Rahim Said, Üren Aykut
Lombardi Comprehensive Cancer Center, Georgetown University.
J Vis Exp. 2011 Apr 1(50):2792. doi: 10.3791/2792.
Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host(1-3). Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors(4,5). The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process(6). Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells. Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes(7,8). The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance(9-12). The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density. The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.
恶性细胞的转移扩散需要基底膜降解、肿瘤细胞与血管内皮细胞附着、内皮细胞连接收缩，最终肿瘤细胞通过内皮层侵袭和迁移进入血液循环，以此作为转移至宿主远处部位的一种运输方式（1 - 3）。一旦进入循环系统，癌细胞会黏附于毛细血管壁并外渗至周围组织以形成转移性肿瘤（4,5）。肿瘤细胞与内皮细胞相互作用的各个环节可在体外通过用癌细胞挑战人脐静脉内皮细胞（HUVEC）单层来模拟。利用电子显微镜和相差显微镜进行的研究表明，体外事件序列相当程度上代表了体内转移过程（6）。在此，我们描述一种基于电阻抗的技术，该技术可实时监测和量化恶性肿瘤细胞对内皮细胞的侵袭。Giaever和Keese首先描述了一种当一群细胞在电极表面生长时测量阻抗波动的技术（7,8）。罗氏公司生产的xCELLigence仪器利用类似技术测量当细胞在覆盖有金微电极阵列的培养皿中附着和铺展时的电阻抗变化，该阵列覆盖了孔底部约80%的面积。当细胞在电极表面附着和铺展时，会导致电阻抗增加（9 - 12）。阻抗以一个无量纲参数即细胞指数显示，该参数与细胞覆盖的组织培养孔总面积成正比。因此，细胞指数可用于监测细胞黏附、铺展、形态和细胞密度。本文所述的侵袭测定基于电极/细胞界面处电阻抗的变化，因为一群恶性细胞侵袭穿过HUVEC单层（图1）。内皮细胞连接的破坏、内皮单层的收缩以及被肿瘤细胞取代会导致阻抗发生巨大变化。这些变化与肿瘤细胞的侵袭能力直接相关，即高侵袭性细胞的侵袭会导致细胞阻抗发生巨大变化，反之亦然。与现有的测量侵袭的方法如博伊登小室法和基质胶测定法相比，该技术具有双重优势：1）内皮细胞 - 肿瘤细胞相互作用更紧密地模拟体内过程；2）数据是实时获取的，并且与其他方法的终点分析相比更易于量化。
