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In vivo imaging of ligand receptor binding with Gaussia luciferase complementation

Abstract

Studies of ligand-receptor binding and the development of receptor antagonists would benefit greatly from imaging techniques that translate directly from cell-based assays to living animals. We used Gaussia luciferase protein fragment complementation to quantify the binding of chemokine (C-X-C motif) ligand 12 (CXCL12) to chemokine (C-X-C motif) receptor 4 (CXCR4) and CXCR7. Studies established that small-molecule inhibitors of CXCR4 or CXCR7 specifically blocked CXCL12 binding in cell-based assays and revealed differences in kinetics of inhibiting chemokine binding to each receptor. Bioluminescence imaging showed CXCL12-CXCR7 binding in primary and metastatic tumors in a mouse model of breast cancer. We used this imaging technique to quantify drug-mediated inhibition of CXCL12-CXCR4 binding in living mice. We expect this imaging technology to advance research in areas such as ligand-receptor interactions and the development of new therapeutic agents in cell-based assays and small animals.

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Figure 1: Development of GLuc complementation for CXCL12 binding to CXCR4 or CXCR7.
Figure 2: Small-molecule inhibitors block GLuc complementation between CXCL12 and CXCR7.
Figure 3: Specific inhibition of CXCL12-CXCR4 complementation in cell-based assays.
Figure 4: Imaging of CXCL12-CXCR7 binding in primary and metastatic breast tumors.
Figure 5: In vivo imaging of CXCL12-CXCR4 binding and inhibition.

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Acknowledgements

This research was supported by the US National Institutes of Health grants R01 CA136553, R01 CA136829, R01 CA142750 and P50 CA93990 (G.D.L.) and Measures to Attract Leading Scientists to Russian Educational Institutions grant 11.G34.31.0017 and Russian Ministry of Education and Science grants 16.740.11.0367 and 16.512.11.2139 (D.M.C.). We thank ChemoCentryx for the monoclonal antibody 11G8 and the small-molecule inhibitors of CXCR7. We acknowledge support from the US National Institutes of Health grant U24 CA083099 to B.D. Ross for the Michigan Small Animal Imaging Resource. We thank J. Jaen, M. Penfold and D. Dairaghi for helpful discussions.

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K.E.L., L.A.M., B.T.S., S.A.L., P.R. and G.D.L. performed the cell culture and animal experiments and analyzed the data. K.E.L., D.S., D.M.C. and G.D.L. provided new reagents. K.E.L., B.T.S., D.M.C. and G.D.L. prepared the manuscript. K.E.L. and G.D.L. supervised the project.

Corresponding author

Correspondence to Gary D Luker.

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The authors declare no competing financial interests.

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Supplementary Figures 1–12 and Supplementary Methods. (PDF 1271 kb)

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Luker, K., Mihalko, L., Schmidt, B. et al. In vivo imaging of ligand receptor binding with Gaussia luciferase complementation. Nat Med 18, 172–177 (2012). https://doi.org/10.1038/nm.2590

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