ORIGINAL ARTICLE Cellular and molecular events associated with the antitumor response induced by the cytosine deaminase/5-fluorocytosine suicide gene therapy system in a rat liver metastasis model S Bertin 1,2,8 , S Neves 3,8 , A Gavelli 1,2,4 , P Baque ´ 1,2,5 , N Brossette 1,2 , S Simo ˜es 3,6 , MC Pedroso de Lima 3,7 and V Pierrefite-Carle 1,2 1 INSERM, Unite ´ 638, Nice, France; 2 Faculte ´ de Me´decine, Universite´ de Nice Sophia Antipolis, Nice, France; 3 Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 4 Service de Chirurgie Ge ´ne ´rale et Digestive, Centre Hospitalier Princesse Grace, Monaco; 5 Service de Chirurgie d’Urgence, Ho ˆpital St Roch, Nice, France; 6 Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal and 7 Department of Biochemistry, University of Coimbra, Coimbra, Portugal The bacterial cytosine deaminase (CD) gene converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil. We have previously shown, in a rat liver metastasis model from colon carcinoma, that intratumoral injection of a CD-expressing plasmid into the animals followed by 5-FC treatment results in the regression of the treated tumor as well as distant uninjected tumors. The aim of this study was to further analyze the mechanisms associated with tumor regression induced upon application of suicide CD/5-FC strategy. Tumor regression was associated with an increased apoptosis, the recruitment of natural killer cells, CD4- and CD8 T lymphocytes within the tumors and an increased expression of several cytokines/chemokines mRNAs. These data indicate that the CD/5-FC suicide strategy is associated with the triggering of cellular and molecular events leading to an efficient antitumor immune response involving both innate and acquired immunity. Cancer Gene Therapy (2007) 14, 858–866; doi:10.1038/sj.cgt.7701075; published online 22 June 2007 Keywords: suicide gene therapy; colon carcinoma; cytosine deaminase; cationic liposomes Introduction The liver is the major site of metastases from colorectal cancer, and hepatic metastases are generally responsible for the patient’s death. 1,2 Despite recent progress of chemotherapy, 3 surgical resection of hepatic metastases constitutes the only curative treatment, with a 5-year survival from 25 to 30%. 1 Since resection is applicable in only 10–20% of patients, many efforts have been devoted to the development of alternative treatments. In this context, cancer gene therapy constitutes a promising therapeutic approach. Among the different strategies, suicide gene therapy, which consists in the transfer within the tumor of a viral or bacterial gene able to convert a non-toxic compound into a lethal drug, 4 is particularly interesting for several reasons. First, this strategy results in a tumor-targeted chemotherapy, allowing to bypass the toxic side effects generally associated to systemic chemo- therapy. Second, the existence of a local bystander effect allows to induce complete tumor regression despite a low gene transfer efficiency. 5,6 Finally, regression of distant non-transfected tumors can also be observed because of the triggering of a systemic antitumor immune response, which results in the destruction of the suicide gene- expressing tumor and tumor antigen release (distant bystander effect). 7–12 The induction of an antitumor immune response is essential for the treatment of a metastatic disease since introduction of the suicide gene in a single tumor can theoretically result in the regression of all the metastasis. According to the selected animal model and the applied suicide gene therapy system, T lympho- cytes 9,10 and natural killer (NK) cells 8,11,12 have been reported to be involved in distant antitumor effect. Over the past several years, the use of naked DNA or in association with liposomes 13,14 for in vivo gene transfer has significantly increased, and these non-viral vectors are now used, respectively, in 18 and 7.9% of the clinical trials worldwide. 15 Naked DNA and cationic liposomes are attractive for in vivo gene transfer because of their favorable characteristics, such as biodegradability, low toxicity and immunogenicity, versatility and simplicity of use. 16–18 The two major problems encountered with these Received 11 March 2007; revised 26 April 2007; accepted 26 May 2007; published online 22 June 2007 Correspondence: Dr V Pierrefite-Carle, INSERM, Unite´ 638, Faculte´ de Me´decine, Universite´ de Nice Sophia Antipolis, Avenue de Valombrose, 06107 Nice ce´dex 2, France. E-mail: pierrefi@unice.fr 8 These authors contributed equally to this work. Cancer Gene Therapy (2007) 14, 858–866 r 2007 Nature Publishing Group All rights reserved 0929-1903/07 $30.00 www.nature.com/cgt