Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells
Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells
复制标题
DOI：
10.1074/jbc.m109.041152
复制
发表时间：
2009-12-04
影响因子：
4.8
通讯作者：
Fais, Stefano
中科院分区：
文献类型：
--
作者：
Parolini, Isabella;Federici, Cristina;Fais, Stefano
正常细胞和癌细胞分泌的外泌体携带和传递各种分子。迄今为止，涉及肿瘤外泌体运输的机制，包括释放和细胞-细胞传递，尚未被描述。为了深入了解这一点，从转移性黑色素瘤细胞培养基中纯化的外泌体用脂质荧光探针R18标记，并通过荧光光谱法和共聚焦显微镜进行分析。低pH值是恶性肿瘤的标志，可能影响癌细胞的外泌体释放和摄取。使用不同的pH条件作为外泌体流量的调节剂，我们发现(i)与缓冲条件相比，低pH条件下外泌体释放和摄取增加，（ii）黑色素瘤细胞通过融合发生外泌体摄取。膜生物物理分析，如流动性和脂质组成，表明在低ph下释放的外泌体具有高刚性和鞘磷脂/神经节苷脂GM3 （n -乙酰神经酰胺半乳糖糖基神经酰胺）含量。这可能是融合效率提高的原因。与这些结果一致，质子泵抑制剂预处理导致黑色素瘤细胞外泌体摄取的抑制。肿瘤外泌体在转移源细胞中的融合效率高于原发肿瘤细胞或正常细胞。此外，我们发现参与黑色素瘤进展的蛋白小窝蛋白-1在酸性条件下通过释放的外泌体高度传递。我们的研究结果提供了外泌体可能被用作肿瘤恶性肿瘤旁分泌扩散的递送系统的证据，反过来支持外泌体和肿瘤pH作为未来抗癌策略的关键靶点的重要性。
Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.