Histological observations and Biodistribution of bacteria in
normal and tumor tissues
Evaluation of Bacterial biodistribution in tumor and normal tissues was
carried out 48 h after tail vain injection of 108cfu/g BW25133 bacteria in mice. livers as the highest perfused normal
organ and tumors were first excised and fixed in 10% formalin solution.
The specimens were then paraffin embedded, sectioned and stained with
gram stain and visualized at 100× using a light microscope. For counting
the number of bacteria in normal and tumor tissues, liver and tumor
samples were carefully weighed, homogenized in PBS and plated on LB
agar. Bacterial concentration was determined by counting the number of
CFUs after 24- to 48 h incubation of plates at 37 °C. Some of the
sections obtained from specimens were stained with hematoxylin and eosin
(H&E) staining solution to evaluate cytotoxic damages induced by
cardiac peptide. Presence of BW25133 E. coli in sections was also
confirmed by visualization of GFP under fluorescent microscope.
In vivo fluorescence
reflectance imaging
Fluorescence reflectance imaging
was utilized to initially evaluate whether bacteria could specifically
localize in tumor sites and then to monitor time-course of GFP
expression as the indicator of successfull expression of cardiac
peptides in tumor site under hypoxic condition. Briefly, mice were IV
injected with either PBS (control group) or
E. coli BW25133 strain bearing
construct with concentration of 108 cfu/g and images
were taken on days 0, 1, 3 and 6, post injection using the FlouVison
fluorescence planar imaging system in vivo (Tajhizafarinan Noori
Parseh Co., Tehran, Iran). Images from whole body were taken by placing
anesthetized mice in the supine position, in the center of the imaging
cassette, inside the scanning field of the system. After proper
positioning of animals, the imaging cassette was set to the appropriate
depth to finely confine anesthetized mice. Finally, the animal body was
subjected to the laser beam and emitted non-uniform fluorescent beams
were recorded using a highly sensitive thermoelectrically cooled CCD
camera located on the same side of the imaged animal.