An integrated multiomic and quantitative label-free microscopy-based approach to study pro-fibrotic signalling inex vivohuman precision-cut lung slices

Abstract

Fibrosis can affect any organ resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by expansion of connective tissue due to excessive deposition of extracellular matrix proteins (ECM), including the fibrillar forms of collagen. A significant limitation for discovering cures for fibrosis is the availability of suitable human models and techniques to quantify mature fibrillar collagen deposition as close as possible to human physiological conditions. Here we have extensively characterised an ex vivo cultured human lung tissue-derived, precision-cut lung slices model (hPCLS) using label-free second harmonic (SHG) light microscopy to quantify fibrillar collagen deposition and mass spectrometry-based techniques to obtain a proteomic and metabolomic fingerprint of hPCLS in ex vivo culture.We demonstrate that hPCLS are viable and metabolically active with mesenchymal, epithelial, endothelial, and immune cell types surviving for at least 2 weeks in ex vivo culture. Analysis of hPCLS-conditioned supernatants showed a strong induction of pulmonary fibrosis-related ECM proteins upon TGFß1 stimulation. This upregulation of ECM proteins was not translated into an increased deposition of fibrillar collagen. In support of this observation, we revealed the presence of a pro-ECM degradation activity in our ex vivo cultures of hPCLS, inhibition of which by metalloproteinase inhibitor resulted in increased collagen deposition in response to TGFß1 stimulation. Together the data show that an integrated approach of measuring soluble pro-fibrotic markers alongside quantitative SHG-based analysis of fibrillar collagen is a valuable tool for studying pro-fibrotic signalling and testing antifibrotic agents.

The Challenge of Long-Term Cultivation of Human Precision-Cut Lung Slices

“…via LPS and vaccine antigens) when cultured for up to 14 days 12 . Even though the data is very promising and points to a long-term experimental window for human PCLS cultivation 12 , some functional and cellular changes could be observed during long term cultivation 5,12,13,36 . Long-term cultivated PCLS show decreased cytokine and chemokine production in response to external stimuli, such as LPS 12,13 .…”

“…Long-term cultivated PCLS show decreased cytokine and chemokine production in response to external stimuli, such as LPS 12,13 . Additionally, proteomic data revealed a persistent inflammatory activity during long-term cultivation, especially present during the first 4 days in culture 36 . Histomorphologically, a reduced number of pneumocytes and a slight degradation of connective tissue fibers has been described 13 .…”

“…The benefit of PCLS as a model is that a single lung can provide hundreds of slices for both control and experimental conditions while mimicking most of the structural and functional aspects of the respiratory tract and including all the relevant cell types, except those recruited by the circulatory system 5,12,14,15,31-33 . In the majority of studies PCLS were cultured for less than one week, which is suitable for the investigation of most toxicological and pharmacological outcomes 7,17,34,35 , but not when it comes to long-term investigation of repeated exposure to a substance or disease modeling. Indeed, recent studies could show that human PCLS are viable, preserve structural integrity, contain all lung-relevant cell types and are metabolically active for up to 15 days of cultivation 12,13,36 . Recently, it could also been shown, that the embedding of PCLS into specifically engineered hydrogels is able to significantly increase viability and pneumocyte type 2 functionality over a course of 21 days of cultivation when compared with non-encapsulated controls 37 .…”

Targeting Alveolar Repair in Idiopathic Pulmonary Fibrosis

“…How relevant either the PCLS approach, or even TGF-beta stimulation alone on PCLS, might be in predicting clinical efficacy remains unknown. However, several omics-based approaches have recently been optimized for PCLS and will most likely accelerate the characterization of this ex vivo model (156)(157)(158)(159). In addition, other small molecules in Phase I and Phase II (e.g.…”

“…How relevant either the PCLS approach or even the use of TGF-β stimulation alone on PCLSs might be in predicting clinical efficacy remains unknown. However, several omics-based approaches have recently been optimized for PCLSs and will most likely accelerate the characterization of this ex vivo model ( 156 – 159 ). In addition, the use of other small molecules in phase I and phase II trials (e.g., GB0139) and the use of direct modulation of targets expressed in epithelial cells (e.g., JNK) have been shown to be capable of regulating epithelial-cell behavior ( 126 , 138 , 160 ).…”