Rauschenberger Lisa, Staar Doreen, Thom Kathleen, Scharf Christian, Venz Simone, Homuth Georg, Schlüter Rabea, Brandenburg Lars-Ove, Ziegler Patrick, Zimmermann Uwe, Weitschies Werner, Völker Uwe, Lendeckel Uwe, Walther Reinhard, Burchardt Martin, Stope Matthias B
Department of Urology, University Medicine Greifswald, Greifswald, Germany.
Department of Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald, Greifswald, Germany.
Prostate. 2016 Mar;76(4):409-24. doi: 10.1002/pros.23132. Epub 2015 Dec 8.
Remodeling of the tumor environment and the modulation of tumor associated non-malignant cells are essential events in tumor progression. Exosomes are small membranous vesicles of 50-150 nm in diameter, which are secreted into the extracellular space and supposedly serve as vehicles for signal and effector molecules to modulate adjacent target cells. We characterized the mRNA and protein composition as well as cellular functions of prostate cancer cell-derived exosomes.
Exosomes were prepared from prostate cancer cell culture supernatant by ultracentrifugation and subsequently characterized by dynamic light scattering and electron microscopy. Exosomal mRNA and protein composition were analyzed by DNA microarrays and gel electrophoresis coupled with mass spectrometry. Physiological effects of exosomes were studied by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release cell assays. Using a SILAC approach, putative uptake of exosomal human proteins in canine cells and canine de novo synthesis of proteins specified by exosome-transferred human mRNA was analyzed in MDCK cells via mass spectrometry.
Preparations of exosomes revealed typical cup shaped particles of 150 nm in diameter. Analysis of mRNA and protein composition of exosomes exhibited a wide range of mRNA and protein species. Interestingly, the packaging of at least small proteins into exosomes was apparently unspecific, as shown with the example of two model proteins. In cell culture incubation experiments exosomal preparations of prostate cancer cells caused anti-proliferative effects. MS analysis revealed the uptake of exosomal human proteins into canine cells after 6 hr of incubation.
The results reveal a distinct exosomal functionality in the modulation of the prostatic tumor adjacent environment. The multitude of translocated factors implies the induction of numerous effects in tumor-associated target cells, including impact on cellular growth.
肿瘤微环境的重塑以及肿瘤相关非恶性细胞的调节是肿瘤进展中的关键事件。外泌体是直径为50 - 150纳米的小膜泡，分泌到细胞外空间，被认为是信号和效应分子调节相邻靶细胞的载体。我们对前列腺癌细胞衍生的外泌体的mRNA和蛋白质组成以及细胞功能进行了表征。
通过超速离心从前列腺癌细胞培养上清液中制备外泌体，随后通过动态光散射和电子显微镜进行表征。通过DNA微阵列以及凝胶电泳结合质谱分析外泌体的mRNA和蛋白质组成。通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐和乳酸脱氢酶释放细胞试验研究外泌体的生理效应。使用稳定同位素标记氨基酸法（SILAC），通过质谱分析在MDCK细胞中分析犬细胞中外泌体人蛋白的假定摄取以及外泌体转移的人mRNA指定的犬蛋白质的从头合成。
外泌体制备物显示出典型的直径为150纳米的杯状颗粒。外泌体的mRNA和蛋白质组成分析显示出广泛的mRNA和蛋白质种类。有趣的是，如两种模型蛋白的例子所示，至少小蛋白包装到外泌体中显然是非特异性的。在细胞培养孵育实验中，前列腺癌细胞的外泌体制备物具有抗增殖作用。质谱分析显示孵育6小时后犬细胞中外泌体人蛋白的摄取。
结果揭示了外泌体在调节前列腺肿瘤相邻环境中的独特功能。大量易位因子意味着在肿瘤相关靶细胞中诱导多种效应，包括对细胞生长的影响。
