Pancreatic Stellate Cells (high Expression Of Galectin 1 Promote Pancreatic Cancer, Immunosuppression And Progress Of Research

Backgroud&Aim：Pancreatic cancer microenvironment is composed by stromal cells and extracellularcomponents, in which the main stromal cells included activated pancreatic stellatecells （PSC, one of the most important stromal cells）, endothelial cells andmacrophages etc., and the major extracellular components included matrixmetalloproteinases-2（MMP-2）, matrix metalloproteinases-9（MMP-9）, transforminggrowth factor-（TGF-β）, vascular endothelial growth factor （VEGF） and othercytokines. PSC in pancreatic cancer microenvironment can promote tumor cellgrowth, and increase the tumor cells resistance to chemical drugs in vitro. In addition,these cytokines can also activate PSC in turn and ultimately promote the formation ofbeneficial micro-environment for pancreatic cancer growth.Galectin-1is a lectin protein with high affinity to β-galactose, and presentintracellular and extracellular with biologically active dimer form. Galectin-1combines with membrane receptors and extracellular matrix proteins such as integrins,laminin and fibronectin etc., and plays relevant functional effects.Galectin-1binds tocytoplasmic protein and nuclear protein intracellular, and made the functions of celltransformation and mRNA shear. Galectin-1can inhibit T cell proliferation andinduce tumor infiltrating T-cell apoptosis. Recently, it has founded that Galectin-1was significantly expressed in cultured activated PSC. As a lot of activated PSC existing in pancreatic cancer microenvironment, the relationship between endogenousGalectin-1of PSC and the pancreatic cancer immunosuppression is unclear.In this section, PSC were isolated from resected fresh pancreatic tissue and Galectin-1was knocked down using a small hairpin RNA （shRNA） or overexpressed usingrecombinant lentiviruses. In order to investigate the relationship between Galectin-1expression and tumor immune suppression in pancreatic cancer, the impacts on Tcells function and apoptosis by primary PSC with different levels of Galectin-1expression were studied, and the expression of Galectin-1and CD₃in pathologicalspecimens of pancreatic cancer, chronic pancreatitis and normal pancreas tissues wereanalyzed.Methods:1. Isolation and culture of primary human PSC, and immunohistochemical stainingdentification was performed for Desmin （quiescent mark for PSCs） and α-SMA（activated mark for PSCs）.2. Galectin-1over-expression/RNA interference lentiviral vectors were constructed,PSC were infected by recombinant lentiviruses and purified. Real-timequantitative RT-PCR and Western Blot were performed to identify the Galectin-1mRNA and protein expression levels of infected PSC, and the secreted Galectin-1protein levels in supernatants of PSC were detected by ELISA.3. Peripheral blood T lymphocytes of healthy volunteers were isolated and cultured,and co-cultured with PSC with different Galectin-1expression levels （overexpressed, silenced and normal expressed）. T-cell apoptosis was detected by flowcytometry and cytokines （IL-2、INF-γ、IL-4and IL-5） in supernatant secreted byT cells were detected by ELISA. 4. The Galectin-1and CD3expression of66pancreatic cancer,18chronic pancreatitisand10normal pancreas tissues were detected by immunohistochemical staining.Results:1. Compared with normal PSCs, PSCs with Galectin-1over-expression significantlyinduced apoptosis of CD3+T cells （p <0.01）, CD4+T cells （p <0.01） and CD8+Tcells （p <0.05）, and CD3, CD4and CD8T cells apoptosis was significantlydecreased in PSCs with Galectin-1silenced （p <0.05）.2. Compared with normal PSCs, PSCs with Galectin-1over-expression significantlyinhibited secretion of Th1cytokines （IL-2and INF-γ）（p <0.01）, and inducedsecretion of Th2cytokines （IL-4and IL-5）（p <0.01）, and PSCs with Galectin-1silenced increased Th1cytokines （IL-2and INF-γ） secretion （p <0.01） anddecreased Th2cytokines （IL-4and IL-5） secretion （p <0.01and p <0.05,respectively）.3. Expression of Galectin-1and CD3inpancreatic cancer tissues were located aroundthe pancreatic cancer cells and significantly high than chronic pancreatitis andnormal pancreas tissues （p <0.01）.Conclusion:1. PSC with Galectin-1high expression promoted the T-cell apoptosis, andsignificantly inhibited the secretion of Th1cytokines （IL-2and INF-γ） andinduced secretion of Th2cytokines （IL-4and IL-5） which skewed Th1/Th2balance.2. High expressed Galectin-1of PSC in pancreatic cancer microenvironment mightform a tumor immunosuppression barricade which induced apoptosis of T cells and inhibited the infiltration of T cells, and results in development ofimmunosuppression of pancreatic cancer. Backgroud&Aim：Galectin-1as a member of Galectin family was the first to be discovered, and closelyrelated to tumor immunosuppression, tumor angiogenesis and tumor cell proliferation,invasion and metastasis. Galectin-1high expressed in pancreatic stellate cells ofpancreatic cancer microenvironment. This part of the study was to detect Galectin-1and CD3expression in pancreatic cancer tissue specimens, and discuss therelationship between Galectin-1expression in pancreatic cancer microenvironmentand immunosuppression, invasion, metastasis and prognosis of pancreatic cancer.Methods:1. A retrospective analysis of66cases clinical data of patients with surgically resectedpancreatic ductal adenocarcinoma in the First Affiliated Hospital of NanjingMedical University from January2006to December2010, in which did notinclude patients with widespread metastasis, unresectable of pancreatic cancer, orwith other pancreatic tumor.2. The relevant Galectin-1and CD3expression of tumor specimens were detected byimmunohistochemical staining.3. Kaplan-Meier curves described the survival of patients with pancreatic cancer, andclinical data was statistical analyzed by Log-rank test.4. Univariate and multivariate analysis was performed to analyze the pancreatic cancer prognosis and the clinical parameters with Cox regression hazardsregression model.Results:1. After statistical analysis for clinical data of66patients, the results of follow-upshowed that the survival time was ranged from4.7-47.0months and mediansurvival was16.2months.2. Galectin-1expression was not directly related to patient’s age, gender and tumorperineural invasion, but closely related to tumor T stage, lymph node metastasis,UICC staging and histological grading, and increased gradually with thehistological degree increased from well differentiation to poorly differentiated（Galectin-1strong/weak expression proportion were:4/6cases inwell-differentiated,16/8cases in moderately differentiated,27/5cases in poorlydifferentiated, p <0.05）. CD3expression was not directly related to patient’s age,gender, tumor neural invasion, T stage and lymph node metastasis, but closelyrelated to UICC stage and histological grade, and decreased gradually with thehistological degree decreased from well differentiation to poorly differentiated（CD3strong/weak proportion were:8/2cases in well-differentiated,13/11casesin moderately differentiated,9/23cases in poorly differentiated, p<0.01）.Galectin-1expression was negatively correlated with CD3expression （p=0.001）.3. Pancreatic cancer patients with Galectin-1overexpression and low expression ofCD3had poorly prognosis. The median survival time of patients with Galectin-1high expression and low expression was9.1months and19.8months （p <0.001）,respectively. The median survival time of patients with CD3high expressionand low expression was14.3months and10.9months （p <0.05）, respectively.4. Univariate analysis showed that the survival of pancreatic cancer patients was independent of patient’s age, gender and tumor neural invasion, and depended ontumor T stage, lymph node metastasis, UICC staging, histological grade,Galectin-1and CD3expression. Multivariate analysis showed that lymph nodemetastasis of tumors, UICC stage, histological grade, Galectin-1and CD3expression is an independent risk factor for pancreatic cancer prognosis.Conclusion:1. Galectin-1expression closely related to tumor T stage, lymph node metastasis,UICC staging and histological grading, and increased gradually with thehistological degree decreased from well differentiation to poorly differentiation.CD3expression closely related to UICC stage and histological grade, anddecreased gradually with the histological degree decreased from welldifferentiation to poorly differentiation.2. The levels of Galectin-1expression and CD3expression in pancreatic cancer wereclosely related to patient’s prognosis. With the increased expression of Galectin-1,the CD3expression was decreased and the worse prognosis become in patientswith pancreatic cancer.3. Tumor lymph node metastasis, UICC staging, histological grade, Galectin-1andCD3expression are the independent prognostic risk factors for pancreatic cancer.