Author Affiliations: Department of Biliary-Pancreatic Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China (Xu XD, Wang M, Peng F, Tian R, Guo XJ, Xie Y and Qin RY); Department of Colon Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China (Hu J)

Corresponding Author: Ren-Yi Qin, PhD, Department of Biliary-Pancreatic Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China (Tel/Fax: +86-27-83665294; Email: ryqin@tjh.tjmu.edu.cn)

 

© 2016, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60413-1

Published online September 9, 2015.

 

 

Acknowledgement: We thank Professor Jian-Xin Jiang for his help in preparation of this manuscript.

Contributors: WM and QRY proposed the study and revised the article. XXD and HJ performed the research and wrote the first draft. PF, TR, GXJ and XY collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. QRY is the guarantor.

Funding: This study was supported by grants from the National Natural Science Foundation of China (81071775, 81272659, 81101621, 81160311, 81172064, 81001068, 81272425 and 81101870), National “Eleventh Five-Year” Scientific and Technological Support Projects (2006BAI02A13-402), Key Projects of Science Foundation of Hubei Province (2011CDA030), and Research Fund of Young Scholars for the Doctoral Program of Higher Education of China (20110142120014).

Ethical approval: These studies were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of the article.

 

 

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are heterogeneous cell types that suppress T-cell responses in cancer patients and animal models, some MDSC subpopulations are increased in patients with pancreatic cancer. The present study was to investigate a specific subset of MDSCs in patients with pancreatic cancer and the mechanism of MDSCs increase in these patients.

 

METHODS: Myeloid cells from whole blood were collected from 37 patients with pancreatic cancer, 17 with cholangiocarcinoma, and 47 healthy controls. Four pancreatic cancer cell lines were co-cultured with normal peripheral blood mononuclear cells (PBMCs) to test the effect of tumor cells on the conversion of PBMCs to MDSCs. Levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and arginase activity in the plasma of cancer patients were analyzed by enzyme-linked immunosorbent assay.

 

RESULTS: CD14⁺/CD11b⁺/HLA-DR^- MDSCs were increased in patients with pancreatic or bile duct cancer compared with those in healthy controls, and this increase was correlated with clinical cancer stage. Pancreatic cancer cell lines induced PBMCs to MDSCs in a dose-dependent manner. GM-CSF and arginase activity levels were significantly increased in the serum of patients with pancreatic cancer.

 

CONCLUSIONS: MDSCs were tumor related: tumor cells induced PBMCs to MDSCs in a dose-dependent manner and circulating CD14⁺/CD11b⁺/HLA-DR^- MDSCs in pancreatic cancer patients were positively correlated with tumor burden. MDSCs might be useful markers for pancreatic cancer detection and progression.

 

KEY WORDS: pancreatic cancer; myeloid-derived suppressor cells; granulocyte-macrophage colony-stimulating factor; arginase

Pancreatic cancer is a highly aggressive disease that is usually at an advanced stage at diagnosis in most patients and therefore, the prognosis is extremely poor. It is the sixth most common cause of cancer-related mortality in China. Novel therapies, such as immunotherapy, are therefore urgently needed for treatment of this disease. Pancreatic cancer-associated antigens have spurred the development of vaccination-based treatment strategies. Although animal models have offered promising results, most clinical studies found only limited success,^[1] which may be due to immune suppression mechanisms.^[2] Abnormal accumulation of myeloid-derived suppressor cells (MDSCs) is thought to play a critical immunosuppressive role in tumor immune evasion and promotion.^[3]

 

MDSCs expand during cancer, inflammation and infection in both preclinical models and human patients. In mouse models, MDSCs are described as CD11b⁺/Gr-1⁺ cells. More recently, however, MDSCs have been divided into monocytic and granulocytic subsets, reflecting differential expression of Ly6C and Ly6G markers.^{[4, 5]} Although CD15⁺ granulocytic MDSCs and CD33⁺ MDSCs have been widely studied,^[6-11] the origin of monocytic MDSCs in pancreatic cancer is still not clear. It was demonstrated that in patients with hepatocellular carcinoma, CD14⁺ MDSCs induce T-cell anergy through multiple mechanisms.^[12,13] Tumor-secreted proinflammatory molecules induce MDSCs, leading to the hypothesis that inflammation promotes accumulation of MDSCs, which down-regulates immune surveillance and anti-tumor immunity and therefore, facilitates tumor growth.^[14] A recent study^[15] showed that monocytic MDSC subset has the immunoregulatory properties in many solid tumors and in vitro assays. Aberrantly expressed granulocyte-macrophage colony stimulating factor (GM-CSF) represents a key inflammatory component that facilitates monocytic MDSC accumulation.^[16] MDSCs have a remarkable ability to suppress T-cell reactivity by production of arginase, reactive oxygen species, inducible NO synthase, IL-10, IL-6, transforming growth factor-β, and sequestration of cysteine.^[17-21] The present study focused on the accumulation of monocytic MDSCs, their relevance to tumor burden and the possible mechanisms.

 

 

Peripheral blood specimens were collected from 37 patients with pancreatic cancer (mean age: 57 years; range: 50-71) and 17 patients with cholangiocarcinoma (mean age: 55 years; range: 53-68) who were newly diagnosed at the Department of Biliary-Pancreatic Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from May 2011 to October 2012 (Table). All cancer patients were grouped in accordance with the American Joint Committee on Cancer (AJCC) Cancer Staging Manual, 7th edition (2010). We collected 4 mL of venous blood from each patient in heparin-lithium-green top tubes (BD Biosciences, Bedford, MA, USA), before surgery, radiation, or any systemic chemotherapy. Forty-seven age-matched normal healthy volunteers served as controls. These studies were approved by the Ethics Committee of Tongji Medical College. Informed consent was obtained from all subjects.

 

Pancreatic cancer cell lines were obtained from the Cell Repository, Chinese Academy of Sciences. BxPC-3 and SW-1990 were cultured in RPMI-1640 (Gibco, Life Technologies Corporation, Shanghai, China), Mia PaCa-2 and Panc-1 were in Dulbecco’s Modified Eagle’s Media (Gibco), supplemented with 10% FBS (Gibco), at 37 �� in a humidified 5% CO₂ incubator, and passaged 2-3 times per week by brief trypsinization.

 

All monoclonal antibodies used in the study were purchased from BD Biosciences. Staining was performed on fresh venous blood collected in heparin-lithium-green tubes. Briefly 100 µL of blood was mixed with 10 µL of each antibody or isotype control. Tubes were incubated at 4 �� away from light for 30 minutes. After incubation, each sample was mixed with 2 mL of red blood cell lysing buffer and incubated for 10 minutes twice. Samples were washed with BD Pharmingen Stain Buffer. Pellets were resuspended in 200 µL of the buffer. Samples were acquired and analyzed by flow cytometry using LSR II (BD Biosciences). Fc receptors were blocked by pre-incubating cells with 10 µL of Fc receptor blocker (Miltenyi Biotec, Gladbach, Germany). Freshly drawn whole blood from patients was then labeled with FITC-conjugated CD14, PE-Cy7-conjugated CD11b, and APC-conjugated HLA-DR monoclonal antibodies. After lysing the red blood cells, flow cytometry samples were taken. Target cells were mainly found among peripheral blood mononuclear cells (PBMCs; P1), which were then gated based on their CD14⁺and CD11b⁺ expressions (P2); the HLA-DR^- cell fraction was determined from this population. In our study, MDSCs were thus defined as CD14⁺/CD11b⁺/HLA-DR^-, based on the study performed by Rodrigues et al.^[15]

 

PBMCs were isolated from the peripheral blood of healthy controls and cancer patients by density gradient centrifugation. In brief, blood was collected in heparin-treated tubes, diluted with RPMI 1640 medium, and carefully layered onto a density gradient Ficoll-Hypaque (Hao Yang, Tianjin, China). After centrifugation, the PBMC band was aspirated and washed three times with ice-cold phosphate buffered saline containing 1% human serum.

 

In 12-well plates, 1×10⁵ healthy control PBMCs were separately incubated with four pancreatic cancer cell lines for 48 hours at 37 �� in AIM V serum-free media (Gibco). Controls were cultured alone. The induced PBMCs were then harvested by scraping loosely attached cells. In co-culture experiments, isolated PBMCs were mixed with tumor cell lines.

 

Part samples of pancreatic cancer patients (n=27) and controls (n=27) was analyzed for plasma arginase activity using a commercially available ELISA kit, specific for human arginase (Boster, Wuhan, China). Hemolyzed samples were excluded. Samples were assayed in triplicate. Mean absorbance was calculated from the standard curve. Arginase activity was expressed as units per liter.

 

Part samples of pancreatic cancer patients (n=29) and controls (n=25) were analyzed for plasma GM-CSF levels using a commercially available ELISA kit specific for human GM-CSF (Boster). Hemolyzed samples were excluded. Samples were assayed in triplicate. Mean absorbance was calculated from the standard curve.

 

Data were analyzed using GraphPad Prism version 5.0 (GraphPad Software Inc, La Jolla, CA, USA), and Student’s t test was performed using SPSS 17.0 statistical software when two conditions were compared. A P<0.05 was considered statistically significant. Graphical data were presented as means with standard errors.

 

 

Since MDSCs apparently increased in the peripheral blood of patients with pancreatic cancer,^[6-11] we analyzed CD14⁺/CD11b⁺/HLA-DR^- MDSCs in freshly drawn whole blood from patients with varying stages of pancreatic cancer (Table), and calculated them as percentages of PBMCs. Representative flow cytometry plots of a healthy control and two patients with stage I and III cancer respectively are shown in Fig. 1. More circulating MDSCs were seen in the patients with pancreatic cancer (3.05%±2.71%) than in normal controls (0.20%±0.21%; P<0.01; Fig. 2A). A similar expansion of CD14⁺/CD11b⁺/HLA-DR^- MDSCs was observed in 17 patients with cholangiocarcinoma (P<0.05).

 

We then grouped patients by clinical cancer stage. The differences between healthy controls (n=47; 0.20%±0.21%) and patients with stages I/II (n=8; 1.08%±0.34%; P=0.03), stage III (n=19; 2.25%±1.21%; P=0.009), and stage IV (n=10; 5.42%±2.76%; P=0.0007) disease were statistically significant (Fig. 2B). Moreover, the mean percentage of MDSC increased significantly at each advance in disease stage (P<0.05), and stage IV cancer patients had the greatest percentage of MDSC (Fig. 2C).

 

Patients with pancreatic cancer had increased MDSCs in PBMCs compared with their healthy counterparts. To see if tumor burden and tumor-derived factors are responsible for MDSC accumulation, four pancreatic cancer cell lines were co-cultured with PBMCs from four healthy controls to produce MDSCs. CD14⁺/CD11b⁺/HLA-DR^- MDSCs were generated, and increased in number, in all four pancreatic cancer cell lines (Fig. 3A). We then compared the induction ability of the four cell lines with the same healthy PBMCs at the ratio of 10:1. The SW-1990 cells had the strongest MDSC-inducing capacity (Fig. 3B).

 

Multiple cytokines and growth factors, such as GM-CSF, VEGF, IL-6, and granulocyte-CSF, secreted in the tumor microenvironment promoted MDSC expansion and activation. However, serum GM-CSF levels in patients with pancreatic cancer were 54 pg/mL (n=29), not significantly different from those in healthy controls (62 pg/mL, n=25; Fig. 4A). L-arginine catabolismis was a primary mediator of MDSC immune suppression. Serum arginase activity was significantly elevated in patients with pancreatic cancer, compared with that in healthy controls (P=0.006; Fig. 4B).

 

 

MDSCs possess strong immunosuppressive activities which limit immune-based cancer therapies, and identifying and characterizing these cells are of vital importance. Previous studies^[6-11] confirmed the presence of CD33⁺ and CD15⁺ circulating MDSCs that suppress T-cell proliferation and cytokine production in patients with pancreatic cancer. Considering the heterogeneity of MDSC subgroups, we extended previous findings by analyzing the percentage, origin and immunosuppressive function of circulating CD14⁺ MDSCs in blood samples from patients with pancreatic cancer at various clinical stages.

 

Our results indicated that the percentage of monocytic MDSCs cells was significantly increased in patients with pancreatic cancer compared with that in healthy controls.^[6-11] This increase is parallel to the cancer stage. The results were not consistent with those from another study^[22] on colon cancer demonstrating that patients with stage I/II colon cancer had a moderate increase in CD33⁺ MDSCs.

 

Increased MDSCs have been reported in a variety of cancers and their accumulation appears to correlate with increased tumor burden in both animal models and cancer patients.^{[3, 7]} We hypothesize that exposure of PBMCs to cancer cells results in the conversion from normal PBMCs to a MDSC-like phenotype. To test this hypothesis, four human pancreatic cancer cell lines were co-cultured with PBMCs from healthy controls. A previous study found that three of 10 pancreatic cancer cell lines induce PBMCs to CD33⁺ or CD11b⁺ MDSCs,^[23] whereas all four pancreatic cancer cell lines in our study could induce CD14⁺ MDSCs. We found that CD14⁺ MDSCs were increased in parallel with disease stage in pancreatic cancer patients. Our data implied that CD14⁺ MDSCs were more specific for pancreatic cancer and might be a useful marker for pancreatic cancer detection and monitoring.

 

In both animal tumor models and cancer patients, tumor-derived factors, such as GM-CSF, granulocyte-CSF, IL-6, IL-13, prostaglandin E2 and VEGF, are responsible for the accumulation and activation of MDSC, which in turn contributed to tumor immune escape and the failure of anti-tumor immunotherapy.^[24-30] GM-CSF is important in mobilization of hematopoietic stem cells, progenitors and mature cells. It also plays a role in regulating MDSC accumulation in both animal pancreatic cancer models and cancer patients.^{[10, 15]} Furthermore, oncogenic K-ras-induced GM-CSF production may actively promote the development of MDSC.^{[24, 25]} We therefore determined whether GM-CSF mediated induction of MDSC, and thus we examined whether patients with pancreatic cancer had increased serum GM-CSF levels. However, no difference was found between patients with pancreatic cancer and the healthy controls. Among studies of GM-CSF as a tumor marker in pancreatic cancer, one found that serum GM-CSF is increased in patients with pancreatic cancer,^[31] whereas another study showed no difference.^[32] Research showed that IL-6 had a similar effect on GM-CSF.^[26] IL-6 derived from pancreatic cancer stroma was essential for MDSC generation, and IL-6 was critical for the development of pancreatic cancer in inducible K-ras driven mouse models.^[27] Furthermore, one study showed a synergistic effect of inducing MDSC between GM-CSF and IL-6.^[28] Therefore, multiple humoral factors produced by pancreatic cancer cells are likely responsible for MDSC proliferation. Two recent studies revealed that cytokines are diverse in patients with pancreatic cancer. Increased IL-6 other than granulocyte-CSF promoted systemic trafficking of immunosuppressive mesenchymal stem cells and other populations of bone marrow-derived cells.^{[33, 34]}

 

Expression of high levels of arginase is the first major mechanism of MDSC immune suppression. Lack of L-arginine in the microenvironment significantly inhibits T-cell function.^[35] Elevated arginase activity in serum and the tumor microenvironment has been reported in several cancers, including renal, melanoma and glioblastoma.^{[16, 36, 37]} We proposed that the increased serum arginase in patients with pancreatic cancer is due to elevated MDSCs. Understanding these mechanisms should provide insights into novel therapeutic approaches to block immunosuppression and enhance the effect of immunotherapy.

 

In conclusion, pancreatic cancer cells induced the conversion from PBMCs to MDSCs which suppressed the immunoresponse via the catabolism of L-arginine. Circulating CD14⁺/CD11b⁺/HLA-DR^- MDSCs correlated with clinical tumor stage. Pancreatic cancer burden was responsible for the abnormal accumulation of MDSC. MDSCs were a target in the treatment of patients with pancreatic cancer.

 

 

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