For almost two decades, human lung cancer xenografts have been established in the lungs of immunodeficient rodents. Because of the anatomical location and microenvironment surrounding the tumor, these models have been very helpful in evaluating cancer therapies. These models were further improved after genetic manipulation of a reporter luciferase gene in the human cancer cell genome, providing a mechanism for monitoring tumor progression and response using bioluminescence imaging. However, the stringent requirement of an appropriate orthotopic lung tumor model for stereotactic body radiation therapy, in which an ablative radiation dose can be administered to the tumor while minimizing normal lung toxicity, required the ability to reproducibly produce a solitary nodule within a desired location in the lung. Current orthotopic lung models, developed primarily in mice, are inappropriate for this application due to widespread dissemination of tumors growing in both lungs. Additionally, the small size of the mouse lung precludes delivery of large doses without excessive morbidity. Our studies demonstrate that a single tumor can be introduced into a specific location in the rat lung and treated effectively with high radiation doses without dose-limiting toxicity.
Several models describing the formation of orthotopic lung tumors have been reported. Yamaura et al.
developed a model for mediastinal lymph node metastasis in the C57/BL6 mouse by implantation of Lewis lung carcinoma cells in the intercostal space (31
). Onn et al.
developed an orthotopic lung model in nude mice to study the efficacy of chemotherapeutic drugs (32
). Injection of tumor cells in saline was quickly followed by diffuse dissemination. Cells suspended in matrigel initially produced a solitary lesion, which then spread progressively within animals. March et al.
described a rat orthotopic lung model in which NSCLC cells with EDTA or elastase were implanted surgically through the trachea (33
). They observed that co-administration of tumor cells with EDTA significantly enhanced the tumor uptake rate but was also associated with the rapid development of multiple tumors within the lung (33
). An intrabronchial instillation approach to develop a rat lung cancer was also described by Byhardt et al.
Like Onn et al., our model requires no surgical intervention. Serial bioluminescence imaging provides an excellent mechanism for tracking tumor development over several weeks until tumor characteristics are suitable for therapy. Based on bioluminescence imaging, our success rate in developing a solitary tumor is essentially 100%. However, on subsequent CT and dissection, this rate falls to 60%. The two common sources of failure are injection at the wrong level, either too superior or too inferior, and failure to insert the needle to the appropriate depth. The latter produces multiple disease foci, which is suboptimal for stereotactic irradiation. To improve our success rate, we are now developing a technique for image-guided tumor cell implantation. This will enable us to visualize the precise needle location prior to injection of the matrigel mixture.
Since the end point of this study is either complete ablation or long-term control of tumor growth, noninvasive monitoring of tumor progression before and after therapeutic interventions is necessary. Bioluminescence imaging is based on oxidation of substrate D-luciferin by luciferase in the presence of O2
and ATP resulting in the emission of light (35
). The comparison of bioluminescence imaging with other imaging modalities including CT and MRI has been reported (36
). In this study we used both bioluminescence imaging and CT for identification, localization and targeting of SBRT delivery. We demonstrated that bioluminescence imaging and CT are necessary for successful SBRT as well as for verifying response to therapy (). Additionally, the response was validated in irradiated tumors by demonstrating the localization of bavituximab to PS that becomes exposed on tumor vascular endothelium after irradiation (23
). With the success of this model, a number of further studies are anticipated.
One shortcoming of our current irradiation system is the inability to easily focus beams from multiple directions. We are presently developing a rotational stage to facilitate multibeam delivery. Nevertheless, we believe the physical limitation does not present any biological limitations; that is, all of the relevant biology can be probed and peripheral toxicity minimized, even without the efficient multibeam capability.