T.H. Booij1, H. Bange1, K. Yan4, G. van Westen3, W.N. Leonhard2, B. van de Water1, D.J.M. Peters2 and L.S. Price1,4
1Division of Toxicology, LACDR, Leiden University, The Netherlands; 2Department of Human Genetics, LUMC, The Netherlands; 3Division of Medicinal Chemistry, LACDR, Leiden University, The Netherlands; 4OcellO B.V., Leiden, The Netherlands
Introduction. Polycystic kidney diseases (PKDs) are inherited diseases characterized by the formation of fluid-filled cysts in the kidney. Although caused by mutations in the PKD genes, the mechanism of cyst formation is poorly understood and there very limited treatment options. 2D cell-culture models are not able to recapitulate cysts and are therefore unsuitable for phenotypic screening for PKD. 3D cell culture allows the formation of kidney epithelial cysts and therefore presents an opportunity to screen for compounds that inhibit cyst growth.
Methods. Immortalized wild type and PKD1-/- mouse kidney epithelial cells were cultured in 3D hydrogels to recapitulate renal cysts in vitro. This methodology was implemented for use with 384-well plates using automated liquid handling and automated microscopy. In-house 3D image analysis software was used to extract phenotypic information from the cell cultures to measure cyst size, count individual cells/nuclei and discriminate compounds that induced cytotoxicity from compounds that only reduced cyst size. This method was used to screen a library of 273 published kinase inhibitors and validate many of the hit compounds.
Results/Conclusion. We used the PKD assay to screen a compound library of kinase inhibitors which generally contained several compounds reported to inhibit each target. Consistent with published literature, we found that the majority of (but not all) mTOR inhibitors profoundly inhibited cyst growth. In contrast, only a minority of PI3K inhibitors inhibited cyst growth in vitro. In addition to these well-described targets in PKD, we identified several novel and potentially drugable targets.
In conclusion, high-content screening of 3D cultures offers a more physiologically relevant context for the screening and identification of active compounds against PKD in vitro, in a high-throughput setting.