Isolation of large soft agar clones from HBEC3p53,KRAS and HBEC3p53,KRAS,MYC identifies tumorigenic and nontumorigenic clones and genome-wide mRNA expression.

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Isolation of large soft agar clones from HBEC3p53,KRAS and HBEC3p53,KRAS,MYC identifies tumorigenic and nontumorigenic clones and genome-wide mRNA expression profiling of HBEC3p53,KRAS soft agar clones identifies a clinically applicable signature of prognos... Isolation of large soft agar clones from HBEC3p53,KRAS and HBEC3p53,KRAS,MYC identifies tumorigenic and nontumorigenic clones and genome-wide mRNA expression profiling of HBEC3p53,KRAS soft agar clones identifies a clinically applicable signature of prognosis. A, uncloned, parental populations of HBEC3p53,KRAS and HBEC3p53,KRAS,MYC form very large (>1-mm diameter) soft agar colonies (arrowhead). These large colonies were isolated, expanded, and retested for soft agar colony formation where they maintained the ability to form large soft agar colonies (representative soft agar pictures of 2 clones; ×4 magnification). B, immunoblot of HBEC3p53,KRAS,MYC soft agar clones confirming p53, KRASV12, and c-MYC manipulations. The presence (+) or absence (−) of each manipulation is shown. C, anchorage-independent soft agar colony formation ability of HBEC3p53,KRAS soft agar clones. D, unsupervised hierarchical clustering of whole-genome mRNA expression profiles of HBEC3p53,KRAS soft agar clones harvested at 2 time points [denoted “(1)” and “(2)”] spanning a 3-week interval. E, a supervised analysis comparing HBEC3p53,KRAS tumorigenic (clone 1, 5, and 11) with HBEC3p53,KRAS nontumorigenic (clone 6, 7, 8, and 9) clones identified 203 probes, representing 171 unique genes, significantly differentially expressed (SAM, FDR = 5%). Samples (represented horizontally: red, tumorigenic clones; green, nontumorigenic clones) and probes (represented vertically) were clustered using centered Pearson clustering. F, Kaplan–Meier log-rank analysis of overall survival in patients with lung cancer predicted to have good (black) or poor (red) outcome using the 171 probe signature HBEC3p53,KRAS soft agar signature. A supervised principal component analysis was used to train the model in one dataset (Consortium) and test in a second dataset (SPORE; top) then the datasets were reversed to test for model robustness (bottom). Full-length blots are presented in Supplementary Fig. S8. Mitsuo Sato et al. Mol Cancer Res 2013;11:638-650 ©2013 by American Association for Cancer Research