of the myriad cancer types , malignant gliomas are probably the most devastating and difficult - to - treat cancer . although enormous advances have been made in the past decade in treating other solid cancers , such as lung and breast cancer , the median survival for the glioblastoma , the most malignant type of gliomas , stayed nearly the same with average about 1 year2 ) . regardless of advances in surgical and imaging techniques , we still face numerous obstacles when treating brain tumors . in brain tumors , the blood - brain barrier , the blood - cerebrospinal fluid barrier and the blood - tumor barrier may act as obstacles in drug delivery . one of the main reasons for the continuing dismal outlook in treatment of malignant gliomas is the lack of effective methods for drug delivery . most conventional methods for delivering drug molecules , such as oral administration or intravenous injection , fail to achieve therapeutic concentrations of drugs within intracranial tumors , despite reaching toxic systemic levels . live bacteria were first used in the treatment of cancer about 150 years ago . today , bacterial strains have been demonstrated specifically targeting in cancers , and supported by genomic sequencing and genetic engineering , there is new interest in the use of bacteria as a vector for cancer treatment7,8 ) . a number of recent publications have revealed that bacteria are capable of targeting both primary cancers and metastases , enabling them to be imaged noninvasively in mouse tumor models3,4,12,13,14 ) . salmonella grow under both aerobic and anaerobic conditions , and so are able to colonize small and large tumors , respectively . our review of prior publications revealed evidence that attenuated salmonella hinder tumor growth in a broad range of human and mouse tumors implanted in mice . in the present study , we engineered an attenuated salmonella typhimurium ( s. typhimurium ) defective in guanosine 5'-diphosphate-3'-diphosphate ( ppgpp ) synthesis ( ppgpp strain ) , which expresses the bacterial luciferase gene ( lux ) as a means of providing an imaging signal . the lux operon was integrated into the salmonella chromosome ; therefore the attenuated bacteria produce bioluminescence which does not require an exogenous source of substrate . and then , we established mouse glioma xenograft models by stereotactic injection of glioma cell line , which could be visualized using bioluminescence to evaluate tumor growth , and investigated the targeting of the glioma xenograft by the genetically engineered s. typhimurium . the bioluminescence emitted from the salmonella was optically detected in the mouse glioma xenograft models . u87 malignant glioma cells ( u87-mg ) cells were grown in high - glucose dulbecco 's modified eagle 's medium containing 10% fetal bovine serum and 1% penicillin - streptomycin . u87-mg cell line was transfected with firefly luciferase ( fluc ) , as shown in previous paper6 ) . bacterial doses were 310 colony - forming units per mouse suspended in 100 l of phosphate buffered saline and injected into the tail vein of each mouse . the brains of sacrificed mice were removed , fixed in formalin , and embedded in paraffin . for immunofluorescent staining , the primary and secondary antibody was rabbit anti - salmonella ( abcam , cambridge , ma , usa ) and alexa fluor 488 chicken anti - rabbit ( 1:100 ; invitrogen , carlsbad , ca , usa ) , respectively . the samples were mounted with 4',6-diamidino-2-phenylindole / antifade ( 1:200 ; invitrogen , carlsbad , ca , usa ) . images of fluorescently immunolabeled sections were acquired using an 80i microscope ( nikon , tokyo , japan ) . images were captured using cell - p imaging software ( olympus , tokyo , japan ) in both the fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate channels . all animal care and procedures were approved by animal care and use committees of our institute . u87-fluc was implanted in 6-week old male balb / cann athymic nu / nu mice ( orient bio , seoul , korea ) by stereotaxic injection into the left striatum . mice were anesthetized with isoflurane ( 1.5% in air ) and secured in a stereotaxic apparatus ( stoelting europe , dublin , ireland ) . a small hole ( 0.5 mm diameter ) was drilled 3 mm posterior to and 3 mm left from the bregma . u87-fluc cells ( 510 ) respectively dissolved in 5 l of minimum essential medium were injected with a 10 l hamilton syringe into the left hemisphere at a depth of 3.5 mm below the brain surface . on retreat of the injection needle , the hole in the skull was sealed with bone wax and the scalp was sutured . tumor growth was followed using the ivis100-system ( caliper , hopkinton , ma , usa ) until the region of interest ( roi ) reached around 210 ( photons / s / cm / sr ) ( about day 15 in u87-fluc - bearing nude mouse ) , whereupon s. typhimurium-ppgp - plux intravenously into the tail vein . rio signal was analyzed at ivis - system every day . to obtain images of bacterial bioluminescence , anesthetized animals were placed in a light - tight chamber of the ivis100 imaging system equipped with a cooled charge - coupled device ( ccd ) camera . the dose of luciferin ( caliper , hopkinton , ma , usa ) that was intraperitoneally ( i.p . ) injected was 3 mg per mouse . pseudo - color images indicating photon counts were overlaid on photographs of the mice using the living image software v. 2.25 ( caliper , hopkinton , ma , usa ) . a roi was selected manually based on the signal intensity measured at the same site . the data of the roi was normalized to peak signal intensity of each time course . u87 malignant glioma cells ( u87-mg ) cells were grown in high - glucose dulbecco 's modified eagle 's medium containing 10% fetal bovine serum and 1% penicillin - streptomycin . u87-mg cell line was transfected with firefly luciferase ( fluc ) , as shown in previous paper6 ) . bacterial doses were 310 colony - forming units per mouse suspended in 100 l of phosphate buffered saline and injected into the tail vein of each mouse . the brains of sacrificed mice were removed , fixed in formalin , and embedded in paraffin . for immunofluorescent staining , the primary and secondary antibody was rabbit anti - salmonella ( abcam , cambridge , ma , usa ) and alexa fluor 488 chicken anti - rabbit ( 1:100 ; invitrogen , carlsbad , ca , usa ) , respectively . the samples were mounted with 4',6-diamidino-2-phenylindole / antifade ( 1:200 ; invitrogen , carlsbad , ca , usa ) . images of fluorescently immunolabeled sections were acquired using an 80i microscope ( nikon , tokyo , japan ) . images were captured using cell - p imaging software ( olympus , tokyo , japan ) in both the fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate channels . all animal care and procedures were approved by animal care and use committees of our institute . u87-fluc was implanted in 6-week old male balb / cann athymic nu / nu mice ( orient bio , seoul , korea ) by stereotaxic injection into the left striatum . mice were anesthetized with isoflurane ( 1.5% in air ) and secured in a stereotaxic apparatus ( stoelting europe , dublin , ireland ) . a small hole ( 0.5 mm diameter ) was drilled 3 mm posterior to and 3 mm left from the bregma . u87-fluc cells ( 510 ) respectively dissolved in 5 l of minimum essential medium were injected with a 10 l hamilton syringe into the left hemisphere at a depth of 3.5 mm below the brain surface . on retreat of the injection needle , the hole in the skull was sealed with bone wax and the scalp was sutured . tumor growth was followed using the ivis100-system ( caliper , hopkinton , ma , usa ) until the region of interest ( roi ) reached around 210 ( photons / s / cm / sr ) ( about day 15 in u87-fluc - bearing nude mouse ) , whereupon s. typhimurium-ppgp - plux intravenously into the tail vein . to obtain images of bacterial bioluminescence , anesthetized animals were placed in a light - tight chamber of the ivis100 imaging system equipped with a cooled charge - coupled device ( ccd ) camera . the dose of luciferin ( caliper , hopkinton , ma , usa ) that was intraperitoneally ( i.p . ) injected was 3 mg per mouse . pseudo - color images indicating photon counts were overlaid on photographs of the mice using the living image software v. 2.25 ( caliper , hopkinton , ma , usa ) . a roi was selected manually based on the signal intensity measured at the same site . the data of the roi was normalized to peak signal intensity of each time course . injection of d - luciferin , and the mean photon flux relative to peak signal was determined . 1a shows the bioluminescence images of a nude mouse with orthotopic glioma xenograft from this experiment . bioluminescence images of nude mouse , displayed as a pseudocolor image in the brain , revealed intense signals arising from the tumor . bioluminescence images of u87-fluc tumors developed in mice ( n=8 ) from day 10 to day 24 after implantation were obtained ( fig . modification of cells by fluc tranfection allowed stable , long - term expression of the luciferase gene by the transduced cells . this optical bioluminescence of brain tumor model was non - invasive , reproducible , repeated detection using the cooled - ccd camera . we repeated this experiment using the mouse implanted with lewis lung cancer - fluc ( llc - fluc ) cell line ( data not shown ) . expression of the lux gene was monitored using a cooled ccd camera . we determined the spatial distribution of s. typhimurium-ppgpp carrying fluc , using nude mice in which the u87-fluc cell line had been implanted in the brain 15 days ( fig . 2a - a ) before s. typhimurium-ppgpp ( 310 cfu ) were intravenously injected into each mouse via the tail vein . bioluminescence images were then collected . during the first few hours , bioluminescence signals were detected mainly in the spleen and liver of these mice . however , 5 days post - inoculum , signals were detected exclusively in the tumor region , and persisted more than 6 days . strong bioluminescence signals from the chromosomal lux operon of salmonellae were detected specificially in the tumor area in 6 mice ( fig . however , these mice displayed signal after removing out brain for direct detecting under the a cooled ccd camera . mouse was sacrificed to perform hematoxylin & eosin and immunofluorescence staining at day 9 after bacteria injection . immuofluorescence staining revealed that the genetically engineered bacteria maintained their ability to target glioma xenograft injected with the u87-fluc cell line ( fig . 2c - a - d ) . we repeated this experiment in llc - fluc bearing - tumor model after tail vein injection with salmonella , the bacteria also targeted the brain tumor ( data not shown ) . injection of d - luciferin , and the mean photon flux relative to peak signal was determined . 1a shows the bioluminescence images of a nude mouse with orthotopic glioma xenograft from this experiment . bioluminescence images of nude mouse , displayed as a pseudocolor image in the brain , revealed intense signals arising from the tumor . bioluminescence images of u87-fluc tumors developed in mice ( n=8 ) from day 10 to day 24 after implantation were obtained ( fig . modification of cells by fluc tranfection allowed stable , long - term expression of the luciferase gene by the transduced cells . this optical bioluminescence of brain tumor model was non - invasive , reproducible , repeated detection using the cooled - ccd camera . we repeated this experiment using the mouse implanted with lewis lung cancer - fluc ( llc - fluc ) cell line ( data not shown ) . we determined the spatial distribution of s. typhimurium-ppgpp carrying fluc , using nude mice in which the u87-fluc cell line had been implanted in the brain 15 days ( fig . 2a - a ) before s. typhimurium-ppgpp ( 310 cfu ) were intravenously injected into each mouse via the tail vein . however , 5 days post - inoculum , signals were detected exclusively in the tumor region , and persisted more than 6 days . strong bioluminescence signals from the chromosomal lux operon of salmonellae were detected specificially in the tumor area in 6 mice ( fig . however , these mice displayed signal after removing out brain for direct detecting under the a cooled ccd camera . mouse was sacrificed to perform hematoxylin & eosin and immunofluorescence staining at day 9 after bacteria injection . immuofluorescence staining revealed that the genetically engineered bacteria maintained their ability to target glioma xenograft injected with the u87-fluc cell line ( fig . 2c - a - d ) . we repeated this experiment in llc - fluc bearing - tumor model after tail vein injection with salmonella , the bacteria also targeted the brain tumor ( data not shown ) . the overall prognosis of brain cancer patients has remained virtually unchanged for the last 20 years11 ) , with minimal improvement despite advances in surgery , chemotherapy , and radiation treatment . therefore , it is necessary to develop novel drug delivery system . for this purpose , we considered the targeted use of bacteria as drug delivery vehicles . orthotopic brain tumors are inaccessible for direct measurements of tumor growth , therefore in vivo bioluminescence imaging is particularly attractive in evaluating brain tumor status . one study concluded that bioluminescence imaging is a highly sensitive technique for assessment of tumor burden and response to therapy , making it a superior option for longitudinal monitoring in intracranial rodent models of primary brain tumors1 ) . two studies compared bioluminescence signal of intracranial supratentorial tumors with magnetic resonance imaging - based measurements using complementary models - the 9l gliosarcoma model in rat9 ) and the u87 model in nude mice10)- and found an excellent correlation . so , bioluminescence imaging is a useful tool to evaluate the brain tumor generation and growth . but , the bioluminescence signal of the tumor is suddenly lost during penetration through tissue and skull , making comparisons between mice is difficult because of the variable depth of targeting in each tumor . using each mouse as its own control normalizes these variations that make data more believable . therefore , we established a glioma xenograft model that could be traced using a cooled ccd camera . attenuated s. typhimurium reportedly has a propensity to naturally accumulate and replicate in a wide variety of solid tumors7 ) . although there are some doubt in safety of bacterial injection , previous investigations demonstrated that salmonella injection with the amount used in this study ( 310 cfu ) did not decline body weight and survival time13,14 ) . they checked gfp - labeled bacteria under the ccd camera without immunofluorescence or ihc staining . furthermore , without treatment effect after tail vein injection , inhibition of tumor growth was only observed after directly intracranial injection of bacteria . the main reason of these results could be explained by lower sensitivity of gfp to be detected under ccd camera . our paper focuses on bacteria targeting in glioma tumor model using more sensitive reporter gene lux . in this report , we have demonstrated that s. typhimurium-ppgpp - lux targeted glioma xenograft in a mouse model after tail vein injection . in sharp contrast , three days later , s. typhimurium was undetectable in the liver , lung , spleen , and kidney , but it continued to proliferate in the brain tumor for over 6 days . therefore , genetically engineered s. typhimurium may be a useful probe for targeting therapy glioma in the future . the present study successfully established a valuable tool of bacteria targeting gene therapy in a glioma xenograft model . this genetically engineered s. typhimurium could be useful for pre - clinical evaluation of new therapeutic regimens for the treatment of brain tumors .
objectivewith the growing interests of bacteria as a targeting vector for cancer treatment , diverse genetically engineered salmonella has been reported to be capable of targeting primary or metastatic tumor regions after intravenous injection into mouse tumor models . 
 the purpose of this study was to investigate the capability of the genetically engineered salmonella typhimurium ( s. typhimurium ) to access the glioma xenograft , which was monitored in mouse brain tumor models using optical bioluminescence imaging technique.methodsu87 malignant glioma cells ( u87-mg ) stably transfected with firefly luciferase ( fluc ) were implanted into balb / cann nude mice by stereotactic injection into the striatum . after tumor formation , attenuated s. typhimurium expressing bacterial luciferase ( lux ) was injected into the tail vein . 
 bioluminescence signals from transfected cells or bacteria were monitored using a cooled charge - coupled device camera to identify the tumor location or to trace the bacterial migration . 
 immunofluorescence staining was also performed in frozen sections of mouse glioma xenograft.resultsthe injected s. typhimurium exclusively localized in the glioma xenograft region of u87-mg - bearing mouse . 
 immunofluorescence staining also demonstrated the accumulation of s. typhimurium in the brain tumors.conclusionthe present study demonstrated that s. typhimurium can target glioma xenograft , and may provide a potentially therapeutic probe for glioma .