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- Detection of the interactions of tumour derived extracellular vesicles with immune cells is dependent on EVālabelling methods
Detection of the interactions of tumour derived extracellular vesicles with immune cells is dependent on EVālabelling methods
Auteurs
Luisa Loconte, Davinia Arguedas, Rojbin El, Alix Zhou, Anna Chipont, Lea Guyonnet, Coralie Guerin, Ester Piovesana, JosĆ© Luis VĆ”zquezāIbar, Alain Joliot, Clotilde ThĆ©ry, Lorena MartĆnāJaular
RƩsumƩ
Abstract
Cellācell communication within the complex tumour microenvironment is critical to cancer progression. Tumorāderived extracellular vesicles (TDāEVs) 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 TDāEVs and immune cells is essential to understand immune modulation by cancer cells. Fluorescent labelling of TDāEVs 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 tripleānegative breast carcinoma cell line MDAāMBā231 were labelled either with the lipophilic dye MemGlowā488 (MGā488), Carboxyfluorescein diacetate, succinimidyl ester (CFDAāSE) or through ectopic expression of a MyrPalmāsuperFolderGFP reporter (mpāsfGFP), 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 MGā488ālabelled EVs incorporate in all cell types, CFSEālabelled EVs are restricted to a minor subset of cells and mpāsfGFPālabelled 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, MGā488, CFSE and mpāsfGFP result in observation suggesting, respectively, transient EVāPM 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 EVācell interaction and EV fate.
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