Tumor cell extravasation in an in vitro microvascular network platform

Abstract

A deeper understanding of the mechanisms of tumor cell extravasation is essential in creating therapies that target this crucial step in cancer metastasis. Extravasation assays exist, but with limitations; data from in vivo models are frequently inferred from low-resolution end-point assays while most in vitro platforms are limited in their physiological relevance of the tumor microenvironment. To address this need, we developed a microfluidic platform to study tumor cell extravasation from in vitro microvascular networks formed via vasculogenesis. Various techniques to yield optimal networks were assessed in order to achieve an appropriate balance between vascular growth, remodeling and stabilization. These include the application of various soluble biochemical factors and both paracrine and juxtacrine co-culture with stromal cells. We demonstrate that out of all methods attempted, paracrine non-contact co-culture with human lung fibroblasts yield the most interconnected and stable networks. Vasculatures developed exhibit tight endothelial cell-cell junctions, basement membrane deposition and physiological values of vessel permeability. Employing our assay, we demonstrate impaired endothelial barrier function and increased extravasation efficiency with inflammatory cytokine stimulation, as well as positive correlations between the metastatic potentials of tumor cells lines and their extravasation capabilities. High-resolution time-lapse microscopy reveals the highly dynamic nature of extravasation events, beginning with thin tumor cell protrusions across the endothelium followed by extrusion of the remainder of the cell body through the formation of sub nuclear sized openings in the endothelial barrier. No disruption to endothelial cell-cell junctions is discernible at 60X, or by changes in local barrier function after completion of transmigration. Using our platform, we also elucidate the extravasation patterns of different tumor cell subpopulations, including mechanically lodged cells, single arrested non-trapped cells, and tumor cell clusters. Our platform offers key advantages over existing in vitro extravasation models by enabling all of the following: (1) high temporal and spatial resolution of extravasation events, (2) the ability to perform parametric studies in a tightly controlled and high throughput microenvironment and (3) increased physiological relevance compared to 2D and 3D planar monolayer models. Findings from our platform result in a deeper understanding of tumor cell extravasation mechanisms and demonstrate our assay's potential to be employed for the discovery of factors that could inhibit this crucial step in metastasis.