André Maria do Rosário, Pedro Ana, Lyden David
Champalimaud Foundation, Lisbon, Portugal.
Department of Genetics, Oncology and Human Toxicology, NOVA Medical School/Faculty of Medical Sciences, NOVA University, Lisbon, Portugal.
Methods Mol Biol. 2016;1395:229-39. doi: 10.1007/978-1-4939-3347-1_13.
In the last decades, several studies demonstrated that the tumor microenvironment is a critical determinant not only of tumor progression and metastasis, but also of resistance to therapy. Exosomes are small membrane vesicles of endocytic origin, which contain mRNAs, DNA fragments, and proteins, and are released by many different cell types, including cancer cells. Mounting evidence has shown that cancer-derived exosomes contribute to the recruitment and reprogramming of constituents associated with the tumor microenvironment. Understanding how exosomes and the tumor microenvironment impact drug resistance will allow novel and better strategies to overcome drug resistance and treat cancer. Here, we describe a technique for exosome purification from cell culture, and fresh and frozen plasma, and further analysis by electron microscopy, NanoSight microscope, and Western blot.
在过去几十年中,多项研究表明肿瘤微环境不仅是肿瘤进展和转移的关键决定因素,也是治疗耐药性的关键决定因素。外泌体是内吞起源的小膜泡,含有mRNA、DNA片段和蛋白质,由包括癌细胞在内的许多不同细胞类型释放。越来越多的证据表明,癌症来源的外泌体有助于肿瘤微环境相关成分的募集和重编程。了解外泌体和肿瘤微环境如何影响耐药性将有助于制定新的更好的策略来克服耐药性并治疗癌症。在这里,我们描述了一种从细胞培养物、新鲜和冷冻血浆中纯化外泌体,并通过电子显微镜、纳米可视显微镜和蛋白质免疫印迹进行进一步分析的技术。