Bioluminescence has been used for the detection of primary tumor growth and tumor metastasis in animal models of several tumors, but, to our knowledge, this is the first report on the use of bioluminescence in animal models of pheochromocytoma. The use of this technology allows for the non-invasive and real-time assessment of tumor burden in the same group of animals over time [19
]. This type of model is particularly well suited for evaluating the efficacy of novel therapy and has been developed with the intent to create a platform for pre-clinical evaluation of new targeted therapy for pheochromocytoma.
Previous studies in our group have established a metastatic model of pheochromocytoma by mouse passages of the murine pheochromocytoma cell line MPC [21
]. Through disaggregation and culturing of liver tumor metastasis, we established the MTT cell line, which displays a reproducible metastatic phenotype when injected intravenously. In order to follow the localization of these cells in the intact animal longitudinally, we generated a bioluminescent MTT cell line and compared the bioluminescent signal with serial MRI imaging.
In our study we demonstrated a strong correlation between the detection of photons and the radiologic examination. BLI offers several advantages over more traditional radiologic techniques, such as MRI and CT scanning, which require long scan times and expensive instrumentation. Bioluminescence offers a signal with practically no background, as other source of significant bioluminescence are absent in mammals, and the light generated easily penetrates mammalian tissues and can be detected by sensitive charge-coupled device (CCD) cameras and quantified more precisely by the conversion of the luminescence signal into a digital value. This is in contrast with the use of fluorescent tags, that require an excitation signal (which penetrate tissue layers with difficulty), and are limited by the presence of tissue autofluorescence and photobleaching. Moreover, the luciferase gene can be stably integrated into the chromosomes of target cells, and so carries over subsequent cell divisions and is not lost over time.
Luciferase-transduced cells can be easily monitored in virtually any location in the body, including sanctuary sites, where only a few cells are sufficient to generate a detectable signal that would be undetectable by MRI or CT scanning; thus, it is by far the most sensitive of the noninvasive techniques. Consequently, among the several imaging techniques available for in vivo studies, bioluminescence is the more sensitive to detect minimal residual disease, which is one of the more daunting and elusive entities in clinical oncology.
Moreover, BLI can represent quantitatively the amount of viable tumor cells in the body, allowing comparison not only within the same experiment but also across several experiments. The ability to non-invasively track the growth of tumors and metastases in vivo
also permits a better understanding of the mechanisms of cancer development and intervention. Several investigations in other types of cancers have already demonstrated the power of BLI in longitudinal therapy intervention studies for the follow-up of tumor growth after treatment with experimental drugs [23
]. In these types of studies the BLI signal in the animal injected with tumor cells is determined prior to intervention with the drug of interest to establish a reference/starting measurement. Subsequent scans are then normalized relative to the reference signal in the same animal and differences are calculated between the control group (receiving vehicle alone) versus the group receiving the tested drug.
We have also established a spontaneous metastasis model of pheochromocytoma, in which cells from a subcutaneous implant send micrometastases to the lungs. These models are rare in the literature, and represent a unique opportunity to explore steps of the metastatic cascade that are not testable in an experimental metastasis model [24
]. Indeed, spontaneous metastases spread following a natural mechanism of invasion of the surrounding tissue, and allow the examination of all steps of the metastatic cascade. This model could in particular be more clinically relevant, when testing for drugs that target more advanced disease stages. For example other groups using a spontaneous metastasis model of the breast cancer cell line MDA-MB-435 [25
] was able to demonstrate the responsiveness of metastasis to therapies that were initially found to be ineffective in the treatment of the primary tumor. On the same line other groups have successfully used these animal models for testing novel molecular targeted therapies [26
Besides the study of intact animals along the course of the study, ex vivo analysis at the end of experiments allows for additional important information to be collected from the same animal. Namely, ex vivo bioluminescence can help to identify very small metastatic lesions that are not easily detectable by in vivo imaging, more accurately assessing metastatic burden. For example, while it was relatively easy to detect signals from the abdominal cavity, brain lesions were undetectable by both MRI imaging and in vivo BLI. In contrast, ex vivo BLI was able to promptly detect a small number of tumor cells that crossed the blood-brain barrier causing micrometastatic brain disease.
A technical point of interest was that we were able to use G418 to exert selective pressure on transduced MTT cells. This was somewhat unexpected because the primary tumor from which MPC and MTT are derived arose in a Nf1
knockout mouse generated by insertion of a neomycin resistance gene in reverse orientation into the Nf1
] and the original MPC line was intrinsically G418-resistant (JF Powers, unpublished). The apparent loss of intrinsic resistance might have resulted from somatic recombination, a possibility that itself has implications for design of targeted cancer therapy.
In summary, these experiments demonstrate the ability of bioluminescent imaging to follow the progression of pheochromocytoma cells in live animals in order to study the course of tumor progression and to test clinically relevant antitumor treatments in a mouse model of metastatic pheochromocytoma. The sites of metastasis in this model are also favored for metastases of human pheochromocytomas [6
]. No comparable human cell-based model currently exists.