In Vivo Stable Tumor-Specific Painting in Various Colors Using Dehalogenase-Based Protein-Tag Fluorescent Ligands
In Vivo Stable Tumor-Specific Painting in Various Colors Using Dehalogenase-Based Protein-Tag Fluorescent Ligands
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DOI：
10.1021/bc9001344
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发表时间：
2009-07-01
影响因子：
4.7
通讯作者：
Kobayashi, Hisataka
中科院分区：
文献类型：
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作者：
Kosaka, Nobuyuki;Ogawa, Mikako;Kobayashi, Hisataka
体内荧光癌症成像是理解肿瘤生长和治疗监测的重要工具，并且可以用内源性产生的荧光蛋白或外源性引入的与靶向分子结合的荧光探针进行。然而，内源性荧光蛋白在转染后不能改变，因此需要针对每种期望的颜色重新衍生细胞系，而外源靶向荧光探针受到天然存在的细胞靶标的异质表达的限制。在这项研究中，我们适应了脱卤酶为基础的蛋白质标签（HaloTag）系统在体内癌症成像，通过引入高度表达的HaloTag受体（HaloTagR）在癌细胞耦合的范围内的外部注射的荧光团共轭脱卤酶反应性反应性接头。从单个转染细胞系产生的肿瘤结节用发射光谱前绿色至近红外变化的荧光稳定标记。在建立和验证用HaloTagR稳定转染的SHIN 3细胞系（HaloTagR-SHIN 3）后，使用腹膜播散模型在活动物中进行体内光谱荧光成像研究。由HaloTagR-SHIN 3产生的肿瘤结节可以被四种不同的荧光团缀合的HaloTag配体成功标记，每种荧光团缀合的HaloTag配体以不同的波长发光。这些荧光团可以在允许评估间隔生长的连续成像会话上交替。固定后，组织学标本中保留了荧光。因此，该标记系统证明是通用的，在体内和体外成像，而不需要修改的基础细胞系。因此，这种策略可以克服与目前使用的内源性荧光蛋白和外源性靶向光学试剂的使用相关的一些限制。
In vivo fluorescence cancer imaging is an important tool in understanding tumor growth and therapeutic monitoring and can be performed either with endogenously produced fluorescent proteins or with exogenously introduced fluorescent probes bound to targeting molecules. However, endogenous fluorescence proteins cannot be altered after transfection, thus requiring rederivation of cell lines for each desired color, while exogenously targeted fluorescence probes are limited by the heterogeneous expression of naturally occurring cellular targets. In this study, we adapted the dehalogenase-based protein-Tag (HaloTag) system to in vivo cancer imaging, by introducing highly expressed HaloTag receptors (HaloTagR) in cancer cells coupled with a range of externally injected fluorophore-conjugated dehalogenase-reactive reactive linkers. Tumor nodules arising from a single transfected cell line were stably labeled with fluorescence varying in emission spectra front green to near-infrared. After establishing and validating a SHIN3 cell line stably transfected with HaloTagR (HaloTagR-SHIN3), in vivo spectral fluorescence imaging Studies were performed in live animals using a peritoneal dissemination model. The tumor nodules arising from HaloTagR-SHIN3 could be successfully labeled by four different fluorophore-conjugated HaloTag-ligands each emitting light at different wavelengths. These fluorophores could be alternated on serial imaging sessions permitting assessment of interval growth. Fluorescence was retained in histological specimens after fixation. Thus, this tagging system proves versatile both for in vivo and in vitro imaging without requiring modification of the underlying cell line. Thus, this strategy can overcome some of the limitations associated with the use of endogenous fluorescent proteins and exogenous targeted optical agents in current use.