Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.

Tumor hypoxia has been considered to be a potential therapeutic problem because it renders solid tumors more resistant to sparsely ionizing radiation (IR) and chemotherapeutic drugs. Moreover, recent laboratory and clinical data have shown that tumor hypoxia is also associated with a more malignant phenotype and poor survival in patients suffering from various solid tumors. Therefore, selective targeting of hypoxic tumor cells has been explored, and since severe hypoxia (pO 2 < < < <0.33%, 2.5 mmHg) does not occur in normal tissue, tumor hypoxia could be exploited for therapeutic advantage. However, the following three characteristics of hypoxic tumor regions present obstacles in targeting hypoxic cells. First, it is difficult to deliver a sufficient amount of drug to a region that is remote from blood vessels. Second, one must specifically target hypoxic tumor cells while sparing normal well-oxygenated tissue from damage. Finally, the severely hypoxic tumor cells to be attacked have often stopped dividing. Therefore, high delivery efficiency, high specificity and selective cytotoxicity are all necessary to target and combat hypoxic tumor cells. The current review describes progress on the biological aspects of tumor hypoxia and provides a compilation of the recent molecular approaches used to target hypoxic tumors. These approaches include our work with a unique hypoxia-targeting protein drug, TOP3, with which we have sought to address the above three difficulties. (Cancer Sci 2003; 94: 1021-1028) n solid tumors, oxygen delivery to the respiring neoplastic and stromal cells is frequently reduced or even abolished by deteriorating diffusion geometry, severe structural abnormalities of tumor microvessels, and damaged microcirculation.1) It has been known for some time that tumor cells can survive and even grow in such a deteriorated microenvironment. Moreover, as described in detail below, tumor hypoxia has been shown to be a cause of malignant transformation and a source of resistance to current cancer therapies. For these reasons, approaches to targeting hypoxic tumor cells have been sought over a period of years. Recent progress in understanding the molecular mechanisms of hypoxic response, and especially the discovery of a key molecule, Hypoxia Inducible Factor (HIF), as well as improvements in diagnostic techniques through which the tumor microenvironment can be assessed, has opened the door to a solution of this longstanding issue.

A tumor-specific microenvironment is characterized by hypoxia, in which oxygen tension is considerably lower than in normal tissues.

In preclinical cancer research, bioluminescence imaging with firefly luciferase and D-luciferin has become a standard to monitor biological processes both in vitro and in vivo. However, the emission maximum (λmax) of bioluminescence produced by D-luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λmax=677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with D-luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models.