NINTEDANIB AND PIRFENIDONE AFFECT GROWTH AND DIFFERENTIATION OF HUMAN ALVEOLAR TYPE 2 CELLS

Abstract

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease characterized by epithelial cell senescence. Pirfenidone and nintedanib are approved drugs for the treatment of IPF. They significantly slow disease progression, but their mechanisms of action, especially on alveolar type 2 (AT2) cells, are poorly understood. We addressed this question by evaluating colony formation and growth of human AT2 cells co-cultured with fibroblasts in organoid culture in the presence of pirfenidone and nintedanib. We further evaluated molecular changes induced by these drugs via single cell RNA-seq of treated organoids. MethodsAT2 cells isolated from normal donor lungs or IPF patients were mixed with human fibroblasts in 3D culture and grown in the absence or presence of pirfenidone or nintedanib. After 14 days in culture, the organoids were quantified and cells extracted from Matrigel for single cell RNA-seq. ResultsAT2 cell organoids cultured in the presence of pirfenidone or nintedanib resulted in increased colony formation and, in the case of nintedanib, in larger colonies. We observed that untreated or pirfenidone treated AT2 cells lost surfactant protein C (SFTPC) expression and acquired an expression profile consistent with keratin (KRT)17high/KRT5- basaloid cells, whereas a larger proportion of nintedanib treated cells retained SFTPC expression. In contrast, AT2 cells treated with TGF{beta} inhibitor exhibited intermediate (SFTPC-/KRT17low) gene expression profile. ConclusionThese results suggest that nintedanib maintains an AT2-like expression state in culture and acts proximal to TGF{beta}. Conflict of Interest StatementPJW was supported by grants from Boehringer Ingelheim, Roche, Sanofi, Pliant and Arda Therapeutics and received personal fees from Boehringer Ingelheim and Sanofi. None of these companies had a role in the design or analysis of the study or in the writing of the manuscript. ASM, GP, JRR and DG are employees of Genetech. The other authors have no conflicts of interest to declare.