Abstract 2472: Interdisciplinary method to elucidate the interaction between brain microenvironment and cancer cells
Authors: Tsuji Takahiro , Wake Hiroaki , Shindo Mariko , Hartantio Rahadian Y. , Horikoshi Misuzu , Kato Daisuke , Ozasa Hiroaki , Hirai Toyohiro 
Published: April 2023

Abstract
Cancer cells may escape from host immunity to form distant metastases. We have made an original imaging model of brain metastasis (IBrM) to show a heterogeneous interaction between microglia, the tissue macrophages in the brain, and cancer cells in the brain niche. Here we report that enhanced microglial elimination of cancer cells is a new potential treatment for metastatic brain tumors by inhibiting their engraftment. First, to explore factors that favor survival of cancer cells in the brain microenvironment, mouse lung cancer cells (CMT167) were injected through the internal carotid artery, and after 4, 7, 10, and 13 days, surviving cancer cells in the mouse brain were collected and RNAseq was performed. In particular, we focused on CD24/CD7/PD-L1, a known factor that escapes phagocytosis by macrophages. The expression of CD24/CD47 in cancer cells gradually increased, while that of PD-L1 decreased. Next, we injected adenocarcinoma lung cancer cells (LUCs, CMT167) or breast cancer cells (BCCs, E0711) expressing mCherry via the internal carotid artery of the CX3CR1-EGFP mice, whose microglia had been specifically labeled with EGFP. The microglia and LUCs/BCCs were visualized in vivo simultaneously by two-photon microscopy for 14 days. The tumor fate and microglial response against cancer cells were evaluated at single cell resolution in the IBrM. Microglial phagocytosis was significantly increased by single deletion of CD47 or CD24 compared with WT cells. Combined deletion of CD47 and CD24 also synergically increased microglial phagocytosis, resulting in reduced IBrM and prolonged mouse survival. By contrast, deletion of PD-L1 did not enhance microglial phagocytosis or prolong survival. The pharmacological (PLX3397) or genetic conditional (Siglechdtr het/CX3CR1GFP het) conditional depletion of microglia canceled both the increased phagocytosis and the extended survival time, suggesting that the effect of “don't eat me” signals was microglia dependent. The extended survival time was also observed in IBrM using BALB-c nu/nu, that suggest the effect is not dependent on matured T-lymphocytes.These results indicate that novel function of microglia against cancer cells, and suppression of both CD24 and CD47 in cancer cells and enhances microglial phagocytic activity and suppresses the metastatic formation. We are further developing a method for isolating LUCs fluorescently labeled in vivo using holographic 2-photon microscopy for evaluating single cell gene expression profiles. Using the novel techniques, the spatio-temporal information can be integrated across the molecular, cellular and tissue levels that allow us to further investigate the dynamic behavior of not only tumors, but also microglia and neurons.
Citation Format: Takahiro Tsuji, Hiroaki Wake, Mariko Shindo, Rahadian Y. Hartantio, Misuzu Horikoshi, Daisuke Kato, Hiroaki Ozasa, Toyohiro Hirai. Interdisciplinary method to elucidate the interaction between brain microenvironment and cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2472.