Cancer-cell killing by engineered Salmonella imaged by multiphoton tomography in live mice

Our laboratory has previously developed a bacterial cancer therapy strategy by targeting tumors using engineered Salmonella typhimurium auxotrophs (S. typhimurium A1-R) that were generated to grow in viable as well as necrotic areas of tumors but not in normal tissue. The mechanism by which A1-R kills cancer cells is unknown. In the present report, high-resolution multiphoton tomography was used to investigate the cellular basis of bacteria killing of cancer cells in live mice. Lewis lung cancer cells (LLC) were genetically labeled with red fluorescent protein (RFP) and injected subcutaneously in nude mice. After tumor growth was observed, the mice were treated with A1-R bacteria expressing GFP, via tail-vein injection. Mice without A1-R treatment served as untreated controls. The imaging system was 3D scan head mounted on a flexible mechano-optical articulated arm. A tunable 80 MHz titanium:sapphire femtosecond laser (710-920 nm) was used for the multiphoton tomography. We applied this high-resolution imaging tool to visualize A1-R bacteria targeting the Lewis lung cancer cells growing subcutaneously in nude mice. The tomographic images revealed that bacterially-infected cancer cells greatly expanded and burst and thereby lost viability. Similar results were seen in vitro using confocal microscopy. The bacteria targeted the tumor within minutes of tail-vein injection. Using mice in which the nestin-promoter drives GFP and in which blood vessels are labeled with GFP, the bacteria could be imaged in and out of the blood vessels. Collagen scaffolds within the tumor were imaged by second harmonic generation (SHG). The multiphoton tomographic system described here allows imaging of cancer cell killing by bacteria and can therefore be used to further understand its mechanism and optimization for clinical application.