Establishment Of Mouse Lymphoma Model With EG7 Cells Expressing Human CD19 Molecule And Luciferase

Background:In recent years,Chimeric Antigen Receptor T-cell(CAR-T)Immunotherapy has achieved unprecedented success in the field of malignant hematological tumor biotherapy.The principle of CAR-T is that CAR-modified T cells can specifically recognize hematological tumor-associated antigens,and the targeting,killing activity and persistence of CAR-T cells are higher than those of conventionally applied immune cells.Our group's previous work has constructed a new type anti-human CD19-CAReffector cells and observed its antitumor effects.After reinfusion of these effector cells in a mouse xenogeneic leukemia model constructed from tumor cells of leukemia patients,tumor burden in mice peripheral blood was significantly reduced and mice survived for prolonged period and survival rate improved greatly.However,in the xenogeneic leukemia mouse model,in order to ensure the survival and tumorigenesis of human tumor cells,we used T cell,B cell,and NK cell-deficient NOD/Scid/?2 genedeficient mice which cannot simulate the normal immune environment.So it is difficult to detect the anti-tumor effects of the CAR-effector cells,and also it is impossible to elucidate the collaborative anit-tumor mechanism by other immune cells.Objective:To construct a mouse lymphoma model induced by EG7 cell which can stably express human CD19 molecule and luciferase in immunocompetent wild type C57BL/6 mice.This lymphoma model will provide a suitable research platform for elucidating the role and mechanism of mouse-derived anti-human CD19-CAR-effector cells in lymphoma.Methods:1)Gene vector containing human CD19 molecule and luciferase was constructed,and EG7 cell line was infected by lentivirus to obtain an EG7 cell line stably expressing human CD19 molecule and luciferase.2)Mouse subcutaneous lymphoma model was constructed by inoculating EG7 cells which stably expressing human CD19 molecule and luciferase subcutaneously into C57BL/6 mice.Body weight and tumor volume were measured,and in vivo tumor growth was detected by IVIS Lumina XR instrument.Percentages of tumor cell and immune cells in mouse peripheral blood,spleen,lymph nodes,bone marrow,and tumor tissues were detected by flow cytometry.3)Mouse peritoneal lymphoma model was constructed by inoculating EG7 cells which stably expressing human CD19 molecule and luciferase subcutaneously into C57BL/6 mice.Body weight and tumor volume were measured every day,and in vivo tumor growth was detected by IVIS Lumina XR instrument.Percentages of tumor cell and immune cells in mouse peripheral blood,spleen,lymph nodes,bone marrow,and tumor tissues were detected by flow cytometry..Results:1)Mouse lymphoma model induced by EG7 cell line stably expressing human CD19 and luciferase was successfully constructed.2)EG7 cell line stably expressing human CD19 molecule and luciferase can form tumors subcutaneously in C57BL/6 mice with healthy immune system.Tumor volume and tumor growth rate were positively correlated with the number of inoculated cells.Human CD19 molecules expression in EG7 cells was reduced greatly.However,the proportion of various immune cells in the tumor tissue and other organs did not change significantly compared with control group.3)Intraperitoneal inoculation of EG7 cell line stably expressing human CD19 and luciferase can form tumors in the mesentery and intestinal wall of healthy immune system C57BL/6 mice,but human CD19 expression on the surface of tumor cells cannot be detected by flow cytometry.Conclusion:1)Using the EG7 cell line stably expressing human CD19 molecule and luciferase,subcutaneous inoculation method can be used to establish mouse lymphoma model in C57BL/6 mice for the study of anti-tumor effects of our new type anti-human CD19-CAR effector cells and collaborative anti-tumor mechanism by other immune cells under normal immune conditions.2)Intraperitoneal inoculation of EG7 cells stably expressing human CD19 and luciferase can form tumors in the mesentery and intestinal wall in C57BL/6 mice.However,no tumor cells express human CD19 molecules were detected in tumor tissues.The conditions for constructing lymphoma models in the abdominal cavity need further exploration and optimization.