Abstract 1983: Enriching EpCAM+/Sca-1+ mouse bronchioalveolar stem cells to generate lung adenocarcinoma

Abstract

Abstract Background: Clarifying the cells of origin in human lung adenocarcinoma will contribute to a better understanding of oncogene-induced lung adenocarcinomas and to the development of new treatments. Alveolar type II cells and club cells (Clara cells) have been shown to be the cells of origin by lineage-tracing approaches and conditional transgenic mice. Additionally, bronchioalveolar stem cells (BASCs), which are adult murine distal lung epithelial stem cells, are considered to be the cells of origin in lung adenocarcinomas because BASCs have both self-renewal abilities and differentiation properties. We attempted to enrich BASCs from mouse lungs and determine whether or not BASCs have the potential to form tumors driven by the oncogene. Methods: The epithelial cell adhesion molecule (EpCAM)+/CD45-/CD31- lung cells were isolated from the Ink4a/Arf-/- C57BL/6 mice by using a fluorescence activated cell sorting (FACS). The cells were then subjected to 3D culture on Matrigel with B27, EGF, KGF, ROCK inhibitor in serum-free DMEM/F-12. For detection of BASCs, we investigated the expressions of EpCAM and Sca-1 by flow cytometric analyses. We also investigated the expressions of SP-C and CC-10 by immunofluorescence analyses. The cells were then infected with bicistronic retroviruses having green fluorescent protein (GFP) and either KRASG12V or EML4-ALK genes. The GFP-positive oncogene-expressing cells were sorted by FACS and expanded under 3D culture conditions. Cells were then transplanted into the recipient C57BL/6 mice so that we could investigate their tumorigenic activities. Results: Flow cytometric analyses revealed that the EpCAM+/Sca-1+ cells, which are putative BASCs, were enriched in the 3D culture of the isolated EpCAM+/CD45-/CD31- lung cells from Ink4a/Arf-/- mice. The percentage of EpCAM+/Sca-1+ cell fraction was 36.97%, 92.63%, 99.98%, at primary EpCAM+/CD45-/CD31- lung cells and after the first and second passage, respectively. We also detected a small population of SP-C+/CC-10+ cells in colonies formed under our 3D culture conditions. The oncogene-expressing cells maintained the population of EpCAM+/Sca-1+ cells; 88.64-99.89% and 72.72-99.12%, KRASG12V and EML4-ALK, respectively. These cells formed differentiated tumors which were similar to human lung adenocarcinomas, having papillary, acinar, and solid components. Conclusions: The EpCAM+/Sca-1+ lung cells, putative BASCs, from Ink4a/Arf-/- mice were enriched in 3D culture. These cells formed histologically different types of lung adenocarcinomas after the transduction of KRASG12V or EML4-ALK, suggesting that we had established the source of the lung adenocarcinoma cells. Citation Format: Takashi Semba, Ryo Sato, Akiyoshi Kasuga, Makoto Suzuki, Hideyuki Saya, Yoshimi Arima. Enriching EpCAM+/Sca-1+ mouse bronchioalveolar stem cells to generate lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1983.