Interleukin-6 Trans-Signaling Pathway Promotes Immunosuppressive Myeloid-Derived Suppressor Cells via Suppression of Suppressor of Cytokine Signaling 3 in Breast Cancer

Interleukin-6 (IL-6) has been reported to stimulate myeloid-derived suppressor cells (MDSCs) in multiple cancers, but the molecular events involved in this process are not completely understood. We previously found that cancer-derived IL-6 induces T cell suppression of MDSCs in vitro via the activation of STAT3/IDO signaling pathway. In this study, we aimed to elucidate the underlying mechanisms. We found that in primary breast cancer tissues, cancer-derived IL-6 was positively correlated with infiltration of MDSCs in situ, which was accompanied by more aggressive tumor phenotypes and worse clinical outcomes. In vitro IL-6 stimulated the amplification of MDSCs and promoted their T cell suppression ability, which were fully inhibited by an IL-6-specific blocking antibody. Our results demonstrate that IL-6-dependent suppressor of cytokine signaling 3 (SOCS3) suppression in MDSCs induced phosphorylation of the JAK1, JAK2, TYK2, STAT1, and STAT3 proteins, which was correlated with T cell suppression of MDSCs in vitro. Therefore, dysfunction in the SOCS feedback loop promoted long-term activation of the JAK/STAT signaling pathway and predominantly contributed to IL-6-mediated effects on MDSCs. Furthermore, IL-6-induced inhibition of SOCS3 and activation of the JAK/STAT pathway was correlated with an elevated expression of IL-6 receptor α (CD126), in which the soluble CD126-mediated IL-6 trans-signaling pathway significantly regulated IL-6-mediated effects on MDSCs. Finally, IL-6-induced SOCS3 dysfunction and sustained activation of the JAK/STAT signaling pathway promoted the amplification and immunosuppressive function of breast cancer MDSCs in vitro and in vivo, and thus blocking the IL-6 signaling pathway is a promising therapeutic strategy for eliminating and inhibiting MDSCs to improve prognosis.

Interleukin-6 (IL-6) has been reported to stimulate myeloid-derived suppressor cells (MDSCs) in multiple cancers, but the molecular events involved in this process are not completely understood. We previously found that cancer-derived IL-6 induces T cell suppression of MDSCs in vitro via the activation of STAT3/IDO signaling pathway. In this study, we aimed to elucidate the underlying mechanisms. We found that in primary breast cancer tissues, cancer-derived IL-6 was positively correlated with infiltration of MDSCs in situ, which was accompanied by more aggressive tumor phenotypes and worse clinical outcomes. In vitro IL-6 stimulated the amplification of MDSCs and promoted their T cell suppression ability, which were fully inhibited by an IL-6-specific blocking antibody. Our results demonstrate that IL-6-dependent suppressor of cytokine signaling 3 (SOCS3) suppression in MDSCs induced phosphorylation of the JAK1, JAK2, TYK2, STAT1, and STAT3 proteins, which was correlated with T cell suppression of MDSCs in vitro. Therefore, dysfunction in the SOCS feedback loop promoted long-term activation of the JAK/STAT signaling pathway and predominantly contributed to IL-6-mediated effects on MDSCs. Furthermore, IL-6-induced inhibition of SOCS3 and activation of the JAK/STAT pathway was correlated with an elevated expression of IL-6 receptor α (CD126), in which the soluble CD126-mediated IL-6 trans-signaling pathway significantly regulated IL-6-mediated effects on MDSCs. Finally, IL-6-induced SOCS3 dysfunction and sustained activation of the JAK/STAT signaling pathway promoted the amplification and immunosuppressive function of breast cancer MDSCs in vitro and in vivo, and thus blocking the IL-6 signaling pathway is a promising therapeutic strategy for eliminating and inhibiting MDSCs to improve prognosis.
Keywords: breast cancer, interleukin-6, myeloid-derived suppressor cells, suppressor of cytokine signaling 3, the JaK/sTaT signaling pathway, trans-signaling pathway IL-6 Trans-Signaling Pathway Promotes MDSCs Frontiers in Immunology | www.frontiersin.org December 2017 | Volume 8 | Article 1840 inTrODUcTiOn Increasing evidence has highlighted the importance of crosstalk between cancer cells and the surrounding microenvironment in the initiation and progression of various cancers (1)(2)(3). The tumor microenvironment is composed of multiple immunosuppressive cells, among which myeloid-derived suppressor cells (MDSCs) play a vital role in promoting tumor invasion and metastasis (3). Myeloid-derived suppressor cells are a heterogeneous population of immature myeloid cells with suppressive effects on both innate and adaptive immunity; therefore, they are regarded as a major obstacle in antitumor immunotherapy (4). Different MDSCs subsets display varied phenotypes in mice or in humans. For example, monocytic MDSCs (M-MDSCs) express CD11b + Ly6G − Ly6C hi in mice and CD11b + HLA-DR −/lo CD14 + CD15 − in human; while polymorphonuclear MDSCs (PMN-MDSCs) express CD11b + Ly6G + Ly6C lo in mice and CD11b + HLA-DR −/lo CD14 − CD15 + in human. Additionally, early-stage MDSCs (eMDSCs), which comprised more immature progenitors than M-MDSCs and PMN-MDSCs, are defined as specific MDSCs subset expressing Lin − HLA-DR − CD33 + in human tumors (5). In our previous study, we identified a subset of poorly differentiated eMDSCs in breast cancer that expressed an immature phenotype of Lin − HLA-DR − CD45 + CD33 + CD13 + CD14 − CD15 − and displayed potent suppression of T cells in vitro and in vivo (6). We found that cancer-derived interleukin-6 (IL-6) induces the immunosuppressive ability of MDSCs by activating the STAT3/IDO signaling pathway, but the detailed molecular events are unclear (7).
Interleukin-6 is known as a key regulator of immunosuppression in advanced cancer and is responsible for the development of pro-inflammatory and metastatic tumor microenvironments (8). Numerous studies have reported significant correlations between IL-6 and circulating MDSCs in both human and mouse models (9)(10)(11)(12)(13)(14). IL-6 increased circulating CD11b + CD14 + HL A − DR − cells in squamous carcinoma of the esophagus (9) and prostate cancer (13). Though previous studies reported that IL-6 restored MDSC accumulation in a mouse model of mammary carcinoma (14), a few studies have focused on the relationship between IL-6 and MDSCs in human breast cancer. Our previous study demonstrated that in breast cancers, IL-6 stimulates STAT3-dependent, nuclear factor-κB-mediated indoleamine 2,3-dioxygenase (IDO) upregulation in MDSCs (7); this triggers immunosuppressive effects of MDSCs in vitro and vivo (6). Although abnormal accumulation of MDSCs via the IL-6/ STAT3 pathway was reported in multiple cancers (9,13,15), the major regulatory mechanisms remain unclear.
It is well-established that the interaction between IL-6 and IL-6R initiates the activation of the JAK/STAT signaling pathway, which transduces the IL-6 signal in both normal and malignant cells. In contrast to the rapid and reversible activation of STAT proteins in normal cells, phosphorylation of STAT proteins is sustained for a long time in malignant cells (16,17). The dysfunctional negative feedback loop in the JAK/ STAT signaling pathway induces constitutive activation of STAT proteins, oncogenic transformation, tumor invasion, and metastasis (18).
Suppressor of cytokine signaling (SOCS) proteins, particularly SOCS3, are major negative feedback regulators of the JAK/STAT signaling pathway (19). Under physiological conditions, IL-6 stimulates the expression of SOCS3 and inhibits phosphorylation of STAT proteins (20). This attenuates IL-6-induced activation of the JAK/STAT signaling pathway and inhibits expression of downstream functional genes (17,21). It has been reported that constitutive defects in the expression of SOCS3 protein is frequent in malignant cells and is associated with dysregulation of cell growth, migration, and apoptosis (19).
However, only short-term and reversible suppression of SOCS was detected in certain types of immune cells in cancer, such as tumor-infiltrated T cells, dendritic cells (DCs), and macrophages (22,23). It has been demonstrated that knockdown of SOCS3 in macrophages is beneficial for inhibiting tumor metastases in mice (24). However, it has also been reported that SOCS3 deficiency in myeloid cells promotes tumor development by inducing MDSCs in the tumor microenvironment (25). Therefore, it is urgent to elucidate the biological significance of SOCS3 deficiency in MDSC development and tumor progression, which may provide insight into potential therapeutic targets for breast cancer.
In this study, we evaluated the expression of SOCS proteins and their effects on IL-6-induced activation of the JAK/STAT signaling pathway in breast cancer MDSCs. We found that more MDSCs were recruited in IL-6 high-expressing breast cancer tissues, in which SOCS3 inhibition was detected. IL-6 promoted the amplification of MDSCs and enhanced their suppressive effects on T cells immunity in vitro. IL-6 stimulated SOCS3 suppression and thus induced long-term activation of the JAK/ STAT signaling pathway in breast cancer MDSCs. IL-6-induced SOCS3 dysfunction and sustained activation of the JAK/STAT signaling pathway predominantly contributed to IL-6-mediated effects on MDSCs. Furthermore, we observed that IL-6-induced suppression of SOCS3 and activation of the JAK/STAT pathway were correlated with an elevated expression of IL-6 receptor α (IL-6-Rα, CD126), which was regulated by the soluble CD126mediated IL-6 trans-signaling pathway. Summarily, we found that IL-6-induced SOCS3 dysfunction and sustained activation of the JAK/STAT signaling pathway promoted the amplification and immunosuppressive function of breast cancer MDSCs in vitro and vivo. Thus, blocking IL-6 signaling pathway is a promising therapeutic strategy for eliminating and inhibiting MDSCs to improve prognosis.

MaTerials anD MeThODs clinical samples and healthy Donors
In this study, we collected 253 primary breast cancer tissue samples from two cohorts for immunohistochemistry (IHC) analysis. Cohort 1 included 113 primary breast cancer patients who received surgical resection at the Department of Breast Oncology of Tianjin Medical University Cancer Institute and Hospital from October 2012 to October 2014. Cohort 2 included 140 breast cancer cases whose tumor tissues were assembled on tissue arrays after surgical removal between January 2001 and isolation of Primary MDscs and cD33 + Progenitors Twenty primary breast cancer tissues and their adjacent tissues were collected during surgery and cut into small pieces before being ground and filtered using a filter mesh to prepare a singlecell suspension. CD33 + cells were isolated using human CD33 MicroBeads (130-045-501; Miltenyi Biotec, Bergisch Gladbach, Germany) as previously reported (6). The negative-selected cells were defined as breast cancer cells and analyzed for the expression of IL-6. Thirty cases of PB samples were collected from healthy donors for PBMC isolation. CD33 + and CD14 + cells were enriched using human CD33 (130-045-501; Miltenyi Biotec) and human CD14 MicroBeads (130-050-201; Miltenyi Biotec), respectively, according to the manufacturer's instructions. Trypan blue staining was performed to ensure the full viability of each cell fraction.

ihc assay
Fresh tissues were immediately fixed with formalin after surgical removal; tissues were made into paraffin embedded blocks and then sliced into 4-µm serial sections. The samples were heated for 1 h at 70°C, deparaffinized in xylene, and rehydrated using graded alcohol. Antigens were retrieved in citrate buffer (pH 6.0) for 2 min. Endogenous peroxidase activity was quenched in 3% hydrogen peroxide for 20 min. We previously examined the expression a series of pan-myeloid and differentiated markers of myeloid linage and confirmed high expression of CD33 and CD13, low expression of HLA-DR and CD14, and negative expression of CD15 on the surface of breast cancer MDSCs (6). We also detected the expression of 3 pan-myeloid markers, including CD33 (26), CD13 (27), and CD11b (28) and found that non-specific staining of CD13 and CD11b on cancer cells, endothelial cells, and fibroblasts interfered with the specific staining on MDSCs (Figures S1B-C in Supplementary Material), indicating the feasibility of using CD33 to detect breast cancer MDSCs in an IHC assay. Therefore, all samples were incubated with mouse anti-human IL-6 (PeproTech, Rocky Hill, NJ, USA) and CD33 (Abcam, Cambridge, UK) monoclonal antibody (McAb) at a concentration of 1 µg/mL overnight at 4°C. A secondary antibody conjugated with streptavidin-horseradish peroxidase (Santa Cruz, Biotech, Dallas, TX, USA) was then added, and the mixture was incubated for 30 min before adding diaminobenzidine staining buffer (Maixin Biotechnology, Fuzhou, China). All images were captured using an Olympus BX51 microscope (Olympus, Tokyo, Japan). Five representative high-power fields (400× magnification) from each tissue section were selected for histology evaluation as previously described (6). anti-human CD13 and CD33, and fluorescein isothiocyanateconjugated anti-human CD14 and CD15 (BD Biosciences) antibodies were used to label the MDSCs. An isotype-matched IgG1 antibody (BD Biosciences) was used as a negative control. After incubation, cells were washed and resuspended in buffer, and the expression of cell surface markers was detected using the flow cytometer. The leukocyte population was gated using PerCp-labeled anti-human CD45, and breast cancer MDSCs were defined as CD33 + CD13 + CD14 − CD15 − in the CD45 + population. Furthermore, to detect the production of interferon (IFN)-γ and IL-10 in T cells co-cultured with or without MDSCs, we conducted an intracellular staining assay using flow cytometry. After co-culture, T cells were distinguished by allophycocyanin- cell line and cell culture

Flow cytometry analysis
The human breast cancer cell line MDA-MB-231 was obtained from the Chinese Academy of Medical Sciences (Resource number: 3111C0001CCC000014). The cell line was cultured in complete RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum in a 5% CO2 incubator at 37°C. CD33 + progenitors isolated from PBMCs of healthy donors were co-cultured with breast tumor cells to induce MDSCs with or without IL-6 antibody. CD33 + and CD14 + control cells were cultured in complete RPMI 1640 medium. After co-culture with MDSCs for 3 days, the proliferation, apoptosis, and cytokine secretion of T cells were studied to evaluate the immunosuppressive ability of MDSCs pretreated with or without IL-6 antibody.

induction of MDscs In Vitro
CD33 + myeloid progenitors (2 × 10 6 /mL) isolated from healthy PBMCs were added to multi-well plates and co-cultured with MDA-MB-231 breast cancer cells to induce MDSCs with or without IL-6 antibody (EMD Millipore, Billerica, MA, USA) at a concentration of 50 µg/mL. CD33 + progenitors were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum as negative controls. After 48 h of culture, MDSCs were harvested for further analysis, and the supernatants were collected to detect soluble CD126 using the Human IL-6R ELISA Kit (GenWay Biotech, Inc., San Diego, CA, USA). The phenotype of harvested cells was examined by flow cytometry as previously described; the proportion of CD45 + CD13 + CD33 + CD14 − CD15 − MDSCs was examined (6).

cell counting Kit 8 (ccK8) assay
Cell Counting Kit 8 assay was used to detect the proliferation of T cells co-cultured with or without MDSCs. CD3 + T cells were isolated from PBMCs of 10 healthy donors using the Human Pan T cell Isolation Kit II (130-091-156; Miltenyi Biotec). Both MDSCs and T cells with viability >95% were used for functional assays. Purified T cells (2 × 10 5 ) were plated in a 96-well plate and co-cultured with CD33 + cells or MDSCs in the presence or absence of IL-6 antibody at ratio of 1:3 in triplicate. Cells were cultured in complete medium supplemented with 1,000 IU/mL recombinant human IL-2 (PeproTech) or anti-CD3/CD28 Abs (at bead/cell ratio of 1:1, Gibco) at 37°C in a 5% CO2 incubator for 3 days. Next, 10 µL CCK8 (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) was added to each well and incorporated into living cells during cell proliferation. Blank wells without cells were used as negative controls. T cells stimulated with IL-2 or anti-CD3/CD28 Abs were used as the T cell control. After 4 h of incubation, the optical density at 450 nm was measured using an enzyme immunoassay analyzer (Thermo Fisher Scientific, Waltham, MA, USA). Cell proliferation was evaluated using stimulation index (SI), which was calculated using the following formula: SI = [(experimental counts)/(responder control counts + stimulator control counts)].

annexin V assay
Purified T cells (5 × 10 5 ) were plated in a 24-well plate and cocultured with CD33 + cells or MDSCs in the presence or absence of IL-6 antibody at ratio of 1:3 in triplicate. Cells were cultured in complete medium supplemented with 1,000 IU/mL recombinant human IL-2 at 37°C in a 5% CO2 incubator for 3 days. The Annexin V assay was used to detect the apoptosis of T cells. We initially gated lymphocytes according to SSC and FSC features and then gated T cells using allophycocyanin-labeled anti-CD3 McAb (BioLegend, San Diego, CA, USA). The cells were stained with FITC-Annexin V and propidium iodide provided in an Apoptosis Detection Kit (BD Biosciences) as previously described (6). The positive expression of Annexin V and negative expression of PI represent apoptotic T cells.

enzyme-linked immunosorbent assay (elisa)
Purified T cells (5 × 10 5 ) were plated in a 24-well plate and cocultured with CD33 + cells or MDSCs in the presence or absence of IL-6 antibody at ratio of 1:3 in triplicate. Cells were cultured in complete medium supplemented with 1,000 IU/mL recombinant human IL-2 or anti-CD3/CD28 Abs (at a bead/cell ratio of 1:1) at 37°C in a 5% CO2 incubator for 3 days. The corresponding T cell culture supernatants were collected to detect cytokine levels in an ELISA assay. Levels of T cell-secreted cytokines, including IFN-γ and IL-10, were analyzed using the ELISA kits (Dakewe Biotech Co., Ltd., Shenzhen, China) as per the manufacturer's instructions.

Quantitative real-time rT-Pcr (qrT-Pcr) analysis
Interleukin-6, CD126, gp130, SOCS1, SOCS2, SOCS3, ADAM10, and ADAM17 mRNA expression in MDSCs isolated from primary breast cancer tissues and in vitro-induced MDSCs was analyzed by qRT-PCR. The mRNA levels of target genes were quantified using the SYBR Premix Ex Taq TM system (Takara Bio, Shiga, Japan) with an ABI 7500 instrument (Applied Biosystems, Foster City, CA, USA). The primers for IL-6, CD126, gp130, SOCS1-3, ADAM10, ADAM17, and β-actin are shown in Table 2. Relative mRNA levels in each sample were calculated based on their threshold cycle (Ct) values normalized to the Ct value of β-actin using the formula: 2 −ΔCt (ΔCt = Cttarget gene − Ctβactin). All tests were conducted at least five times.

Western Blot analysis
Western blot analysis was performed to detect the levels of CD126, gp130, ADAM10, ADAM17, and SOCS1-3 proteins, as well as total and phosphorylated JAK1, JAK2, TYK2, STAT1, and STAT3 in MDSCs and CD33 + control cells. Cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes for western blot analysis using mouse anti-human CD126, gp130 (R&D Systems, Inc., Minneapolis, MN, USA), SOCS1 (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), SOCS2-3 (R&D Systems, Inc.), and β-actin. Rabbit antihuman antibodies were used to detect JAK1, JAK2, TYK2, STAT1, STAT3, p-STAT1, p-JAK1, p-JAK2, p-TYK2, and p-STAT3 (Cell Signaling Technology, Danvers, MA, USA). Membranes were incubated with primary antibodies overnight at 4°C, as described previously (7). Membranes were then incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG Ab (Zhongshanjinqiao, Beijing, China), and protein bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce Biotechnology, Rockford, IL, USA). The relative densities of protein bands were determined by comparing the band densities of proteins of interest to those of β-actin, using Quantity One software. We used the density ratio of phosphorylated protein to total protein to compare the expression of these phosphorylated proteins.

statistical analysis
Statistical analyses were performed using the SPSS 20.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 5.0 software (GraphPad, Inc., La Jolla, CA, USA). Measured data were presented as the mean ± SD; one-way analysis of variance and least significant difference tests were used to compare quantitative data. Categorical data were presented as the median, and the nonparametric χ 2 test was used to compare qualitative data. The cumulative survival probability was determined by the Kaplan-Meier method, and the log-rank test was used to compare overall survival (OS) of each subgroup of patients. P-values for each analysis are reported in the figure legends, and the level of statistical significance was set to P < 0.05.

resUlTs
Tumor-Derived il-6 and local MDscs infiltration are significantly correlated with lymph node Metastasis and Poor Prognosis in Breast cancer Patients Interleukin-6 was mainly expressed in the cytoplasm of breast cancer cells, as well as in some mesenchymal cells ( Figure 1A). Based on the staining intensity and extent of IL-6 expression, breast cancer patients were divided into an IL-6 low expression group (IL-6 low ) and high expression group (IL-6 high ). The IL-6 high cases accounted for 44.4% (48/108) of patients in cohort 1 and 50.5% (50/99) of patients in cohort 2. CD33 + MDSCs were scattered in the stroma of breast cancer tissues with varying sizes and shapes ( Figure 1B). According to the number of CD33 + MDSCs that infiltrated locally, breast cancer patients were divided into lowly infiltrated MDSC group (MDSCs low ) and highly infiltrated MDSC group (MDSCs high ). The MDSCs high cases accounted for 52.0% of patients in cohort 1 and 50.9% of patients in cohort 2. We next compared the correlations between IL-6 expression, MDSCs infiltration, other clinical pathological features (age, agenda, tumor size, tumor pathologic stage, tumor histological grade, and lymph node invasion), and expression of hormone receptors (estrogen receptor, progesterone receptor, and HER2) in the two cohorts. As shown in Table 3, tumor-derived IL-6 expression was significantly correlated with lymph node invasion and tumor histological grade; compared to IL-6 low patients, IL-6 high patients suffered from more aggressive histological features (cohort 1: P < 0.001; cohort 2: P = 0.001) and a higher risk of early lymph node invasion (cohort 1: P < 0.001; cohort 2: P = 0.012). Similar trends were observed in MDSC high patients as compared with MDSC low patients, where more infiltrated MDSCs were detected in cancer tissues at more advanced pathological stages, with higher histological grade, more lymph node metastasis, and larger tumor size (P < 0.001, P < 0.001, P = 0.025, P = 0.018; P < 0.001, P = 0.007; P = 0.022, P = 0.032, respectively, Table 4). These results demonstrated that breast cancers with higher IL-6 expression and greater MDSC infiltration possess a higher potential for invasion and metastasis.
Next, we compared the OS of the 140 patients in cohort 2 with IL-6 expression and MDSC infiltration. We found that the OS, 3-, 5-, and 10-year survival of IL-6 high patients, was significantly shorter than those of IL-6 low patients (P < 0.001, Figure 1C). Similarly, IL-6 high patients displayed worse overall breast cancer-specific survival, 3-, 5-, and 10-year breast cancer-specific survival compared to IL-6 low patients (P < 0.001, Figure 1C). Similar results were observed in MDSC high patients compared to MDSC low patients (P < 0.001, Figure 1D). Thus, these findings indicate that tumor-derived IL-6 and MDSC infiltration are both unfavorable prognostic factors in breast cancer and are significantly correlated with aggressive tumor behavior and poor clinical outcomes in patients. Tumor-Derived il-6 is significantly correlated with the number of infiltrated MDscs In Situ Both at the mrna and Protein levels We compared the correlation between the expression of IL-6 and number of infiltrated MDSCs in situ to evaluate the effects of IL-6 on MDSC accumulation in breast cancer tissues. We first studied the expression of IL-6 protein in 253 paraffin-embedded breast tissues from cohorts 1 and 2 by IHC. We found greater MDSC infiltration in cancer tissues with a high level of IL-6 ( Figure 2A). The average number of MDSCs in the IL-6 low group was significantly lower than that in the IL-6 high group in both cohorts 1 and 2 [(1.95 ± 0.26) vs. (6.40 ± 0.48), P < 0.001; (1.31 ± 0.27) vs. (6.43 ± 0.79), P < 0.001, Figure 2B]. Pearson correlation analysis revealed a positive correlation between the expression of IL-6 and the number of MDSCs in situ in both cohorts (cohort 1, R 2 = 0.3974, P < 0.0001; cohort 2, R 2 = 0.2812, P < 0.0001, Figure 2B). Twenty fresh breast cancer tissue samples were collected to study the correlation between RNA levels of tumor-derived IL-6 and percentages of CD45 + CD33 + CD13 + CD14 − CD15 − MDSCs in breast cancer tissues by flow cytometry analysis. We observed a cluster of CD33 + CD13 + CD14 − CD15 − cells in breast cancer tissue, which represented the predominant phenotype of MDSCs in breast cancer ( Figure 2C). The percentage of CD45 + CD33 + CD13 + CD14 − CD15 − MDSCs was 15.3-58.1% with a mean value of 29.82 ± 11.463%. Based on the median relative RNA level of IL-6, breast cancer samples were divided into IL-6 high and IL-6 low groups. The average IL-6 mRNA level in the IL-6 high group was 37.25-fold higher than that in the IL-6 low group (P = 0.0093, Figure 2D). Higher frequency of MDSCs was detected in the IL-6 high group compared to in the IL-6 low group [(13.75 ± 3.44%) vs. (4.31 ± 1.50%), P = 0.03, Figure 2E]. Furthermore, Pearson correlation analysis revealed a strong positive correlation between the expression of IL-6 mRNA and number of MDSCs in fresh breast cancer tissues (R 2 = 0.4399, P = 0.0014, Figure 2F).

il-6 enhanced the generation and T cell immunosuppressive ability of MDscs In Vitro
To mimic the breast cancer cell-conditioned microenvironment in vitro, CD33 + myeloid progenitors were isolated from healthy donors' PMBCs and co-cultured with MDA-MB-231 breast   Figure 3A).

il-6 stimulated sustained activation of the JaK/sTaT Pathway in MDscs Displaying Persistent Phosphorylation of Downstream sTaT Proteins
To elucidate the molecular mechanisms regulating IL-6-induced MDSC differentiation and immunosuppressive activities, we studied the activation status of the JAK/STAT pathway downstream of IL-6 signaling. We assessed the expression and phosphorylation of multiple functional proteins along the JAK/STAT pathway, such as JAK1, JAK2, TYK2, STAT1, and STAT3, using western blot assays. Comparable increases in phosphorylated JAK1, JAK2, TYK2, STAT1, and STAT3 proteins were detected in MDSCs as compared to that in CD33 + controls ( Figure 4A). Furthermore, sustained phosphorylation of STAT1 and STAT3 proteins was observed in MDSCs, which was maintained for a longer time than in normal IL-6-stimulated PBMCs (2 vs. 4 h, Figure 4B). In IL-6 (100 ng/mL)-stimulated PBMCs, the levels of phosphorylated STAT1 and STAT3 proteins were increased at 30 min, but decreased at 2 h, disappearing entirely at 4 h ( Figure 4B). In contrast, persistent IL-6-induced STAT1 and STAT3 phosphorylation in MDSCs lasted for more than 4 h. After adding an IL-6 blocking antibody, phosphorylation levels of the above proteins were reduced significantly in MDSCs, including p-JAK1 (1.059 ± 0.06000 vs.

il-6-induced suppression of sOcs3 in MDscs Was Determined at both the mrna and Protein levels
Because the loss of SOCS proteins has been reported to induce continuous activation of the JAK/STAT pathway in malignancy, we compared the expression of SOCS1, SOCS2, and SOCS3 between MDSCs and normal myeloid controls at both the mRNA and protein levels. Primary MDSCs isolated from 20 cases of primary breast cancer tissues were studied. CD33 + and CD14 + cells from healthy donors were regarded as normal myeloid-derived cell controls. We first detected the mRNA levels of SOCS1-3 and found that mRNA level of SOCS1 increased in MDSCs compared to in both CD33 + (P = 0.006) and CD14 + (P = 0.003) controls; the mRNA level of SOCS3 significantly decreased in MDSCs compared to in CD33 + (P < 0.001) and CD14 + (P < 0.001, Figure 5A) controls. An undetectable level of SOCS2 mRNA was observed in both MDSCs and controls ( Figure 5A). We then compared the mRNA levels of SOCS1-3 between MDSCs from IL-6 high tissues (MDSC IL-6h ) and MDSCs from IL-6 low tissues (MDSC IL-6l ). The results demonstrated that the mRNA level of SOCS1 increased in MDSC IL-6h (P = 0.0459), while the mRNA level of SOCS3 decreased in MDSC IL-6h compared to that in MDSC IL-6l (P = 0.0089, Figure 5B). Linear regression analysis demonstrated that IL-6 expression was not correlated with SOCS1 (R 2 = 0.09071, P = 0.2102) but was negatively correlated with SOCS3 expression (R 2 = 0.2205, P = 0.0367, Figure 5B). We then detected the mRNA levels of SOCS1-3 in induced MDSCs in vitro. Untreated CD33 + myeloid progenitors were used as negative controls, while CD14 + monocyte-derived immature DCs (iDC) were used as positive controls. The results were consistent with those observed in primary MDSCs, where the mRNA level of SOCS1 increased, while that of SOCS3 decreased in induced MDSCs (Figure 5C). The disparity between SOCS1 and SOCS3 expression was confirmed at the protein level. The expression of SOCS1 protein notably increased, while that of SOCS3 protein dramatically decreased in MDSCs compared to CD33 + negative controls and iDC positive controls (Figure 5D). To further verify the effect of IL-6 on SOCS3 expression, an IL-6-neutralizing antibody was added to block IL-6 signaling in MDSCs, and the synthesis and expression of SOCS was detected. Ab-treated MDSCs displayed slightly lower mRNA level of SOCS1 (P = 0.0917, Figure 5E), but significantly higher mRNA level of SOCS3 compared to that in Ab-untreated MDSCs

FigUre 3 | Interleukin-6 (IL-6) enhanced the generation and T cells immunosuppressive ability of myeloid-derived suppressor cells (MDSCs) in vitro.
(a) The proportion of healthy people's untreated CD33 + myeloid progenitors and the treated CD33 + cells was compared using flow cytometry method. Cells in Q4 represent MDSCs. (1) The subpopulation was gated using anti-CD45 mAb; (2) isotype control was used; (3) the proportion range of CD33 + was 15.6 ± 2.6%; (4) when treated with cancer cells the proportion of MDSCs was highly increased which was 30.83 ± 1.595%; (5) an IL-6 neutralizing antibody was added to the cancer-conditioned MDSCs culture and MDSCs decreased to 11.98 ± 3.479%; (6) the result of statistical analysis (n = 6). The effects of MDSCs and CD33 + cells on T cell proliferation, apoptosis and cytokine secretion were examined (B-e). (B) T cells stimulated with 1,000 IU/ml IL-2 were co-cultured with CD33 + control cells or MDSCs at ratio of 1:3 for detecting apoptosis. T cells were gated using APC-labeled anti-CD3 mAb, and apoptotic cells were stained with FITC-labeled Annexin V. Cells in Q4 represent apoptotic T cells. (1) The lymphocytes were gated according to SSC and FSC features; (2) CD3 mAb labeled T cells; (3) isotype control; (4) T cells only; (5) CD33 + controls stimulated T cells; (6) MDSCs stimulated T cells; (7) T cells were co-cultured with MDSCs in the presence of IL-6 antibody; (8) Summary of (4-7) MDSCs stimulated greater T cell apoptosis compared to in CD33 + controls. In addition, the IL-6 antibody dramatically abolished MDSCs-induced T cell apoptosis (n = 5). (c) The proliferation of T cells was detected using CCK8 method. Compared to in CD33 + controls, MDSCs significantly inhibited IL-2 or anti-CD3/CD28 Abs-induced proliferation of T cells at a ratio of 1:3, which was attenuated by IL-6 blocking antibody (n = 5). (D,e) Supernatants were collected for detecting interferon (IFN)-γ and IL-10 level using ELISA assay (n = 5). (D) MDSCs inhibited IL-2 or anti-CD3/CD28 Abs-induced IFN-γ secretion, which was increased after IL-6 antibody pretreatment. (e) In contrast, MDSCs stimulated IL-10 secretion in IL-2 or anti-CD3/CD28 Abs simulated T cells compared to in CD33 + controls, which was dropped significantly after IL-6 blocking. (F) Flow cytometry was used to detect IFN-γ expression by intracellular staining. The proportion of IFN-γ-positive T cells decreased after co-culture with MDSCs compared to in CD33 + controls, but MDSCs-mediated inhibition of IFN-γ production in T cells was reversed by blocking of IL-6 (n = 5). (g) In contrast, the percentages of IL-10-positive T cells increased after co-culture with MDSCs and blocking of IL-6 inhibited IL-10 production in T cells (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001. (P = 0.0117, Figure 5E). Consistent results were confirmed at the protein level ( Figure 5F). Therefore, our study suggests that IL-6 induces inhibition of SOCS3 expression in MDSCs at both the mRNA and protein levels in vivo and vitro.
il-6-Dependent sOcs3 suppression and sustained activation of the JaK/sTaT Pathway Was correlated with cD126 Upregulation The IL-6 signal is transduced as a result of the interaction between IL-6 and IL-6R, which includes 2 subunits, CD126 and gp130 (8). We analyzed the expression of CD126 and gp130 in primary MDSCs and found that the mRNA levels of CD126 and gp130 in primary MDSCs were higher than those in CD33 + and CD14 + controls ( Figure 6A). Furthermore, the mRNA levels of CD126 and gp130 in primary MDSCs IL-6h were higher than those in MDSCs IL-6l (P = 0.010, Figure 6B). Linear regression analysis demonstrated that CD126, rather than gp130, was positively correlated with IL-6 levels (R 2 = 0.6717, P < 0.0001, Figure 6B). Similarly, we examined expression of CD126 and gp130 in induced MDSCs, and found that these MDSCs exhibited higher mRNA levels of CD126 and gp130 than CD33 + controls (P = 0.0145, P = 0.0011, Figure 6C). Similar results were obtained at the protein level, in which CD126 expression was significantly enhanced, while gp130 showed no significant changes in the expression (Figure 6D). An anti-IL-6R (CD126) neutralizing antibody was used to block the interaction between IL-6 and IL-6R in MDSCs to study the effect of elevated CD126 on the JAK/STAT pathway.
Phosphorylation levels of JAK1, JAK2, TYK2, and STAT3 proteins decreased after the addition of the anti-IL-6R neutralizing antibody ( Figure 6E). This result indicates that CD126 plays a significant role in IL-6-dependent activation of the JAK/STAT pathway. Furthermore, the mRNA level of SOCS3 increased, while the mRNA level of SOCS1 decreased after blocking CD126 FigUre 4 | Interleukin-6 (IL-6) stimulated the sustained activation of the JAK/STAT pathway in myeloid-derived suppressor cells (MDSCs) displaying persistent phosphorylation of downstream STAT proteins. The activation status of the JAK/STAT pathway was measured using Western blot. (a) Comparable increases in phosphorylated JAK1, JAK2, TYK2, STAT1, and STAT3 proteins were detected in MDSCs as compared to in CD33 + controls (n = 3). (B) Furthermore, sustained phosphorylation of STAT1 and STAT3 proteins was observed in MDSCs, which was maintained for a longer time than in normal IL-6-stimulated PBMCs. In IL-6 (100 ng/mL)-stimulated PBMCs, the levels of phosphorylated STAT1 and STAT3 proteins were increased at 30 min, but decreased at 2 h, disappearing entirely at 4 h. In contrast, persistent IL-6-induced STAT1 and STAT3 phosphorylation in MDSCs lasted for more than 4 h (n = 3). (c) In contrast, persistent IL-6-induced STAT1 and STAT3 phosphorylation in MDSCs lasted for more than 4 h. After adding an IL-6-blocking antibody, phosphorylation levels of the above proteins were reduced significantly in MDSCs. They were compared using the density ratio of phosphorylated protein to total protein (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. (P = 0.0318, P = 0.0190, Figure 6F). The expression of corresponding proteins in MDSCs displayed the same trend as that of mRNA after CD126 blocking ( Figure 6G). These results indicate that suppressed expression of SOCS3 is significantly correlated with CD126 upregulation, which induces long-term activation of the JAK/STAT pathway.
soluble cD126-Mediated il-6 Transsignaling regulated il-6 Dependent sOcs3 suppression and sustained activation of the JaK/sTaT Pathway in MDscs Signaling through membrane-bound and soluble IL-6R (CD126) is known as the cis-and trans-mediated signaling pathways, respectively (29). To investigate which type of CD126 mainly regulates IL-6-dependent activation of the JAK/STAT pathway, we measured the levels of membrane-bound and soluble CD126 in MDSCs. The results showed that MDSCs expressed lower levels of membrane-bound CD126 (7.667 ± 1.808 vs. 15.63 ± 1.200%, P = 0.0214, Figure 7A), but generated more soluble CD126 than those in CD33 + controls (249.1 ± 24.35 vs. 165.6 ± 21.83 pg/mL, P = 0.0236, Figure 7B). These results demonstrate that soluble CD126 is significantly increased in MDSCs and may play major roles in suppressing SOCS3 expression and activating the JAK/ STAT pathway in MDSCs.
To determine if soluble CD126 regulates SOCS3 expression and activation of the JAK/STAT pathway, we added ADAM proteases to the MDSC culture system in vitro. ADAM proteases, particularly ADAM10 and ADAM17, can induce shedding of membrane CD126 (30). We firstly detected the expression of ADAM10 and ADAM17 in MDSCs by RT-PCR and western blotting and found that the mRNA levels of ADAM10 and ADAM17 were clearly enhanced in MDSCs compared to those in CD33 + controls (P = 0.0064; P = 0.0297, Figure 7C). But at the protein level, exclusively ADAM10 rather than ADAM17 significantly increased ( Figure 7D). We then treated MDSCs with exogenous recombinant ADAM10 protein and measured the levels of soluble CD126 at different time points. The level of soluble CD126 in MDSCs significantly increased at 30 min (184.7 ± 5.066 vs.
We next detected the activation of STAT and SOCS in MDSCs at different time points after adding exogenous ADAM10. We found that the levels of phosphorylated STAT1 and STAT3 proteins increased after ADAM10 treatment in MDSCs (Figure 7F).
A slight increase in SOCS1 and decrease in SOCS3 protein were also detected after adding ADAM10 in MDSCs (Figure 7F). These results revealed that ADAM10 promotes the suppression of SOCS3 expression and phosphorylation of STAT proteins in MDSCs. This indicates that IL-6 trans-signaling is predominately mediated by soluble CD126 to regulate IL-6-dependent SOCS3 (D) The whole cellular lysates were collected for western blot analysis. SOCS1 protein was notably increased, but SOCS3 protein was dramatically decreased in MDSCs as compared with those in CD33 + negative controls and iDC positive controls (n = 5). (e) RT-PCR show that Ab-treated MDSCs displayed slightly decreased mRNA level in SOCS1 (P = 0.0917), but significantly higher mRNA levels in SOCS3 compared to in Ab-untreated MDSCs (P = 0.0117) (n = 5). (F) Consistent results were confirmed at the protein level (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.
suppression and sustained activation of the JAK/STAT pathway in MDSCs, as well as coordinates the differentiation and immunosuppressive activity of MDSCs in breast cancer.

DiscUssiOn
Multiple immunocytes recruited into the tumor microenvironment play pivotal roles in tumorigenesis (31). However, MDSCs represent a specific subset of heterogeneous immunosuppressive cells that enable cancer cells to escape immune surveillance and inhibit the host immune system attack on cancer cells (32). Bronte et al. recommended the characterization standards and nomenclature of MDSCs and indicated that MDSCs are often divided into two subtypes in humans: PMN-MDSCs and MO-MDSCs (5). In addition to these MDSCs subtypes, the eMDSC subtype is marked with Lin − HLA-DR − CD33 + and comprised of more immature progenitors than M-MDSCs and PMN-MDSCs (5).
However, the MDSC subset is tumor-dependent. Previous studies of breast cancer examined MDSCs in mouse models rather than in humans because of the uncertainty of cell phenotypes and complicated regulatory mechanisms in human MDSCs (33)(34)(35). Determining the precise phenotype of breast cancer MDSCs in humans improves the understanding of the crosstalk between cancer cells and the microenvironment in the initiation and progression of breast cancer.
In our previous study, we identified a subset of poorly differentiated eMDSCs in breast cancer displaying potent suppression of T cells in vitro and vivo (6). As a pan-myeloid marker, CD33 is expressed earlier and more extensively in the myeloid lineage, and we found that CD33 + HLA-DR − cells rather than CD14 + HLA-DR − cells and CD11b + HLA-DR − cells were increased in patient blood samples compared to in healthy donor blood samples (6). We further detected the expression of a series of markers of myeloid linage, including HLA-DR, CD15, CD14, CD13, and CD11b. We . We found the mRNA levels of CD126 and gp130 in primary myeloid-derived suppressor cells (MDSCs) were higher than those in CD33 + and CD14 + controls (n = 5). (B) The mRNA levels of CD126 and gp130 in primary MDSCs IL-6h were higher than those in MDSCs  . Linear regression analysis demonstrated that CD126, rather than gp130, was positively correlated with IL-6 levels (R 2 = 0.6717, P < 0.0001) (n = 20). (c) The expressions of CD126 and gp130 in induced MDSCs were also examined. MDSCs exhibited higher mRNA levels of CD126 and gp130 compared to in CD33 + controls (n = 3). (D) Similar results were obtained at the protein level, in which CD126 expression was significantly increased, while gp130 showed no significant changes in the expression (n = 3). (e-g) IL-6R antibody was used to block IL-6 signal and the downstream signaling pathway. (e) Phosphorylation levels of JAK1, JAK2, TYK2, and STAT3 proteins decreased after the addition of the anti-IL-6R neutralizing antibody (n = 3). (F) The mRNA level of SOCS3 was increased, while the mRNA level of SOCS1 was decreased after blocking of CD126 (n = 3). (g) The expression of corresponding proteins in MDSCs displayed the same trend as that of mRNA after CD126 blocking. β-actin blots were used as protein loading controls (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. In this study, we demonstrated the positive correlations between MDSCs in situ and numbers of metastatic lymph nodes, tumor volume, pathological stage, and histology grade. Furthermore, we confirmed the negative correlation between MDSCs and OS in breast cancer patients and found that patients with more MDSCs showed worse clinical outcomes. Similar findings were reported in other tumors, such as in digestive system malignant tumors (38), prostate cancer (39), and advanced melanoma (40). Our results indicate that MDSCs are unfavorable prognostic factors in breast cancer patients.
In this study, we evaluated the correlation between tumor-derived IL-6 and MDSC infiltration in 253 paraffin-embedded primary breast tissues and 20 fresh breast cancer tissues. We found that more MDSCs infiltrated IL-6 high-expressing cancer tissues, and that tumor-derived IL-6 displayed a strong positive correlation with the number of infiltrating MDSCs in situ at both the mRNA and protein levels. Furthermore, we demonstrated that tumor-derived IL-6 was essential for MDSCs amplification and function in vitro, including promoting T cells apoptosis, inhibiting T cell proliferation, decreasing IFN-γ secretion, and increasing IL-10 production. Therefore, determining the detailed molecular mechanisms that regulate IL-6-dependent recruitment and amplification of MDSCs in breast cancer may help screen for potential therapeutic targets to eradicate MDSCs and reverse MDSCs-mediated immune tolerance in breast cancer patients. Interleukin-6 signals are transduced via the JAK/STAT signaling pathway in most cell types (44)(45)(46). Aberrant activation of the JAK/STAT signaling pathway in MDSCs has been reported in pancreatic cancer (15) and multiple myeloma (47). Physiologically, cytokine signal transduction can be switched off by SOCS proteins (48). Therefore, the activation of the JAK/STAT signaling pathway is rapid and reversible in normal cells. However, defects in SOCS expression frequently occur in malignant cells (16,19), causing sustained phosphorylation of key proteins along the JAK/ STAT signaling pathway (16,17). In this study, we found that tumor-derived IL-6 triggers the differentiation and immunosuppressive activity of MDSCs. This was accompanied by sustained activation of the JAK/STAT signaling pathway, which led to phosphorylation of the STAT1, STAT3, JAK1, JAK2, and TYK2 proteins. Furthermore, the activation of the JAK/STAT signaling pathway in MDSCs was persistent, and lasted longer than that in normal myeloid controls. Accordingly, significant suppression of SOCS3 at both the RNA and protein levels was observed in MDSCs. Therefore, significant defects in the SOCS feedback loop may participate in the regulation of IL-6-dependent, sustained activation of the JAK/STAT signaling pathway in MDSCs.
The SOCS protein family consists of SOCS1-7 and CIS, which are divided into three subgroups: CIS and SOCS1-3, SOCS4/5, and SOCS6/7. CIS and SOCS1-3 are associated with the control of cytokine signaling, whereas the SOCS4-7 subgroup regulates the growth factor-induced receptor tyrosine kinase signaling (19). As reported previously, the expression of SOCS proteins is rapidly upregulated by IL-6, among which SOCS3 is the most important, and in turn, inhibits IL-6 cytokine signaling (48,49). Numerous reports showed that SOCS3 defects are responsible for sustained IL-6/STAT3 signaling in human cancers (16,50,51). However, few studies have examined the expression of SOCS3 in immune cells. SOCS3 can also regulate the activation and differentiation of naïve CD4 + T cells, preferentially by promoting Th2 and inhibiting Th1 differentiation (52). In addition, SOCS3 can regulate the activation of DCs and polarization of macrophages (53,54).
Regarding MDSCs, recent studies demonstrated that SOCS3 negatively regulates the development and function of MDSCs via inhibition of STAT3 activation in prostate cancer (25). SOCS3deficient mice showed elevated Gr-1 + CD11b + MDSCs in tumors and exhibited heightened STAT3 activation (25). Consistent with the above results, we found that SOCS3 was significantly decreased in primary breast cancer MDSCs and induced MDSCs and was significantly correlated with sustained activation of the JAK/ STAT signaling pathway and enhanced T cells immunosuppression in MDSCs. Furthermore, in a co-culture system in vitro, we demonstrated that suppressed expression of SOCS3 was initiated by IL-6. This explains the phenomenon observed in our previous study, which showed that cancer-derived IL-6-induced T cell suppression in primary MDSCs by activating STAT3-dependent, IL-6 Trans-Signaling Pathway Promotes MDSCs Frontiers in Immunology | www.frontiersin.org December 2017 | Volume 8 | Article 1840 nuclear factor-κB-mediated long-term IDO overexpression (7). Thus, SOCS3 defects may be the main cause of IL-6-induced persistent activation of the JAK/STAT signaling pathway and consequent enhanced differentiation and immunosuppressive activity of MDSCs. Interestingly, in this study, we also found synchronous yet opposing changes in SOCS1 and SOCS3 expression at both the mRNA and protein levels. In contrast to SOCS3, SOCS1 expression was dramatically increased by IL-6-dependent sustained activation of the JAK/STAT signaling pathway in MDSCs. Both SOCS1 and SOCS3 have been demonstrated to inhibit phosphorylation of gp130, STATs, and JAK proteins along the JAK/ STAT signaling pathway (48,49). However, for IFN-α and IFN-γ secretion, SOCS1 is not as efficient as SOCS3 in inhibiting IL-6dependent activation of the JAK/STAT signaling pathway (55). SOCS3 is associated with specific phosphotyrosine motifs within the activated IL-6 receptor gp130 (56)(57)(58), which directly inhibit the catalytic domains of JAK1, JAK2, and TYK2 (59). This may explain the relative specificity of SOCS3 in inhibiting IL-6 pathways. Therefore, the increase in SOCS1 may be a consequence of sustained IL-6 stimulation, which is consistent with the results of other studies (60,61).
We further demonstrated that IL-6-induced inhibition of SOCS3 and activation of the JAK/STAT pathway was correlated with the elevated expression of CD126 via the IL-6 trans-signaling pathway. The IL-6 signaling complex assembly is composed of IL-6, CD126, and the shared signaling receptor gp130. CD126 exists in two forms, membrane-bound and soluble CD126. IL-6 signal transduction via membrane-bound CD126 is known as the cis-signaling pathway, while signal transduction via soluble CD126 is known as the trans-signaling pathway (8). The IL-6 cissignaling pathway is mainly limited to hepatocytes, megakaryocytes, neutrophils, and certain T cell subsets (62). In contrast, the IL-6 trans-signaling pathway can potentially stimulate all types of cells that do not express membrane-bound IL-6R. During IL-6 trans-signaling, the soluble form of CD126 is generated either by alternative splicing or shedding of membrane-bound IL-6R, which is mediated by the metalloproteases ADAM10 and ADAM17 (29,62,63).
Previous studies of breast cancer indicated that MDSCs express ADAM-family proteases and IL-6Rα, which contribute to breast cancer cell invasiveness and distant metastasis through the IL-6 trans-signaling pathway in murine models (10). In our study, we compared the expression of IL-6R in MDSCs and found that both CD126 and gp130 were increased in MDSCs. However, while the soluble form of CD126 was increased, membranebound CD126 was decreased. Importantly, we reported that MDSCs express higher levels of ADAM10 as compared to that in CD33 + controls. These results indicate that a higher level of soluble CD126 in MDSCs may be derived from enhanced shedding of membrane-bound IL-6R by ADAM10. To verify the effect of ADAM10, we added exogenous ADAM10 to increase the level of soluble CD126. This resulted in enhanced suppression of SOCS3 and phosphorylation of STAT1 and STAT3 in MDSCs. Although a previous study demonstrated that reduced expression of membrane-bound CD126 may result in impaired IL-6 classic signaling, followed by decreased phosphorylation of STAT3 and STAT1 (64), our results indicated that soluble CD126-mediated IL-6 trans-signaling pathway is sufficient for IL-6 signal transduction in MDSCs. Downstream effects include persistent activation of the JAK/STAT pathway and generation of more immunosuppressive MDSCs via suppression of SOCS3.
Taken together, this study provides insight into the cross-talk between breast cancer cells and regulatory immunocytes in local microenvironments. In breast cancer, tumor-derived IL-6 predominantly modulates the differentiation and immunosuppressive ability of MDSCs at both the tissue and cellular levels in which the soluble CD126-mediated IL-6 trans-signaling pathway and SOCS3 suppression are the most crucial molecular events orchestrating IL-6-dependent sustained activation of the JAK/ STAT pathway in breast cancer MDSCs. Therefore, blocking the IL-6 signaling pathway is a promising therapeutic strategy for eliminating and inhibiting MDSCs, as well as reversing MDSCsmediated immune escape in breast cancer.