The Mechanism Of IL-15Gene Therapy Of Peritoneal Cancer Metastases In Mice

Objective:To explore the mechanism of IL-15to inhibit the growth of metastases and provide experimental basis IL-15-based tumor immunogene therapy. Interleukin-15(IL-15) expression plasmid pHi2-spIL15-CMV-TAT (L3) was used to control colon cancer peritoneal metastasis model in mice, The growth of tumor and the survival of mice were monitored. The effect of IL-15gene therapy on the immune cells within peritoneum were determined.Methods:In this study, mice model of peritoneal cancer metastases was established using the murine colon carcinoma CT26through intraperitoneal injection, and the green fluorescent protein-expressing plasmid pHi2-EGFP-CMV-TAT (L6) was injected into the mice peritoneal cavity for in vivo transfection. Small animal in vivo imaging techniques were used to assess the efficiency of transfection and expression of target genes.Animals inoculated with CT26tumor cell were devided into several groups, mice in treatment group were injected intraabdominally with L3, mice without treatment or mice injected with empty plasmid M7, or PBS were control groups. Mice in blank group were not inoculated with tumor cell. Tumor size, weight of mice, ascites, the influence of survival on mice were evaluated. The expression of CD4, CD8, CD25, NKG2D, CD11b on the surface of cells harvested from mice peritoneal cavity were analyzed by flow cytometry.Results:1.The tumor-bearing mouse injected intraperitoneally with plasmid L6expressed green fluorescence indicated by in vivo imaging.2.Most of mice in the control groups (M7group, PBS group, tumor untreated group) gained weight rapidly due to tumor ascites and intra-abdominal tumor growth and died within seven weeks. The majority of mouse in the treatment group (L3group) showed prolonged survival and slowly weight gain. 3.In the treatment group (L3group), the tumor-bearing mice showed prolonged survival. Compared with other groups, the difference was statistically significant (P＜0.05).4.In the first and second week, the absolute number of CD4+T cells in the treatment group (L3group) was significantly different from that of the control group (M7group, PBS group, the tumor untreated group), and that of the blank control group (no tumor group)(P<0.05), and the value of the treatment group (L3group) was higher than that of the other groups.In the first week, the number CD8+T cells of the treatment group (L3group) was significantly different from that of the control groups (M7group, PBS group, the tumor untreated group), and that of the blank group (no tumor group)(p<0.05), and the value of the treatment group was higher than that of the other groups. In the second week, the difference between the absolute value of CD8+T cells of the treatment group (L3group) and that of the control groups (M7group, the PBS group, the tumor untreated group), and the blank group (tumor group) was not statistically significant(p>0.05).The difference between the CD8+/CD4+cells ratio of the treatment group (L3group) and that of the control groups (M7group, PBS group, tumor untreated group) and blank group (tumor group) was not statistically significant(P>0.05) in the first week and the secend week.In the first week, the difference between the number of CD25+CD4+T cells of the treatment group (L3group) and that of the control groups (M7group, PBS group, tumor untreated group) and blank control group(no tumor group) was statistically significant (P<0.05), and the value of the treatment group was higher than that of the control groups.In the second week,the difference between the number of CD25+CD4+T cells of treatment group (L3group) and that of the control groups(M7group, PBS group, the tumor untreated group) and blank control group(no tumor group) was not statistically significant(P>0.05).The difference between the number of CD11b+macrophages of the treatment group(L3group) and that of the control groups(M7group, PBS group, the tumor untreated group) and blank control group(no tumor group) was statistically significant(p＜0.05), and the value of the treatment group (L3group) was higher than that of the other groups at first week and second week.The difference between the number of NKG2D+NK cells of the treatment group (L3group), percentage of lymphocytes and that of the control groups(M7group, PBS group, tumor untreated group) had no statistical significance(P>0.05) in the first week and the second week.The difference between the total number of peritoneal cells of the treatment group(L3group) and that of the control groups(M7group, PBS group, tumor untreated group) and the blank control group(no tumor group) was statistically significant (P<0.05),and the value of the treatment group was higher than that of the control group in the first week and the second week.5.In the treatment group (L3group), the number of CD8+T cells, the number of CD25+CD4+T cells, the number of CDllb+cells, the percentage of lymphocytes and the total number of the peritoneal cells in the first week were significantly different from that of the second week(P<0.05). The proportion of lymphocytes and the number of CD25+CD4+T cells in the first week were less than that of in the second week, but the number of CD8+T cells, the number of CD11b+cells, and the total number of peritoneal cells in the first week were more than that of in the second week. In addition, the number of the CD4+T cells, the CD8+/CD4+cell ratio, and the number of NKG2D+NK cells had no statistical difference(p＞0.05) between the first week and the second week.Conclusion:1.Intraperitoneal injection of plasmid vector can result in vivo gene transfection and express the gene.2.The pHi2-spIL15-CMV-TAT (L3) gene therapy for the treatment of the peritoneal cancer metastases in mice could significantly delay the tumor growth rate and extend the survival of tumor-bearing mice.3.The pHi2-spIL15-CMV-TAT (L3) gene therapy could strengthen the ability of the tumor-bearing mice to kill tumor cells. The mechanism include inhibiting the infiltration of CD4+CD25+T cells and promoting the infiltration of CDllb+macrophages, the CD4+helper T cells and CD8+cytotoxic T cells in the abdominal cavity.4.The pHi2-spIL15-CMV-TAT (L3) gene therapy don’t increase the infiltration of the NKG2D+NK cells, which suggest that in this model, IL-15do not enhance tumor resistance by increasing the number of NKG2D+NK cells.This experiment provides the basis to investigate the mechanism of IL-15tumor immunogene therapy, and lay a solid foundation for further experimental study.