Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake.
Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake.
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DOI：
10.1186/s13550-016-0213-8
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发表时间：
2016-12
期刊：
影响因子：
3.2
通讯作者：
Sel-tag imaging project
中科院分区：
文献类型：
--
作者：
Cheng Q;Wållberg H;Grafström J;Lu L;Thorell JO;Hägg Olofsson M;Linder S;Johansson K;Tegnebratt T;Arnér ES;Stone-Elander S;Ahlzén HS;Ståhl S;Sel-tag imaging project
尽管表皮生长因子受体（EGFR）在几种形式的癌症中的过表达被认为是与不良预后相关的重要预后生物标志物，但生物标志物测定和患者管理之间的明确相关性一直难以建立。在这里，我们利用直接靶向，然后基于非靶向示踪剂的正电子发射断层扫描（PET）方法来检查确定肿瘤中特异性EGFR结合的一些方面。EGFR结合亲和体分子ZEGFR：2377和其大小匹配的非结合对照ZTaq：3638与C末端含硒代半胱氨酸的Sel-tag（ZEGFR：2377-ST和ZTaq：3638-ST）重组融合。蛋白质用DyLight 488位点特异性标记用于流式细胞术和离体组织分析，或用11 C位点特异性标记用于体内PET研究。在健康小鼠和携带来自人FaDu（鳞状细胞癌）和A431（表皮样癌）细胞系的异种移植物的小鼠中进行11 C标记的蛋白质的动力学扫描。还监测A431异种移植物中示踪剂摄取随时间的变化，随后进行EGFR表达的离体邻近连接测定（PLA）。流式细胞术和离体组织分析证实了ZEGFR：2377-ST-DyLight 488的EGFR靶向作用。[甲基-11 C]-标记的ZEGFR：2377-ST-CH 3和ZTaq：3638-ST-CH 3在体内显示出相似的分布，除了前者的浓度显著更高，特别是在肝脏和血液中。[甲基-11 C]-ZEGFR：2377-ST-CH 3成功地使分别具有中等和高EGFR表达水平的FaDu和A431异种移植物可视化。然而，在FaDu肿瘤中，非特异性摄取很大，有时同样大，说明了适当控制的重要性。在纵向观察的A431组中，非特异性摄取在观察期内保持相同水平。特异性摄取随肿瘤大小增加而增加，但个体肿瘤随时间变化很大。切除切片中的总（膜和胞质）EGFR随肿瘤生长而增加。总EGFR和特异性示踪剂摄取之间没有正相关性，这是因为ZEGFR：2377在细胞外结合并缓慢内化，表明可用的膜和总EGFR表达水平之间不一致。通过Sel-tag技术和11 C标记实现的同一天体内双示踪剂成像提供了一种非侵入性监测膜定位EGFR以及影响PET配体非特异性摄取的因素的方法。本文的在线版本（doi：10.1186/s13550-016-0213-8）包含补充材料，可供授权用户使用。
Though overexpression of epidermal growth factor receptor (EGFR) in several forms of cancer is considered to be an important prognostic biomarker related to poor prognosis, clear correlations between biomarker assays and patient management have been difficult to establish. Here, we utilize a targeting directly followed by a non-targeting tracer-based positron emission tomography (PET) method to examine some of the aspects of determining specific EGFR binding in tumors. The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377-ST and ZTaq:3638-ST). The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with 11C for in vivo PET studies. Kinetic scans with the 11C-labeled proteins were performed in healthy mice and in mice bearing xenografts from human FaDu (squamous cell carcinoma) and A431 (epidermoid carcinoma) cell lines. Changes in tracer uptake in A431 xenografts over time were also monitored, followed by ex vivo proximity ligation assays (PLA) of EGFR expressions. Flow cytometry and ex vivo tissue analyses confirmed EGFR targeting by ZEGFR:2377-ST-DyLight488. [Methyl-11C]-labeled ZEGFR:2377-ST-CH3 and ZTaq:3638-ST-CH3 showed similar distributions in vivo, except for notably higher concentrations of the former in particularly the liver and the blood. [Methyl-11C]-ZEGFR:2377-ST-CH3 successfully visualized FaDu and A431 xenografts with moderate and high EGFR expression levels, respectively. However, in FaDu tumors, the non-specific uptake was large and sometimes equally large, illustrating the importance of proper controls. In the A431 group observed longitudinally, non-specific uptake remained at same level over the observation period. Specific uptake increased with tumor size, but changes varied widely over time in individual tumors. Total (membranous and cytoplasmic) EGFR in excised sections increased with tumor growth. There was no positive correlation between total EGFR and specific tracer uptake, which, since ZEGFR:2377 binds extracellularly and is slowly internalized, indicates a discordance between available membranous and total EGFR expression levels. Same-day in vivo dual tracer imaging enabled by the Sel-tag technology and 11C-labeling provides a method to non-invasively monitor membrane-localized EGFR as well as factors affecting non-specific uptake of the PET ligand. The online version of this article (doi:10.1186/s13550-016-0213-8) contains supplementary material, which is available to authorized users.