On-chip tumor vasculatures reconstitute endothelial immune barrier expressing FasL

Abstract

Here we propose a microfluidic tumor vasculature model that recreates physiologically relevant tumor microenvironments, inducing apoptosis in immune cells through FasL-Fas ligation. A perfusable tumor microvascular network (MVN) was constructed in 3D hydrogel inside a microfluidic chip. Human umbilical vein endothelial cells transfected with red fluorescence protein (RFP-HUVEC) and hepatocarcinoma cells (HepG2) were mixed in fibrin gel (10:1 ratio) and were injected into a central gel region of the chip. With 4-day culture and additional 20-hour treatment in the humidified incubator, FasL-expressed MVN with HepG2 was successfully established. We quantitatively measured on-chip endothelial FasL expression levels under TME-specific conditions using immuno-fluorescently labeled antibodies. In particular, replacement of oxygen level from 18% (Control condition) to 1.5% increased endothelial FasL intensity by 1.9-fold in the MVN w/o HepG2 under EBM culture. We further investigated whether the cell-to-cell interaction between HepG2 and HUVECs influences endothelial FasL expression. Notably, co-culture with HepG2 under 18% O2 EBM significantly increased the relative HUVEC FasL intensity by 1.5-fold. Hypoxic microenvironment amplified the tendency that the relative HUVEC FasL intensity increased 1.4- and 2.1-fold, to the MVN w/ HepG2 under 18% O2 EBM and to the control condition, respectively. We further investigated whether the upregulated endothelial FasL in TME-specific conditions promotes apoptosis of target Jurkat cells via Fas-FasL binding. As expected, the percentage of Jurkat apoptosis significantly increased in the MVN w/ HepG2 chips where oxygen level was maintained at 1.5% instead of 18%, suggesting the possible correlation between the increased apoptosis rates and the FasL enhancement (55.0±8.5% v.s. 12.5±5.6%, p-value<0.001). Taken together, these results support that our microfluidic assay could be utilized in evaluating anti-tumor immunity of patient-derived T cells and screening of novel therapeutic blockades.