Purpose

We recently developed a new instrument called “diffuse in vivo flow cytometry” (DiFC) for enumeration of rare fluorescently labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins or were pre-labeled with a fluorescent dye ex vivo. In this work, we investigated the use of a folate receptor (FR)-targeted fluorescence molecular probe for in vivo labeling of FR+ CTCs for DiFC.

Procedures

We used EC-17, a FITC-folic acid conjugate that has been used in clinical trials for fluorescence-guided surgery. We studied the affinity of EC-17 for FR+ L1210A and KB cancer cells. We also tested FR− MM.1S cells. We tested the labeling specificity in cells in culture in vitro and in whole blood. We also studied the detectability of labeled cells in mice in vivo with DiFC.

Results

EC-17 showed a high affinity for FR+ L1210A and KB cells in vitro. In whole blood, 85.4 % of L1210A and 80.9 % of KB cells were labeled above non-specific background with EC-17, and negligible binding to FR− MM.1S cells was observed. In addition, EC-17-labeled CTCs were readily detectable in circulation in mice with DiFC.

Conclusions

This work demonstrates the feasibility of labeling CTCs with a cell-surface receptor-targeted probe for DiFC, greatly expanding the potential utility of the method for pre-clinical animal models. Because DiFC uses diffuse light, this method could be also used to enumerate CTCs in larger animal models and potentially even in humans.

中文翻译：

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用叶酸受体靶向分子探针对循环肿瘤细胞进行荧光标记，用于体内扩散流式细胞术。

目的

我们最近开发了一种名为“体内扩散流式细胞术”(DiFC)的新仪器，用于在不抽取血液样本的情况下对小动物中罕见的荧光标记循环肿瘤细胞 (CTC) 进行计数。到目前为止，我们使用的细胞系表达荧光蛋白或预先用荧光染料离体标记。在这项工作中，我们研究了使用叶酸受体 (FR) 靶向荧光分子探针在体内标记 DiFC 的 FR+ CTC。

程序

我们使用 EC-17，一种 FITC 叶酸结合物，已用于荧光引导手术的临床试验。我们研究了 EC-17 对 FR+ L1210A 和 KB 癌细胞的亲和力。我们还测试了 FR-MM.1S 电池。我们测试了体外培养和全血细胞中的标记特异性。我们还使用 DiFC研究了小鼠体内标记细胞的可检测性。

结果

EC-17在体外对 FR+ L1210A 和 KB 细胞显示出高亲和力。在全血中，85.4% 的 L1210A 和 80.9% 的 KB 细胞用 EC-17 标记在非特异性背景之上，观察到与 FR-MM.1S 细胞的结合可以忽略不计。此外，在 DiFC 小鼠的循环中很容易检测到 EC-17 标记的 CTC。

结论

这项工作证明了用细胞表面受体靶向探针为 DiFC 标记 CTC 的可行性，极大地扩展了该方法在临床前动物模型中的潜在效用。由于 DiFC 使用漫射光，因此该方法还可用于在较大的动物模型中甚至在人类中枚举 CTC。