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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. [ABSTRACT FROM AUTHOR]

Natural Killer (NK) cells are innate cytotoxic lymphoid cells that play a crucial role in cancer immunosurveillance. NKG2D is an activating receptor that binds to MIC and ULBP molecules typically induced on damaged, transformed, or infected cells. The secretion of NKG2D ligands (NKG2DLs) through protease-mediated cleavage or in an extracellular vesicle (EV) is a mode to control their cell surface expression and a mechanism used by cancer cells to evade NKG2D-mediated immunosurveillance. EVs are emerging as important players in mediating cell-to-cell communication due to their ability to transfer biological material to acceptor cells. Herein, we investigated the spreading of NKG2DLs of both MIC and ULBP molecules through the EV-mediated cross-dressing on multiple myeloma (MM) cells. We focused our attention on two MICA allelic variants, namely MICA*008 and MICA*019, representing the prototype of short and long MICA alleles, respectively, and on ULBP-1, ULBP-2, and ULBP-3. Our findings demonstrate that both ULBP and MICA ligands can be acquired from tumor cells through EVs enhancing NK cell recognition and killing. Moreover, besides MICA, EVs expressing ULBP-1 but not ULBP-2 and 3 were detected in bone marrow aspirates derived from a cohort of MM patients. Our findings shed light on the role of EV-associated MICA allelic variants and ULBP molecules in the modulation of NKG2D-mediated NK cell immunosurveillance in the tumor microenvironment. Moreover, the EV-mediated transfer of NKG2DLs could suggest novel therapeutic approaches based on the usage of engineered nanoparticles aimed at increasing cancer cell immunogenicity. [ABSTRACT FROM AUTHOR]