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

超免疫缺陷NOG小鼠人源性结肠癌转移肝脏的活体微循环观察和代谢组分析

基本信息

批准号：
18591524
负责人：
SHIMZAU Motohide
金额：
$ 2.57万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591524/
关键词：
Live Metastasis Metabolome Immunodeficient mouse 肝臓外科学肝転移代謝癌抗がん剤

项目摘要

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

尽管在荷瘤条件下维持器官能量代谢稳态的机制仍然很大程度上未知，但门静脉循环播散性转移导致的肝衰竭是结肠癌最严重的临床并发症之一。我们的目的是通过使用超级免疫缺陷型 NOD/scid/Y^null (NOG) 小鼠建立人类结肠癌肝转移的实验模型来剖析此类机制，该模型允许对肝脏中表达突变型 GFP (venus) 的 HCT116 结肠癌细胞的生长产生免疫耐受。通过新开发的荧光共聚焦显微镜成功地观察到肝脏中的转移性癌细胞。这种显微镜可以实现肿瘤微循环的高分辨率图像，且光毒性很小。所提出的金星转染结肠癌细胞的 NOG 小鼠模型代表门静脉转移途径，使我们能够动态观察转移中的耳部事件。我们通过传统的胶原酶灌注方法从肝脏收集肝细胞。将通过FACS分离的肝细胞(对照)和表达金星的荷瘤细胞与含有^ 13 C13_6-葡萄糖-、^ 13 C_3-丙酮酸或^ 15 N_2-谷氨酰胺的培养基一起温育，并检查对照细胞和荷瘤细胞之间肝细胞代谢谱的差异。使用 CE/MS 对这些细胞的代谢组分析使我们揭示了糖酵解、TCA 循环、磷酸戊糖途径和氨基酸谱中代谢物的显着变化。使用^ 15 N-标记的谷氨酰胺的攻击测试揭示，来自荷瘤肝脏的肝细胞加速氨基酸与核苷酸的掺入以进行DNA合成；该事件与显示增殖标记物（如 PCNA）激活的组织学观察相匹配。结果总体表明，肿瘤条件触发了肝细胞的代谢重塑，这可能满足细胞对 DNA 合成和翻译后蛋白质修饰的代谢需求，但会损害通过糖异生传递葡萄糖的能力。较少的