Figure 1.
(a,b) Bioluminescence from B. breve UCC2003, E. coli MG1655 or S. Typhimurium SL7207 in s.c. LLC tumour bearing mice 11 days following IV delivery. Representative mice are shown. Increase in bacterial bioluminescence in tumours was observed over time (n = 6). Data graphed represent the mean ± S.E. There was no detectable bioluminescence in organs of treated animals, except for S. Typhimurium (c) Representative S. Typhimurium administered mice displaying non-tumour-specific bacterial bioluminescence. Ventral image – Day 3, Dorsal – Day 11. (d) All tumour types examined were colonised by the various strains. (i) B. breve, B16, Day 11 (ii) E. coli, FaDu, Day 7 (iii) B. breve, U87, Day 14.
Figure 2.
Relationship Between Bacterial Numbers And Bioluminescence.
Viable bacteria in tumours were enumerated by ex vivo bacterial culture from LLC tumours subsequent to BLI at various time-points post IV administration of B. breve or E. coli (n = 6). (a) Bacterial replication in tumours. Increases over time in viable bacterial numbers and bioluminescence. Data graphed represent the mean ± S.E. (b) Correlation between bacterial numbers and bioluminescence in tumours. Direct comparison between photon flux and bacterial colony counts. Log values of bacterial numbers relative to in vivo bioluminescent units are graphed. Correlation between bacterial counts and bacterial bioluminescence signals: R2 = 0.84 for B. breve: R2 = 0.97 for E. coli which correlates well with previous studies using E. coli MG1655 where R2 = 0.94, P<0.001 [57].
Figure 3.
Bacterial Bioluminescence in the GIT.
The GIT of mice was colonised with either B. breve or E. coli by oral administration of 109 cfu of the relevant strain for three consecutive days. (a) 2D image of athymic mouse 9 days post final feed with B. breve UCC2003/lux. (b) IVIS XR 2D bioluminescence overlayed with X-ray image of mouse 27 days-post feeding with E. coli MG1655/lux. (c) Bacterial counts in specific regions of the GIT 14 days post feeding. Abdominal bioluminescence corresponding to average cfu is also shown (black dots). Data graphed represent the mean ± S.E. (d) Sample isolated images from 3D tomography of mouse from (b). 3D images show a digital mouse atlas of the skeleton to provide anatomical registration. (Movie available in Movie S1). E. coli MG1655 bioluminescence is visible in blue at lower, and white or green at higher levels.
Figure 4.
Co-localisation of Tumour And Bacterial Bioluminescence.
(a) 2D bioluminescence co-registration. Athymic mouse bearing s.c. FLuc expressing FaDu tumour 7 days post IV administration of B. breve. Tumour FLuc = Green, Bacterial lux = orange. (b) 3D bioluminescence co-registration. 3D overlay of B. breve lux (orange) 10 days post administration to athymic mice bearing HCT116 FLuc (green) expressing tumours.
Figure 5.
Ex vivo histological analysis.
Fluorescent bacterial and tumour cells were imaged using fluorescence microscopy (Nuance). Blue- DAPI (nuclei), Green- U87/GFP, Magenta- B. breve/pCheMC.
Figure 6.
(a) Whole Body 3D co-registration of lux, FLuc and μCT. Combined luminescence and μCT demonstrating co-localisation of B. breve (bacterial lux - orange) and subcutaneous HCT116-luc2 tumour (FLuc - green). (b) Intratumoural Imaging. Combined μCT and luminescence imaging. Magnification of subcutaneous tumour from mouse in (a), top, side and base view. Viable tumour (FLuc green/blue), vasculature (contrast agent – red) and bacterial (orange/yellow) signals are visualised. Movie on website (Movie S3).