Since the mid twentieth century, when the discovery of X-radiation and its development as a diagnostic aid provided the first opportunity to noninvasively image a living being, the detection and evaluation of tumors in vivo have been an area of ongoing research. In the latter half of the twentieth century, a wide array of noninvasive, live-tissue imaging options were developed to complement and even replace traditional X-radiography [1]. Rapid development and improvement in our understanding of ultrasonic imaging [2], computed tomography [3], positron emission tomography [4], magnetic resonance imaging [5], and nuclear scintigraphy [6] has provided us with a host of new tools to observe and measure tumors in vivo. These techniques provide access to a realm of detailed and important information, but to properly obtain and interpret this information, we must rely upon the expertise of board-certified radiologists within the medical profession who possess extensive training and experience.

In the laboratory, efforts are constantly underway to improve our understanding of tumor biology. To this end, scientists have developed a variety of tools, including the use of immunocompromised mice as hosts for human xenografts, thus providing researchers a method with which they may observe the growth and development of human tumors within the complex biological milieu of a living animal. These models, while tremendously useful, also lack some key properties that would allow them to better represent the development of tumors in their natural environment [7]. For instance, often xenografts behave in a different manner when transplanted in mice than they would in their native host [8, 9].

The recognized deficiencies in xenograft models have resulted in efforts to develop improved murine tumor models and imaging systems. Advances in genetic manipulation provide us with the ability to selectively knockout tumor suppressor genes and to overexpress oncogenes, thus allowing scientists to generate tumors in specific tissues which mimic the genetic changes found in human cancers [9–13]. Although murine tumors, whether induced or naturally occurring, are not always perfect analogs of their human counterparts, they provide valuable information on tumor behavior. In addition, they can be excellent systems for determining the efficacy of many anti-cancer therapeutics. One example of murine tumor models successfully used to this end is the development of transgenic mice predisposed to the development of mammary tumors [11, 14, 15].

While the development of transgenic mice has provided us with excellent models of stochastic, spontaneous tumorigenesis and development in vivo, the noninvasive imaging techniques available to observe these tumors remain largely inadequate. Human imaging modalities, including ultrasonography, computed tomography, and magnetic resonance imaging, often pose technical, financial, and regulatory challenges to the typical scientist. As a result, the scientific community has come to rely on more “user friendly” alternatives, the most popular of which is the use of bioluminescent or fluorescent imaging. Many effective in vivo imaging models have now been developed which depend either upon the conditional co-expression of bioluminescent reporter genes with specific tumor-associated genes, or upon the use of fluorescent reporter molecules directed towards specific cancer-associated cell proteins [16]. These models, however, are inherently limited in use to a single type or subtype of cancer, and may not be broadly applied to tumors of differing origins.

With the model described here, we attempted to alleviate many of the deficiencies of murine mammary tumor models by combining the ease of use of bioluminescent imaging with the superior biological model of stochastically occurring in vivo tumors in immunocompetent mice. This method, as with all tumor imaging techniques, depended upon the differentiation of tumor cells from normal tissue. It has long been known that there are differences between cancerous tissue and normal tissue that, if exploited, might provide a foundation for a more broadly applicable tumor imaging system [17]. For instance, all solid tumors have been shown to experience a certain degree of relative hypoxia when compared with surrounding tissue [18–20].

Recently, a transgenic mouse expressing the oxygen-dependent degradation (ODD) domain of the Hypoxia Inducible Factor 1-α (HIF1-α) gene fused to a luciferase bioluminescent reporter gene was developed by Safran et al. [21]. This model allows for the bioluminescent imaging of regions in which hypoxia is present. The HIF1-α gene is ubiquitously transcribed and translated, but under normoxic conditions, the enzyme HIF prolyl hydroxylase utlilizes oxygen to hydroxylate the HIF ODD. The hydroxylated ODD then recruits von Hippel–Lindau protein (pVHL), a ubiquitin E3 ligase, leading to poly-ubiquitination of HIF1-α and its subsequent degradation by the proteasome. Conversely, under hypoxic conditions, lack of hydroxylation of the ODD domain leads to the accumulation of HIF1-α which acts as a transcriptional regulator for a number of hypoxia-induced genes. By universally expressing the ODD-luciferase gene in mice, Safran et al. developed an organism in which luciferase would be continuously transcribed and translated in all cells, but would also be rapidly degraded under normoxic conditions. Only under conditions of hypoxia can luciferase accumulate to the point that it can act upon the substrate luciferin and generate a bioluminescent signal that may be imaged.

Our technique is based upon the hypothesis that solid tumors should be visible in ODD-luciferase expressing mice due to the relative hypoxia experienced by the tumor cells, essentially allowing for the use of the ODD-luciferase transgenic model as a platform for noninvasive imaging of spontaneous solid tumors in mice [22]. For our work, we utilize mice predisposed to the development of mammary gland tumors (MMTV-neu and Beclin1 +/−). These mice were cross-bred with ODD-luciferase mice. The resultant cross-breeds yielded tumors which generated substantial bioluminescent signals clearly discernable from background tissue luminescence. This technique may be used to track tumor growth and development longitudinally over several weeks.