In the United States, colorectal cancer is the third leading cause of cancer-related deaths in both men and women1. The pathogenesis of colorectal cancer begins with the development of polyps in the innermost colorectal lining and progresses to the final stage when metastasis occurs2. Metastasis is the spread of cancer cells to other organs, and has been shown to be organ specific rather than a random process. Genes that direct tumor cells to target specific organs have been identified. The primary tumor is thought to secrete molecules that promote the establishment of liver metastasis even before the arrival of cancer cells into this organ.
We have previously isolated a highly liver metastatic cell line, CT26-FL3 by in vivo selection of CT26 cells in balb/c mice. Microarray analyses showed that the CT26-FL3 cells expressed 34-fold higher levels of the Interleukin-33 (IL-33) cytokine as compared to the parental and less metastatic CT26 cells. Over-expression of IL-33 was shown to potently promote tumor proliferation and metastasis to the liver5 indicating that IL-33 plays a crucial role in the pathogenesis of colon cancer.
In this study, we will use C57Bl/6 mice and the murine colon cancer cell line, MC38, to increase circulating serum levels of IL-33 using a gene therapy approach by in vivo electroporation of a plasmid expressing IL-33, pV1J-IL33. We hypothesized that the increased secretion of IL-33 in MC38 tumor cells will increase cell metastasis to the liver, and that increasing circulating levels of IL-33 in mice bearing MC38 tumors will be sufficient to enhance its ability to metastasize to the liver.
Here we report the construction of plasmids expressing IL-33 and verification of the expression of IL-33 from these plasmids in transfected cells by Western Blot analyses. We also discuss the introduction of pV1J-IL-33 into C57Bl/6 mice by quadriceps injections and in vivo electroporation. Lastly, we report ELISA assays on mouse sera after splenic injections of MC38 tumor cells to determine the levels of secreted IL-33 and how long the IL-33 levels stayed up regulated after in vivo electroporation.
We concluded that the elevated serum levels of IL-33 increased tumor proliferation and their ability to metastasize to the liver in C57Bl/6 mice
