Studying extracellular vesicle transfer by a Cre-loxP method

Abstract

Extracellular vesicle (EV) transfer is increasingly recognized as an important mode of intercellular communication by transferring a wide variety of biomolecules between cells. The characterization of in vitro- or ex vivo-isolated EVs has considerably contributed to the understanding of biological functions of EV transfer. However, the study of EV release and uptake in an in vivo setting has remained challenging, because cells that take up EVs could not be discriminated from cells that do not take up EVs. Recently, a technique based on the Cre-loxP system was developed to fluorescently mark Cre-reporter cells that take up EVs released by Cre recombinase-expressing cells in various in vitro and in vivo settings. Here we describe a detailed protocol for the generation of Cre(+) cells and reporter(+) cells, which takes ∼ 6 weeks, and subsequent assays with these lines to study functional EV transfer in in vitro and in vivo (mouse) settings, which take up to ∼ 2 months.

Cancer cells copy migratory behavior and exchange signaling networks via extracellular vesicles

“…We observed that the pool of EVs enriched at a lower centrifugation speed (16,500 g ) and the pool of EVs enriched at a higher speed (100,000 g ; Thery et al , ; Greening et al , ; Szatanek et al , ) are both taken up by recipient cells in vitro (Fig E). To test whether the mutual uptake of EVs also led to the functional release of the content in the recipient cells, we employed the Cre‐LoxP system (Ridder et al , ; Zomer et al , , ). In the EVs released by Cre‐expressing B16 cells, the mRNA of Cre was present (Fig F).…”

“…15070‐063). Cre + and reporter + cells were made as previously described (Zomer et al , ). In short, B16F1 or B16F10 cells were transfected with plasmid pcDNA3.1‐CFP; Cre25nt; Zeo (Addgene, plasmid number 65727) using Lipofectamine 2000 (Life Technologies, cat.…”

“…After 3 weeks, fluorescent images of the co‐cultures were obtained with a Leica AF7000 microscope for CFP (excitation 430/24, emission 470/40), GFP (excitation 470/40, emission 520/40), and DsRed (excitation 572/35, emission 640/50). Percentages of eGFP + reporter + cells were determined as described before (Zomer et al , ).…”

“…For example, upon EV uptake, vesicular mRNA is translated into functional proteins (Valadi et al , ), and vesicular miRNAs suppress target genes in recipient cells (Hergenreider et al , ; Fong et al , ; Zhang et al , ). Moreover, EV‐RNA‐based reporter systems have confirmed the transport of functional mRNA for Cre (Ridder et al , ; Zomer et al , , ) or GlucB (Lai et al , ) into recipient cells. Thus, by exchanging EVs, cells can transfer biomolecules to recipient cells, thereby potentially influencing the recipient cell's behavior (Valadi et al , ) and tumor heterogeneity (Zomer & van Rheenen, ).…”

Frontiers in intravital multiphoton microscopy of cancer

“…A strategy based on the Cre‐LoxP was developed to distinguish cells that have taken up EVs from those that have not using IV‐MPM . When the EV‐mediated Cre transfer occurs and the cargo is released into the cytoplasm, cells switch from DsRed to eGFP labeling (see Zomer et al for details). One major advantage of this protocol is that the Cre + and the reporter + cell populations can be defined a priori, allowing the study of EVs originating from specific cell types.…”