Abstract 2728: Novel in vivo imaging method to evaluate “Don't eat me” signal of tumor against microglia
Authors: Tsuji Takahiro , Wake Hiroaki , Shindo Mariko , Haruwaka Koichiro , Ajimizu Hitomi , Yamazoe Masatoshi , Funazo Tomoko , Yasuda Yuto , Yoshida Hironori , Sakamori Yuichi , Kim Young Hak , Ozasa Hiroaki , Hirai Toyohiro 
Published: September 2020

Abstract
The brain metastasis of lung cancer is a crucial problem that causes poor clinical outcome and alters patient's quality of life. Previous reports have indicated that tumor formation is promoted by increased expression of “Don't eat me” signals in cancer cells that inhibit phagocytic action of macrophages. Inhibiting the signals and promoting phagocytosis, such as anti-CD47 or anti-CD24a neutralize antibody, have been focused as a novel therapeutic method, but whether the signals can regulate generation of brain metastasis is still unclear. Here, we have developed an in-vivo imaging method that can allow us to follow the tumor fate in single cell resolution to evaluate microglial phagocytosis of tumor. Using this method, we classified various fates of cancer cells invaded into the brain which is partially determined by microglial phagocytotic functions as a therapeutic target. An mCherry-labeled lung cancer cell line (CMT167mC) was established from CMT167, a mice lung cancer cell line derived from C57BL. A craniotomy was performed on CX3CR1-EGFP mice, in which EGFP is specifically expressed in microglia. The skull of the mice was replaced with a glass coverslip for chronic imaging, and the fluorescently labeled cancer cells were injected via the internal carotid artery of the mice to visualize simultaneously with microglia using two-photon microscopy. To detect a microglial phagocytosis of cancer cells in micro-metastasis region and to follow the tumor fate after phagocytosis, we first evaluate the natural fate of the cancer cells after injection into the brain. Arrested cancer cells in the brain vascular were identified immediately after the cancer cell injection. The fate of each arrested cell could be followed for 14 days. Monitoring of 105 arrested cancer cells (N=3 mice) in the brain revealed 54% of cancer cells displaced from their original site, development of micro-metastasis and attached with microglia in 34.3% of cells, and phagocytosis of 13.3% of cells by microglia resulting in cell death, suggesting their heterogeneous fate. To focus on the microglial phagocytosis of cancer cells that may be one of the candidate for therapeutic target, we evaluated the functional role of a “Don't eat me” signal, which is a surface protein CD47 that inhibited the phagocytic action of macrophages when the cancer cells were invaded to the brain. A CD47 gene knockout cell line (CMT167mC-CD47-/-) was established from CMT167mC cells using CRISPR/Cas9. CMT167mC-CD47-/- cells provided into the brain developed micro-metastasizes less frequently as compared with CMT167mC. Further study to address the role of CD47 in microglial phagocytosis is in progress. The result suggests that this system potentially provide a novel screening method for validation of crucial target inhibiting “Don't eat me” signal. Targeting the microglial phagocytosis could be a potent novel therapy to prevent the development of brain metastasis. In future, other potential “Don't eat me” signals, such as CD24a and PD-L1, will be evaluated.
Citation Format: Takahiro Tsuji, Hiroaki Wake, Mariko Shindo, Koichiro Haruwaka, Hitomi Ajimizu, Masatoshi Yamazoe, Tomoko Funazo, Yuto Yasuda, Hironori Yoshida, Yuichi Sakamori, Young Hak Kim, Hiroaki Ozasa, Toyohiro Hirai. Novel in vivo imaging method to evaluate “Don't eat me” signal of tumor against microglia [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 2728.