Integration of bulk and single-cell transcriptomics via the Scissor algorithm reveals APOE-dependent cholesterol metabolic reprogramming in macrophages associated with osimertinib resistance in non-small cell lung cancer

Background The tumor microenvironment contributes to acquired resistance against osimertinib in non-small cell lung cancer (NSCLC), yet the key cell subpopulations and the underlying metabolic mechanisms remain incompletely elucidated. Methods The Scissor algorithm was applied to integrate bulk osimertinib-resistance transcriptomic profiles with single-cell RNA-seq data to pinpoint cell subpopulations functionally associated with the osimertinib resistant phenotype. AddModuleScore was applied to calculate scores for cholesterol metabolism-related modules. Gene-set enrichment analysis was carried out to uncover pathway alterations in osimertinib resistance-related macrophages. Cross-validation was performed in an independent dataset comparing treatment-naive and EGFR-tyrosine kinase inhibitors (EGFR-TKIs)-treated progressive-disease lesions. EGFR-mutant lung adenocarcinoma cells were treated with corresponding conditioned medium combined with osimertinib, followed by a panel of cell function assays, including cell proliferation, colony formation, and apoptosis. The gain- and loss- of function studies of APOE were conducted in THP-1 derived macrophages. Results Osimertinib-resistant associated Scissor + cells were mainly enriched within macrophages, which exhibited prominent APOE-dependent cholesterol metabolic reprogramming. APOE was markedly up-regulated in Scissor + macrophages and macrophages from EGFR-TKI-treated progressive-disease lesions. Macrophage-derived APOE shapes extracellular cholesterol availability in the microenvironment, which may promote intracellular free-cholesterol accumulation in tumor cells and counteract osimertinib-triggered apoptosis. Conclusion APOE-dependent cholesterol metabolic reprogramming in macrophages is associated with osimertinib resistance in NSCLC and may contribute to therapeutic failure. Our results identify APOE as a hypothesis-generating candidate molecule that merits further pre-clinical investigation.