SAA3 specifically affected the Mo subset of the MDSCs through TLR2 signaling, which leads to the secretion of IL-6 and TNF-α and activation of STAT3. Activated STAT3, in turn, was www.eji-journal.eu

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Figure 6. SAA3–TLR2 signaling increases the STAT3-dependent suppressive function of Mo MDSCs through the secretion of IL-6 and TNF-α. (A) Cytokine levels of GM-CSF, IL-1β, IL-6, and TNF-α were analyzed by ELISA in 16 hour supernatant of indicated MDSCs of Her-2/CT26 tumorbearing mice treated with apo-SAA (500 ng/mL). Data are shown as mean + SEM of three samples representative of two independent experiments. (B) IL-6 and TNF-α secretions were analyzed in Mo MDSCs from WT or TLR2−/− mice treated with apo-SAA (500 ng/mL) and are shown as mean + SEM of three samples representative of two independent experiments. (C–E) Protein expression of pSTAT3 and STAT3 was analyzed by Western blotting using Mo and PMN MDSCs of MC38 tumor-bearing mice treated with apo-SAA (500 ng/mL) for (C) the indicated times and (D and E) in Mo MDSCs of MC38 tumor-bearing WT or TLR2−/− mice treated with apo-SAA (500 ng/mL) for 4 hour. For blocking IL-6 and TNF-α cytokines in (E), Mo MDSCs were preincubated with α-IL-6 and/or α-TNF-α neutralizing Abs for 1 hour before apo-SAA treatment. β-Actin was used as a loading control. Blots are representative of two independent experiments. (F) Mo MDSCs of MC38 tumor-bearing mice treated with vehicle, apo-SAA (500 ng/mL), JSI124 (0.25 μM), and STA21 (30 μM) for 24 hour were cocultured with antigen-stimulated DO11.10 cells for 3 days, and 3 H-thymidine incorporation was measured. Data are shown as mean + SEM of three samples representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test).

responsible for enhancing the suppressive function of Mo MDSCs. Furthermore, we showed that SAA3 might enhance the suppressive capacity of Mo MDSCs, thus exacerbating cancer progression in vivo. We also found that endogenous SAA3 expression was specifically increased in Mo MDSCs as the tumor grew and that SAA3 was important for increased survival and suppressive activity of Mo MDSCs, suggesting that the increased immunosuppressive capacity of Mo MDSCs that occurs with tumor growth could be due to SAA3 induction. SAA interacts with at least six distinct receptors, including FPR2 (formyl peptide, like–1, FPRL1/ALX), TLR2, TLR4, CD36 (CLA-1 and LIMPII analogous–1, a human ortholog of rodent scavenger receptor BI), receptor for advanced glycation end products, and Tanis [7]. In addition, SAA binds heparin, heparan sulfate, and certain glycoproteins, although whether these interactions lead to transmembrane signaling remains to be determined [38, 39].

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There is a possibility that SAA3 signaling in Mo MDSCs could be mediated by receptors other than TLR2 because TLR2 blockade significantly, albeit not completely, attenuated the effect of SAA3. However, it seems clear that TLR2 signaling plays a main role in the effects of SAA3 in Mo MDSCs because the apo-SAA treatment in TLR2−/− Mo MDSCs almost completely abrogated the increase of survival (Fig. 3C and D), the defect in differentiation (Fig. 4D), and STAT3 activation (Fig. 6D) compared with WT Mo MDSCs. To the extent of our knowledge, SAA3−/− mice have not been reported thus far. Instead, we investigated the effect of endogenous SAA3-TLR2 signaling in tumor-bearing mice by utilizing TLR2−/− mice (Fig. 7B and C). Tumor-infiltrating Mo MDSCs were significantly decreased, and splenic Mo MDSCs had a lower suppressive activity in TLR2−/− mice compared with WT mice. However, tumor-derived factors, such as Hsp72 and versican, trigger activation of myeloid cells in a TLR2-dependent manner, thus

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Figure 7. The suppressive capacity of Mo MDSCs is increased by SAA3-TLR2 signaling in vivo. (A) Na¨ıve BALB/c mice were s.c. injected with 1 × 105 Her-2/CT26 tumor cells along with 5 × 105 Mo MDSCs of Her-2/CT26 tumor-bearing mice, which were treated with vehicle or apo-SAA (1 μg/mL) for 16 hour. Two days later, Mo MDSCs, which were treated with vehicle or apo-SAA (1 μg/mL) for 16 hour, were i.v. injected into the respective groups. Tumor size was measured three times a week and each symbol represents mean ± SEM of five samples representative of two independent experiments, **p < 0.01, ***p < 0.001 (two-way ANOVA, Bonferroni’s posttest), as compared with Her-2/CT26 + Veh-Mo-treated group. (B and C) Na¨ıve C57BL/6 mice were s.c. injected with 2–3 × 105 MC38 tumor cells along with vehicle or apo-SAA (1 μg/mL) treated 1–2 × 105 Mo MDSCs of WT or TLR2−/− MC38 tumor-bearing mice. Two days later, WT or TLR2−/− Mo MDSCs, which were treated with vehicle or apo-SAA (1 μg/mL) for 16 hour, were i.v. injected into the respective groups. Tumor size was measured three times a week. Each symbol represents mean ± SEM of four to five samples representative of two independent experiments. **p < 0.01 (two-way ANOVA, Bonferroni’s post-test). (D) WT and TLR2−/− mice were s.c. injected with 2 × 105 MC38 tumor cells. Tumor size was measured three times a week. Each symbol represents mean ± SEM of five samples representative of two independent experiments. **p < 0.01, ***p < 0.001 (two-way ANOVA, Bonferroni’s post-test). (E) Cells were isolated from the tumor mass of B16F10 tumor-bearing WT or TLR2−/− mice, and percentages of CD11b+ , Mo MDSCs, and PMN MDSCs in CD11b+ cells were assessed in tumor-infiltrating leukocytes by collagenase treatment. Data are shown as mean + SEM of three samples representative of two independent experiments. (F) Mo and PMN MDSCs isolated from splenocytes of B16F10 tumor-bearing WT or TLR2−/− mice were cocultured with antigen-stimulated DO11.10 cells for 3 days, and [3 H] thymidine incorporation was measured. Data are shown as mean + SEM of three samples representative of two independent experiments. *p < 0.05, **p < 0.01 (Student’s t-test).

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enhancing tumor progression [26, 27]. Hsp72 expressed on tumorderived exosome surface triggers STAT3 activation in MDSCs in a TLR2/Myd88-dependent manner through an autocrine production of IL-6, resulting in restraining tumor immune surveillance by promoting MDSC suppressive functions. Versican secreted by Lewis lung cacinoma cells induces TLR2-mediated myeloid cell activation and TNF-α production, which is necessary for metastatic spreading to the lung, liver, and adrenal gland. Consequently, we cannot exclude the possibility that the effect observed in TLR2−/− mice is not solely dependent on SAA3. In further studies, the relative contributions and interactions among diverse TLR2 ligands in tumor microenvironment need to be examined. A recent study has demonstrated that hepatic acute-phase proteins, including SAA, cooperatively promote MDSC mobilization, accumulation, and survival in a sepsis model [40], suggesting that the positive correlation between SAA and MDSCs can be applied to diverse experimental models. Moreover, SAA3, which is induced in premetastatic lungs by S100A8 and S100A9, contributes to the accumulation of myeloid cells and acts as a positivefeedback regulator for chemoattractant secretion [41]. Therefore, it is possible that other inflammatory factors, including S100A8 and S100A9, contribute to the induction of SAA3 in Mo MDSCs. Also, in this Lewis lung cacinoma model, it is shown that Mac1+ myeloid cells and lung endothelial cells express SAA3. Therefore, there is a possibility that myeloid cells and endothelial cells are the main sources of SAA3 secretion in cancer-bearing mice. Furthermore, TLR2 expression on Mo MDSCs, but not on PMN MDSCs (Supporting Information Fig. 3), was increased with tumor progression, suggesting that SAA3-TLR2 signaling in Mo MDSCs could be dramatically increased as the tumor progresses. Therefore, we suggest that SAA3, which is increasingly secreted by Mo MDSCs with tumor progression, might be a significant contributor to the increased survival and suppressive activity of Mo MDSCs. In further studies, the mechanism by which SAA3 is specifically increased in Mo MDSCs and the main origin of SAA3 secretion needs to be elucidated. In conclusion, the current study demonstrates a causal relationship between the increased production of SAA3 and the immunosuppressive capacity of Mo MDSCs, suggesting that SAA3 may contribute to the Mo MDSC-mediated tumor immune evasion. Our results also provide a novel mechanism by which inflammation augments tumor progression. Taken together, these data suggest that SAA3 could be a promising target for tumor therapy: Targeting SAA3 would not only inhibit inflammation-induced tumor growth, but also regulate MDSC-mediated immune suppression.

Materials and methods Mice BALB/c and C57BL/6 mice at 6 weeks of age were purchased from Charles River Laboratories. OVA-specific CD4+ TCR transgenic DO11.10 breeding pairs were purchased from The  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Cellular immune response

Jackson Laboratory. TLR2−/− mice were obtained from Shizuo Akira (Osaka University, Osaka, Japan). All mice were maintained under specific pathogen-free conditions in the Animal Center for Pharmaceutical Research at Seoul National University, Seoul, South Korea. All experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University.

Reagents All of the in vitro cultures were performed in RPMI culture media supplemented with 10% FBS, 100 μg/mL penicillin/streptomycin, 10 mM HEPES, 1 mM pyruvate, 0.1 mM nonessential amino acids, and 0.05 mM 2-ME. MDSCs were incubated with 500 ng/mL of recombinant human apo-SAA or recombinant human apo-SAA1 (both from Peprotech, Inc., Rocky Hill, NJ) dissolved in PBS. Mo or PMN MDSCs were cultured with 20 ng/mL of recombinant murine GM-CSF (R&D system, Minneapolis, MN) to induce differentiation. To block TLR2, TLR4, or FPR2, MDSCs were preincubated with α-TLR2 Ab, α-TLR4 Ab (10 μg/mL; both from eBioscience, San Diego, CA), or WRW4 (2 μM; Tocris Bioscience, Ellisville, MO) for 1 hour. WRW4 (WRWWWW), which inhibits WKYMVm binding to FPR2, is a selective antagonist of FPR2 signaling. To block STAT3 signaling of MDSCs, JSI124 (0.25 μM; Sigma Aldrich, St. Louis, MO) or STA21 (30μM; Santa Cruz Biotechnology, Santa Cruz, CA) were used.

Cell lines Her-2/CT26 [42], a Her-2/neu expressing transfectoma cell line, and 4T1, a murine mammary carcinoma cell line (ATCC), were used to generate solid tumors in BALB/c mice to produce MDSCs. The murine thymoma cell line EL4, the murine melanoma cell line B16F10, and the murine colorectal adenocarcinoma cell line MC38 (all from ATCC) were also used to form solid tumors in C57BL/6 mice.

Flow cytometry To detect MDSCs, splenocytes from tumor-bearing mice were stained with FITC-conjugated anti-Gr-1 Ab and allophycocyaninconjugated anti-CD11b Ab (both from BioLegend, San Diego, CA). The subsets of MDSCs were analyzed by staining CD11b+ cells with FITC-conjugated anti-Ly6C Ab (BD Bioscience, San Diego, CA) and PE/Cy7-conjugated anti-Ly6G Ab (BioLegend). To examine the diverse cell populations in splenocytes, FITC-conjugated anti-CD3ε Ab and PE/Cy7-conjugated anti-B220 Ab (both from BioLegend) were used. To detect the expression of receptors for SAA3, MDSCs were stained with PE-conjugated anti-TLR2 Ab, anti-TLR4 Ab (both from eBioscience), rabbit anti-mouse FPR2 Ab (Santa Cruz Biotechnology), and PE-conjugated donkey antirabbit Ab (BD Bioscience). To determine the differentiation status of MDSCs, PE-conjugated anti-F4/80 Ab, biotin-conjugated www.eji-journal.eu

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anti-I-A/I-E Ab (BD Bioscience), and PE/Cy7-conjugated streptavidin (BioLegend) were used. The percentage of apoptotic cells was determined by 7-AAD staining (BioLegend), according to the manufacturer’s instructions.

Cytokine ELISA Isolated MDSCs were cultured with indicated reagent for 16 hour, then GM-CSF, IL-1β, IL-6, and TNF-α in the culture supernatants were measured by BD OptEIATM kit (BD Bioscience) according to manufacturer’s instruction.

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Cell survival analysis using the Cell Counting Kit-8 Cell survival analysis was performed using the Cell Counting Kit-8 (CCK-8; Dojindo Technologies, Inc., Rockville, MD) that utilizes a water-soluble tetrazolium salt. Samples were incubated with human SAA or human SAA1 (500 ng/mL) at 37°C in 5% CO2 for 24 hour, and then 10% (v/v) CCK-8 was added. Four hours after the addition of CCK-8, the absorbance at 450 nm was measured using a microplate reader (AT/Spectrafluor plus, Tecan, Austria). Cell viability (%) = [sample (OD) – medium alone (OD)/vehicle group (OD) – medium alone (OD)] × 100.

Ag-specific proliferation assay Western blot Isolated MDSCs were cultured with apo-SAA (500 ng/mL) for 2 or 4 hour, then whole protein lysate was obtained using a lysis buffer (1% Triton X-100 in 50mM Tris-HCl, 150 mM NaCl, 1 mM PMSF, and proteinase inhibitor cocktail). Whole lysates were loaded and electrophoresed on a 10% SDS-PAGE gel. Separated proteins were transferred to PVDF membrane, then Western blotted Abs, using following Abs: anti-rabbit STAT3, anti-rabbit pSTAT3 (pY705), and anti-mouse β-actin (all from Cell Signaling, Inc., MA), were used as primary Abs. HRP-conjugated goat anti-rabbit and goat anti-mouse (both from Santa Cruz Biotechnology) were used as secondary Abs.

Isolation of MDSC subsets To obtain the Mo and PMN MDSC subsets, na¨ıve BALB/c or C57BL/6 mice were s.c. injected with 2 × 105 tumor cells, then spleens from tumor-bearing mice were isolated when the tumor size reached a diameter of 20–25 mm (approximately 4–5 weeks (2–3 weeks) after BALB/c (C57BL/6) background tumor challenge). For each experiment, more than three tumor-bearing mice were used to isolate MDSCs. After depleting the B cells, CD4+ T cells, and CD8+ T cells with antiB220, anti-CD4, and anti-CD8 microbeads, the remaining cells were stained with allophycocyanin-conjugated anti-CD11b Ab, FITC-conjugated anti-Ly6C Ab, and PE/Cy7-conjugated anti-Ly6G Ab. Mo MDSCs (Ly-6Chigh Ly-6Glow CD11b+ ) and PMN MDSCs (Ly-6Clow Ly-6Ghigh CD11b+ ) were sorted using a FACSAriaII (BD Bioscience).

Morphological analysis MDSC subsets were cytocentrifuged onto precoated microscope slides (Thermo Shandon, Pittsburgh, PA; 400 cells/slide), followed by staining with Diff-Quick (DADE Behring, Dudingen, Switzerland). Images were obtained using a Nikon Eclipse E600 microscope (Nikon, Melville, NY).  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Total splenocytes (3 × 105 ) from na¨ıve DO11.10 mice were stimulated with 250 μg/mL OVA protein and 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256 of 3 × 105 Mo MDSCs were added in vitro. After 3 days of culture, T-cell proliferation was determined using an 18 hour pulse with 4 μCi/mL [3 H] thymidine.

siRNA nucleofection We used a commercially available siRNA composed of a heterogeneous mixture that all target mouse SAA3 sequences (siSAA3; R MISSION esiRNA, Sigma Aldrich) or control siRNA that contained a scrambled sequence that should not lead to the specific degradation of any known cellular mRNA (Santa Cruz Biotechnology). To knock down a target gene, MDSCs were nucleofected with 500 nM siSAA3 or siCtrl using Amaxa nucleofection system (Lonza, Germany).

Quantitative real-time PCR Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA) and then reverse-transcribed using M-MLV RT (Invitrogen, Carlsbad, CA). Quantitative real-time PCR was performed using LightCycler optical system (Roche, California, CA) and SYBR Premix Ex Taq (Takara, Japan). The expression levels of the target genes were calculated relative to hprt expression. The following primers were used: Saa1 sense (CAT TTG TTC ACG AGG CTT TCC); Saa1 antisense (GTT TTT CCA GTT AGC TTC CTT CAT GT); Saa2 sense (TGT GTA TCC CAC AAG GTT TCA GA); Saa2 antisense (TTA TTA CCC TCT CCT CCT CAA GCA); Saa3 sense (CGC AGC ACG AGC AGG AT); Saa3 antisense (CCA GGA TCA AGA TGC AAA GAA TG); iNos sense (AGG AAG TGG GCC GAA GGA); iNos antisense (GAA ACT ATG GAG CAC AGC CAC AT); Arg1 sense (AAC ACG GCA GTG GCT TTA ACC); Arg1 antisense (GTG ATG CCC CAG ATG GTT TTC); Nox2 sense (GAC CCA GAT GCA GGA AAG GAA); Nox2 antisense (TCA TGG TGC ACA GCA AAG TGA); Cebpα sense (CCC CAG TCA GAC CAG AAA GC); Cebpα antisense (TGG TCC CCG TGT CCT CCT A); Cebpβ sense (TGC AAT CCG GAT CAA ACG T); Cebpβ antisense (AAC CCC GCA GGA ACA www.eji-journal.eu

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TCT TT); Pu.1 sense (GCC TCA GTC ACC AGG TTT CC); Pu.1 antisense(CTC TCA CCC TCC TCC TCA TCT G); Hprt sense (AAG ACT TGC TCG AGA TGT CAT GAA); and Hprt antisense (ATC CAG CAG GTC AGC AAA GAA).

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4 Porta, C., Riboldi, E. and Sica, A., Mechanisms linking pathogensassociated inflammation and cancer. Cancer Lett. 2011. 305: 250–262. 5 Vakkila, J. and Lotze, M. T., Inflammation and necrosis promote tumour growth. Nat. Rev. Immunol. 2004. 4: 641–648. 6 Ostrand-Rosenberg, S. and Sinha, P., Myeloid-derived suppressor cells: linking inflammation and cancer. J. Immunol. 2009. 182: 4499–4506.

Tumor model

7 Malle, E., Sodin-Semrl, S. and Kovacevic, A., Serum amyloid A: an acutephase protein involved in tumour pathogenesis. Cell. Mol. Life. Sci. 2009.

To identify the roles of SAA3 in Mo MDSCs in vivo, na¨ıve BALB/c or C57BL/6 mice were s.c. injected with Her-2/CT26 or MC38 tumor cells and WT or TLR2−/− Mo MDSCs that were treated with vehicle or apo-SAA (1 μg/mL) for 16 hour. Two days later, the vehicle or apo-SAA treated Mo MDSCs of the WT- or TLR2−/− mice were i.v. injected, respectively. To investigate the effect of endogenous SAA3-TLR2 signaling in tumor-bearing mice, we used TLR2−/− mice. MC38 tumor cells were s.c. injected into WT or TLR2−/− mice, and tumor size was measured three times a week (n = 4–5).

66: 9–26. 8 Vlasova, M. A. and Moshkovskii, S. A., Molecular interactions of acute phase serum amyloid A: possible involvement in carcinogenesis. Biochemistry (Mosc.) 2006. 71: 1051–1059. 9 Uhlar, C. M. and Whitehead, A. S., Serum amyloid A, the major vertebrate acute-phase reactant. Eur. J. Biochem. 1999. 265: 501–523. 10 Jensen, L. E. and Whitehead, A. S., Regulation of serum amyloid A protein expression during the acute-phase response. Biochem. J. 1998. 334(Pt 3): 489–503. 11 Kumon, Y., Suehiro, T., Hashimoto, K., Nakatani, K. and Sipe, J. D., Local expression of acute phase serum amyloid A mRNA in rheumatoid arthritis synovial tissue and cells. J. Rheumatol. 1999. 26: 785–790.

Statistical analysis

12 Cunnane, G., Amyloid precursors and amyloidosis in inflammatory arthritis. Curr. Opin. Rheumatol. 2001. 13: 67–73.

To compare the differences between two groups, Student’s t-test was used. To compare multiple groups, we carried out two-way ANOVA, followed by the Bonferroni’s posttest.

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Acknowledgments: This study was supported by Public Welfare & Safety Research program (20110020963) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology; National R&D Program for Cancer Control (0720500) funded by the Ministry of Health & Welfare; and BK21 Plus Program (10220130000017) of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology. We thank Dr. Shizuo Akira (Osaka University, Osaka, Japan), Dr. Seung-Yong Seong (Seoul National University, Seoul, Korea), and Eun-Kyeong Jo (Chungnam National University, Daejeon, Korea) for providing TLR2−/− mice.

Y., Matsudo, T. et al., Significance of serum amyloid A on the prognosis in patients with renal cell carcinoma. Cancer 2001. 92: 2072–2075. 16 Yokoi, K., Shih, L. C., Kobayashi, R., Koomen, J., Hawke, D., Li, D., Hamilton, S. R. et al., Serum amyloid A as a tumor marker in sera of nude mice with orthotopic human pancreatic cancer and in plasma of patients with pancreatic cancer. Int. J. Oncol. 2005. 27: 1361–1369. 17 Kristiansson, M. H., Bhat, V. B., Babu, I. R., Wishnok, J. S. and Tannenbaum, S. R., Comparative time-dependent analysis of potential inflammation biomarkers in lymphoma-bearing SJL mice. J. Proteome Res. 2007. 6: 1735–1744. 18 Juan, H. F., Chen, J. H., Hsu, W. T., Huang, S. C., Chen, S. T., Yi-Chung Lin, J., Chang, Y. W. et al., Identification of tumor-associated plasma biomarkers using proteomic techniques: from mouse to human. Proteomics 2004. 4: 2766–2775. 19 Liu, D. H., Wang, X. M., Zhang, L. J., Dai, S. W., Liu, L. Y., Liu, J. F., Wu, S. S. et al., Serum amyloid A protein: a potential biomarker correlated with

Conflict of interest: The authors declare no financial or commercial conflict of interest.

clinical stage of lung cancer. Biomed. Environ. Sci. 2007. 20: 33–40. 20 Cho, W. C., Yip, T. T., Yip, C., Yip, V., Thulasiraman, V., Ngan, R. K., Lau, W. H. et al., Identification of serum amyloid a protein as a potentially useful biomarker to monitor relapse of nasopharyngeal cancer by serum

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Abbreviations: CCK-8: Cell Counting Kit-8 · FPR2: formyl peptide receptor

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 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: 3/7/2013 Revised: 5/2/2014 Accepted: 14/3/2014 Accepted article online: 22/3/2014

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