Interaction and uptake of exosomes by ovarian cancer cells.
Interaction and uptake of exosomes by ovarian cancer cells.
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DOI：
10.1186/1471-2407-11-108
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发表时间：
2011-03-27
期刊：
影响因子：
3.8
通讯作者：
Costa J
中科院分区：
文献类型：
--
作者：
Escrevente C;Keller S;Altevogt P;Costa J
外泌体由膜囊泡组成，由几种细胞类型分泌，包括肿瘤，并在生物液体中发现。外泌体与其他细胞相互作用，并可作为细胞间蛋白质和RNA转移的载体。SKOV3外泌体用羧基荧光素二乙酸丁二酰酯标记，用超离心法收集。免疫荧光显微镜和流式细胞术分析监测了这些囊泡在不同条件下由来自它们起源的相同细胞摄取。通过凝集素分析来研究外泌体和细胞提取物中蛋白质的糖基化特性。在这项工作中，卵巢癌SKOV3细胞系已被证明通过几种内吞途径内化来自相同细胞的外泌体，这些内吞途径在4°C时被强烈抑制，表明它们的能量依赖性。与内核体标记EEA1的部分共定位和氯丙嗪的抑制表明参与了网格蛋白依赖的内吞作用。此外，在5-乙基- n -异丙基酰胺、细胞松弛素D和甲基- β -环糊精的存在下，摄取抑制表明参与了额外的内吞途径。摄取需要来自外泌体和细胞的蛋白质，因为它在蛋白酶K处理后被抑制。外泌体富含特定的甘露糖和唾液酸糖蛋白。唾液酸的去除引起了小但不显著的摄取增加。此外，单糖d -半乳糖、α-L-焦糖、α- d -甘露糖、d - n -乙酰氨基葡萄糖和双糖β-乳糖减少外泌体摄取的程度与对照d -葡萄糖相当。综上所述，卵巢肿瘤细胞通过多种内吞途径将外泌体内化，摄取外泌体和细胞中的蛋白质是必需的。另一方面，外泌体富含可能构成外泌体标记物的特定糖蛋白。这项工作有助于了解外泌体在癌症中的特性和动力学。
Exosomes consist of membrane vesicles that are secreted by several cell types, including tumors and have been found in biological fluids. Exosomes interact with other cells and may serve as vehicles for the transfer of protein and RNA among cells. SKOV3 exosomes were labelled with carboxyfluoresceine diacetate succinimidyl-ester and collected by ultracentrifugation. Uptake of these vesicles, under different conditions, by the same cells from where they originated was monitored by immunofluorescence microscopy and flow cytometry analysis. Lectin analysis was performed to investigate the glycosylation properties of proteins from exosomes and cellular extracts. In this work, the ovarian carcinoma SKOV3 cell line has been shown to internalize exosomes from the same cells via several endocytic pathways that were strongly inhibited at 4°C, indicating their energy dependence. Partial colocalization with the endosome marker EEA1 and inhibition by chlorpromazine suggested the involvement of clathrin-dependent endocytosis. Furthermore, uptake inhibition in the presence of 5-ethyl-N-isopropyl amiloride, cytochalasin D and methyl-beta-cyclodextrin suggested the involvement of additional endocytic pathways. The uptake required proteins from the exosomes and from the cells since it was inhibited after proteinase K treatments. The exosomes were found to be enriched in specific mannose- and sialic acid-containing glycoproteins. Sialic acid removal caused a small but non-significant increase in uptake. Furthermore, the monosaccharides D-galactose, α-L-fucose, α-D-mannose, D-N-acetylglucosamine and the disaccharide β-lactose reduced exosomes uptake to a comparable extent as the control D-glucose. In conclusion, exosomes are internalized by ovarian tumor cells via various endocytic pathways and proteins from exosomes and cells are required for uptake. On the other hand, exosomes are enriched in specific glycoproteins that may constitute exosome markers. This work contributes to the knowledge about the properties and dynamics of exosomes in cancer.
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