Abstract 1161: Visualizing lung cancer heterogeneity by color-coding clonal cell subpopulations
Authors: Liao Yong , Valdecanas David R. , Liu Shangfeng , Molkintene David , Wani Khalida M. , Patel Nalini B. , Wang Li , Dai Chun , Milas Luka , Hittelman Walter N. 
Published: February 2011

Abstract
Tumors are composed of heterogeneous cell populations exhibiting different biological features (e.g., growth rate, metastatic propensity, responsiveness to therapy) that differentially determine disease outcome. However, it has been difficult to map heterogeneity within tumors and correlate it to specific biological features. This study's aim was to develop a model system where clonal tumor subpopulations are differentially color tagged and can be directly correlated with biological features. Two approaches were used to color-code tumor subpopulations: lung tumor H460 cells were stably transfected with either (a) fluorescence-tagged Histone H2b (GFP or RFP) plasmids or (b) Brainbow vectors encoding different fluorescent proteins with intervening loxP and loxP variant recombination sites. Cre induction generated stable, multicolor, clonal cell populations. When these cell populations were mixed and grown in vitro, each color-coded clone expanded and occupied a spatially distinct region discernable by color thresholding imaging techniques. When these same populations were injected into the leg of nude mice, they generated solid tumors exhibiting spatially distinct, color-coded neighborhoods of clonally derived cells. While the various populations exhibited similar cloning efficiencies in vitro, they exhibited different tumor growth capabilities and growth patterns (e.g., compact versus dispersed) in vivo. To further determine whether these clonal variants possess different biological behaviors, we tested their sensitivities to radiation using an in vitro clonogenic survival assay whereby mixed populations were irradiated with 2-6 Gy γ-rays and the identities of surviving clones determined by color thresholding. Color-coded clones significantly varied in their radiosensitivities (i.e., the radiation dose required to reduce survival to 0.1 ranged from 2.4- to 5-Gy, and the survival fraction at 2-Gy ranged from 0.15 to 0.91). While the most sensitive clone lacked a shoulder on the radiation dose-survival curve, the most resistant clone exhibited a broad shoulder indicative of high repair proficiency. The introduction of color did not affect radiation sensitivity. Established tumor xenografts of these mixed cell populations were exposed to 20-Gy radiation and then analyzed for preferential clonal regrowth over 12 days following radiation. Preliminary analyses revealed evidence of preferential clonal regrowth. These results suggest that this color-coding methodology offers a unique capability for visualizing clonal subpopulations within tumors and quantifying their impact on therapeutic outcome. Further molecular characterization (and genetic manipulation) of these subpopulations may facilitate delineation of critical factors determining behavior in heterogeneous tumors. (Supported in part by the Susan G. Komen BCRT0504146, NIH-NCI CA 06294, CA 103820, and DAMD W81XWH-04-1-01)
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1161.