Lorena Martín‐Jaular

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.