Abstract
In vitro studies using cell culture, including three-dimensional cultures without the involvement of tumor vessels, have limitations in simulating complex intratumoral hypoxic conditions in live subjects. To generate experimental hypoxic conditions closer to those observed in humans in clinical settings, in vivo studies are necessary. In addition, visible light generated via bioluminescence and fluorescence is generally unsuitable for in vivo experiments because of low tissue penetration. Furthermore, near-infrared light (NIR), which has the highest tissue penetration among lights of different wavelengths, cannot be assessed precisely in vivo because of the difficulty in correcting tissue absorption and scatter. For in vivo quantitative analyses, imaging modalities that use high tissue-penetrating signals, such as computed tomography (CT) using X-rays, radionuclide imaging using γ-rays, and magnetic resonance imaging (MRI) using electromagnetic waves, are ideal.
Therefore, as an advanced protocol for this research purpose, we provide ex vivo and in vivo methods to investigate the genetic response of multiple copies of hypoxia response elements (HREs) to tumor hypoxia in terms of intensity and intratumoral distribution using a human sodium/iodide symporter (hNIS) reporter gene and radionuclide reporter probes (radioiodine and its chemical analog Tc-99m) based on our previous research. This protocol includes cloning an hNIS reporter construct with multiple copies of HREs, establishing stable cell lines of the reporter construct, preparing a mouse subcutaneous xenograft model, and evaluating the genetic response of multiple HREs to tumor hypoxia using digital autoradiography (ARG) ex vivo and using single-photon emission computed tomography (SPECT) or positron emission tomography (PET) in vivo.
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References
Bristow RG, Hill RP (2008) Hypoxia, DNA repair and genetic instability. Nat Rev Cancer 8(3):180–192
Saxena K, Jolly MK (2019) Acute vs. chronic vs. cyclic hypoxia: their differential dynamics, molecular mechanisms, and effects on tumor progression. Biomolecules 9(8):339
Takeuchi Y, Inubushi M, Jin YN, Murai C, Tsuji AB, Hata H, Kitagawa Y, Saga T (2014) Detailed assessment of gene activation levels by multiple hypoxia-responsive elements under various hypoxic conditions. Ann Nucl Med 28(10):1011–1019
Takeuchi Y, Inubushi M, Jin YN, Xu H, Yamamoto K, Nagatsu K, Tsuji AB, Murai C, Hata H, Kitagawa Y, Saga T (2023) Ex vivo and in vivo molecular imaging of genetic activation of hypoxia response elements in response to hypoxia; for aid of understanding tumor accumulation of 18F-FMISO and 64Cu-ATSM. Ann Nucl Med. in press
National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals (2011) Guide for the care and use of laboratory animals, 8th edn. National Academies Press (US), Washington, DC
Inubushi M, Jin YN, Murai C, Hata H, Kitagawa Y, Saga T (2012) Single-photon emission computed tomography of spontaneous liver metastasis from orthotopically implanted human colon cancer cell line stably expressing human sodium/iodide symporter reporter gene. EJNMMI Res 2(1):46
Burgman P, O’Donoghue JA, Lewis JS, Welch MJ, Humm JL, Ling CC (2005) Cell line-dependent differences in uptake and retention of the hypoxia-selective nuclear imaging agent Cu-ATSM. Nucl Med Biol 32(6):623–630
Acknowledgments
We thank Dr. Masanori Hatakeyama of Microbial Chemistry Research Foundation and Prof. Hideaki Higashi of Hokkaido University for technical guidance. This work was supported by JSPS KAKENHI Grants Number 19659298, 22390239, 22H03285, and 22K19608.
Source of Funding
This work was supported by JSPS KAKENHI Grants Number 19659298, 22390239, 22H03285 and 22K19608.
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Inubushi, M., Takeuchi, Y., Kitagawa, Y. (2024). Radionuclide Reporter Imaging to Visualize Tumor Hypoxia Ex Vivo and In Vivo. In: Gilkes, D.M. (eds) Hypoxia. Methods in Molecular Biology, vol 2755. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3633-6_7
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DOI: https://doi.org/10.1007/978-1-0716-3633-6_7
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-3633-6
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