Abstract 62: Third hit genetic changes and clonal heterogeneity in a genetically engineered mouse lung cancer model
Authors: Um Sang-Won , Xiao Ping-Jie , Hayes David Neil , Kim William Y. , Randell Scott H. 
Published: May 2015

Background: Cancer cells may undergo mutations beyond their original causal changes, resulting in clonal expansion, and potentially driving tumor heterogeneity and evolution. Our hypothesis was that additional, “third hit” genetic changes in clonally derived tumors, originally generated by the same oncogenic stimulus, would have different phenotypes.

Methods: Epithelial cells were isolated from LSL-KrasG12D+/-/Trp53fl/fl/Rosa-CAG-LSL-tdTomato+/- and C57BL/6 control mouse tracheas and were cultured on plastic. On day 3, cells were treated with an adenovirus expressing Cre recombinase. After 7 days, single EPCAM+, 7-aminoactinomycin D (7-AAD)-, and tdTomato+ cells from the LSL-KrasG12D+/-/Trp53fl/fl/Rosa-CAG-LSL-tdTomato+/- mouse, and EPCAM+/ 7-AAD- cells from the control mouse were FACS sorted, and individually seeded in 96-well plates. Clones developing in each well were passaged separately and characterized using growth curves on plastic, staining for lineage markers, and colony forming efficiency (CFE) and colony morphology in 3D culture. Selected clones were further investigated in vivo after subcutaneous injection into the flanks of NOD/SCID mice. Agilent SureSelect targeted exome sequencing was used to evaluate genetic variations in cells derived from each cell clone.

Results: Thirty-five LSL-KrasG12D+/-/Trp53fl/fl/Rosa-CAG-LSL-tdTomato+/- transformed clones and 2 control mouse clones were serially passaged, and 4 transformed cell lines representing distinct phenotypes (named MT1, 2, 3, and 4), were identified based on growth rates, lineage marker staining, CFE, and 3D colony morphology. All 4 clones were pancytokeratin+, 3 were thyroid trasnscription factor-1 (TTF-1)+ and one was p63+. All 4 cell lines generated tumors in NOD/SCID mice, which exhibited variable histology and marker expression. Exome sequencing revealed unique copy number profiles. Specifically, MT1 had gains and losses in chromosome 3, MT2 had losses in chromosome 9, MT3 had losses in chromosomes 7 and 14, and MT4 had losses in chromosome 7. Mutations potentially representing third hits were identified in each cell line, particularly Ss18l1 (synovial sarcoma translocation gene on chromosome 18-like 1) and Gas7 (growth arrest specific 7) in MT1, Runx1 (runt related transcription factor 1) and Dicer1 (dicer 1, ribonuclease type III) in MT2, Apc (adenomatous polyposis coli) in MT3, and Csmd1 (CUB and Sushi multiple domains 1) in MT4.

Conclusions: Multiple single cell derived mouse tracheal epithelial tumor cell clones were generated from a single LSL-KrasG12D+/-/Trp53fl/fl/Rosa-CAG-LSL-tdTomato+/- mouse. Selected clones exhibited unique growth rates, lineage marker expression patterns, CFE, 3D cell morphology and variable in vivo tumor histology and marker expression. Unique copy number aberrations and third hit mutations likely generate distinct clone phenotypes, driving tumor heterogeneity and evolution.

Citation Format: Sang-Won Um, Ping-Jie Xiao, David Neil Hayes, William Y. Kim, Scott H. Randell. Third hit genetic changes and clonal heterogeneity in a genetically engineered mouse lung cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 62. doi:10.1158/1538-7445.AM2014-62