Novel high throughput 3D ECM remodeling assay identifies MEK as key driver of fibrotic fibroblast activity

Abstract

In fibrotic tissues, activated fibroblasts remodel the collagen-rich extracellular matrix (ECM). Intervening with this process represents a candidate therapeutic strategy to attenuate disease progression. Models that generate quantitative data on 3D fibroblast-mediated ECM remodeling with the reproducibility and throughput needed for drug testing are lacking. Here, we develop a model that fits this purpose and produces combined quantitative information on drug efficacy and cytotoxicity. We use microinjection robotics to design patterns of fibrillar collagen-embedded fibroblast clusters and apply automated microscopy and image analysis to quantify ECM remodeling between-, and cell viability within clusters of TGF{beta}-activated primary human skin or lung fibroblasts. We apply this assay to compound screening and reveal actionable targets to suppress fibrotic ECM remodeling. Strikingly, we find that after an initial phase of fibroblast activation by TGF{beta}, canonical TGF{beta} signaling is dispensable and, instead, non-canonical activation of MEK-ERK signaling drives ECM remodeling. Moreover, we reveal that higher concentrations of two TGF{beta} receptor inhibitors while blocking canonical TGF{beta} signaling, in fact stimulate this MEK-mediated profibrotic ECM remodeling activity.