Academic Background and Problem Statement

Idiopathic Pulmonary Fibrosis (IPF) is a progressive interstitial lung disease associated with aging and is an independent risk factor for lung cancer. Epidemiological studies indicate that 3%-22% of IPF patients develop lung cancer during follow-up, with the cumulative incidence increasing over time, making lung cancer a primary cause of mortality in these patients. IPF combined with lung cancer (IPF-LC) exhibits higher invasiveness compared to sporadic lung cancer, with Non-Small Cell Lung Cancer (NSCLC) being the main pathological type of IPF-LC. However, there are currently no specific treatment guidelines for IPF-LC. Standard lung cancer therapies such as chemotherapy, radiotherapy, immunotherapy, and targeted therapy have been found to cause acute exacerbations of IPF in clinical practice, thereby limiting treatment options for IPF-LC patients. Therefore, elucidating the molecular mechanisms underlying IPF-LC progression is crucial for developing new therapeutic targets.

In recent years, Cancer-Associated Fibroblasts (CAFs) in the Tumor Microenvironment (TME) have garnered significant attention. CAFs promote tumor cell proliferation, immune evasion, metastasis, and therapeutic resistance by remodeling the Extracellular Matrix (ECM) and secreting growth factors and cytokines. Studies have found many similarities between myofibroblasts in IPF and CAFs, including sustained overactivation, abnormal proliferation, mesenchymal characteristics, and similar signaling pathway activations. Additionally, fibroblasts in IPF have been shown to promote tumorigenesis and progression. However, the number of available studies is limited.

Cellular senescence, an irreversible process leading to cell cycle arrest, has been increasingly implicated in the pathogenesis of IPF. Although cellular senescence was initially considered a tumor-suppressive mechanism, recent studies suggest that senescent fibroblasts can promote tumor cell proliferation and invasion through the secretion of Senescence-Associated Secretory Phenotype (SASP) factors. Therefore, investigating the impact of senescent fibroblasts in IPF on the malignant behavior of NSCLC cells is of great significance.

Source of the Paper

This paper was co-authored by Yuqiong Lei, Cheng Zhong, Jingyuan Zhang, Qi Zheng, Yongle Xu, Zhoubin Li, Chenwen Huang, and Tao Ren, affiliated with the Department of Respiratory Medicine at Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, the Department of Lung Transplantation and Thoracic Surgery at the First Affiliated Hospital of Zhejiang University School of Medicine, the Clinical Research Center at Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, and the Stem Cell Center at Shanghai Sixth People’s Hospital. The paper was published in 2024 in the journal Oncogene, with the DOI: https://doi.org/10.1038/s41388-024-03236-5.

Research Process and Results

1. Senescence Characteristics of IPF Fibroblasts

The study first assessed senescence markers (γH2AX, p53, p16, p21) in lung tissues from IPF patients using immunofluorescence co-localization. It was found that fibroblasts, alveolar type I (AT1), and alveolar type II (AT2) cells in IPF lung tissues exhibited senescent phenotypes, with fibroblasts showing the highest proportion of senescence. Compared to healthy controls, IPF lung tissues exhibited significantly elevated expression of γH2AX, p53, p16, and p21. Further extraction of primary fibroblasts revealed that IPF fibroblasts (DHLFs) displayed typical senescence features, such as increased cell size, irregular morphology, elevated expression of α-SMA and Vimentin, and significantly reduced proliferative capacity. SA-β-gal activity assays further confirmed the senescent characteristics of DHLFs.

2. Senescent IPF Fibroblasts Promote NSCLC Proliferation and Invasion

Using a Transwell indirect co-culture model, the study found that NSCLC cells (A549 and SK-MES-1) co-cultured with IPF fibroblasts exhibited a morphological transformation from a cobblestone-like appearance to an elongated spindle shape, indicative of Epithelial-Mesenchymal Transition (EMT). CCK8 assays showed that NSCLC cells co-cultured with IPF fibroblasts had significantly enhanced proliferative capacity. Additionally, colony formation and Transwell assays demonstrated that IPF fibroblasts significantly enhanced the clonogenic and invasive/migratory abilities of NSCLC cells. Western blot analysis revealed that IPF fibroblasts promoted changes in the expression of EMT-related proteins in NSCLC cells, such as decreased E-cadherin and increased N-cadherin, Vimentin, and Snail expression.

3. Exosomes from IPF Fibroblasts Promote Malignant Transformation of NSCLC In Vitro

To identify the specific secretory components released by IPF fibroblasts that facilitate phenotypic alterations in NSCLC, the study isolated microvesicles (MVs), exosomes, and extracellular vesicle-free (EVs-free) supernatant from serum-free conditioned media using differential ultracentrifugation. Western blot analysis confirmed the successful extraction of exosomes. CCK8 and colony formation assays showed that exosomes had the most significant pro-tumor effect on NSCLC cell proliferation and clonogenicity. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) confirmed the characteristic bilayer vesicular structure and size distribution (30-150 nm) of exosomes. Fluorescently labeled exosomes were internalized by NSCLC cells within 4 hours, with substantial uptake by 24 hours. Further experiments demonstrated that exosomes secreted by IPF fibroblasts significantly enhanced the malignant phenotype of NSCLC cells.

4. Upregulated Proteins in DHLF-Exosomes Highlight SASP Factors Linked to NSCLC Progression

Proteomic analysis of exosomes revealed that DHLF-exosomes exhibited upregulation of 392 proteins compared to NHLF-exosomes, including key SASP components such as FBLN2, IL-6, MMP1, MMP3, and IGFBP3. Gene Ontology (GO) enrichment analysis showed that “extracellular exosome” was the most enriched cellular component, and KEGG pathway analysis indicated significant enrichment of the PI3K-AKT-mTOR signaling pathway and cellular senescence pathway. Single-cell sequencing data analysis further revealed IPF-specific fibroblast subclusters with higher senescence scores and enriched expression of proteins related to extracellular matrix (ECM) remodeling and fibrosis. Quantitative assessment of SASP factors in exosomes showed a marked increase in the expression of SASP factors within IPF exosomes, with MMP1 exhibiting the most pronounced variance.

5. MMP1 is a Key Factor in DHLF-Exosome-Mediated Progression of NSCLC

The study found that fibroblasts in IPF lung tissues exhibited high expression of MMP1, whereas fibroblasts in normal lung tissues showed minimal expression. Western blot and ELISA assays confirmed that MMP1 expression was significantly higher in DHLF and DHLF-exosomes compared to normal fibroblasts. Knockdown of MMP1 using shRNA significantly reduced the tumor-promoting effects of DHLF-exosomes on NSCLC cell proliferation and colony formation. Further experiments demonstrated that MMP1 exerted its pro-tumor effects by binding to PAR1 and activating the PI3K-AKT-mTOR signaling pathway.

6. DHLF-Exosomes Promote Lung Cancer Cell Proliferation via PAR1-Mediated PI3K-AKT-mTOR Pathway Activation

PAR1 is a widely recognized receptor for MMP1. The study used the PAR1 antagonist SCH79797 to verify that MMP1 exerts its pro-tumor effects through binding to PAR1. RNA-seq analysis of SK-MES-1 cells incubated with and without DHLF-exosomes identified 407 upregulated and 182 downregulated genes. GO and KEGG analysis indicated enrichment in several pro-tumor biological processes, including the PI3K-AKT signaling pathway. Western blot analysis confirmed that DHLF-exosomes significantly activated the PI3K-AKT-mTOR signaling pathway in NSCLC cells, while exosomes from MMP1-knockdown DHLF weakened this activation.

7. Exosomal MMP1 Promotes Tumor Growth In Vivo

A subcutaneous tumorigenesis assay in nude mice showed that DHLF-exosomes significantly promoted tumor growth, while exosomes from MMP1-knockdown DHLF or PAR1 inhibitors effectively mitigated this tumor-promoting effect. Immunohistochemistry (IHC) and Western blot analysis further confirmed that DHLF-exosomes activated the PI3K-AKT-mTOR signaling pathway in subcutaneous tumors, promoting tumor growth.

Conclusions and Significance

This study demonstrates that senescent fibroblasts in IPF promote NSCLC progression by releasing exosomal MMP1, which activates the PI3K-AKT-mTOR signaling pathway via PAR1. These findings provide a novel therapeutic approach for IPF-LC patients by targeting senescent fibroblasts or key factors such as MMP1 and PAR1 to inhibit tumor progression. Additionally, the study highlights the important role of exosomes in IPF-LC progression, offering new directions for future therapeutic strategies.

Research Highlights

1. Key Discovery: First to reveal that senescent fibroblasts in IPF promote NSCLC progression through exosomal MMP1.
2. Methodological Innovation: Systematic identification of SASP factors in IPF fibroblast-derived exosomes using proteomic analysis and single-cell sequencing.
3. Clinical Relevance: Provides new therapeutic targets for IPF-LC patients, with significant potential for clinical application.

Additional Valuable Information

The study’s limitations include focusing solely on the pro-tumor effects of exosomal MMP1 without comprehensively investigating the roles of other active components within exosomes. Future research will address this gap by examining the contributions of additional factors in IPF-LC progression to identify more potential therapeutic targets. Additionally, the sample size of IPF patients and healthy controls was relatively small. Future studies plan to collect more specimens to enhance the reliability and validity of the findings.