Application of an Integrated Single-Cell and Three-Dimensional Spheroid Culture Platform in Investigating Drug Resistance Heterogeneity and EMT in Lung Cancer Subclones

Lung cancer is a leading cause of cancer-related mortality worldwide, largely due to its heterogeneity and intrinsic drug resistance. Malignant pleural effusions (MPE) provide diverse tumor cell populations ideal for studying these complexities. Although chemotherapy and tar-geted therapies can be initially effective, subpopulations of cancer cells with phenotypic plasticity often survive treatment, eventually developing resistance. Here, we integrated single-cell isola-tion and three-dimensional (3D) spheroid culture to dissect subclonal heterogeneity and drug responses, aiming to inform precision medicine approaches. Using A549 lung cancer cells, we established a cisplatin-resistant line and isolated three resistant subclones (Holoclone, Meroclone, Paraclone) via single-cell sorting. In 3D spheroids, Docetaxel and Alimta displayed higher IC50 values than in 2D cultures, suggesting that 3D models better reflect clinical dosing. Additionally, MPE-derived Holoclone and Paraclone subclones exhibited distinct sensitivities to Giotrif and Capmatinib, revealing their heterogeneous drug responses. Molecular analyses confirmed ele-vated MDR-1, ABCG2, cancer stem cells (CSC) markers (OCT4, SOX2, CD44, CD133), and epi-thelial-mesenchymal transition (EMT) markers (E-cadherin downregulation, increased Vimentin, N-cadherin, Twist) in resistant subclones, correlating with enhanced migration and invasion. This integrated approach clarifies the interplay between heterogeneity, CSC/EMT phenotypes, and drug resistance, providing a valuable tool for predicting therapeutic responses and guiding per-sonalized, combination-based lung cancer treatments.