ex-vivo-live-imaging-of-lung-metastasis-and-their-microenvironment

<em>Ex vivo</em> Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

University of California

Hubrecht Institute-Royal Dutch Academy of Science and University Medical Center Utrecht

Most cancer cells release heterogeneous populations of extracellular vesicles (EVs) containing proteins, lipids, and nucleic acids. In vitro experiments showed that EV uptake can lead to transfer of functional mRNA and altered cellular behavior. However, similar in vivo experiments remain challenging because cells that take up EVs cannot be discriminated from non-EV-receiving cells. Here, we used the Cre-LoxP system to directly identify tumor cells that take up EVs in vivo. We show that EVs released by malignant tumor cells are taken up by less malignant tumor cells located within the same and within distant tumors and that these EVs carry mRNAs involved in migration and metastasis. By intravital imaging, we show that the less malignant tumor cells that take up EVs display enhanced migratory behavior and metastatic capacity. We postulate that tumor cells locally and systemically share molecules carried by EVs in vivo and that this affects cellular behavior.

In Vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior.

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.

Carrie Maynard

Department of Anatomy

University of California

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Carrie Maynard

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Anatomy

University of California

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Department of Anatomy