Bitterman, Peter B.

Abstract RATIONALE: Mesenchymal progenitor cells (MPCs) in the lungs of individuals with Idiopathic Pulmonary Fibrosis (IPF) show intrinsic fibrogenic properties, possessing a distinct transcriptomic profile and maintaining fibrogenic activity, which contributes to the fibroblast population in IPF. We hypothesize that IPF MPCs are distinguished from control MPCs by a unique gene interaction network. METHODS: We analyzed bulk RNA-seq data, available on the Gene Expression Omnibus (GEO) under accession numbers GSE97038 and GSE276568. In these experiments, CD44hi MPCs were isolated through flow cytometry from mesenchymal cell lines derived from IPF patients (explanted lungs from live IPF patients after lung transplant) and control subjects (lobectomy for clinical reasons) using antibodies against CD44 and SSEA4. The pooled dataset comprises nine IPF and nine control MPC lines. We identified differentially expressed genes (DEGs) in IPF MPCs compared to controls using DESeq and performed pathway enrichment analysis on DEGs using Gene Ontology (GO). We also conducted Gene Set Enrichment Analysis (GSEA) with gene sets related to stemness, pluripotency, and self-renewal. Weighted gene co-expression network analysis (WGCNA) identified functional modules specifically associated with IPF MPCs. RESULTS: Applying an adjusted p-value threshold of 0.05, we identified 134 DEGs in IPF MPCs compared to controls. GO analysis showed significant enrichment in pathways associated with leukocyte migration, extracellular matrix organization, and cell differentiation and growth. GSEA highlighted significant enrichment in two gene sets related to stemness, with adjusted p-values of 0.002 and 0.048. Using WGCNA, we identified 16 functional modules, one of which reached significance based on an adjusted p-value threshold of 0.05 and a correlation coefficient cutoff of 0.45. This significant module demonstrated a positive correlation with IPF status (correlation coefficient of 0.69, adjusted p-value 0.0085). CONCLUSION: This study is the first to characterize a distinct gene expression profile in IPF MPCs compared to controls, highlighting enriched biological processes relevant to IPF pathogenesis. GSEA results further support the hypothesis that IPF MPCs display stem cell-like gene expression patterns, including genes associated with pluripotency and self-renewal. Additionally, we identified functional gene modules that significantly correlate with the fibrogenic properties of IPF MPCs, including hub genes that may play critical roles in promoting fibrosis in IPF. These findings deepen our understanding of IPF MPC biology and identify potential targets for future research. We plan to expand this approach by increasing the sample size to enhance statistical power, aiming to identify more robust functional modules and their highly connected hub genes.

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease targeting the alveolar gas exchange apparatus, leading to death by asphyxiation. IPF progresses on a tissue scale through aberrant matrix remodeling, enhanced cell contraction, and subsequent microenvironment densification. Although two pharmaceuticals modestly slow progression, IPF patient survival averages less than 5 years. A major impediment to therapeutic development is the lack of high-fidelity models that account for the fibrotic microenvironment. Our goal is to create a three-dimensional (3D) platform to enable lung fibrosis studies and recapitulate IPF tissue features. We demonstrate that normal lung fibroblasts encapsulated in collagen microspheres can be pushed toward an activated phenotype, treated with FDA-approved therapies, and their fibrotic function quantified using imaging assays (extracellular matrix deposition, contractile protein expression, and microenvironment compaction). Highlighting the system's utility, we further show that fibroblasts isolated from IPF patient lungs maintain fibrotic phenotypes and manifest reduced fibrotic function when treated with epigenetic modifiers. Our system enables enhanced screening due to improved predictability and fidelity compared to 2D systems combined with superior tractability and throughput compared to 3D systems.

Abstract Rationale Chronic obstructive pulmonary disease is an independent risk factor for lung cancer, but the underlying molecular mechanisms are unknown. We hypothesized that lung stromal cells activate pathological gene expression programs that support oncogenesis. Objectives To identify molecular mechanisms operating in the lung stroma that support the development of lung cancer. Methods The study included subjects with and without lung cancer across a spectrum of lung-function values. We conducted a multiomics analysis of nonmalignant lung tissue to quantify the transcriptome, translatome, and proteome. Measurements and Main Results Cancer-associated gene expression changes predominantly manifested as alterations in the efficiency of mRNA translation modulating protein levels in the absence of corresponding changes in mRNA levels. The molecular mechanisms that drove these cancer-associated translation programs differed based on lung function. In subjects with normal to mildly impaired lung function, the mammalian target of rapamycin (mTOR) pathway served as an upstream driver, whereas in subjects with severe airflow obstruction, pathways downstream of pathological extracellular matrix emerged. Consistent with a role during cancer initiation, both the mTOR and extracellular matrix gene expression programs paralleled the activation of previously identified procancer secretomes. Furthermore, an in situ examination of lung tissue showed that stromal fibroblasts expressed cancer-associated proteins from two procancer secretomes: one that included IL-6 (in cases of mild or no airflow obstruction), and one that included BMP1 (in cases of severe airflow obstruction). Conclusions Two distinct stromal gene expression programs that promote cancer initiation are activated in patients with lung cancer depending on lung function. Our work has implications both for screening strategies and for personalized approaches to cancer treatment.

Abstract Rationale Chronic obstructive pulmonary disease (COPD) is an independent risk factor for lung cancer, but the underlying molecular mechanisms are unknown. We hypothesized that stromal cells comprising the COPD lung contain pathological gene expression programs supporting oncogenesis. Objectives To identify molecular mechanisms operating in the lung stroma that support the development of lung cancer. Methods Study subjects included patients with COPD with (n = 30) or without (n = 30) lung cancer across a spectrum of COPD severity. We conducted multi-omics analysis of nonmalignant lung tissue to quantify the transcriptome, translatome, and proteome. Results Cancer-associated gene expression changes predominantly manifested as alterations in the efficiency of mRNA translation impacting protein levels in the absence of corresponding changes in mRNA levels. The molecular mechanisms driving these procancer translation programs differed on the basis of COPD severity. In mild or no COPD, the mammalian target of rapamycin pathway served as an upstream driver; whereas in advanced COPD, pathways downstream of pathological extracellular matrix emerged. Both the mammalian target of rapamycin and extracellular matrix gene expression programs paralleled activation of previously identified secretomes that can be independently induced and promote cancer initiation in mouse models. These included the senescence secretome in mild to no COPD and the ETS2 (erythroblast transformation-specific 2) secretome in moderate to severe COPD. Furthermore, in situ examination of COPD lung tissue documented stromal fibroblasts expressing key cancer-associated proteins from procancer secretomes including IL-6 (senescence secretome) in mild COPD and bone morphogenetic protein-1 (ETS2 secretome) in advanced COPD. Conclusions Two distinct stromal gene expression programs promoting cancer initiation are activated in patients with lung cancer depending on the severity of COPD as measured by lung function. Our work has implications both for screening strategies and personalized approaches to prevent cancer in COPD.

Abstract In April 2010, a NIH workshop was convened to discuss the current state of understanding of lung cell plasticity, including the responses of epithelial cells to injury, with the objectives of summarizing what is known, what the field needs to know, and how to get there. The proximal stimulus for this workshop is the body of recent evidence suggesting that plasticity is a prominent but incompletely characterized property of lung epithelial cells, and that a focus on understanding this aspect of epithelial cell biology in particular, may be an important window into disease pathobiology and pathogenesis. In addition to their many vital functions in maintaining tissue homeostasis, epithelial cells have emerged as both a central target of disease initiation and an active contributor to disease progression, making a workshop to investigate the role of cell plasticity in lung injury and repair timely. The workshop was organized around four major themes: lung epithelial cell plasticity, signaling control of plasticity, fibroblast plasticity and crosstalk, and translation to human disease. Although this breakdown was recognized to be somewhat artificial, it was felt that this approach would promote cross-fertilization among groups that ordinarily do not communicate and lend itself to the generation of new approaches. The summary reports of individual group discussions below are followed by consensus priorities and recommendations of the workshop participants.