ASIC3-PLCG1 axis-driven macropinocytosis promotes osimertinib drug-tolerant persistence in malignant pleural effusion-associated NSCLC.

BACKGROUND: Non-small cell lung cancer (NSCLC) frequently develops resistance to EGFR-tyrosine kinase inhibitors (TKIs), particularly in malignant pleural effusion (MPE), where a highly acidic tumor microenvironment promotes the emergence of drug-tolerant persister (DTP) cells. Building upon previous evidence that MPE acidity drives metabolic plasticity, this study investigates how acid-sensing ion channel 3 (ASIC3) coordinates PLCG1-dependent macropinocytosis to maintain osimertinib tolerance.

METHODS: Primary MPE-derived NSCLC cultures underwent next-generation sequencing to define mutational heterogeneity. Acidic (pH 6.5-6.8) conditions were used to generate osimertinib-induced DTP (Osi-DTP) models. Functional assays assessed viability, invasion, colony formation, autophagy, and macropinocytosis. ASIC3 was silenced using shRNA, followed by Seahorse metabolic analysis. Transcriptomic profiling identified differentially expressed genes. ASIC3-targeted therapeutic interventions were evaluated in vitro and in xenograft models.

RESULTS: MPE-derived NSCLC cultures showed substantial genomic diversity, including EGFR exon 19 deletion and T790M mutations. Acid-adapted Osi-DTP cells exhibited EMT-like phenotypes, reduced proliferation, elevated stemness markers, and strong activation of stress-response pathways. ASIC3 was consistently upregulated under acidic conditions and drove PLCG1-mediated macropinocytosis to support nutrient scavenging and survival. ASIC3 knockdown markedly reduced DTP cell viability, invasiveness, colony formation, autophagy, and glycolysis, while inducing a metabolic shift toward oxidative phosphorylation. In vivo, combining an ASIC3 inhibitor with osimertinib significantly delayed tumor progression and improved survival without added toxicity. Clinically, high ASIC3 expression correlated with poor overall survival and increased autophagy-associated markers.

CONCLUSION: ASIC3 is a central regulator of acidosis-driven drug persistence in MPE-associated NSCLC, sustaining osimertinib tolerance through PLCG1-dependent macropinocytosis and metabolic reprogramming. Targeting ASIC3 restores TKI sensitivity by disrupting nutrient acquisition, autophagy, and metabolic adaptation, representing a promising therapeutic strategy to prevent or delay EGFR-TKI resistance.