Abstract 290: Derivation of immortalized human alveolar epithelial cells for lung adenocarcinoma modeling
Authors: Offringa Ite A. , Tran Evelyn , Shi Tuo , St.Pierre Laurence , Puente Pablo E. , Lu Rong , Marconett Crystal N. , Zhou Beiyun , Borok Zea 
Published: June 2022

Abstract
Lung cancer, primarily caused by tobacco smoke exposure, kills more Americans than the next top three cancers combined. The two main histological subtypes are lung adenocarcinoma, which arises in the alveoli, and squamous cell lung cancer, which arises in the airways. In vitro models of squamous cell lung cancer have used human bronchial epithelial cells immortalized with Simian Virus 40 Large T antigen (SV40 LgT) or mutant CDK4R24C combined with telomerase. The derived immortalized human bronchial epithelial cells have been very useful to study the effects of tobacco smoke exposure and oncogene activation. However, to date it has not been possible to develop similar models for lung adenocarcinoma; immortalized human alveolar epithelial cells (AECs) were not available due to the difficulty in establishing alveolar cell lines that proliferate yet remain epithelial. Here we describe a method to derive AEC lines, providing a new resource for in vitro lung adenocarcinoma models. We purified human AECs from de-identified remnant human transplant lungs from deceased subjects and tested numerous conditions to establish immortalized cell lines. We found that the addition of Rho-associated kinase inhibitor (Y27632) to culture media allowed AECs to proliferate so that they could then be transduced with lentivirus. Transduction with CDK4R24C + telomerase did not yield cell lines that grew well. However, transduction with SV40 LgT resulted in well-proliferating cell lines that retained their epithelial morphology. In 2-dimensional (2D) culture the AEC LgT-expressing cell lines grow in cobblestone monolayers and do not express mature alveolar markers. However, in 3-dimensional (3D) culture in Matrigel with fibroblasts the cells form alveolar-like organoids of varying complexity. Immunofluorescence and single cell RNA sequencing indicate that the transition from 2D monolayer to 3D organoids is accompanied by the expression of mature alveolar markers such as aquaporin 5 (AQP5), G-protein-coupled receptor class C, member A (GPRC5A), lysosomal-associated membrane protein 3 (LAMP3) and ATP-binding cassette subfamily A member 3 (ABCA3, involved in surfactant production). In addition, while in 2D culture the cells form a single cluster with homogeneous gene expression patterns, in 3D diverse cell groups appear. Using a 3D culture medium that stimulates alveologenesis induces additional mature alveolar markers. We are using AEC lines to study the effects of environmental exposures such as tobacco smoke and the role of lung cancer driver genes. In addition, we intend to establish AEC lines from individuals of diverse ethnicities to investigate the effects of genetic background on lung cancer development. Our AEC lines provide a valuable new system to model and study the distal lung and associated diseases in vitro.
Citation Format: Ite A. Offringa, Evelyn Tran, Tuo Shi, Laurence St.Pierre, Pablo E. Puente, Rong Lu, Crystal N. Marconett, Beiyun Zhou, Zea Borok. Derivation of immortalized human alveolar epithelial cells for lung adenocarcinoma modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 290.