Detection of tumor-derived extracellular vesicles interactions with immune cells is dependent on EV-labelling methods

Abstract

Cell-cell communication within the complex tumor 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. Understanding the interactions between TD-EVs and immune cells is essential for understanding 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 of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs), analyzed by imaging flow cytometry and multicolor spectral flow cytometry. EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labeled either with the lipophilic dye MemGlow-488 (MG-488), with Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE), or through expression of a MyrPalm-superFolder GFP (sfGFP) that incorporates into EVs during their biogenesis using a genetically engineered cell line. Our results showed that these different labeling strategies, although analyzed 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 sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labeling method. Moreover, MG-488-labeled liposomes behaved similarly to MG-488 EVs, highlighting the predominant role of the labelling strategy on the visualization and analysis of TD-EVs uptake by immune cell types. Consequently, the use of different EV labeling methods has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate.

Molecular mapping of neuronal architecture using STORM microscopy and new fluorescent probes for SMLM imaging

“…MemBright has been widely adopted by the extracellular vesicles community 51 to track extracellular vesicles both in vitro or in vivo 61 – 81 in hippocampal 63 or cortical neurons, 79 zebrafish, 62 , 67 , 72 breast cancer cells or tumours, 66 , 71 , 80 myotubes, 82 and red blood cells 74 , 76 …”

Influence of Breast Cancer Extracellular Vesicles on Immune Cell Activation: A Pilot Study

“…Moreover, EVs contain nucleic acids and proteins that are released into the extracellular environment [14]. They mediate cell-to-cell communication and modulate their function; however, EVs released by cancer cells may play an important role in shaping the tumor immune microenvironment, which is still understudied [15]. In the tumor environment, EVs can have different functions [16].…”