Epithelial−Mesenchymal Transition Induced by Tumor Cell-Intrinsic PD-L1 Signaling Predicts a Poor Response to Immune Checkpoint Inhibitors in PD-L1-High Lung Cancer

Rheumatology and Immunology Respiratory System Oncology Life Sciences Immunology Medicine Cell Biology Biochemistry
PD-L1 Epithelial-Mesenchymal Transition Non-Small-Cell Lung Cancer Immune Checkpoint Inhibitors Biomarkers Tumor Progression

Tumor Cell-Intrinsic PD-L1 Signaling Induces Epithelial-Mesenchymal Transition (EMT) Predicting Poor Response to Immune Checkpoint Inhibitors in PD-L1 High-Expressing Lung Cancer

Research Background and Motivation

In cancer immunotherapy, Programmed Death Ligand-1 (PD-L1) inhibits immune responses by binding to Programmed Death-1 (PD-1) receptors on immune cells. Immune checkpoint inhibitors (ICIs) that block the PD-1/PD-L1 interaction have become a primary treatment method for non-small cell lung cancer (NSCLC). Despite a response rate of approximately 40% in PD-L1 positive patients to PD-1/PD-L1 blockade, more effective biomarkers are still needed to predict the efficacy of ICI treatment.

Most studies have focused on the traditional extracellular role of PD-L1, whereas the intrinsic signaling functions of PD-L1 within tumor cells have gained attention in recent years, involving various functions like cancer cell survival, invasion, stemness, glycolysis, chemotherapy resistance, and DNA damage response. Additionally, intrinsic PD-L1 functions are associated with anti-PD-1 therapy resistance in melanoma and colon cancer mouse models. Nevertheless, the intracellular role and mechanisms of PD-L1 in cancer biology and therapeutic development remain incompletely elucidated.

Epithelial-Mesenchymal Transition (EMT) plays a crucial role in cancer progression and metastasis. A positive correlation between PD-L1 expression and the EMT phenotype has been confirmed in various tumors, including NSCLC, breast cancer, and head and neck squamous cell carcinoma. EMT is a common phenomenon during or after treatment, indicating treatment resistance and cancer progression. However, the association between EMT and immunotherapy remains unclear. Therefore, this study aims to investigate the role of intrinsic PD-L1 signaling in NSCLC EMT and tumor progression and explore the potential of PD-L1-induced EMT as a predictive biomarker for ICI therapy.

Source of the Paper

This study was authored by Dr. Hyein Jeong and others from multiple research institutions in South Korea, including the Cancer Research Institute at Seoul National University, Department of Pathology at Seoul National University College of Medicine, and Korea University Guro Hospital. The paper was published online in the British Journal of Cancer on May 10, 2024, titled “Epithelial−Mesenchymal Transition Induced by Tumor Cell-Intrinsic PD-L1 Signaling Predicts a Poor Response to Immune Checkpoint Inhibitors in PD-L1-High Lung Cancer.”

Research Process

Methods and Experimental Design

  1. Cell Lines and Reagents: The study utilized various human NSCLC cell lines (like A549, H460, H596, etc.) and mouse lung cancer cell line LLC, cultured using media and fetal bovine serum. Various recombinant proteins and antibodies were used in experiments, such as recombinant human PD-1 and Interferon-γ (IFNγ).

  2. Overexpression and Gene Knockdown: PD-L1 expression plasmids or PD-L1 small interfering RNA (siRNA) were transfected into cells through transfection techniques. Lentiviral particles were used to generate stable cell lines for experiments. In mouse models, LLC cells stably expressing or knocking down PD-L1 were used for in vivo studies.

  3. RNA Sequencing and Gene Expression Analysis: RNA extraction, RNA sequencing, and Gene Set Enrichment Analysis (GSEA) were conducted to compare gene expression differences in PD-L1 overexpressing and knockdown cells, with a particular focus on EMT-related pathways.

  4. Experimental Measurements: Methods such as quantitative real-time polymerase chain reaction (qRT-PCR), protein co-immunoprecipitation (Co-immunoprecipitation), Western blot, immunofluorescence staining, and ELISA were used to detect and quantify the expression of EMT markers and related factors.

  5. In Vivo Experiments: Immunodeficient mouse models were used by implanting LLC cells that stably expressed or knocked down PD-L1 to assess tumor growth and metastasis.

  6. Patient Cohort Analysis: Through RNA sequencing and immunohistochemistry (IHC) analysis, 234 and 90 NSCLC patients who received ICI therapy were evaluated, assessing the impact of EMT and PD-L1 expression on the treatment response and survival rates.

Research Results

  1. PD-L1 Intrinsic Signaling Promotes EMT: RNA sequencing results showed significant enrichment of EMT pathways in PD-L1 overexpressing cells, with increased expression of EMT markers such as Twist1, ZEB1, Snail, Slug, and decreased E-cadherin expression. Conversely, PD-L1 knockdown cells showed the opposite pattern.

  2. Relationship Between PD-L1 and TGF-β Production: The study found that PD-L1 promotes EMT by inhibiting protein phosphatase PPM1B, activating p38 MAPK and increasing TGF-β production.

  3. In Vitro Experiments: Overexpression of PD-L1 significantly increased cell proliferation, migration, and invasion abilities, whereas PD-L1 knockdown significantly reduced these abilities.

  4. In Vivo Experiments: In immunodeficient mouse models, PD-L1 overexpressing LLC cells increased tumor growth and lung metastasis, while PD-L1 knockdown LLC cells reduced these characteristics.

  5. Relationship Between EMT and ICI Efficacy: In patient cohort analysis, the EMT signature was poorly correlated with ICI efficacy in PD-L1 high-expressing patients, but not in PD-L1 low-expressing patients. Survival analysis showed that PD-L1 high-expressing patients with a high EMT score had a shorter progression-free survival (PFS) after ICI treatment.

Conclusion and Significance

  1. Scientific Significance: The study elucidates the mechanism by which intrinsic PD-L1 signaling promotes EMT through TGF-β and demonstrates the association of EMT with immune escape and therapeutic resistance in PD-L1 high-expressing NSCLC patients.

  2. Application Value: PD-L1-induced EMT could serve as a predictive biomarker for ICI treatment in PD-L1 high-expressing NSCLC patients, emphasizing the importance of targeting EMT as a potential therapeutic strategy to enhance immunotherapy efficacy.

  3. Research Highlights: This study is the first to detail the impact of the interaction between PD-L1 and TGF-β on EMT, offering new mechanistic insights and potential therapeutic strategies.

This research comprehensively explores the role of intrinsic PD-L1 signaling in cancer progression and immunotherapy, providing valuable theoretical and practical foundations, and it is expected to guide future clinical interventions and treatment strategies.