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Circulating myeloid-derived suppressor cells in patients with pancreatic cancer
MDSCs in pancreatic cancer Original Article / Pancreas
Circulating myeloid-derived suppressor cells in patients with pancreatic cancer Xiao-Dong Xu, Jun Hu, Min Wang, Feng Peng, Rui Tian, Xing-Jun Guo, Yu Xie and Ren-Yi Qin Wuhan, China
BACKGROUND: Myeloid-derived suppressor cells (MDSCs) KEY WORDS: pancreatic cancer; are heterogeneous cell types that suppress T-cell responses in myeloid-derived suppressor cells; cancer patients and animal models, some MDSC subpopulagranulocyte-macrophage colony-stimulating factor; tions are increased in patients with pancreatic cancer. The arginase present study was to investigate a specific subset of MDSCs in patients with pancreatic cancer and the mechanism of MDSCs increase in these patients.
Introduction
P
METHODS: Myeloid cells from whole blood were collected from ancreatic cancer is a highly aggressive disease that is 37 patients with pancreatic cancer, 17 with cholangiocarcinoma, usually at an advanced stage at diagnosis in most paand 47 healthy controls. Four pancreatic cancer cell lines were cotients and therefore, the prognosis is extremely poor. cultured with normal peripheral blood mononuclear cells (PBMCs) to test the effect of tumor cells on the conversion of PBMCs to It is the sixth most common cause of cancer-related morMDSCs. Levels of granulocyte-macrophage colony-stimulating tality in China. Novel therapies, such as immunotherapy, factor (GM-CSF) and arginase activity in the plasma of cancer are therefore urgently needed for treatment of this disease. 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. (Hepatobiliary Pancreat Dis Int 2016;15:99-105)
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: [email protected]) © 2016, Hepatobiliary Pancreat Dis Int. All rights reserved. doi: 10.1016/S1499-3872(15)60413-1 Published online September 9, 2015.
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 granulocytemacrophage colony stimulating factor (GM-CSF) rep-
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resents 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.
Methods Patients 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.
Table. Patient characteristics Pancreatic cancer Resectable* Unresectable* (n=37) (n=22) (n=15) Age (mean±SD, yr) 57±11 56±7 59±10 Gender (Male/Female) 20/17 12/10 8/7 Clinical TNM stage# I 2 2 0 II 6 6 0 III 19 13 6 IV 10 1 9 Tumor site Head, neck 26 16 10 Body, tail 11 6 5 Histological grade△ Well/moderately 7 6 1 Poorly 5 3 2 Resection Yes 12 9 3 No 25 13 12 Variables
*: Resectable pancreatic cancer corresponds to AJCC stages I and II; unresectable to AJCC stages III and V. #: Stage according to the AJCC classification for pancreatic cancer. △: Of the 22 resectable cancer patients, 9 patients received curative resections eventually, 2 abandoned treatment in our hospital, and 3 received palliative operation; 3 of the 15 unresectable patients received curative resection as well.
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 Cell lines and cell culture were mainly found among peripheral blood mononuclear Pancreatic cancer cell lines were obtained from the cells (PBMCs; P1), which were then gated based on their Cell Repository, Chinese Academy of Sciences. BxPC-3 + + CD14 and CD11b expressions (P2); the HLA-DR- cell and SW-1990 were cultured in RPMI-1640 (Gibco, In our study, Life Technologies Corporation, Shanghai, China), Mia fraction was determined from this population. + MDSCs were thus defined as CD14 /CD11b+/HLA-DR-, PaCa-2 and Panc-1 were in Dulbecco’s Modified Eagle’s [15] Media (Gibco), supplemented with 10% FBS (Gibco), at based on the study performed by Rodrigues et al. 37 ℃ in a humidified 5% CO2 incubator, and passaged Cell isolation 2-3 times per week by brief trypsinization. PBMCs were isolated from the peripheral blood of healthy controls and cancer patients by density gradient Antibodies and flow cytometry analysis All monoclonal antibodies used in the study were centrifugation. In brief, blood was collected in heparinpurchased from BD Biosciences. Staining was performed treated tubes, diluted with RPMI 1640 medium, and careon fresh venous blood collected in heparin-lithium- fully layered onto a density gradient Ficoll-Hypaque (Hao green tubes. Briefly 100 µL of blood was mixed with 10 Yang, Tianjin, China). After centrifugation, the PBMC µL of each antibody or isotype control. Tubes were incu- band was aspirated and washed three times with ice-cold bated at 4 ℃ away from light for 30 minutes. After incu- phosphate buffered saline containing 1% human serum. bation, each sample was mixed with 2 mL of red blood cell lysing buffer and incubated for 10 minutes twice. MDSC induction in vitro Samples were washed with BD Pharmingen Stain Buffer. In 12-well plates, 1×105 healthy control PBMCs were Pellets were resuspended in 200 µL of the buffer. Samples separately incubated with four pancreatic cancer cell lines were acquired and analyzed by flow cytometry using LSR for 48 hours at 37 ℃ in AIM V serum-free media (Gibco). 100 • Hepatobiliary Pancreat Dis Int，Vol 15，No 1 • February 15，2016 • www.hbpdint.com
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Controls were cultured alone. The induced PBMCs were Statistical analysis then harvested by scraping loosely attached cells. In co-culData were analyzed using GraphPad Prism version 5.0 ture experiments, isolated PBMCs were mixed with tumor (GraphPad Software Inc, La Jolla, CA, USA), and Student’ s cell lines. t test was performed using SPSS 17.0 statistical software when two conditions were compared. A P<0.05 was conSerum arginase assay sidered statistically significant. Graphical data were prePart samples of pancreatic cancer patients (n=27) sented as means with standard errors. and controls (n=27) was analyzed for plasma arginase activity using a commercially available ELISA kit, specific for human arginase (Boster, Wuhan, China). Hemolyzed Results samples were excluded. Samples were assayed in tripli+ + cate. Mean absorbance was calculated from the standard Elevation of CD14 /CD11b /HLA-DR MDSCs in patients with pancreatic cancer curve. Arginase activity was expressed as units per liter. Since MDSCs apparently increased in the peripheral Serum GM-CSF ELISA blood of patients with pancreatic cancer,[6-11] we ana+ + Part samples of pancreatic cancer patients (n=29) lyzed CD14 /CD11b /HLA-DR MDSCs in freshly drawn and controls (n=25) were analyzed for plasma GM-CSF whole blood from patients with varying stages of pancrelevels using a commercially available ELISA kit specific atic cancer (Table), and calculated them as percentages of for human GM-CSF (Boster). Hemolyzed samples were PBMCs. Representative flow cytometry plots of a healthy excluded. Samples were assayed in triplicate. Mean ab- control and two patients with stage I and III cancer resorbance was calculated from the standard curve. spectively are shown in Fig. 1. More circulating MDSCs
Fig. 1. MDSCs defined by flow cytometry. Fresh whole blood was incubated with monoclonal antibodies against CD14, CD11b, and HLA-DR. MDSC percentage was calculated as the percentage of total nucleated cells in whole blood samples. Representative flow cytometric dot plots of a healthy control (A), and patients with stage I (B) and stage III (C) pancreatic cancer. Hepatobiliary Pancreat Dis Int，Vol 15，No 1 • February 15，2016 • www.hbpdint.com • 101
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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+/HLADR- MDSCs was observed in 17 patients with cholangiocarcinoma (P<0.05).
Correlation of frequency of MDSCs in peripheral blood with clinical cancer stage We then grouped patients by clinical cancer stage.
Fig. 2. Total percentage of circulating MDSCs was correlated with clinical tumor stage. A: Significantly higher percentages of circulating MDSCs were seen in pancreatic cancer patients and bile duct cancer patients than in the normal controls; B: Differences were seen between normal controls and cancer patients of all stages; C: Percentages of MDSCs differed significantly between cancer patients at stage IV cancer patients and those at stages I/II and III. PC: pancreatic cancer; BDC: bile duct cancer.
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).
Induction of tumor-associated MDSCs in vitro 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-DRMDSCs 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
Fig. 3. Four pancreatic cancer cell lines successfully induced normal human PBMCs into MDSCs in vitro. Cancer cell lines were co-cultured with PBMCs from four healthy controls to educate human MDSCs. A: CD14+/CD11b+ /HLA-DR- MDSCs were generated by all four pancreatic cancer cell lines and increased with cell numbers; B: The four pancreatic cancer cell lines had different capacities to induce MDSCs when co-cultured with the same amount of healthy human PBMCs (10:1). *: P<0.05, compared with the PBMC group.
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Fig. 4. Patients with pancreatic cancer elevated levels of plasma GM-CSF and arginase activity. (A) Serum samples from patients with pancreatic cancer (n=29) and healthy donors (n=25) were thawed, and GM-CSF was measured using an ELISA. Data represent mean±standard error from three different experiments (P=0.193). (B) Serum arginase levels for healthy, age-matched donors (n=27) and patients with pancreatic cancer (n=27) were measured. Serum samples from pancreatic cancer patients had significantly higher levels of arginase than did control plasma samples (P=0.006). PC: pancreatic cancer.
cells had the strongest MDSC-inducing capacity (Fig. 3B).
Elevated plasma GM-CSF and arginase activity in patients with pancreatic cancer Multiple cytokines and growth factors, such as GMCSF, 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).
Discussion 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 cocultured 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, granulocyteCSF, 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] GMCSF 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
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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.
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.
References
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Circulating myeloid-derived suppressor cells in patients with pancreatic cancer Xiao-Dong Xu, Jun Hu, Min Wang, Feng Peng, Rui Tian, Xing-Jun Guo, Yu Xie and Ren-Yi Qin Wuhan, China
BACKGROUND: Myeloid-derived suppressor cells (MDSCs) KEY WORDS: pancreatic cancer; are heterogeneous cell types that suppress T-cell responses in myeloid-derived suppressor cells; cancer patients and animal models, some MDSC subpopulagranulocyte-macrophage colony-stimulating factor; tions are increased in patients with pancreatic cancer. The arginase present study was to investigate a specific subset of MDSCs in patients with pancreatic cancer and the mechanism of MDSCs increase in these patients.
Introduction
P
METHODS: Myeloid cells from whole blood were collected from ancreatic cancer is a highly aggressive disease that is 37 patients with pancreatic cancer, 17 with cholangiocarcinoma, usually at an advanced stage at diagnosis in most paand 47 healthy controls. Four pancreatic cancer cell lines were cotients and therefore, the prognosis is extremely poor. cultured with normal peripheral blood mononuclear cells (PBMCs) to test the effect of tumor cells on the conversion of PBMCs to It is the sixth most common cause of cancer-related morMDSCs. Levels of granulocyte-macrophage colony-stimulating tality in China. Novel therapies, such as immunotherapy, factor (GM-CSF) and arginase activity in the plasma of cancer are therefore urgently needed for treatment of this disease. 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. (Hepatobiliary Pancreat Dis Int 2016;15:99-105)
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: [email protected]) © 2016, Hepatobiliary Pancreat Dis Int. All rights reserved. doi: 10.1016/S1499-3872(15)60413-1 Published online September 9, 2015.
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 granulocytemacrophage colony stimulating factor (GM-CSF) rep-
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resents 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.
Methods Patients 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.
Table. Patient characteristics Pancreatic cancer Resectable* Unresectable* (n=37) (n=22) (n=15) Age (mean±SD, yr) 57±11 56±7 59±10 Gender (Male/Female) 20/17 12/10 8/7 Clinical TNM stage# I 2 2 0 II 6 6 0 III 19 13 6 IV 10 1 9 Tumor site Head, neck 26 16 10 Body, tail 11 6 5 Histological grade△ Well/moderately 7 6 1 Poorly 5 3 2 Resection Yes 12 9 3 No 25 13 12 Variables
*: Resectable pancreatic cancer corresponds to AJCC stages I and II; unresectable to AJCC stages III and V. #: Stage according to the AJCC classification for pancreatic cancer. △: Of the 22 resectable cancer patients, 9 patients received curative resections eventually, 2 abandoned treatment in our hospital, and 3 received palliative operation; 3 of the 15 unresectable patients received curative resection as well.
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 Cell lines and cell culture were mainly found among peripheral blood mononuclear Pancreatic cancer cell lines were obtained from the cells (PBMCs; P1), which were then gated based on their Cell Repository, Chinese Academy of Sciences. BxPC-3 + + CD14 and CD11b expressions (P2); the HLA-DR- cell and SW-1990 were cultured in RPMI-1640 (Gibco, In our study, Life Technologies Corporation, Shanghai, China), Mia fraction was determined from this population. + MDSCs were thus defined as CD14 /CD11b+/HLA-DR-, PaCa-2 and Panc-1 were in Dulbecco’s Modified Eagle’s [15] Media (Gibco), supplemented with 10% FBS (Gibco), at based on the study performed by Rodrigues et al. 37 ℃ in a humidified 5% CO2 incubator, and passaged Cell isolation 2-3 times per week by brief trypsinization. PBMCs were isolated from the peripheral blood of healthy controls and cancer patients by density gradient Antibodies and flow cytometry analysis All monoclonal antibodies used in the study were centrifugation. In brief, blood was collected in heparinpurchased from BD Biosciences. Staining was performed treated tubes, diluted with RPMI 1640 medium, and careon fresh venous blood collected in heparin-lithium- fully layered onto a density gradient Ficoll-Hypaque (Hao green tubes. Briefly 100 µL of blood was mixed with 10 Yang, Tianjin, China). After centrifugation, the PBMC µL of each antibody or isotype control. Tubes were incu- band was aspirated and washed three times with ice-cold bated at 4 ℃ away from light for 30 minutes. After incu- phosphate buffered saline containing 1% human serum. bation, each sample was mixed with 2 mL of red blood cell lysing buffer and incubated for 10 minutes twice. MDSC induction in vitro Samples were washed with BD Pharmingen Stain Buffer. In 12-well plates, 1×105 healthy control PBMCs were Pellets were resuspended in 200 µL of the buffer. Samples separately incubated with four pancreatic cancer cell lines were acquired and analyzed by flow cytometry using LSR for 48 hours at 37 ℃ in AIM V serum-free media (Gibco). 100 • Hepatobiliary Pancreat Dis Int，Vol 15，No 1 • February 15，2016 • www.hbpdint.com
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Controls were cultured alone. The induced PBMCs were Statistical analysis then harvested by scraping loosely attached cells. In co-culData were analyzed using GraphPad Prism version 5.0 ture experiments, isolated PBMCs were mixed with tumor (GraphPad Software Inc, La Jolla, CA, USA), and Student’ s cell lines. t test was performed using SPSS 17.0 statistical software when two conditions were compared. A P<0.05 was conSerum arginase assay sidered statistically significant. Graphical data were prePart samples of pancreatic cancer patients (n=27) sented as means with standard errors. and controls (n=27) was analyzed for plasma arginase activity using a commercially available ELISA kit, specific for human arginase (Boster, Wuhan, China). Hemolyzed Results samples were excluded. Samples were assayed in tripli+ + cate. Mean absorbance was calculated from the standard Elevation of CD14 /CD11b /HLA-DR MDSCs in patients with pancreatic cancer curve. Arginase activity was expressed as units per liter. Since MDSCs apparently increased in the peripheral Serum GM-CSF ELISA blood of patients with pancreatic cancer,[6-11] we ana+ + Part samples of pancreatic cancer patients (n=29) lyzed CD14 /CD11b /HLA-DR MDSCs in freshly drawn and controls (n=25) were analyzed for plasma GM-CSF whole blood from patients with varying stages of pancrelevels using a commercially available ELISA kit specific atic cancer (Table), and calculated them as percentages of for human GM-CSF (Boster). Hemolyzed samples were PBMCs. Representative flow cytometry plots of a healthy excluded. Samples were assayed in triplicate. Mean ab- control and two patients with stage I and III cancer resorbance was calculated from the standard curve. spectively are shown in Fig. 1. More circulating MDSCs
Fig. 1. MDSCs defined by flow cytometry. Fresh whole blood was incubated with monoclonal antibodies against CD14, CD11b, and HLA-DR. MDSC percentage was calculated as the percentage of total nucleated cells in whole blood samples. Representative flow cytometric dot plots of a healthy control (A), and patients with stage I (B) and stage III (C) pancreatic cancer. Hepatobiliary Pancreat Dis Int，Vol 15，No 1 • February 15，2016 • www.hbpdint.com • 101
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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+/HLADR- MDSCs was observed in 17 patients with cholangiocarcinoma (P<0.05).
Correlation of frequency of MDSCs in peripheral blood with clinical cancer stage We then grouped patients by clinical cancer stage.
Fig. 2. Total percentage of circulating MDSCs was correlated with clinical tumor stage. A: Significantly higher percentages of circulating MDSCs were seen in pancreatic cancer patients and bile duct cancer patients than in the normal controls; B: Differences were seen between normal controls and cancer patients of all stages; C: Percentages of MDSCs differed significantly between cancer patients at stage IV cancer patients and those at stages I/II and III. PC: pancreatic cancer; BDC: bile duct cancer.
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).
Induction of tumor-associated MDSCs in vitro 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-DRMDSCs 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
Fig. 3. Four pancreatic cancer cell lines successfully induced normal human PBMCs into MDSCs in vitro. Cancer cell lines were co-cultured with PBMCs from four healthy controls to educate human MDSCs. A: CD14+/CD11b+ /HLA-DR- MDSCs were generated by all four pancreatic cancer cell lines and increased with cell numbers; B: The four pancreatic cancer cell lines had different capacities to induce MDSCs when co-cultured with the same amount of healthy human PBMCs (10:1). *: P<0.05, compared with the PBMC group.
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Fig. 4. Patients with pancreatic cancer elevated levels of plasma GM-CSF and arginase activity. (A) Serum samples from patients with pancreatic cancer (n=29) and healthy donors (n=25) were thawed, and GM-CSF was measured using an ELISA. Data represent mean±standard error from three different experiments (P=0.193). (B) Serum arginase levels for healthy, age-matched donors (n=27) and patients with pancreatic cancer (n=27) were measured. Serum samples from pancreatic cancer patients had significantly higher levels of arginase than did control plasma samples (P=0.006). PC: pancreatic cancer.
cells had the strongest MDSC-inducing capacity (Fig. 3B).
Elevated plasma GM-CSF and arginase activity in patients with pancreatic cancer Multiple cytokines and growth factors, such as GMCSF, 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).
Discussion 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 cocultured 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, granulocyteCSF, 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] GMCSF 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
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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.
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.
References
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