Abstract 6342: Ensuring diverse <i>in vitro</i> lung cancer models: development of alveolar epithelial cell lines from individuals of African ancestry

Lung cancer disproportionately affects Black/African American (B/AA) individuals, particularly men, who experience 12% higher incidence and 15% higher death rates than White/European (W/E) men. Lung adenocarcinoma (LUAD) originates in the alveolar epithelium and is the most common histological subtype of lung cancer for all racial groups. Immortalized human alveolar epithelial cells (ihAEC) are key tools to study the etiology and development of LUAD, as well as effects from environmental exposures. We recently established three ihAEC lines from remnant lung tissue of deceased unidentified W/E individuals using lentiviral transduction of simian virus 40 large-tumor antigen (SV40 LgT) and human telomerase reverse transcriptase (TERT) genes. SV40 LgT is an oncogene that promotes constant proliferation, while TERT prevents telomere shortening. Lentiviral vectors randomly integrate into the host genome, causing potential unwanted side effects. In addition, integrated transgenes may become silenced in the long-term. Furthermore, viral vectors that deliver oncogenes are a considerable biosafety concern that warrants avoidance whenever possible. Here, we describe an alternative gene-delivery approach that avoids the use of lentiviral vectors, and we use this new strategy to generate ihAEC lines of diverse ancestry. We have constructed a plasmid carrying SV40 LgT, an internal ribosome entry site, and TERT, as well as a puromycin resistance gene. The SV40 LgT sequence has been engineered with flanking loxP sites to allow its excision by Cre recombinase, if desired. To direct genomic integration by CRISPR/Cas9, the transgene is surrounded by homology arms that target integration into the adeno-associated virus integration site 1 (AAVS1), a safe harbor region that prevents inserted genes from being silenced. We will electroporate this plasmid into AECs isolated from remnant non-tumor lung tissue that was collected with full consent from lung cancer patients of diverse ancestry, including B/AA men. Development of new ihAEC lines will provide valuable tools for studying responses to environmental exposures, carcinogen detoxification processes, oncogenic transformation, and for testing novel therapies in models distinct from W/E genotypes. This work is an important step towards minimizing lung cancer health disparities caused by the absence of diverse model systems. Supported by grants U54CA233396, U54CA233444, and U54CA233465 from the National Institutes of Health (NIH)/National Cancer Institute (NCI), and the Norris Comprehensive Cancer Center core grant, award number P30CA014089 from the NIH/NCI. Citation Format: Bianca Dal Bó, Sofia A. Lugo, David M. Lee, Kweku Ofosu-Asante, Kyle R. Phillips, Benjamin J. Ryder, Yong Huang, Nazarius S. Lamango, Ite A. Offringa. Ensuring diverse in vitro lung cancer models: development of alveolar epithelial cell lines from individuals of African ancestry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6342.