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In Vivo Imaging with Bioluminescence: Ancient Scientific Phenomena Driving Cutting-Edge Tumor Research

heat map depicting bioluminescence

Preclinical In Vivo Imaging is a technique that allows scientists to observe biological processes at the cellular level in real time. Preclinical In Vivo Imaging has been especially useful for imaging the tumor microenvironment (TME) and tracking the complex interactions between tumor cells and immune system cells that can result in tumor growth and development or tumor regression. Preclinical In Vivo Imaging is a noninvasive technique that relies on visualization of specific cells that emit light through bioluminescent reactions that can be detected within living animal models with highly sensitive cameras. Here we provide an overview of bioluminescence-based Preclinical In Vivo Imaging methods that are being used in preclinical oncology research.


Bioluminescence Basics

Bioluminescence is a naturally occurring phenomenon in marine and terrestrial animals that causes them to emit visible light. Fireflies are the most familiar species of bioluminescent animals in addition to several species of snails, zooplankton, sea anemones, jellyfish and bacteria. These organisms express species-specific luciferins, as well as luciferases that catalyze the oxidation of luciferin, resulting in light emission. Firefly luciferase was one of the first enzymes to be understood at a biochemical level and its structure determined, along with its enzymatic substrates and products[1],[2]. Many of these luciferase and luciferin genes have been cloned and studied for development into molecular tools, and three different luciferases have been widely adapted for use in biomedical research, including Photinus pyralis (firefly) luciferase (Fluc), sea pansy Renilla reniformis luciferase (Rluc), and the marine copepod Gaussia princeps luciferase (Gluc)[3]. Gluc does not need external ATP for bioluminescence and can be cell-associated or detected in fluids like blood or urine[4]. In contrast, Fluc and Rluc remain within cells and emit more photons when localized within tissues. Fluc and Rluc use different luciferins and emit photons at different wavelengths so can be used together in cell cultures or animal models. Fluc emits green light at 562 nm where as Rluc and Gluc emit within the blue end of the visible light spectrum and are more easily absorbed by hemoglobin and melanin, thus making Fluc more suitable for Preclinical In Vivo Imaging[5]. Luciferase expression can be introduced by adenoviral vectors or adeno-associated vectors using gene therapy-like approaches of gene transduction. These vectors express luciferase and can be engineered to be driven by cell-specific promoters or transcription activators. These vectors are transfected or transduced into cells, and expression is monitored by light emission upon exogenous treatment with luciferin. Bioluminescence imaging (BLI) measurements can be used to directly measure gene expression in vitro or in vivo.


diagram of visible spectrum of light: gamma rays, x-rays, UV, infrared, radio waves


BLI and Preclinical In Vivo Imaging: A Tumor Tale

BLI and Preclinical In Vivo Imaging have many applications including basic and developmental biology, neurobiology, and infectious disease. BLI-based Preclinical In Vivo Imaging is particularly well-suited to immuno-oncology studies as both immune cells, like T cells, and tumor cells can be independently monitored using different luciferase reporters, such that T cell infiltration into tumors can be measured[6]. These BLI applications are useful for understanding basic tumor biology as well as for the preclinical screening of immunotherapeutics. More recently, modified bioluminescent probes have been designed to be cleaved in the presence of proteases, like urokinase-type plasminogen activator (uPA), which is secreted at high levels by some tumors. A modified luciferin probe was designed to include a tripeptide substrate of uPA, which is cleaved in the presence of the protease and allows for luciferin bioluminescence upon exposure to luciferase[7]. This approach is being evaluated as an in vitro diagnostic for uPA activity as well as a tool to evaluate protease inhibitors in mouse xenograft models.

BLI-based Preclinical In Vivo Imaging continues to be a valuable non-invasive method for studying numerous aspects of biology. New luciferins and luciferases are under development and include emission at different wavelengths. Better cameras and microscopy tools are also being developed toward a goal of more sensitive and longer lasting BLI signals that accurately reflect biological processes.


Preclinical In Vivo Imaging in Systemic Tumor Models - download now

[1] DeLuca M, McElroy WD. Kinetics of the firefly luciferase catalyzed reactions. Biochemistry. 1974 Feb 26;13(5):921-5.

[2] Conti E, Franks NP, Brick P. Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure. 1996 Mar 15;4(3):287-98.

[3] Badr CE, Tannous BA. Bioluminescence imaging: progress and applications. Trends Biotechnol. 2011 Dec;29(12):624-33. doi: 10.1016/j.tibtech.2011.06.010. Epub 2011 Jul 23. PMID: 21788092; PMCID: PMC4314955.

[4] Tannous BA. Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nat. Protoc. 2009;4(4):582-91. doi: 10.1038/nprot.2009.28. PMID: 19373229; PMCID: PMC2692611.

[5] Badr CE, Tannous BA. Bioluminescence imaging: progress and applications. Trends Biotechnol. 2011 Dec;29(12):624-33. doi: 10.1016/j.tibtech.2011.06.010. Epub 2011 Jul 23. PMID: 21788092; PMCID: PMC4314955.

[6] Liu TW, Gammon ST, Fuentes D, Piwnica-Worms D. Multi-Modal Multi-Spectral Intravital Macroscopic Imaging of Signaling Dynamics in Real Time during Tumor-Immune Interactions. Cells. 2021 Feb 25;10(3):489.

[7] Chen Y, Wu C, Wang C, Zhang T, Hua Y, Shen Y, Liang G. Bioluminescence Imaging of Urokinase-Type Plasminogen Activator Activity in Vitro and in Tumors. Anal. Chem. 2021 Jul 27;93(29):9970-9973. doi: 10.1021/acs.analchem.1c02499. Epub 2021 Jul 15. PMID: 34264075.