A phenotypic cell-based extracellular matrix deposition assay for target validation and drug discovery

Background: Idiopathic pulmonary fibrosis (IPF) is a lethal, progressive interstitial lung disease. As approved pharmacological therapies may only slow down disease progression, identification of therapeutic targets and potential pharmacotherapeutics is a major medical need. A central pathogenetic mechanism in IPF is the cytokine-mediated transdifferentiation of lung fibroblasts into myofibroblasts. The consecutive excessive deposition of extracellular matrix (ECM) molecules, such as collagens or ED-A fibronectin, is critical to disease pathogenesis leading to an increased tissue stiffness and ultimately loss of lung function. Inhibiting the excessive accumulation of specific ECM molecules has been shown to alleviate fibrosis in the mouse bleomycin model. Aim: We aimed to develop an assay measuring changes in deposition of distinct ECM molecules for the discovery and validation of possible anti-fibrotic targets and therapeutics. Methods: We measured the ECM deposition of cultured patient-derived primary human lung fibroblasts in 96-well plates by live immunofluorescence labeling and confocal 3D imaging. Results: The assay was validated with collagen synthesis stimulating cytokines, such as TGFβ1, and a small molecule inhibitor of protein secretion Brefeldin A. In a proof of concept study, the assay detected the inhibition of collagen type I deposition by antagonizing the collagen-proline-4-hydroxylase, a therapeutic target confirmed in the bleomycin model. Conclusion: We developed an in-vitro assay that inhibits excessive ECM deposition. By using TGFβ1-activated myofibroblasts, the optimized assay might enable the identification of compounds blocking cytokine-mediated ECM deposition.