Cell-cell communication within the complex tumour microenvironment is critical to cancer progression. Tumor-derived extracellular vesicles (TD-EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Deciphering the interactions between TD-EVs and immune cells is essential to understand immune modulation by cancer cells. Fluorescent labelling of TD-EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection by imaging flow cytometry and multicolour spectral flow cytometry of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs). EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labelled either with the lipophilic dye MemGlow-488 (MG-488), Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) or through ectopic expression of a MyrPalm-superFolderGFP reporter (mp-sfGFP), which incorporates into EVs during their biogenesis. Our results show that these labelling strategies, although analysed with the same techniques, led to diverging results. While MG-488-labelled EVs incorporate in all cell types, CFSE-labelled EVs are restricted to a minor subset of cells and mp-sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labelling method. Furthermore, our results show that the method used for EV labelling influences the detection of the different types of EV interactions with the recipient cells. Specifically, MG-488, CFSE and mp-sfGFP result in observation suggesting, respectively, transient EV-PM interaction that results in dye transfer, EV content delivery, and capture of intact EVs. Consequently, the type of EV labelling method has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate.

中文翻译：

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肿瘤来源的细胞外囊泡与免疫细胞相互作用的检测依赖于 EV 标记方法

复杂的肿瘤微环境中的细胞间通讯对于癌症进展至关重要。肿瘤源性细胞外囊泡（TD-EV）是这一过程的关键参与者。它们可以与免疫细胞相互作用并调节其活性，抑制或激活免疫系统。破译 TD-EV 和免疫细胞之间的相互作用对于了解癌细胞的免疫调节至关重要。TD-EV 的荧光标记是研究这种相互作用的一种选择方法。这项工作旨在确定 EV 标记方法对通过成像流式细胞术和多色光谱流式细胞术检测人外周血单核细胞 (PBMC) 内不同免疫细胞类型的 EV 相互作用和捕获的影响。三阴性乳腺癌细胞系 MDA-MB-231 释放的 EV 用亲脂性染料 MemGlow-488 (MG-488)、羧基荧光素二乙酸酯、琥珀酰亚胺酯 (CFDA-SE) 或通过 MyrPalm- 的异位表达进行标记。 superFolderGFP 报告基因 (mp-sfGFP)，在 EV 的生物发生过程中融入其中。我们的结果表明，尽管使用相同的技术进行分析，这些标记策略却导致了不同的结果。虽然 MG-488 标记的 EV 包含在所有细胞类型中，但 CFSE 标记的 EV 仅限于一小部分细胞，而 mp-sfGFP 标记的 EV 主要在 CD14+ 单核细胞中检测到，CD14+ 单核细胞是 EV 和其他颗粒的主要摄取者，无论如何的标记方法。此外，我们的结果表明，用于 EV 标记的方法会影响不同类型的 EV 与受体细胞相互作用的检测。具体而言，MG-488、CFSE 和 mp-sfGFP 的观察结果分别表明短暂的 EV-PM 相互作用导致染料转移、EV 内容物传递和完整 EV 的捕获。因此，必须考虑 EV 标记方法的类型，因为它们可以提供有关各种类型的 EV 细胞相互作用和 EV 命运的补充信息。