CpG ODN, Toll Like Receptor (TLR)-9 Agonist, Inhibits Metastatic Colon Adenocarcinoma in a Murine Hepatic Tumor Model Ik Yong Kim, M.D.,‡ Xiaohong Yan, Ph.D.,* Samer Tohme, M.D.,* Aqeel Ahmed, M.D.,† Carlos Cordon-Cardo, M.D.,† H. M. C. Shantha Kumara, Ph.D.,* Soo-Ki Kim, M.D.,‡ and Richard L. Whelan, M.D.* ,1 *Department of Surgery, Division of Colon and Rectal Surgery, St Luke-Roosevelt Hospital Center, New York, New York; †Herbert Irving Comprehensive Cancer Center, Columbia University, New York, New York; and ‡Department of Surgery and Institute of Basic Medical Science, Yonsei University, Wonju College of Medicine, Wonju, Korea Originally submitted August 27, 2010; accepted for publication December 15, 2010 Background. Colorectal liver metastases (mets) are often refractory to conventional therapies. CpG oligo- deoxynucleotide 1826 (CpG), a Toll like receptor (TLR)-9 agonist, inhibits murine tumor growth by aug- menting Th1 immunity. The impact of CpG on meta- static colon tumors is unknown. The purpose of this study was to determine the effect of CpG on the growth of hepatic colon cancer mets. Methods. Two studies with separate control groups were performed using 40 Balb/C mice (study A, CpG 50 mg/dose; study B, 100 mg/dose; n [ 9–11/subgroup). Tumors were induced via portal vein injection of 2 3 10 4 CT26 colon tumor cells. After surgery, the mice were randomized; test groups were given 14 daily in- traperitoneal (i.p.) CpG injections (50 or 100 mg/dose) while the control group received i.p. saline. On d 21 mice were sacrificed, the livers and spleens ex- cised and weighed and the mets counted (reported as median ± 95% confidence interval [CI]) and histologi- cally assessed. Results. The CpG mice had significantly fewer he- patic mets/mouse (study A, median two nodules, 95% CI, 0–3; study B, 0 nodules, 95% CI 0–0) than the control mice (study A, 6 nodules, 95% CI, 3–9, P [ 0.002; Study B, 6 nodules, 95% CI, 3–9, P < 0.001). In study B, there were no mets in 9/11 CpG mice (versus 2/10 for CpG 50 mg and 0/19 for control mice). The mean liver/spleen weights of the CpG mice in both studies were signifi- cantly greater than in control mice. Histologically, high mitotic rates were noted in control mets while fewer tumor cells and histiocytic and lymphocytic in- filtrates were found in CpG livers. Conclusions. CpG inhibited liver tumor growth in this model (100 mg/dose more than 50 mg/dose). CpG was associated with increased liver and spleen weights not related to tumor burden. Increased lymphocytic and histiocytic infiltrates were noted in CpG-treated tumor nodules. Ó 2012 Elsevier Inc. All rights reserved. Key Words: toll like receptor (TLR)-9; CpG oligodeox- ynucleotide (CpG); liver metastasis; colon cancer. INTRODUCTION Surgical resection remains the mainstay of treat- ment for colorectal cancer; unfortunately, at least 35% of patients develop metastases (mets) [1]. The liver is the most common site of recurrence; however, most liver mets are not amenable to curative resection and are difficult to treat. In recent years, strides have been made in the chemotherapy arena; combinations of 5-FU, irinotecan, oxaliplatin, Avastin and cetuximab have resulted in prolonged survival for stage 3 and four patients. However, the mortality rate remains high, thus, the search continues for new cancer treatments. Immunotherapy is one area of study [2, 3]. Tumor vaccines have proven difficult to develop be- cause most antigens found in tumors are also expressed by normal host tissues. Also, tumors, via T-regulatory lymphocytes and other mechanisms, can evade, defeat, or prevent the immune system from effectively pene- trating the tumor. Nonspecific immunomodulators are also being investigated. IL-2, granulocyte-macrophage colony-stimulating factor (GMCSF), and other drugs that broadly stimulate an array of native immune cells 1 To whom correspondence and reprint requests should be ad- dressed at Department of Surgery, Division of Colon and Rectal Sur- gery, St. Luke-Roosevelt Hospital Center, 425 West, 59th Street, Suite 7B, New York, NY 10019. E-mail: rlw3@columbia.edu. 0022-4804/$36.00 Ó 2012 Elsevier Inc. All rights reserved. 284 Journal of Surgical Research 174, 284–290 (2012) doi:10.1016/j.jss.2010.12.021