Quantification of fibrosis extend and airspace availability in lung: A semi-automatic ImageJ/Fiji toolbox

PLOS ONE LAB PROTOCOL Quantification of fibrosis extend and airspace availability in lung: A semi-automatic ImageJ/ Fiji toolbox Bertrand-David Ségard ID1*, Kodai Kimura1, Yuimi Matsuoka1, Tomomi Imamura1, Ayana Ikeda1, Takahiro Iwamiya1,2 1 Research and Development Department, Metcela Inc., Kawasaki, Kanagawa, Japan, 2 Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Ségard B-D, Kimura K, Matsuoka Y, Imamura T, Ikeda A, Iwamiya T (2024) Quantification of fibrosis extend and airspace availability in lung: A semi-automatic ImageJ/Fiji toolbox. PLoS ONE 19(2): e0298015. https://doi. org/10.1371/journal.pone.0298015 Editor: Panayiotis Maghsoudlou, University College London Institute of Child Health, UNITED KINGDOM Received: July 18, 2023 Accepted: January 17, 2024 Published: February 29, 2024 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0298015 Copyright: © 2024 Ségard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Macros are available on GitHub: DOI: 10.5281/zenodo.10669353 Repository URL: https://github.com/Metcela-Code/ * Abstract The evaluation of the structural integrity of mechanically dynamic organs such as lungs is critical for the diagnosis of numerous pathologies and the development of therapies. This task is classically performed by histology experts in a qualitative or semi-quantitative manner. Automatic digital image processing methods appeared in the last decades, and although immensely powerful, tools are highly specialized and lack the versatility required in various experimental designs. Here, a set of scripts for the image processing software ImageJ/Fiji to easily quantify fibrosis extend and alveolar airspace availability in Sirius Red or Masson’s trichrome stained samples is presented. The toolbox consists in thirteen modules: sample detection, particles filtration (automatic and manual), border definition, air ducts identification, air ducts walls definition, parenchyma extraction, MT-staining specific pre-processing, fibrosis detection, fibrosis particles filtration, airspace detection, and visualizations (tissue only or tissue and airspace). While the process is largely automated, critical parameters are accessible to the user for increased adaptability. The modularity of the protocol allows for its adjustment to alternative experimental settings. Fibrosis and airspace can be combined as an evaluation of the structural integrity of the organ. All settings and intermediate states are saved to ensure reproducibility. These new analysis scripts allow for a rapid quantification of fibrosis and airspace in a large variety of experimental settings. Introduction Respiratory diseases are one of the leading causes of disability and death worldwide, through a decline in lung function [1–3]. The induction of fibrosis is a major factor in this functional decline as the increase in stiffness of fibrotic tissues leads to mechanical defects. Additionally, the increased volume of these tissues leads to the collapse of alveolar structures and a decrease in gas exchange capacity. The etiology of lung fibrosis is varied and incompletely described. The main disorders known to promote scarring are idiopathic pulmonary fibrosis (IPF), collagen vascular PLOS ONE | https://doi.org/10.1371/journal.pone.0298015 February 29, 2024 1 / 15 PLOS ONE Lung-fibrosis-and-airspace-quantification The protocol is available on Protocols.io (publication synchronized with PLOS ONE): RESERVED DOI: 10.17504/protocols.io.14egn7nqmv5d/v1 Repository URL: https://www.protocols.io/view/ quantification-of-fibrosis-extend-and-airspace-avab9ztr76n Raw data, settings, results, and figures are available on Figshare: Repository URL: https:// figshare.com/projects/Supporting_data_for_ PLOS_ONE_S_gard_2024_/146790". Funding: All authors of the present article are current or former employees of Metcela Inc. This study was funded by grants from Kawasaki City (https://www.city.kawasaki.jp/en/index.html), namely “Kawasaki City New Technology/New Product Development Support Project Subsidy” (Grant No. 84; awarded to TIw) and “Kawasaki City “New Normal” Research and Development Subsidy” (Grant No. 225; awarded to TIw). Sample preparation, imaging, and method development were financed by these grants. Metcela provided support in the form of salaries for authors BDS, KK, YM, TIm, AI, and Tiw. Funders did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the “author contributions” section. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests to declare. All authors of the present article are current or former employees of Metcela Inc. Metcela is developing cell therapies to treat chronic organ diseases. Metcela’s core patented technology involves a particular population of cardiac fibroblasts, namely VCAM-1-positive cardiac fibroblasts. VCAM-1positive cardiac fibroblasts are known to replenish and re-establish the damaged cardiac muscles and the microenvironment surrounding them. Two products for heart failure patients are currently being tested in phase I clinical trial. Takahiro Iwamiya is a co-founder and co-CEO of Metcela Inc. and has ownership of stocks. TIw has the authority to make payment decisions regarding employee salaries. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Quantification of fibrosis extend and airspace availability in lung disorders, telomere disease, Erdheim-Chester disease (ECD), and Hermansky-Pudlak syndrome (HPS) [4, 5]. Additionally, the pandemic of COVID-19 leads to a surge of new cases of lung fibrosis [6, 7]. Therefore, there is an urgent need to develop therapies for pulmonary fibrosis [1, 2]. One important milestone in such endeavor is the standardization of the method to evaluate lung structural integrity in experimental models or clinical specimens. Affordability, speed, and flexibility are desirable to accelerate research globally. The precise quantification of pulmonary fibrosis is challenging, and not standardized [8]. Because of technical challenges, most methods are applied on random fields instead of whole slide scans [5]. Indeed, the most widely used method to estimate lung fibrosis was proposed by Thom (...truncated)