Cancer-associated fibroblasts in a 3D engineered tissue model induce tumor-like matrix stiffening and EMT transition.

Abstract

There is an increasing need for the use of 3D in vitro models over 2D culture. Yet, most models remain unable to fully reproduce the complexity observed in vivo. Tissue-engineered models based on the self-assembly method have the potential to better recapitulate the stroma architecture, composition, and cell heterogeneity. Here, we used the self-assembly method and a bladder tissue model to engineer a tumor-like environment, and validated its relevance as a cancer model. The bioengineered bladder cancer model was reconstituted using healthy primary fibroblasts (HFs) and a sub-population of induced cancer-associated fibroblast-like cells (iCAFs) along with a normal urothelium overlay. The iCAFs-derived extracellular matrix (ECM) composition was found to be more fibrotic with increased collagen remodeling and altered fibronectin content. Moreover, the average stiffness of the ECM scaffolds produced by the iCAFs was 7.4 kPa compared to 1.3 kPa for those produced by HFs. In line with those results, urothelial cells overlaid on the iCAFs-derived ECM was found to be more contractile. We also observed increased nuclear translocation of the mechanosensor YAP1 in urothelial cells overlaid on these iCAFs-derived ECM. Furthermore, urothelial cells seeded on iCAFs-derived stroma displayed invasive behavior compared to urothelial cells on HFs. As increased ECM stiffness and cell contractility are associated with the epithelial-to-mesenchymal transition (EMT), we showed an increase expression of the mesenchymal EMT markers, vimentin and ZEB1, within the urothelium on the iCAFs-derived ECM. Overall, our tissue-engineered tumor model achieved stiffness levels comparable to those observed in bladder cancers along with CAF-mediated ECM remodeling, while the urothelium displayed the biological responses that would be expected within a tumor-like environment. Future experiments will take advantage of the 3D and in vivo-like properties of our engineered model to investigate tumor cell invasion.