Salmonella -mediated tumor-targeting TRAIL gene therapy significantly suppresses melanoma growth in mouse model Jianxiang Chen, 1 Bingya Yang, 1 Xiawei Cheng, 1 Yiting Qiao, 1 Bo Tang, 1,2 Guo Chen, 1 Jing Wei, 1 Xiufeng Liu, 1 Wei Cheng, 1 Pan Du, 1 Xiaofeng Huang, 1 Wenhui Jiang, 1 Qingang Hu, 1 Yiqiao Hu, 1 Jiahuang Li 1,3 and Zi-Chun Hua 1,2,3 1 The State Key Laboratory of Pharmaceutical Biotechnology and School of Stomatology, Affiliated Stomatological Hospital, Nanjing University, Nanjing; 2 Changzhou High-Tech Research Institute of Nanjing University and Jiangsu TargetPharma Laboratories, Changzhou, China (Received June 2, 2011 ⁄ Revised October 19, 2011 ⁄ Accepted October 22, 2011 ⁄ Accepted manuscript online November 6, 2011 ⁄ Article first published online December 15, 2011) Attenuated Salmonella typhimurium (S. typhimurium) strains can selectively grow and express exogenous genes in tumors for targeted therapy. We engineered S. typhimurium strain VNP20009 to secrete tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) under the control of a hypoxia-induced nirB promoter and examined the efficacy of Salmonella-mediated targeted expression of TRAIL in mice bearing melanoma tumor and in TRAIL-resistant RM-1 tumor. We found that VNP preferentially accumulated in tumor tissues and the nirB promoter effectively drove targeted expression of TRAIL. Compared with recombinant TRAIL protein and VNP20009 combination therapy, VNP20009 expressing TRAIL significantly suppressed melanoma growth but failed to suppress RM-1 tumor growth. Furthermore, we confirmed that VNP20009 expressing TRAIL yielded its antitumor effect by inducing mela- noma apoptosis. Our findings indicate that Salmonella-mediated tumor-targeted therapy with TRAIL could reduce tumor growth and extend host survival. (Cancer Sci 2012; 103: 325–333) T umor tissues consume a greater amount of oxygen than that supplied by the tumor-feeding blood vessels. (1) Normal tis- sues typically have median oxygen tensions of 40–70 mmHg, whereas that of most solid tumors is <10 mmHg. Clinically, poor oxygenation is a major indicator of adverse prognosis for solid tumors, which frequently contain large regions with low oxygen concentrations. (2–6) One contributing factor to the adverse outcome is the inadequate distribution of antitumor agents to the hypoxic areas of a tumor where blood flow is slug- gish or highly irregular. (2) Strategies for improving tumor oxy- genation have met with rather limited success. (7) Specific killing of hypoxic tumor cells by hypoxia-selective tumoricidal agents or modalities offers a separate strategy that views the presence of hypoxia in tumors not only as a determinant for poor progno- sis but also as an opportunity for tumor-specific treatment. Selective targeting of tumors where hypoxia exists enhances targeted concentration of therapeutic agents of interest in cancer cells while minimizing damage to the normal tissues, regardless of whether these therapeutic agents are chemotherapeutic drugs or recombinant proteins or ectopic genes. Attenuated Salmonella typhimurium (S. typhimurium) strains have been shown to selec- tively grow and express exogenous genes in tumors for targeted therapy. (8–12) We have previously shown that the facultative anaerobe S. typhimurium strain VNP20009 can replicate in hyp- oxic tumors (13–16) and is synergistic with cyclophosphamide against melanoma in a mouse tumor model. (17) Because the bac- teria could preferentially accumulate in the hypoxic tumor microenvironment, there have been attempts to engineer control- lable expression of therapeutic molecules such as prodrug- converting enzymes and antigens. (18–21) Tumor necrosis fac- tor-related apoptosis-inducing ligand can selectively induce apoptosis in cancer cells and is effective against many cancers, including melanoma. (22) Although S. typhimurium can thrive in the hypoxic environ- ment of solid tumors after systemic infection, it can also survive in the oxygenated environment of normal tissues. (23,24) By sur- viving in normal tissues such as the liver, S. typhimurium engi- neered to secrete TRAIL may generate hepatotoxicity, as TRAIL has been found to induce hepatic cell death. (25) There- fore, selective accumulation of S. typhimurium in tumors not only enhances localized expression of therapeutic genes of inter- est but also avoids unintended toxicities from extra-tumor growth of S. typhimurium. The anaerobic-inducible nirB pro- moter can be heterologously expressed in facultative anaerobes and shows excellent stability, (26) and has been explored in Salmonella-mediated cancer immunotherapy. (27–33) Here, we engineered S. typhimurium strain VNP20009 to carry nirB promoter-driven vectors expressing our therapeutic gene of interest encoding TRAIL and we evaluated the distribution of VNP20009 and the effectiveness of the nirB promoter in mice. We further assessed the efficacy of VNP20009 engineered to produce TRAIL in a mouse melanoma model and mouse RM-1 prostate tumor. We found that VNP preferentially accumulated in tumor tissues and the nirB promoter effectively drove targeted expression of TRAIL. We also showed that Salmonella-medi- ated tumor-targeted TRAIL gene therapy could reduce tumor growth and extend host survival, suggesting the preclinical util- ity of Salmonella-mediated tumor-targeted therapy against mela- noma. Materials and Methods Expression vectors. The prokaryotic pGFP expression vector (LacZ promoter, pLacZ-GFP) was purchased from Clonetech (Mountain View, CA, USA). The PCR primers and their sequences are listed in Table 1. sTRAIL was amplified from B6 mouse liver cDNA using primers TRAIL-F (nirB) and TRAIL- R. The amplified products were ligated with the nirB promoter by overlap PCR using primers nirB-F and TRAIL-R, which were subsequently cloned into the HindIII and SalI site of the plasmid pBR322 (Takara, Kusatsu, Japan) and the construct was designated pTRAIL. The gene encoding eGFP was amplified using primers GFP-F (nirB) and GFP-R, respectively, from the plasmid pEGFP (Clontech). The amplified products were then ligated to the nirB promoter and digested with HindIII and SalI, or directly digested with HindIII and SalI then subcloned into 3 To whom correspondence should be addressed. E-mail: zchua@nju.edu.cn; lijiah@nju.edu.cn doi: 10.1111/j.1349-7006.2011.02147.x Cancer Sci | February 2012 | vol. 103 | no. 2 | 325–333 ª 2011 Japanese Cancer Association