Abstract LB-006: In vivo imaging of HIF-active cells in tumors using transgenic mice with a HIF-dependent reporter gene
Authors: Miyabara Hitomi , Hirano Ryuichiro , Kuchimaru Takahiro , Kadonosono Tetsuya , Watanabe Hitomi , Kondoh Gen , Kizaka-Kondoh Shinae 
Published: September 2020

Abstract
Hypoxia inducible factor (HIF) is a transcription factor essential for hypoxia adaptation. In tumors, cells increase HIF activity through excess growth factor stimulation and oncogene activation as well as hypoxia, leading to the expression of many genes involved in malignancy. Since elevated HIF activity is one of the key features of malignancy, changes based on HIF activity can be very important therapeutic targets for cancer therapy, especially in the treatment of refractory cancer. Therefore, understanding HIF-active cells in tumors during tumor development and malignant progression is important in developing cancer treatment strategies.Various types of immune cells such as lymphocytes, neutrophils and monocytes infiltrate into tumors at different times. These immune cells have antitumor functions, but have also been reported to promote tumor immunosuppression and malignant progression. Although HIF activation in these cells has been suggested to play a key role in these events, it is not yet clear how and when the HIF activity is involved in the process.We have been developing transgenic mice (Tg) to monitor HIF activity by in vivo imaging. So far, however, no Tg can achieve enough sensitivity to detect cell-based HIF activity by in vivo imaging. Recently, we have constructed a new Tg (HVA-Tg) with a reporter transgene that exresses AkaLuc and Venus proteins in a HIF activity-dependent manner. AkaLuc [1] is a mutant firefly luciferase that achieves 100- - 1000-fold higher detection sensitivity with artificial luciferin AkaLumine-HCl [2] compared to natural firefly luciferase and D-luciferin combination. The bioluminescence produced by AkaLuc and AkaLumine-HCl is called AkaBLI and has a peak emission at 670-680 nm, in the near infrared region, leading to much higher biopermeability compared to the firefly luciferase-D-luciferin system, whose bioluminescence peak is 560 nm.In this study, we monitored HIF activity in tumors syngenically transplanted into HVA-Tg: E0771 (a breast cancer cell line) was orthotopically transplanted and bioluminescence form the tumors were monitored by in vivo imaging overtime. As expected, the host derived luminescence ware detected from the tumor site at two weeks post transplantation. Ex vivo imaging confirmed that this signal was generated from the tumor. Immunohistochemical analysis of tumor sections revealed that AkaLuc was detected in the same cells as Gr-1 and F4/80-positive cells. These data suggest that HVA-Tg is a useful tool to investigate HIF activity in tumor stromal cells, and its analysis will provide useful information to further explore the mechanism how HIF activity contributes to tumor immunosuppression.[1] Iwano S, Kuchimaru T, Kizaka-Kondoh S, et al. Science 359:935-939 (2018).[2] Kuchimaru T., Kadonosono T., Kizaka-Kondoh S. et al. Nat Commun, 7, 11856 (2016).
Citation Format: Hitomi Miyabara, Ryuichiro Hirano, Takahiro Kuchimaru, Tetsuya Kadonosono, Hitomi Watanabe, Gen Kondoh, Shinae Kizaka-Kondoh. In vivo imaging of HIF-active cells in tumors using transgenic mice with a HIF-dependent reporter gene [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-006.