The James Buchanan Brady Urological Institute and Department of Urology, The Johns Hopkins School of Medicine, 600 N Wolfe St, Baltimore, MD 21287.
The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Prostate. 2014 Sep;74(13):1286-1296. doi: 10.1002/pros.22845. Epub 2014 Jul 25.
Circulating tumor cells (CTCs) hold great promise as biomarkers and are a direct source of tumor cells through a simple blood draw. However, CTCs are rare and their detection requires sensitive and specific methods to overcome the overwhelming hematocyte population. Therefore, CTC detection remains technically challenging.
An assay was developed for detecting viable and tissue-specific CTCs using a tropism-enhanced and conditionally replicating reporter adenovirus (CTC-RV). Adenoviral replication was made prostate-specific by placing the E1A gene under the control of the probasin promoter and prostate-specific antigen enhancer (PSE-PBN). Viral tropism was expanded through capsid-displayed integrin targeting peptides. A secreted reporter, humanized Metridia Luciferase (hMLuc), was engineered for expression during the major late phase of viral replication. The assay involves red blood cell lysis, cell collection, viral infection, and subsequent quantification of reporter activity from cellular media. Assay and reporter stability, cell specificity and sensitivity were evaluated in cell dilution models in human blood.
A conditionally replicating prostate-selective adenovirus reporter and CTC assay system were generated. The secreted reporter, MLuc, was found to be stable for at least 3 days under assay conditions. CTC detection, modeled by cell dilution in blood, was selective for androgen receptor positive prostate cancer (PCa) cells. Serial dilution demonstrated assay linearity and sensitivity to as few as three cells. Prostate cancer cell viability declined after several hours in anticoagulated blood at ambient temperatures.
Conditionally replicative adenoviral vectors and secreted reporters offer a functional method to detect viable CTCs with cell specificity and high sensitivity.
循环肿瘤细胞（CTCs）作为生物标志物具有巨大的潜力，通过简单的抽血即可直接获得肿瘤细胞。然而，CTCs 数量稀少，其检测需要敏感和特异的方法来克服压倒性的血细胞群体。因此，CTC 的检测仍然具有技术挑战性。
我们开发了一种使用趋化性增强和条件复制报告腺病毒（CTC-RV）检测有活力和组织特异性 CTCs 的方法。通过将 E1A 基因置于前列腺特异性结合蛋白启动子和前列腺特异性抗原增强子（PSE-PBN）的控制下，使腺病毒复制具有前列腺特异性。通过展示整合素靶向肽来扩展病毒趋化性。构建了一个分泌型报告基因，人源化 Metridia 荧光素酶（hMLuc），用于在病毒复制的主要晚期表达。该检测法包括红细胞裂解、细胞收集、病毒感染以及随后从细胞培养基中定量报告基因活性。在人血的细胞稀释模型中评估了检测法和报告基因的稳定性、细胞特异性和敏感性。
生成了一种条件复制的前列腺选择性腺病毒报告基因和 CTC 检测系统。发现分泌型报告基因 MLuc 在检测条件下至少稳定 3 天。CTC 检测是通过细胞稀释在血液中模拟的，对雄激素受体阳性前列腺癌（PCa）细胞具有选择性。连续稀释显示检测法具有线性和对低至 3 个细胞的敏感性。在环境温度下，抗凝血液中前列腺癌细胞的活力在数小时后下降。
条件复制的腺病毒载体和分泌型报告基因为检测有活力的 CTC 提供了一种具有细胞特异性和高灵敏度的功能性方法。
