Event Abstract Back to Event On-chip human microvasculature for real-time visualization and quantification of tumor cell extravasation and early metastatic seeding Michelle Chen1, Jordan A. Whisler1, Alexandra Boussommier1 and Roger D. Kamm1, 2 1 MIT, Mechanical Engineering, United States 2 MIT, Biological Engineering, United States Introduction: Metastasis - the cause of about 90% of cancer related deaths - condists of hematogenous dissemination of single tumor cells via the blood circulation to distant sites. However, the mechanisms involved in this early metastatic seeding remain poorly defined due to the technical challenges associated with detection, tissue isolation, imaging and analysis of single disseminated tumor cells in the vasculature and stroma of distant sites in vivo[1]. Here we present the application of an on-chip human microvasculature platform, developed and charaterized perviously[2], to allow real-time and quantitative study of the extravasation and early metastatic seeding microenvironment, including tumor cell interaction with host bloodbourne immune cells, initial arrest, extravasation, and survival/proliferation. Methods: The early seeding microenvironement is recapitulated by forming perfusable human microvascular networks with human umbilical cord vein cells and human lung fibroblasts as supporting stromal cells. Soft lithography techniques are used to form channels to separate HUVECs from NLFs, which are seeded in fibrin gels and contained via surface tension provided by microposts (Fig 1A). The microvasculature is then perfused with tumor cells and the dynamics of transendothelial migration can be readily visualized, scored and quantified at low magnifications (20X) via confocal microscopy (Fig 1B). Results: At higher magnifications (30-60X), cytoskeletal dynamics of both tumor and endothelial cells can be observed. Tumor cells protrude actin rich fillipodial-like projections into the surrounding stroma. Platelets and bone marrow derived cells (e.g. neutrophils, monocytes) can be isolated from human blood and perfused simultaneously with tumor cells to visualize and assess the importance of host-tumor interactions in the circulation and during extravasation. We demonstrate the addition of activated platelets and neutrophils separately increases both tumor cell transmigration efficiency and kinetics (Fig 2A). Tumor cell interactions with the endothelial basement membrane can also be visualized via immunofluorescent staining of different ECM proteins. Via siRNA interference, we find that extravasation is highly dependent on laminin receptors alpha 3 and alpha 6 integrins, which is supported by the close association of extravasated tumor cells to the laminin basement membrane layer (Fig 2B). Lastly, survival and proliferation of cells post-extravasation can be quantified on a single cell level up to 48 hours post seeding to mimic the dynamics of initial micrometastases formation (Fig 3). We show that extravasated cells continue to proliferate within the parenchyma while intravascular cells proliferate at a lesser extent, suggesting that cells which extravasate and gain access to the stroma are more prone to survival. Conclusion: We have demonstrated the numerous capabilities and potential of this on-chip microvacsulature platform to investigate and gain molecular mechanistic insights into the extravasation and early seeding cascades. Further, the platform has the potential to test and screen therapies targeted towards various aspects of hematogenous dissemination.