KRAS、TP53和LKB1突变在人类和小鼠LUADs中的差异共发生和影响(A) MSK在人肺腺癌中KRAS、TP53和LKB1突变共发生的影响,Fisher精确测试了具有KRAS突变或野生型背景的LUAD中LKB1和TP53突变共发生的统计可能性。(B) Fisher对KRAS突变或野生型背景中LKB1和TP53突变同时发生的统计可能性的精确检验。(C)用tdTomato荧光报告细胞标记的H358细胞系等基因克隆在Matrigel中三维球形生长,并表达CAS9和非靶向对照(sgNT1.4和sgNT1.6)或lkb1特异性(sgLKB1-2.1和sgLKB1-3.2)引导rna。将5000个细胞植入Matrigel,在每24小时更换一次的培养基中培养10天。在EVOS荧光显微镜下,在4倍放大和滤波下拍摄tdTomato信号强度和亮场。(D)用tdTomato荧光报告基因标记的H2009细胞系等基因克隆在Matrigel中三维球形生长,表达CAS9和非靶向对照(sgNT1.1和sgNT1.2)或lkb1特异性(sgLKB1-3.1和sgLKB1-3.7)引导rna。将5000个细胞播种到Matrigel中,在每24小时更换一次的培养基中培养10天。在尼康荧光显微镜下4倍放大和滤波处理tdTomato信号强度和亮场的图像。(E)在CMV启动子控制下,gemm衍生的mLUAD细胞系含有转基因慢病毒表达的GFP,在基质中3D球形生长。将5000个细胞接种到Matrigel中,在每24小时更换一次的培养基中进行10天的检测。 Images taken on EVOS fluorescence microscope under 10x magnification and filter to resolve GFP signal intensity and brightfield. (F) Mean (-/+ s.e.m.) volumes of mouse 634T (KP) and Lkb1-t2 (KPL) lung adenocarcinoma allograft tumors. 1 x 104 cells implanted in right hind flank (n = 10 per cohort). (G) 3D spheroid growth in Matrigel of isogenic clones of the H358 cell line labeled with a tdTomato fluorescent reporter and expressing Cas9 and non-targeting controls (sgNT1.6) or LKB1-specific (sgLKB1-3.2) guide RNAs. 1,000 cells were seeded into Matrigel and grown for 14 days in media changed every 72 hours with addition of fresh AMG-510. Dose range for AMG-510 was 1 nM—1 µM Images taken on EVOS fluorescence microscope under 4x magnification and filter to resolve tdTomato signal intensity. (H) Western blot analysis of H2009 (KRAS;TP53) isogenic clones (KP: sgNT1.1 and sgNT1.2; KPL: sgLKB1-3.1 and sgLKB1-3.7) and lines with additional transgenic expression of guide RNA resistant LKB1 wildtype (WT) (sgLKB1-3.1 + LKB1 WT and sgLKB1-3.7 + LKB1 WT) or LKB1 kinase inactive (KI) (sgLKB1-3.1 + LKB1 KI and sgLKB1-3.7 LKB1 KI) and treated with 11.1 mM or 0.5 mM glucose for 6 hours as indicated. Restoration of AMPK signaling in LKB1 WT lines in response to 0.5 mM glucose validated by blotting for P-AMPK Thr172 and downstream substrates (P-ACC S79, P-ULK1 S555, P-Raptor S792). Similar results observed in three independent experiments. Credit:癌症的发现(2023)。DOI: 10.1158 / 2159 - 8290. - cd - 22 - 0805
目前在达纳法伯癌症中心(Dana Farber Cancer Center)工作的瓦姆斯博士、坎特利博士,以及威尔康奈尔大学(Weill Cornell)和其他机构的其他合作者,开始深入研究这三种基因产物在小鼠模型和培养中的表现人类细胞.该小组采用的技术包括基因工程、蛋白质组学、代谢组学和小鼠模型的前沿技术,以及经典的生化和细胞生物学实验。