Engineered microvasculature platforms to study tumor-host-matrix interactions during metastatic seeding

Abstract

Distant metastases, which result in >90% of cancer related deaths, is enabled by hematogenous dissemination of tumor cells via the circulation. In particular, tumor cell extravasation is thought to be an essential and potential rate-limiting step, as most metastases are found in the extravascular space rather than intraluminal at distant organs. However, mechanistic insights into the cellular and molecular players during extravasation are limited due to technical challenges in observing real-time events in vivo. Increased understanding of the extravasation cascade is critical in uncovering new opportunities for therapeutic intervention during early metastatic dissemination. In this thesis, we develop an in vitro model of the human microcirculation with the capability to recapitulate several discrete steps of hematogenous dissemination, including tumor cell circulatory transport, arrest, and transendothelial migration. The microdevice features self-organized human microvascular networks through which tumor cells can be perfused and tracked over time via standard confocal microscopy. In addition to improved throughput for parametric studies, robust and rapid scoring of extravascular cells combined with high spatio-resolution imaging for deciphering cell morphological dynamics can be easily achieved due to excellent optical accessibility. To demonstrate the ability to obtain novel biological insights, we apply the assay to decipher the roles of tumor integrins in modulating extravasation. In particular, we deplete integrin beta-1 in tumor cells and isolate the specific defects in the extravasation cascade. Dynamic imaging revealed that [beta]1-depleted cells lacked the ability to sustain protrusions into the subendothelial matrix in contrast to control cells. Specifically, adhesion via [alpha]3[beta]1 and [alpha]6[beta]1 to subendothelial laminin was a critical prerequisite for successful transmigration, as well as basement membrane breaching. Combined with validation from in vivo metastasis assays, we find that tumor beta-1 integrin is a critical mediator of extravasation and metastases formation. Furthermore, we demonstrate the potential of our assay to recapitulate the complexities of the host microenvironment via modular addition of non-cancer host cells. Specifically, we explore the interactions of circulating human neutrophils with tumor cells and demonstrate that their interactions can exert pro-extravasation effects through neutrophil-derived IL-8. Through high spatio-temporal resolution imaging, we further identify novel mechanisms through which neutrophils are sequestered and confined at the vicinity of trapped tumor cells during flow conditions, and how the spatial localization of their secreted factors can act to facilitate tumor transmigration. Key words: Metastasis, extravasation, microfluidics, tumor cell migration, neutrophils, integrins.