Efficient detection of human circulating tumor cells without significant production of false-positive cells by a novel conditionally replicating adenovirus.
Efficient detection of human circulating tumor cells without significant production of false-positive cells by a novel conditionally replicating adenovirus.
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DOI：
10.1038/mtm.2016.1
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Mizuguchi H
中科院分区：
文献类型：
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作者：
Sakurai F;Narii N;Tomita K;Togo S;Takahashi K;Machitani M;Tachibana M;Ouchi M;Katagiri N;Urata Y;Fujiwara T;Mizuguchi H
循环肿瘤细胞(CTCs)是几种癌症中很有前景的生物标志物，因此血液中CTCs的检测和定量的方法和装置一直是人们积极追求的对象。最近发展了一种利用绿色荧光蛋白(GFP)表达的条件复制腺病毒(Ad)(Rad-GFP)检测CTC的新系统，但使用Rad-GFP时会产生假阳性细胞(GFP阳性的正常血细胞)，特别是在高滴度时。此外，由于rad-GFP是基于识别柯萨奇病毒-腺病毒受体(CAR)的Ad血清型5而构建的，因此Rad-GFP不能检测到缺乏或表达低水平柯萨奇病毒-腺病毒受体(CAR)的CTCs。为了抑制假阳性细胞的产生，将与血细胞特异性microRNA完全互补的序列miR-142-3p整合到E1B和GFP基因的3‘-非翻译区。此外，纤维蛋白被识别人CD46的Ad血清型35取代，产生了rAdF35-142T-GFP。RAdF35-142T-GFP不仅能有效地标记CAR阳性的肿瘤细胞，还能有效地标记CAR阴性的肿瘤细胞。RAdF35-142T-GFP的假阳性细胞数显著低于rad-GFP。用rAdF35-142T-GFP检测癌症患者外周血中的CTCs，假阳性细胞明显减少。
Circulating tumor cells (CTCs) are promising biomarkers in several cancers, and thus methods and apparatuses for their detection and quantification in the blood have been actively pursued. A novel CTC detection system using a green fluorescence protein (GFP)–expressing conditionally replicating adenovirus (Ad) (rAd-GFP) was recently developed; however, there is concern about the production of false-positive cells (GFP-positive normal blood cells) when using rAd-GFP, particularly at high titers. In addition, CTCs lacking or expressing low levels of coxsackievirus–adenovirus receptor (CAR) cannot be detected by rAd-GFP, because rAd-GFP is constructed based on Ad serotype 5, which recognizes CAR. In order to suppress the production of false-positive cells, sequences perfectly complementary to blood cell–specific microRNA, miR-142-3p, were incorporated into the 3′-untranslated region of the E1B and GFP genes. In addition, the fiber protein was replaced with that of Ad serotype 35, which recognizes human CD46, creating rAdF35-142T-GFP. rAdF35-142T-GFP efficiently labeled not only CAR-positive tumor cells but also CAR-negative tumor cells with GFP. The numbers of false-positive cells were dramatically lower for rAdF35-142T-GFP than for rAd-GFP. CTCs in the blood of cancer patients were detected by rAdF35-142T-GFP with a large reduction in false-positive cells.