Cellular activation of the self-quenched fluorescent reporter probe in tumor microenvironment
Cellular activation of the self-quenched fluorescent reporter probe in tumor microenvironment
复制标题
DOI：
10.1038/sj.neo.7900238
复制
发表时间：
2002-05-01
期刊：
影响因子：
4.8
通讯作者：
Weissleder, R
中科院分区：
文献类型：
--
作者：
Bogdanov, AA;Lin, CP;Weissleder, R
近红外荧光（NIRF）自猝灭大分子探针（PGC-Cy5.5）的溶酶体内蛋白水解作用先前已被报道并用于肿瘤成像。在这里，我们证明可以在细胞水平上在体内无创地检测蛋白水解。在注射 PGC-Cy5.5 的荷瘤动物中进行 GFP 荧光（使用双光子激发）和 NIRF 的联合检测。皮下组织层（最多 160 mm）肿瘤细胞的体内显微镜显示，在 GFP 阴性细胞中具有强烈的 Cy5.5 去猝灭作用。这一观察结果得到了肿瘤分离细胞的流式细胞术、分选和逆转录聚合酶链反应分析的证实。 GFP 阳性（总共 81%）和 GFP 阴性（总共 19%）群体均含有 Cy5.5 阳性细胞。通过缺乏大鼠组织蛋白酶 mRNA 信号，确认 GIFP 阴性细胞是宿主小鼠细胞。 GFP 阴性细胞亚组分 (2.5-3.0%) 的 NIRF 强度比大多数 GFP 阳性或 GFP 阴性细胞（分别为 372 和 55 AU）高七倍。高度 NIRF 阳性、FP 阴性细胞为 CD45 和 MAC3 阳性。我们的结果表明：1）可以使用组织蛋白酶敏感探针在细胞水平上对细胞内蛋白水解进行体内成像； 2) 造血来源的肿瘤招募细胞最积极地参与长循环大分子探针的摄取和降解。
The effect of intralysosomal proteolysis of near-infrared fluorescent (NIRF) self-quenched macromolecular probe (PGC-Cy5.5) has been previously reported and used for tumor imaging. Here we demonstrate that proteolysis can be detected noninvasively in vivo at the cellular level. A codetection of GFP fluorescence (using two-photon excitation) and NIRF was performed in tumor-bearing animals injected with PGC-Cy5.5. In vivo microscopy of tumor cells in subdermal tissue layers (up to 160 mum) showed a strong Cy5.5 dequenching effect in GFP-negative cells. This observation was corroborated by flow cytometry, sorting, and reverse transcription polymerase chain reaction analysis of tumor-isolated cells. Both GFP-positive (81% total) and GFP-negative (19% total) populations contained Cy5.5-positive cells. The GIFP-negative cells were confirmed to be host mouse cells by the absence of rat cathepsin mRNA signal. The subfraction of GFP-negative cells (2.5-3.0%) had seven times higher NIRF intensity than the majority of GFP-positive or GFP-negative cells (372 and 55 AU, respectively). Highly NIRF-positive, FP-negative cells were CD45- and MAC3-positive. Our results indicate that: 1) intracellular proteolysis can be imaged in vivo at the cellular level using cathepsin-sensitive probes; 2) tumor-recruited cells of hematopoetic origin participate most actively in uptake and degradation of long-circulating macromolecular probes.