NoGlow mice reveal unappreciated metastatic dynamics independent of the primary tumor. A, Tumor growth of triple transgenic (3 ×) B16-F10 cells (105) after subcutaneous implantation into the flank of WT C57Bl/6 (n = 2), CAG-driven Full-body WT GFP/Luc expressing mice (n = 3), CMV cre littermates positive (n = 8) or negative (n = 5) for the NoGlow construct, or SCID beige (n = 5) mice. B, Bioluminescence imaging of lungs at the time of euthanasia for each mouse from A. Robust signal is detected only in NoGlow+ lungs. *Indicates no detectable primary tumor at the end of experiment. C, Bioluminescence intensity of individual lungs from B. NoGlow+ lungs contained significantly more positive Luc signal than SCID, WT, or NoGlow− animals. D, Plot of tumor volume at time of euthanasia (x) by total lung radiance (y) for individual animals from A. P values were determined by one-way ANOVA with Tukey correction. All P values represent mean ± SEM.
ARTICLE ABSTRACT
The ability to temporally regulate gene expression and track labeled cells makes animal models powerful biomedical tools. However, sudden expression of xenobiotic genes [e.g., GFP, luciferase (Luc), or rtTA3] can trigger inadvertent immunity that suppresses foreign protein expression or results in complete rejection of transplanted cells. Germline exposure to foreign antigens somewhat addresses these challenges; however, native fluorescence and bioluminescence abrogates the utility of reporter proteins and highly spatiotemporally restricted expression can lead to suboptimal xenoantigen tolerance. To overcome these unwanted immune responses and enable reliable cell tracking/gene regulation, we developed a novel mouse model that selectively expresses antigen-intact but nonfunctional forms of GFP and Luc, as well as rtTA3, after CRE-mediated recombination. Using tissue-specific CREs, we observed model and sex-based differences in immune tolerance to the encoded xenoantigens, illustrating the obstacles of tolerizing animals to foreign genes and validating the utility of these “NoGlow” mice to dissect mechanisms of central and peripheral tolerance. Critically, tissue unrestricted NoGlow mice possess no detectable background fluorescence or luminescence and exhibit limited adaptive immunity against encoded transgenic xenoantigens after vaccination. Moreover, we demonstrate that NoGlow mice allow tracking and tetracycline-inducible gene regulation of triple-transgenic cells expressing GFP/Luc/rtTA3, in contrast to transgene-negative immune-competent mice that eliminate these cells or prohibit metastatic seeding. Notably, this model enables de novo metastasis from orthotopically implanted, triple-transgenic tumor cells, despite high xenoantigen expression. Altogether, the NoGlow model provides a critical resource for in vivo studies across disciplines, including oncology, developmental biology, infectious disease, autoimmunity, and transplantation.
Multitolerant NoGlow mice enable tracking and gene manipulation of transplanted tumor cells without immune-mediated rejection, thus providing a platform to investigate novel mechanisms of adaptive immunity related to metastasis, immunotherapy, and tolerance.
