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Updated: Aug 6, 2026
Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids
Published on: November 22, 2021
PLCG1 and RAC1 orchestrate adaptive cellular programs driving osimertinib-tolerant persister cell formation in
Tung-Yu Tiong1, Chieh-Yung Wang2, Ying Chen3
1Division of Thoracic Surgery, Department of Surgery, School of Medicine, College of Medicine, Taipei Medical University, Taipei, 110, Taiwan; Division of Thoracic Surgery, Department of Surgery, Taipei Medical University-Shuang Ho Hospital, New Taipei City 235, Taiwan.
Abstract:
Acquired resistance to epidermal growth factor receptor (EGFR)-targeted therapies remain a major challenge in non-small cell lung cancer (NSCLC), particularly in patients with malignant pleural effusion (MPE). The MPE microenvironment, characterized by acidic, cytokine- and metabolite-rich conditions, promotes the emergence of osimertinib-tolerant persister cells (OTPCs), contributing to disease relapse. In this study, we investigated the role of MPE in driving OTPC formation and identified key molecular regulators underlying this adaptive phenotype. MPE samples from advanced EGFR-mutant NSCLC were used to generate OTPCs through coculture with PC9 and H1975 cell lines. In contrast, non-malignant pleural effusions induced only limited tolerance. Transcriptomic profiling revealed extensive reprogramming in OTPCs, with PLCG1 and RAC1 among the most significantly upregulated genes, enriched in pathways related to glycolysis, hypoxia, and epithelial-mesenchymal transition. Functional analyses demonstrated that OTPCs exhibit enhanced macropinocytosis, metabolic flexibility, and invasive capacity. Mechanistically, PLCG1 and RAC1 formed a co-dependent signaling network, as supported by reciprocal knockdown and protein interaction studies. Inhibition of PLCG1 significantly impaired both mitochondrial respiration and glycolytic activity, reduced mesenchymal marker expression, and decreased OTPC viability by >60%, thereby restoring sensitivity to osimertinib. In vivo, combined inhibition of EGFR and PLCG1 resulted in sustained tumor suppression and improved survival without detectable toxicity. Collectively, these findings identify a co-dependent PLCG1-RAC1 signaling network that integrates metabolic adaptation and phenotypic plasticity to sustain drug tolerance in MPE-associated NSCLC. Targeting this pathway represents a promising strategy to overcome resistance to EGFR-directed therapies.
Insights
Malignant pleural effusion drives drug resistance in non-small cell lung cancer by promoting persister cells. Targeting the PLCG1-RAC1 pathway restores sensitivity to EGFR-targeted therapies.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Research
Background:
- Acquired resistance to epidermal growth factor receptor (EGFR)-targeted therapies is a significant challenge in non-small cell lung cancer (NSCLC).
- The malignant pleural effusion (MPE) microenvironment fosters drug tolerance and relapse in NSCLC patients.
- Osimertinib-tolerant persister cells (OTPCs) emerge in MPE, contributing to therapeutic failure.
Purpose of the Study:
- To investigate the role of MPE in driving OTPC formation in EGFR-mutant NSCLC.
- To identify molecular regulators responsible for the adaptive phenotype of OTPCs.
- To explore therapeutic strategies targeting MPE-driven drug resistance.
Main Methods:
- Generation of OTPCs from MPE samples of advanced EGFR-mutant NSCLC via co-culture with NSCLC cell lines.
- Transcriptomic profiling to identify differentially expressed genes and enriched pathways in OTPCs.
- Functional assays assessing macropinocytosis, metabolic flexibility, and invasive capacity of OTPCs.
- In vitro and in vivo studies evaluating the efficacy of PLCG1 inhibition in combination with EGFR inhibitors.
Main Results:
- MPE significantly promoted OTPC formation compared to non-malignant effusions.
- Transcriptomic analysis revealed upregulation of PLCG1 and RAC1 in OTPCs, associated with glycolysis, hypoxia, and epithelial-mesenchymal transition pathways.
- OTPCs exhibited enhanced macropinocytosis, metabolic flexibility, and invasiveness.
- Inhibition of PLCG1 impaired mitochondrial respiration and glycolysis, reduced mesenchymal markers, decreased OTPC viability, and restored osimertinib sensitivity.
- Combined inhibition of EGFR and PLCG1 achieved sustained tumor suppression and improved survival in vivo without toxicity.
Conclusions:
- A co-dependent PLCG1-RAC1 signaling network integrates metabolic adaptation and phenotypic plasticity to sustain drug tolerance in MPE-associated NSCLC.
- Targeting the PLCG1-RAC1 pathway is a promising strategy to overcome acquired resistance to EGFR-directed therapies in NSCLC.
- Understanding the MPE microenvironment's role is crucial for developing effective treatments for resistant NSCLC.
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