Coexistence of Osimertinib-Resistant and Osimertinib-Sensitive cell states in an EGFR-Mutant NSCLC model derived at osimertinib resistance

Background: Osimertinib is a standard first-line treatment for EGFR-mutant Non-Small Cell Lung Cancer (NSCLC), but acquired resistance remains a clinical challenge. Resistance involves genetic mechanisms and non-genetic cell-state transitions, including epithelial-to-mesenchymal (EMT) plasticity and drug-tolerant persister phenotypes. Patient-derived models established at disease progression may better capture clinically relevant resistance mechanisms. Methods: We established LIBM-ADK-11 from the pleural effusion of a patient with EGFR exon 19p.Leu747_Pro753delinsSer-mutant NSCLC progressing on osimertinib. Cells were studied at low and high passages and fractionated by CD44 expression into CD44-positive/P2 and CD44-negative/P4 subpopulations. EGFR mutation status, CD44 expression, transcriptomics, growth, osimertinib sensitivity, EGFR signaling, sphere formation, cell- cycle distribution and pemetrexed response were evaluated. Results: LIBM-ADK-11 retained the EGFR mutation during long-term culture. CD44 sorting identified two phenotypically distinct cell states. CD44-positive P2 cells were enriched for extracellular matrix remodeling, EMT-like, inflammatory, CAF-like and macrophage/TAM-mimicry programs, with increased glycolysis-related signatures, slower growth, higher metabolic activity and osimertinib resistance. CD44-negative P4 cells displayed a more epithelial profile, cholesterol-associated gene expression and greater osimertinib sensitivity. Osimertinib inhibited EGFR phosphorylation in both populations, indicating that P2 resistance was not explained by persistent EGFR signaling. Long-term culture favored loss of CD44-positive cells, whereas osimertinib delayed this loss and preferentially depleted the CD44-negative fraction. Pemetrexed partially reduced both compartments. Conclusions: LIBM-ADK-11 is a clinically relevant model of osimertinib-resistant EGFR-mutant NSCLC in which CD44-positive drug-tolerant and CD44-negative drug-sensitive states coexist. These findings highlight phenotypic heterogeneity as a potential determinant of osimertinib resistance and support strategies addressing distinct cellular states at disease progression.