Abstract 1768: Real time in vivo imaging of brain metastasis visualized dynamic reaction of microglia against cancer cells
Authors: Tsuji Takahiro , Wake Hiroaki , Shindo Mariko , Kato Daisuke , Ozasa Hiroaki , Hirai Toyohiro 
Published: July 2021

Abstract
The brain metastasis of lung cancer is a crucial problem that causes poor clinical outcome and alters patient's quality of life. Previous evidences showed that cancer cells may escape from host immunity to form distant metastasis. However, the detailed interaction between lung cancer cells and immune cells in the brain niche is still unclear. Microglia and perivascular macrophages are the only resident immune cells in the brain parenchyma in physiological state. Here, we develop an in-vivo imaging method that can allow us to follow the immune cells and cancer progression in single cell resolution to evaluate immune reaction against cancer. Using this method, we evaluated microglial role in the development of brain-metastasis.Lung cancer cell line (CMT167mC) expressing mCherry was established from CMT167, a mice lung cancer cell line derived from C57BL/6. To generate brain-metastasis model mice, the cancer cells were injected via the internal carotid artery of the CX3XR1-GFP mice (Day 0), in which EGFP is specifically expressed in microglia and macrophage. The skull of the mice was replaced with a glass coverslip for chronic imaging (Day 1), and the fluorescently labeled cancer cells and microglia was simultaneously visualized using two-photon microscopy (Day 2-). The median survival of the brain-metastasis model mice after cancer cell injection was 15.0 days, and the fate of each embolized cell was followed for 14 days. Monitoring of 277 embolized cancer cells in the brain revealed 49.8% of cancer cells displaced from their original site, 40.4% developed micro-metastasis (MMs), and 6.9% were phagocytosed by microglia. We focused on the microglial role around the cancer cells that can form MMs, then the morphology and behavior of microglia around MMs was evaluated. The size of microglia around MMs (&lt;100 µm from cancer cells) was significantly increased on day 6 and subsequently the density was increased on day 10, compared with microglia far from (&gt;100 µm) cancer cells, suggesting that cancer cells can escape from activated microglia without being phagocytosed. Microglia depletion using pexidartinib can reduce the number and size of MMs that implicates activated microglia can inversely help tumor progression. Then we evaluated the functional role of a “Don't eat me” signal against microglia, which is a surface protein CD47 that inhibit the phagocytic action of macrophages. A knockout of the CD47 gene from CMT167mC significantly reduced the formation of MMs, increased phagocytosis by microglia, and prolonged the survival time of the model mice. The result suggests that this model provide a method to visualize immune reaction against cancer and validation of crucial target that reduce MMs. We are further exploring another critical factor that regulate development of brain metastasis using multi-omics analysis both on the activated microglia and on the cancer cells that formed MMs.
Citation Format: Takahiro Tsuji, Hiroaki Wake, Mariko Shindo, Daisuke Kato, Hiroaki Ozasa, Toyohiro Hirai. Real time in vivo imaging of brain metastasis visualized dynamic reaction of microglia against cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1768.