Abstract 380: The quantified immune response against brain metastasis of lung cancer in vivo
Authors: Shindo Mariko , Tsuji Takahiro , Haruwaka Koichiro , Kato Daisuke , Tachibana Yoshihisa , Ozasa Hiroaki , Hirai Toyohiro , Wake Hiroaki 
Published: September 2020

Abstract
Brain metastasis of lung cancers is clinically important to impair the quality of life. Lung cancer cells invade hematogenously into the brain, penetrating blood-brain barrier (BBB) and forming metastasis in the brain parenchyma. Traditionally, these phenomena had been investigated by biochemical methods, which lacks spatio-temporal information. Using recent in vivo imaging 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 brain metastasis. Here, we established an in vivo imaging system to visualize the temporal information of lung cancer brain metastasis with spatio-temporal information to quantify immune response by brain immune cells; microglia. We observed extravasation of lung cancer cells from the blood vessels into the brain that activate microglia to gather around cancer cells. We first performed craniotomy on the mouse and replaced the skull with a glass coverslip for chronic in vivo imaging of the brain metastasis. mCherry or tag-BFP expressing fluorescence labeled mouse lung stable cancer cells (CMT167 and LLC1 cells) were injected into the internal carotid artery of CX3CR1-GFP mice, in which EGFP was expressed specifically in microglia and macrophage to visualize the brain metastasis of lung cancer cells using in vivo two-photon microscope. To visualize brain vessels in vivo, fluorescence conjugated dextran (10kDa-70kDa) was injected into the peripheral vein before observation. Fluorescently labeled cancer cells were trapped at cranial vessels after injection via the internal carotid artery. Then the cancer cells occluded the vessels and proliferated along the vascular lumen. On day 6-8 after injection, the morphology of cancer cells was changed to sphere-like form, suggesting cancer cells invading into brain from the vessels. Next, to evaluate the immune response of brain against the cancer cells, we measured the activation of microglia when the cancer cells were injected. The density and size of the microglia near the tumor were significantly increased as compared with pre-injection of cancer cells. Surprisingly, some microglia phagocyted cancer cells as with macrophages. In addition, microglia start to response against cancer cells simultaneously with the leakage of cancer cells from the blood vessels. In conclusion, we succeeded to construct the experimental system to visualize the micro environment and niche of the micro-metastatic cancer cells with the detailed dynamic interaction among cancer cells, microglia, and micro vascular in the mice brain. We are exploring the molecular mechanisms of immune response in micro-metastasis site that regulate the progression of brain metastasis pathology. In future, integration of spatio-temporal information with molecular profile will provide further insight into the brain metastasis.
Citation Format: Mariko Shindo, Takahiro Tsuji, Koichiro Haruwaka, Daisuke Kato, Yoshihisa Tachibana, Hiroaki Ozasa, Toyohiro Hirai, Hiroaki Wake. The quantified immune response against brain metastasis of lung cancer in vivo [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 380.