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Cross-talk among myeloid-derived suppressor cells, macrophages, and tumor cells impacts the inflammatory milieu of solid tumors.

Author information

Affiliations

  • 1. Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, Maryland, USA.
      Authors
    • Beury DW 1
    • Parker KH 1
    • Nyandjo M 1
    • Sinha P 1
    • Carter KA 1
    (5 authors)
  • 2. Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, Maryland, USA
      Authors
    • Ostrand-Rosenberg S 2
    (1 author)

ORCIDs linked to this article

Journal of Leukocyte Biology, 28 Aug 2014, 96(6):1109-1118
https://doi.org/10.1189/jlb.3a0414-210r PMID: 25170116 PMCID: PMC4226789

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Abstract 

MDSC and macrophages are present in most solid tumors and are important drivers of immune suppression and inflammation. It is established that cross-talk between MDSC and macrophages impacts anti-tumor immunity; however, interactions between tumor cells and MDSC or macrophages are less well studied. To examine potential interactions between these cells, we studied the impact of MDSC, macrophages, and four murine tumor cell lines on each other, both in vitro and in vivo. We focused on IL-6, IL-10, IL-12, TNF-α, and NO, as these molecules are produced by macrophages, MDSC, and many tumor cells; are present in most solid tumors; and regulate inflammation. In vitro studies demonstrated that MDSC-produced IL-10 decreased macrophage IL-6 and TNF-α and increased NO. IL-6 indirectly regulated MDSC IL-10. Tumor cells increased MDSC IL-6 and vice versa. Tumor cells also increased macrophage IL-6 and NO and decreased macrophage TNF-α. Tumor cell-driven macrophage IL-6 was reduced by MDSC, and tumor cells and MDSC enhanced macrophage NO. In vivo analysis of solid tumors identified IL-6 and IL-10 as the dominant cytokines and demonstrated that these molecules were produced predominantly by stromal cells. These results suggest that inflammation within solid tumors is regulated by the ratio of tumor cells to MDSC and macrophages and that interactions of these cells have the potential to alter significantly the inflammatory milieu within the tumor microenvironment.

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J Leukoc Biol. 2014 Dec; 96(6): 1109–1118.
PMCID: PMC4226789
PMID: 25170116

Cross-talk among myeloid-derived suppressor cells, macrophages, and tumor cells impacts the inflammatory milieu of solid tumors

Daniel W. Beury, Katherine H. Parker, Maeva Nyandjo, Pratima Sinha, Kayla A. Carter, and Suzanne Ostrand-Rosenberg1
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Abstract

MDSC and macrophages are present in most solid tumors and are important drivers of immune suppression and inflammation. It is established that cross-talk between MDSC and macrophages impacts anti-tumor immunity; however, interactions between tumor cells and MDSC or macrophages are less well studied. To examine potential interactions between these cells, we studied the impact of MDSC, macrophages, and four murine tumor cell lines on each other, both in vitro and in vivo. We focused on IL-6, IL-10, IL-12, TNF-α, and NO, as these molecules are produced by macrophages, MDSC, and many tumor cells; are present in most solid tumors; and regulate inflammation. In vitro studies demonstrated that MDSC-produced IL-10 decreased macrophage IL-6 and TNF-α and increased NO. IL-6 indirectly regulated MDSC IL-10. Tumor cells increased MDSC IL-6 and vice versa. Tumor cells also increased macrophage IL-6 and NO and decreased macrophage TNF-α. Tumor cell-driven macrophage IL-6 was reduced by MDSC, and tumor cells and MDSC enhanced macrophage NO. In vivo analysis of solid tumors identified IL-6 and IL-10 as the dominant cytokines and demonstrated that these molecules were produced predominantly by stromal cells. These results suggest that inflammation within solid tumors is regulated by the ratio of tumor cells to MDSC and macrophages and that interactions of these cells have the potential to alter significantly the inflammatory milieu within the tumor microenvironment.

Keywords: cancer, cytokines, tumor microenvironment, IL-6, IL-10, nitric oxide

Introduction

Solid tumors are a complex and frequently inflamed environment. The inflammation is driven by proinflammatory mediators that are secreted by tumor cells, various tumor-infiltrating lymphocytes, tumor-associated fibroblasts, and myeloid cells, such as macrophages, dendritic cells, and MDSC [1]. Some of these cells engage in cross-talk with each other, resulting in the release of proinflammatory cytokines (e.g., IL-1, IL-6, IL-17, TNF-α), chemokines (e.g., CCL2, CXCL5, CXCL12), growth factors (e.g., TGF-β, GM-CSF, VEGF), and other effector molecules (e.g., S100A8/A9, high-mobility group box 1) [2,–4]. These factors, in turn, induce the accumulation and enhance the function of immune-suppressive cells, such as regulatory T cells, plasmacytoid dendritic cells, tumor-associated macrophages, and MDSC [3, 5, 6]. Although the cellular interactions contributing to some of the protumor factors present in the tumor microenvironment have been identified, the etiology of others remains unknown.

Macrophages and MDSC are present within most solid tumors, where they are major drivers of immune suppression and inflammation [3]. We have reported previously that these cells participate in cross-talk with each other that results in increased MDSC production of IL-10 and decreased macrophage production of IL-12, thereby polarizing the immune system toward a protumor type 2 environment [7, 8]. Additional factors are also likely to be impacted by cross-talk between MDSC and macrophages, as well as by interactions with tumor cells. Therefore, we have investigated how tumor cells, macrophages, and MDSC interact with respect to IL-6, TNF-α, IL-10, and NO. We have focused on these four molecules, as they are chronically present in many solid tumors and play important roles in tumor progression. IL-6 promotes tumor progression by enhancing tumor cell development, growth, and metastasis and by inhibiting apoptosis and enhancing tumor vascularization [9,–11]. TNF-α causes DNA damage, inhibits apoptosis, and induces the production of matrix metalloproteases, cytokines, and chemokines that facilitate tumor cell invasion and metastasis [12]. In contrast to IL-6 and TNF-α, which when chronically present, are exclusively protumor, NO can have pro- and anti-tumor activity. When produced by M1-like macrophages, NO induces tumor cell apoptosis [13]. However, when produced by MDSC, NO drives immune suppression [14]. IL-10 has also been associated with pro- and anti-tumor activity [15]. Here, we report that macrophages, MDSC, and tumor cells participate in a network of cross-talk, resulting in differential production of IL-6, IL-10, TNF-α, and NO, suggesting that the interaction of these cells has the potential to alter significantly the inflammatory milieu within the tumor microenvironment.

MATERIALS AND METHODS

Mice, tumor cells, tumor growth

BALB/c, C57BL/6, BALB/c IL-6−/−, and BALB/c IL-10−/− mice were bred in the UMBC animal facility from stock obtained from The Jackson Laboratory (Bar Harbor, ME, USA; C57BL/6 and BALB/c) or provided by Dr. Manfred Kopf (Zürich, Switzerland; IL-6−/−). BALB/c-derived 4T1 and TS/A mammary carcinomas, CT26 colon carcinoma, and C57BL/6-derived MC38 colon carcinoma were maintained as described [16]. Mice were inoculated in the abdominal mammary gland with 100 μl DMEM containing 7 × 103 (WT and IL-10−/−mice) or 105 (WT and IL-6−/− mice) 4T1 cells or 106 TS/A cells or s.c. in the flank with 5 × 105, 1 × 105, or 1 × 104 CT26 cells. Primary tumors were measured as described [17]. Survival time was recorded when mice became moribund and were euthanized. All animal procedures were approved by the UMBC Institutional Animal Care and Use Committee.

Flow cytometry and antibodies

Gr1-FITC, Gr1-allophycocyanin, Ly6C-FITC, Ly6G-PB, CD11b-PE, CD11b-PB, F4/80-allophycocyanin, F4/80-PB, pSTAT3-PB, IL-6R-PE, and IL-10R-PE mAb and rat IgG1-PE and IgG2b-PE isotypes were from BD PharMingen (San Diego, CA, USA) or BioLegend (San Diego, CA, USA). Cells were stained for surface markers as described [18]. For phosphoflow experiments, cells were stimulated with 50 ng/mL rIL-10 (BioLegend) or supernatants from MDSC and macrophage cocultures, fixed with Lyse/Fix Buffer (BD Biosciences, San Jose, CA, USA), permeabilized with Perm Buffer III (BD Biosciences), and stained with antibodies diluted in Stain Buffer (BD Biosciences). Samples were analyzed on a Beckman/Coulter CyAn ADP flow cytometer using Summit software.

T Cell proliferation assays

CD4+ and CD8+ T cell proliferation assays were performed as described [18]. Briefly, DO11.10 (ovalbumin peptide323–339-specific, I-Ad-restricted) or Clone 4 (hemagglutinin peptide518–526-specific, H-2Kd-restricted) splenocytes were cultured with their respective cognate peptides and irradiated blood MDSC from 4T1-bearing WT, IL-6−/−, or IL-10−/− mice. Cultures were pulsed with 3H-thymidine on Day 4 and harvested on Day 5. Peptides were synthesized at the UMB Biopolymer Core Facility.

MDSC, macrophage, MDSC-macrophage-tumor cell cross-talk

MDSC were isolated from the peripheral blood of 4T1 tumor-bearing mice [16]. Peritoneal macrophages were prepared from tumor-free mice [8]. MDSC and macrophages in all experiments were >90% Gr1+CD11b+ cells and >95% CD11b+ F4/80+ cells, respectively, as assessed by flow cytometry. MDSC and macrophage cross-talk experiments were performed as described [7] with the following modifications: 4T1, MC38, TS/A, or CT26 tumor cells (1×105 cells) were cultured with or without 7.5 × 105 MDSC and/or macrophages in 500 μl macrophage media (5% FCS in DMEM, 1% penicillin-streptomycin, 1% glutamax, 0.1% gentamycin) for 16 h at 37°C with 100 ng/mL LPS (Difco Laboratories, Franklin Lakes, NJ, USA) and 20 U/mL IFN-γ (R&D Systems, Minneapolis, MN, USA). In some experiments, macrophages and/or MDSC were cultured with LPS, IFN-γ, rIL-6 and rIL-10 (both from BioLegend) and IL-10 that was denatured by boiling at 95°C for 15 min or in the presence of neutralizing antibodies to IL-10 (1 μg/ml; Clone JES5-2A5; eBioscience, San Diego, CA, USA). Cells were harvested by scraping and analyzed by flow cytometry. Supernatants were analyzed for IL-10, IL-6, and TNF-α using ELISA kits (R&D Systems and eBioscience), per the manufacturers' protocol, or by multiplex analysis in the UMB Cytokine Core Facility. NO production was quantified by Griess assay [18]. Values were normalized between experiments using the following formulas:

  • production of IL-6 by macrophages or MDSC in response to tumor cells = (IL-6 from WT MDSC or macrophages with tumor cells) − (IL-6 from IL-6−/− macrophages or MDSC with tumor cells)

  • percent increase in IL-6 or NO by MDSC or macrophages in response to tumor cells = {[(IL-6 or NO from macrophages and MDSC±tumor cells)/(IL-6 or NO from macrophages or MDSC)]×100%} − 100%

  • percent decrease in IL-6 or TNF-α by macrophages in response to tumor cells and/or MDSC = 1 − [(IL-6 or TNF-α from macrophages±tumor cells)/(IL-6 or TNF-α from WT macrophages±tumor cells±MDSC)] × 100%

  • percent increase in IL-10 by MDSC in response to macrophages = {[(IL-10 from macrophages+MDSC)/(IL-10 from MDSC)]×100%} − 100%

If IL-6 was not detected, then the lowest value detectable on the standard curve was used for the calculations.

Macrophages and MDSC were stained with 5 μM CellTrace Violet (Life Technologies, Carlsbad, CA, USA) and 4T1 tumor cells with 1 μM CFSE (Life Technologies). MDSC or macrophages were cultured for 16 h in macrophage media with 100 ng/mL LPS and 20 U/mL IFN-γ in a six-well dish at 3 × 106 cells/well/2 mL, with or without 4 × 105 4T1 cells. Cells were then harvested using Detachin (Genlantis, San Diego, CA, USA) and scraping, washed, and stained for Gr1, CD11b, and with 7-amino-actinomycin D; and analyzed by flow cytometry.

Ex vivo tumor cultures

4T1, CT26, and TS/A tumors >8 mm in diameter were surgically resected from euthanized mice and placed on sterile #50 Whatman filter paper to remove excess liquid. The tumors were then transferred to 6 cm culture dishes and finely minced using a sterile scalpel, and the resulting pieces weighed. 4T1 and TS/A pieces were resuspended in 5 mL prewarmed 4T1 media (10% Fetal Clone I in IMDM, 1% penicillin-streptomycin, 1% glutamax, 0.1% gentamycin) containing 100 ng/mL LPS and 20 U/mL IFN-γ for IL-10 studies or without LPS and IFN-γ for IL-6 studies. Resuspended tumor pieces were incubated for 16 h at 37°C, 5% CO2, and supernatants were analyzed for cytokine production by ELISA. Cytokine levels were normalized to one gram of tumor tissue/mL media using the following formula: cytokine production (normalized) = cytokine (pg/mL) × [(tumor weight/1 g)×5 mL].

Statistical analyses

Student's t-test and Tukey's HSD test were performed using Microsoft Excel 2013. Values denoted with different letters (e.g., a, b, c, etc.) are significantly different from each other; values with the same letter are not significantly different. Tumor growth and exogenous IL-10 data were analyzed using the Mann-Whitney test on the VassarStats website (www.VassarStats.net). Survival data were analyzed using the log-rank test from the Walter and Eliza Hall Institute of Medical Research Bioinformatics webpage (http://bioinf.wehi.edu.au/software/russell/logrank/). Values of P < 0.05 were considered statistically significant.

RESULTS

IL-6 and IL-10 promote tumor progression

Increased levels of serum IL-6 are correlated with chronic inflammation, increased tumor burden, and poor prognosis in some human and mouse systems [19]. IL-6 also promotes MDSC-mediated inhibition of Th1 responses in mice [20]. In contrast, IL-10 correlates with tumor progression in some systems but with tumor regression in other systems [15, 21,–25]. To determine if IL-6 and/or IL-10 contribute to progression of the 4T1 mammary carcinoma or CT26 colon carcinoma, we inoculated syngeneic WT, IL-6−/−, and IL-10−/− mice with 4T1 (Fig. 1A) or CT26 (Fig. 1B) tumor cells and followed the mice for tumor onset, growth, and engraftment. In the absence of host-produced IL-6, 4T1 tumor progression was delayed, and survival time was increased. IL-10−/− mice showed a similar, although less dramatic, delay in tumor progression and extension of survival time. 4T1 tumor engraftment in WT BALB/c and IL-10−/− mice was 90–100%, whereas only 40% of IL-6−/− mice developed tumor. Tumor progression was also delayed, and survival time increased in IL-6−/− mice with CT26 tumors. In contrast, IL-10−/− mice inoculated with 5 × 105 CT26 tumor cells had similar tumor progression, survival time, and percent engraftment as WT mice. Tumor progression, survival time, and engraftment were also similar in WT and IL-10−/− BALB/c mice inoculated with 1 × 105 or 1 × 104 cells (Supplemental Fig. 1A). These results demonstrate that stromal cell-derived IL-6 and IL-10 facilitate progression of 4T1 and CT26 tumors in their syngeneic hosts.

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IL-6 and IL-10 produced by host cells enhance primary tumor growth and decrease survival time.

WT, IL-6−/−, and IL-10−/− BALB/c mice were inoculated with (A) 4T1 or (B) CT26 tumor cells and monitored for tumor diameter, survival, and tumor engraftment. Mice in the WT versus IL-6−/− graphs and WT versus IL-10−/− graphs (tumor diameter and percent survival) were inoculated with 1 × 105 and 7000 4T1 cells, respectively. Mice in the engraftment graph were inoculated with 1 × 105 4T1 cells. All CT26 inoculations were 5 × 105 cells. For tumor engraftment, n = 7 for each 4T1 group; n = 6 for each CT26 group. Statistical significance was tested by Mann-Whitney (tumor growth) or log-rank test (survival). Data are pooled from three independent experiments.

MDSC production of IL-10 decreases macrophage IL-6 and TNF-α and increases NO; IL-6 indirectly regulates MDSC production of IL-10

We have shown previously that MDSC production of IL-10 is enhanced by cross-talk with macrophages and polarizes macrophages toward a tumor-promoting phenotype by inhibiting macrophage production of IL-12 [7, 8]. To determine if IL-10 produced by MDSC impacts the production of additional proinflammatory mediators, we cocultured CD11b+F4/80+ peritoneal macrophages and 4T1-induced Gr1+CD11b+ immune-suppressive MDSC (Fig. 2A) and assayed the supernatants for IL-10 and the proinflammatory cytokine IL-6 (Fig. 2B). Consistent with our previous reports, production of IL-10 was increased significantly in the presence of macrophages (average increase in IL-10 of 116±19.4% for 30 experiments). IL-10 was produced exclusively by MDSC, as macrophage cultures containing IL-10−/− MDSC produced no IL-10. In the same cocultures, macrophages were the sole producers of IL-6, and MDSC decreased macrophage IL-6 (average decrease in IL-6 of 24±3.8% for 30 experiments).

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Cross-talk between MDSC and macrophages regulates production of IL-10, IL-6, and NO.

(A) Peritoneal macrophages from healthy mice and MDSC from tumor-bearing mice were stained with mAb to CD11b, F4/80, Gr1, Ly6C, and/or Ly6G and analyzed by flow cytometry. MDSC from WT, IL-10−/−, and IL-6−/− BALB/c mice with 4T1 tumors were assayed for their ability to suppress the antigen-driven activation of peptide-specific, MHC-restricted, transgenic CD4+ (DO11.10) and CD8+ (Clone 4) T cells. (B–D) 4T1-induced MDSC and peritoneal macrophages (Mac) from WT, IL-10−/− (10−/−), or IL-6−/− (6−/−) BALB/c mice were cocultured, and supernatants were assayed for IL-10, IL-6, and NO. (B) Macrophages enhance MDSC IL-10, and MDSC decrease macrophage IL-6. (C) IL-6 decreases MDSC IL-10. (D) IL-10 production by MDSC decreases macrophage IL-6 and TNF-α and increases macrophage NO. (E) Neutralizing antibodies to IL-10 prevent the down-regulation of macrophage IL-6 and IL-12. (F) Exogenous IL-10 decreases MDSC and macrophage TNF-α, decreases macrophage IL-6, and enhances macrophage NO. Macrophages or MDSC were cultured with IL-10 or denatured IL-10. (G) Macrophages activate STAT3 in response to IL-10. Macrophages (left) were cultured for 5 min in the presence or absence of exogenous IL-10 (middle) or with supernatants (media) from MDSC-macrophage cocultures (right), subsequently fixed and permeabilized, and then stained for F4/80, CD11b, and pSTAT3. (H) Macrophages and MDSC express the receptors for IL-10 and IL-6, respectively. (A–H) Data are from one of two, 30, three, five, four, three, two, and two experiments, respectively. Statistical significance was determined by (A–E) Tukey's HSD test and (F) the Mann-Whitney test. Different lower case letters above each value indicate that those values are statistically, significantly different; values that share the same lowercase letter are not statistically, significantly different.

To determine if IL-6 regulates MDSC production of IL-10, we cocultured WT or IL-6−/− macrophages with WT or IL-6−/− MDSC (Fig. 2C). IL-6−/− MDSC produced significantly more IL-10 than WT MDSC. Macrophage cocultures with IL-6−/− MDSC had significantly more IL-10 than cocultures with WT MDSC. Macrophage IL-6 had no effect on MDSC IL-10, as WT MDSC cocultured with WT or IL-6−/− macrophages produced similar amounts of IL-10. The lack of a direct effect by IL-6 on MDSC IL-10 was confirmed by incubation of MDSC with exogenous IL-6 (Supplemental Fig. 1B). These results indicate that MDSC do not produce IL-6 in the coculture setting; however, their development in vivo in the presence of IL-6 down-regulates their production of IL-10.

To determine if IL-10 produced by MDSC decreased macrophage IL-6 or regulated other molecules characteristic of tumor-rejecting M1 macrophages, WT or IL-10−/− MDSC were cocultured with WT macrophages (Fig. 2D). There was no decrease in IL-6 in the presence of IL-10−/− MDSC, suggesting that IL-10 from WT MDSC reduced macrophage IL-6. To confirm the role of IL-10, neutralizing antibodies to IL-10 were added to MDSC-macrophage cocultures. As previous studies demonstrated that MDSC IL-10 also decreases macrophage IL-12 [8], IL-12 levels served as a positive control (Fig. 2E). IL-10 neutralizing antibodies reduced the MDSC-mediated decrease of IL-6 and IL-12. Thus, a feedback loop exists between macrophages and MDSC, in which macrophages increase MDSC production of IL-10, and MDSC IL-10 regulates macrophage synthesis of IL-6.

We also assessed the role of MDSC IL-10 on macrophage NO and TNF-α production (Fig. 2D). MDSC IL-10 decreased TNF-α in the cocultures; however, this decrease was minimal. In contrast, macrophage production of NO was increased by coculture with MDSC. The increase was predominantly a result of MDSC IL-10, as only a minimal increase in NO was observed in the presence of IL-10−/− MDSC.

To confirm further that IL-10 regulated macrophage production of IL-6 and NO, and macrophage and MDSC production of TNF-α, macrophages or MDSC were cultured in the presence of exogenous IL-10, and culture supernatants were assessed for TNF-α, IL-6, and NO (Fig. 2F). Exogenous IL-10 reduced MDSC and macrophage TNF-α and macrophage IL-6 but increased macrophage NO. As STAT3 is activated by signaling through IL-10R, macrophages were cultured with exogenous IL-10 or with supernatants from MDSC-macrophage cocultures and subsequently stained for phosphorylated STAT3 (Fig. 2G). STAT3 was phosphorylated under both conditions, further confirming the regulatory role of IL-10 produced by MDSC.

MDSC and macrophages express IL-6R and IL-10R, respectively (Fig. 2H), so these cells have the potential to respond directly to these cytokines. The results of Fig. 2F suggest that IL-10 directly impacts macrophages. However, IL-10-deficiency and IL-6-deficiency could also cause other changes in MDSCs and/or macrophages so that the effects are only mediated indirectly by IL-10 or IL-6. To distinguish these possibilities, we compared cytokine/chemokine production by WT, IL-10−/−, and IL-6−/− MDSC to determine if gene deficiency impacts MDSC phenotype (Supplemental Table 1). TGF-β3, GM-CSF, IL-4, IL-13, and IL-23 were not detectable in WT MDSC. TGF-β2, IL-1β, CCL2, and VEGF production was similar for WT, IL-10−/−, and IL-6−/− MDSC. TGF-β1 trended higher in IL-10−/− and IL-6−/− MDSC, and MIP-1α trended lower in IL-10−/− and IL-6−/− MDSC compared with WT MDSC. These results suggest that IL-10-deficiency and IL-6-deficiency may alter the phenotype of MDSC.

These results, together with our earlier studies on IL-12 [7, 8], demonstrate that MDSC production of IL-10 increases some M2-like characteristics of macrophages (i.e., IL-12lowIL-6low) but also increases some M1-like properties (NOhigh).

Other cytokines are also impacted by interactions between MDSC and macrophages

In addition to IL-10, TNF-α, IL-12, NO, and IL-6, other immune-regulatory molecules are present in solid tumors. Of particular note are cytokines that drive effector and regulatory T cells (e.g., IL-23, IL-27, IL-4, and IL-13), growth factors that regulate neovascularization (e.g., VEGF) and myeloid cell differentiation (e.g., GM-CSF), proinflammatory mediators (e.g., IL-1β), and immune-suppressive molecules (e.g., TGF-β). To determine if any of these molecules are affected by cross-talk between MDSC and macrophages, supernatants from cocultures of 4T1-induced WT MDSC and WT BALB/c macrophages were assayed by multiplex analysis (Supplemental Table 1). Neither MDSC nor macrophages produced TGF-β3, GM-CSF, IL-4, IL-13, or IL-23, whereas both cell types produced TGF-β1, TGF-β2, IL-1β, CCL2, MIP-1α, and VEGF. Cocultures using WT MDSC reduced the production of TGF-β1, TGF-β2, and MIP-1α and modestly increased the production of VEGF. Cocultures of WT macrophages with IL-10−/− or IL-6−/− MDSC displayed similar trends, except for CCL2, where we observed a decrease in CCL2 production.

Tumor cells increase MDSC production of IL-6 and vice versa

Tumor cells produce proinflammatory mediators and therefore, may contribute to the polarization of myeloid cells in the tumor microenvironment. To assess if there is cross-talk between MDSC and tumor cells, 4T1, CT26, TS/A, or MC38 murine tumor cells were cultured by themselves or cocultured with MDSC (Fig. 3). When cultured alone, 4T1 and CT26 cells produced IL-6, and TS/A, MC38, and MDSC produced no detectable IL-6. Cultures containing WT MDSC plus 4T1, CT26, TS/A, or MC38 tumor cells contained more IL-6 than cultures of tumor cells alone, whereas cultures of 4T1, CT26, and TS/A tumor cells plus IL-6−/− MDSC produced intermediate levels of IL-6. Cultures of MC38 tumor cells plus IL-6−/− MDSC produced very low levels of IL-6. Increases in IL-6 production in the presence of IL-6−/− MDSC indicate that in vitro, MDSC enhanced tumor cell production of IL-6. However, as IL-6 levels in cocultures of WT MDSC plus tumor cells were even higher than IL-6 production in cocultures with IL-6−/− MDSC, MDSC may also be induced by tumor cells to synthesize IL-6. Interestingly, MDSC, but not tumor cells, proliferated during the overnight culture (Supplemental Fig. 1C), so the increase in IL-6 in this setting could be a result of higher numbers of MDSC. In contrast, tumor cells did not impact MDSC production of TNF-α, IL-12, or IL-10 (Supplemental Fig. 2). These results demonstrate that in vitro, reciprocal cross-talk between MDSC and most tumor cells increases IL-6 production, and there is no cross-talk between MDSC and tumor cells with respect to IL-10, IL-12, or TNF-α.

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Tumor cells induce MDSC to produce IL-6 and vice versa.

WT or IL-6−/− 4T1-induced MDSC were cultured with or without 4T1, CT26, TS/A, or MC38 tumor cells, and the supernatants were assayed for IL-6 by ELISA. One of three independent experiments (left four graphs); average percent increase of three independent experiments comparing tumor cells with IL-6−/− and WT MDSC (right two graphs). Statistical significance for the independent experiments was determined by Tukey's HSD test.

Tumor cells increase macrophage IL-6 and NO and decrease macrophage TNF-α

To assess if there is cross-talk between macrophages and tumor cells, 4T1, CT26, TS/A, or MC38 tumor cells were cultured with macrophages and the culture supernatants assayed for IL-6, NO, and TNF-α (Fig. 4). All four tumor lines increased macrophage production of IL-6. Macrophages also increased IL-6 produced by 4T1, CT26, and TS/A tumor cells, as cultures containing tumor cells plus IL-6−/− macrophages produced more IL-6 than tumor cells alone. In cocultures of WT or IL-6−/− macrophages with 4T1, TS/A, or MC38 tumor cells, macrophages were the dominant producers of IL-6 (Fig. 4A). In contrast, tumor cells were the dominant producers of IL-6 in cultures of macrophages plus CT26 tumor cells, indicating that some tumor cells have a greater response to macrophages. Cross-talk-induced increases in IL-6 ranged from 43% to 230%. These results indicate that tumor cell production of IL-6 is differentially affected by macrophages and that macrophages produce IL-6 in response to tumor cells.

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Tumor cells induce macrophages to produce IL-6 and NO but decrease macrophage TNF-α.

WT or IL-6−/− macrophages were cultured with or without 4T1, CT26, TS/A, or MC38 tumor cells, and the supernatants were assayed for (A) IL-6, (B) NO, or (C) TNF-α. Representative data (left graphs) from one of four, three, and four independent experiments, respectively. Average percent change of pooled data from all experiments (right graphs). (A) Comparison of tumor cells with IL-6−/− and WT macrophages. (B and C) Comparison of tumor cells with WT macrophages. (A–C) Statistical significance was determined by t-test.

4T1, CT26, and TS/A cells also increased macrophage production of NO, and increases in NO ranged from 36% to 72% (Fig. 4B). In contrast, macrophage production of TNF-α was decreased significantly in the presence of the four tumors, as cultures of macrophages plus tumor cells produced significantly less TNF-α compared with macrophages cultured alone. Tumor cell-mediated decreases in TNF-α ranged from 24% to 53% (Fig. 4C). Macrophage production of IL-10 and IL-12 was not affected by tumor cells (Supplemental Fig. 2). Increases in macrophage NO and IL-6 were a result of increased production by individual macrophages, as the macrophages did not proliferate during the overnight culture period (Supplemental Fig. 1C). These results show that macrophages and tumor cells participate in cross-talk with each other, resulting in differential production of proinflammatory mediators, which are characteristic of M1 (NOhi) and M2 (TNF-αlow) macrophages.

MDSC prevent most tumor cells from increasing macrophage IL-6

As MDSC and macrophages are present in the tumor microenvironment, we next tested if MDSC alter cross-talk between tumor cells and macrophages. MDSC, macrophages, and/or tumor cells were cocultured, and IL-6 levels were assessed (Fig. 5A). Cultures containing 4T1, TS/A, or MC38 tumor cells plus MDSC and macrophages produced less IL-6 than cultures without MDSC. MDSC-mediated decreases of IL-6 ranged from 0% to 37%. In contrast, MDSC did not decrease IL-6 in cultures of macrophages and CT26 tumor cells. These results demonstrate that in the presence of most tumors, MDSC modestly reduce macrophage IL-6.

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MDSC decrease tumor cell-mediated enhancement of IL-6 and increase tumor cell-mediated enhancement of macrophage NO.

WT macrophages were cultured with or without 4T1-induced MDSC and/or 4T1, CT26, TS/A, or MC38 tumor cells. Supernatants were assayed for (A) IL-6 and (B) NO. (A and B, left four graphs of each) One of three independent experiments. (Right) Average of the three independent experiments. (A and B) Statistical significance was determined by t-test.

MDSC increase macrophage NO in the presence of tumor cells

To determine if MDSC affect the tumor-driven increase in macrophage NO, tumor cells, macrophages, and MDSC were cocultured (Fig. 5B). Cultures of 4T1 or CT26 tumor cells with macrophages and MDSC contained more NO than cultures without MDSC. MDSC-mediated increases in NO ranged from 0% to 30%. In contrast, TNF-α, IL-10, and IL-12 were not affected by MDSC (Supplemental Fig. 2). These results indicate that MDSC alter the dynamic of tumor cell and macrophage cross-talk by enhancing NO production.

Stromal cells are the dominant producers of IL-6 and IL-10 in the tumor microenvironment

Our in vitro findings suggest that tumor-infiltrating cells and not tumor cells are the dominant producers of IL-6 and IL-10. To determine if this in vitro finding occurs in vivo, we harvested 4T1, CT26, and TS/A tumors from WT, IL-6−/−, and IL-10−/− mice and assayed the tumors for IL-6 and IL-10 (Fig. 6). Tumors in all WT mice contained IL-6 and IL-10, whereas all tumors from IL-10−/− mice contained very little or no IL-10. With the exception of one mouse with CT26 tumor, tumors from IL-6−/− mice did not have IL-6. Isolated tumors did not contain detectable levels of TNF-α or NO (data not shown). These results demonstrate that in vivo in the tumor microenvironment, stromal cells and not tumor cells are the dominant sources of IL-6 and IL-10.

An external file that holds a picture, illustration, etc. Object name is zgb0121462040006.jpg
Host cells are the dominant producers of IL-6 and IL-10 in the tumor microenvironment.

Eight- to 10-mm diameter 4T1, CT26, and TS/A tumors were excised from WT, IL-6−/−, and IL-10−/− BALB/c mice, manually teased into small pieces, and incubated overnight and the supernatants analyzed by ELISA for IL-6 and IL-10. Cytokine levels were normalized to 1 g tumor tissue/mL media. Data are pooled from four independent experiments. Statistical significance was assessed by t-test.

DISCUSSION

Solid tumors include multiple, diverse host cells that contribute to an inflammatory tumor microenvironment and facilitate tumor progression. As macrophages and MDSC are present in most solid tumors, we have examined the interplay of these cells to determine if and how their interactions may influence the intratumor environment. The studies reported here on IL-6, IL-10, TNF-α, and NO, plus our previous reports on IL-12, address some of the most common molecules produced by MDSC and macrophages that contribute to tumor progression. Our findings are summarized in Fig. 7A. Collectively, our results indicate that the levels of IL-6, IL-10, IL-12, TNF-α, and NO are modulated by interactions among MDSC, macrophages, and tumor cells. MDSC induce some M2 macrophage characteristics (IL-6lowIL-12lowTNF-αlow) but simultaneously induce NO, which is characteristic of M1 macrophages. These apparently opposing activities are both regulated by MDSC production of IL-10. Tumor cells also regulate macrophage expression of molecules characteristic of M1 (IL-6hiNOhi) and M2 (TNF-αlow) phenotypes, whereas tumor cells and macrophages enhance MDSC production of IL-6 and IL-10, respectively. As stromal cells are the dominant producers in vivo of several of these cytokines, the complex pattern of cross-talk among MDSCs, macrophages, and tumor cells is likely to have profound effects on tumor progression.

An external file that holds a picture, illustration, etc. Object name is zgb0121462040007.jpg
Summary of cross-talk among MDSC, macrophages (MΦ), and tumor cells.

(A) Cross-talk with respect to IL-10, IL-6, IL-12, TNF-α, and NO. Solid arrows indicate direct effects mediated by the cell type or IL-10. Dashed arrow indicates an indirect effect by IL-6. (B) Potential cycle by IL-6, IL-10, and MDSC cross-talk promotes inflammation and tumor progression.

NO is an important effector molecule that is differentially impacted by IL-10 and promotes or inhibits tumor progression depending on the tumor model. NO is produced by eNOS and iNOS, which are up-regulated [26] and down-regulated [27], respectively, by macrophage-produced IL-10. Pro- and anti-tumor roles have been attributed to NO/iNOS in multiple tumor systems (Supplemental Table 2). It is likely that the apparent conflicting effects of NO are a result of many variables, including, but not limited to, the production of NO by different types of cells, the location of the producer cells, neighboring cells that might be altered by the released NO, and the concentration of NO. As a result of the complexity of NO on tumor progression and the presence of multiple cell types in the tumor microenvironment that may participate in cross-talk, elucidating the role of NO in tumor progression will be challenging.

IL-6 is a pivotal cytokine that promotes tumor progression directly by enhancing tumor cell development, growth, metastasis, vascularization, and inhibiting apoptosis [9,–11]. MDSC were reported to be a primary producer of IL-6 in the tumor microenvironment [20]. This observation is consistent with our finding that stromal cells and not tumor cells are the major producer of IL-6 in vivo and that tumor cells drive MDSC IL-6 production. IL-6 also enhances MDSC accumulation and suppressive activity [28,–30] and decreases MDSC production of IL-10, an anti-inflammatory cytokine [31]. Therefore, positive feedback between MDSC and tumor cells will potentially maintain chronic inflammation and promote tumor progression through the cycle shown in Fig. 7B.

Pro- and anti-tumor roles have been attributed to IL-10. It down-regulates numerous immune-modulatory molecules that are essential for an anti-tumor immune response and is considered an anti-inflammatory cytokine [15, 31]. For example, IL-10 impairs antigen presentation by dendritic cells and macrophages by down-regulating expression of MHC class II, CD80, and CD86. IL-10 also decreases production of IFN-γ and IL-12, cytokines that are characteristic of and facilitate the development of type I anti-tumor effector and helper cells, and IL-10 overexpressing tumor cells have increased growth rates in vivo [32]. In cancer patients, secretion of IL-10 from basal or squamous cell carcinoma cells prevents in vitro lysis of tumor cells by tumor-infiltrating lymphocytes. In vitro, pretreatment of tumor cells (e.g., melanoma, lymphoma) with IL-10 confers resistance to CTL-mediated lysis by decreasing expression of transporter associated with antigen processing 1 and 2 and subsequent surface expression of MHC I. IL-10 also contributes to tumor progression by enhancing angiogenesis and tumor cell proliferation. As MDSC IL-10 is enhanced by macrophage cross-talk, and IL-10 is produced predominantly by tumor-infiltrating stromal cells, cross-talk by macrophages and MDSCs is most likely a source of IL-10 in the tumor microenvironment.

However, IL-10 has also been linked to enhancing anti-tumor immunity [15]. For example, the reduction in MHC I by IL-10 renders tumor cells more susceptible to NK-mediated killing, and a tumor cell-based glioma vaccine induced more effective anti-tumor immunity in WT mice than in IL-10−/− mice. IL-10 also activated tumor-resident CD8+ T cells directly, facilitated tumor rejection of PDV6 squamous carcinoma [23], and served as an adjuvant in immunotherapy. Treatment of mice with pegylated IL-10, a form of IL-10 that has an increased serum half-life, induced IFN-γ and granzyme-B production by tumor-infiltrating CD8+ T cells in a mouse mammary tumor virus tumor model [24]. IL-10 also inhibited tumorigenesis in mice with colon carcinoma and patients with B cell lymphoma [21, 22]. Ablation of IL-10 from CD4+ T cells enhanced tumor burden in APCΔ468 mice [33], whereas IL-10−/− mice bearing MC38 tumors displayed increased tumor growth, metastasis, MDSC accumulation, and enhanced susceptibility to chemical carcinogenesis [34]. Therefore, as reported in the literature and shown in this report, the role of IL-10 in the promotion of tumor progression is dependent on the tumor model.

STAT3 is activated by IL-6 and IL-10; however, the two cytokines can result in different biological effects as a result of the complexity of the STAT3 pathway [35]. There are 1.3 × 106 potential binding sites for STAT3 in the mouse genome [36]; however, STAT3 only binds a few thousand sites in a given cell type [37]. STAT3 is a pleiotropic transcription factor that regulates target genes by acting in conjunction with a variety of transcriptional coactivators. The expression of these coactivators is dependent on the cell type and signaling events that occur in a cell's lifetime. Many of these coactivators are prebound to STAT3 target sites (reviewed in ref. [37]). Therefore, a cell's phenotype following STAT3 signaling depends on its previous history with respect to STAT3 activation. The tumor microenvironment is a complex milieu, so differential expression of transcriptional coactivators is likely. As MDSC-macrophage-tumor cell cross-talk involves activation of STAT3 via IL-6 and IL-10, and the relative amounts of these cytokines differ depending on the type of tumor, cross-talk is likely to contribute to the differential effects of IL-10 on tumor progression.

In addition to the cells examined here, other stromal cells also contribute to inflammation within the tumor microenvironment through their cross-talk with MDSC [38, 39]. However, MDSC and macrophages are present at significant levels in most solid tumors, and therefore, their contributions to the inflammatory milieu are likely to be important.

Supplementary Material

Supplemental Data:

ACKNOWLEDGMENTS

This work was supported by grants from the U.S. National Institutes of Health (RO1CA115880;, RO1CA84232). D.W.B. was partially supported by a predoctoral fellowship from the Congressionally Directed Medical Research Programs Breast Cancer Program (W81XWH-11-1-0115).

We thank Dr. Manfred Kopf for providing breeding stock of BALB/c IL-6−/−, Ms. Lisa Burkheimer for her excellent care of our mice, Ms. Virginia Clements for excellent technical support, and Ms. Katelyn Beury for help with the schematic figures.

The online version of this paper, found at www.jleukbio.org, includes supplemental information.

−/−
deficient
HSD
honestly significant difference
MDSC
myeloid-derived suppressor cell or cells
PB
Pacific Blue
UMB
University of Maryland Baltimore
UMBC
University of Maryland Baltimore County
VEGF
vascular endothelial growth factor
WT
wild-type

AUTHORSHIP

D.W.B., K.H.P., P.S., and S.O-R. designed experiments and analyzed data. D.W.B., K.H.P., M.N., K.A.C., and P.S. performed experiments. D.W.B. and S.O-R. wrote the manuscript. All authors approved the manuscript.

DISCLOSURES

The authors declare no conflicts of interest.

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