GFP and luciferase dual reporter cell lines for non-invasive in vivo fluorescence and bioluminescence imaging in mouse tumor xenograft and syngeneic models

Background: Whole animal in vivo optical imaging is widely used for the ease of operation in visualizing in vivo biological events, eliminating the requirement for animal subject sacrifice, allowing for continuous monitoring/imaging of a single individual animal, and reducing the amount of inter-animal variation. Although xenograft and syngeneic models are both useful in vivo models for studying tumor formation and development, metastases, measuring tumor burden in whole animals, the syngeneic models are particularly valuable for studying the interplay between tumor cells and host immune system and monitoring responses to immunotherapy. Here we report on the generation of dual reporter syngeneic cell lines which stably express GFP and luciferase with broad applications for in vitro and in vivo cancer immunology studies. These GFP and Luciferase dual reporters provide a relatively simple, robust, and highly sensitive means to measure biological processes and to assess therapeutic efficacy in animal models through non-invasive in vivo fluorescence and bioluminescence imaging. 

Methods: These dual reporter cell lines were derived from mouse breast and colon cancer cell lines. After the introduction of Lenti-GFP-LUC2 dual reporter into the parental cell lines and antibiotic selection, single cell cloning was performed to isolate stable clones with high GFP and luciferase expression. The isolated clones were characterized by cell morphology, growth kinetics, and stable expression of GFP and luciferase. The established cell lines were tested for their tumorigenicity in immunodeficient mice and subsequent whole-body in vivo bioluminescent and fluorescent imaging were performed by Xenogen IVIS imaging system. At the endpoint, the tumors were excised for additional ex vivo bioluminescent and fluorescent imaging. 

Results: We confirmed high level of GFP and luciferase expression in selected clones via fluorescence and luminescence imaging and flow cytometric analysis. The fluorescence and bioluminescence intensity showed positive linear correlation with the cell numbers. The GFP and luciferase expression remained high after 30 population doublings. The morphology and the growth rate were comparable to the parental cell lines. Fluorescence and bioluminescence imaging of mouse xenograft models displayed positive correlation of fluorescence or bioluminescence intensity to tumor size. Ex vivo imaging of tumors also showed high intensity GFP and bioluminescence.

Conclusions: In vivo bioluminescence and fluorescence imaging provide complementary non-invasive approaches for real-time monitoring and studying of immune responses and tumor progression in preclinical models. The newly developed dual reporter syngeneic cell lines offer a powerful imaging tool for studying multiple aspects of complex cellular interactions during preclinical investigation and facilitating development of more effective immunotherapeutic strategies.