metastatic mouse models of human prostate cancer cures orthotopicSalmonella typhimuriumMonotherapy with a tumor-targeting mutant of

Ming Zhao, Jack Geller, Huaiyu Ma, Meng Yang, Sheldon Penman, and Robert M. Hoffman

doi:10.1073/pnas.0703867104 published online Jun 4, 2007; PNAS

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Monotherapy with a tumor-targeting mutant ofSalmonella typhimurium cures orthotopic metastaticmouse models of human prostate cancerMing Zhao*, Jack Geller*, Huaiyu Ma*, Meng Yang*, Sheldon Penman†‡, and Robert M. Hoffman*‡§

*AntiCancer, Inc., 7917 Ostrow Street, San Diego, CA 92111; †Department of Surgery, University of California, San Diego, CA 92103;and §Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307

Contributed by Sheldon Penman, April 30, 2007 (sent for review March 15, 2007)

Bacterial infection occasionally has a marked therapeutic effect onmalignancies, as noted as early as the 19th century. Recently, therehave been attempts to develop cancer treatment by using tumor-targeting bacteria. These treatments were developed to delivertherapeutic molecules specifically to tumors. Researchers usedanaerobic microorganisms that preferentially grew in necrotictumor areas. However, the resulting tumor killing was, at best,limited. We have developed a far more effective bacterial cancertherapy by targeting viable tumor tissue by using Salmonellatyphimurium leu-arg auxotrophs. Although these bacteria grow inviable as well as necrotic areas of tumors, the nutritional auxo-trophy severely restricts growth in normal tissue. In the currentstudy, we measured the antitumor efficacy of the S. typhimuriumA1-R mutant, which is auxotrophic for leu-arg and has increasedantitumor virulence selected by tumor passage. A1-R was used totreat metastatic PC-3 human prostate tumors that had been or-thotopically implanted in nude mice. GFP was used to image tumorand metastatic growth. Of the 10 mice with the PC-3 tumors thatwere injected weekly with S. typhimurium A1-R, 7 were alive andwell at the time the last untreated mouse died. Four A1-R-treatedmice remain alive and well 6 months after implantation. Tenadditional nontumor-bearing mice were injected weekly to deter-mine the toxicity of S. typhimurium A1-R. No toxic effects wereobserved. The approach described here, where bacterial mono-therapy effectively treats metastatic prostate tumors, is a signifi-cant improvement over previous bacterial tumor-therapy strate-gies that require combination with toxic chemotherapy.

amino acid auxotrophy � bacterial therapy � antitumor efficacy � imaging

In the 1890s, Coley observed that certain cancer patients withpostoperative bacterial infection were cured of their tumors

(1). It has been known since the 1940s that anaerobic bacteriacan selectively grow in hypoxic and necrotic areas of tumors(2–17). Recently, a number of novel approaches to developingtumor-therapeutic bacteria have been described.

Yazawa et al. (18) demonstrated that the anaerobic bacteriumBifidobacterium longum could selectively germinate and grow inthe hypoxic regions of solid tumors after i.v. injection. B. longumis a nonpathogenic Gram-positive bacterium found in the lowersmall intestine and large intestine of humans and other mam-mals. Yazawa et al. showed that B. longum selectively grew in7,12-dimethylbenzanthracene-induced rat mammary tumors af-ter i.v. administration.

Vogelstein et al. (19) compared B. longum and Clostridiumnovyi, an obligate anaerobic, for their capacity to grow withintransplanted tumors. Vogelstein et al. created a strain of C. novyidepleted of its lethal toxin termed C. novyi NT. They showed thati.v.-administered C. novyi NT spores germinated within theavascular regions of tumors in mice and destroyed surroundingviable tumor cells (19). C. novyi NT grew to a greater extent inthe B16 melanoma than B. longum. When C. novyi NT sporeswere administered together with conventional chemotherapeu-tic drugs or radioactivity, extensive hemorrhagic necrosis of

tumors often developed within 24 h, resulting in significantand prolonged antitumor effects (19, 20). Vogelstein et al. (1, 20)subsequently observed that C. novyi NT treatment of tumorselicits an immune response that can, in combination withchemotherapy, result in tumor cure by using syngeneic modelswith immunocompetent mice.

In another approach, attenuated Salmonella typhimuriumstrains, which are facultative anaerobes, have been described asanticancer agents (21). Salmonella strains have been shown topreferentially amplify within tumors and express prodrug-converting enzymes such as the herpes simplex thymidine kinase(21). Initially, S. typhimurium was attenuated by auxotrophicmutations (22, 23). The internal tumor environment apparentlymeets the auxotrophic requirements of the mutated Salmonella,where they replicated 1,000 times the concentration found innormal tissues (21). Within the tumor, there may be areas ofhypoxia that favor the growth of facultative anaerobes. Inaddition, the presence of tumor necrosis provides additionalnutrients, such as purines, required by the organism. Geneticmodification of the Salmonella lipid A was done to reduce septicshock (21). Disruption of the Salmonella msbB gene reducedTNF� induction and increased the LD50 of Salmonella by 10,000-fold (21). The msbB mutant prevents the addition of a terminalmyristyl group to the lipid A domain (21). Melanomas in micetreated with this Salmonella were 6% the size of tumors inuntreated controls (23). Attenuated strains of Salmonella havealso been modified to secrete immunostimulatory cytokines thatmay have the potential to stimulate host antitumor responses (24).

Recently, the lipid A-attenuated S. typhimurium has beenevaluated in a phase I clinical trial (25). Twenty-four patientswith metastatic melanoma and one patient with metastatic renalcell carcinoma received i.v. bolus infusions containing 106 to 109

cfu/m2 of the lipid A-mutant Salmonella (VNP20009). To in-crease safety and reduce toxicity, S. typhimurium was attenuatedby deletion of both the purI and msbB genes. The purI mutantrequires an external source of adenine (25). The resultingVNP20009 strain of S. typhimurium could be safely administeredto patients. At the highest tolerated dose, tumor colonizationwas observed. Additional studies are required to reduce dose-related toxicity and improve tumor localization (25) to obtainefficacy.

To increase the potential of attenuated Salmonella as agene-delivery vector for cancer treatment, Mengesha et al. (26)developed a hypoxia-inducible promoter (HIP-1) to limit geneexpression specifically to the tumor. Mengesha et al. demon-strated that HIP-1 can drive hypoxia-mediated gene expression

Author contributions: M.Z., J.G., M.Y., and R.M.H. designed research; M.Z., H.M., and M.Y.performed research; M.Z., J.G., M.Y., S.P., and R.M.H. analyzed data; and M.Z., M.Y., S.P.,and R.M.H. wrote the paper.

The authors declare no conflict of interest.

‡To whom correspondence may be addressed. E-mail: all@anticancer.com or 27penman@mit.edu.

© 2007 by The National Academy of Sciences of the USA

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in bacteria that have colonized human tumor xenografts inmouse models. Expression of both GFP and red fluorescentprotein (RFP) under control of HIP-1 demonstrated an �15-fold increase relative to a constitutive promoter when tumorswere made hypoxic. Moreover, the use of a constitutive pro-moter resulted in reporter gene expression in both tumorsand normal tissues, whereas reporter gene expression usingHIP-1 was confined to the tumor (26).

Yu et al. (27, 28) used GFP-labeled bacteria to visualizetumors and metastases in mouse models. Three attenuatedpathogens, Vibrio cholerae, S. typhimurium, and Listeria mono-cytogenes, targeted and replicated in tumors. However, no anti-tumor efficacy was observed.

Very recently, Vogelstein’s group (29) showed that treatmentof mice bearing large, established tumors infected with C. novyiNT plus a single dose of liposomal doxorubicin could lead toeradication of the tumors. The bacterial factor responsible forthe enhanced drug release was identified as a previously unrec-ognized protein termed ‘‘liposomase.’’ However, C. novyi NTwas ineffective as monotherapy.

The relative ease and efficacy of externally imaging implantedtumors made fluorescent with GFP or RFP suggested applyingthe technique to other foreign agents such as infecting bacteria(30). Escherichia coli was transfected with a high-expressionplasmid containing the GFP gene. The GFP-expressing E. coli(E. coli-GFP) was administered to mice by various routes,

and the fate of the bacteria was readily visualized in real time bywhole-body imaging. Real-time images were acquired by using acolor CCD video camera by simply illuminating mice at 470 nm(30). We used the tumor and bacterial imaging technologies todevelop therapeutic bacteria capable of targeting tumors asmonotherapy (31, 32).

We initially developed an S. typhimurium strain, A1, whichgrows in tumor xenografts. In sharp contrast, normal tissuerapidly clears these bacteria even in immunodeficient athymicmice. S. typhimurium A1 is auxotrophic (leu/arg-dependent), butapparently receives sufficient support from the neoplastic tissueto grow locally. In vivo, the bacteria caused PC-3 tumor inhibi-tion and regression of s.c. xenografts (31) visualized by whole-body imaging. By day 15, S. typhimurium A1 was undetectable inthe liver, lung, spleen, and kidney, but it continued to proliferatein the PC-3 tumor, which stopped growing. The S. typhimuriumA1 strain grew throughout the tumor, including viable malignanttissue, which may account, in part, for the strain’s uniqueantitumor efficacy (31). This result is in marked contrast toresults with anaerobic bacteria that grew only in tumor-necroticareas.

To increase the tumor-targeting capability of S. typhimuriumA1, the strain was reisolated after infection of a human colontumor growing in nude mice. The tumor-isolated strain, A1-R,had increased targeting for tumor cells in vivo as well as in vitrocompared to A1. Treatment with A1-R resulted in highlyeffective tumor targeting, including viable tumor tissue and sig-nificant tumor shrinkage in mice with s.c. or orthotopic humanbreast cancer xerographs. Survival of the treated animals wassignificantly prolonged (32).

In the present study, we report that S. typhimurium A1-R,administered systemically, can cure metastatic human prostatecancer in orthotopic mouse models, suggesting the clinicalpotential of this cancer-therapeutic approach.

ResultsAnti-Tumor Virulence of S. typhimurium A1-R in Vitro. Dual-colorPC-3 tumor cells, growing on plates, were labeled with GFP inthe nucleus and RFP in the cytoplasm. Fluorescence microscopyshowed their interaction with the GFP-expressing S. typhi-murium A1-R. The bacteria attached to the cells within 30 min.The infected cells initially rounded up. At 60 min after infection,cell membranes appeared broken, and the cytoplasm had begunto fragment. The nucleus also fragmented, and by 200 min thered fluorescent cytoplasm completely dispersed (Fig. 1).

Primary Tumor Growth Suppression in Vivo. PC-3 tumor cells weretransplanted orthotopically into nude mice. In the first experi-ment, the animals were injected with A1-R at day 16 after tumor

Fig. 1. Real-time imaging of S. typhimurium A1-R-induced cell death of PC-3human prostate cancer cells in vitro. Dual-color PC-3 human prostate tumorcells, labeled with RFP in the cytoplasm and GFP in the nucleus, were grown in24-well tissue culture plates to a density of �104 cells per well. A1-R bacteriaexpressing GFP were grown in LB and added to the tumor cells (1 � 105 cfu perwell). After 1 h incubation at 37°C, the cells were rinsed and cultured inmedium containing gentamycin sulfate (20 �g/ml) to kill external but notinternal bacteria. Cells started to die by 60 min after infection. By 200 min, theRFP cytoplasm completely lysed and dispersed, and nuclei fragmented.

Fig. 2. Time course of tumor progression after two treatments with S. typhimurium A1-R. Mice orthotopically implanted with PC-3 human prostate tumorswere given A1-R at 15 and 27 days after tumor implantation. The tumor-bearing mice were imaged at the indicated time points and compared with untreatedcontrol.

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transplantation. The second injection of A1-R was given 2 weekslater. Tumor growth in the treated animals slowed compared tothe untreated group (Fig. 2). However, after these two doses ofA1-R, the tumors resumed growth and eventually killed theanimals.

In a second experiment, tumor-bearing animals were inocu-lated with bacteria weekly, and efficacy was enhanced dramat-ically (Fig. 3). At day 30 after tumor transplantation, the tumorsize in the treated group was significantly smaller than theuntreated group (Fig. 3A). Weekly treatment resulted in inhi-bition of tumor growth and metastasis for a long period andeventual cures (please see below).

Metastasis Suppression. The PC-3 tumor orthotopic model ishighly metastatic. Metastasis occurred by day 21 after tumortransplantation in the untreated animals. By day 30, metastasesspread to the peritoneal cavity and other organs, and the micewere very sick. In contrast, tumors in the treated group grewslowly or disappeared (Fig. 3 B and C). Most untreated animals

died of metastatic disease at 4 to 5 weeks after tumor trans-plantation. However, in the weekly treated group, metastasis wassuppressed (Fig. 4). Treating animals only twice initially inhib-ited tumor growth, but tumor growth later resumed, and me-tastasis eventually resulted in the animals’ death. These datashow the importance of weekly treatment.

Survival Efficacy. The PC-3 tumor orthotopic model results inrapid death of untreated animals. All 10 untreated mice withorthotopic PC-3 tumors died within 6 weeks of tumor implan-tation (Fig. 5A). However, weekly bacteria treatment greatlyprolonged survival of the tumor-bearing mice. Of the 10 micewith the orthotopic PC-3 tumors treated with A1-R, seven werealive at the time the last untreated mouse died, and foureventually appeared cured.

Cures. Forty percent of the tumor-bearing mice apparently curedof detectable disease by weekly bacteria treatment were exam-ined further (Fig. 5A). Tumors and metastases were imaged inreal time to determine cures. It took 10 to 12 weekly injectionsof bacteria to completely cure the mice. After treatment stopped,tumors did not recur. In contrast, after two injections, only 1 of10 mice was cured (Fig. 5B). Further study will examine theeffects of different protocols.

Safety of Bacterial Treatment. Nontumor-bearing mice weretreated with weekly injections of bacteria to establish the toxicityof bacteria treatment. Body weight and survival time weremonitored with no deaths observed. The weekly bacteria treat-ment had no effect on body weight and no apparent toxicity (Fig.6). These results indicated that the animals well tolerated theweekly infection with S. typhimurium A1-R.

Fig. 3. Efficacy of weekly S. typhimurium A1-R treatment on PC-3 primaryand metastatic tumor growth. Mice were given S. typhimurium A1-R weeklyi.v. Primary and metastatic tumor size was measured by GFP imaging at day 30after tumor transplantation. (A) GFP areas of A1-R-treated and untreatedtumors were compared at day 30. The tumor size in the treated group was�75% smaller than the untreated group (n � 10; P � 0.04). (B) Imaging oftumor growth in eight mice of the untreated group at day 30. (C) Imaging oftumor growth of 10 mice in the A1-R-treated group at day 30.

Fig. 4. Time course of tumor growth and regression of A1-R-treated ortho-topic PC-3 in nude mice. The mice were implanted orthotopically with PC-3tumors expressing GFP. Approximately 2 weeks after implantation, the tumorswere externally imageable macroscopically. Ten mice were given S. typhi-murium A1-R weekly i.v. Ten mice served as untreated controls. Primary andmetastatic tumor size was measured by GFP imaging at different time pointsafter tumor transplantation. (A) Whole-body images of an A1-R-treatedmouse. (B) Whole-body images of an untreated mouse.

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DiscussionFrom the results described above, the following conclusions canbe reached:

1. PC-3 human prostate tumor growth and metastasis in nudemice were targeted by i.v.-administered S. typhimurium A1-R,a leu-arg auxotroph.

2. A1-R treatment of PC-3 tumors resulted in a doubling of the50% survival time of the treated mice.

3. Four of the 10 A1-R-treated mice with PC-3 orthotopictumors were cured.

4. Weekly treatment of bacteria is more effective than two-dosetreatment.

5. A1-R treatment resulted in cures of metastatic cancer withoutcombination with toxic chemotherapy, which distinguishesour approach to bacterial therapy of tumors by using afacultative anaerobic from those using obligate anaerobessuch as Clostridia.

Future experiments will involve designing bacteria focusing ontumor-environment-specific gene expression of the infectingbacteria. The particular auxotroph, A1-R, although effective,may not be optimum for all tumor types, and we will examinefurther mutations. We will eventually consider whether therapycan be individualized by tailoring bacteria to target individualpatient tumors.

Materials and MethodsGFP Gene Transfection of S. typhimurium (31). S. typhimurium(ATCC 14028) was grown at 37°C to midlogarithmic phase inliquid LB and harvested at 4°C. Bacteria (2.0 � 108) in 40 �l of10% glycerol were mixed with 2 �l of the pGFP (Clontech,Mountain View, CA) vector containing the hr-GFP gene (Strat-agene, La Jolla, CA) and placed on ice for 5 min beforeelectroporation with a Gene Pulser apparatus (Bio-Rad, Her-cules, CA) according to the manufacturer’s instructions. Elec-troporation was done at 1.8 kV with the pulse controller at1,000-� parallel resistance.

Induction of Bacterial Mutations with Nitrosoguanidine (NTG) andSelection for Auxotrophs (31). Freshly prepared NTG (1 mg/ml insterile water) was added to the washed culture to a finalconcentration of 100 �g/ml in Tris-maleic acid buffer (pH 6.0).The bacteria were incubated with NTG for 30 min. The NTG-treated cells were grown in nutrient broth to express anymutations that were induced. Bacterial colonies were replica-plated in supplemented minimal agar plates containing specificamino acids to identify the requirements of the auxotrophs.Auxotroph A1, which required leu and arg, was identified.

Reisolation of S. typhimurium A1 (32). S. typhimurium A1 auxo-trophs expressing GFP were reisolated as follows: The A1bacteria were injected into the tail vein of an HT-29 human colontumor-bearing nude mouse. Three days after infection, thetumor tissue was removed from the infected mouse. The tumortissue was then homogenized and diluted with PBS. The resultingsupernatant of the tumor tissue was cultured in LB agar platesat 37°C overnight. The bacteria colony with the brightest greenfluorescence was picked up and cultured in 5 ml of LB medium.This strain was termed ‘‘A1-R.’’

Preparation of Bacteria (32). The A1-R bacteria were grownovernight on LB medium and then diluted 1:10 in LB medium.Bacteria were harvested at late-log phase, washed with PBS, andthen diluted in PBS. Bacteria were then injected into the tail veinof nude mice (5 � 107 cfu per 100 �l PBS).

RFP or GFP Gene Transduction of the PC-3 Human Prostate Cancer CellLine (31). For RFP or GFP gene transduction, �60% confluentPC-3 human prostate cancer cells were incubated with a 1:1precipitated mixture of retroviral supernatants of PT67 pack-aging cells and F12K medium containing 7% FBS (Gemini,Calabasas, CA) for 72 h. The packaging cells produced thepLNCX2 vector (Clontech) containing the DsRed2-expressingRFP or the pLEIN vector containing GFP and the neomycinresistance gene. RFP- or GFP-expressing PC-3 cancer cells wereselected in medium containing 200 to 1,000 �g/ml neomycin

Fig. 5. Survival efficacy of S. typhimurium A1-R. (A) Survival efficacy of S.typhimurium A1-R with weekly treatment (n � 10). (B) Survival efficacy of S.typhimurium A1-R with twice-only treatment (n � 10).

Fig. 6. Toxicity of S. typhimurium A1-R. Nontumor-bearing mice were testedfor the possible toxicity of S. typhimurium A1-R. The mice were injected i.v.weekly with the bacteria (5 � 107 cfu). The survival time and body weight werecompared with the untreated mice. A1-R-infected animals treated with re-peated weekly injections of S. typhimurium A1-R survived as long as untreatedmice with no significant body weight change.

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increased in a stepwise manner. Clones expressing RFP or GFPwere isolated with cloning cylinders (Bel-Art Products, Pequ-annock, NJ) by trypsin/EDTA and were amplified and trans-ferred by conventional culture methods in the absence of selec-tive agent.

Establishment of Dual-Color Cells Expressing GFP and RFP. For es-tablishing dual-color cells, PC-3-RFP cells were then incubatedwith a 1:1 precipitated mixture of retroviral supernatants ofPT67 packaging cells expressing GFP linked to histone H2B (33)and culture medium. The histone H2B-GFP fusion gene wasinserted into the pLHCX vector (Clontech) that also containsthe hygromycin resistance gene. To select the double transfor-mants, cells were incubated with hygromycin 72 h after trans-fection. The level of hygromycin was increased stepwise up to 400�g/ml. These sublines stably expressed GFP in the nucleus andRFP in cytoplasm.

Surgical Orthotopic Implantation of Prostate Tumors. Tumor frag-ments (1 mm3) were prepared from a PC-3-GFP tumor growings.c. in nude mice. Two tumor fragments were implanted bysurgical orthotopic implantation in the lateral lobe of theprostate, which was exposed after a lower midline abdominalincision. After proper exposure of the bladder and prostate, thecapsule of the prostate was opened and the two tumor fragments(1 mm3) were inserted into the capsule. The capsule was thenclosed with an 8–0 surgical suture. The incision in the abdominalwall was closed with a 6–0 surgical suture in one layer. Theanimals were kept under isoflurane anesthesia during surgery.All procedures of the operation described above were performedwith a �7 magnification microscope (Olympus, Tokyo, Japan) (34).

Bacterial Infection. When the implanted tumors were externallyimaginable, 10 mice were administered S. typhimurium A1-R(5 � 107) i.v. Schedules are described below along with theindividual experiments.

Analysis of Antitumor Efficacy. Twenty mice were implanted or-thotopically with PC-3 tumors expressing GFP as describedabove. Ten of the 20 mice served as controls and were followeduntil death without treatment. Ten mice were administered S.typhimurium A1-R i.v. weekly, and survival time was comparedto the untreated mice. Progressive tumor enlargement andmetastasis were externally imaged by their GFP fluorescence.

Host Safety. Ten nontumor-bearing mice served as controls forthe possible toxicity of S. typhimurium A1-R. The mice wereweekly injected i.v. with the bacteria (5 � 107 cfu). Survival timeafter bacteria infection was monitored and body weight wasmeasured.

Imaging in Live Mice. The Olympus OV100 Small Animal ImagingSystem (Olympus) containing an MT-20 light source (Olympus)and DP70 CCD camera (Olympus) was used for imaging in livemice. The optics of the OV100 fluorescence imaging system havebeen specially developed for macroimaging as well as microim-aging with high light-gathering capacity. The instrument incor-porates a unique combination of high numerical aperture andlong working distance. Five individually optimized objectivelenses, parcentered and parfocal, provide a 105-fold magnifica-tion range for seamless imaging of the entire body down to thesubcellular level without disturbing the animal. The OV100 hasthe lenses mounted on an automated turret with a high magni-fication range of �1.6 to �16 and a field of view ranging from6.9 to 0.69 mm. The optics and antireflective coatings ensureoptimal imaging of multiplexed fluorescent reporters in smallanimals. High-resolution images were captured directly on a PC(Fujitsu Siemens, Munich, Germany). Images were processed forcontrast and brightness and analyzed with the use of Paint ShopPro 8 and Cell (Olympus) (33).

This work was supported by the U.S. Army Medical Research andMateriel Command Prostate Cancer Research Program AwardW81XWH-06-1-0117 and National Institutes of Health GrantsCA103563 and CA119841.

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