High Resolution In Vivo Bioluminescent Imaging for the Study of Bacterial Tumour Targeting

Abstract

The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT) for interpretation.In this study, the non-pathogenic commensal bacteria E.coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (IV) administered to mice bearing subcutaneous (s.c) FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post IV-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and μCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT) was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.

Bacterial vectors for imaging and cancer gene therapy: a review

“…26 Numerous publications provide convincing evidence that genera of bacteria including Bifidobacterium and commensal Escherichia coli have potential in cancer therapy. 9,10,12,16,[27][28][29][30] This vector class compares favorably with other vectors in terms of key attributes such as safety, ease of manipulation, and production. While nonpathogenic bacteria have yet to enter clinical trial in the context of tumor targeting vectors, existing knowledge strongly indicates their suitability as a vector system for clinical use.…”

“…To date, the genera of bacteria that have been exploited as gene delivery vehicles include Salmonella, Escherichia, Listeria, Clostridium and Bifidobacterium. [9][10][11][12][13][14][15][16][17] These bacteria can be delivered to the tumor via multiple routes such as intra-tumoral 1 injection, intravenous injection or in certain instances orally. Preclinical studies have shown the ability of different bacterial strains to locally produce therapeutic agents and mediate highly effective and specific therapeutic responses.…”

“…63 Fluorescence imaging is also valuable for fine-detail postmortem histological study of bacterial localization. 9 To date, almost all the published reports of fluorescent imaging for bacteria utilize GFP, despite its unsuitable excitation and emission wavelengths for tissue penetration. However, the relative explosion in the number of red-shifted FPs developed in recent years should see this exciting field begin to flourish.…”

Bioluminescent Bacterial Imaging <em>In Vivo</em>

“…We have engineered a number of strains to express the luxABCDE cassette 1,[8][9][10][11] . The protocol outlined in the video animation uses lux-tagged E. coli as an example.…”

“…E. coli MG1655 containing the integrated luxABCDE was grown aerobically at 37°C in LB medium (Sigma-Aldrich, Ireland) supplemented with 300 μg/ml erythromycin (Em). The bioluminescent derivative of MG1655 was created using the plasmid p16Slux which contains the constitutive P HELP luxABCDE operon 1 . 2.…”