Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
Mol Med. 2011;17(7-8):628-34. doi: 10.2119/molmed.2011.00078. Epub 2011 Apr 8.
Cytological analysis of body fluids is currently used for detecting cancer. The objective of this study was to determine if the herpes virus carrying an enhanced green fluorescent protein (EGFP) could detect rare cancer cells in body fluids against millions of normal cells. Human cancer cells suspended with normal murine cells were infected with NV1066 at a multiplicity of infection (MOI) of 0.5 and 1.0 for 18 h. Fluorescent microscopy and flow cytometry were used for EGFP detection of cancer cells. EGFP-expressing cells were confirmed as cancer cells with specific markers by immunohistochemistry staining. Limits of detection of cancer cells in body fluid were measured by serial dilutions. Applicability of technique was confirmed with samples from patients with malignant pleural effusions. NV1066 expressed EGFP in 111 human cancer cell lines detected by fluorescent microscopy at an MOI of 0.5. NV1066 selectively infected cancer cells and spared normal cells as confirmed by immunohistochemistry. Sensitivity of detecting fluorescent green cells was 92% (confidence interval [CI] 83% to 97%) at a ratio of 1 cancer cell to 1 million normal cells. EGFP-positive cells were detected by fluorescent microscopy in patients' malignant pleural effusion samples. Our data show proof of the concept that NV1066-induced EGFP expression allows detection of a single cancer cell against a background of 1 million normal cells. This method was demonstrated to be a reliable screening tool for human cancer cells in a suspension of normal murine cells as well as clinical specimens of malignant pleural effusions.
体液细胞学分析目前用于检测癌症。本研究的目的是确定携带增强型绿色荧光蛋白（EGFP）的疱疹病毒是否能够在数以百万计的正常细胞中检测到罕见的癌细胞。将与人癌细胞混合的正常鼠细胞用 NV1066 在感染复数（MOI）为 0.5 和 1.0 的条件下感染 18 小时。用荧光显微镜和流式细胞术检测 EGFP 对癌细胞的检测。用免疫组织化学染色法用特定标记物证实表达 EGFP 的细胞为癌细胞。通过连续稀释来测量体液中癌细胞的检测下限。通过对恶性胸腔积液患者的样本进行技术适用性验证。在 MOI 为 0.5 的情况下，荧光显微镜检测到 111 个人癌细胞系表达 EGFP。NV1066 选择性感染癌细胞而不感染正常细胞，这一点通过免疫组织化学得到证实。在 1 个癌细胞对 100 万个正常细胞的比例下，检测到荧光绿色细胞的灵敏度为 92%（置信区间[CI]为 83%至 97%）。在患者的恶性胸腔积液样本中，通过荧光显微镜检测到 EGFP 阳性细胞。我们的数据证明了这样一个概念，即 NV1066 诱导的 EGFP 表达允许在 100 万个正常细胞的背景下检测到单个癌细胞。该方法已被证明是一种可靠的筛选工具，可用于悬浮在正常鼠细胞中的人癌细胞以及恶性胸腔积液的临床标本。
