Efficient Isolation and Accurate InSitu Analysis of Circulating Tumor Cells Using Detachable Beads and a High-Pore-Density Filter
Efficient Isolation and Accurate InSitu Analysis of Circulating Tumor Cells Using Detachable Beads and a High-Pore-Density Filter
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DOI：
10.1002/anie.201302278
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发表时间：
2013-08-05
影响因子：
16.6
通讯作者：
Huh, Nam
中科院分区：
文献类型：
--
作者：
Lee, Hun Joo;Oh, Jin Ho;Huh, Nam
肿瘤患者血液循环中的肿瘤细胞(CTCs)可提示肿瘤转移进展的可能性和严重程度。CTCs的鉴定、计数和鉴定可能为评估患者的癌症状况和制定个性化的抗癌治疗提供一种微创方法。[1]然而，CTCs的检测需要从患者的全血中分离这些细胞，这是困难的，因为它们的数量很少(大约每109个患者血液中有一个CTC)。[2]许多技术被用于分离CTC，包括密度梯度离心法和介电泳法，但使用基于大小的排除或基于亲和力的浓缩的方法很常见。[3]基于大小的排除假设CTCs比大多数造血细胞大，并去除所有小于预定大小阈值的细胞。[4]基于亲和力的浓缩依赖于肿瘤细胞特有的表面蛋白的表达，而造血细胞中不存在。这些方法通常使用抗体结合的磁珠，并通过磁分离来丰富CTCs，如CellSearch系统。[5]由于它们的异质性，使用上述方法可能实际上不可能分离出高分离效率的CTCs。据报道，一些CTC的大小几乎相同，甚至小于白细胞，这使得它们很难区分大小。[6]至于蛋白表达，在上皮向间充质转化(EMT)过程中，上皮标志物，如EPCAM(上皮细胞黏附分子)下调。[7]此外，表面蛋白的类型和表达水平可能因癌症组织亚型而有很大差异。[8]考虑到这些差异，找到正确的抗体或抗体组合来持续捕获所有CTC可能被证明是困难的。为了最大限度地提高分离效率，我们设计了一种双模式分离策略，结合了基于亲和力的浓缩和基于大小的排斥。[9]通过使用与CTC特异性抗体结合的微珠，CTC的大小可以扩大，以实现对白细胞的更好识别。随后的尺寸过滤隔离了结合珠粒的CTC，允许回收更小尺寸的CTC。然而，所有基于珠粒的捕获方法都有固有的局限性，即无法进行准确的图像分析，这是由于附着在细胞上的珠子的存在造成的光学失真。附着的珠子不仅阻碍对细胞形态的观察，而且实际上可以改变荧光信号强度(图1)，表明原位定量分析与基于珠子的捕获方法不兼容(参见支持信息)。
Circulating tumor cells (CTCs) in the bloodstream of cancer patients may indicate the likelihood and severity of metastatic progression. Identification, enumeration, and characterization of CTCs may provide a minimally invasive method for assessing cancer status of patients and prescribing personalized anticancer therapy.[1] However, examination of CTCs requires isolation of these cells from whole blood of patients, which is difficult owing to their low quantity (around one CTC per 109 non-cancerous hematopoietic cells in patient blood).[2] Many techniques are used to isolate CTCs, including density gradient centrifugation and dielectrophoresis, but methods using either size-based exclusion or affinity-based enrichment are common.[3] Size-based exclusion assumes that CTCs are larger than most hematopoietic cells and removes all cells smaller than a pre-determined size threshold.[4] Affinity-based enrichment relies on the expression of surface proteins specific to cancer cells and absent in hematopoietic cells. These methods generally use antibody-conjugated magnetic beads and enrich for CTCs by magnetic separation, such as the CellSearch system.[5] Owing to their heterogeneous nature, it may be practically impossible to isolate CTCs with high isolation efficiency using the aforementioned methods. Some CTCs are reported to be nearly identical in size or even smaller than leukocytes, making them difficult to discriminate by size.[6] As for protein expression, epithelial markers, such as EpCAM (epithelial cell adhesion molecule), are downregulated during epithelialto-mesenchymal transition (EMT).[7] Furthermore, the type and expression levels of surface proteins may vary greatly depending on cancer histological subtype.[8] Considering these variations, finding the right antibody or combination of antibodies that consistently captures all CTCs may prove to be difficult.To maximize isolation efficiency, we devised a dual-mode isolation strategy that combines affinity-based enrichment and size-based exclusion.[9] By using microbeads conjugated with CTC-specific antibodies, the size of CTCs can be augmented to enable better discrimination against leukocytes. Subsequent size filtration isolates bead-bound CTCs, allowing the recovery of even smaller-sized CTCs. However, all beadbased capture methods have the inherent limitation of prohibiting accurate image analysis, which is due to optical distortion created by the presence of beads attached to cells. The attached beads not only impede observation of cellular morphology but can actually alter fluorescence signal intensities (Figure 1), demonstrating the incompatibility of in situ quantitative analysis with bead-based capture methods (see the Supporting Information).