Molecular Fluorescent Imaging of Metastatic Tumors with Conditionally Replication-Selective Adenovirus and GFP Gene

目前可用的体内肿瘤检测方法，如x射线、计算机断层扫描和超声检查是无创的，并且研究得很好；然而，这些图像并不是针对肿瘤的。活体肿瘤细胞的直接光学成像可以清楚地将其与周围的正常组织区分开来，这在临床上可能是有用的。在这里，我们描述了一种可视化肿瘤的新方法，其荧光可以使用肿瘤特异性复制能力腺病毒（OBP-301，“端粒酶”）与表达GFP的复制缺陷腺病毒（Ad-GFP）联合检测。人端粒酶逆转录酶（hTERT）是端粒酶的催化亚基，在癌细胞中高度活跃，而在大多数正常体细胞中处于静止状态。我们构建了一个腺病毒5载体，其中hTERT启动子元件驱动与IRES相关的E1A和E1B基因的表达，并表明OBP-301仅在人类癌细胞中有效复制，而在正常细胞中却不能复制。以低感染倍数（MOI）感染人肺癌细胞株H1299和结肠癌细胞株SW620时，荧光显微镜下无法检测到GFP的表达；然而，当OBP-301存在时，Ad-GFP在这些肿瘤细胞中复制并显示出强烈的绿色信号。相比之下，OBP-301和Ad-GFP共同感染在正常细胞如WI-38成纤维细胞中未显示任何信号。我们还发现，在瘤内注射OBP-301和Ad-GFP后，已建立的皮下肿瘤可以可视化。将A549人肺肿瘤和SW620人结肠肿瘤移植到BALB/c nu/nu小鼠体内，瘤内注射8 × 10^5斑块形成单位（PFU）的Ad-GFP和8 × 10^6 PFU的OBP-301。在治疗3天内，在这些肿瘤中表达的GFP的荧光可以通过3CCD相机看到，而单独在瘤内注射Ad-GFP不能诱导GFP荧光。此外，胸廓内给药Ad-GFP和OBP-301可以显示胸廓内植入小鼠弥散性A549肿瘤结节。我们的结果表明，瘤内或胸内注射Ad-GFP联合OBP-301可能是一种有用的诊断方法，为未来的临床应用提供基础。少

Currently available methods for detection of tumors in vivo such as X-ray, computed tomography, and ultrasonography are noninvasive and well studied ; the images, however, are not specific for tumors. Direct optical imaging of tumor cells in vivo that can clearly distinguish them from surrounding normal tissues may be clinically useful. Here, we describe a new approach to visualizing tumors whose fluorescence can be detected using tumor-specific replication-competent adenovirus (OBP-301, "Telomelysin") in combination with replication-deficient adenovirus expressing GFP (Ad-GFP). Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, which is highly active in cancer cells, but is quiescent in most normal somatic cells. We constructed an adenovirus 5 vector, in which the hTERT promoter element drives expression of E1A and E1B genes linked with an IRES, and showed that OBP-301 replicated efficiently exclusively in human cancer cells, but not in normal cells … More such as human fibroblasts. When the human lung and colon cancer cell lines H1299 and SW620 were infected with Ad-GFP at low multiplicity of infection(MOI), GFP expression could not be detected under a fluorescence microscope ; in the presence of OBP-301, however, Ad-GFP replicated in these tumor cells and showed strong green signals. In contrast, co-infection of OBP-301 and Ad-GFP did not show any signals in normal cells such as WI-38 fibroblasts. We also found that established subcutaneous tumors could be visualized following intratumoral injection of OBP-301 and Ad-GFP. A549 human lung tumors and SW620 human colon tumors transplanted into BALB/c nu/nu mice were intratumorally injected with 8 x 10^5 plaque forming units(PFU) of Ad-GFP in combination with 8 x 10^6 PFU of OBP-301. Within 3 days of treatment, the fluorescence of the expressed GFP became visible by 3CCD camera in these tumors, whereas intratumoral injection of Ad-GFP alone could not induce GFP fluorescence. Moreover, intrathoracic administration of Ad-GFP and OBP-301 could visualize disseminated A549 tumor nodules in mice following intrathoracic implantation. Our results indicate that intratumoral or intrathoracic injection of Ad-GFP in combination with OBP-301 might be a useful diagnostic method that provides a foundation for future clinical application. Less