A novel 3D high-content assay identifies compounds that prevent fibroblast invasion into tissue surrogates

•A novel 3D high content invasion assay using living tissue surrogates.•Identified hits prevented tissue invasion of fibroblasts into living spheroids.•Two major hit classes, prostaglandins and ROCK inhibitors were identified.

Invasion processes underlie or accompany several pathological processes but only a limited number of high-throughput capable phenotypic models exist to test anti-invasive compounds in vitro. We here evaluated 3D co-cultures as a high-content phenotypic screening system for fibrotic invasive processes. 3D multicellular spheroids were used as living tissue surrogates in co-culture with fluorescently labeled lung fibroblasts to monitor invasion processes by automated microscopy. This setup was used to screen a compound library containing 480 known bioactive substances. Identified hits prevented fibroblast invasion and could be subdivided into two hit classes. First, Prostaglandins were shown to prevent fibroblast invasion, most likely mediated by the prostaglandin EP2 receptor and generation of cAMP. Additionally, Rho-associated protein kinase (ROCK) inhibitors prevented fibroblast invasion, possibly by inactivation of myosin II. Importantly, both Prostaglandins and ROCK inhibitors are potential treatment options shown to be effective in in vitro and in vivo models of fibrotic diseases. This validates the presented novel phenotypic screening approach for the evaluation of potential inhibitors and the identification of novel compounds with activity in diseases that are associated with fibroblast invasion.