Abstract B09: Lineage tracing of tumor-propagating cells in LUAD
Authors: Marini Kieren D. , Hwang Elizabeth , Lee Alex , Sayles Leanne , Sweet-Cordero Alejandro 
Published: June 2020

Lung cancer is the leading cause of cancer-related death worldwide. Non-small lung cancer accounts for 85% of diagnoses, and the predominant subtype is lung adenocarcinoma (LUAD). LUAD has a less than 20% response rate to conventional chemotherapy, and despite identification of several therapeutic targets, the overall 5-year survival is only 23%. Previously, we identified a population of tumor propagating cells (TPCs) in the Kras; TP53 GEM model of LUAD. We demonstrated that TPCs have a greater capacity for secondary tumor formation and are enriched after treatment with cisplatin. The TPC population is defined by the expression of CD24, ITGB4, and Notch high, with further functional validation showing that Notch3 contributes to this propagation phenotype in a nonredundant fashion. Currently, we are attempting to use mouse modeling to get a more granular understanding of Notch3 signaling and TPC biology. Specifically, we want to examine the cell types/progeny generated by Notch3 expressing cells and the effects of removing these cells on the tumor microenvironment. Further to this, we want to determine the dynamism of Notch3 expression in lung adenocarcinoma and how this affects cell state. To investigate the biology of these TPCs, we have a KrasFSFG12D; Tp53FRT/FRT; Notch3CreERT2; RFP; inducible diphtheria toxin receptor mouse (KPFFNRiDTR) to perform lineage tracing and ablation of Notch3-expressing tumor cells. In this model, adenovirus expressing FLP is delivered intratracheally, leading to expression of an oncogenic form of Kras and deletion of TP53, initiating LUAD tumors. Subsequently, upon administration of tamoxifen, Notch3-expressing cells are specifically labeled with RFP and simultaneously induce expression of the DTR. Dosing with diphtheria toxin then eliminates cells that were Notch positive at the time of tamoxifen labeling. By labeling/ablating cells at different disease progressions, we can infer the importance of Notch signaling at key timepoints and assess the contribution of Notch3-expressing cells to the tumor microenvironment. Preliminary experiments show that Notch3 is not expressed in the epithelial compartment of the adult lung but instead is expressed in the lung pericytes. The Notch3CreER was crossed into the standard KP model to test if Notch3-expressing cells had the capacity to form LUAD, and 12 weeks after initiation with tamoxifen, no formation of LUAD tumors was observed, suggesting that the cell of origin for LUAD does not express Notch3, even if the TPC does. Despite Notch3 not being epithelial in the adult lung, we do see colocalization with known AT1/AT2 cell type markers after tumor initiation in the KPFFNRiDTR model, suggesting that Notch3 expression is gained by tumors after initiation. Interestingly, from the patterning of Notch3 positivity observed in these tumors, it does not seem that the TPCs are maintained in a quiescent state, suggesting that the TPC state is not dormant and instead may be an active signaling process.

Citation Format: Kieren D. Marini, Elizabeth Hwang, Alex Lee, Leanne Sayles, Alejandro Sweet-Cordero. Lineage tracing of tumor-propagating cells in LUAD [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B09.