Abstract 6347: Epithelial stem cells in tumorigenesis of the lung: lessons from a murine model of lung adenocarcinoma

Abstract Introduction: Lung cancer is the leading cause of cancer-related deaths, with a poor five-year survival rate. The period of premalignancy is marked by structural and functional alterations in the epithelium that eventually progress to cancer. Epithelial stem/progenitor cells are essential for the maintenance and repair of the airway epithelium. Prior research from our group demonstrated a reduction in the function of normal progenitors in human lung premalignancy. However, studying progressive changes in the epithelium as it advances from normal to premalignant is challenging in humans. This led us to conduct a time-course study of urethane-induced lung adenocarcinoma (ADC) in mice to better understand the role of normal tissue stem cells in tumorigenesis. Methods: Tumorigenesis was induced in A/J mice (12 mice/group) by a single intraperitoneal injection urethane (1mg/gm), and control mice were injected with saline. Lung tissues were harvested at 2-, 4-, 10-, 16-, and 22-week post-injection; the left lobe was used for histological analysis, and the rest of the lung tissue was digested proteolytically to generate single-cell suspensions. Stem cell function was determined by organoid-forming assay, and molecular analysis of organoids was performed to identify genetic changes in stem cell populations. Results & Conclusions: Histological analysis revealed that the premalignant changes induced by urethane closely resembled the lesions observed in human lung ADC, including adenomatous hyperplasia, nodular hyperplasia, and adenoma. Immunofluorescence staining (IF) with cell type-specific markers to track the progression of these changes identified hyperplasia of club cells (CCSP+) at 2 weeks, followed by proliferation of type II (SPC+) cells, the formation of adenomas by 10 weeks, and the development of adenocarcinoma at 22 weeks. Quantification of organoids at all time points showed fewer organoids in urethane-treated mice compared to saline at 2 weeks, but the reverse was true at subsequent time points when greater numbers of organoids were made by the urethane-treated mice (p<0.01 for both directions). IF staining of organoids identified both airway and alveolar organoids, and there was no difference in their proportions between saline and urethane-treated mice. Interestingly, a small number of bronchoalveolar organoids containing both club and type II cells were detected exclusively in the saline-treated mice that were absent in the urethane group. Significantly, whole exome sequencing of organoids identified several oncogenic mutations in organoids from urethane-treated mice but did not detect mutation in Kras, the known driver for urethane-induced ADC. Thus, our results pointed to an early response of stem cells following urethane exposure, with notable shifts in their behavior during premalignant progression to cancer. Citation Format: Bridget K. Greuel, Thomas Danhorn, Caroline Hauer, Meredith Tennis, Alison Bauer, Daniel T. Merrick, Debashis Ghosh, Robert L. Keith, York E. Miller, Moumita Ghosh. Epithelial stem cells in tumorigenesis of the lung: lessons from a murine model of lung adenocarcinoma [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 6347.