Research Of Chemotactic Phenomenon Of Foxp3 Positive Pancreatic Ductal Adenocarcinoma Cell To Treg

Objective Pancreatic cancer is one of the highly malignant tumors, ductal adenocarcinoma is the most common pathological pattern. Pancreatic Ductal Adenocarcinoma(PDAC)accounts for 90% of pancreatic cancer. About 60% of pancreatic cancer patients present metastases when diagnose PDAC. Otherwise, 25% of PDAC patients show locally advanced,which can not be radical resected. The median survival of this kind of patients is only six to nine months. 5-year overall survival rate of patients with unresectable diseases is less than 5%. Tumorigenesis and metastasis have a close relationship with internal and external environment in which tumor cells live. This environment is called the tumor microenvironment, specifically refers to the local internal environment which consist of the infiltrating immune cells and stromal cells,secretion of active substance, and self tumor cells. As an important part of tumor microenvironment, immune cells is increasingly emphasized by people.Regulatory T cells(Treg) are a subset of T cell controlling the body own immune response, which play an immunosuppression role in the tumor microenvironment. Treg cells characteristically express CD4+CD25+Foxp3+. The transcription factor Foxp3 is not only the sign molecule of CD4+CD25+Treg, but also key gene of CD4 + CD25 + Treg deciding its function. According to the literature and our previous experiment, we have found that PDAC express higher Foxp3 than normal pancreatic tissue. And we also found that Treg infiltration is also more in tissue samples with higher Foxp3 expression. So we speculate that ductal adenocarcinoma cells may through express Foxp3, start the expression of certain genes, signaling pathways, or some event and then attract circulating Treg cells into the tumor microenvironment which plays immunosuppressive effects resulting poor prognosis.This study, as preliminary basis of Foxp3 to Treg chemotaxis, focusing on the clinical significance of Foxp3, Foxp3 to Treg chemotaxis in patient samples and in vitro experiments, building Foxp3 overexpression plasmids and stably transfected cell lines laying a solid foundation for subsequent experiments.Methods1.The corelation between the expression of Foxp3 in tumor cells, Treg numbers and patient prognosis. The relationship between the expression of Foxp3 in tumor cells and Treg infiltrating in the tumor microenvironment.1) Using immunohistochemical methods to detect a variety of pancreatic tumors and normal pancreatic tissues Foxp3 expression.To investigate the relationship between Foxp3 expression and prognosis of PDAC patients.2) Using immunohistochemical methods to detect ductal adenocarcinoma tumor microenvironment Treg infiltration and analyze its relationship with the patient prognosis.3) Using Spearman correlation analysis to test whether Foxp3 expression and infiltrating Treg have statistical association in the same patient tumor tissue.2.The establishment and validation of human Foxp3 overexpression plasmid, human Foxp3 overexpression stably transfected cell line, murine Foxp3 knockdown stably transfected cell line.1) Using genetic engineering technology to connect human Foxp3 protein coding sequence to the transient transfection vector and lentivirus vector.2) Using Ca Cl2 transfect 293 T cells to produce virus solution, using three packaging system to infect target cell, and then construct stably transfected cell lines.3) Purchase murine Sh Foxp3 vector, using three packaging system to infect murine pancreatic cancer cell line, and then build Foxp3 knockdown stably transfected cell line.4) Respectively using realtime PCR and Western Blot method to detect the effect of transient and stably transfected cell lines.3. Pancreatic cancer cell lines to Treg chemotaxis in vitro1) Using stably transfected cell lines, transient transfer technology and small interfering RNA technology to collect the supernatant of pancreatic cancer cell lines Foxp3 overexpression or interference.2) Using ficoll density gradient centrifugation to obtain a large number of lymphocytes from umbilical cord blood, and using magnetic beads to isolate CD4+CD25 + Foxp3+ Treg cells.3) Using Transwell chamber to verify different cell culture supernatant to Treg chemotaxisResults In PDAC, Foxp3 expression in tumor cells is significantly increased. Foxp3 expression and the degree of histological differentiation of patient specimens have statistical association. Besides Foxp3 expression and patient overall survival andrelapse-free survival also have statistical association. There are Treg expression in PDAC tumor microenvironment and its expression have statistical link with overall survival and relapse-free survival of patient.Spearman correlation analysis show the expression of Foxp3 have correlation with Treg infiltration surrounding the tumor.By sequencing analysis, we successfully constructed Foxp3 overexpression plasmid and lentivirus plasmids. Using Realtime PCR and Western Blot to detect the effect of transient and stably transfected cell lines, we have found Foxp3 can be obviously up-regulation or down-regulation. The stably transfected cell line was successfully constructed which laid the foundation of in vitro experiment and animal experiment.Using flow cytometry detection to verify the method of ficoll density gradient centrifugation and magnetic beads can separate sufficient amount of Treg to ensure subsequent experiments.Transwell chemotaxis experment has proven that whether transient transfect overexpression plasmid or stably transfected cell lines, the supernatant of Foxp3 overexpressing have significantly enhanced chemotaxis to Treg,and the supernatant of Foxp3 interference Treg have significantly weakened chemotaxis to Treg. The difference was statistically significant.Conclusion PDAC highly express Foxp3, and have a relationship with patient overall survival and relapse-free survival. Foxp3 expression and Treg infiltration have statistical contact suggesting that Foxp3 in tumor cells may have chemotaxis to Treg.On the basis of constructing overexpression plasmids and stably transfected cell lines,chemotaxis experiments show Foxp3 have chemotaxis to purified Treg.