Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan; Laboratory of Regulatory Sciences for Oligonucleotide Therapeutics, Clinical Drug Development Unit, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University , Osaka, Japan.
Mol Ther Methods Clin Dev. 2016 Mar 2;3:16001. doi: 10.1038/mtm.2016.1. eCollection 2016.
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.
循环肿瘤细胞（CTCs）是几种癌症中有前途的生物标志物，因此人们积极寻求在血液中检测和定量它们的方法和设备。最近开发了一种使用绿色荧光蛋白（GFP）表达的条件复制腺病毒（Ad）（rAd-GFP）的新型 CTC 检测系统；然而，当使用 rAd-GFP 时，人们担心会产生假阳性细胞（GFP 阳性的正常血细胞），尤其是在高滴度时。此外，由于 rAd-GFP 是基于识别 CAR 的 Ad 血清型 5 构建的，因此缺乏或表达低水平 Coxsackie 病毒-腺病毒受体（CAR）的 CTCs无法被 rAd-GFP 检测到。为了抑制假阳性细胞的产生，将与血细胞特异性 microRNA（miR-142-3p）完全互补的序列整合到 E1B 和 GFP 基因的 3'-非翻译区。此外，还将纤维蛋白替换为识别人 CD46 的 Ad 血清型 35，从而创建 rAdF35-142T-GFP。rAdF35-142T-GFP 不仅能有效地标记 CAR 阳性肿瘤细胞，还能标记 GFP 的 CAR 阴性肿瘤细胞。rAdF35-142T-GFP 的假阳性细胞数量明显低于 rAd-GFP。rAdF35-142T-GFP 可检测癌症患者血液中的 CTC，假阳性细胞数量大大减少。
