Integrative multi-omics and network perturbation analysis in human airway organoids reveals product-specific toxicity profiles of heated tobacco products

The respiratory toxicity of heated tobacco products (HTPs) remains incompletely characterized, and traditional models often fail to capture human-specific responses. Here, we established a human pluripotent stem cell (hPSC)-derived airway organoid (AO) platform and systematically compared the toxicological profiles of two HTP aerosols using an integrated framework encompassing conventional cytotoxicity assays, lineage-specific analysis, network perturbation modeling and multi-omics profiling. Both HTPs induced time- and concentration-dependent cytotoxicity, oxidative stress, DNA damage, and apoptosis in AOs. Exposure also triggered epithelial chemokine response characterized by elevated IL-8, MCP-1, MIP-1β, GM-CSF, and RANTES, with concomitant suppression of IP-10, indicating epithelial-derived inflammatory alarm signals. Lineage-specific transcriptional changes revealed mucociliary dysfunction characterized by goblet cell hyperplasia (MUC5AC upregulation) and ciliated cell impairment (FOXJ1 downregulation), key features of airway remodeling in chronic respiratory diseases. To delineate underlying mechanisms, we employed Network Perturbation Amplitude (NPA) analysis, which uncovered qualitatively distinct toxicity architectures: HTP-1 exhibited higher overall toxicity and elicited broad-spectrum network perturbations involving cell stress, proliferation, and immune regulation, correlating with greater apoptotic induction; HTP-2 triggered focused activation of damage-sensing pathways, consistent with its earlier membrane disruption and more pronounced genotoxicity. Multi-omics analysis further linked these mechanistic perturbations to human disease-relevant pathways, with HTP-1 showing stronger enrichment for COPD-associated expression patterns and HTP-2 for lung cancer-related signatures, suggesting the acute molecular response to each product exhibits similarity to specific pulmonary disease-associated molecular signatures. These findings establish human-derived airway organoids as a sensitive, human-relevant platform within the New Approach Methodologies‌ (NAMs) framework for qualitative comparison and mechanistic interrogation of product-specific toxicity.