Intravital microcircular observation and metabolome analysis of the liver bearing human-derived metastatic colon cancer metastasis in the superimmunodeficient NOG mice

经门静脉循环播散性转移引起的肝功能衰竭是结肠癌最严重的临床并发症之一，但在荷瘤条件下维持器官能量代谢稳态的机制仍不清楚。我们的目的是通过建立人类结肠癌肝转移的实验模型来剖析这种机制，该模型使用的是超免疫缺陷NOD/scid/Y^null （NOG）小鼠，这些小鼠允许对表达HCT116的突变GFP （venus）结肠癌细胞在肝脏中的生长产生免疫耐受。通过一种新开发的荧光共聚焦显微镜成功地观察了肝脏中的转移癌细胞。这种显微镜可以获得高分辨率的肿瘤微循环图像，并且光毒性很小。本文建立的结肠癌细胞金星转染的NOG小鼠模型代表了门静脉转移途径，使我们能够对转移过程中的更多事件进行动态观察。采用常规的胶原酶灌注法提取肝细胞。将FACS分离的肝细胞（对照组）和静脉表达的荷瘤细胞分别用含有^<13> c13_6 -葡萄糖-、^<13> c_3 -丙酮酸-或^<15> n_2 -谷氨酰胺的培养基孵育，观察对照组和荷瘤细胞代谢谱的差异。利用CE/MS对这些细胞进行代谢组分析，我们发现糖酵解、TCA循环、戊糖磷酸途径和氨基酸谱的代谢物发生了显著变化。使用^<15> n标记谷氨酰胺的激发试验显示，来自荷瘤肝脏的肝细胞加速了氨基酸与核苷酸的结合，用于DNA合成；该事件与组织学观察相符，显示增殖标志物如PCNA的激活。这些结果共同表明，荷瘤条件触发肝细胞的代谢重塑，这可能满足细胞对DNA合成和翻译后蛋白修饰的代谢需求，但损害了通过糖异生传递葡萄糖的能力。少

Liver failure through disseminated metastasis through portal circulation is one of the most serious clinical complications of the colon cancer, although mechanisms for maintaining the organ homeostasis of energy metabolism under the tumor-bearing conditions remain largely unknown. We aimed to dissect such mechanisms by establishing an experimental model of the liver metastasis of human colon cancer using the superimmunodeficient NOD/scid/Y^null (NOG) mice that allow immunological tolerance for the growth of mutant GFP (venus)-expressing HCT116 colon cancer cells in the liver. The metastatic cancer cells in the liver were successfully visualized through a newly developed fluorescence confocal microscopy. This microscopy made it possible to achieve high resolution images of the tumor microcirculation with little phototoxicity. The presented NOG mouse model with Venus transfected colon cancer cells stands for the portal metastatic route and enables us to perform dynamic observation of ear … More ly events in metastasis.We collected the hepatocytes from the livers through the conventional collagenase perfusion method. The isolated hepatocyte(control)and venus-expressing tumor-bearing cells by FACS were incubated with ^<13>C13_6-glucose-, ^<13>C_3-pyruvate-, or ^<15>N_2-glutamine-contained medium, and the differences in metabolic profiling of hepatocytes between the control and tumor-bearing cells were examined. The metabolome analysis of these cells using CE/MS led us to reveal remarkable alterations in metabolites in glycolysis, TCA cycle, pentose phosphate pathway, and profiles of amino acids. The challenge test using ^<15>N-labelled glutamine revealed that hepatocytes from the tumor-bearing liver accelerate incorporation of the amino acid to nucleotides for DNA synthesis; the event matched histological observation showing activation of the proliferation marker such as PCNA.The results collectively suggest that tumor-bearing conditions trigger metabolic remodeling of the hepatocytes that might satisfy metabolic demand of the cells for DNA synthesis and post-translational protein modification but compromise the ability to deliver glucose through gluconeogenesis. Less