Bacterial strains, plasmids, and cell lines
strain SL7838 has been described previously [7
]. This strain contains deletions in the aroA
genes, which make it non-virulent and more specific for targeting tumors. The bacterium was transformed by electroporation [13
] with expression plasmids (Table S1
) encoding the bacterial Lux operon (pCGSL1), GFP (pFVP25.1), and ChrR6 (pET28a+
); ChrR6 [7
]) is an improved version of the Escherichia coli
ChrR wild type nitroreductase with markedly increased capacity for the activation of reductive prodrugs CB 1954 and MMC [7
]. Pure ChrR and ChrR6 were generated as described previously [7
]. JC (murine mammary cancer) cells were obtained from Cancer Research UK; 4T1 (murine mammary cancer), MCF-7 (human mammary cancer), HeLa (human cervical cancer) HCT 116 (human colorectal cancer), and 293T human embryonic kidney cancer cells were obtained from ATCC (Manassas, VA). Cells were grown as adherent cultures in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin (0.5 U and 0.5 µg/ml, respectively; Invitrogen, Carlsbad, CA). CNOB/ChrR6 regime was equally effective against these cell lines; the studies presented here focus on three of them: JC (in vitro
and in vivo
killing and visualization), 4T1 (in vivo
killing, visualization, and MCHB killing mechanism); and HCT 116 (MCHB killing mechanism). Results presented are representative of different cell lines.
In vitro viability assays
The effect of converted prodrug on cell lines was measured as described before [7
]. Prodrug reduction mixtures containing CNOB (Molecular Probes-Invitrogen, Carlsbad, CA) and pure ChrR6, 1 mM NADPH, and DMEM added to a final volume of 0.5 ml were allowed to carry out drug reduction at 37°C for 30 minutes before addition to cancer cells. The extent of drug activation was inferred from the loss of cell viability after 30 minutes. Cell viability was determined by MTS assay according to the manufacturer’s instructions (CellTiter 96®AqueousOne, Promega, Madison, WI). A490
was measured in an ASYS UVM 340 plate reader (Asys Hitech, Cambridge, UK). In control experiments, CNOB was added to cells without ChrR6 and viability was determined by the MTS assay as before.
ChrR6 was also delivered to cancer cells, as specified from a GDEPT system involving Salmonella typhimurium strain SL7838-chrR6, with strain SL7838 being used as control. The strains also expressed GFP which permitted visualization of cell infection. Different multiplicities of infection [MOI; bacterial colony forming units (CFU)/cancer cell] were added to cells in black walled 96 well plates along with 15 µM CNOB. After one hour incubation, fluorescence (GFP: excitation 488 nm, emission 525 nm; MCHB: excitation 575 nm, emission 625 nm) was measured using a plate reader with appropriate filters (SpectraMax, Molecular Devices/MDA Analytic Technologies, Sunnyvale, CA). Cell viability was determined as described above.
Kinetic constants and killing mechanism
To determine enzyme kinetics, 100 µl of 0.1 M Tris-HCl buffer (50 mM; pH 7), 20 µg ml−1 pure ChrR6 and 100 µM NADPH were mixed. The concentration of CNOB was varied from 0.5–2.5 µM and the progress of the reaction was followed by measuring fluorescence at intervals of 1 minute for 10 minutes. Kinetic parameters were estimated using Excel plots of linear regression of reciprocals of Vmax and Km. Standard curves relating fluorescence and MCHB (ChemBridge, San Diego, CA) concentrations were generated and used to determine its levels. CNOB reduction product was determined by high performance liquid chromatography (HPLC), using the following conditions: flow rate, 3 ml/min; detection wavelength, 280 nm (for CNOB), 500 nm (for MCHB); mobile phase, RP C18, 5 µm; column-packing, Phenomenex Luna 5μ C18(2); dimensions: 250×10 mm; injection volumn: 100 µL. Gradients of acetonitrile, containing 0.1% TFA (“A”) and of water containing 0.1%TFA (‘B’) used were as follows: 0, 3, 28, and 30 min, A: 10% B: 90%; 25 and 28 min, A: 100% , B:0%.
The redox balance method to determine the proportion of electrons utilized in CNOB reduction and reactive oxygen species (ROS) generation was determined by quantifying the NADPH consumed and H2
produced during ChrR6-catalysed CNOB reduction. The former was done spectrophotometrically; the latter fluorometrically, using the Amplex Red kit (Molecular Probes/Invitrogen), as before [14
For DNA binding assays, 1 µg of pUC19 plasmid DNA was mixed with 1 µM MCHB, or Tris buffer (control) for 10 minutes. The DNA was then purified by phenol:chloroform extraction, ethanol precipitated and re-dissolved in Tris-EDTA buffer. The differently treated DNA samples were run on 1% agaraose in parallel; bands were then cutout from the gel and MCHB fluorescence was read in a plate reader (SpectraMax, Molecular Devices).
To conduct cell cycle assays, non-confluent cells were treated with 0.1 µM MCHB for 24 hours before collection and staining with 7-amino-actinomycin D (7-AAD). Flow cytometry was used to remove doublets and gates representing G1, S and G2/M phase cells were set. Annexin-PE staining was used to examine apoptosis 3 hours after MCHB (1 µM) addition to cells, using flow cytometry.
For assaying caspase activity, MCHB was added to cancer cells (1 µM) for 1h, and caspase activities assayed using Apoalert Caspase assay (Clontech, Mountain View, CA). MCHB-mitochondrial co-localization was determined using MitoTracker™ green FM dye (Molecular Probes). Cells were incubated for 1 hour with 1 µM MCHB; 200 nM of MitoTracker™ green FM was then added, followed by an additional 1 hour incubation. Confocal microscopy (TCS SP2 Leica Microsystems) was used to visualize the co-localization of MCHB and the mitochondria. Mitochondrial membrane potential was assayed using the JC Mitochondrial Assay (Molecular Probes) 3 hours after addition of MCHB (1 µM).
In vitro visualization of CNOB reduction in live cell
Cells were grown as adherent cultures and mixed with SL7838 expressing both GFP and ChrR6 at specified MOI values (Results). One hour later, the cells were washed to remove extracellular bacteria and CNOB was added (15 µM) along with gentamycin (20 µg ml−1) to suppress bacterial growth. The conversion of CNOB to fluorescent product (MCHB) and location of the GFP-expressing bacteria were followed using a confocal microscope (TCS SP2, Leica). Spheroids were formed by growing JC cells on non-tissue culture treated plates, transferred to chamber well slides (Lab-Tek, Nunc/Thermo Fisher, Rochester, NY), and treated with bacteria, CNOB and gentamycin as before. Prodrug conversion and bacterial GFP were imaged using a 2-photon microscope (Carl Zeiss Inc., Thornwood, NY).
In vivo qualitative visualization of MCHB and efficacy of CNOB/ChrR6 tumor treatment
Immunocompetent BALB/c mice were subcutaneously implanted with 4T1 cells (1×105) expressing luciferase. Tumors were allowed to form for 14 days, at which point they were approximately 100 mm3 in size. Animals were then intratumorally injected with 1×105 CFU of SL7838 expressing the Lux operon and ChrR6. 1, 3 and 5 days later, they were injected intravenously with 0.1 mg of CNOB (in 100 µl; 3.3 mg/kg) each day (ca. 10 mg total CNOB/kg), or PBS (n = 8 mice/group). (CNOB was initially dissolved in DMSO and then diluted 1:10 in PBS immediately prior to injection.) MCHB production was qualitatively imaged in living animals using a Maestro system (CRI Inc., Woburn, MA) with dsRed filter sets and spectral unmixing. Imaging of bacterial Lux activity was performed using an IVIS100 system (Xenogen/Caliper, Alameda, CA); imaging of firefly luciferase (Luc)-expressing 4T1 cells was performed 5 minutes after intraperitoneal injection of luciferin (150 µl of 30 mg ml−1). The signal generated after luciferin addition (Luc expression) includes the signal due to the bacterial Lux expression; however, since the former was more than 50 fold greater, the latter was negligible.
Generating 4T1 cells transfected to express ChrR6
In one experimental regime the efficacy of CNOB/ChrR6 treatment was determined by initiating tumors with bioluminescent 4T1 cells with constitutive capacity to produce ChrR6. These cells were generated by transposon-mediated gene transfer of human codon optimized chrR6
gene (encoding “HChrR6” enzyme; GeneScript Corp. NJ USA); and firefly luciferase (luc
) in the transposon vector pKT2/BsdR=EGFP-fLuc, (pKT2/BGL for short) [16
]. Briefly HchrR6
gene was cloned into the Sleeping Beauty
transposon, pKT2/UXbG (Table S1
), using HindIII/ApaI restriction sites creating "pKT2/hU-HchrR6
-SN". Cells were grown to 90–95% confluency in DMEM without antibiotics in a 6 well plate. 0.8 µg transposase vector (pUb-SB11) and 7.2 µg transposon DNA (pKT2/hU-HchrR
-SN and pKT2/BGL) were added to 0.5 ml OptiMem (Invitrogen). In a second vial 20 µl of Lipofectamine 2000 (Invitrogen) was added to 0.5 ml of OptiMem, and incubated at room temperature for 5 minutes.
The medium was aspirated and cells were washed once with PBS. The above solutions were combined (total 1 ml/well), added to each well and incubated for 18–24 hours. The transfection solution was then aspirated and replaced by regular complete DMEM. Transfection efficiency was monitored by adding 2–5 µl of luciferin (30 mg/ml) per well and imaged immediately using the IVIS50 system; while the untranfected cells expressed no Luc luminescence, the transfected ones showed high expression. Cells were incubated for an additional 48 hours, selected with blasticydin and geneticin (Invitrogen) (5 and 2 µg/ml, respectively; these concentrations were predetermined as the minimal killing dose for 4T1 cells). To ensure homogeneity of HchrR6 expression, cells expressing luciferase were diluted to about 30 cells per 10 ml DMEM, supplemented with the selection antibiotics and 100 µl aliquots were dispensed into a 96 well plate. This dilution generates a ~30% probability of a well receiving a cell, ensuring that colonies developing in a well originated from a single cell. Tumors from these cells were generated as before.
Tumor burden was measured by caliper measurement at specified times after CNOB treatment. Whole blood counts, and the chemistry panel measurements were performed by the Stanford Veterinary Service Center. All animal studies were performed according to approved institutional IACUC and biosafety committee protocols.
Student’s T test was performed for all statistical analyses, except for the Kaplan-Meier survival curves, where Logrank tests were used. p-values are indicated (significance was assigned at p-values less than 0.05).