三株荧光素酶标记的小鼠乳腺癌细胞在小鼠体内生长及转移的比较

Comparison of the tumor growth and metastasis of tumors from three luciferase-labeled mouse breast cancer cell lines

目的采用活体成像技术比较三株荧光素酶标记的小鼠乳腺癌细胞在小鼠体内生长及转移情况,为研究肿瘤转移提供理想的动物模型以及活体分析方法。方法以荧光素酶(luciferase,Luc)作为报告基因导入小鼠乳腺癌细胞4T1、66c14和4TO7中,经G418筛选获得稳定表达荧光素酶的细胞克隆并扩大培养。标记细胞稀释成1×107cells/mL,取0.1 mL进行乳腺原位及尾静脉接种BALB/c小鼠,制作小鼠乳腺原位和尾静脉移植瘤模型,比较三株细胞在小鼠体内生长及转移情况。结果获得稳定表达荧光素酶基因的细胞克隆,将Luc标记的4T1、66c14、4TO7细胞对BALB/c小鼠乳腺原位接种后7 d,均有肿瘤生长,接种后28 d,4T1细胞乳腺原位移植瘤最大,66c14细胞瘤体次之,4TO7细胞瘤体最小;接种后35 d,三株细胞乳腺原位移植瘤大小较一致,但4T1和66c14原位移植瘤均发生转移,其中4T1细胞较66c14细胞转移严重,而4TO7细胞未见转移;接种后42 d,三株细胞乳腺原位移植瘤大小无明显差别,而4T1和66c14细胞随天数的增加,移植瘤转移程度逐渐严重,4T1较66c14细胞转移更严重,呈广泛性转移,4TO7细胞仍未见转移。将Luc标记的4T1、66c14、4TO7细胞对BALB/c小鼠尾静脉接种后7 d,小动物活体成像发现小鼠肺部均能检测到荧光,其中4T1细胞接种的小鼠肺部荧光信号最强,且小鼠陆续死亡;4TO7细胞接种小鼠肺部荧光信号次之;66c14细胞接种小鼠肺部荧光信号最弱。尾静脉接种后14 d,4TO7和66c14细胞随着观察天数的增加,转移程度逐渐严重,4TO7细胞接种小鼠肺部荧光信号较66c14细胞强且小鼠陆续死亡。结论乳腺原位自发转移模型较尾静脉转移模型更真实反应了肿瘤细胞在体的转移特性,且能完整地呈现肿瘤转移的全过程,可作为研究肿瘤转移的最理想模型。

Objective To compare the in vivo growth and metastasis of tumors from three luciferase-labeled mouse breast cancer cell lines （4T1-Luc, 66c14-Luc and 4TO7-Luc） by bioluminescence imaging system, and to develop ideal tumor models and living animal analysis technique for research of tumor metastasis and anti-metastatic therapy. Method A vector containing ]uciferasc gene was constructed and transfected into mouse breast cancer cells and selected with C,418 to obtain stable Luc-expressing clones. The cells in logarithmic phase were collected and diluted to 1 ~ 107cells/ v mL. Then 0. 1 mL cell suspension was inoculated into the second mammary fat pad on the right side of normal BALB/c mice to establish orthotopic tumor models. Another 0. 1 mL cell suspension was intravenously injected into the tail vein of BALB/c mice to establish metastasis models. Their tumorigenesis and metastasis were analyzed in vivo. Result Stable Luc-expressing cell lines were obtained. The whole-body optical imaging revealed that orthotopical tumors formed 7 days after cell inoculation. At 28 days after inoculation, the tumor size of 4T1 cells was the biggest, that of 66c14 cells was the second, and that of 4TO7 cells was the smallest. At 35 days after intravenous inoculation, the tumor sizes were similar among the three cell lines, while tumor metastasis were found from the 4T1 and 66c14 tumors. The metastasis of 4T1 tumors was more serious than that of 66e14 tumors. 4TO7 tumors didn＇t cause metastasis. At 42 days after intravenous inoculation, the tumor sizes were similar among the three cell lines. The metastasis of 4T1 and 66c14 tumors became more serious with the time passed, and the metastasis of 4T1 tumors were more extensive than that of 66c14 tumors. 4TO7 tumors still didn＇t show metastasis. At 7 days after intravenous inoculation, the whole-body optical imaging showed the presence of tumor formation in the lungs. The strongest fluorescence signal was observed in 4T1 tumors, followed by 4TO7 tumors, and weakest in the 66c14 tumors. At 14 days after intravenous inoculation, the metastases of 4TO7 and 66c14 tumors became more serious with time passed, and the fluorescence signal of 4T07 tumors was stronger than that of 66c14 tumors, and death of mice occurred in succession. Conclusions Mouse models of spontaneous metastasis from orthotopic breast cancer can better show metastatic characteristics and the whole metastatic process in vivo than the tail vein xenografted models, and provide an ideal model for studies of tumor metastasis.