Naakka Erika, Tuomainen Katja, Wistrand Henrik, Palkama Miila, Suleymanova Ilida, Al-Samadi Ahmed, Salo Tuula
Department of Oral and Maxillofacial Diseases, Clinicum, Faculty of Medicine, University of Helsinki.
Department of Oral and Maxillofacial Diseases, Clinicum, Faculty of Medicine, University of Helsinki; Cancer and Translational Medicine Research Unit, University of Oulu; Medical Research Center, Oulu University Horspital; Helsinki University Hospital;
J Vis Exp. 2019 May 7(147). doi: 10.3791/59567.
Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.
基于二维细胞培养的检测方法常用于体外癌症研究。然而，它们缺乏构成肿瘤微环境的几个基本要素。为了获得更可靠的体外结果，人们引入了几种三维（3D）细胞培养检测方法。这些检测方法使癌细胞能够与细胞外基质相互作用。这种相互作用会影响细胞行为，如增殖和侵袭，以及细胞形态。此外，这种相互作用可能诱导或抑制几种促肿瘤和抗肿瘤分子的表达。球体侵袭检测方法的开发是为了提供一种合适的三维体外方法来研究癌细胞侵袭。目前，球体侵袭检测主要使用动物来源的基质，如小鼠肉瘤来源的基质（MSDM）和大鼠尾部I型胶原蛋白。考虑到人类肿瘤微环境与动物来源基质之间的差异，人们从良性子宫平滑肌瘤组织中开发出了人肌瘤来源的基质（HMDM）。研究表明，HMDM比MSDM更能诱导癌细胞的迁移和侵袭。本方案提供了一种使用HMDM/纤维蛋白基质的简单、可重复且可靠的基于三维人类肿瘤的球体侵袭检测方法。它还包括成像和分析的详细说明。球体在HMDM/纤维蛋白基质中的U形超低附着板中生长并穿过该基质进行侵袭。每天使用ilastik和Fiji ImageJ软件对侵袭进行成像、测量和分析。该检测平台已用人喉原发性和转移性鳞状细胞癌细胞系进行了验证。然而，该方案也适用于其他实体癌细胞系。
