Hughes Andrew D, Mattison Jeff, Powderly John D, Greene Bryan T, King Michael R
Department of Biomedical Engineering, Cornell University, USA.
J Vis Exp. 2012 Jun 15(64):e4248. doi: 10.3791/4248.
Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of >50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.
循环肿瘤细胞（CTC）是从原发性肿瘤扩散至整个循环系统，并最终可能在远处形成继发性肿瘤的细胞。基于血液中CTC浓度与疾病严重程度之间的相关性，CTC计数可用于追踪疾病进展。作为一种治疗工具，可在实验室中对CTC进行研究以开发个性化疗法。为此，CTC分离过程中不得造成细胞损伤，并且必须尽可能避免其他细胞类型（尤其是白细胞）的污染。包括唯一获得美国食品药品监督管理局（FDA）批准用于CTC计数的设备在内，当前的许多技术在分离过程中都会破坏CTC（更多信息见参考文献2）。本文描述了一种用于捕获活CTC的微流控设备，该设备表面除了有针对上皮标志物的抗体外，还功能化有E-选择蛋白糖蛋白。为提高设备性能，应用了一种由埃洛石纳米管组成的纳米颗粒涂层，埃洛石纳米管是一种从粘土中提取的铝硅酸盐纳米颗粒。E-选择蛋白分子提供了一种捕获快速流动的CTC的方法，这些CTC被泵入设备，这比其他微流控设备具有优势，在其他微流控设备中需要更长的处理时间，以便为靶细胞提供足够的时间与表面相互作用。针对上皮靶点的抗体赋予了该设备CTC特异性，同时还提供了一个易于调节的参数来调整分离效果。最后，与其他技术相比，埃洛石纳米管涂层通过帮助捕获快速移动的细胞、增加蛋白质吸附的表面积以及排斥污染的白细胞，显著增强了分离效果。该设备采用现成材料通过一种简单的技术生产，已成功用于从转移性癌症患者的血液中捕获癌细胞。分离后捕获的细胞在培养中可维持长达15天，这些样本通常包含来自每位患者的>50%的活原发性癌细胞。该设备已用于从稀释的全血和血沉棕黄层样本中捕获活的CTC。最终，我们展示了一种在临床环境中具有功能的技术，用于开发个性化癌症疗法。
