RT Journal Article
SR Electronic
T1 Novel high throughput 3D ECM remodeling assay identifies MEK as key driver of fibrotic fibroblast activity
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2024.08.26.609699
DO 10.1101/2024.08.26.609699
A1 Liao, Chen-Yi
A1 Hundscheid, Jasmijn HM
A1 Crawford, Justin
A1 ten Dijke, Peter
A1 Coorneaert, Beatrice
A1 Danen, Erik HJ
YR 2024
UL http://biorxiv.org/content/early/2024/08/27/2024.08.26.609699.abstract
AB 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 TGFβ-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 TGFβ, canonical TGFβ signaling is dispensable and, instead, non-canonical activation of MEK-ERK signaling drives ECM remodeling. Moreover, we reveal that higher concentrations of two TGFβ receptor inhibitors while blocking canonical TGFβ signaling, in fact stimulate this MEK-mediated profibrotic ECM remodeling activity.Competing Interest StatementThis research was partly funded by Galapagos and J.C. and B.C. are employees of Galapagos.