Abstract Lung adenocarcinoma is closely associated with chronic inflammation, but the underlying mechanism has not been fully elucidated yet. As a mucosal organ, lung was thought to be sterile in the past due to the limitation of bacteria culture techniques, but recent 16s RNA sequencing analysis has proved that lung is colonized by a diverse bacterial community. Recent studies suggest that the dysbiosis of lung microbiota plays a critical role in lung cancer tumorigenesis. However, there is no direct evidence addressing the complex interactions between local microbiota, immune cells and tumor cells due to the limitation of traditional imaging methods. Here we developed an advanced imaging technique combining RNA fluorescent in situ hybridization, immunostaining and Clearing-enhanced 3D (Ce3D), and applied it in a genetically engineered mouse model harboring the most common mutations (activation of Kras and p53 loss of function) in patients with lung adenocarcinoma. It revealed a strong spatial-temporal correlation between lung microbiota and tissue-resident immune cells during lung adenocarcinoma development. This research was supported by the Intramural Research Programs of NIAID and NHLBI, NIH. Citation Format: Chen Zhao, Weizhe Li, Chengcheng Jin, Ronald N. Germain. Directly visualizing interactions between lung microbiota, tissue-resident immune cells and tumor cells in a genetically engineered lung adenocarcinoma mouse model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A62.