ARTICLES The presence of bacteria and viruses in human tumors has been recog- nized for more than 50 years 1 . Large numbers of bacteria 2,3 as well as viral particles have been found in tumors excised from patients 4–7 . To demonstrate the survival of bacteria in tumors, spores of the obligate anaerobe Clostridium pasteurianum have been injected intra- venously into tumor-bearing mice and found to replicate in the hypoxic center of the tumor 8 . Intratumoral and intravenous (i.v.) injection of auxotrophic mutants of S. typhimurium results in elevated bacterial titers in the tumor tissues 9 . Engineered retroviral and aden- oviral vectors have been administered intratumorally and systemically to tumorous animals, resulting in reduction of tumor size 10,11 and metastatic activity 10,12,13 , as well as in decreased angiogenesis 13 . To determine the location of viral particles in rodents with tumors, vac- cinia virus carrying the firefly luciferase expression cassette has been injected intravenously 14–17 . Subsequent luciferase assays of homo- genates of excised individual organs and tumors reveal a 3- to 500-fold enhancement of light emission in tumor samples, showing the accu- mulation of viral particles. To determine the spatial and temporal progression of infections in live animals with implanted tumors, tracing the movement of bacteria or virions is crucial. Isolated structural genes encoding light-emitting proteins, such as luciferases and fluorescent proteins 18–24 , allow the detection of bacteria based on luciferase-catalyzed light emission or fluorescence 25–31 . Transfer of the luxCDABE gene cluster 32,33 into bacteria results in continuous light emission without the need of exogenous substrate 34 . Injection of light-emitting bacteria allows the visualization and localization of bacteria in live mice 35 , even through hard tissues 36 . The Renilla reniformis luciferase–Aequorea victoria green fluorescent (GFP) fusion protein (RUC-GFP) 37,38 allows real- time monitoring of gene activation in live animals based on luciferase activity and GFP fluorescence 39 . The activity of the RUC-GFP expres- sion cassette inserted into vaccinia virus DNA (rVV-RUC-GFP) has been imaged in both virus-infected mammalian cell cultures and in virus-infected live animals 40 . In this paper, we describe the monitoring of the movement of light-emitting bacteria and vaccinia virus from the injection site to tumor tissue in live animals. We show that bacteria or vaccinia virus survived and replicated in the tumors for weeks without causing bacteremia or viremia. This was observed in both immunocompro- mised and immunocompetent animals with allogeneic and syngeneic tumors. RESULTS Clearance of bacteria and vaccinia virus from nude mice To follow the fate of bacteria injected intravenously into the animals, we monitored each animal by low-light imaging at 2-d time intervals (n > 10). Injection of an attenuated S. typhimurium, an intracellular bacterium, immediately resulted in a disseminated light throughout the body of the animal (Fig. 1a). In contrast, injection of V. cholerae, an extracellular bacterium, resulted in light emission localized in the liver 1 Department of Biochemistry, 2 Center for Molecular Biology and Gene Therapy, School of Medicine, Loma Linda University, Loma Linda, California 92350, USA. 3 Genelux Corp., San Diego Science Center, 3030 Bunker Hill St., Ste. 310, San Diego, California 92109, USA. 4 Department of Microbiology, Biocenter 5 Rudolf-Virchow-Center, DFG-Research Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg D97074, Germany. Correspondence should be addressed to A.A.S. (Msz1998@aol.com). Published online 8 February 2003; doi:10.1038/nbt937 Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins Yong A Yu 1,3 , Shahrokh Shabahang 1 , Tatyana M Timiryasova 2,3 , Qian Zhang 3 , Richard Beltz 1 , Ivaylo Gentschev 4 , Werner Goebel 4 & Aladar A Szalay 1,4,5 We have shown that bacteria injected intravenously into live animals entered and replicated in solid tumors and metastases. The tumor-specific amplification process was visualized in real time using luciferase-catalyzed luminescence and green fluorescent protein fluorescence, which revealed the locations of the tumors and metastases. Escherichia coli and three attenuated pathogens (Vibrio cholerae, Salmonella typhimurium, and Listeria monocytogenes) all entered tumors and replicated. Similarly, the cytosolic vaccinia virus also showed tumor-specific replication, as visualized by real-time imaging. These findings indicate that neither auxotrophic mutations, nor vaccinia virus deficient for the thymidine kinase gene, nor anaerobic growth conditions were required for tumor specificity and intratumoral replication. We observed localization of tumors by light-emitting microorganisms in immunocompetent and in immunocompromised rodents with syngeneic and allogeneic tumors. Based on their ‘tumor-finding’ nature, bacteria and viruses may be designed to carry multiple genes for detection and treatment of cancer. NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 3 MARCH 2004 313 © 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology