器官特异性转移肺癌细胞株的筛选及建立

Screening and Establishment of Human Lung Cancer Cell Lines with Organ-specific Metastasis Potential

背景与目的肺癌转移是肺癌的恶性标志和特征,也是肺癌病人治疗失败和死亡的主要原因。肺癌转移具有器官特异性,最常转移的部位是淋巴结、大脑、骨、肝脏和肾上腺。本研究的目的是应用我们实验室的人高转移大细胞肺癌细胞株L9981,筛选鉴定出具有器官特异性转移的肺癌亚代细胞株,为进一步研究肺癌细胞器官特异转移提供科学可靠的细胞模型。方法通过裸鼠实验,将母系细胞株L9981-Luc皮下接种,每周一次动物活体成像观察肺癌器官转移情况,数周后构建出具有肺、脊柱、纵隔淋巴结和大脑等器官特异性转移的小鼠模型;处死裸鼠,切取肺、脊柱、纵隔淋巴结和大脑器官肺癌转移瘤进行原代培养,构建具有器官靶向特异性转移潜能的肺癌亚代细胞株;将第一代器官特异性转移肺癌细胞株接种裸鼠皮下,再次构建肺癌器官特异性转移小鼠模型;通过反复多次将肺癌器官特异性转移瘤构建肺癌细胞株,再裸鼠接种,最终获得具有肺、脊柱、纵隔淋巴结和大脑器官特异性转移的肺癌细胞株。结果经过裸鼠反复接种,动物活体成像、动物体内反复筛选鉴定,成功构建了4株分别特异性转移到肺、脊柱、纵隔淋巴结和大脑的器官特异性转移肺癌细胞株,分别命名为L9981-LuM、L9981-BoM、L9981-LnM和L9981-BrM。结论成功构建出具有肺、纵隔淋巴结、脊柱和大脑特异性转移的人大细胞肺癌细胞模型,为进一步研究肺癌器官特异转移的分子机制、信号调控途径,以及未来研究和开发抑制或/和阻断肺癌转移的分子靶向药物提供了可靠的细胞模型。

Background and objective Cancer metastasis is not only the malignant marker and characteristics, but also the main cause of failure to cure and lose their life in the patients with lung cancer. Lung cancer metastasis has organ- specific characteristics. The most common sites of lung cancer metastasis are mediastinal lymph node, brain, bone, liver and adrenal gland. The aim of this study is to screen and establish lung cancer cell model with organ-specific metastasis potential with human high-metastatic large cell lung cancer cell line L9981 established by our laboratory previously, and to provide cell models for studying the mechanisms and signal regulation of organ-specific metastasis of lung cancer. Materials and meth- ods The parent lung cancer cell line, L9981-Luc, was inoculated in the armpit of nude mice. The live animal imaging system, MS-200, was used to detect the lung cancer organ-specific metastasis every week. When the organ-specific metastasis were established, the nude mites bearing the lung cancer were sacrificed when they became moribund. Under sterile conditions, the organs （mediastinal lymph nodes, lung, spinal column and brain） with lung cancer organ-specific metastasis were removed and the metastasized nodules were dissected free of connective tissue and blood dots, and rinsed twice with medium. The metas- tasized nodules were finely minced using sterile scalpel blades in medium, and the cells were seeded in tissue culture dishes. Then, the cells with organ-specific metastasis potential were reinoculated into the armpit of nude mice, respectively. Thisprocesses were repeated to establish the organ-specific metastatic sublines of L9981-Luc cell line more than 10 times. Finally, the organ-specific metastasis sublines of L9981-Luc were screened and established, which the four cell lines have the characteristics only metastasized to brian, lung, bone and mediastinal lymph node. Results A group of organ-specific metastasis cell lines which only metastasized to brian, lung, bone and mediastinal lymph node were successfully established through repeating reinocnlatation, live animal imaging in nude mice, and screening and identification in vitro. We named the four cell lines as L9981-BoM, L9981-LuM, L9981-BrM and L9981-LnM, respectively. The L9981-BoM cell was only metastasized to bone. The 19981-LuM cell was only metastasized to lung. The L9981-BrM only metastasized to brain. The L9981-LnM cell was only metastasized to midiastinal lymph nodes. Conclusions A human large cell lung cancer cell model with bone, lung, brain and lymph node-specific metastasis potential was successfully established. It will be helpful to further study the molecular mechanisms and signal regulation of lung cancer organ- specific metastasis. It will be to also provide reliable cell model for developing new techniques and molecular targeting drugs of inhibiting or reversing lung cancer metastasis.