The Study Of Psma Expression In Lung Cancer And Used For Immunotherapy

ObjectiveTo study the expression of prostate specific membrane antigen (PSMA) in lung cancer by immunohistochemistry. Analyze the correlation between PSMA expression and the clinicopathologic features of lung cancer. To construct the plasmid of AAV/PSMA and generate Adenovirus-free AAV/PSMA virus stocks. AAV/PSMA trsansfecting the DC and generating the CTL.Materials and Methods:We got the human lung cancer tissue and normal lung tissue specimens from Tianjin Medical University Cancer Hospital.The specimens was including30squamous carcinoma.29adenocarcinoma,28large cell carcinoma,30small cell carcinoma and33normal lung tissue specimens. These specimens were immunostained for PSMA and a vascular endothelial marker CD31. prostatic adenocarcinoma specimens known to be positive for PSMA expression served as a positive control for PSMA. PBS was utilized in place of the first antibody for the negative control.Total RNA was isolatcd from LNcap which was PSMA positive. The mRNA Was separated. PSMA cDNA was generated by RT-PCR amplification. The PSMA cDNA product was then gel-purified. The PSMA cDNA was determined to be identical to the published sequence by PCR and sequence.The plasmid of AAV/PSMA was constructed. Adenovirus-free virus was generated using the complementor plasmid.AAV/PSMA trsamfecting the DC and generating the CTLPeripheral blood mononuclearcells (PBMC) was isolated by density gradient centrifugation from healthy donors. PBMCs were inoculated into culture dish according to be about8×107～12×107cells/dish.The culture dishes were put in carbon dioxide gas incubator for3hours. Then adde AAV/PSMA virus and continue culture for8hours. After8hours, the suspended T lymphocytes was transferred into T75cell culture flasks and added IL-2. The adherent cells were dendritic cell (DC) AIM-V medium、GM-CSF and IL-4were added into culture dishes for DC. On day6T lymphocytes were added to DCs(ratio of1:20, dendritics:lymphocytes). The cells were observed under inverted microscope every day until CTL were activated. Cell surface markers analysis of DCs was by flow cytometry, including CD86-Cell surface marker analysis of stimulated T cells was by flow cytometry, including CD4,CD8, CD25and CD69-FITC-anti-IFN-γ was used for analysis of CTL for intracellular cytokines.Optimum MOI dose was studied by flow cytometry.Result:For non-small cell lung cancer,positive PSMA staining was detected in54.02%of tumor cells, while PSMA staining was detected in85.06%of the tumor-associated neovasculature. For small cell lung cancer, positive PSMA staining was not detected in tumor cells, while PSMA staining was detected in73.33%of the tumor-associated neovasculature. For normal lung tissue adjacent to tumor, positive PSMA staining was not detected in any cells. Vessels within the tumor,both CD31and PSMA exhibit endothelial expression.However,Vessels in normal lung tissue, showed only CD31staining, and PSMA did not express.AAV/PSMA plasmid was successfully constructed. AAV/PSMA virus was generated. PSMA was successfully put into AAV/PSMA plasmid. The rates of CD8o+,CD83+and CD86+for dendritic cell were54%,83%and72%; The rates of CD25+CD4+, CD69+CD8+and IFN-γ for CTL were9.052%,42.76%and39%.Conclusion:For NSCLC, PSMA expressed not only in the tumor-associated neovasculature but also in the tumor cells; For SCLC, PSMA only expressed in tumor-associated neovasculature; For normal lung tissue adjacent to tumor, positive PSMA staining was not detected in any cells. We found PSMA expression was positively correlated with age at diagnosis, pathological type and stage.Our findings suggests that PSMA may potentially be used as a novel diagnostic marker and therapeutic target for lung cancer.The study suggested that rAAV-loading of DCs can successfully generating the CTL and that the cell-mediated immunotherapeutic protocols aimed PSMA antigen may be a new therapeutic Method for lung cancer.