Bacterial strains and growth conditions
) was kindly supplied by Dr. Arash Komeili at the University of California, Berkeley. The bacteria were grown at 30°C with modified Magnetospirillum
growth medium (16
). Cultures were grown in sealed tubes with 7% headspace of air. Bacterial cell density was determined by optical density measurements (Shimadzu BioSpec-1601 spectrophotometer) correlated to a standard curve. Iron (40 μmol/L) was supplied either as ferric malate or FeCl3
, as described in Results. (Magnetospirillum
growth medium from here on refers to the medium without supplemental iron.)
Magnetic moment measurements
AMB-1 cells were washed three times and suspended in Magnetospirillum
growth medium at a range of concentrations. Their magnetic moment was measured with a Princeton MicroMag 2900 alternating gradient magnetometer by applying fields of ±5,000 Oe in 20 Oe steps. Dividing the magnetic moment values by the known magnetic moment of magnetite (480/mL; ref. 17
) and multiplying by magnetite density (5.2 g/mL; ref. 18
) provided the amount of magnetite per sample. Because 99.5% of iron consumed by magnetotactic bacteria is incorporated into magnetite (19
), all cellular iron was assumed to be in magnetite. Iron content is simply the molar ratio (168/232) of iron in magnetite (Fe3
Magnetic resonance imaging
For in vitro (phantom) studies, AMB-1 samples were washed twice and suspended in 3% gelatin (Sigma G9382) in plastic tubes. Feridex I.V. (Advanced Magnetics, Inc.) phantoms were prepared similarly. The phantoms were aligned inside a 50-mL screw-cap tube, which was subsequently filled with 0.7% agar. The gelatin was snap solidified (4°C) to maintain a homogenous cellular distribution.
For in vivo studies, female athymic nu−/nu− mice (6-8 wk old; Charles River) were used. S.c. tumors were initiated by injecting 3 × 106 293T human embryonic immortalized kidney cells; this cell line was chosen because it produces firm tumors, permitting i.t. injection. Palpable tumors formed within ~2 wk. Twice washed AMB-1 cells suspended in Magnetospirillum growth medium were injected either i.t. at concentrations specified in the Results section or i.v. (tail vein injection) at 1 × 109 cells in 100 μL. For magnetic resonance imaging, animals were anesthetized with isoflurane (2%) plus oxygen (1 L/min) delivered through a nose cone. These studies were done in compliance with a Stanford-approved protocol.
For all magnetic resonance measurements, a GE 3T magnetic resonance scanner equipped with self-shielded gradients (40 mT/m, 150 mT/m/ms) was used. A custom-made radiofrequency quadrature coil was used for radiofrequency excitation and signal reception (Ø = 44 mm for in vivo and Ø = 64 mm for in vitro). A three-dimensional spoiled gradient recalled (SPGR) sequence (TE/TR = 4/27 ms) with axial slice orientation was used to acquire T1-weighted images over 15 min (nominal resolution, 0.25 × 0.25 × 0.5 mm3). A two-dimensional gradient recalled echo (GRE) sequence (TE/TR = 9/1,000 ms; 1-mm slice thickness) was used for T2-weighted imaging.
For T1 measurements, an inversion-recovery fast-spin echo sequence (TE/TR = 8.2/10,000 ms; FOV, 64 mm; 128 × 128 matrix; 6-mm slice thickness) was used, with inversion times of 50, 100, 150, 200, 400, 800, 1,500, 2,500, and 4,000 ms. T1 values were estimated by a nonlinear least squares fit of the data to a modified IR curve. A fitting parameter was used to account for the imperfect inversion along the Z axis caused by flip angle deviations due to B1
). For T2 measurements, an SE sequence was used (TR = 10,000 ms; FOV, 64 mm; 128 × 128 matrix; 6-mm slice thickness) with TEs of 10, 15, 20, 40, 60, 100, 150, 200, 250, and 400 ms. T2 values were estimated by fitting the data to a monoexponential decay curve. Relaxation rate constants (R1
= 1/T1 and r2
= 1/T2) were plotted versus millimole per liter Fe of AMB-1, and the slope was used to determine relaxivity.
Signal intensity was measured from axial-slice 16-bit images using ImageJ (1.39u with the Dicom input/output Plug-in, NIH freeware), background corrected with muscle intensity, and normalized to preinjection values or contralateral controls. Signal intensities were averaged among five consecutive axial-slice images, using mean values from regions of interest (ROI) drawn on the in vitro images and maximum values from the in vivo images. Maximum values were used for in vivo images because of the need to arbitrarily choose ROIs because of localized tumor colonization by AMB-1 following i.t. or i.v. injection. Among the maximum values from five consecutive slices, the SD was consistently <10% of the mean.
Micro-positron emission tomography (PET)
Cu was produced at the University of Wisconsin by cyclotron irradiation of an enriched 64
Ni target according to published methods (21
). The 64
-methylthiosemicarbazone) (PTSM) was prepared as described previously (22
). Briefly, the PTSM was prepared by mixing 10 μL of PTSM (dissolved in 1 mg/mL DMSO) with 150 μL of 64
Cu (dissolved in 1 mol/L NaOAc; pH 5.5), as described (23
). After 3 to 5 min, the mixture was added to an ethanol-preconditioned C-18 SepPak Light column and the 64
Cu-PTSM was eluted off with 500 μL ethanol after the first 150 μL fraction.
To optimize labeling conditions, the uptake and efflux of 64Cu-PTSM was examined. For uptake, cells were incubated with 33 μCi for 0.5, 1, 2, 4, and 18 h. At each time point, triplicate samples were washed twice, and activity was counted with a gamma counter. After 2 h, cells had taken up 56.7 ± 2.4% activity, which increased only minimally by 4 h (57.4 ± 2.0%); thus, 2-h incubation was chosen for labeling. For efflux, cells were incubated with 123 μCi for 18 h then resuspended in ice-cold PBS. At 0, 0.5, 2, 4, and 24 h, triplicate samples were pelleted, the supernatant was aspirated, and the activity of the pellet was counted; 24 h later, the samples were found to retain 74.4 ± 2.3% activity. Lack of toxicity of 64Cu-PTSM to AMB-1 cells was verified after 24 h of incubation by microscopic observation of motile cells and by staining with the LIVE/DEAD BacLight viability stain (Molecular Probes).
For the in vivo
experiment, AMB-1 cells were labeled with 64
Cu-PTSM by coincubation for 2 h. 1 × 109
AMB-1 cells suspended in 100 μL (~220 μCi of activity) were injected i.v. through the tail vein to three mice and i.t. to a second group of three mice to serve as positive controls. (1 × 109
cells were used for the positive controls to match the i.v. group, not as a comparison to the i.t. group of the magnetic resonance imaging studies). A third group of mice was injected i.v. with 100 μL of 64
Cu-PTSM alone (negative controls). The mice were anesthetized (as described above) and imaged at 0.5, 1, 2, 4, 16, 24, 42, and 64 h postinjection with a Siemens/Concorde Microsystems MicroPET rodent R4. The images were collected with static scans of 3 min (at 0.5, 1, 2, and 4 h), 5 min (at 16 and 24 h), or 10 min (at 42 and 64 h). The microPET images were analyzed using ASIPro VM 18.104.22.168 (Acquisition Sinogram Image PROcessing using the Virtual Machine of IDL). ROIs were drawn on decay-corrected whole-body coronal images (0.845-mm thickness) and converted to percent injected dose per gram of tissue, as described previously (22
). We have previously shown that quantification by this method agrees with biodistribution data (24
Electron microscopy and size analysis of magnetite particles
Suspensions of AMB-1 were fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.0) for 1 h, then washed 2× with wash buffer for 10 min. Postfixation was done with 1% osmium tetroxide in fixative buffer for 1 h and rinsed 2× with double-distilled water. The samples were left on 1% uranyl acetate in 20% acetone for 30 min and dehydrated with a graded acetone series. Samples were then infiltrated and embedded in Spurr’s resin. Ultrathin sections were cut with a diamond knife and mounted onto uncoated copper grids. The sections were poststained with 2% uranyl acetate for 15 min and 1% lead citrate for 5 min. The samples were examined with a CM-12 Phillips electron microscope. Magnetite particle diameter was measured for >100 magnetite particles per group from digitized transmission electron microscopy (TEM) micrographs using ImageJ 1.39u. The particle diameter histogram was made with 5 nm bins using MATLAB (The Mathworks).
Tumors were harvested from sacrificed animals and fixed in 10% buffered formalin overnight. Histology preparation was done by Histology Research Core Laboratory at Stanford University Hospital. Slices of 5-mm thickness were embedded in paraffin and longitudinally cut into sections of 5-μm thickness. Neighboring sections were stained with Perl’s Prussian blue (for visualizing iron) and Gram stain (for visualizing bacteria), respectively.
Viable plate counts
Nude mice bearing 293T s.c. tumor xenografts were injected with 1 × 109 AMB-1 in 100 μL medium through the tail vein. Groups of three animals were sacrificed 1, 3, and 6 d after injection, and the tumor, liver, and spleen were aseptically removed from each animal. The samples were rinsed with sterile PBS, weighed, and homogenized, then centrifuged at 1,000 rpm for 5 min. Samples from the supernatant were diluted, suspended in 5 mL of warmed Magnetospirillum growth medium with 0.7% agar, and plated on Magnetospirillum growth medium plates in duplicate. The plates were incubated in bags flushed with N2 gas at 30°C for 2 wk. Colony forming units were counted and normalized by tissue mass.
Two-tailed unpaired t tests were done for in vitro comparisons, and paired tests were used to compare contrast differences between experimental and control tumors; in paired t tests, each tumor was compared with its contralateral control (if applicable) or its own preinjection value. The Mann-Whitney significance test6 was used to evaluate the difference between distributions (of magnetite particle size or particle number). Statistical significance was determined by P < 0.05.