High Expression Of Galectin-1in Pancreatic Stellate Cells Promote Tumor Angiogenesis Of Pancreatic Cancer

Backgroud&aim：Pancreatic cancer microenvironment is composed by stromal cells and extracellularcomponents. The mainly stromal cells included activated pancreatic stellate cells(PSC), endothelial cells and macrophages et al. The major extracellular componentsincluded extracellular matrix (ECM), matrix metalloproteinases-2(MMP-2), matrixmetalloproteinases-9(MMP-9), transforming growth factor-(TGF-β), vascularendothelial growth factor (VEGF) and other cytokines. PSC in pancreatic cancermicroenvironment can promote tumor cell growth, and increase the tumor cellsresistance to chemical drugs in vitro. In addition, these cytokines can also activatePSC in turn and ultimately promote the formation of micro-environment which willbenefit to pancreatic cancer growth.Galectin-1is a lectin protein with high affinity to β-galactose. Galectin-1expressionlevels were significantly increased in most tumors. Galectin-1produced by Tumorcells can significantly promote the proliferation of endothelial cells and induce tumormicrovessel formation, and which will promote tumor growth and metastasis. Recentstudies show that Galectin-1is significantly expressed in cultured activated PSC.There is large amount of activated PSC in pancreatic cancer microenvironment. But the relationship between Galectin-1which secreted by PSC and angiogenesis ofpancreatic tumor is not clear.We firstly used immunohistochemical staining to analysis the Galectin-1expressionin normal pancreas and pancreatic cancer tissues in this section. And then weconstructed and packaged lentivitral expression vector/RNA interference vector forhuman Galectin-1gene and infected the human primary PSC in vitro culture.Galectin-1expression level was detected by quantitative realtime RT-PCR (qRT-PCR)and Western-blot. These will benefit to further study about the relationship betweenthe high expression of Galectin-1in pancreatic stellate cells and angiogenesis ofpancreatic cancer.Methods:1. Isolation and culture of human primary PSC, and immunohistochemical stainingidentification was performed for Desmin (quiescent mark for PSC) and α-SMA(activated mark for PSC). Galectin-1expression in quiescent/actived PSC was alsostained.2. Differential expression of Galectin-1in normal pancreas and pancreatic cancertissue was detected by immunohistochemistry.3. Constructed over-expression/RNA interference lentiviral vector for Galectin-1and infected PSC and purified. Galectin-1mRNA and protein expression levels inPSC were detected by quantitative realtime RT-PCR and Western-blot. Galectin-1protein secretion in PSC supernatants was identified by ELISA.Results:1. Human primary PSC were successfully isolated and cultured.Immunohistochemical identification showed quiescent PSC Desmin (positive), α-SMA (negative), Galectin-1(weak); actived PSC Desmin (positive), α-SMA(strong), Galectin-1(strong).2. Immunohistochemical staining showed that Galectin-1expression in pancreaticcancer tissue compared with normal pancreatic tissue was significantly increased.3. Over-expression/RNA interference lentiviral vectors for Galectin-1wereSuccessfuliy constructed and infected PSC. Quantitative Realtime RT-PCR showedthat the Galectin-1expression in over-expression group (Over) was significantlyincreased compared with the normal group (Na ve) and bank group (GFP)(P<0.01);the Galectin-1expression in interference group (Sh-1and Sh-2) was obviouslyreduced by more than85%compared to the Naive group and blank group (Scr)(P<0.01). The Galectin-1expression in Scr group and the GFP group compared withthe Na ve group was not statistically significant (P>0.05). Western-blot and ELISAresults were consistent with the above results.Conclusion:1. Human primary PSC were successfully isolated, cultured and identified.2. Galectin-1expression in the activated PSC and pancreatic cancer tissues weresignificantly increased compared with that in quiescent PSC and normal pancreatictissue（P<0.01）.3. Over-expression/RNA interference lentiviral vector for Galectin-1weresuccessfully constructed, and could significantly increase/decrease Galectin-1expression in PSC. Backgroud&aim：Galectin-1as a member of the Galectin family has high affinity with theglycoconjugates which contain β-galactose glycosidic receptors, involved in a varietyof biological functions such as cell adhesion, growth regulation and immune responseand so on. Galectin-1has a high expression in tumor tissues wich relates to tumorimmune suppression, tumor angiogenesis and tumor cell proliferation, invasion andmetastasis. Primary studies showed that there are an amount of fibrous connectivetissue around pancreatic cancer and activation of pancreatic stellate cells express highlevel of Galectin-1in the pancreatic cancer microenvironment. This part of the studywe detect the impact of angiogenesis ability under the PSC expressing different levelof Galectin-1or which was co-cultured with pancreatic cancer cells in vivo and vitroexperiments. Detect the relationship between Galectin-1expression and microvesseldensity (MVD) in pancreatic cancer tissue Compared with normal pancreatic tissue.Investigate the correlation between Galectin-1expressions of PSC and the formationof MVD in pancreatic cancer.Methods:1. The impact of cell supernatant from different Galectin-1expression levels of PSCor co-cultured with pancreatic tumor cells on HUVEC proliferation, migration andtube formation ability were observed in vitro experiments.2. The relationship between Galectin-1expression and the number of vessels formation was detected in nude mice subcutaneous transplanted tumors experiments.3. Galectin-1expression and quantity of MVD were detected in differentpathological grades of pancreatic cancer tissues and normal pancreatic tissues. Thecorrelation of MVD with Galectin-1expression in pancreatic cancer was analysised.Results:1. PSC supernatant with high expression of Galectin-1significantly enhancedHUVEC proliferation, migration and tube formation ability compared with Galectin-1interference group (P<0.01). The HUVEC proliferation, migration and tube formationcapacity was significantly enhanced by the group of PSC with Galectin-1highexpression co-cultured with pancreatic cell lines CFPAC-1, BXPC-3supernatantcompared with Galectin-1interference co-culture group (P<0.01).2. In nude mice subcutaneous implanted tumors experiments, the ability of inducingangiogenesis in PSC with high expression of Galectin-1mixtured with CFPAC-1andBXPC-3implanted group was significantly increased compared with normal PSCmixtured with CFPAC-1/BXPC-3implanted group. But the ability in interferenceGalectin-1PSC mixtured with CFPAC-1/BXPC-3implanted group and pureCFPAC-1/BXPC-3implanted group were significantly decreased compared withnormal PSC mixtured with CFPAC-1/BXPC-3implanted group. Pure PSC group hadno solid tumor formation in nude mice subcutaneous tumor transplanted experiments.3. Galectin-1expression in pancreatic cancer tissues was positively correlated withthe quantity of MVD（P<0.0001）. When the degree of differentiation is worse, theexpression of Galectin-1is higher, and the quantity of MVD was also significantlyincreased. MVD of pancreatic cancer tissues was significantly increased comparedwith normal pancreatic tissue (P<0.05). Conclusion:1. The vitro experiments results showed that the PSC with high expression ofGalectin-1significantly promoted HUVEC proliferation, migration and tubeformation. The effects were opposite when decreased Galectin-1expression in PSC.2. Galectin-1expression was positively related to the number of vessels in nude micesubcutaneous implanted tumor.3. Galectin-1expression in pancreatic cancer tissue was positively correlated withthe quantity of MVD. Galectin-1expression and quantity of MVD in pancreaticcancer were closely related to the histological degree of pancreatic cancer. Itsexpression level is higher with the lower histological degree.