Kuirong Jiang

High expression of Galectin-1 in pancreatic stellate cells plays a role in the development and maintenance of an immunosuppressive microenvironment in pancreatic cancer

Galectin‐1 is implicated in making tumor cells immune privileged, in part by regulating the survival of infiltrating T cells. Galectin‐1 is strongly expressed in activated pancreatic stellate cells (PSCs); however, whether this is linked to tumor cell immune escape in pancreatic cancer is unknown. Galectin‐1 was knocked down in PSCs isolated from pancreatic tissues using small interfering RNA (siRNA), or overexpressed using recombinant lentiviruses, and the PSCs were cocultured with T cells. CD3+, CD4+ and CD8+ T cell apoptosis was detected by flow cytometry; T cell IL‐2, IL‐4, IL‐5 and INF‐γ production levels were quantified using ELISA. Immunohistochemical analysis showed increased numbers of PSCs expressed Galectin‐1 (p < 0.01) and pancreatic cancers had increased CD3+ T cell densities (p < 0.01) compared to normal pancreas or chronic pancreatitis samples. In coculture experiments, PSCs that overexpressed Galectin‐1 induced apoptosis of CD4+ T cells (p < 0.01) and CD8+ T cells (p < 0.05) significantly, compared to normal PSCs. Knockdown of Galectin‐1 in PSCs increased CD4+ T cell (p < 0.01) and CD8+ T cell viability (p < 0.05). Supernatants from T cells cocultured with PSCs that overexpressed Galectin‐1 contained significantly increased levels of Th2 cytokines (IL‐4 and IL‐5, p < 0.01) and decreased Th1 cytokines (IL‐2 and INF‐γ, p < 0.01). However, the knockdown of PSC Galectin‐1 had the opposite effect on Th1 and Th2 cytokine secretion. Our study suggests that the overexpression of Galectin‐1 in PSCs induced T cell apoptosis and Th2 cytokine secretion, which may regulate PSC‐dependent immunoprivilege in the pancreatic cancer microenvironment. Galectin‐1 may provide a novel candidate target for pancreatic cancer immunotherapy.

Persistent activation of pancreatic stellate cells creates a microenvironment favorable for the malignant behavior of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumors with poor prognosis due to extremely high malignancy, low rate of eligibility for surgical resection and chemoradiation resistance. Increasing evidence indicate that the interaction between activated pancreatic stellate cells (PSCs) and PDAC cells plays an important role in the development of PDAC. By producing high levels of cytokines, chemotactic factors, growth factors and excessive extracellular matrix (ECM), PSCs create desmoplasia and a hypoxic microenvironment that promote the initiation, development, evasion of immune surveillance, invasion, metastasis and resistance to chemoradiation of PDAC. Therefore, targeting the interaction between PSCs and PDAC cells may represent a novel therapeutic approach to advanced PDAC, especially therapies that target PSCs of the pancreatic tumor microenvironment.

Comprehensive analysis of the percentage of surface receptors and cytotoxic granules positive natural killer cells in patients with pancreatic cancer, gastric cancer, and colorectal cancer

BackgroundDigestive malignancies, especially pancreatic cancer (PC), gastric cancer (GC), and colorectal cancer (CRC), still occur at persistently high rates, and disease progression in these cancers has been associated with tumor immunosurveillance escape. Natural killer (NK) cell dysfunction may be responsible for this phenomenon, however, the exact relationship between tumor immunosurveillance escape in digestive malignancies and NK cell dysfunction remains unclear.MethodsPercentage of the surface receptors NKG2A, KIR3DL1, NKG2D, NKp30, NKp44, NKp46, and DNAM-1, as well as the cytotoxic granules perforin and granzyme B positive NK cells were determined in patients with pancreatic cancer (n = 31), gastric cancer (n = 31), and CRC (n = 32) prior to surgery and healthy controls (n = 31) by multicolor flow cytometry. Independent t-tests or Mann-Whitney U-tests were used to compare the differences between the patient and healthy control groups, as well as the differences between patients with different pathologic features of cancer.ResultsPercentage of NKG2D, NKp30, NKp46, and perforin positive NK cells was significantly down-regulated in patients with PC compared to healthy controls, as well as GC and CRC; reduced levels of these molecules was associated with indicators of disease progression in each malignancy (such as histological grade, depth of invasion, lymph node metastasis). On the contrary, percentage of KIR3DL1 positive NK cells was significantly increased in patients with PC, as well as GC and CRC, but was not associated with any indicators of disease progression.ConclusionsAltered percentage of surface receptors and cytotoxic granules positive NK cells may play a vital role in tumor immunosurveillance escape by inducing NK cell dysfunction in patients with PC, GC, and CRC.

Galectin-1 secreted by activated stellate cells in pancreatic ductal adenocarcinoma stroma promotes proliferation and invasion of pancreatic cancer cells: an in vitro study on the microenvironment of pancreatic ductal adenocarcinoma.

Objectives: This study aimed to clarify that the activated pancreatic stellate cells (PaSCs) are the origin of the highly expressed galectin-1 in the stroma of pancreatic ductal adenocarcinoma (PDAC) tissue and to evaluate the effect of the secreted galectin-1 on proliferation and invasion ability of pancreatic cancer cell line CFPAC-1 in vitro. Methods: Different kinds of PaSCs were isolated from the normal or cancerous pancreatic tissues and cultured. Immunohistochemistry study, quantitative polymerase chain reaction, and Western blot were carried out to check the cellular origin of galectin-1 in PDAC tissue. By using modified Boyden chambers, in vitro coculture system of PaSCs was established with the pancreatic cancer cell line CFPAC-1 and based on which we assessed the proliferation and invasion ability of CFPAC-1 with or without galectin-1 antagonists. Results: We identified PaSCs as the primary source of the highly expressed galectin-1 in PDAC stroma. Galectin-1 secreted by PaSCs increased CFPAC-1 proliferative rate in the proliferation assay and facilitated CFPAC-1 infiltration in the invasion assay. Conclusions: Under malignant circumstances, PaSCs express and secret galectin-1, which could further promote the proliferation and invasion of cancer cells.

RNA interference‐mediated silencing of the polo‐like kinase 1 gene enhances chemosensitivity to gemcitabine in pancreatic adenocarcinoma cells

Gemcitabine is the first‐line chemotherapeutic agent for advanced adenocarcinoma of the pancreas; however, chemoresistance to gemcitabine remains a major cause of failure for the clinical treatment of this disease. Polo‐like kinase 1 (Plk‐1) is highly expressed in pancreatic cancer cell lines and pancreatic tumour tissues, and is involved in a wide variety of cell cycle processes. Nevertheless, its biological role and implication for gemcitabine resistance are not clearly defined. In this study, we used RNA‐interference (RNAi)‐mediated depletion of Plk‐1 to determine its potential for sensitizing pancreatic tumour cells to gemcitabine. We showed that the level of Plk‐1 protein was correlated significantly with gemcitabine resistance in human pancreatic adenocarcinoma cells and that overexpression of Plk‐1 reduced sensitivity to gemcitabine in these cells. In addition, small interfering RNA (siRNA)‐mediated knockdown of Plk‐1 caused cell cycle arrest at G2/M and the reduction of cellular proliferation. More importantly, the treatment of pancreatic cancer cells with Plk‐1 siRNA followed by exposure to gemcitabine dramatically decreased cell viability and increased cellular apoptosis, as compared with treatment with either agent alone. These observations indicate that down‐regulation of Plk‐1 expression by RNAi enhances gemcitabine sensitivity and increases gemcitabine cytotoxicity in pancreatic tumour cells. This is the first demonstration that the combination of Plk‐1 gene therapy and gemcitabine chemotherapy has synergistic anti‐tumour activity against pancreatic carcinoma in vitro. This combination treatment warrants further investigation as an effective therapeutic regimen for patients with resistant pancreatic cancer and other tumours.

Yin Yang-1 suppresses invasion and metastasis of pancreatic ductal adenocarcinoma by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism

BackgroundIncreasing evidence indicates an important role of transcription factor Yin Yang-1 (YY1) in human tumorigenesis. However, its function in cancer remains controversial and the relevance of YY1 to pancreatic ductal adenocarcinoma (PDAC) remains to be clarified.MethodsIn this study, we detected YY1 expression in clinical PDAC tissue samples and cell lines using quantitative RT-PCR, immunohistochemistry and western blotting. We also detected MUC4 and MMP10 mRNA levels in 108 PDAC samples using qRT-PCR and analyzed the correlations between YY1 and MUC4 or MMP10 expression. The role of YY1 in the proliferation, invasion and metastatic abilities of PDAC cells in vitro was studied by CCK-8 assay, cell migration and invasion assays. In vivo pancreatic tumor growth and metastasis was studied by a xenogenous subcutaneously implant model and a tail vein metastasis model. The potential mechanisms underlying YY1 mediated tumor progression in PDAC were explored by digital gene expression (DGE) sequencing, signal transduction pathways blockage experiments and luciferase assays. Statistical analysis was performed using the SPSS 15.0 software.ResultsWe found that the expression of YY1 in PDACs was higher compared with their adjacent non-tumorous tissues and normal pancreas tissues. However, PDAC patients with high level overexpression of YY1 had better outcome than those with low level overexpression. YY1 expression levels were statistically negatively correlated with MMP10 expression levels, but not correlated with MUC4 expression levels. YY1 overexpression suppressed, whereas YY1 knockdown enhanced, the proliferation, invasion and metastatic properties of BXPC-3 cells, both in vitro and in vivo. YY1 suppresses invasion and metastasis of pancreatic cancer cells by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism.ConclusionsThe present study suggested that YY1 plays a negative role, i.e. is a tumor suppressor, in PDAC, and may become a valuable diagnostic and prognostic marker of PDAC.

Elevation of MMP-9 and IDO induced by pancreatic cancer cells mediates natural killer cell dysfunction

BackgroundNatural killer (NK) cells play a key role in non-specific immune response in different cancers, including pancreatic cancer. However the anti-tumor effect of NK cells decreases during pancreatic cancer progression. The regulatory pathways by which NK cells facilitate tumor immune escape are unclear, therefore our purpose was to investigate the roles of the contributory factors.MethodsNK cells isolated from fresh healthy peripheral blood were co-cultured with normal human pancreatic ductal cells hTERT-HPNE and human pancreatic cancer cell lines SW1990 and BxPc-3 in vitro. Then NK cell function was determined by Flow cytometric analysis of surface receptors and cytotoxic granules in NK cells, NK cell apoptosis and cytotoxicity, and Enzyme-linked immunosorbent assay of cytokines. Expression level of MMP-9, IDO and COX-2 in hTERT-HPNE and SW1990 cells were detected by quantitative RT-PCR. Statistical differences between data groups were determined by independent t-tests using SPSS 19.0 software.ResultsOur results showed that NK cell function was significantly downregulated following exposure to pancreatic cancer cells compared to normal pancreatic cells, as demonstrated by lower expressions of activating surface receptors (NKG2D, DNAM-1, NKp30 and NKp46) and cytotoxic granules (Perforin and Granzyme B); decreased secretion of cytokines (TNF-α and IFN-γ); and reduced cytotoxicity against myelogenous leukemia K562 cells. Further investigations revealed that MMP-9 and IDO may be implicated in SW1990 cell-induced NK cell dysfunction by facilitating tumor immune evasion. Blockade by TIMP-1 and/or 1-MT could partially restore NK function.ConclusionsTaken together, elevation of MMP-9 and IDO induced by pancreatic cancer cells mediates NK cell dysfunction. Our findings could contribute to the development of NK cell-based immunotherapy in patients with pancreatic cancer.

Apoptosis and anergy of T cell induced by pancreatic stellate cells-derived galectin-1 in pancreatic cancer

Galectin-1, a β-galactoside-binding protein implicated in cancer cell immune privilege, was highly expressed in activated pancreatic stellate cells (PSCs). This study was designed to investigate the relationship between PSC-derived galectin-1 and tumor immunity in pancreatic cancer. Isolated PSCs were identified as normal pancreas cells (hNPSCs) or pancreatic cancer cells (hCaPSCs) by immunohistochemical staining for α-SMA and vimentin, and galectin-1 expression was evaluated by Western blotting and quantitative RT-PCR. Apoptosis, caspase activity, and cytokine production (IL-6, IL-10, TNF-β, and IFN-γ) of T cells co-cultured with PSCs were evaluated, and immunohistochemical staining of galectin-1 was correlated with CD3 and clinicopathological variables in 66 pancreatic cancer and 10 normal pancreatic tissue samples. hCaPSCs exhibited higher galectin-1 expression than did hNPSCs, and hCaPSCs induced higher levels of apoptosis in T cells following co-culture. hCaPSCs activated caspase-9 and caspase-3 in the mitochondrial apoptotic pathway and stimulated secretion of Th2 cytokines (IL-6 and IL-10) but decreased secretion of Th1 cytokines (TNF-β and IFN-γ), compared with hNPSCs. Immunohistochemical staining indicated that galectin-1 and CD3 were more highly expressed in pancreatic cancer tissue. Galectin-1 expression was highest in poorly differentiated pancreatic cancer cells and lowest in well-differentiated pancreatic cancer cells and was associated with tumor size, lymph node metastasis, differentiation, and UICC stage. However, CD3 expression showed the opposite trend and was highest in well-differentiated pancreatic cancer cells and was associated with tumor differentiation and UICC stage. High expression of galectin-1 was associated with short survival, as was low expression of CD3. hCaPSC-derived galectin-1 enhanced apoptosis and anergy of T cells in pancreatic cancer, which contributes to the immune escape of pancreatic cancer cells.

Pancreatic Satellite Cells Derived Galectin-1 Increase the Progression and Less Survival of Pancreatic Ductal Adenocarcinoma

Background Galectin-1, a member of carbohydrate-binding proteins with a polyvalent function on tumor progression, was found strongly expressed in pancreatic satellite cells (PSCs), which partner in crime with cancer cells and promote the development of pancreatic ductal adenocarcinoma (PDAC). We evaluated the effects of PSCs derived Galectin-1 on the progression of PDAC, as well as the tumor establishment and development in mouse xenografts. Methods The relationship between immunohistochemistry staining intensity of Galectin-1 and clinicopathologic variables were assessed in 66 PDAC tissues, 18 chronic pancreatitis tissues and 10 normal controls. The roles of PSCs isolated from PDAC and normal pancreas on the proliferative activity, MMP2 and MMP9 expression, and the invasion of CFPAC-1 in the co-cultured system, as well as on the tumor establishment and development in mouse xenografts by mixed implanting with CFPAC-1 subcutaneously were evaluated. Results Galectin-1 expression was gradually increased from normal pancreas (negative), chronic pancreatitis (weak) to PDAC (strong), in which Galectin-1 expression was also increased from well, moderately to poorly differentiated PDAC. Galectin-1 staining intensity of pancreatic cancer tissue was associated with increase in tumor size, lymph node metastasis, perineural invasion and differentiation and UICC stage, and served as the independent prognostic indicator of poor survival of pancreatic cancer. In vitro and in vivo experiments indicated that TGF-β1 upregulated Galectin-1 expression in PSCs, which could further promotes the proliferative activity, MMP2 and MMP9 expression, and invasion of pancreatic cancer cells, as well as the tumor establishment and growth. Conclusion Galectin-1 expression in stromal cells of pancreatic cancer suggests that this protein plays a role in the promotion of cancer cells invasion and metastasis and provides a therapeutic target for the treatment of pancreatic cancer.

CEACAM6 induces epithelial-mesenchymal transition and mediates invasion and metastasis in pancreatic cancer.

Pancreatic cancer is a disease with an extremely poor prognosis. The acquisition of invasion properties in pancreatic cancer is accompanied by the process of epithelial-mesenchymal transition (EMT). Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is emerging as an important determinant of the malignant phenotype in a range of cancers, including pancreatic cancer. Therefore, the aim of this study was to evaluate the potential involvement of CEACAM6 in the invasion and metastasis of pancreatic cancer cells via EMT regulation. The results of our study showed a positive association between CEACAM6 expression and poor prognosis of pancreatic cancer, differentiation and lymph node metastasis. Elevated levels of CEACAM6 in pancreatic cancer cells promoted EMT, migration and invasion in vitro and metastasis in animal models, whereas shRNA-mediated CEACAM6 knockdown had the opposite effect. Furthermore, we demonstrated that miR-29a/b/c specific for CEACAM6 could regulate its expression at the post-transcriptional level. Collectively, our findings identified CEACAM6, which is regulated by miR-29a/b/c, as an important positive regulator of EMT in pancreatic cancer offering an explanation for how elevated levels of CEACAM6 are likely to contribute to the highly metastatic phenotype of pancreatic cancer.