Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine
Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine
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DOI：
10.1080/20013078.2019.1579541
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发表时间：
2019-01-01
影响因子：
16
通讯作者：
Shiba, Kiyotaka
中科院分区：
文献类型：
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作者：
Matsumura, Sachiko;Minamisawa, Tamiko;Shiba, Kiyotaka
磷脂酰丝氨酸（PS）在质膜中分布不均匀，并且优先位于正常细胞的内叶中。然而，肿瘤细胞在细胞表面的外层暴露 PS，从而可能调节细胞的生物信号传导。有趣的是，外泌体——或更准确地说，小细胞外囊泡（sEV）——由肿瘤细胞分泌，富含外化的 PS，已被认为参与癌症的进展，并可用作肿瘤诊断的标志物。然而，通过各种方法制备的 sEV 级分由不同的囊泡亚型组成，并且关于富含暴露的 PS 的亚型的知识仍然有限。在这里，我们通过密度梯度离心将 sEV 与癌细胞系区分开来，并通过在原子力显微镜、凝血酶生成测定、大小和 zeta 电位测量以及蛋白质印迹分析中使用 PS 金标记来表征分离的部分。这些分析揭示了一种先前未报道的富含 PS+ 的 sEV 亚型，其特征是密度低于典型外泌体（1.06 g/ml 与 1.08 g/ml）、尺寸更大（122 nm 与 105 nm）、更负的 zeta 电位（-28 mV 与 -21 mV）以及典型外泌体标记丰度较低。 sEV 暴露于 PS 的亚型的鉴定将有助于更深入地了解 EV 在肿瘤生物学中的作用，并促进基于 EV 的肿瘤诊断和治疗的发展。
Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes - or, more properly, small extracellular vesicles (sEVs) - which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (-28 mV vs. -21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy.