Dual-labeled trastuzumab-based imaging agent for the detection of human epidermal growth factor receptor 2 overexpression in breast cancer
Dual-labeled trastuzumab-based imaging agent for the detection of human epidermal growth factor receptor 2 overexpression in breast cancer
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DOI：
10.2967/jnumed.107.042234
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发表时间：
2007-09-01
影响因子：
9.3
通讯作者：
Sevick-Muraca, Eva M.
中科院分区：
文献类型：
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作者：
Sampath, Lakshmi;Kwon, Sunkuk;Sevick-Muraca, Eva M.
人类表皮生长因子受体(HER)家族的过度表达与癌症有关，因为它参与了调节细胞增殖、分化、运动和生存的信号通路。在这项工作中，我们利用曲妥珠单抗的胞外结合特性，设计了一种诊断显像剂(In-111-DTPA)(N)-曲妥珠单抗-(IRDye 800CW)(M)，它被伽马发射体In-111和近红外(NIR)荧光染料IRDye 800CW双重标记，以检测HER2在乳腺癌细胞中的过表达。化学计量比“n”和“m”分别是指每个曲妥珠单抗分子中结合的二乙三胺五乙酸二酐(DTPA)和IRDye 800CW分子的数量。方法：用荧光显微镜和共聚焦显微镜检测(DTPA)(N)-曲妥珠单抗(IRDye800)(M)体外对乳腺癌细胞SKBr3(HER2阳性)和MDA-MB-231(HER2阴性)的分子特异性。将SKBr3细胞与(DTPA)(N)-曲妥珠单抗(IRDye800)(M)或IRDye800CW孵育，或先用曲妥珠单抗或人免疫球蛋白处理，再用(DTPA)(N)-曲妥珠单抗-(IRDye800)(M)处理，在荧光显微镜下观察。为进行体内研究，将荷瘤裸鼠皮下移植瘤HER2高表达SKBr3-Luc的裸鼠静脉注射(In-111-DTPA)(N)-trastuzumab-(IRDye800)(M)，48h后进行SPECT和近红外荧光成像，荷瘤小鼠在注射(In-111-DTPA)(N)-trastuzumab-(IRDye800)(M)前24 h静脉注射曲妥珠单抗。通过给小鼠注射IRDye 800CW和(In-111-DTPA)(P)-Ig G-(IRDye800)(Q)来分析SKBr3-Luc肿瘤的非特异性摄取，其中“p”和“q”分别是每个抗体结合的DTPA和IRDye 800CW的化学计量比。结果：(DTPA)(N)-曲妥珠单抗(IRDye800)(M)与SKBr3细胞的结合显著高于与MDA-MB-231细胞的结合。共聚焦成像显示这种结合主要发生在细胞膜周围。用过量的曲妥珠单抗与(DTPA)(N)-曲妥珠单抗(IRDye800)(M)孵育前的竞争结合研究取消了这种结合亲和力，但用非特异性免疫球蛋白(Ig G)预处理不改变结合。体内注射(In-111-DTPA)(N)-trastuzumab-(IRDye800)(M)的SKBr3-Luc移植瘤的核成像和光学成像显示，肿瘤区域的摄取明显多于对侧肌肉区域。用曲妥珠单抗预处理的小鼠和注射IRDye 800CW和(In-111-DTPA)(P)-Ig G(IRDye800)(Q)的小鼠，肿瘤与肌肉的比率降低。解剖器官的体外成像证实了这些结果。组织学苏木精-伊红染色与来自肿瘤和肌肉组织切片的放射自显影信号的联合配准表明，(In-111-DTPA)(N)-trastuzumab-(IRDye800)(M)仅结合在肿瘤组织中，而不结合在肌肉中。结论：双标记(In-111-DTPA)(N)-曲妥珠单抗-(IRDye800)(M)可能是一种能追踪乳腺癌患者HER2过表达的有效生物标志物。
Overexpression of the human epidermal growth factor receptor (HER) family has been implicated in cancer because of its participation in signaling pathways regulating cellular proliferation, differentiation, motility, and survival. In this work, we exploited the extracellular binding property of trastuzumab, a clinically therapeutic monoclonal antibody to the second member of the HER family (HER2), to design a diagnostic imaging agent, (In-111-DTPA)(n)-trastuzumab-(IRDye 800CW)(m), that is dual labeled with In-111, a gamma-emitter, and a near-infrared (NIR) fluorescent dye, IRDye 800CW, to detect HER2 overexpression in breast cancer cells. The stoichiometric ratios "n" and "m" refer to the number of diethylenetriaminepentaacetic acid dianhydride (DTPA) and IRDye 800CW molecules bound per trastuzumab molecule, respectively. Methods: Fluorescence microscopy and confocal microscopy were used to determine the molecular specificity of (DTPA)(n)-trastuzumab-(IRDye800)(m) in vitro in SKBr3 (HER2-positive) and MDA-MB-231 (HER2-negative) breast cancer cells. SKBr3 cells were incubated with (DTPA)(n)-trastuzumab-(IRDye800)(m) or IRDye800CW or pretreated with trastuzumab or human IgG followed by (DTPA)(n)-trastuzumab-(IRDye800)(m) and examined under a fluorescence microscope. For in vivo characterization, athymic nude mice bearing HER2-overexpressing SKBr3-luc subcutaneous xenografts were injected intravenously with (In-111-DTPA)(n)-trastuzumab-(IRDye800)(m) and imaged with SPECT and NIR fluorescence imaging at 48 h. Tumor-bearing mice were also injected intravenously with trastuzumab 24 h before administration of (In-111-DTPA)(n)-trastuzumab-(IRDye800)(m). Nonspecific uptake in the SKBr3-luc tumors was analyzed by injecting the mice with IRDye 800CW and (In-111-DTPA)(p)-IgG-(IRDye800)(q), where "p" and "q" are the stoichiometric ratios of DTPA and IRDye 800CW bound per IgG antibody, respectively. Results: (DTPA)(n)-trastuzumab-(IRDye800)(m) showed significantly greater binding to SKBr3 cells than to MDA-MB-231 cells. Confocal imaging revealed that this binding occurred predominantly around the cell membrane. Competitive binding studies with excess trastuzumab before incubation with (DTPA)(n)-trastuzumab-(IRDye800)(m) abolished this binding affinity, but pretreatment with nonspecific IgG did not alter binding. In vivo nuclear and optical imaging of SKBr3-luc xenografts injected with (In-111-DTPA)(n)-trastuzumab-(IRDye800)(m) revealed significantly more uptake in the tumor region than in the contralateral muscle region. The tumor-to-muscle ratio decreased in mice pretreated with trastuzumab and in mice injected with IRDye 800CW and (In-111-DTPA)(p)-IgG(IRDye800)(q). Ex vivo imaging of dissected organs confirmed these results. Finally, coregistration of histologic hematoxylin-eosin stains with autoradiography signals from tumor and muscle tissue slices indicated that (In-111-DTPA)(n)-trastuzumab-(IRDye800)(m) bound only in tumor tissue and not to muscle. Conclusion: Dual-labeled (In-111-DTPA)(n)-trastuzumab-(IRDye800)(m) may be an effective diagnostic biomarker capable of tracking HER2 overexpression in breast cancer patients.