Telomerase-specific fluorescent navigation system for lung cancer surgery

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，其荧光可以通过表达绿色荧光蛋白（GFP） （OBP-401）的端粒酶特异性复制能力腺病毒来检测。OBP-401包含复制盒，其中人类端粒酶逆转录酶（hTERT）启动子驱动El基因的表达，以及用于监测病毒复制的GFP基因。hTERT是端粒酶的催化亚基，端粒酶在癌细胞中高度活跃，但在大多数正常体细胞中处于静止状态。当OBP-401感染人肺癌细胞系H1299时，病毒在肿瘤细胞中复制，并表现出强烈的绿色信号。相比之下，OBP-401感染在正常细胞如成纤维细胞中未显示任何信号。我们还发现，瘤内注射OBP-401后，已建立的皮下肿瘤可以可视化。移植到BALB/c nu/nu小鼠体内的H1299人肺肿瘤瘤内注射1 × 10^8斑块形成单位（PFU）的OBP-401。在治疗24小时内，通过3CCD相机在这些肿瘤中可以看到表达的GFP的荧光。此外，胸廓内给药OBP-401可以显示胸廓内植入小鼠弥散性A549肿瘤结节。结果表明，瘤内或胸内注射OBP-401可能是一种有效的诊断方法，为今后的临床应用提供了基础。

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 telomerase-specific replication-competent adenovirus expressing green fluorescent protein (GFP) (OBP-401). OBP-401 contains the replication cassette, in which the human telomerase reverse transcriptase (hTERT) promoter drives expression of El genes, and the GFP gene for monitoring viral replication. The hTERT is the catalytic subunit of telomerase, which is highly active in cancer cells, but is quiescent in most normal somatic cells. When the human lung cancer cell line H1299 was infected with OBP-401, the virus replicated in tumor cells and showed strong green signals. In contrast, infection of OBP-401 did not show any signals in normal cells such as fibroblasts. We also found that established subcutaneous tumors could be visualized following intratumoral injection of OBP-401. H1299 human lung tumors transplanted into BALB/c nu/nu mice were intratumorally injected with 1 x 10^8 plaque forming units (PFU) of OBP-401. Within 24 hours of treatment, the fluorescence of the expressed GFP became visible by 3CCD camera in these tumors. Moreover, intrathoracic administration of OBP-401 could visualize disseminated A549 tumor nodules in mice following intrathoracic implantation. Our results indicate that intratumoral or intrathoracic injection of OBP-401 might be a useful diagnostic method that provides a foundation for future clinical application.