Abstract Human cancers are composed of genetically and phenotypically heterogeneous subpopulations organized into a hierarchy of tumor initiating stem cells and various differentiated tumor cell. Cancer organoids seem to be more suitable in vitro models for targeting tumor heterogeneity than cell lines. We established cancer organoid lines from lung cancer patient tissues, recapitulating histology of human cancers in three dimensional culture conditions. Thirteen organoid lines from various lung cancer subtypes including adenocarcinoma, squamous cell carcinoma and small cell carcinoma were compared with their original human tumors in histologic features and genetic profiles. In the IHC staining, it revealed that organoids of adenocarcinomas have typical glandulo-papillary structures or mucin-containing tumor cells. Organoids of squamous cell carcinomas showed keratinization and intercellular bridges. Organoids of small cell carcinoma displayed typical neuroendocrine morphology. Using NGS cancer panel sequencing, the organoid lines maintained genetic characteristics of the original human tumors in major driver genes including EGFR, TP53, and RB mutation. In xenograft experiments, the organoid lines revealed stronger tumorigenesity than direct graft of human tumor tissues. Cancer organoids showed resistance to various anticancer drug treatments. In conclusion, our lung cancer organoid lines are alternative cancer model for preclinical researches recapitulating genotypic and phenotypic heterogeneity of original human tumors. Note: This abstract was not presented at the meeting. Citation Format: Minsuh Kim, Sung-Min Chun, Hyemin Mun, Young-Ah Suh, Se jin Jang. Establishment of lung cancer organoid lines as a new preclinical model for lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4832. doi:10.1158/1538-7445.AM2017-4832