(1242-C) Cell and tissue-based in vitro pulmonary fibrosis assays for identification of compounds with potential anti-fibrotic activity
Tuesday, February 6, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Introduction Pulmonary fibrosis (PF) is a complex disease that is difficult to reproduce in vitro. Various cell types are involved in pathogenesis of PF making it quite difficult to mimic the key aspects of disease with a single in vitro assay. Better and more predictive screening assays are needed for evaluation of potency and efficacy of novel antifibrotic treatments in addition to the only two approved drugs, nintedanib and pirfenidone. Aim The aim of the study was to develop a comprehensive set of in vitro assays using human primary cells and lung tissue and to validate them by control compounds. Methods Human primary lung fibroblasts from healthy or diseased (IPF) donors, primary bronchial epithelial cells and precision cut lung slices (PCLS) were stimulated with TGFβ and induction of fibrotic markers like collagen 1, fibronectin 1 and alpha-smooth muscle actin (αSMA), was analyzed by RT-qPCR, ELISA or immunostaining. Production of components of extracellular matrix was measured after 48 h for up to 8 days, depending on the assay. TGFβ receptor type I inhibitor (SB525334) and tyrosine kinase receptors inhibitor (nintedanib) were tested in listed in vitro models. Results In lung primary cells, as well as in PCLS, expression of biomarkers of extracellular matrix, αSMA, collagen 1 and fibronectin 1, was highly increased by TGFβ on RNA level. Protein levels of pro-collagen 1 and fibronectin 1, detected by ELISA, and αSMA and collagen, detected by immunostaining, were also increased. Reference compounds tested, SB525334 and nintedanib, concentration dependently inhibited fibrotic markers production. Conclusions In vitro TGFβ-driven assays relevant to pulmonary fibrosis were developed in lung primary fibroblasts, epithelial cells, and tissue. Fibrotic markers production was inhibited using SB525334, which activity confirmed that the process is activated through TGFβ receptor. Nintedanib, oral treatment for idiopathic lung fibrosis, inhibited production of fibrotic markers and confirmed relevance of developed in vitro assays. Overall, we present platform of different assays, using relevant primary cells and tissues, which provide comprehensive evaluation of new potential anti-fibrotic treatments in specific cellular systems (fibroblasts, epithelial) important for fibrosis developments as well as in more complex (PCLS) biological systems.