Department of Radiology, University of Wisconsin - Madison Madison, WI, USA.
Am J Transl Res. 2013 Apr 19;5(3):291-302. Print 2013.
THE GOAL OF THIS STUDY IS TO EMPLOY THE HALOTAG TECHNOLOGY FOR POSITRON EMISSION TOMOGRAPHY (PET), WHICH INVOLVES TWO COMPONENTS: the HaloTag protein (a modified hydrolase which covalently binds to synthetic ligands) and HaloTag ligands (HTLs). 4T1 murine breast cancer cells were stably transfected to express HaloTag protein on the surface (termed as 4T1-HaloTag-ECS, ECS denotes extracellular surface). Two new HTLs were synthesized and termed NOTA-HTL2G-S and NOTA-HTL2G-L (2G indicates second generation, S stands for short, L stands for long, NOTA denotes 1,4,7-triazacyclononane-N,N'N''-triacetic acid). Microscopy studies confirmed surface expression of HaloTag in 4T1-HaloTag-ECS cells, which specifically bind NOTA-HTL2G-S/L. Uptake of (64)Cu-NOTA-HTL2G-L in 4T1-HaloTag-ECS tumors (4.3 ± 0.5, 4.1± 0.2, 4.0 ± 0.2, 2.3 ± 0.1, and 2.2 ± 0.1 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection respectively; n = 4) was significantly higher than that in the 4T1 tumors (3.0 ± 0.3, 3.0± 0.1, 3.0 ± 0.2, 2.0 ± 0.4, and 2.4 ± 0.3 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection respectively; n = 4) at early time points. In comparison, (64)Cu-NOTA-HTL2G-S did not demonstrate significant uptake in either 4T1-HaloTag-ECS or 4T1 tumors. Blocking studies and autoradiography of tumor lysates confirmed that (64)Cu-NOTA-HTL2G-L binds specifically to HaloTag protein in the 4T1-HaloTag-ECS tumors, corroborated by histology. HaloTag protein-specific targeting and PET imaging in vivo with (64)Cu-NOTA-HTL2G-L serves as a proof-of-principle for future non-invasive and sensitive tracking of HaloTag-transfected cells with PET, as well as many other studies of gene/protein/cell function in vivo.
本研究的目的是将 HaloTag 技术应用于正电子发射断层扫描（PET），该技术包含两个组成部分： HaloTag 蛋白（一种经过修饰的水解酶，可与合成配体共价结合）和 HaloTag 配体（HTLs）。4T1 鼠乳腺癌细胞被稳定转染以在表面表达 HaloTag 蛋白（称为 4T1-HaloTag-ECS，ECS 表示细胞外表面）。合成了两种新的 HTLs，并分别命名为 NOTA-HTL2G-S 和 NOTA-HTL2G-L（2G 表示第二代，S 表示短，L 表示长，NOTA 表示 1,4,7-三氮杂环壬烷-N,N'N''-三乙酸）。显微镜研究证实，4T1-HaloTag-ECS 细胞表面表达 HaloTag，可特异性结合 NOTA-HTL2G-S/L。4T1-HaloTag-ECS 肿瘤中（64）Cu-NOTA-HTL2G-L 的摄取（分别在注射后 0.5、3、6、18 和 24 小时为 4.3±0.5、4.1±0.2、4.0±0.2、2.3±0.1 和 2.2±0.1 %ID/g；n=4）明显高于 4T1 肿瘤（分别在 0.5、3、6、18 和 24 小时为 3.0±0.3、3.0±0.1、3.0±0.2、2.0±0.4 和 2.4±0.3 %ID/g；n=4）。相比之下，（64）Cu-NOTA-HTL2G-S 在 4T1-HaloTag-ECS 或 4T1 肿瘤中均无明显摄取。肿瘤裂解物的阻断研究和放射自显影证实，（64）Cu-NOTA-HTL2G-L 特异性结合 4T1-HaloTag-ECS 肿瘤中的 HaloTag 蛋白，这与组织学结果一致。（64）Cu-NOTA-HTL2G-L 对 HaloTag 蛋白的特异性靶向和体内 PET 成像为未来利用 PET 对 HaloTag 转染细胞进行非侵入性和敏感跟踪以及对体内基因/蛋白/细胞功能进行许多其他研究提供了原理验证。
