Incubation and application of transgenic green fluorescent nude mice in visualization studies on glioma tissue remodeling

• Published in: Chinese medical journal 2012-12, Vol.125 (24), p.4349-4354
• Main Author: DONG Jun, DAI Xing-liang LU Zhao-hui FEI Xi-feng CHEN Hua ZHANG Quan-bin ZHAO Yao-dong WANG Zhi-min WANG Ai-dong LAN Qing HUANG Qiang
• Format: Article
• Language: English
• Subjects: Animals; Cell Line, Tumor; Female; Glioma - metabolism; Glioma - pathology; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Humans; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Mice, Transgenic; Red Fluorescent Protein; 共聚焦激光扫描显微镜; 可视化; 增强型绿色荧光蛋白; 病理组织学; 红色荧光蛋白; 脑胶质瘤; 裸鼠; 转基因
• ISSN: 0366-6999; 2542-5641
• DOI: 10.3760/cma.j.issn.0366-6999.2012.24.006

Background The primary reasons for local recurrence and therapeutic failure in the treatment of malignant gliomas are the invasion and interactions of tumor cells with surrounding normal brain cells. However, these tumor cells are hard to be visualized directly in histopathological preparations, or in experimental glioma models. Therefore, we developed an experimental human dual-color in vivo glioma model, which made tracking solitary invasive glioma cells possible, for the purpose of visualizing the interactions between red fluorescence labeled human glioma cells and host brain cells. This may offer references for further studying the roles of tumor microenvironment during glioma tissue remodeling. Methods Transgenic female C57BL/6 mice expressing enhanced green fluorescent protein （EGFP） were crossed with male Balb/c nude mice. Then sib mating was allowed to occur continuously in order to establish an inbred nude mice strain with 50% of their offspring that are EGFP positive. Human glioma cell lines U87-MG and SU3 were transfected with red fluorescent protein （RFP） gene, and a rat C6 glioma cell line was stained directly with CM-Dil, to establish three glioma cell lines emitting red fluorescence （SU3-RFP, U87-RFP, and C6-CM-Dil）. Red fluorescence tumor cells were inoculated via intra-cerebral injection into caudate nucleus of the EGFP nude mice. Tumor-bearing mice were sacrificed when their clinical symptoms appeared, and the whole brain was harvested and snap frozen for further analysis. Confocal laser scanning microscopy was performed to monitor the mutual interactions between tumor cells and host brain cells. Results Almost all the essential tissues of the established EGFP athymic Balb/c nude mice, except hair and erythrocytes, fluoresced green under excitation using a blue light-emitting flashlight with a central peak of 470 nm, approximately 50% of the offsprings were nu/nu EGFP＋. SU3-RFP, U87-RFP, and C6-CM-Dil almost 100% expressed red fluorescence under the fluorescence microscope. Under fluorescence microscopic view, RFP＋ cells were observed growing wherever they arrived at, locating in the brain parenchyma, ventricles, and para-vascular region. The interactions between the transplanted tumor cells and host adjacent cells could be classified into three types： （1） interweaving; （2） mergence; and （3） fusion. Interweaving was observed in the early stage of tumor remodeling, in which both transplantable tumor cells and host cells were observed scatt