Il-15 Gene Therapy Of Colon Cancer In Mice

Although great progress has been made in the biomedical field in recent decades, the treatment and prognosis of malignant tumors is still far from optimistic. Gene therapy has become an important component of cancer treatment in recent years, especially the cytokine-based immunogene therapy. Many achievements have been made in gene therapy of cancer; however, problems still exist, such as the safety of gene transfer vector and the efficacy of transfection which need further improved. We had constructed the plasmid vector pHi2-IL15-CMV-tat (L1) with dual-promoter amplified expressing of human IL-15. The IL-15 signal sequence was replaced by IL-2 signal sequence, resulting plasmid pHi2-spIL15-CMV-tat (L3). The CEA promoter was cloned into plasmid L1,L3 and replaced CMV promoter to construct plasmid pHi2-IL15-CEA-tat (L2),pHi2-spIL15-CEA-tat (L4). Current study will further evaluate these vectors in vitro and in vivo.Plasmid vectors expressing IL-15 or encoding enhanced green fluorescence protein (EGFP) were transfected by electroporation into SW480, a CEA-positive human colon cancer cell line, and MCF-7, a CEA-negative human breast cancer cell line. The transfection efficiency was determined by fluorescence microscope and flow cytometry. IL-15 expression was detected by ELISA. In in vivo study, plasmid L6 was injected into the peritoneum of BALB/c mice and transfected into the subcutaneously growing CT-26 tumor to evaluate the transfection efficacy by in vivo imaging techniques. BALB/c mice were inoculated with CT-26 cells by peritoneal injection, and the plasmid vectors L3, L4 were transfected by intraperitoneal injection after tumor inoculation. Empty plasmid and PBS were used as control. Weight change and life span of the mice were. In addition, the therapeutic effects of L3 were studied through electroporation to control the growth of subcutaneously inoculated CT-26 tumor.Results showed that green fluorescent protein (GFP) could be detected in the transfected cells 24 hours after transfection. The percentage of transfection was 10%-30%. The IL-15 expression in culture supernatant of L3 and L4 transfectant was significantly higher than that of L1 and L2 (P＜0.05). In transfected SW480 cells, there is no significant deference for IL-15 expression between the CEA promoter positively controlled plasmids L2, L4 and the CMV promoter positively controlled plasmids L1, L3 respectively (P>0.05). In transfected MCF-7 cells, CEA promoter positively controlled plasmids L2, L4 gave rise to lower IL-15 expression than the CMV promoter positively controlled plasmids L1, L3, respectively (P<0.01). GFP expression was observed after intraperitoneal injection of plasmid L6 and electroporation into the subcutaneously inoculated CT-26 tumor. Comparing to the control group, and repeated injection of plasmid L3 (pHi2-spIL15-CMV-tat) and L4 (pHi2-spIL15-CEA-tat) DNA were able to inhibit tumor growth, and prolong the life of tumor-bearing mice. In addition, in vivo transfection of plasmid L3 through electroporation slowed the subcutaneously inoculated CT-26 growth.In conclusions, electroporation can efficiently transfer IL-15 expression plasmids into tumor cells in vitro and in vivo, in addition, intraperitoneal injection results in efficient transgene expression by epithelial cells in the peritoneum. IL-15 expression plasmid can decrease the growth of inoculated tumor cells after intrapertoneal injection. In addition, electroporation of IL-15 expression plasmid can inhibit the growth of subcutaneously inoculated mice CT-16 tumor. Therefore, this study provides evidence for the clinical application of IL-15 base gene therapy.