优化三维球体培养的酶谱方案,研究肿瘤细胞中MMP-2和-9的活性。

IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Sandra Majo, Chloe Redoute-Timonnier, Aurelie Lacour, Laurine Challeat, Eva Epinette, Jeremie Teillon, Christophe F Grosset, Patrick Auguste
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引用次数: 0

摘要

三维球体比细胞培养的单层更能代表肿瘤。与肿瘤一样,在球形培养物中发现了氧、营养物质和废物的梯度,而在传统的单层培养物中则没有。另一方面,对后者的基于细胞的测定几乎不适用于球形培养。酶谱分析通常用于测量MMP-2和MMP-9的活性,免疫印迹则用于测量正常细胞和肿瘤细胞中与配体诱导的细胞内信号传导有关的蛋白质的磷酸化。在这项研究中,我们使用两种肾癌细胞系作为模型,第一种来自儿童横纹肌样肿瘤,第二种来自成人透明细胞肾细胞癌。利用这两种细胞系,我们成功地开发了一种简单廉价的方法来测量甲基纤维素存在下球体中MMP-2和MMP-9的活性。洗净后,将1 - 5个球体聚在一起,用I型胶原蛋白刺激24小时后进行分析。使用标准但增强的酶谱法测定上清液中MMP-2和MMP-9的活性。在两种细胞系建立的球体中均检测到前mmp -9和MMP-2活性。与我们之前使用经典培养单层的数据相比,胶原I刺激降低了mmp -9的活性,但不影响MMP-2的活性。另一方面,我们无法准确测量胶原i刺激的球体中AKT的细胞内信号通路。最后,我们采用3D方案分析了EGF诱导后肾肿瘤细胞中的MAPK/ERK通路。总之,该酶谱分析方法可用于分析球状体中MMP-2和MMP-9的活性,为肿瘤生物学的新实验铺平了道路。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Optimized zymogram protocol from 3D spheroid cultures to study MMP-2 and -9 activities in tumor cells.

Three-dimensional spheroids are more representative of tumors than cell-cultured monolayers. As in tumors, gradients of oxygen, nutrients and wastes are found in spheroid cultures but not in classical cultured monolayers. On the other hand, cell-based assays on the latter are hardly applicable to spheroid cultures. Such is the case for zymogram assays, which are classically used to measure MMP-2 and MMP-9 activities, and for immunoblots to measure the phosphorylation of proteins involved in ligand-induced intracellular signaling in normal and tumor cells. In this study we used two renal cancer cell lines as models, the first derived from a pediatric rhabdoid tumor and the second from an adult clear cell renal cell carcinoma. Using these two cell lines, we successfully developed a simple inexpensive assay to measure MMP-2 and MMP-9 activities in spheroids established in the presence of methylcellulose. After washing, 1 to 5 spheroids were pooled and stimulated with collagen I for 24 h before analysis. MMP-2 and MMP-9 activities were measured in supernatants using a standard but enhanced zymogram assay. Both pro-MMP-9 and MMP-2 activities were detected in spheroids established from both cell lines. In contrast with our previous data using classical cultures monolayers, collagen I stimulation decreased pro-MMP-9 activity without affecting MMP-2 activity. On the other hand, we could not accurately measure AKT intracellular signaling pathways from spheroids stimulated with collagen I. Finally, we adapted our 3D protocol to analyze the MAPK/ERK pathway in kidney tumor cells following induction by EGF. In conclusion, this zymogram assay for analyzing MMP-2 and MMP-9 activities in spheroids paves the way for novel experimentations in tumor biology.

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来源期刊
BMC Biotechnology
BMC Biotechnology 工程技术-生物工程与应用微生物
CiteScore
6.60
自引率
0.00%
发文量
34
审稿时长
2 months
期刊介绍: BMC Biotechnology is an open access, peer-reviewed journal that considers articles on the manipulation of biological macromolecules or organisms for use in experimental procedures, cellular and tissue engineering or in the pharmaceutical, agricultural biotechnology and allied industries.
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