近红外荧光染料标记表皮生长因子探针在荷人乳腺癌裸鼠移植瘤中的分子成像研究

Molecular imaging of human breast cancer xenografts in nude mice by epidermal growth factor labelled by near infrared fluorescent dye

目的探讨近红外荧光（NIRF）染料Cy5．5标记的表皮生长因子（EGF）在乳腺癌EGF受体（EGFR）特异性显像中的价值。方法流式细胞仪检测人乳腺腺癌细胞株（MDA—MB-231）和人乳腺导管腺癌细胞株（MDA—MB-435S）的EGFR表达水平。激光共聚焦显微镜观察MDA—MB-231和MDA—MB-435S对EGF—Cy5．5的摄取。建立MDA-MB-231和MDA-MB-435S裸鼠胸部乳腺癌移植瘤模型，经尾静脉注射EGF-Cy5．5（1nmol／0．2ml）后，即刻进行NIRF活体成像，在不同时间点连续采集图像，利用ROI技术检测EGF-Cy5．5在MDA-MB-231和MDA-MB-435S肿瘤组织摄取情况，通过体外及活体阻断实验观察抗EGFR单克隆抗体C225对细胞摄取EGF—Cy5．5的阻断作用。对离体肿瘤进行荧光成像并进行病理切片HE染色。采用t检验比较肿瘤区与对侧正常区平均荧光强度及阻断实验对荧光强度的影响。结果流式细胞学检测显示MDA—MB-231 EGFR阳性表达百分比为41．96％，MDA—MB-435S为0．12％。激光共聚焦显微镜显示MDA—MB-231对EGF—Cy5．5的摄取可被C225阻断，MDA—MB-435S对EGF—Cy5．5无特异性摄取。静脉注射EGF—Cy5．524h后MDA-MB-231肿瘤区平均荧光强度为（38220±3144）all，明显高于对侧正常组织区的（11980±1496）au（t=17．491，P〈0．01）及MDA—MB-435S肿瘤区的（11885±1144）all（t=17．600，P〈0．01）。用C225预处理后的MDA—MB-231肿瘤平均荧光强度为（10472±842）au，与MDA-MB-231未阻断组相比平均荧光强度明显减弱（t=16．772，P〈0．01）；而阻断组MDA．MB-435S肿瘤区平均荧光强度为（11460±896）au，与MDA—MB-435S未阻断组的平均荧光强度差异无统计学意义（t=0．6504，P=0．551）。离体MDA-MB-231肿瘤内可探测到明显的荧光信号，HE染色证实为低分化腺癌。结论EGF-Cy5．5能与乳腺癌细胞的EGFR特异性结合，NIRF成像能实时、无创示踪EGF—Cy5．5在EGFR阳性肿瘤中的分布。

Objective This work was to determine the feasibility of using epidermal growth factor （EGF） labeled with a near-infrared fluorescent （NIRF） dye （Cy5. 5） to selectively localize and image epidermal growth factor receptor （EGFR）of the human breast cancer. Methods MDA-MB-231 of human mammary adenocarcinoma and MDA-MB-435S of human mammary ductal carcinoma were detected using a flow eytometry. Laser confocal microscopy was used to examine the intake of EGF-Cy5. 5 by MDA-MB-231 and MDA-MB-435S cells. MDA-MB-231 and MDA-MB-435S human breast cancer orthotopic xenograft nude mice models were established. In vivo NIRF imaging was acquired after intravenous injection of EGF-Cy5. 5 （1 nmoL/0. 2 ml）immediately and different time intervals. The up-take of EGF-CyS. 5 in MDA-MB-231 and MDA-MB-435S was detected using ROI technique. The blockage of the monocolonal antibody C225 to the EGF-Cy5. 5 uptake were observed in viovo and ex vivo. Ex vivo tumor tissue fluorescent imaging was executed and the histological sections were stained by HE method for pathobiology assay. Student t test was used for statistical analysis with SPSS for Windows. Results Flow cytometry indicated the EGFR expression percentage of MDA-MB-231 was gl. 96% and the percentage of MDA-MB-435S was 0. 12% . The fluorescence showed that the uptake of EGF-Cy5. 5 by MDA-MB-231 cells could be observed by the laser confocal microscopy. There was no specific uptake of EGF-CyS. 5 by MDA-MB-435S. In vivo NIRF images showed mean fluorescence intensity in MDA-MB-231 tumors was （ 38 220 ± 3 144 ） au, which was significantly higher than MDA-MB-435S＇ s （ 11 885 ± 1 144） au （ t = 17. 600, P 〈 0. 01 ） or normal region＇ s （ 11 980 ± 1 496） au （ t = 17. 491, P 〈 0. 01 ） at 24 h postinjection of EGF-CyS. 5. The mean fluorescence intensity was significantly reduced in MDA-MB-231 group with preadministration of C225, whose mean intensity was （ 10 472 ± 842 ） an （ t = 16. 772, P 〈 0. 01 ）, while the mean intensity was not apparently decreased in MDA-MB-435S block group, whose mean intensity was （11 460 ± 896） au （t = 0. 6504 ,P = 0. 551 ）. Apparent fluorescence signal can be detected in the ex vivo MDA-MB-231 tumor. The presence of poorly differentiated adenocarcinoma was confirmed by HE stain and pathology morphology. Conclusion This study suggested that EGF-Cy5.5 can specifically combined with EGFR, which may be applied to noninvasive NIRF imaging of tumors highly expressed EGFR in vivo.