Optimized in vivo multispectral bioluminescent imaging of tumor biology using engineered BRET reporters
Bryan Labra,
Kshitij Parag-Sharma,
John J. Powers,
Sonal Srivastava,
Joel R. Walker,
Thomas A. Kirkland,
Caroline K. Brennan,
Jennifer A. Prescher,
Antonio L. Amelio
Affiliations
Bryan Labra
Lineberger Comprehensive Cancer Center, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Kshitij Parag-Sharma
Graduate Curriculum in Cell Biology & Physiology, Biological & Biomedical Sciences Program, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
John J. Powers
Department of Tumor Microenvironment and Metastasis, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USA
Sonal Srivastava
Department of Tumor Microenvironment and Metastasis, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USA
Joel R. Walker
Promega Biosciences, LLC, San Luis Obispo, CA, USA
Thomas A. Kirkland
Promega Biosciences, LLC, San Luis Obispo, CA, USA; Promega Corporation, Madison, WI, USA
Caroline K. Brennan
Department of Chemistry, University of California, Irvine, Irvine, CA, USA
Jennifer A. Prescher
Department of Chemistry, University of California, Irvine, Irvine, CA, USA; Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA; Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA
Antonio L. Amelio
Department of Tumor Microenvironment and Metastasis, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USA; Cancer Cell Biology Program, Lineberger Comprehensive Cancer Center, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Cell Biology and Physiology, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Head and Neck-Endocrine Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USA; Corresponding author
Summary: The ability to visualize and track multiple biological processes in vivo in real time is highly desirable. Bioluminescence imaging (BLI) has emerged as an attractive modality for non-invasive cell tracking, with various luciferase reporters enabling parallel monitoring of several processes. However, simultaneous multiplexed imaging in vivo is challenging due to suboptimal reporter intensities and the need to image one luciferase at a time. We report a multiplexed BLI approach using a single substrate that leverages bioluminescence resonance energy transfer (BRET)-based reporters with distinct spectral profiles for triple-color BLI. These luciferase-fluorophore fusion reporters address light transmission challenges and use optimized coelenterazine substrates. Comparing BRET reporters across two substrate analogs identified a green-yellow-orange combination that allows simultaneous imaging of three distinct cell populations in vitro and in vivo. These tools provide a template for imaging other biological processes in vivo during a single BLI session using a single reporter substrate.
