Tumor metastases develop when disseminated intravascular cancer cells acquire the ability to arrest by adhering to the capillary walls of distant organs, actively extravasate into their parenchyma, proliferate and establish secondary colonies. The extravasation assay described here is an in vivo technique aimed to analyze the ability of tumor cells to achieve early colonization of the lungs following tail vein injection in mice. Importantly, tumor cells need to be easily visible, therefore either they are fluorescent (e.g. expressing RFP or GFP) or they have to be pre-labelled with a fluorescent tracker prior to injection. Lungs are analyzed at different time points, experimentally determined by the researcher, depending on cell features and malignancy. Generally, an early time point is required to check equal lodging in the pulmonary vasculature for the various cells injected. At one or more later time points (from 6 to 48 h) extravasated cells dispersed in the lung parenchyma are quantitated. With our protocol extravasation is directly evaluated in the whole lungs ex vivo considering cell fluorescence. However, immunofluorescence stainings for endothelial markers and microscopic analyses of lung sections are recommended to evaluate positioning and status of tumor cells (i.e. inside, outside the vessels or associated to them; single cells or clusters). Since extravasation is not only influenced by tumor cell motility but also by their survival ability, the results obtained with this technique should be complemented with proliferation and apoptosis analyses.
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