Willetts Lian, Bond David, Stoletov Konstantin, Lewis John D
Department of Oncology, University of Alberta, 5-142C Katz Group Building, 114th St and 87th Ave, Edmonton, AB, Canada, T6G 2E1.
Methods Mol Biol. 2016;1458:27-37. doi: 10.1007/978-1-4939-3801-8_3.
Metastasis, or the spread of cancer cells from a primary tumor to distant sites, is the leading cause of cancer-associated death. Metastasis is a complex multi-step process comprised of invasion, intravasation, survival in circulation, extravasation, and formation of metastatic colonies. Currently, in vitro assays are limited in their ability to investigate these intricate processes and do not faithfully reflect metastasis as it occurs in vivo. Traditional in vivo models of metastasis are limited by their ability to visualize the seemingly sporadic behavior of where and when cancer cells spread (Reymond et al., Nat Rev Cancer 13:858-870, 2013). The avian embryo model of metastasis is a powerful platform to study many of the critical steps in the metastatic cascade including the migration, extravasation, and invasion of human cancer cells in vivo (Sung et al., Nat Commun 6:7164, 2015; Leong et al., Cell Rep 8, 1558-1570, 2014; Kain et al., Dev Dyn 243:216-28, 2014; Leong et al., Nat Protoc 5:1406-17, 2010; Zijlstra et al., Cancer Cell 13:221-234, 2008; Palmer et al., J Vis Exp 51:2815, 2011). The chicken chorioallantoic membrane (CAM) is a readily accessible and well-vascularized tissue that surrounds the developing embryo. When the chicken embryo is grown in a shell-less, ex ovo environment, the nearly transparent CAM provides an ideal environment for high-resolution fluorescent microcopy approaches. In this model, the embryonic chicken vasculature and labeled cancer cells can be visualized simultaneously to investigate specific steps in the metastatic cascade including extravasation. When combined with the proper image analysis tools, the ex ovo chicken embryo model offers a cost-effective and high-throughput platform for the quantitative analysis of tumor cell metastasis in a physiologically relevant in vivo setting. Here we discuss detailed procedures to quantify cancer cell extravasation in the shell-less chicken embryo model with advanced fluorescence microscopy techniques.
转移，即癌细胞从原发性肿瘤扩散至远处部位，是癌症相关死亡的主要原因。转移是一个复杂的多步骤过程，包括侵袭、血管内渗、循环存活、血管外渗以及转移瘤形成。目前，体外试验在研究这些复杂过程方面能力有限，无法如实地反映体内发生的转移情况。传统的体内转移模型在可视化癌细胞扩散的地点和时间这种看似随机的行为方面能力有限（雷蒙德等人，《自然评论：癌症》13:858 - 870，2013年）。禽类胚胎转移模型是一个强大的平台，可用于研究转移级联反应中的许多关键步骤，包括体内人类癌细胞的迁移、血管外渗和侵袭（宋等人，《自然通讯》6:7164，2015年；梁等人，《细胞报告》8，1558 - 1570，2014年；凯恩等人，《发育动力学》243:216 - 228，2014年；梁等人，《自然方法》5:1406 - 1417，2010年；齐尔斯特拉等人，《癌细胞》13:221 - 234，2008年；帕尔默等人，《可视实验杂志》51:2815，2011年）。鸡胚绒毛尿囊膜（CAM）是一种易于获取且血管丰富的组织，环绕着发育中的胚胎。当鸡胚在无壳的体外环境中生长时，近乎透明的CAM为高分辨率荧光显微镜方法提供了理想环境。在这个模型中，可以同时观察胚胎鸡的脉管系统和标记的癌细胞，以研究转移级联反应中的特定步骤，包括血管外渗。当与适当的图像分析工具结合使用时，体外鸡胚模型为在生理相关的体内环境中对肿瘤细胞转移进行定量分析提供了一个经济高效且高通量的平台。在此，我们讨论使用先进荧光显微镜技术在无壳鸡胚模型中量化癌细胞血管外渗的详细步骤。
