Single-cell mass cytometry reveals cell type- and cluster-specific heterogeneity in silver nanoparticle responses in a 3D alveolar tetra-culture model

Abstract

Silver nanoparticles (AgNPs) are widely used in medicine, environmental science, and industry. However, their heterogeneous interactions with complex biological systems, especially at the single-cell level, are not fully understood yet. Conventional toxicity assessment methods are typically conducted on oversimplified in vitro models that fail to replicate actual physiological conditions, and measure collective responses across cell populations, obscuring differences among individual cells. To overcome these limitations, we utilized single-cell mass cytometry (CyTOF) to investigate individual cell responses to AgNP-induced stress, combined with a 3D alveolar tetra-culture model designed to better reflect the complexity of biological systems. Single-cell mass cytometry of a 3D alveolar model revealed heterogeneous, cell type–specific responses to AgNP exposure. Specifically, PMA-differentiated THP-1, A549 and EA.hy926 cells exhibited high AgNP association but limited cytotoxicity, indicating activation of stress-mitigation pathways, while THP-1 cells showed early inflammatory activation despite minimal AgNP association, suggesting an indirect mechanism. Single-cell analysis and FlowSOM clustering revealed distinct subpopulations exhibiting diverse intracellular signaling profiles of inflammatory cytokines, anti-inflammatory mediators, and stress-response proteins, which unveiled common cellular responses and unique cell-type specific pathways determining cell fate (survival, transitional states, or apoptosis) upon AgNP exposure. This study introduces a novel framework for studying heterogeneous interactions of nanoparticles with complex biological systems by integrating a 3D alveolar tetra-culture model with single-cell mass cytometry analysis, enabling the dissection of nanoparticle-induced stress responses at an unprecedented level of detail. These insights have broad implications for nanotoxicology and nanomedicine, underscoring the need to account for cellular heterogeneity when evaluating nanoparticle-induced toxicity.