Reconstructing drug-tolerant persister (DTP) niches in iPSC-derived alveolar lung cancer organoids for spatio-temporal dissection of cell-cell interactions and ligand-receptor targets

Purpose: TKIs and ADCs have transformed NSCLC therapy, yet most patients relapse without detectable resistant mutations due to drug-tolerant persister (DTP) cells. Although immune evasion and CAF-mediated remodeling are well recognized, the contribution of lung-specific epithelial and ECM components to protective niche formation remains unclear. Systems that recapitulate alveolar architecture are needed to reveal targetable mechanisms. Methods: We developed a three-dimensional system, termed MicroEnvironment-Embedded Tumor in Fibroblast-integrated Organoid Alveolar MicroStructure (MEET-FOAms), by integrating human iPSC-derived alveolar organoids with NSCLC lines harboring EGFR exon19 deletion or ERBB2 exon20 insertion. Organoids were exposed to osimertinib, erlotinib, or trastuzumab-deruxtecan. Longitudinal single-cell RNA-seq and Xenium 5K spatial transcriptomics mapped lineage trajectories, ligand-receptor networks, and ECM remodeling. Results: Analysis of cancer cell clusters revealed that, even before drug exposure, a distinct subset persisted within the alveolar organoid background. These clusters lacked resistance mutations and were transcriptionally aligned with persister-like states, suggesting that the alveolar niche may inherently sustain DTP populations independently of drug pressure. Following TKI treatment, a stress-adaptive DTP subpopulation remained stable, relying on stromal-epithelial communication and ECM-receptor engagement. Under ADC exposure, an epithelium-associated DTP program was maintained via adhesion networks originating from alveolar epithelial cells, enabling protection from cytotoxic payload delivery. Spatial multi-omics profiling delineated organized multicellular interactions underpinning these responses. Intervention studies showed: (1) ADCs with bystander effect eliminated DTPs along with surrounding niche cells; (2) neutralizing CAF-derived factors suppressed DTP expansion; and (3) blocking ECM-related adhesion pathways disrupted persister maintenance. Conclusions: With MEET-FOAms, we experimentally reconstructed lung cancer tissue architecture by introducing oncogene-driven NSCLC cells into a human iPSC-derived alveolar background, elucidating that persister-like clusters exist prior to therapy and are sustained by lung-specific niche interactions. Unlike prior models emphasizing CAFs or immune escape alone, this organoid platform highlights the underexplored role of alveolar epithelial cells and ECM in priming and maintaining DTP states. These findings suggest that such persister niches may act as reservoirs from which genetically resistant clones eventually emerge, supporting a paradigm expansion into therapeutic strategies that target multicellular remodeling in drug-tolerant NSCLC.