3D lung organoid platform for pre-clinical testing of active substances targeting senescence

Abstract

Chronic lung diseases such as chronic obstructive pulmonary disease, cystic fibrosis and idiopathic lung disease are incurable. Epithelial senescence, a state of dysfunctional cell cycle arrest, contributes to the progression of such diseases. Therefore, lung epithelial cells are an interesting target for therapeutic intervention. Here, we present a 3D lung organoid platform for the preclinical testing of active substances with regard to senescence, toxicity, and inflammation under standardized conditions. Senescence was induced with doxorubicin in 3D lung organoids, and measured by galactosidase activity using a fluorescence-based assay and p21 protein abundance by immunostaining and Western blot. Viability was assessed using LDH and MTT tests as well as microscopic observation of the organoids. Pharmaceutical compounds such as quercetin and dasatinib antagonized doxorubicin-induced senescence and selectively decreased the release of epithelial mediators without compromising organoid integrity. Undifferentiated airway epithelial cells cultured in conventional 2D culture were less responsive to the tested drugs. In conclusion, the 3D lung organoid platform enables for the analysis of senescence-related processes and will allow the pre-selection of a wide range of compounds (e.g. natural products) in preclinical studies, thus reducing the need for animal testing.