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Cancer metastasis is a multistep process during which tumor cells leave the primary tumor mass and form distant secondary colonies that are lethal. Circulating tumor cells (CTCs) are transported by body fluids to reach distant organs, where they will extravasate and either remain dormant or form new tumor foci. Development of methods to study the behavior of CTCs at the late stages of the intravascular journey is thus required to dissect the molecular mechanisms at play. Using recently developed microfluidics approaches, we have demonstrated that CTCs arrest intravascularly, through a two-step process: (a) CTCs stop using low energy and rapidly activated adhesion receptors to form transient metastable adhesions and (b) CTCs stabilize their adhesions to the endothelial layer with high energy and slowly activated adhesion receptors. In this methods chapter, we describe these easy-to-implement quantitative methods using commercially available microfluidic channels. We detail the use of fast live imaging combined to fine-tuned perfusion to measure the adhesion potential of CTC depending on flow velocities. We document how rapidly engaged early metastable adhesion can be discriminated from slower activated stable adhesion using microfluidics. Finally, CTC extravasation potential can be assessed within this setup using long-term cell culture under flow. Altogether, this experimental pipeline can be adapted to probe the adhesion (to the endothelial layer) and extravasation potential of any circulating cell.

Most cancers end up with the death of patients caused by the formation of secondary tumors, called metastases. However, how these secondary tumors appear and develop is only poorly understood. A fine understanding of the multiple steps of the metastasis cascade requires in vivo models allowing high spatiotemporal analysis of the behavior of metastatic cells. Zebrafish embryos combine several advantages such as transparency, small size, stereotyped anatomy, and easy handling, making it a very powerful model for cell and cancer biology, and in vivo imaging analysis. In the following chapter, we describe a complete procedure allowing in vivo imaging methods, at high throughput and spatiotemporal resolution, to assess the behavior of circulating tumor cells (CTCs) in an experimental metastasis assay. This protocol provides access, for the first time, to the earliest steps of tumor cell seeding during metastasis formation.