Design and development of new approaches for targeted radiotherapy of cancer and improvement of therapeutic index by more local radiation therapy are very important issues. Adenovirus-mediated delivery of the sodium iodide symporter (NIS) gene to cancer cells is a powerful technique to concentrate lethal radiation in tumor cells and eradicate tumors with increased therapeutic index. A replicationdefective adenoviral vector expressing the rat NIS gene (Ad-rNIS) was used for in vitro gene delivery and into human prostate cancer xenografts to study antitumor effect. Robust function of the rat symporter was detected in DU145, T47D, and HCT-15 human cancer cell lines transduced with Ad-rNIS. All three cancer cell lines successfully transferred functionally active rat symporter to the plasma membrane, resulting in very high levels of iodine-125 accumulation. Three-dimensional multicellular tumor spheroids derived from DU145 human prostate cancer cells were transduced with Ad-rNIS and incubated with 131 I for 24 hours. After treatment, spheroids rapidly decreased in size and disappeared within 10 days. In vivo data revealed an inhibition of tumor growth in athymic nude mice after intratumoral Ad-rNIS injection followed by 131 I administration. Eightyeight percent of experimental mice survived >30 days, whereas control groups had only 18% survival >30 days. This is the first report that demonstrates the rat NIS gene can effectively induce growth arrest of human tumor xenografts after in vivo adenoviral gene delivery and 131 I administration. The data confirm our hypothesis that the rat NIS gene is an attractive suicide gene candidate for cancer treatment.

Efficient gene delivery is a critical obstacle for gene therapy that must be overcome. Until current limits of gene delivery technology are solved, identification of systems with bystander effects is highly desirable. As an anticancer agent, radioactive iodine 131 I has minimal toxicity. The physical characteristics of 131 I decay allow radiation penetration within a local area causing bystander killing of adjacent cells. Accumulation of 131 I mediated by the sodium iodide symporter (NIS) provides a highly effective treatment for well-differentiated thyroid carcinoma. Other types of cancer could also be treated by NIS-mediated concentration of lethal 131 I radiation in tumor cells. Our group and others previously reported that a significant antitumor effect in mice was achieved after adenoviral delivery of rat or human NIS gene following administration of 3 mCi of 131 I. We have also demonstrated 5-6-fold greater uptake of 125 I by rat NIS over human NIS in human cancer cells. Recently, we reported the capability of the rat NIS and 131 I to effectively induce growth arrest of relatively large tumors (approximately 800 mm 3 ) in an animal model. In the present work tumor growth inhibition was achieved using adenoviral delivery of the rat NIS gene and 1 mCi of 131 I (one-third of the dose used in earlier reports). We also demonstrated that a higher concentration of 123 I was accumulated in the NIS-expressing tumors than in the thyroid 20 min after radioiodine administration. The highest intratumoral radioiodine concentration was observed along the needle track; however, the rat NIS-131 I effectively induced growth arrest of tumor xenografts in mice through its radiological bystander effect. Importantly, the rat NIS allowed reducing the injected radioiodine dose by 70% with the same antitumor efficacy in pre-established tumors. These results suggest that the rat NIS gene may be advantageous compared to the human gene in its ability to enhance intratumoral 131 I uptake.

We have demonstrated that the rat sodium iodide symporter (rNIS) and 131I can effectively induce growth arrest of human prostate tumor xenografts [Mitrofanova, E., Unfer, R., Vahanian, N., Daniels, W., Roberson, E., Seregina, T., Seth, P., and Link, C. (2004). Rat sodium iodide symporter (rNIS) for radioiodide therapy of cancer. Clin. Cancer Res. 10, 6969-6976]. In that study the average size of tumors established in athymic nude mice was 200 +/- 50 mm3 when treated. Testing under more rigorous and extreme in vitro conditions will better evaluate the ability of an anticancer approach to induce tumor regression or killing capacity in preclinical studies. In this work the ability of the rNIS and 131I system to inhibit the growth of relatively large (about 800 mm3 when treated with 131I) and rapidly growing colon tumors in an animal model was examined. in vitro experiments demonstrated that transduction of human colon cancer cells with Ad-rNIS resulted in a 100- to 150-fold increase in 125I uptake compared with nontransduced cells. Western blot analysis revealed robust expression of rNIS protein in cells 72-120 hr posttransduction with Ad-rNIS. Immunocytochemical analysis demonstrated that intracellular localization of rNIS-specific staining was observed mainly in plasma membranes of cells. in vitro studies revealed an immediate inhibition of growth of rapidly expanding tumors after radioiodine injection in the rNIS and 131I treatment group of mice. Twenty-seven percent of experimental mice survived more than 30 days (p = 0.019), whereas control groups had only 7% survival over 30 days. This is the first report demonstrating that rat NIS and 131I can effectively induce growth arrest of relatively large tumors in an animal model.

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