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1
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Chemla Y, Levin I, Fan Y, Johnson AA, Coley CW, Voigt CA. Hyperspectral reporters for long-distance and wide-area detection of gene expression in living bacteria. Nat Biotechnol 2025:10.1038/s41587-025-02622-y. [PMID: 40216953 DOI: 10.1038/s41587-025-02622-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2024] [Accepted: 02/27/2025] [Indexed: 04/27/2025]
Abstract
Genetically encoded reporters are suitable for short-distance imaging in the laboratory but not for scanning wide outdoor areas from a distance. Here we introduce hyperspectral reporters (HSRs) designed for hyperspectral imaging cameras that are commonly mounted on unmanned aerial vehicles and satellites. HSR genes encode enzymes that produce a molecule with a unique absorption signature that can be reliably distinguished in hyperspectral images. Quantum mechanical simulations of 20,170 metabolites identified candidate HSRs, leading to the selection of biliverdin IXα and bacteriochlorophyll a for their distinct absorption spectra and biosynthetic feasibility. These genes were integrated into chemical sensor circuits in soil (Pseudomonas putida) and aquatic (Rubrivivax gelatinosus) bacteria. The bacteria were detectable outdoors under ambient light from up to 90 m in a single 4,000-m2 hyperspectral image taken using fixed and unmanned aerial vehicle-mounted cameras. The dose-response functions of the chemical sensors were measured remotely. HSRs enable large-scale studies and applications in ecology, agriculture, environmental monitoring, forensics and defense.
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Affiliation(s)
- Yonatan Chemla
- Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Itai Levin
- Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Yueyang Fan
- Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Anna A Johnson
- Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Connor W Coley
- Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
- Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Christopher A Voigt
- Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
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2
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Liu L, Tan Z, Wei Y, Sun Q. A multi-perspective deep learning framework for enhancer characterization and identification. Comput Biol Chem 2025; 114:108284. [PMID: 39577030 DOI: 10.1016/j.compbiolchem.2024.108284] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2024] [Revised: 11/02/2024] [Accepted: 11/13/2024] [Indexed: 11/24/2024]
Abstract
Enhancers are vital elements in the genome that boost the transcriptional activity of neighboring genes and are essential in regulating cell-specific gene expression. Therefore, accurately identifying and characterizing enhancers is essential for comprehending gene regulatory networks and the development of related diseases. This study introduces MPDL-Enhancer, a novel multi-perspective deep learning framework aimed at enhancer characterization and identification. In this study, enhancer sequences are encoded using the dna2vec model along with features derived from the structural properties of DNA sequences. Subsequently, these representations are processed through a novel dual-scale deep neural network designed to discern subtle correlations and extended interactions embedded within the semantic content of DNA. The predictive phase of our methodology employs a Support Vector Machine classifier to render the final classification. To rigorously assess the efficacy of our approach, a comprehensive evaluation was executed utilizing an independent test dataset, thereby substantiating the robustness and accuracy of our model. Our methodology demonstrated superior performance over existing computational techniques, with an accuracy (ACC) of 81.00 %, a sensitivity (SN) of 79.00 %, and specificity (SP) of 83.00 %. The innovative dual-scale deep neural network and the unique feature representation strategy contributed to this performance improvement. MPDL-Enhancer has effectively characterized enhancer sequences and achieved excellent predictive performance. Building upon this foundation, we conducted an interpretability analysis of the model, which can assist researchers in identifying key features and patterns that affect the functionality of enhancers, thereby promoting a deeper understanding of gene regulatory networks.
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Affiliation(s)
- Liwei Liu
- College of Science, Dalian Jiaotong University, Dalian 116028, China.
| | - Zhebin Tan
- College of Software, Dalian Jiaotong University, Dalian 116028, China
| | - Yuxiao Wei
- College of Software, Dalian Jiaotong University, Dalian 116028, China
| | - Qianhui Sun
- College of Software, Dalian Jiaotong University, Dalian 116028, China
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3
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Cotta GC, Teixeira dos Santos RC, Costa GMJ, Lacerda SMDSN. Reporter Alleles in hiPSCs: Visual Cues on Development and Disease. Int J Mol Sci 2024; 25:11009. [PMID: 39456792 PMCID: PMC11507014 DOI: 10.3390/ijms252011009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2024] [Revised: 10/05/2024] [Accepted: 10/08/2024] [Indexed: 10/28/2024] Open
Abstract
Reporter alleles are essential for advancing research with human induced pluripotent stem cells (hiPSCs), notably in developmental biology and disease modeling. This study investigates the state-of-the-art gene-editing techniques tailored for generating reporter alleles in hiPSCs, emphasizing their effectiveness in investigating cellular dynamics and disease mechanisms. Various methodologies, including the application of CRISPR/Cas9 technology, are discussed for accurately integrating reporter genes into the specific genomic loci. The synthesis of findings from the studies utilizing these reporter alleles reveals insights into developmental processes, genetic disorder modeling, and therapeutic screening, consolidating the existing knowledge. These hiPSC-derived models demonstrate remarkable versatility in replicating human diseases and evaluating drug efficacy, thereby accelerating translational research. Furthermore, this review addresses challenges and future directions in refining the reporter allele design and application to bolster their reliability and relevance in biomedical research. Overall, this investigation offers a comprehensive perspective on the methodologies, applications, and implications of reporter alleles in hiPSC-based studies, underscoring their essential role in advancing both fundamental scientific understanding and clinical practice.
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Affiliation(s)
| | | | | | - Samyra Maria dos Santos Nassif Lacerda
- Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, 31270-901 Belo Horizonte, Brazil; (G.C.C.); (R.C.T.d.S.); (G.M.J.C.)
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4
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Fröse J, Rowley J, Farid AS, Rakhshandehroo T, Leclerc P, Mak H, Allen H, Moravej H, Munaretto L, Millan-Barea L, Codet E, Glockner H, Jacobson C, Hemann M, Rashidian M. Development of an antigen-based approach to noninvasively image CAR T cells in real time and as a predictive tool. SCIENCE ADVANCES 2024; 10:eadn3816. [PMID: 39292778 PMCID: PMC11409975 DOI: 10.1126/sciadv.adn3816] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/07/2023] [Accepted: 08/12/2024] [Indexed: 09/20/2024]
Abstract
CAR T cell therapy has revolutionized the treatment of a spectrum of blood-related malignancies. However, treatment responses vary among cancer types and patients. Accurate monitoring of CAR T cell dynamics is crucial for understanding and evaluating treatment efficacy. Positron emission tomography (PET) offers a comprehensive view of CAR T cell homing, especially in critical organs such as lymphoid structures and bone marrow. This information will help assess treatment response and predict relapse risk. Current PET imaging methods for CAR T require genetic modifications, limiting clinical use. To overcome this, we developed an antigen-based imaging approach enabling whole-body CAR T cell imaging. The probe detects CAR T cells in vivo without affecting their function. In an immunocompetent B cell leukemia model, CAR-PET signal in the spleen predicted early mortality risk. The antigen-based CAR-PET approach allows assessment of CAR T therapy responses without altering established clinical protocols. It seamlessly integrates with FDA-approved and future CAR T cell generations, facilitating broader clinical application.
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Affiliation(s)
- Julia Fröse
- David H. Koch Institute for Integrative Cancer Research, Cambridge, MA 02142, USA
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
| | - Jennifer Rowley
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
- Harvard Medical School, Boston, MA 02215, USA
| | - Ali Salehi Farid
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Taha Rakhshandehroo
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Paul Leclerc
- David H. Koch Institute for Integrative Cancer Research, Cambridge, MA 02142, USA
| | - Howard Mak
- David H. Koch Institute for Integrative Cancer Research, Cambridge, MA 02142, USA
| | - Harris Allen
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Heydar Moravej
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Leila Munaretto
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Luis Millan-Barea
- David H. Koch Institute for Integrative Cancer Research, Cambridge, MA 02142, USA
| | - Elisabeth Codet
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Hannah Glockner
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Caron Jacobson
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Michael Hemann
- David H. Koch Institute for Integrative Cancer Research, Cambridge, MA 02142, USA
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
| | - Mohammad Rashidian
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
- Harvard Medical School, Boston, MA 02215, USA
- Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02215, USA
- Parker Institute for Cancer Immunotherapy, San Francisco, CA 94129, USA
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Hong S, Lee DS, Bae GW, Jeon J, Kim HK, Rhee S, Jung KO. In Vivo Stem Cell Imaging Principles and Applications. Int J Stem Cells 2023; 16:363-375. [PMID: 37643761 PMCID: PMC10686800 DOI: 10.15283/ijsc23045] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2023] [Revised: 07/13/2023] [Accepted: 07/21/2023] [Indexed: 08/31/2023] Open
Abstract
Stem cells are the foundational cells for every organ and tissue in our body. Cell-based therapeutics using stem cells in regenerative medicine have received attracting attention as a possible treatment for various diseases caused by congenital defects. Stem cells such as induced pluripotent stem cells (iPSCs) as well as embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), and neuroprogenitors stem cells (NSCs) have recently been studied in various ways as a cell-based therapeutic agent. When various stem cells are transplanted into a living body, they can differentiate and perform complex functions. For stem cell transplantation, it is essential to determine the suitability of the stem cell-based treatment by evaluating the origin of stem, the route of administration, in vivo bio-distribution, transplanted cell survival, function, and mobility. Currently, these various stem cells are being imaged in vivo through various molecular imaging methods. Various imaging modalities such as optical imaging, magnetic resonance imaging (MRI), ultrasound (US), positron emission tomography (PET), and single-photon emission computed tomography (SPECT) have been introduced for the application of various stem cell imaging. In this review, we discuss the principles and recent advances of in vivo molecular imaging for application of stem cell research.
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Affiliation(s)
- Seongje Hong
- Department of Anatomy, College of Medicine, Chung-Ang University, Seoul, Korea
| | - Dong-Sung Lee
- Department of Life Sciences, University of Seoul, Seoul, Korea
| | - Geun-Woo Bae
- Department of Anatomy, College of Medicine, Chung-Ang University, Seoul, Korea
| | - Juhyeong Jeon
- Department of Anatomy, College of Medicine, Chung-Ang University, Seoul, Korea
| | - Hak Kyun Kim
- Department of Life Science, Chung-Ang University, Seoul, Korea
| | - Siyeon Rhee
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA
| | - Kyung Oh Jung
- Department of Anatomy, College of Medicine, Chung-Ang University, Seoul, Korea
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Liao S, Zhou M, Wang Y, Lu C, Yin B, Zhang Y, Liu H, Yin X, Song G. Emerging biomedical imaging-based companion diagnostics for precision medicine. iScience 2023; 26:107277. [PMID: 37520706 PMCID: PMC10371849 DOI: 10.1016/j.isci.2023.107277] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/01/2023] Open
Abstract
The tumor heterogeneity, which leads to individual variations in tumor microenvironments, causes poor prognoses and limits therapeutic response. Emerging technology such as companion diagnostics (CDx) detects biomarkers and monitors therapeutic responses, allowing identification of patients who would benefit most from treatment. However, currently, most US Food and Drug Administration-approved CDx tests are designed to detect biomarkers in vitro and ex vivo, making it difficult to dynamically report variations of targets in vivo. Various medical imaging techniques offer dynamic measurement of tumor heterogeneity and treatment response, complementing CDx tests. Imaging-based companion diagnostics allow for patient stratification for targeted medicines and identification of patient populations benefiting from alternative therapeutic methods. This review summarizes recent developments in molecular imaging for predicting and assessing responses to cancer therapies, as well as the various biomarkers used in imaging-based CDx tests. We hope this review provides informative insights into imaging-based companion diagnostics and advances precision medicine.
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Affiliation(s)
- Shiyi Liao
- State Key Laboratory for Chemo, Biosensing and Chemometrics, College of Chemistry and Chemical, Engineering, Hunan University, Changsha 410082, China
| | - Mengjie Zhou
- State Key Laboratory for Chemo, Biosensing and Chemometrics, College of Chemistry and Chemical, Engineering, Hunan University, Changsha 410082, China
| | - Youjuan Wang
- State Key Laboratory for Chemo, Biosensing and Chemometrics, College of Chemistry and Chemical, Engineering, Hunan University, Changsha 410082, China
| | - Chang Lu
- State Key Laboratory for Chemo, Biosensing and Chemometrics, College of Chemistry and Chemical, Engineering, Hunan University, Changsha 410082, China
| | - Baoli Yin
- State Key Laboratory for Chemo, Biosensing and Chemometrics, College of Chemistry and Chemical, Engineering, Hunan University, Changsha 410082, China
| | - Ying Zhang
- State Key Laboratory for Chemo, Biosensing and Chemometrics, College of Chemistry and Chemical, Engineering, Hunan University, Changsha 410082, China
| | - Huiyi Liu
- State Key Laboratory for Chemo, Biosensing and Chemometrics, College of Chemistry and Chemical, Engineering, Hunan University, Changsha 410082, China
| | - Xia Yin
- State Key Laboratory for Chemo, Biosensing and Chemometrics, College of Chemistry and Chemical, Engineering, Hunan University, Changsha 410082, China
| | - Guosheng Song
- State Key Laboratory for Chemo, Biosensing and Chemometrics, College of Chemistry and Chemical, Engineering, Hunan University, Changsha 410082, China
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7
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McMorrow R, Zambito G, Nigg A, Lila K, van den Bosch TPP, Lowik CWGM, Mezzanotte L. Whole-body bioluminescence imaging of T-cell response in PDAC models. Front Immunol 2023; 14:1207533. [PMID: 37497236 PMCID: PMC10367003 DOI: 10.3389/fimmu.2023.1207533] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Accepted: 06/12/2023] [Indexed: 07/28/2023] Open
Abstract
Introduction The location of T-cells during tumor progression and treatment provides crucial information in predicting the response in vivo. Methods Here, we investigated, using our bioluminescent, dual color, T-cell reporter mouse, termed TbiLuc, T-cell location and function during murine PDAC tumor growth and checkpoint blockade treatment with anti-PD-1 and anti-CTLA-4. Using this model, we could visualize T-cell location and function in the tumor and the surrounding tumor microenvironment longitudinally. We used murine PDAC clones that formed in vivo tumors with either high T-cell infiltration (immunologically 'hot') or low T-cell infiltration (immunologically 'cold'). Results Differences in total T-cell bioluminescence could be seen between the 'hot' and 'cold' tumors in the TbiLuc mice. During checkpoint blockade treatment we could see in the tumor-draining lymph nodes an increase in bioluminescence on day 7 after treatment. Conclusions In the current work, we showed that the TbiLuc mice can be used to monitor T-cell location and function during tumor growth and treatment.
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Affiliation(s)
- Roisin McMorrow
- Erasmus Medical Centre, Department of Radiology and Nuclear Medicine, Rotterdam, Netherlands
- Erasmus Medical Centre, Department of Molecular Genetics, Rotterdam, Netherlands
- Percuros BV, Leiden, Netherlands
| | - Giorgia Zambito
- Erasmus Medical Centre, Department of Radiology and Nuclear Medicine, Rotterdam, Netherlands
- Erasmus Medical Centre, Department of Molecular Genetics, Rotterdam, Netherlands
| | - Alex Nigg
- Erasmus Medical Centre, Department of Pathology, Erasmus MC Cancer Institute, Rotterdam, Netherlands
| | - Karishma Lila
- Erasmus Medical Centre, Department of Pathology, Erasmus MC Cancer Institute, Rotterdam, Netherlands
| | | | - Clemens W. G. M. Lowik
- Erasmus Medical Centre, Department of Radiology and Nuclear Medicine, Rotterdam, Netherlands
| | - Laura Mezzanotte
- Erasmus Medical Centre, Department of Radiology and Nuclear Medicine, Rotterdam, Netherlands
- Erasmus Medical Centre, Department of Molecular Genetics, Rotterdam, Netherlands
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8
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Echeverria I, Braberg H, Krogan NJ, Sali A. Integrative structure determination of histones H3 and H4 using genetic interactions. FEBS J 2023; 290:2565-2575. [PMID: 35298864 PMCID: PMC9481981 DOI: 10.1111/febs.16435] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2021] [Revised: 02/11/2022] [Accepted: 03/15/2022] [Indexed: 11/28/2022]
Abstract
Integrative structure modeling is increasingly used for determining the architectures of biological assemblies, especially those that are structurally heterogeneous. Recently, we reported on how to convert in vivo genetic interaction measurements into spatial restraints for structural modeling: first, phenotypic profiles are generated for each point mutation and thousands of gene deletions or environmental perturbations. Following, the phenotypic profile similarities are converted into distance restraints on the pairs of mutated residues. We illustrate the approach by determining the structure of the histone H3-H4 complex. The method is implemented in our open-source IMP program, expanding the structural biology toolbox by allowing structural characterization based on in vivo data without the need to purify the target system. We compare genetic interaction measurements to other sources of structural information, such as residue coevolution and deep-learning structure prediction of complex subunits. We also suggest that determining genetic interactions could benefit from new technologies, such as CRISPR-Cas9 approaches to gene editing, especially for mammalian cells. Finally, we highlight the opportunity for using genetic interactions to determine recalcitrant biomolecular structures, such as those of disordered proteins, transient protein assemblies, and host-pathogen protein complexes.
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Affiliation(s)
- Ignacia Echeverria
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
- Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94158, USA
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Hannes Braberg
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
- Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Nevan J. Krogan
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
- Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94158, USA
- Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA 94158, USA
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Andrej Sali
- Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94158, USA
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94158, USA
- Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA
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Chmelyuk NS, Oda VV, Gabashvili AN, Abakumov MA. Encapsulins: Structure, Properties, and Biotechnological Applications. BIOCHEMISTRY (MOSCOW) 2023; 88:35-49. [PMID: 37068871 PMCID: PMC9937530 DOI: 10.1134/s0006297923010042] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/19/2023]
Abstract
In 1994 a new class of prokaryotic compartments was discovered, collectively called "encapsulins" or "nanocompartments". Encapsulin shell protomer proteins self-assemble to form icosahedral structures of various diameters (24-42 nm). Inside of nanocompartments shells, one or several cargo proteins, diverse in their functions, can be encapsulated. In addition, non-native cargo proteins can be loaded into nanocompartments, and shell surfaces can be modified via various compounds, which makes it possible to create targeted drug delivery systems, labels for optical and MRI imaging, and to use encapsulins as bioreactors. This review describes a number of strategies of encapsulins application in various fields of science, including biomedicine and nanobiotechnologies.
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Affiliation(s)
- Nelly S Chmelyuk
- National University of Science and Technology "MISIS", Moscow, 119049, Russia
- Pirogov Russian National Research Medical University, Ministry of Health of the Russian Federation, Moscow, 117977, Russia
| | - Vera V Oda
- National University of Science and Technology "MISIS", Moscow, 119049, Russia
| | - Anna N Gabashvili
- National University of Science and Technology "MISIS", Moscow, 119049, Russia
| | - Maxim A Abakumov
- National University of Science and Technology "MISIS", Moscow, 119049, Russia.
- Pirogov Russian National Research Medical University, Ministry of Health of the Russian Federation, Moscow, 117977, Russia
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10
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Application of Genetically Encoded Molecular Imaging Probes in Tumor Imaging. CONTRAST MEDIA & MOLECULAR IMAGING 2022; 2022:5473244. [PMID: 36101803 PMCID: PMC9440812 DOI: 10.1155/2022/5473244] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/25/2021] [Revised: 04/05/2022] [Accepted: 07/14/2022] [Indexed: 11/17/2022]
Abstract
In recent years, imaging technology has made rapid progress to improve the sensitivity of tumor diagnostic. With the development of genetic engineering and synthetic biology, various genetically encoded molecular imaging probes have also been extensively developed. As a biomedical imaging method with excellent detectable sensitivity and spatial resolution, genetically encoded molecular imaging has great application potential in the visualization of cellular and molecular functions during tumor development. Compared to chemosynthetic dyes and nanoparticles with an imaging function, genetically encoded molecular imaging probes can more easily label specific cells or proteins of interest in tumor tissues and have higher stability and tissue contrast in vivo. Therefore, genetically encoded molecular imaging probes have attracted increasing attention from researchers in engineering and biomedicine. In this review, we aimed to introduce the genetically encoded molecular imaging probes and further explained their applications in tumor imaging.
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11
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Abstract
Molecular imaging is used to improve the disease diagnosis, prognosis, monitoring of treatment in living subjects. Numerous molecular targets have been developed for various cellular and molecular processes in genetic, metabolic, proteomic, and cellular biologic level. Molecular imaging modalities such as Optical Imaging, Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), and Computed Tomography (CT) can be used to visualize anatomic, genetic, biochemical, and physiologic changes in vivo. For in vivo cell imaging, certain cells such as cancer cells, immune cells, stem cells could be labeled by direct and indirect labeling methods to monitor cell migration, cell activity, and cell effects in cell-based therapy. In case of cancer, it could be used to investigate biological processes such as cancer metastasis and to analyze the drug treatment process. In addition, transplanted stem cells and immune cells in cell-based therapy could be visualized and tracked to confirm the fate, activity, and function of cells. In conventional molecular imaging, cells can be monitored in vivo in bulk non-invasively with optical imaging, MRI, PET, and SPECT imaging. However, single cell imaging in vivo has been a great challenge due to an extremely high sensitive detection of single cell. Recently, there has been great attention for in vivo single cell imaging due to the development of single cell study. In vivo single imaging could analyze the survival or death, movement direction, and characteristics of a single cell in live subjects. In this article, we reviewed basic principle of in vivo molecular imaging and introduced recent studies for in vivo single cell imaging based on the concept of in vivo molecular imaging.
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Affiliation(s)
- Seongje Hong
- Department of Anatomy, College of Medicine, Chung-Ang University, Seoul 06974, Korea
| | - Siyeon Rhee
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Kyung Oh Jung
- Department of Anatomy, College of Medicine, Chung-Ang University, Seoul 06974, Korea
- Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA
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12
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Hong S, Rhee S, Jung KO. In vivo molecular and single cell imaging. BMB Rep 2022; 55:267-274. [PMID: 35651326 PMCID: PMC9252890] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2022] [Revised: 04/11/2022] [Accepted: 04/29/2022] [Indexed: 02/21/2025] Open
Abstract
Molecular imaging is used to improve the disease diagnosis, prognosis, monitoring of treatment in living subjects. Numerous molecular targets have been developed for various cellular and molecular processes in genetic, metabolic, proteomic, and cellular biologic level. Molecular imaging modalities such as Optical Imaging, Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), and Computed Tomography (CT) can be used to visualize anatomic, genetic, biochemical, and physiologic changes in vivo. For in vivo cell imaging, certain cells such as cancer cells, immune cells, stem cells could be labeled by direct and indirect labeling methods to monitor cell migration, cell activity, and cell effects in cell-based therapy. In case of cancer, it could be used to investigate biological processes such as cancer metastasis and to analyze the drug treatment process. In addition, transplanted stem cells and immune cells in cell-based therapy could be visualized and tracked to confirm the fate, activity, and function of cells. In conventional molecular imaging, cells can be monitored in vivo in bulk non-invasively with optical imaging, MRI, PET, and SPECT imaging. However, single cell imaging in vivo has been a great challenge due to an extremely high sensitive detection of single cell. Recently, there has been great attention for in vivo single cell imaging due to the development of single cell study. In vivo single imaging could analyze the survival or death, movement direction, and characteristics of a single cell in live subjects. In this article, we reviewed basic principle of in vivo molecular imaging and introduced recent studies for in vivo single cell imaging based on the concept of in vivo molecular imaging. [BMB Reports 2022; 55(6): 267-274].
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Affiliation(s)
- Seongje Hong
- Department of Anatomy, College of Medicine, Chung-Ang University, Seoul 06974, Korea, CA 94305, USA
| | - Siyeon Rhee
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Kyung Oh Jung
- Department of Anatomy, College of Medicine, Chung-Ang University, Seoul 06974, Korea, CA 94305, USA
- Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA
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13
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Woong Yoo S, Young Kwon S, Kang SR, Min JJ. Molecular imaging approaches to facilitate bacteria-mediated cancer therapy. Adv Drug Deliv Rev 2022; 187:114366. [PMID: 35654213 DOI: 10.1016/j.addr.2022.114366] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2022] [Revised: 05/06/2022] [Accepted: 05/25/2022] [Indexed: 12/14/2022]
Abstract
Bacteria-mediated cancer therapy is a potential therapeutic strategy for cancer that has unique properties, including broad tumor-targeting ability, various administration routes, the flexibility of delivery, and facilitating the host's immune responses. The molecular imaging of bacteria-mediated cancer therapy allows the therapeutically injected bacteria to be visualized and confirms the accurate delivery of the therapeutic bacteria to the target lesion. Several hurdles make bacteria-specific imaging challenging, including the need to discriminate therapeutic bacterial infection from inflammation or other pathologic lesions. To realize the full potential of bacteria-specific imaging, it is necessary to develop bacteria-specific targets that can be associated with an imaging assay. This review describes the current status of bacterial imaging techniques together with the advantages and disadvantages of several imaging modalities. Also, we describe potential targets for bacterial-specific imaging and related applications.
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Affiliation(s)
- Su Woong Yoo
- Department of Nuclear Medicine, Chonnam National University Hwasun Hospital, Hwasun, Jeonnam, Korea
| | - Seong Young Kwon
- Department of Nuclear Medicine, Chonnam National University Hwasun Hospital, Hwasun, Jeonnam, Korea; Department of Nuclear Medicine, Chonnam National University Medical School, Hwasun, Jeonnam, Korea
| | - Sae-Ryung Kang
- Department of Nuclear Medicine, Chonnam National University Hwasun Hospital, Hwasun, Jeonnam, Korea
| | - Jung-Joon Min
- Department of Nuclear Medicine, Chonnam National University Hwasun Hospital, Hwasun, Jeonnam, Korea; Department of Nuclear Medicine, Chonnam National University Medical School, Hwasun, Jeonnam, Korea.
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14
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Liu T, Li Z, Li X, Zhao R, Wei X, Wang Z, Xin SX. In vivo visualization of murine melanoma cells B16-derived exosomes through magnetic resonance imaging. Biochim Biophys Acta Gen Subj 2022; 1866:130062. [PMID: 34822924 DOI: 10.1016/j.bbagen.2021.130062] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2021] [Revised: 10/22/2021] [Accepted: 11/17/2021] [Indexed: 10/19/2022]
Abstract
BACKGROUND Numerous studies demonstrated that exosomes play a powerful role in mediating intercellular communication to induce a pro-tumoral environment to promote tumor progression, including pre-metastatic niche formation and metastasis. Noninvasive imaging could determine the in vivo kinetics of exosomes in real time to provide better understanding of the mechanisms of the tumor formation, progression and metastasis. Magnetic resonance imaging (MRI) is an ideal technique which provides excellent anatomical resolution, intrinsic soft tissue contrast, unlimited penetration depth and no radiation exposure. METHODS A fusion protein composed of ferritin heavy chain (FTH1) and lactadherin was designed for visualizing exosomes through MRI. FTH1 was served as MRI reporter protein and lactadherin is a membrane-associated protein that is distributed on exosome surface. The characterizations of labeled exosomes were validated through transmission electron microscopy, western blot, nanoparticle tracking analysis and finally visualized in vitro and in vivo through MRI. RESULTS MR imaging showed that the labeled exosomes are able to be visualized in vitro and in vivo. Verification of the characterizations of exosomes observed no significant difference between labeled and unlabeled exosomes. CONCLUSION The proposed FTH1 labeling method was useful for visualizing exosomes through MRI. GENERAL SIGNIFICANCE The present study first reported a novel self-label method for imaging labeled exosomes of tumor cells in vivo through MR with cell endogenous MRI reporter protein. It may be further used as a tool to enhance understanding the role of exosomes in various pathophysiological conditions.
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Affiliation(s)
- Tianqi Liu
- School of Biomedical Engineering, Southern Medical University, Guangzhou 510515, Guangdong, China
| | - Zhenlin Li
- Department of Histology and Embryology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, Guangdong, China
| | - Xiaodong Li
- School of Medicine, South China University of Technology, Guangzhou 510006, Guangdong, China
| | - Ruiting Zhao
- School of Biomedical Engineering, Southern Medical University, Guangzhou 510515, Guangdong, China
| | - Xinhua Wei
- Department of Radiology, Guangzhou First People's Hospital, South China University of Technology, Guangzhou 510180, Guangdong, China
| | - Zixin Wang
- School of Electronics and Information Technology, Sun Yat-Sen University, Xingang Xi Road 135, Guangzhou 510275, Guangdong, China
| | - Sherman Xuegang Xin
- School of Biomedical Engineering, Southern Medical University, Guangzhou 510515, Guangdong, China; School of Medicine, South China University of Technology, Guangzhou 510006, Guangdong, China.
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15
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Hu Y, Lv S, Wan J, Zheng C, Shao D, Wang H, Tao Y, Li M, Luo Y. Recent advances in nanomaterials for prostate cancer detection and diagnosis. J Mater Chem B 2022; 10:4907-4934. [PMID: 35712990 DOI: 10.1039/d2tb00448h] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Despite the significant progress in the discovery of biomarkers and the exploitation of technologies for prostate cancer (PCa) detection and diagnosis, the initial screening of these PCa-related biomarkers using current...
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Affiliation(s)
- Yongwei Hu
- Laboratory of Biomaterials and Translational Medicine, Center for Nanomedicine, Department of Urology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China.
| | - Shixian Lv
- School of Materials Science and Engineering, Peking University, Beijing 100871, China
| | - Jiaming Wan
- Laboratory of Biomaterials and Translational Medicine, Center for Nanomedicine, Department of Urology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China.
| | - Chunxiong Zheng
- Laboratory of Biomaterials and Translational Medicine, Center for Nanomedicine, Department of Urology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China.
| | - Dan Shao
- Institutes of Life Sciences, School of Medicine, South China University of Technology, Guangzhou 510006, China
| | - Haixia Wang
- Laboratory of Biomaterials and Translational Medicine, Center for Nanomedicine, Department of Urology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China.
| | - Yu Tao
- Laboratory of Biomaterials and Translational Medicine, Center for Nanomedicine, Department of Urology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China.
| | - Mingqiang Li
- Laboratory of Biomaterials and Translational Medicine, Center for Nanomedicine, Department of Urology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China.
- Guangdong Provincial Key Laboratory of Liver Disease, Guangzhou 510630, China
| | - Yun Luo
- Laboratory of Biomaterials and Translational Medicine, Center for Nanomedicine, Department of Urology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China.
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16
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Liu S, Su Y, Lin MZ, Ronald JA. Brightening up Biology: Advances in Luciferase Systems for in Vivo Imaging. ACS Chem Biol 2021; 16:2707-2718. [PMID: 34780699 PMCID: PMC8689642 DOI: 10.1021/acschembio.1c00549] [Citation(s) in RCA: 54] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
![]()
Bioluminescence imaging
(BLI) using luciferase reporters is an
indispensable method for the noninvasive visualization of cell populations
and biochemical events in living animals. BLI is widely performed
with preclinical rodent models to understand disease processes and
evaluate potential cell- or gene-based therapies. However, in vivo BLI remains constrained by low photon production
and tissue attenuation, limiting the sensitivity of reporting from
small numbers of cells in deep locations and hindering its application
to larger animal models. This Review highlights recent advances in
the development of luciferase systems that improve the sensitivity
of in vivo BLI and discusses the expanding array
of biological applications.
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Affiliation(s)
- Shirley Liu
- Robarts Research Institute, The University of Western Ontario, London, Ontario N6A3K7, Canada
- Department of Medical Biophysics, The University of Western Ontario, London, Ontario N6A3K7, Canada
| | - Yichi Su
- Department of Neurobiology, Stanford University, Stanford, California 94305, United States
- Department of Bioengineering, Stanford University, Stanford, California 94305, United States
| | - Michael Z. Lin
- Department of Neurobiology, Stanford University, Stanford, California 94305, United States
- Department of Bioengineering, Stanford University, Stanford, California 94305, United States
| | - John A. Ronald
- Robarts Research Institute, The University of Western Ontario, London, Ontario N6A3K7, Canada
- Department of Medical Biophysics, The University of Western Ontario, London, Ontario N6A3K7, Canada
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17
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Liu S, Nyström NN, Kelly JJ, Hamilton AM, Fu Y, Ronald JA. Molecular Imaging Reveals a High Degree of Cross-Seeding of Spontaneous Metastases in a Novel Mouse Model of Synchronous Bilateral Breast Cancer. Mol Imaging Biol 2021; 24:104-114. [PMID: 34312806 PMCID: PMC8760205 DOI: 10.1007/s11307-021-01630-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Revised: 05/18/2021] [Accepted: 07/05/2021] [Indexed: 11/16/2022]
Abstract
Purpose Synchronous bilateral breast cancer (SBBC) patients present with cancer in both breasts at the time of diagnosis or within a short time interval. They show higher rates of metastasis and lower overall survival compared to women with unilateral breast cancer. Here we established the first preclinical SBBC model and used molecular imaging to visualize the patterns of metastasis from each primary tumor. Procedures We engineered human breast cancer cells to express either Akaluc or Antares2 for bioluminescence imaging (BLI) and tdTomato or zsGreen for ex vivo fluorescence microscopy. Both cell populations were implanted into contralateral mammary fat pads of mice (n=10), and dual-BLI was performed weekly for up to day 29 (n=3), 38 (n=4), or 42 (n=3). Primary tumors and lungs were fixed, and ex vivo fluorescence microscopy was used to analyze the cellular makeup of micrometastases. Results Signal from both Antares2 and Akaluc was first detected in the lungs on day 28 and was present in 9 of 10 mice at endpoint. Ex vivo fluorescence microscopy of the lungs revealed that for mice sacrificed on day 38, a significant percentage of micrometastases were composed of cancer cells from both primary tumors (mean 37%; range 27 to 45%), while two mice sacrificed on day 42 showed percentages of 51% and 70%. Conclusions A high degree of metastatic cross-seeding of cancer cells derived from bilateral tumors may contribute to faster metastatic growth and intratumoral heterogeneity. We posit that our work will help understand treatment resistance and optimal planning of SBBC treatment. Supplementary Information The online version contains supplementary material available at 10.1007/s11307-021-01630-z.
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Affiliation(s)
- Shirley Liu
- Robarts Research Institute, University of Western Ontario, London, ON, Canada
- Department of Medical Biophysics, University of Western Ontario, London, ON, Canada
| | - Nivin N Nyström
- Robarts Research Institute, University of Western Ontario, London, ON, Canada
- Department of Medical Biophysics, University of Western Ontario, London, ON, Canada
| | - John J Kelly
- Robarts Research Institute, University of Western Ontario, London, ON, Canada
| | - Amanda M Hamilton
- Robarts Research Institute, University of Western Ontario, London, ON, Canada
| | - Yanghao Fu
- Robarts Research Institute, University of Western Ontario, London, ON, Canada
- Department of Medical Biophysics, University of Western Ontario, London, ON, Canada
| | - John A Ronald
- Robarts Research Institute, University of Western Ontario, London, ON, Canada.
- Department of Medical Biophysics, University of Western Ontario, London, ON, Canada.
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18
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Abstract
Apoptosis is a process in which cells are genetically regulated to cause a series of changes in morphology and metabolic activity, which ultimately lead to cell death. Apoptosis plays a vital role in the entire life cycle of an organism. Too much or too little apoptosis can cause a variety of diseases. Therefore, efficient and convenient methods for detecting apoptosis are necessary for clinical treatment and drug development. Traditional methods for detecting apoptosis may cause damage to the body during sample collection, such as for flow cytometry analysis. So it is necessary to monitor apoptosis without invasion in vivo. Optical imaging technique provides a more sensitive and economical way for apoptosis visualization. A subset of engineered reporter genes based on fluorescent proteins or luciferases are currently developed to monitor the dynamic changes in apoptotic markers, such as activation of caspases and exposure of phosphatidylserine on the surface of dying cells. These reporters detect apoptosis when cells have not undergone significant morphological changes, providing conditions for early diagnosis of tumors. In addition, these reporters show considerable value in high-throughput screening of apoptosis-related drugs and evaluation of their efficacy in treating tumors. In this review, we will discuss the recent research progress in the optical imaging of apoptosis based on the genetically encoded reporter genes.
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19
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Lonser RR, Akhter AS, Zabek M, Elder JB, Bankiewicz KS. Direct convective delivery of adeno-associated virus gene therapy for treatment of neurological disorders. J Neurosurg 2021; 134:1751-1763. [PMID: 32915526 DOI: 10.3171/2020.4.jns20701] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2020] [Accepted: 04/16/2020] [Indexed: 11/06/2022]
Abstract
Molecular biological insights have led to a fundamental understanding of the underlying genomic mechanisms of nervous system disease. These findings have resulted in the identification of therapeutic genes that can be packaged in viral capsids for the treatment of a variety of neurological conditions, including neurodegenerative, metabolic, and enzyme deficiency disorders. Recent data have demonstrated that gene-carrying viral vectors (most often adeno-associated viruses) can be effectively distributed by convection-enhanced delivery (CED) in a safe, reliable, targeted, and homogeneous manner across the blood-brain barrier. Critically, these vectors can be monitored using real-time MRI of a co-infused surrogate tracer to accurately predict vector distribution and transgene expression at the perfused site. The unique properties of CED of adeno-associated virus vectors allow for cell-specific transgene manipulation of the infused anatomical site and/or widespread interconnected sites via antero- and/or retrograde transport. The authors review the convective properties of viral vectors, associated technology, and clinical applications.
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Affiliation(s)
- Russell R Lonser
- 1Department of Neurological Surgery, The Ohio State University Wexner Medical Center, Columbus, Ohio; and
| | - Asad S Akhter
- 1Department of Neurological Surgery, The Ohio State University Wexner Medical Center, Columbus, Ohio; and
| | - Mirosław Zabek
- 2Department of Neurological Surgery, Bródno Hospital, Warsaw, Poland
| | - J Bradley Elder
- 1Department of Neurological Surgery, The Ohio State University Wexner Medical Center, Columbus, Ohio; and
| | - Krystof S Bankiewicz
- 1Department of Neurological Surgery, The Ohio State University Wexner Medical Center, Columbus, Ohio; and
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20
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Shao F, Long Y, Ji H, Jiang D, Lei P, Lan X. Radionuclide-based molecular imaging allows CAR-T cellular visualization and therapeutic monitoring. Am J Cancer Res 2021; 11:6800-6817. [PMID: 34093854 PMCID: PMC8171102 DOI: 10.7150/thno.56989] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2020] [Accepted: 04/20/2021] [Indexed: 02/07/2023] Open
Abstract
Chimeric antigen receptor T cell (CAR-T) therapy is a new and effective form of adoptive cell therapy that is rapidly entering the mainstream for the treatment of CD19-positive hematological cancers because of its impressive effect and durable responses. Huge challenges remain in achieving similar success in patients with solid tumors. The current methods of monitoring CAR-T, including morphological imaging (CT and MRI), blood tests, and biopsy, have limitations to assess whether CAR-T cells are homing to tumor sites and infiltrating into tumor bed, or to assess the survival, proliferation, and persistence of CAR-T cells in solid tumors associated with an immunosuppressive microenvironment. Radionuclide-based molecular imaging affords improved CAR-T cellular visualization and therapeutic monitoring through either a direct cellular radiolabeling approach or a reporter gene imaging strategy, and endogenous cell imaging is beneficial to reflect functional information and immune status of T cells. Focusing on the dynamic monitoring and precise assessment of CAR-T therapy, this review summarizes the current applications of radionuclide-based noninvasive imaging in CAR-T cells visualization and monitoring and presents current challenges and strategic choices.
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21
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Kelly JJ, Saee-Marand M, Nyström NN, Evans MM, Chen Y, Martinez FM, Hamilton AM, Ronald JA. Safe harbor-targeted CRISPR-Cas9 homology-independent targeted integration for multimodality reporter gene-based cell tracking. SCIENCE ADVANCES 2021; 7:eabc3791. [PMID: 33523917 PMCID: PMC7817109 DOI: 10.1126/sciadv.abc3791] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/22/2020] [Accepted: 11/25/2020] [Indexed: 05/12/2023]
Abstract
Imaging reporter genes provides longitudinal information on the biodistribution, growth, and survival of engineered cells in vivo. A translational bottleneck to using reporter genes is the necessity to engineer cells with randomly integrating vectors. Here, we built homology-independent targeted integration (HITI) CRISPR-Cas9 minicircle donors for precise safe harbor-targeted knock-in of fluorescence, bioluminescence, and MRI (Oatp1a1) reporter genes. Our results showed greater knock-in efficiency using HITI vectors compared to homology-directed repair vectors. HITI clones demonstrated functional fluorescence and bioluminescence reporter activity as well as significant Oatp1a1-mediated uptake of the clinically approved MRI agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. Contrast-enhanced MRI improved the conspicuity of both subcutaneous and metastatic Oatp1a1-expressing tumors before they became palpable or even readily visible on precontrast images. Our work demonstrates the first CRISPR-Cas9 HITI system for knock-in of large DNA donor constructs at a safe harbor locus, enabling multimodal longitudinal in vivo imaging of cells.
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Affiliation(s)
- John J Kelly
- Robarts Research Institute, University of Western Ontario, London, Ontario, Canada
- Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada
| | - Moe Saee-Marand
- Robarts Research Institute, University of Western Ontario, London, Ontario, Canada
| | - Nivin N Nyström
- Robarts Research Institute, University of Western Ontario, London, Ontario, Canada
- Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada
| | - Melissa M Evans
- Robarts Research Institute, University of Western Ontario, London, Ontario, Canada
| | - Yuanxin Chen
- Robarts Research Institute, University of Western Ontario, London, Ontario, Canada
| | - Francisco M Martinez
- Robarts Research Institute, University of Western Ontario, London, Ontario, Canada
| | - Amanda M Hamilton
- Robarts Research Institute, University of Western Ontario, London, Ontario, Canada
| | - John A Ronald
- Robarts Research Institute, University of Western Ontario, London, Ontario, Canada.
- Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada
- Lawson Health Research Institute, London, Ontario, Canada
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22
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Phonbuppha J, Tinikul R, Chaiyen P. Use of Bacterial Luciferase as a Reporter Gene in Eukaryotic Systems. Methods Mol Biol 2021; 2274:53-65. [PMID: 34050462 DOI: 10.1007/978-1-0716-1258-3_6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/25/2023]
Abstract
Reporter gene assays are powerful tools for monitoring dynamic molecular changes and for evaluating the responses that occur at the genetic elements within cells in response to exogenous molecules. In general, various protein systems can be used as reporter genes, including luciferases. Here, the present protocol introduces a unique reporter gene system for monitoring molecular events in cells using bacterial luciferase (lux), which can generate blue-green light suitable for gene reporter applications with the highest cost performance. The protocol also guides the assay conditions and necessary components for using of lux gene (lux) as a eukaryotic reporter system. The lux system can be applied to monitor variety of molecular events inside mammalian cellular systems.
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Affiliation(s)
- Jittima Phonbuppha
- School of Biomolecular Science & Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, Thailand
| | - Ruchanok Tinikul
- Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok, Thailand
| | - Pimchai Chaiyen
- School of Biomolecular Science & Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, Thailand.
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23
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Jacobs AH, Schelhaas S, Viel T, Waerzeggers Y, Winkeler A, Zinnhardt B, Gelovani J. Imaging of Gene and Cell-Based Therapies: Basis and Clinical Trials. Mol Imaging 2021. [DOI: 10.1016/b978-0-12-816386-3.00060-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022] Open
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24
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Molecular Imaging of Gene Therapy. Mol Imaging 2021. [DOI: 10.1016/b978-0-12-816386-3.00064-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
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25
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Chandy M, Wu JC. Molecular Imaging of Stem Cell Therapy in Ischemic Cardiomyopathy. Mol Imaging 2021. [DOI: 10.1016/b978-0-12-816386-3.00065-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
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26
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Rajendran RL, Jogalekar MP, Gangadaran P, Ahn BC. Noninvasive in vivo cell tracking using molecular imaging: A useful tool for developing mesenchymal stem cell-based cancer treatment. World J Stem Cells 2020; 12:1492-1510. [PMID: 33505597 PMCID: PMC7789123 DOI: 10.4252/wjsc.v12.i12.1492] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Revised: 10/05/2020] [Accepted: 10/21/2020] [Indexed: 02/06/2023] Open
Abstract
Mounting evidence has emphasized the potential of cell therapies in treating various diseases by restoring damaged tissues or replacing defective cells in the body. Cell therapies have become a strong therapeutic modality by applying noninvasive in vivo molecular imaging for examining complex cellular processes, understanding pathophysiological mechanisms of diseases, and evaluating the kinetics/dynamics of cell therapies. In particular, mesenchymal stem cells (MSCs) have shown promise in recent years as drug carriers for cancer treatment. They can also be labeled with different probes and tracked in vivo to assess the in vivo effect of administered cells, and to optimize therapy. The exact role of MSCs in oncologic diseases is not clear as MSCs have been shown to be involved in tumor progression and inhibition, and the exact interactions between MSCs and specific cancer microenvironments are not clear. In this review, a multitude of labeling approaches, imaging modalities, and the merits/demerits of each strategy are outlined. In addition, specific examples of the use of MSCs and in vivo imaging in cancer therapy are provided. Finally, present limitations and future outlooks in terms of the translation of different imaging approaches in clinics are discussed.
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Affiliation(s)
| | | | - Prakash Gangadaran
- Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu 41944, South Korea
- BK21 Plus KNU Biomedical Convergence Program, Department of Biomedical Science, School of Medicine, Kyungpook National University, Daegu 41944, South Korea
| | - Byeong-Cheol Ahn
- BK21 Plus KNU Biomedical Convergence Program, Department of Biomedical Science, School of Medicine, Kyungpook National University, Daegu 41944, South Korea
- Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, Daegu 41944, South Korea.
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27
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Identification of Lymphatic and Hematogenous Routes of Rapidly Labeled Radioactive and Fluorescent Exosomes through Highly Sensitive Multimodal Imaging. Int J Mol Sci 2020; 21:ijms21217850. [PMID: 33105908 PMCID: PMC7660226 DOI: 10.3390/ijms21217850] [Citation(s) in RCA: 35] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2020] [Revised: 10/09/2020] [Accepted: 10/19/2020] [Indexed: 12/14/2022] Open
Abstract
There has been considerable interest in the clinical use of exosomes as delivery vehicles for treatments as well as for promising diagnostic biomarkers, but the physiological distribution of exosomes must be further elucidated to validate their efficacy and safety. Here, we aimed to develop novel methods to monitor exosome biodistribution in vivo using positron emission tomography (PET) and optical imaging. Exosomes were isolated from cultured mouse breast cancer cells and labeled for PET and optical imaging. In mice, radiolabeled and fluorescently labeled exosomes were injected both via lymphatic and hematogenous metastatic routes. PET and fluorescence images were obtained and quantified. Radioactivity and fluorescence intensity of ex vivo organs were measured. PET signals from exosomes in the lymphatic metastatic route were observed in the draining sentinel lymph nodes. Immunohistochemistry revealed greater exosome uptake in brachial and axillary versus inguinal lymph nodes. Following administration through the hematogenous metastasis pathway, accumulation of exosomes was clearly observed in the lungs, liver, and spleen. Exosomes from tumor cells were successfully labeled with 64Cu (or 68Ga) and fluorescence and were visualized via PET and optical imaging, suggesting that this simultaneous and rapid labeling method could provide valuable information for further exosome translational research and clinical applications.
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28
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Song Y, Xu Z, Wang F. Genetically Encoded Reporter Genes for MicroRNA Imaging in Living Cells and Animals. MOLECULAR THERAPY. NUCLEIC ACIDS 2020; 21:555-567. [PMID: 32721876 PMCID: PMC7390858 DOI: 10.1016/j.omtn.2020.06.021] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/23/2020] [Revised: 06/12/2020] [Accepted: 06/24/2020] [Indexed: 12/13/2022]
Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by base paring with the complementary sequences of the target mRNAs, and then exert their function through degrading mRNA or inhibiting protein translation. They play a significant role as a regulatory factor in biological processes of organism development, cell proliferation, differentiation, and cell death. Some of the traditional methods for studying miRNAs, such as northern blot, real-time PCR, or microarray, have been extensively used to investigate the biological properties and expression patterns of miRNAs. However, these methods often require considerable time, cell samples, and the design of effective primers or specific probes. Therefore, in order to gain a deeper understanding of the role of miRNAs in biological processes and accelerate the clinical application of miRNAs in the field of disease treatment, non-invasive, sensitive, and efficient imaging methods are needed to visualize the dynamic expression of miRNAs in living cells and animals. In this study, we reviewed the recent progress in the genetically encoded reporter genes for miRNA imaging.
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Affiliation(s)
- Yingzhuang Song
- Institute of Medical Research, Northwestern Polytechnical University, Xi'an, Shaanxi 710072, China
| | - Zhijing Xu
- Institute of Medical Research, Northwestern Polytechnical University, Xi'an, Shaanxi 710072, China
| | - Fu Wang
- Institute of Medical Research, Northwestern Polytechnical University, Xi'an, Shaanxi 710072, China; Engineering Research Center of Molecular and Neuro Imaging, Ministry of Education, School of Life Science and Technology, Xidian University, Xi'an, Shaanxi 710071, China.
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29
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Lee SB, Park H, Lee JE, Kim KS, Jeon YH. In Vivo Optical Reporter-Gene-Based Imaging of Macrophage Infiltration of DNCB-Induced Atopic Dermatitis. Int J Mol Sci 2020; 21:ijms21176205. [PMID: 32867320 PMCID: PMC7503337 DOI: 10.3390/ijms21176205] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2020] [Revised: 08/24/2020] [Accepted: 08/25/2020] [Indexed: 11/16/2022] Open
Abstract
This study was conducted to monitor the macrophage infiltration of atopic dermatitis (AD)-like skin lesions and to evaluate the effects of anti-AD therapeutic agents in immunocompetent mice via optical reporter-gene-based molecular imaging. The enhanced firefly luciferase (effluc)-expressing macrophage cell line (Raw264.7/effluc) was intravenously introduced into mice with 2,4-dinitrochlorobenzene (DNCB)-induced AD, followed by bioluminescent imaging (BLI). After in vivo imaging, AD-like skin lesions were excised, and ex vivo imaging and Western blotting were conducted to determine the presence of infused macrophages. Finally, the therapeutic effect of dexamethasone (DEX), an AD-modulating agent, was evaluated via macrophage tracking. In vivo imaging with BLI revealed the migration of the reporter macrophages to DNCB-induced AD-like skin lesions on day 1 post-transfer. The greatest recruitment was observed on day 3, and a decline in BLI signal was observed on day 14. Notably, in vivo BLI clearly showed the inhibition of the reporter macrophage infiltration of DNCB-induced AD-like skin lesions by DEX, which was consistent with the reduced AD symptoms observed in DEX-treated mice. We successfully visualized the macrophage migration to DNCB-induced AD-like skin lesions, proving the feasibility of macrophage imaging for evaluating AD-regulating drugs in living organisms.
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Affiliation(s)
- Sang Bong Lee
- Korea Institute of Medical Microrobotics (KIMIRo), Gwangju 61011, Korea;
| | - Hyeonsoo Park
- Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 700-721, Korea; (H.P.); (J.-E.L.); (K.-S.K.)
- Research Center of Stickus Corporation, Haeundae-gu jaesong-dong 1050-21, Busan 48054, Korea
| | - Jae-Eon Lee
- Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 700-721, Korea; (H.P.); (J.-E.L.); (K.-S.K.)
- Department of Biomaterials Science, College of Natural Resources and Life Science/Life and Industry Convergence Research Institute, Pusan National University, Pusan 50463, Korea
| | - Kil-Soo Kim
- Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 700-721, Korea; (H.P.); (J.-E.L.); (K.-S.K.)
- College of Veterinary Medicine, Kyungpook National University, Daegu 700-721, Korea
| | - Yong Hyun Jeon
- Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 700-721, Korea; (H.P.); (J.-E.L.); (K.-S.K.)
- Leading-Edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University Hospital, Daegu 700-721, Korea
- Correspondence: ; Tel.: +82-53-790-5726
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30
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Pauwels E, Van Aerde M, Bormans G, Deroose CM. Molecular imaging of norepinephrine transporter-expressing tumors: current status and future prospects. THE QUARTERLY JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING : OFFICIAL PUBLICATION OF THE ITALIAN ASSOCIATION OF NUCLEAR MEDICINE (AIMN) [AND] THE INTERNATIONAL ASSOCIATION OF RADIOPHARMACOLOGY (IAR), [AND] SECTION OF THE SOCIETY OF RADIOPHARMACEUTICAL CHEMISTRY AND BIOLOGY 2020; 64:234-249. [PMID: 32397701 DOI: 10.23736/s1824-4785.20.03261-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
The human norepinephrine transporter (hNET) is a transmembrane protein responsible for reuptake of norepinephrine in presynaptic sympathetic nerve terminals and adrenal chromaffin cells. Neural crest tumors, such as neuroblastoma, paraganglioma and pheochromocytoma often show high hNET expression. Molecular imaging of these tumors can be done using radiolabeled norepinephrine analogs that target hNET. Currently, the most commonly used radiopharmaceutical for hNET imaging is meta-[123I]iodobenzylguanidine ([123I]MIBG) and this has been the case since its development several decades ago. The γ-emitter, iodine-123 only allows for planar scintigraphy and single photon emission computed tomography imaging. These modalities typically have a poorer spatial resolution and lower sensitivity than positron emission tomography (PET). Additional practical disadvantages include the fact that a two-day imaging protocol is required and the need for thyroid blockade. Therefore, several PET alternatives for hNET imaging are actively being explored. This review gives an in-depth overview of the current status and recent developments in clinical trials leading to the next generation of clinical PET ligands for imaging of hNET-expressing tumors.
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Affiliation(s)
- Elin Pauwels
- Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, University Hospitals Leuven, Leuven, Belgium.,Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, KU Leuven, Belgium
| | - Matthias Van Aerde
- Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, University Hospitals Leuven, Leuven, Belgium.,Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, KU Leuven, Belgium
| | - Guy Bormans
- Radiopharmaceutical Research, Department of Pharmacy and Pharmacology, KU Leuven, Leuven, Belgium
| | - Christophe M Deroose
- Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, University Hospitals Leuven, Leuven, Belgium - .,Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, KU Leuven, Belgium
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31
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Tunç E. Biyolüminesans ışıma ve biyolüminesans görüntüleme tekniklerinin moleküler biyoloji araştırmaları bakımından önemi. CUKUROVA MEDICAL JOURNAL 2019. [DOI: 10.17826/cumj.535811] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022] Open
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Abstract
Regenerative medicine with the use of stem cells has appeared as a potential therapeutic alternative for many disease states. Despite initial enthusiasm, there has been relatively slow transition to clinical trials. In large part, numerous questions remain regarding the viability, biology and efficacy of transplanted stem cells in the living subject. The critical issues highlighted the importance of developing tools to assess these questions. Advances in molecular biology and imaging have allowed the successful non-invasive monitoring of transplanted stem cells in the living subject. Over the years these methodologies have been updated to assess not only the viability but also the biology of transplanted stem cells. In this review, different imaging strategies to study the viability and biology of transplanted stem cells are presented. Use of these strategies will be critical as the different regenerative therapies are being tested for clinical use.
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Affiliation(s)
- Fakhar Abbas
- Department of Cardiovascular Medicine, Mayo Clinic, Rochester, MN, USA
| | - Joseph C. Wu
- Molecular Imaging Program at Stanford, Stanford University, Stanford, CA, USA
- Department of Medicine (Cardiology), Stanford University, Stanford, CA, USA
| | - Sanjiv Sam Gambhir
- Molecular Imaging Program at Stanford, Stanford University, Stanford, CA, USA
- Department of Bio-Engineering, Stanford University, Stanford, CA, USA
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33
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Zhao X, Long J, Liang F, Liu N, Sun Y, Xi Y. Dynamic profiles, biodistribution and integration evaluation after intramuscular/intravenous delivery of a novel therapeutic DNA vaccine encoding chicken type II collagen for rheumatoid arthritis in vaccinated normal rodent. J Nanobiotechnology 2019; 17:94. [PMID: 31492169 PMCID: PMC6729025 DOI: 10.1186/s12951-019-0528-5] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2019] [Accepted: 08/28/2019] [Indexed: 01/23/2023] Open
Abstract
BACKGROUND The persistence, biodistribution, and risk of integration into the host genome of any new therapeutic DNA vaccine must be established in preclinical studies. We previously developed the DNA vaccine pcDNA-CCOL2A1 encoding chicken type II collagen (CCII) for the treatment of rheumatoid arthritis (RA). In the present study, we characterized its dynamic profile, biodistribution, and potential for genomic DNA integration in normal vaccinated rodent. RESULTS A real-time quantitative PCR analysis (RT-qPCR) of animals administered a single dose of pcDNA-CCOL2A1 (300 μg/kg by intramuscular injection) showed that CCOL2A1 mRNA level in the blood peaked between 2 and 6 h post-immunization and then rapidly declined, and was undetectable between day 1-42. CCOL2A1 transcript was detected at the muscle injection site on days 3-14 post-immunization. Starting from day 14, the transcript was detected in the heart, liver, lung, and kidney but not in the spleen or thymus, and was expressed only in the lung on day 28. There was no CCOL2A1 mRNA present in the testes or ovaries at any time point. Non-invasive in vivo fluorescence imaging revealed CCII protein expression from 2 h up to day 10 and from 2 h up to day 35 after administration of pcDNA-CCOL2A1 via the intravenous and intramuscular routes, respectively; the protein had disappeared by day 42. Importantly, CCOL2A1 was not integrated into the host genome. CONCLUSIONS These results indicate that pcDNA-CCOL2A1 vaccine is rapidly cleared within a short period of time and is therefore safe, and merits further development as a therapeutic vaccine for RA treatment.
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Affiliation(s)
- Xiao Zhao
- Department of Immunology and National Center for Biomedicine Analysis, the Fifth Medical Center (formerly known as Beijing 307 Hospital), Chinese PLA General Hospital, No. 8, Dongda Ave, Fengtai District, Beijing, 100071, People's Republic of China
| | - Juan Long
- Department of Immunology and National Center for Biomedicine Analysis, the Fifth Medical Center (formerly known as Beijing 307 Hospital), Chinese PLA General Hospital, No. 8, Dongda Ave, Fengtai District, Beijing, 100071, People's Republic of China
| | - Fei Liang
- Department of Immunology and National Center for Biomedicine Analysis, the Fifth Medical Center (formerly known as Beijing 307 Hospital), Chinese PLA General Hospital, No. 8, Dongda Ave, Fengtai District, Beijing, 100071, People's Republic of China
| | - Nan Liu
- Department of Immunology and National Center for Biomedicine Analysis, the Fifth Medical Center (formerly known as Beijing 307 Hospital), Chinese PLA General Hospital, No. 8, Dongda Ave, Fengtai District, Beijing, 100071, People's Republic of China
| | - Yuying Sun
- Department of Immunology and National Center for Biomedicine Analysis, the Fifth Medical Center (formerly known as Beijing 307 Hospital), Chinese PLA General Hospital, No. 8, Dongda Ave, Fengtai District, Beijing, 100071, People's Republic of China
| | - Yongzhi Xi
- Department of Immunology and National Center for Biomedicine Analysis, the Fifth Medical Center (formerly known as Beijing 307 Hospital), Chinese PLA General Hospital, No. 8, Dongda Ave, Fengtai District, Beijing, 100071, People's Republic of China.
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Zheng N, Su P, Liu Y, Wang H, Nie B, Fang X, Xu Y, Lin K, Lv P, He X, Guo Y, Shan B, Manyande A, Wang J, Xu F. Detection of neural connections with ex vivo MRI using a ferritin-encoding trans-synaptic virus. Neuroimage 2019; 197:133-142. [DOI: 10.1016/j.neuroimage.2019.04.039] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Revised: 03/06/2019] [Accepted: 04/11/2019] [Indexed: 12/11/2022] Open
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35
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Liu SW, Hsu CH, Chen MR, Chiu IM, Lin KM. A Tri-fusion Reporter Mouse Reveals Tissue-Specific FGF1B Promoter Activity in vivo. Sci Rep 2019; 9:11143. [PMID: 31367001 PMCID: PMC6668445 DOI: 10.1038/s41598-019-47641-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2019] [Accepted: 07/18/2019] [Indexed: 01/15/2023] Open
Abstract
Transgenic mice harboring imaging reporters take full advantage of imaging technologies in studies using living mice. Here, we established a tri-fusion multimodal reporter gene containing fragments from firefly luciferase, enhanced green fluorescent protein, and herpes simplex virus type 1 thymidine kinase and generated tri-fusion reporter Tg mice. Fibroblast growth factor type 1 (FGF1), a multifunctional mitogen to a wide range of tissues, regulates proliferation of neural stem cells of the brain, where FGF1 expression is initiated through activation of the FGF1B (F1B) promoter. The reporter mouse under the control of the human F1B promoter enables visualization in vivo where F1B activity is elevated, including tissues not only in the brain but also in the nasopharynx, skull, spine, and testes, particularly in Leydig cells. Treating Tg mice with the alkylating agent busulfan, which is known to eradicate Leydig cells and disrupt spermatogenesis in mice, eliminated the reporter signals. Restoring Leydig cells recovered reporter expression, indicating that the reporter can be used as a surrogate marker for Leydig cells. The F1B tri-fusion reporter mouse model can be utilized in longitudinal monitoring of the health status of the male reproductive system, such as in studies exploring the toxicity of chemicals to spermatogenesis.
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Affiliation(s)
- Shan-Wen Liu
- Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan.,Department of Biomedical Engineering and Environmental Science, National Tsing-Hua University, Hsinchu, Taiwan
| | - Ching-Han Hsu
- Department of Biomedical Engineering and Environmental Science, National Tsing-Hua University, Hsinchu, Taiwan
| | - Mei-Ru Chen
- Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan
| | - Ing-Ming Chiu
- Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan
| | - Kurt M Lin
- Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan. .,Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan.
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36
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Liu Y, Bhattarai P, Dai Z, Chen X. Photothermal therapy and photoacoustic imaging via nanotheranostics in fighting cancer. Chem Soc Rev 2019; 48:2053-2108. [PMID: 30259015 PMCID: PMC6437026 DOI: 10.1039/c8cs00618k] [Citation(s) in RCA: 1768] [Impact Index Per Article: 294.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The nonradiative conversion of light energy into heat (photothermal therapy, PTT) or sound energy (photoacoustic imaging, PAI) has been intensively investigated for the treatment and diagnosis of cancer, respectively. By taking advantage of nanocarriers, both imaging and therapeutic functions together with enhanced tumour accumulation have been thoroughly studied to improve the pre-clinical efficiency of PAI and PTT. In this review, we first summarize the development of inorganic and organic nano photothermal transduction agents (PTAs) and strategies for improving the PTT outcomes, including applying appropriate laser dosage, guiding the treatment via imaging techniques, developing PTAs with absorption in the second NIR window, increasing photothermal conversion efficiency (PCE), and also increasing the accumulation of PTAs in tumours. Second, we introduce the advantages of combining PTT with other therapies in cancer treatment. Third, the emerging applications of PAI in cancer-related research are exemplified. Finally, the perspectives and challenges of PTT and PAI for combating cancer, especially regarding their clinical translation, are discussed. We believe that PTT and PAI having noteworthy features would become promising next-generation non-invasive cancer theranostic techniques and improve our ability to combat cancers.
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Affiliation(s)
- Yijing Liu
- Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA
| | - Pravin Bhattarai
- Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China
| | - Zhifei Dai
- Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China
| | - Xiaoyuan Chen
- Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA
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37
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Chefer S, Seidel J, Cockrell AS, Yount B, Solomon J, Hagen KR, Liu DX, Huzella LM, Kumar MR, Postnikova E, Bohannon JK, Lackemeyer MG, Cooper K, Endlich-Frazier A, Sharma H, Thomasson D, Bartos C, Sayre PJ, Sims A, Dyall J, Holbrook MR, Jahrling PB, Baric RS, Johnson RF. The Human Sodium Iodide Symporter as a Reporter Gene for Studying Middle East Respiratory Syndrome Coronavirus Pathogenesis. mSphere 2018; 3:e00540-18. [PMID: 30541777 PMCID: PMC6291621 DOI: 10.1128/msphere.00540-18] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2018] [Accepted: 10/26/2018] [Indexed: 12/12/2022] Open
Abstract
Single photon emission computed tomography (SPECT) is frequently used in oncology and cardiology to evaluate disease progression and/or treatment efficacy. Such technology allows for real-time evaluation of disease progression and when applied to studying infectious diseases may provide insight into pathogenesis. Insertion of a SPECT-compatible reporter gene into a virus may provide insight into mechanisms of pathogenesis and viral tropism. The human sodium iodide symporter (hNIS), a SPECT and positron emission tomography reporter gene, was inserted into Middle East respiratory syndrome coronavirus (MERS-CoV), a recently emerged virus that can cause severe respiratory disease and death in afflicted humans to obtain a quantifiable and sensitive marker for viral replication to further MERS-CoV animal model development. The recombinant virus was evaluated for fitness, stability, and reporter gene functionality. The recombinant and parental viruses demonstrated equal fitness in terms of peak titer and replication kinetics, were stable for up to six in vitro passages, and were functional. Further in vivo evaluation indicated variable stability, but resolution limits hampered in vivo functional evaluation. These data support the further development of hNIS for monitoring infection in animal models of viral disease.IMPORTANCE Advanced medical imaging such as single photon emission computed tomography with computed tomography (SPECT/CT) enhances fields such as oncology and cardiology. Application of SPECT/CT, magnetic resonance imaging, and positron emission tomography to infectious disease may enhance pathogenesis studies and provide alternate biomarkers of disease progression. The experiments described in this article focus on insertion of a SPECT/CT-compatible reporter gene into MERS-CoV to demonstrate that a functional SPECT/CT reporter gene can be inserted into a virus.
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Affiliation(s)
- Svetlana Chefer
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Jurgen Seidel
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Adam S Cockrell
- Department of Epidemiology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA
| | - Boyd Yount
- Department of Epidemiology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA
| | - Jeffrey Solomon
- Clinical Research Directorate/Clinical Monitoring Research Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA
| | - Katie R Hagen
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - David X Liu
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Louis M Huzella
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Mia R Kumar
- Emerging Viral Pathogens Section, Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Elena Postnikova
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - J Kyle Bohannon
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Matthew G Lackemeyer
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Kurt Cooper
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Ariel Endlich-Frazier
- Emerging Viral Pathogens Section, Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Heema Sharma
- Emerging Viral Pathogens Section, Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - David Thomasson
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Christopher Bartos
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Philip J Sayre
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Amy Sims
- Department of Epidemiology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA
| | - Julie Dyall
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Michael R Holbrook
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Peter B Jahrling
- Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
- Emerging Viral Pathogens Section, Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
| | - Ralph S Baric
- Department of Epidemiology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA
| | - Reed F Johnson
- Emerging Viral Pathogens Section, Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, USA
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Bose RJC, Mattrey RF. Accomplishments and challenges in stem cell imaging in vivo. Drug Discov Today 2018; 24:492-504. [PMID: 30342245 DOI: 10.1016/j.drudis.2018.10.007] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2018] [Revised: 09/24/2018] [Accepted: 10/13/2018] [Indexed: 02/08/2023]
Abstract
Stem cell therapies have demonstrated promising preclinical results, but very few applications have reached the clinic owing to safety and efficacy concerns. Translation would benefit greatly if stem cell survival, distribution and function could be assessed in vivo post-transplantation, particularly in patients. Advances in molecular imaging have led to extraordinary progress, with several strategies being deployed to understand the fate of stem cells in vivo using magnetic resonance, scintigraphy, PET, ultrasound and optical imaging. Here, we review the recent advances, challenges and future perspectives and opportunities in stem cell tracking and functional assessment, as well as the advantages and challenges of each imaging approach.
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Affiliation(s)
- Rajendran J C Bose
- Department of Radiology and Advanced Imaging Research Center, 5323 Harry Hines Blvd, UT Southwestern Medical Center, Dallas, TX 75390-8514, USA; Current affiliation: Molecular Imaging Program at Stanford (MIPS) and the Canary Center at Stanford for Cancer Early Detection, Department of Radiology, School of Medicine, Stanford University, Stanford, CA 94305-5427, USA
| | - Robert F Mattrey
- Department of Radiology and Advanced Imaging Research Center, 5323 Harry Hines Blvd, UT Southwestern Medical Center, Dallas, TX 75390-8514, USA.
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39
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Chapelin F, Capitini CM, Ahrens ET. Fluorine-19 MRI for detection and quantification of immune cell therapy for cancer. J Immunother Cancer 2018; 6:105. [PMID: 30305175 PMCID: PMC6180584 DOI: 10.1186/s40425-018-0416-9] [Citation(s) in RCA: 68] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2018] [Accepted: 09/21/2018] [Indexed: 01/01/2023] Open
Abstract
Over the past two decades, immune cell therapy has emerged as a potent treatment for multiple cancers, first through groundbreaking leukemia therapy, and more recently, by tackling solid tumors. Developing successful therapeutic strategies using live cells could benefit from the ability to rapidly determine their in vivo biodistribution and persistence. Assaying cell biodistribution is unconventional compared to traditional small molecule drug pharmacokinetic readouts used in the pharmaceutical pipeline, yet this information is critical towards understanding putative therapeutic outcomes and modes of action. Towards this goal, efforts are underway to visualize and quantify immune cell therapy in vivo using advanced magnetic resonance imaging (MRI) techniques. Cell labeling probes based on perfluorocarbon nanoemulsions, paired with fluorine-19 MRI detection, enables background-free quantification of cell localization and survival. Here, we highlight recent preclinical and clinical uses of perfluorocarbon probes and 19F MRI for adoptive cell transfer (ACT) studies employing experimental T lymphocytes, NK, PBMC, and dendritic cell therapies. We assess the forward looking potential of this emerging imaging technology to aid discovery and preclinical phases, as well as clinical trials. The limitations and barriers towards widespread adoption of this technology, as well as alternative imaging strategies, are discussed.
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Affiliation(s)
- Fanny Chapelin
- Department of Bioengineering, University of California San Diego, 2880 Torrey Pines Scenic Drive, La Jolla, CA, 92037, USA
| | - Christian M Capitini
- Department of Pediatrics and Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, 1111 Highland Avenue, Madison, WI, 53705, USA.
| | - Eric T Ahrens
- Department of Radiology, University of California of San Diego, 9500 Gilman Dr. #0695, La Jolla, CA, 92093-0695, USA.
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Jiang H, DeGrado TR. [ 18F]Tetrafluoroborate ([ 18F]TFB) and its analogs for PET imaging of the sodium/iodide symporter. Theranostics 2018; 8:3918-3931. [PMID: 30083270 PMCID: PMC6071519 DOI: 10.7150/thno.24997] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2018] [Accepted: 03/16/2018] [Indexed: 12/27/2022] Open
Abstract
Sodium/iodide symporter (NIS)-mediated iodide uptake in thyroid follicular cells is the basis of clinical utilization of radioiodines. The cloning of the NIS gene enabled applications of NIS as a reporter gene in both preclinical and translational research. Non-invasive NIS imaging with radioactive iodides and iodide analogs has gained much interest in recent years for evaluation of thyroid cancer and NIS reporter expression. Although radioiodines and [99mTc]pertechnetate ([99mTc]TcO4-) have been utilized in positron emission tomography (PET) and single photon emission computed tomography (SPECT), they may suffer from limitations of availability, undesirable decay properties or imaging sensitivity (SPECT versus PET). Recently, [18F]tetrafluoroborate ([18F]TFB or [18F]BF4-) and other fluorine-18 labeled iodide analogs have emerged as a promising iodide analog for PET imaging. These fluorine-18 labeled probes have practical radiosyntheses and biochemical properties that allow them to closely mimic iodide transport by NIS in thyroid, as well as in other NIS-expressing tissues. Unlike radioiodides, they do not undergo organification in thyroid cells, which results in an advantage of relatively lower uptake in normal thyroid tissue. Initial clinical trials of [18F]TFB have been completed in healthy human subjects and thyroid cancer patients. The excellent imaging properties of [18F]TFB for evaluation of NIS-expressing tissues indicate its bright future in PET NIS imaging. This review focuses on the recent evolution of [18F]TFB and other iodide analogs and their potential value in research and clinical practice.
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Li M, Wang Y, Liu M, Lan X. Multimodality reporter gene imaging: Construction strategies and application. Theranostics 2018; 8:2954-2973. [PMID: 29896296 PMCID: PMC5996353 DOI: 10.7150/thno.24108] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2017] [Accepted: 03/06/2018] [Indexed: 12/11/2022] Open
Abstract
Molecular imaging has played an important role in the noninvasive exploration of multiple biological processes. Reporter gene imaging is a key part of molecular imaging. By combining with a reporter probe, a reporter protein can induce the accumulation of specific signals that are detectable by an imaging device to provide indirect information of reporter gene expression in living subjects. There are many types of reporter genes and each corresponding imaging technique has its own advantages and drawbacks. Fused reporter genes or single reporter genes with products detectable by multiple imaging modalities can compensate for the disadvantages and potentiate the advantages of each modality. Reporter gene multimodality imaging could be applied to trace implanted cells, monitor gene therapy, assess endogenous molecular events, screen drugs, etc. Although several types of multimodality imaging apparatus and multimodality reporter genes are available, more sophisticated detectors and multimodality reporter gene systems are needed.
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Affiliation(s)
- Mengting Li
- Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology
- Hubei Province Key Laboratory of Molecular Imaging
| | - Yichun Wang
- Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology
- Hubei Province Key Laboratory of Molecular Imaging
| | - Mei Liu
- Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology
- Hubei Province Key Laboratory of Molecular Imaging
| | - Xiaoli Lan
- Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology
- Hubei Province Key Laboratory of Molecular Imaging
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Oh EJ, Lee HW, Kalimuthu S, Kim TJ, Kim HM, Baek SH, Zhu L, Oh JM, Son SH, Chung HY, Ahn BC. In vivo migration of mesenchymal stem cells to burn injury sites and their therapeutic effects in a living mouse model. J Control Release 2018; 279:79-88. [PMID: 29655989 DOI: 10.1016/j.jconrel.2018.04.020] [Citation(s) in RCA: 75] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2017] [Revised: 04/06/2018] [Accepted: 04/11/2018] [Indexed: 12/18/2022]
Abstract
Mesenchymal stem cell (MSC)-based therapy has emerged as a promising therapeutic strategy for tissue regeneration and repair. In this study, we non-invasively monitored the tracking of MSCs toward burn injury sites using MSCs expressing firefly luciferase (Fluc) gene in living mice, and evaluated the effects of the MSCs at the injury site. Murine MSCs co-expressing Fluc and green fluorescent protein (GFP) were established using a retroviral system (referred to as MSC/Fluc). To evaluate the ability of MSC migration toward burn injury sites, cutaneous burn injury was induced in the dorsal skin of mice. MSC/Fluc was intravenously administrated into the mice model and bioluminescence imaging (BLI) was performed to monitor MSC tracking at designated time points. BLI signals of MSC/Fluc appeared in burn injury lesions at 4 days after the cell injection and then gradually decreased. Immunoblotting analysis was conducted to determine the expression of neovascularization-related genes such as TGF-β1 and VEGF in burnt skin. The levels of TGF-β1 and VEGF were higher in the MSC/Fluc-treated group than in the burn injury group. Our observations suggested that MSCs might assist burn wound healing and that MSCs expressing Fluc could be a useful tool for optimizing MSC-based therapeutic strategies for burn wound healing.
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Affiliation(s)
- Eun Jung Oh
- Department of Plastic and Reconstructive Surgery, School of Medicine, Kyungpook National University, Daegu, South Korea; Department of Plastic and Reconstructive Surgery, Kyungpook National University Hospital, Daegu, South Korea; Cell & Matrix Research Institute, Kyungpook National University, Daegu, South Korea
| | - Ho Won Lee
- Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu, South Korea; Department of Nuclear Medicine, Kyungpook National University Hospital, Daegu, South Korea
| | - Senthilkumar Kalimuthu
- Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu, South Korea; Department of Nuclear Medicine, Kyungpook National University Hospital, Daegu, South Korea
| | - Tae Jung Kim
- Department of Plastic and Reconstructive Surgery, School of Medicine, Kyungpook National University, Daegu, South Korea; Department of Plastic and Reconstructive Surgery, Kyungpook National University Hospital, Daegu, South Korea
| | - Hyun Mi Kim
- Department of Plastic and Reconstructive Surgery, School of Medicine, Kyungpook National University, Daegu, South Korea; Department of Plastic and Reconstructive Surgery, Kyungpook National University Hospital, Daegu, South Korea
| | - Se Hwan Baek
- Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu, South Korea; Department of Nuclear Medicine, Kyungpook National University Hospital, Daegu, South Korea
| | - Liya Zhu
- Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu, South Korea; Department of Nuclear Medicine, Kyungpook National University Hospital, Daegu, South Korea
| | - Ji Min Oh
- Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu, South Korea; Department of Nuclear Medicine, Kyungpook National University Hospital, Daegu, South Korea
| | - Seung Hyun Son
- Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu, South Korea; Department of Nuclear Medicine, Kyungpook National University Hospital, Daegu, South Korea
| | - Ho Yun Chung
- Department of Plastic and Reconstructive Surgery, School of Medicine, Kyungpook National University, Daegu, South Korea; Department of Plastic and Reconstructive Surgery, Kyungpook National University Hospital, Daegu, South Korea.
| | - Byeong-Cheol Ahn
- Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu, South Korea; Department of Nuclear Medicine, Kyungpook National University Hospital, Daegu, South Korea.
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Gurung S, Deane JA, Darzi S, Werkmeister JA, Gargett CE. In Vivo Survival of Human Endometrial Mesenchymal Stem Cells Transplanted Under the Kidney Capsule of Immunocompromised Mice. Stem Cells Dev 2018; 27:35-43. [DOI: 10.1089/scd.2017.0177] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Affiliation(s)
- Shanti Gurung
- Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia
- The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Victoria, Australia
| | - James A. Deane
- Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia
- The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Victoria, Australia
| | - Saeedeh Darzi
- Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia
- The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Victoria, Australia
| | - Jerome A. Werkmeister
- Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia
- The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Victoria, Australia
- CSIRO Manufacturing, Clayton South, Victoria, Australia
| | - Caroline E. Gargett
- Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia
- The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Victoria, Australia
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Sum CH, Shortall SM, Nicastro JA, Slavcev R. Specific Systems for Imaging. EXPERIENTIA SUPPLEMENTUM (2012) 2018; 110:69-97. [PMID: 30536227 DOI: 10.1007/978-3-319-78259-1_3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/09/2023]
Abstract
Microscopy allows for the characterization of small objects invisible to the naked eye, a technique that, since its conception, has played a key role in the development across nearly every field of science and technology. Given the nanometer size of the materials explored in the field of nanotechnology, the contributions of modern microscopes that can visualize these materials are indispensable, and the ever-improving technology is paramount to the future success of the field. This chapter will focus on four fundamental areas of microscopy used in the field of nanotechnology including fluorescence microscopy (Sect. 3.1), particle tracking and photoactivated localization microscopy (Sect. 3.2), quantum dots and fluorescence resonance energy transfer (Sect. 3.3), and cellular MRI and PET labeling (Sect. 3.4). The functionality, as well as the current and recommended usage of each given imaging system, will be discussed.
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Slavcev RA, Sum CH, St Jean J, Huh H, Nafissi N. Specific Systems for Evaluation. EXPERIENTIA SUPPLEMENTUM (2012) 2018; 110:99-123. [PMID: 30536228 DOI: 10.1007/978-3-319-78259-1_4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/09/2023]
Abstract
Fluorescent-based visualization techniques have long been used to monitor biological activity. This chapter explores the delivery of reporter genes as a means to assay and track activity in biological systems. Bioluminescence is the production of light due to biochemical processes. By encoding genes for bioluminescence, biological processes can be visualized based on gene expression. This chapter also discusses the primary applications of bioluminescence as seen through bioluminescent imaging techniques, flow cytometry, and PCR-based methods of gene detection. These techniques are described in terms of researching gene expression, cancer therapy, and protein interactions.
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Affiliation(s)
| | - Chi Hong Sum
- University of Waterloo, School of Pharmacy, Waterloo, ON, Canada
| | - Jesse St Jean
- University of Waterloo, School of Pharmacy, Waterloo, ON, Canada
| | - Haein Huh
- University of Waterloo, School of Pharmacy, Waterloo, ON, Canada
| | - Nafiseh Nafissi
- University of Waterloo, School of Pharmacy, Waterloo, ON, Canada
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Rathod M, Mal A, De A. Reporter-Based BRET Sensors for Measuring Biological Functions In Vivo. Methods Mol Biol 2018; 1790:51-74. [PMID: 29858783 DOI: 10.1007/978-1-4939-7860-1_5] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Genetic reporter systems provide a good alternative to monitor cellular functions in vitro and in vivo and are contributing immensely in experimental research. Reporters like fluorescence and bioluminescence genes, which support optical measurements, provide exquisite sensitivity to the assay systems. In recent years several activatable strategies have been developed, which can relay specialized molecular functions from inside the cells. The application of bioluminescence resonance energy transfer (BRET) is one such strategy that has been proved to be extremely valuable as an in vitro or in vivo assay to measure dynamic events such as protein-protein interactions (PPIs).The BRET assay using RLuc-YFP was introduced in biological research in the late 1990s and demonstrated the interaction of two proteins involved in circadian rhythm. Since then, BRET has become a popular genetic reporter-based assay for PPI studies due to several inherent attributes that facilitate high-throughput assay development such as rapid and fairly sensitive ratio-metric measurement, the assessment of PPI irrespective of protein location in cellular compartment and cost effectiveness. In BRET-based screening, within a defined proximity range of 10-100 Å, the excited energy state of the luminescent molecule excites the acceptor fluorophore in the form of resonance energy transfer, causing it to emit at its characteristic emission wavelength. Based on this principle, several such donor-acceptor pairs, using Renilla luciferase or its mutants as donor and either GFP2, YFP, mOrange, TagRFP or TurboFP as acceptor, have been reported for use.In recent years, the applicability of BRET has been greatly enhanced by the adaptation of the assay to multiple detection devices such as a luminescence plate reader, a bioluminescence microscope and a small animal optical imaging platform. Apart from quantitative measurement studies of PPIs and protein dimerization, molecular spectral imaging has expanded the scope for fast screening of pharmacological compounds that modulate PPIs by unifying in vitro, live cell and in vivo animal/plant measurement, all using one assay. Using examples from the literature, we will describe methods to perform in vitro and in vivo BRET imaging experiments and some of its applications.
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Affiliation(s)
- Maitreyi Rathod
- KS325, Molecular Functional Imaging Lab, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre(TMC), Navi Mumbai, Maharashtra, India
| | - Arijit Mal
- KS325, Molecular Functional Imaging Lab, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre(TMC), Navi Mumbai, Maharashtra, India
| | - Abhijit De
- KS325, Molecular Functional Imaging Lab, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre(TMC), Navi Mumbai, Maharashtra, India.
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Gaur S, Bhargava-Shah A, Hori S, Afjei R, Sekar TV, Gambhir SS, Massoud TF, Paulmurugan R. Engineering Intracellularly Retained Gaussia Luciferase Reporters for Improved Biosensing and Molecular Imaging Applications. ACS Chem Biol 2017; 12:2345-2353. [PMID: 28767220 DOI: 10.1021/acschembio.7b00454] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Gaussia luciferase (GLUC) is a bioluminescent reporter protein of increasing importance. As a secretory protein, it has increased sensitivity in vitro and in vivo (∼20 000-fold, and ∼1000-fold, respectively) over its competitor, secreted alkaline phosphatase. Unfortunately, this same advantageous secretory nature of GLUC limits its usefulness for many other possible intracellular applications, e.g., imaging signaling pathways in intact cells, in vivo imaging, and in developing molecular imaging biosensors to study protein-protein interactions and protein folding. Hence, to widen the research applications of GLUC, we developed engineered variants that increase its intracellular retention both by modifying the N-terminal secretory signal peptide and by tagging additional sequences to its C-terminal region. We found that when GLUC was expressed in mammalian cells, its N-terminal secretory signal peptide comprising amino acids 1-16 was essential for GLUC folding and functional activity in addition to its inherent secretory property. Modification of the C-terminus of GLUC by tagging a four amino acid (KDEL) endoplasmic reticulum targeting peptide in multiple repeats significantly improved its intracellular retention, with little impact on its folding and enzymatic activity. We used stable cells expressing this engineered GLUC with KDEL repeats to monitor chemically induced endoplasmic reticulum stress on cells. Additionally, we engineered an apoptotic sensor using modified variants of GLUC containing a four amino acid caspase substrate peptide (DEVD) between the GLUC protein and the KDEL repeats. Its use in cell culture resulted in increased GLUC secretion in the growth medium when cells were treated with the chemotherapeutic drugs doxorubicin, paclitaxel, and carboplatin. We thus successfully engineered a new variant GLUC protein that is retained inside cells rather than secreted extracellularly. We validated this novel reporter by incorporating it in biosensors for detection of cellular endoplasmic reticulum stress and caspase activation. This new molecularly engineered enzymatic reporter has the potential for widespread applications in biological research.
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Affiliation(s)
- Shuchi Gaur
- Departments of Radiology,
and Bioengineering, the Bio-X Program, Molecular Imaging Program at
Stanford (MIPS), Stanford University School of Medicine, 3155 Porter
Drive, Palo Alto, California 94304-1110, United States
| | - Aarohi Bhargava-Shah
- Departments of Radiology,
and Bioengineering, the Bio-X Program, Molecular Imaging Program at
Stanford (MIPS), Stanford University School of Medicine, 3155 Porter
Drive, Palo Alto, California 94304-1110, United States
| | - Sharon Hori
- Departments of Radiology,
and Bioengineering, the Bio-X Program, Molecular Imaging Program at
Stanford (MIPS), Stanford University School of Medicine, 3155 Porter
Drive, Palo Alto, California 94304-1110, United States
| | - Rayhaneh Afjei
- Departments of Radiology,
and Bioengineering, the Bio-X Program, Molecular Imaging Program at
Stanford (MIPS), Stanford University School of Medicine, 3155 Porter
Drive, Palo Alto, California 94304-1110, United States
| | - Thillai V. Sekar
- Departments of Radiology,
and Bioengineering, the Bio-X Program, Molecular Imaging Program at
Stanford (MIPS), Stanford University School of Medicine, 3155 Porter
Drive, Palo Alto, California 94304-1110, United States
| | - Sanjiv S. Gambhir
- Departments of Radiology,
and Bioengineering, the Bio-X Program, Molecular Imaging Program at
Stanford (MIPS), Stanford University School of Medicine, 3155 Porter
Drive, Palo Alto, California 94304-1110, United States
| | - Tarik F. Massoud
- Departments of Radiology,
and Bioengineering, the Bio-X Program, Molecular Imaging Program at
Stanford (MIPS), Stanford University School of Medicine, 3155 Porter
Drive, Palo Alto, California 94304-1110, United States
| | - Ramasamy Paulmurugan
- Departments of Radiology,
and Bioengineering, the Bio-X Program, Molecular Imaging Program at
Stanford (MIPS), Stanford University School of Medicine, 3155 Porter
Drive, Palo Alto, California 94304-1110, United States
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Pei Z, Zeng J, Song Y, Gao Y, Wu R, Chen Y, Li F, Li W, Zhou H, Yang Y. In vivo imaging to monitor differentiation and therapeutic effects of transplanted mesenchymal stem cells in myocardial infarction. Sci Rep 2017; 7:6296. [PMID: 28740146 PMCID: PMC5524783 DOI: 10.1038/s41598-017-06571-8] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2016] [Accepted: 06/14/2017] [Indexed: 01/04/2023] Open
Abstract
Here, we used a noninvasive multimodality imaging approach to monitor differentiation of transplanted bone marrow mesenchymal stem cells (BMSCs) and recovery of cardiac function in an in vivo model of myocardial infarction (MI). We established a rat MI model by coronary artery ligation. Ninety rats were randomly assigned into four groups: sham-operated, MI model, and α-MHC-HSV1-tk-transfected or un-transfected BMSCs-treated MI model. We used 18F-Fluro-deoxyglucose (18F-FDG) positron emission tomography (PET) to monitor recovery of cardiac function, and 18F-FHBG PET/CT imaging to monitor transplanted BMSCs differentiation 24 h after 18F-FDG imaging. The uptake of 18F-FDG at 3, 16, 30 and 45 days after BMSCs injection was 0.39 ± 0.03, 0.57 ± 0.05, 0.59 ± 0.04, and 0.71 ± 0.05% ID/g, respectively. Uptake of 18F-FHBG increased significantly in large areas in the BMSCs-treated group over time. Ex vivo experiments indicated that expression of the cardiomyocyte markers GATA-4 and cardiac troponin I markedly increased in the BMSCs-treated group. Additionally, immunohistochemistry revealed that HSV-tk-labelled BMSCs-derived cells were positive for cardiac troponin I. Multimodal imaging systems combining an α-MHC-HSV1-tk/18F-FHBG reporter gene and 18F-FDG metabolism imaging could be used to track differentiation of transplanted BMSCs and recovery of cardiac function in MI.
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Affiliation(s)
- Zhijun Pei
- Department of PET Center and Institute of Anesthesiology and Pain, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, China.
| | - Jing Zeng
- Department of Infection Control, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, China
| | - Yafeng Song
- Department of PET Center and Institute of Anesthesiology and Pain, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, China
| | - Yan Gao
- Department of PET Center and Institute of Anesthesiology and Pain, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, China
| | - Ruimin Wu
- Department of PET Center and Institute of Anesthesiology and Pain, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, China
| | - Yijia Chen
- Department of PET Center and Institute of Anesthesiology and Pain, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, China
| | - Fuyan Li
- Department of PET Center and Institute of Anesthesiology and Pain, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, China
| | - Wei Li
- Department of PET Center and Institute of Anesthesiology and Pain, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, China
| | - Hong Zhou
- Department of PET Center and Institute of Anesthesiology and Pain, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, China
| | - Yi Yang
- Department of PET Center and Institute of Anesthesiology and Pain, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, China
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Hao D, Sun L, Hu X, Hao X. 99mTc-LHRH in tumor receptor imaging. Oncol Lett 2017; 14:569-578. [PMID: 28693207 PMCID: PMC5494691 DOI: 10.3892/ol.2017.6246] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2016] [Accepted: 12/13/2016] [Indexed: 11/24/2022] Open
Abstract
Detection of gonadotropin-releasing hormone (GnRH) also known as luteinizing hormone-releasing hormone (LHRH) in the relevant tumor tissue and normal tissues and organs in vivo expression was investigated. To examine the method of direct radio labeling of LHRH by 99mTc with relatively high radiochemical purity and stability, screening the best labeling conditions, to establish a simple and reliable method of preparation of 99mTc-LHRH was undertaken. The detection of radioisotope-labeled LHRH distribution in mice, LHRH receptor imaging for the study and treatment of cancer basis were evaluated. i) Immunohistochemical staining test was used in 23 patients with hepatocellular carcinoma (HCC), 20 patients with breast cancer, 10 patients with prostate cancer, 20 patients with lung cancer, 20 patients with endometrial cancer tumor cells and normal tissue LHRH-R De Biaoda levels; ii) pre-tin method use direct labeling of LHRH, marking completion of saline or human serum were added at room temperature, the chromatography was measured at different times, to calculate the rate of labeled product and the radiochemical purity of the label, in vivo observation of its stability, and comparative analysis of selected optimal condition; iii) rat pituitary cell membrane protein, the product of in vitro radio-receptor marker analysis, through the saturation and inhibition experiments, was used to test its receptor binding activity; iv) Ch-T method labeled 125I-LHRH, tail vein injection of normal mice at different times were sacrificed, blood and major organs were determined and calculated per gram organization percentage injected dose rate (%, ID/g). Detected by immunohistochemistry in 23 cases of HCC in the LHRH-positive rate was 82.61%, in the corresponding normal tissues, the positive rate was 15%; 20 cases of breast cancer positive rate of 95%, the corresponding normal tissues, the positive rate was 20%; 10 cases of prostate cancer positive rate of 70%, the corresponding normal tissues, the positive rate of 40%; 20 cases of lung cancer positive rate of 85%, the corresponding normal tissues, the positive rate of 15.79%; 20 cases of endometrial cancer positive rate of 80% in the corresponding normal tissues was 16.67% positive. 99mTc-LHRH mark was 97.9–100.0%, the radiochemical purity of 93.9–96.4%, marking the reaction gel content of <5%. Great product receptor marker analysis showed 99mTc-LHRH with saturable receptor binding characteristics and inhibition, and high affinity, RT = 23.2174 pmol, KD = 0.4348 nmol; intravenous injection of 131I-LHRH within 72 h after the mice rapidly cleared the blood radioactivity, the major radioactive accumulation in the liver and kidneys and by the liver, renal clearance, and other tissues and organs of the radioactivity gradually decreased with time. In conclusion, i) the liver, lung, breast, prostate, endometrial cancer exist in both LHRHR; ii) 99mTc-LHRH preparation is simple, rapid, radiochemical purity product obtained higher marks, better stability, no further purification; and iii) LHRH 99mTc labeled, still has a high receptor binding ability, biological activity; and has an ideal and realistic dynamics in animals, there is hope, as with the clinical value of imaging agent of GnRH receptors.
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Affiliation(s)
- Dawei Hao
- Department of Radiotherapy, Xuzhou Central Hospital, Xuzhou, Jiangsu 221009, P.R. China
| | - Lingfei Sun
- Department of Radiotherapy, Xuzhou Central Hospital, Xuzhou, Jiangsu 221009, P.R. China
| | - Xiang Hu
- Department of Radiotherapy, Xuzhou Central Hospital, Xuzhou, Jiangsu 221009, P.R. China
| | - Xiaowen Hao
- Department of Radiotherapy, Xuzhou Central Hospital, Xuzhou, Jiangsu 221009, P.R. China
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Bai Z, Shi Y, Wang J, Qiu L, Teng G, Zhang F, Yang X. Multi-modality imaging-monitored creation of rat orthotopic pancreatic head cancer with obstructive jaundice. Oncotarget 2017; 8:54277-54284. [PMID: 28903340 PMCID: PMC5589579 DOI: 10.18632/oncotarget.17347] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2017] [Accepted: 04/07/2017] [Indexed: 02/07/2023] Open
Abstract
Purpose To investigate the feasibility of using multi-modality imaging to monitor the creation of rat models with orthotopic pancreatic head cancer with obstructive jaundice. Results 27 of 52 rats (51.92%) developed pancreatic head cancer. The tumor formation rate was significantly higher in the animal group receiving bioluminescent tumor, compared to the group receiving non-bioluminescent donor tumors [78.1% (25/32 rats) vs 10.0% (2/20 rats), P = 0.0001]. Both ultrasound imaging and MRI clearly characterized the orthotopic tumors. Laboratory biochemistry test for those rats with obstructive jaundice showed elevated levels of bilirubin, aspartate transaminase (AST), alkaline phosphatase (ALT) and gamma-glutamyl transpeptidase (λ-GGT), compared with those rats without jaundice (P < 0.05). Correlative pathology confirmed that all tumors were ductal adenocarcinomas, and located in pancreatic head regions. Materials and Methods Rat pancreatic adenocarcinoma cells (DSL-6A/C1) were first transfected with lentivirus/mCherry-luciferase genes, and then subcutaneously implanted into flanks of donor immunocompetent Lewis rats, to create pancreatic tumor tissues. The tumor tissues from donor rats with either bioluminescence signal or without the signal were then transplanted into the pancreatic heads of 52 recipient Lewis rats. Bioluminescence optical and ultrasound imaging, as well as magnetic resonance imaging (MRI), were performed to follow up the tumor formation and growth in these tumor-transplanted rats. Physical examination and biochemistry test were used to discern the rats with obstructive jaundice. The rats were euthanized for subsequent histologic correlation and confirmation. Conclusions We successfully created a new rat model with orthotopic pancreatic head cancer, which can be accurately monitored and visualized by different imaging modalities.
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Affiliation(s)
- Zhibin Bai
- Image-Guided Biomolecular Intervention Research, Section of Interventional Radiology, Department of Radiology, University of Washington School of Medicine, Seattle, WA, USA.,Department of Radiology, Zhongda Hospital, Southeastern University, Nanjing, China
| | - Yaoping Shi
- Image-Guided Biomolecular Intervention Research, Section of Interventional Radiology, Department of Radiology, University of Washington School of Medicine, Seattle, WA, USA
| | - Jianfeng Wang
- Image-Guided Biomolecular Intervention Research, Section of Interventional Radiology, Department of Radiology, University of Washington School of Medicine, Seattle, WA, USA
| | - Longhua Qiu
- Image-Guided Biomolecular Intervention Research, Section of Interventional Radiology, Department of Radiology, University of Washington School of Medicine, Seattle, WA, USA
| | - Gaojun Teng
- Department of Radiology, Zhongda Hospital, Southeastern University, Nanjing, China
| | - Feng Zhang
- Image-Guided Biomolecular Intervention Research, Section of Interventional Radiology, Department of Radiology, University of Washington School of Medicine, Seattle, WA, USA
| | - Xiaoming Yang
- Image-Guided Biomolecular Intervention Research, Section of Interventional Radiology, Department of Radiology, University of Washington School of Medicine, Seattle, WA, USA.,Department of Radiology, Sir Run Run Show Hospital, Zhejiang University School of Medicine, Hangzhou, China
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