Abstract Tumor formation is promoted by increased expression of “Don’t eat me” signals such as CD47 in cancer cells that inhibit phagocytic action of macrophages. However, the signals can regulate generation of brain metastasis is still unclear. In the brain, microglia and perivascular macrophages are the only resident immune cells of the brain parenchyma. Here, we have developed an in-vivo imaging method that can allow us to evaluate microglial phagocytosis of tumor and to detemine the fates of cancer cells in the brain. The skull of the CX3CR1-EGFP mice, in which microglia is specifically labeled with EGFP, was replaced with a glass coverslip, and the fluorescently labeled lung cancer cells (CMT167-mCherry) were injected via the internal carotid artery of the mice. The fate of arrested cancer cells in the brain vascular was monitored for 12-16 days. Of the 324 cancer cells (4 mice), 49.8% (N=138) of cells displaced from their original site, 40.4% (N=112) of cells developed micro-metastasis, and 6.9% (N=27) of cells were processed by microglia resulting in cell death. To evaluate the “Don’t eat me” signal against microglia, a CD47 gene knockout cell line was monitored as well, and these cells developed significantly less micro-metastasizes compared with CD47 positive cells. This in vivo imaging of the brain metastasis potentially provide a method for monitoring of tumor fate and validation of crucial target. Citation Format: Takahiro Tsuji, Hiroaki Wake, Mariko Shindo, Daisuke Kato, 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 AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO039.