Cancer-FOXP3 Recruits FOXP3+Treg Cells Via CCL5 In Pancreatic Ductal Adenocarcinoma

Background Forkhead Box Protein3(FOXP3) is an important transcription factor mainly expressed in CD4+CD25+ Regulatory T cells(Treg cells). It major functions in the differention of T cells towards regualtory T cells, thus further enhance the immuno-inhibitory effect in pancreatic cancer. However, FOXP3 has also been discovered in various types of tumor cells besides Treg cells. The investigation of FOXP3 expression and function in pancreatic ductal adenocarcinoma may provide potent novel targets in the treatment. In our previous experiments, we discovered that FOXP3 is highly expressed in pancreatic ductal adenocarcinoma tissues and cell lines in compare to the normal ones. Therefore, this project was designed to further investigate the expression of FOXP3 in pancreatic ductal adenocarcinoma tissues and cell lines; to explore the function of cancer-FOXP3 in the progression of pancreatic ductal adenocarcinoma and the regulation mechanism lying under from clinical level, cell level and animal level. Method1. Immunohistochemistry staining of consecutive paraffin-fixed sections was performed to detect the expression of cancer-FOXP3(referred as c-FOXP3 hereafter) in pancreatic ductal adenocarcinoma tissue and its adjacent normal tissue. In-hospital clinical information and follow-up information for each patient was collected and recorded by special assigned person. The correlation between c-FOXP3 expression and clinical and pathological parameters was analyzed by SPSS software.2. The correlation of c-FOXP3 expression and Treg cells accumulation in pancreatic ductal adenocarcinoma, and the correlation of clinical and pathological parameters and the synthetic effect of c-FOXP3 expression together with Treg cells accumulation in tumor microenvironment for each patient were analyzed by SPSS software as well.3. Western blot experiment was used for the detection of c-FOXP3 base expression level in four pancreatic cancer cell lines(Panc-1, As PC-1, MIA Pa Ca-2 and Bx PC-3) and normal pancreatic ductal cell line(HPDE6C7). FOXP3 overexertion plasmid and FOXP3-sh RNA plasmid were constructed. FOXP3 overexpression and downexpression stable transfected cell lines were established by lentiviral stable transfection system. Western blog experiment was further used to confirm FOXP3 expression in these cell lines. Cell apoptosis, cell cycle, Edu-incorporation proliferation assay and subcutaneous pancreatic tumor model in NOD-SCID mice were used for the investigation of c-FOXP3 direct function on pancreatic ductal adenocarcinoma cells. Subcutaneous pancreatic tumor model in C57/BL mice model was used for the investigation of c-FOXP3 function on the immunological microenvironmentof pancreatic ductal adenocarcinoma.4. Human Peripheral Mononuclear Cells(PBMCs) were isolated from Peripheral blood by Ficoll. Regulatory T cells(Treg cells) were further isolated from human peripheral Mononuclear Cells. Co-culture system were used to investigate the prolferation stimulation on Treg cells by c-FOXP3 in vitro. Transwell assays were used to study the chemotaxis function of Treg cells towards FOXP3-positive pancreatic tumor cells in vitro. Orthotopic pancreatic cancer model in nude mice with PBMCs injection from tail vein was used to confirm the chemotaxis function of Treg cells by c-FOXP3 in vivo.5. RT-PCR experiment was used to screen out tumor-Treg cells-related chemokine. Western blot, ELISA assays and immunohistochemistry were used to further confirm the results of RT-PCR. Chromatin immunoprecipitation assay and dual luciferase reporter gene assay were used to investigate the regulation mechanism of c-FOXP3 on its targeted chemokine.6. Bottle-neck experiments were performed in vitro to further identical the very chemokine function to recruit Treg cells towards tumor microenvironment. Chemokine blocking in subcutaneous pancreatic tumor model in C57/BL mice was used to validate the role of chemokine blocking in Treg cells recruitment by c-FOXP3 and in promoting pancreatic ductal adenocarcinoma growth in vivo. The results1. Immunohistochemistry staining of consecutive paraffin-fixed sections was performed to detect the expression of c-FOXP3 in pancreatic ductal adenocarcinoma tissue and its adjacent normal tissue. In-hospital clinical information and follow-up information for each patient was collected and recorded by special assigned person. The correlation between c-FOXP3 expression and clinical and pathological parameters was analyzed by SPSS software.2. The correlation of c-FOXP3 expression and Treg cells accumulation in pancreatic ductal adenocarcinoma, and the correlation of clinical and pathological parameters and the synthetic effect of c-FOXP3 expression together with Treg cells accumulation in tumor microenvironment for each patient were analyzed by SPSS software as well.3. Western blot experiment was used for the detection of c-FOXP3 base expression level in four pancreatic cancer cell lines(Panc-1, As PC-1, MIA Pa Ca-2 and Bx PC-3) and normal pancreatic ductal cell line(HPDE6C7). FOXP3 overexertion plasmid and FOXP3-sh RNA plasmid were constructed. FOXP3 overexpression and downexpression stable transfected cell lines were established by lentiviral stable transfection system. Western blog experiment was further used to confirm FOXP3 expression in these cell lines. Cell apoptosis, cell cycle, Edu-incorporation proliferation assay and subcutaneous pancreatic tumor model in NOD-SCID mice were used for the investigation of c-FOXP3 direct function on pancreatic ductal adenocarcinoma cells. Subcutaneous pancreatic tumor model in C57/BL mice model was used for the investigation of c-FOXP3 function on theimmunological microenvironmentof pancreatic ductal adenocarcinoma.4. Human Peripheral Mononuclear Cells(PBMCs) were isolated from peripheral blood by Ficoll. Regulatory T cells(Treg cells) were further isolated from human Peripheral Mononuclear Cells. Co-culture system were used to investigate the prolferation stimμLation on Treg cells by c-FOXP3 in vitro. Transwell assays were used to study the chemotaxis function of Treg cells towards FOXP3-positive pancreatic tumor cells in vitro. Orthotopic pancreatic cancer model in nude mice with PBMCs injection from tail vein was used confirm the chemotaxis function of Treg cells by c-FOXP3 in vivo.5. RT-PCR experiment was used to screen out tumor-Treg cells-related chemokine. Western blot, ELISA assays and immunohistochemistry were used to IX further confirm the results of RT-PCR. Chromatin immunoprecipitation assay and dual luciferase reporter gene assay were used to investigate the regulation mechanism of c-FOXP3 on its targeted chemokine.6. Bottle-neck experiments were performed in vitro to further identical the very chemokine function to recruit Treg cells towards tumor microenvironment. Chemokine blocking in subcutaneous pancreatic tumor model in C57/BL mice was used to validate the role of chemokine blocking in Treg cells recruitment by c-FOXP3 and in promoting pancreatic ductal adenocarcinoma growth in vivo. Conclusions1. c-FOXP3 expression is positive in both pancreatic adenocarcinoma tissue and pancreatic cancer cell lines. The expression of c-FOXP3 is positively correlated with Treg cells infiltration in tumor microenvironment. Patients with high expression of c-FOXP3 and high infiltration of Treg cells often appears with a poor prognosis.2. c-FOXP3 directly bind to the promoter of CCL5 gene to activate its transcripttion and translation. Over accumμLation of CCL5 intracellμLar stimμLated its secretion.3. The recruitment of Treg cells by c-FOXP3 is mediated by CCL5. High expression of c-FOXP3 and high infiltration of Treg cells in tumor site cooperated to promote the growth of pancreatic ductal adenocarcinoma.4. CCL5 blocking in c-FOXP3 high expression tumor decreased local Treg cells infiltration and further inhibited the growth of tumor itself.