Noninvasive Optical Tracking of Red Fluorescent Protein-Expressing Cancer Cells in a Model of Metastatic Breast Cancer

Winnard, Paul T.; Kluth, Jessica; Raman, Venu

doi:10.1593/neo.06304

Cited by 80 publications

(77 citation statements)

References 31 publications

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“…Several newer mutants of mrfp1 with increased photostability and brightness (mCherry and tdTomato) or far-red emission spectra (mPlum and mRaspberry at 649 and 625 nm) have been reported (29). Based on recent tests in xenograft models of metastatic breast cancer (30), tdTomato would be the most likely to provide a further improvement over mrfp1.…”

Section: Discussionmentioning

confidence: 99%

Construction and Validation of Improved Triple Fusion Reporter Gene Vectors for Molecular Imaging of Living Subjects

2007

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Multimodality imaging using several reporter genes and imaging technologies has become an increasingly important tool in determining the location(s), magnitude, and time variation of reporter gene expression in small animals. We have reported construction and validation of several triple fusion genes composed of a bioluminescent, a fluorescent, and a positron emission tomography (PET) reporter gene in cell culture and in living subjects. However, the bioluminescent and fluorescent components of fusion reporter proteins encoded by these vectors possess lesser activities when compared with the bioluminescent and fluorescent components of the nonfusions. In this study, we first created a mutant (mtfl) of a thermostable firefly luciferase (tfl) bearing the peroxisome localization signal to have greater cytoplasmic localization and improved access for its substrate, D-luciferin. Comparison between the three luciferases [mtfl, tfl, and firefly luciferase ( fl)] both in cell culture and in living mice revealed that mtfl possessed 6-to 10-fold (in vitro) and 2-fold (in vivo) higher activity than fl. The improved version of the triple fusion vector carrying mtfl as the bioluminescent reporter component showed significantly (P < 0.05) higher bioluminescence than the previous triple fusion vectors. Of the three different red fluorescent reporter genes ( jred, hcred, and mrfp1, isolated from jellyfish chromophore, coral Heteractis crispa, and coral Discosoma, respectively) evaluated, mrfp1 was able to preserve highest expression as a component of the triple fusion reporter gene for in vivo fluorescence imaging. A truncated version of wild-type herpes simplex virus 1 (HSV1) thymidine kinase gene (wttk) retained a higher expression level than the truncated mutant HSV1-sr39 TK (ttk) as the third reporter component of this improved triple fusion vector. Multimodality imaging of tumor-bearing mice using bioluminescence and microPET showed higher luciferase activity [(2.7 F 0.1 versus 1.9 F 0.1) Â (10 6 p/s/cm 2 /sr)] but similar level of fluorine-18-labeled 2 ¶-fluoro-2 ¶-deoxyarabinofuranosyl-5-ethyluracil (18F-FEAU) uptake (1.37 F 0.15 versus 1.37 F 0.2) percentage injected dose per gram] by mtfl-mrfp1-wttk-expressing tumors compared with the fl-mrfp1-wttkexpressing tumors. Both tumors showed 4-to 5-fold higher accumulation (P < 0.05) of 18F-FEAU than fluorine-18-labeled 9-(4-fluoro-3-hydroxymethylbutyl)guanine. This improved triple fusion reporter vector will enable high sensitivity detection of lower numbers of cells from living animals using the combined bioluminescence, fluorescence, and microPET imaging techniques. [Cancer Res 2007;67(7):3085-93]

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Section: Discussionmentioning

confidence: 99%

Construction and Validation of Improved Triple Fusion Reporter Gene Vectors for Molecular Imaging of Living Subjects

2007

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“…This is particularly pertinent for monitoring the behavior of static cell populations (e.g., tumor xenografts) (463), and for investigating the bio- distribution, underlying mechanisms, and functional benefits of cell-based therapies, thus expediting the clinical translation of these promising treatment strategies. Molecular imaging will likely play a key role in the clinical translation of cell-based therapies.…”

Section: E Cell Trackingmentioning

confidence: 99%

A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications

James

Gambhir

2012

Physiological Reviews

942

954

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Molecular imaging is revolutionizing the way we study the inner workings of the human body, diagnose diseases, approach drug design, and assess therapies. The field as a whole is making possible the visualization of complex biochemical processes involved in normal physiology and disease states, in real time, in living cells, tissues, and intact subjects. In this review, we focus specifically on molecular imaging of intact living subjects. We provide a basic primer for those who are new to molecular imaging, and a resource for those involved in the field. We begin by describing classical molecular imaging techniques together with their key strengths and limitations, after which we introduce some of the latest emerging imaging modalities. We provide an overview of the main classes of molecular imaging agents (i.e., small molecules, peptides, aptamers, engineered proteins, and nanoparticles) and cite examples of how molecular imaging is being applied in oncology, neuroscience, cardiology, gene therapy, cell tracking, and theranostics (therapy combined with diagnostics). A step-by-step guide to answering biological and/or clinical questions using the tools of molecular imaging is also provided. We conclude by discussing the grand challenges of the field, its future directions, and enormous potential for further impacting how we approach research and medicine.

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“…1 In addition, the MDA-MB-231 cells that stably express the bright red fluorescence protein used in that study were easily identified in post mortem tissue preparations. Thus, macroscopic to microscopic detection was achieved.…”

Section: Discussionmentioning

confidence: 95%

“…In addition, fluorescence based optical imaging is limited to detection depths of, at best, several millimeters below the surface. 1 On the other hand MRI can not resolve microscopic structures in ex-vivo samples. This exemplifies that multi-modality detection systems are inherently more flexible and supply complementary data sets.…”

Section: Discussionmentioning

confidence: 99%

“…The ideal experimental models are those that can recapitulate the human disease and allow for both non-invasive imaging of live animals followed by characterization of post mortem preparations. 1 A number of molecular imaging schemes have been developed to track human disease progression in animals 2 but not without limitations. Our approach in this area of research has focused on targeting cell surface markers of human cancer cells in xenograft mouse model systems.…”

Section: Introductionmentioning

confidence: 99%

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Development of novel chimeric transmembrane proteins for multimodality imaging of cancer cells

Winnard¹,

Kluth²,

Kato³

et al. 2007

Cancer Biology & Therapy

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Tracking the migration of cancer cells is essential to understanding the metastatic process. In order to facilitate the tracking of metastatic progression, we have generated transgenic cancer cell lines that express novel chimeric proteins composed of truncated human type II transmembrane proteins fused in-frame to a red fluorescence protein. These chimeric proteins have been engineered to display the fluorescence domain on the surface of cells. The three novel chimeric proteins exhibited high transient expression in several cancer cell lines. Membrane expression was well characterized in MCF-7 cell lines that stably express the chimeric proteins. Indirect immunocytochemistry of non-premeablized cells demonstrated co-localization of endogenous red and green fluorescence labeled secondary antibody bound on the cell surfaces. Immunoblots of total protein prepared from membrane, cytosolic and nuclear fractions indicated that the chimeric proteins were mainly associated with the membrane fraction. Further evidence of the membrane expression of these proteins was confirmed by confocal microscopy. Moreover, the chimeric protein was detected on the cell surface by T2-weighted magnetic resonance imaging using anti-red fluorescence protein antibody and superparamagnetic iron oxide particles in vitro and by optical imaging in vivo. The development of this non-mammalian cell surface marker, that can be detected by multi-modality imaging, will find utility in non-invasive longitudinal tracking of biological processes including metastatic progression, solid tumor treatment regimes and the fate of cells used in cell therapies such as islet or stem cells.

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Noninvasive Optical Tracking of Red Fluorescent Protein-Expressing Cancer Cells in a Model of Metastatic Breast Cancer

Cited by 80 publications

References 31 publications

Construction and Validation of Improved Triple Fusion Reporter Gene Vectors for Molecular Imaging of Living Subjects

Construction and Validation of Improved Triple Fusion Reporter Gene Vectors for Molecular Imaging of Living Subjects

A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications

Development of novel chimeric transmembrane proteins for multimodality imaging of cancer cells

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