Problem or question being addressed Irreversible pulmonary damage, including remodeling, fibrosis and emphysema are characteristic features of respiratory diseases, including systemic sclerosis-associated interstitial lung disease (SSc-ILD) (1), idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) (2, 3). Impaired lung epithelial repair responses and dysregulated immune cell activation may contribute to development of this lung tissue damage. Lung damage results in reduced lung function which ultimately restricts the patient’s quality of life and in the worst scenario requires lung transplantation or results in death. Recent studies demonstrate unique cell populations in lungs of patients with IPF, SSc-ILD (4-6) and COPD (7), which are characterized by loss of classical markers of basal cells such as KRT5 and KRT15, show expression of KRT17 (6), and are named “aberrant basaloid cells” (8) (KRT5−/KRT17+ cells). These cells were observed at the interface of areas of ‘normal’ alveolar lung tissue and fibrotic tissue, but origin and role of basaloid cells in pathogenesis are not understood. KRT17 expression has been shown in lungs of COPD patients and to some extent in severe COVID-19 patients (9), however whether these cells contribute to these diseases, appear at a specified location and/or interact with other cell-types is currently unknown. Issue and aim of the project The role of KRT17+ cells in ILD and emphysema development and progression is currently unclear, despite their localization at the interface of affected and unaffected tissue in SSc-ILD. We hypothesize that the aberrant basaloid cells play a key role in the aberrant lung repair in ILD and emphysema. Therefore, in this project, we aim to characterize the microenvironment and cellular interactions of these aberrant basaloid cells in ILD and emphysema by applying imaging mass cytometry (IMC), which will provide us a detailed look at this unknown but important cell-type and the cells they interact with. Rational for approach We confirmed the presence of the KRT17+ aberrant basaloid cells in SSc-ILD explanted lung tissue using Multiplex-fluorescence imaging (max. 7 markers) in explanted lung tissue from these patients undergoing lung transplantation (n=6). Furthermore, we have access to emphysematous lung tissue and to autopsy material from COVID-19 patients (n= 6-8; PI). To characterize the localization of these aberrant basaloid cells in different types of lung damage environments, and unravel their role, we need much more in-depth analysis. Therefore, we will apply state-of-the-art high-dimensional imaging mass cytometry (IMC), which allows the simultaneous detection of up to 40 markers in a single tissue slide. We will therefore not only obtain information on expression of these proteins, but also on their localization and microenvironment. Details of suggested approach The protocols for antibody staining on lung tissue sections, data acquisition and data analysis tools for IMC have been optimized via a collaborative project. We will use markers for not only abnormal basaloid cells, but also major immune lineages, remodeling, lung epithelial cells and cellular senescence, to gain insight into the inflammatory and structural alterations in affected lung tissue. We will follow the following procedure: o Immunohistochemical staining to confirm the presence of KRT17+ cells in emphysematous and COVID-19 tissue; optimization of IMC panel o For each sample, we will use sequential FFPE sections: o H&E stained sections will be used for selection of regions of interest (ROIs) o We will measure the ROIs using the Hyperion system in the FCF facility (LUMC). o Analysis on the ROIs using Imacyte by identification of the aberrant basaloid cells, and characterization of the localization and cellular interactions How it will affect the broader field The obtained datasets on the expression levels of > 38 cell-specific markers and their specific localization will provide detailed insight into the localization of aberrant basaloid cells, their microenvironment and the cellular interactions of these basaloid cells in lung tissue from patients with emphysema, ILD and severe COVID-19. This information is essential for understanding if these cells could be used as a new therapeutic target, to either eliminate or support their function. This information is highly needed as currently there are no drugs available for these patient that can reverse or even halt their disease. By extending our knowledge on the localization of aberrant basaloid cells and their interactions with surrounding cells, we will expect to gain important insight into the dysregulated repair process in lung diseases. This provides us with targets that we can study in our models to identify how this dysregulated repair can be normalized again. Furthermore, we will be able to study the origin of the aberrant basaloid cell and investigate whether these cells occur in an early disease-state in emphysema and ILD. Currently, alveolar damage in emphysema and fibrosis in ILD are only identified in a late progressive state. Therefore, there is a need for an early biomarker, which can be used to monitor disease progression and therapy efficiency. References 1. Khedoe P, Marges E, Hiemstra P, Ninaber M, Geelhoed M. Interstitial Lung Disease in Patients With Systemic Sclerosis: Toward Personalized-Medicine-Based Prediction and Drug Screening Models of Systemic Sclerosis-Related Interstitial Lung Disease (SSc-ILD). Front Immunol. 2020;11:1990. 2. Broekman W, Khedoe P, Schepers K, Roelofs H, Stolk J, Hiemstra PS. Mesenchymal stromal cells: a novel therapy for the treatment of chronic obstructive pulmonary disease? Thorax. 2018;73(6):565-74. 3. Khedoe P, Wu X, Gosens R, Hiemstra PS. Repairing damaged lungs using regenerative therapy. Curr Opin Pharmacol. 2021;59:85-94. 4. Jaeger B, Schupp JC, Plappert L, Terwolbeck O, Kayser G, Engelhard P, et al. Airway Basal Cells show a dedifferentiated KRT17^highPhenotype and promote Fibrosis in Idiopathic Pulmonary Fibrosis. bioRxiv. 2020:2020.09.04.283408. 5. Habermann AC, Gutierrez AJ, Bui LT, Yahn SL, Winters NI, Calvi CL, et al. Single-cell RNA sequencing reveals profibrotic roles of distinct epithelial and mesenchymal lineages in pulmonary fibrosis. Science Advances. 2020;6(28):eaba1972. 6. Carraro G, Mulay A, Yao C, Mizuno T, Konda B, Petrov M, et al. Single-Cell Reconstruction of Human Basal Cell Diversity in Normal and Idiopathic Pulmonary Fibrosis Lungs. Am J Respir Crit Care Med. 2020;202(11):1540-50. 7. Sauler M, McDonough JE, Adams TS, Kothapalli N, Barnthaler T, Werder RB, et al. Characterization of the COPD alveolar niche using single-cell RNA sequencing. Nat Commun. 2022;13(1):494. 8. Adams TS, Schupp JC, Poli S, Ayaub EA, Neumark N, Ahangari F, et al. Single-cell RNA-seq reveals ectopic and aberrant lung-resident cell populations in idiopathic pulmonary fibrosis. Science Advances. 2020;6(28):eaba1983. 9. Consortia CZIS-CC-, Ballestar E, Farber DL, Glover S, Horwitz B, Meyer K, et al. Single cell profiling of COVID-19 patients: an international data resource from multiple tissues. medRxiv. 2020:2020.11.20.20227355. 10. Eenjes E, van Riet S, Kroon AA, Slats AM, Khedoe P, Boerema-de Munck A, et al. Disease modeling following organoid-based expansion of airway epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2021;321(4):L775-L86.