Experimental Study Of Gemcitabine Combined With Anti PD-1 Monoclonal Antibody In The Treatment Of Pancreatic Cancer

Objective:By transplanting human peripheral blood mononuclear cells(PBMC)and CD34~+hematopoietic stem cells(HSC)to construct the immune humanized mouse models;Further injection of human pancreatic cancer cells to obtain tumor immune system dual humanized mouse model;Using these two models to explore the efficacy of gemcitabine combined with immune checkpoint inhibitor(ICI)in the treatment of pancreatic cancer,in order to provide data support for co-immunotherapy of pancreatic cancer.Methods:The expression of PD-L1 in two human pancreatic cancer cell lines Aspc-1 and Bxpc-3 was detected by Western Blot.The IC50 value of human pancreatic cancer cell line Bxpc-3 was measured by CCK8.The expression of PD-L1 on Bxpc-3cell treated with gemcitabine at a concentration of 15μg/m L was determined using Western-Blot assay and flow cytometry.The Ficoll density gradient centrifugation method was conducted to separate fresh peripheral blood mononuclear cells(PBMCs)from peripheral blood of healthy volunteers.This PBMC was injected into Five-to six-week-old female severely immunodeficient NCG mice through the tail vein at a dose of 1×10~7/200μL per mouse.One week after PBMC implantation,the human pancreatic cancer cell line Bxpc-3(1×10~7/200μL/mouse)was subcutaneously implanted into the severe combined immunodeficiency NCG mice.So as to establish a double humanized mouse model both with human PBMC immune system and pancreatic cancer(Hu-PBMC).After 3 weeks of implantation,peripheral blood of these mice were collected using the tail cutting method.PBMCs were separated from the peripheral blood of these mice using Ficoll density gradient centrifugation method.The proportion of human CD45~+immune cells in the peripheral blood of these mice was analyzed by flow cytometry.When the CD45~+content of human immune cells was more than 20%,we confirmed the successful construction of a dual humanized mouse model of PBMCs(Hu-PBMC).While the symptom of graft-versus-host disease(GVHD)in the mouse becomes apparent,the experiment was terminated.After the successful construction of Hu-PBMC model,the mice were divided into different groups for treatment and the tumor growth was regularly monitored to evaluate the efficacy of gemcitabine combined with anti-PD-1 monoclonal antibody.The infiltration of human immune cells in the tumor tissue was analyzed using immunohistochemical staining and immunofluorescence staining.Fresh mononuclear cell(MNC)from fetal umbilical cord blood were isolated using Ficoll density gradient centrifugation,and purify human CD34~+HSC was obtained using the Mini MACS method.The CD34~+HSC was injected into three-four week-old female NCG mice by a tail vein method.The injection volume was 1×10~5/200μL.Twelve weeks after CD34~+HSC implantation,the human pancreatic cancer cell line Bxpc-3 was(1×10~7/200μL/mouse)was subcutaneously implanted into the severe combined immunodeficiency NCG mice to construct a dual humanized mouse model both with human CD34~+HSC immune system and pancreatic cancer(Hu-HSC).After 3months of implantation,peripheral blood of these mice was collected using the tail cutting method.PBMCs were separated from the peripheral blood of mice by Ficoll density gradient centrifugation.The proportion of human CD45~+immune cells in the peripheral blood of these mice were analyzed using flow cytometry.When the CD45~+content was more than 20%in the peripheral blood,we confirmed the successful construction of a dual humanized mouse model of CD34~+HSC(Hu-HSC).After the successful construction of Hu-HSC model,the tumor growth was monitored regularly after treatment to evaluate the therapeutic effect of gemcitabine combined with anti PD-1monoclonal antibody.At the end of the experiment,the tumor tissue of these mice was taken for immunohistochemical staining and immunofluorescence staining,and the infiltration of human immune cells in the tumor tissue of these mice were assayed.The characteristics of the tumor immune microenvironment of mice were analyzed to explore the possible mechanism of gemcitabine combined with anti PD-1 monoclonal antibody in the treatment of pancreatic cancer.Results:PD-L1 was highly expressed in human pancreatic cancer cell line Bxpc-3,while Aspc-1 hardly expressed PD-L1.The results of Western Blot and flow cytometry displayed that gemcitabine could up-regulate the expression of PD-L1 in human pancreatic cancer Bxpc-3 cells after 72 hours of treatment.Three weeks after PBMC implantation,the content of human immune molecule CD45~+in the peripheral blood of double humanized mice model(Hu-PBMC)was more than 20%.Immunohistochemical staining showed that h CD45~+,h CD8~+and h CD4~+cells infiltrated in tumor tissue of these mice,indicating that the dual humanized mouse model both with human immune system and pancreatic cancer tumor(Hu-PBMC)was successfully constructed.Twelve weeks after human CD34~+HSC implantation,the content of human immune cells h CD45~+in the peripheral blood of dual humanized mice model both with human CD34~+HSC immune system and pancreatic cancer(Hu-HSC)was more than 20%.Immunohistochemical staining showed that h CD45~+,h CD8~+and h CD4~+cells infiltrated in the tumor tissue of these mice,indicating that the dual humanized mouse model both with human immune system and pancreatic cancer tumor(Hu-HSC)was successfully constructed.Whether in the Hu-PBMC dual humanization model or in the Hu-HSC dual humanization,the results of animal experiments revealed that gemcitabine combined with anti-PD-1 monoclonal antibody could effectively inhibit the growth of pancreatic cancer.Immunohistochemical staining and immunofluorescence staining demonstrated that gemcitabine combined with anti PD-1 monoclonal antibody therapy could increase the contents of CD45~+cells,CD8~+cells and PD-L1~+cells in the tumors significantly,while decrease the contents of Ki67~+cells significantly.These results indicated that the therapy strategy of gemcitabine combined with anti PD-1 monoclonal antibody can achieve the goal of treating pancreatic cancer by enhancing the infiltration and activation of CD8~+T cells in pancreatic cancer tissue.Conclusion:We successfully established Hu-PBMC and Hu-HSC double humanized mouse models of pancreatic cancer respectively.In those models,gemcitabine combined with anti-PD-1 monoclonal antibody can enhance the infiltration and activation of CD8~+T cells in pancreatic cancer tissue,play a specific anti-tumor effect,and thus inhibit the growth of pancreatic tumor.