Quantitative bioluminescence imaging of tumor-targeting bacteria in living animals

Abstract

We describe a protocol for imaging bacterial luciferase (Lux)-expressing bacteria in small living animals. In this protocol, light emitted by Lux-expressing bacteria is detected and monitored by a cooled charge-coupled device detector. When these bacteria are administered to animals, it provides a potentially valuable approach to generate sensitive whole-body images with extremely low background. This imaging technology should enable the real-time monitoring of bacterial migration into both primary and metastatic tumors in several different mouse tumor models at a strong quantification power.

Molecular Imaging of Biological Gene Delivery Vehicles for Targeted Cancer Therapy: Beyond Viral Vectors

“…The bacterial luciferase gene (lux) operon encodes five gene clusters, lux C, D, A, B, and E. Lux A and B control luciferase enzyme expression, while lux C, D, and E control fatty aldehyde enzyme complex production, which synthesizes the substrates. Because the lux operon encodes all of the proteins necessary for lightemitting systems to function in bioluminescent bacteria, including luciferase, substrate, and substrate-regenerating enzymes, bacteria that express the lux operon only require oxygen and do not require an exogenous substrate to produce bioluminescence [9,24]. The advantage of bioluminescence is its minimal background noise, since luciferase is not a natural constituent of mammalian organisms.…”

“…The advantage of bioluminescence is its minimal background noise, since luciferase is not a natural constituent of mammalian organisms. Bioluminescence-based approaches currently lack detailed tomographic information and are limited to relatively small animals [9,25,26].…”

“…. In particular, S. typhimurium has been bioengineered to generate bioluminescence [9,48] or fluorescence [49] signals and has been employed to monitor bacterial migration to tumors in living small-animal models. These data may facilitate the prediction of the therapeutic efficacy of bacterio-therapy through visualization of bacterial accumulation and replication in specific organs.…”

“…The bioluminescence emitted from the bacteria was detected in various tumor models after IV bacterial injection using a cooled-CCD camera. The lux operon can also be cloned into a plasmid, and transformed bacteria will generate light [9,24]. However, using this strategy, E. coli fails to maintain the pLux expression plasmid in the absence of selection, particularly in infected animals [48].…”

“…Furthermore, most current GDVs lack sufficient specificity for cancer tissue, which limits their gene delivery capability. This is particularly important given that optimizing the specific affinity of GDVs would improve the efficiency of gene/protein delivery and reduce unwanted transfection of noncancerous tissues [9].…”