Here we report a genetically modified bacteria strain, Salmonella typhimurium A1, selected for anticancer activity in vivo. The strain grows in tumor xenografts. In sharp contrast, normal tissue is cleared of these bacteria even in immunodeficient athymic mice. S. typhimurium A1 is auxotrophic (Leu͞Arg-dependent) but apparently receives sufficient support from the neoplastic tissue to grow locally. Whether additional genetic lesions are present is not known. In in vitro infection, the GFP-expressing bacteria grew in the cytoplasm of PC-3 human prostate cancer cells and caused nuclear destruction. These effects were visualized in cells labeled with GFP in the nucleus and red fluorescent protein in the cytoplasm. In vivo, the bacteria caused tumor inhibition and regression of xenografts visualized by whole-body imaging. The bacteria, introduced i.v. or intratumorally, invaded and replicated intracellularly in PC-3 prostate cancer cells labeled with red fluorescent protein grafted into nude mice. By day 15, S. typhimurium A1 was undetectable in the liver, lung, spleen, and kidney, but it continued to proliferate in the PC-3 tumor, which stopped growing. When the bacteria were injected intratumorally, the tumor completely regressed by day 20. There were no obvious adverse effects on the host when the bacteria were injected by either route. The S. typhimurium A1 strain grew throughout the tumor, including viable malignant tissue. This result is in marked contrast to bacteria previously tried for cancer therapy that were confined to necrotic areas of the tumor, which may account, in part, for the strain's unique antitumor efficacy. red fluorescent protein ͉ bacterial therapy ͉ auxotrophy ͉ leucine ͉ arginine

With the use of dual-color fluorescent cells and a highly sensitive whole-mouse imaging system with both macro-optics and micro-optics, we report here the development of subcellular real-time imaging of cancer cell trafficking in live mice. To observe cytoplasmic and nuclear dynamics in the living mouse, tumor cells were labeled in the nucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with an Olympus OV100 whole-mouse imaging system with a sensitive CCD camera and five objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dualcolor imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. With the dual-color cancer cells and the highly sensitive whole-mouse imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time. This imaging technology will enable further understanding of the critical steps of metastasis and provide visible targets for antimetastasis drug development. (Cancer Res 2006; 66(8): 4208-14)

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The mechanism of cancer cell deformation and migration in narrow vessels is incompletely understood. In order to visualize the cytoplasmic and nuclear dynamics of cells migrating in capillaries, red fluorescent protein was expressed in the cytoplasm, and green fluorescent protein, linked to histone H2B, was expressed in the nucleus of cancer cells. Immediately after the cells were injected in the heart of nude mice, a skin flap on the abdomen was made. With a color CCD camera, we could observe highly elongated cancer cells and nuclei in capillaries in the skin flap in living mice. The migration velocities of the cancer cells in the capillaries were measured by capturing images of the dual-color fluorescent cells over time. The cells and nuclei in the capillaries elongated to fit the width of these vessels. The average length of the major axis of the cancer cells in the capillaries increased to approximately four times their normal length. The nuclei increased their length 1.6 times in the capillaries. Cancer cells in capillaries over 8 Mm in diameter could migrate up to 48.3 Mm/hour. The data suggests that the minimum diameter of capillaries where cancer cells are able to migrate is approximately 8 Mm. The use of the dual-color cancer cells differentially labeled in the cytoplasm and nucleus and associated fluorescent imaging provide a powerful tool to understand the mechanism of cancer cell migration and deformation in small vessels. (Cancer Res 2005; 65(10): 4246-52)

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