Kanda, N; (2015) Fibroblast spheroids: a useful assay for drug screening in idiopathic pulmonary fibrosis? Doctoral thesis , UCL (University College London).

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Abstract

Introduction: Idiopathic pulmonary fibrosis (IPF) is characterised by excessive deposition of extracellular matrix proteins and destruction of the lung architecture. The aetiology of this disorder is unknown and few effective therapies are available. Several models have been established to identify key pathological cells and mediators that may be important in IPF, however these models lack the classical histopathological features seen in IPF, such as fibrotic foci. The aim of this project was to develop a novel 3-D in vitro assay system which more closely mimics a fibrotic focus, for pre-clinical drug evaluation. Methods: Primary human lung fibroblasts were isolated as outgrowths from small (<1 mm3) lung explants (non-IPF and IPF patients). Non-IPF (n = 10) and IPF (n = 10) fibroblasts were cultured in non- adherent 96-well plates to generate fibroblastic spheroids. Spheroid formation and phenotypic features were characterised using time-lapse videomicroscopy, histological analysis, (including TUNEL assay) and, electron microscopy. RNA was extracted from the spheroids and microarray analysis and qRT-PCR were used to analyse mRNA levels. Total collagen was measured using HPLC analysis of hydroxyproline levels while active TGFβ within the spheroid homogenates and supernatants were measured using the transformed mink lung epithelial cell bioassay. A medium-throughput screen of potential anti-fibrotic compounds (using a focused GSK compound library known as the fibrosis toolbox) was also performed, using hydroxyproline levels as the endpoint measure. Results: Non-IPF and IPF fibroblasts were able to form non-proliferating spheroids within 24 hours of incubation, with clear organisation and orientation of cells within the spheroid. IPF spheroids had a myofibroblastic phenotype with increase expression of αSMA. TUNEL assay identified increased numbers of apoptotic cells in non-IPF spheroids in comparison to IPF spheroids, which may be due, in part, to autocrine/paracrine COX1-mediated PGE2 generation. The mink lung cell assay demonstrated that non-IPF and IPF spheroids spontaneously produced high levels of active TGFβ, which was partially dependent on β3 and β8 integrins. Antagonising TGFβ signalling did not however affect spheroid collagen production. Microarray data analysis illustrated a limited number of differentially expressed genes, with the majority involved in encoding proteins that play a key role in metabolic pathways. The fibrosis toolbox identified potential target molecules that impact on collagen biosynthesis including EP2/4 compounds, an integrin αv inhibitor, Smo antagonists, MCP-1 inhibitor, and mTORC 1/2 inhibitors. Conclusions: Fibrotic fibroblast spheroids mimic some of the key characteristics of fibroblasts in fibrotic foci of IPF lungs (i.e. increased collagen production, elevated levels of active TGFβ and resistance to apoptosis). In addition, microarray and medium-throughput screening identified several potential targets. Therefore, fibrotic fibroblast spheroids may represent a novel assay system for pre-clinical drug evaluation, and warrant further investigation.

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