The major goal of this project is to develop and exploit a tractable, preclinical mouse model of metastatic melanoma that recapitulates the clinical progression of surgical removal of primary tumor, treatment with adjuvant therapy and clinical recurrence at distant sites. We see a translational opportunity to devise a novel strategy to prevent the clinical recurrence of metastatic disease. The model will be designed to incorporate normal immune system function to maintain an appropriate tumor microenvironment and provide superior tumor-host interactions, an approach that is significantly more likely to yield biologically and clinically relevant data. Since metastasis often targets internal organs and the timing of its recurrence may vary greatly, the preclinical model will allow for the non-invasive, long-term monitoring of disease progression within the immunocompetent mouse. To meet these criteria, we have developed as a proof-of-concept a syngeneic immunocompetent mouse model of Lewis Lung Carcinoma (LLC) using tissue that was never adapted to cell culture and labeled using a high-titer lentivirus encoding a luciferase/GFP fusion reporter protein, developed by this laboratory in collaboration with Dom Esposito at the Protein Expression Laboratory, Advanced Technology Program (Day et al., Pig Cell Melanoma Res 22:283-295, 2009). Our decision to employ the NSCLC mouse LLC model is well aligned with the new NCI Lung Cancer Program, and provides a robust, well-characterized system to validate our preclinical approach. LLC exhibits highly favorable growth kinetics and virtually 100% take rate of lung metastases in C57BL/6 mice, and was instrumental in the successful development of early conventional chemotherapies that are still used in the clinical today. LLC has been subcloned in vitro and many sublines have been established since being discovered in 1952;however, for this preclinical model we felt it was essential to use an in vivo-maintained LLC stock that has never been adapted to cell culture, an approach that has been reported to yield more reliable preclinical data. In full collaboration with Dr. Melinda Hollingshead (DTP, NCI), we have now established a stably labeled bank of highly metastatic LLC tissue (LLC-Luc/GFP) through multiple rounds of subcutaneous transplantation, resection, and selection of Luc/GFP+ metastatic clones in syngeneic C57BL/6 mice. Our preclinical approach will be to subcutaneously inoculate Luc/GFP-labeled LLC cells into albino C57BL/6 mice and resect consistently at a selected tumor size, at which time drug treatment can be initiated. Recurrence of clinically significant LLC macrometastases is evident in the lungs as Luc/GFP+ lesions within a week, and an entire drug study can be completed in less than one month. The BL signal allows the monitoring of disease after resection of the primary tumor, the progression of metastatic growth over time, and the response of the tumor to therapy;moreover, the BL intensity is well correlated with the pathological score of the metastatic disease. We have found that this resection/recurrence metastasis model fulfills multiple requirements for a tractable and robust syngeneic preclinical model of advanced stage disease, one that we anticipate may more accurately predict therapeutic response. To date we have treated mice in a setting akin to post-surgical first-line adjuvant chemotherapy using cisplatin, paclitaxel and/or antiangiogenic agents. As in the clinic these drugs were found to be most effective against progression in combination. However, the response of metastases to agents could not be predicted from, and often opposed, their effects on subcutaneous tumors. Moreover, time to macrometastasis onset, rather than growth, correlated with both mouse survival and treatment efficacy. This work was recently published (Day et al., 2011, Int. J. Cancer, Epub ahead of print). We have also noted that even low expression of the xenobiotic reporters GFP and/or luciferase can render growth of labeled metastatic tumors unpredictable in syngeneic mice and severely limit the utility of any immunocompetent preclinical model. We have developed transgenic mice in both FVB/N and C57BL/6 backgrounds that express the Luc/GFP fusion gene at an irrelevant site (in this case the anterior pituitary). These mice are thus pre-tolerized to both xenobiotic markers, and we note that clearly labeled metastatic lesions arise much more regularly in the lungs of inoculated, resected host mice. These mice are currently being employed in our improving preclinical model (Day et al., manuscript in preparation). The primary goal now is to combine all the reagents we have developed to study the mechanisms by which BRafV600E-containing melanoma (found in 50% of melanoma patients), recurs at metastatic sites as invariably happens in the clinic today. This extensive preclinical effort with be in collaboration with extramural scientists Dr. Marcus Bosenberg (Yale), Dr. Martin McMahon (UCSF) and Dr. Keith Flaherty (Harvard Medical School), and intramural scientists Drs. Nick Restifo and Terry Van Dyke.
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