Metastatic Dissemination Mimicked in a Multicellular Transwell Assay.

Tumor progression and metastasis are multistep processes that are critically dependent on the interaction of metastasizing tumor cells with other cells of the local microenvironment. Mimicking the single steps of the metastatic cascade in vitro is therefore challenging when investigating not only tumor cell behavior alone but also cellular crosstalk between different cell populations. In particular, the crosstalk of tumor cells with pericytes and endothelial cells when accessing the bloodstream is of great importance for successful intravasation and metastatic dissemination. To examine metastatic intravasation and analyze the interaction of tumor cells with pericytes, which reside within the basement membrane and endothelial cells, aligning the vessel wall, we have designed a 3D in vitro transwell assay mimicking tumor cell intravasation into a vessel. Modifying the Boyden chamber transwell assay by seeding first an endothelial cell layer onto the transwell membrane and covering it with pericytes before adding the tumor cells allows us to investigate the role of pericytes and endothelial cells on tumor cell intravasation and at the same time to study their crosstalk at the molecular level and how this interaction influences tumor cell behavior. It further allows the manipulation of the system for proof-of-principle experimentation.