Lung Fibrosis: Drug Screening and Disease Biomarker Identification with a Lung Slice Culture Model and Subtracted cDNA Library

Abstract

Pulmonary fibrosis is a progressive and irreversible disorder with no appropriate cure. A practical and effective experimental model that recapitulates the disease will greatly benefit the research community and, ultimately, patients. In this study, we tested the lung slice culture (LSC) system for its potential use in drug screening and disease biomarker identification. Fibrosis was induced by treating rat lung slices with 1ng/ml TGF-ß1 and 2.5μM CdCl2, quantified by measuring the content of hydroxyproline, and confirmed by detecting the expression of collagen type III alpha 1 (Col3α1) and connective tissue growth factor (CTGF) genes. The anti-fibrotic effects of pirfenidone, spironolactone and eplerenone were assessed by their capability to reduce hydroxyproline content. A subtractive hybridisation technique was used to create two cDNA libraries (subtracted and unsubtracted) from lung slices. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed to assess the subtraction efficiency of the subtracted cDNA library. Clones from the two libraries were sequenced and the genes were identified by performing a BLAST search on the NCBI GenBank database. Furthermore, the relevance of the genes to fibrosis formation was verified. The results presented here show that fibrosis was effectively induced in cultured lung slices, which exhibited significantly elevated levels of hydroxyproline and Col3α1/CTGF gene expression. Several inhibitors have demonstrated their anti-fibrotic effects by significantly reducing hydroxyproline content. The subtracted cDNA library, which was enriched for differentially expressed genes, was used to successfully identify genes associated with fibrosis. Collectively, the results indicate that our LSC system is an effective model for the screening of drug candidates and for disease biomarker identification.

Fibrotic microtissue array to predict anti-fibrosis drug efficacy

“…The contraction of fibroblast/myofibroblast spheroids has been used as a phenotypic parameter in anti-fibrosis screening 8 , 39 ; however, the lack of morphological control and stiffness measurement of this model resulted in limited prediction power and accuracy for drug efficacy. Tissue slices prepared from different organs allowed the study of anti-fibrosis drug efficacy on multiple cell types in well-retained anatomical structures, but the low-throughput of this model limited its application in drug screening 40 , 41 . The fibrotic lung microtissue array system presented in the current study recapitulated key biomechanical properties of both healthy and fibrotic lung alveolar tissues, allowing for simultaneous biochemical and biomechanical analysis of the efficacy of the anti-fibrosis drugs; therefore, it represents a novel approach for drug screening with improved physiological relevance, accuracy and throughput.…”

Taking Advantage of 3D Human Cell Culture Platforms to Study Pulmonary Fibrotic Diseases

“…Thus, this "mini lung" has been used as a model of lung diseases, and as a tool for toxicology studies, drug screening, and potential therapeutic targets [89][90][91][92][93] . For example, PCLT exposed to cadmium chloride and TGF-β1 demonstrated pathohistological changes similar to the histological patterns seen in early lung fibrogenesis, such as an increase in pro-fibrotic genes, myofibroblasts activation leading to higher ECM deposition and changes in protein patterns 90,94 . Recently, non-IPF/ILD human PCLT were exposed to a profibrotic cocktail (with TGF-β, PDGF-AB, TNF-α, and LPA), which resulted in fibrotic-like changes in the lung tissue, such as higher production of profibrotic and proinflammatory cytokines, ECM secretion, deposition, and alveolar epithelium injury 84 .…”

“…PCLT have demonstrated to be a useful tool to correlate cellular function with organ physiology as they have been used to study responses to stimuli such as contraction of the airways and the immune response 85,87,88 . Thus, this "mini lung" has been used as a model of lung diseases, and as a tool for toxicology studies, drug screening, and potential therapeutic targets [89][90][91][92][93] . For example, PCLT exposed to cadmium chloride and TGF-β1 demonstrated pathohistological changes similar to the histological patterns seen in early lung fibrogenesis, such as an increase in pro-fibrotic genes, myofibroblasts activation leading to higher ECM deposition and changes in protein patterns 90,94 .…”

Use of precision cut lung slices as a translational model for the study of lung biology

“…PCLS have been successfully used to study the early onset of lung fibrosis in IPF. By exposure to TGF-β1 and cadmium chloride, PCLS from human or rat have shown relevant pathohistological changes observed in early lung fibrosis, including upregulation of important pro-fibrotic genes, increased thickness of alveolar septa, and aberrant activation of pulmonary cells [38, 85, 86]. More recently, Alsafadi et al established an ex vivo human PCLS model of early fibrosis, which requires exposure of PCLS to a combination of profibrotic growth factors and signaling molecules (TGF-β1, TNF-α, platelet-derived growth factor-AB, and lysophosphatidic acid), paving a way to study early-stage IPF pathomechanisms and evaluate novel therapies [29].…”