115 Integrative transcriptomics to map carbon based nanomaterial–induced toxicity pathways in human lung organoids

Abstract Exposure to airborne carbon-based nanomaterials (CBN) is associated with several respiratory diseases. Our recent in vivo study showed that carbon nanoparticles (CNP), double-walled carbon nanotubes (DWCNT), and multi-walled carbon nanotubes (MWCNT) trigger different modes of action, involving epithelial injury signals, macrophage activation, and the release of various chemokines after 12 h of exposure (Voss and Han et al., 2025). Due to the interspecies differences and the need for a more realistic representation of human lung physiology in vitro, stem-cell derived lung organoids might provide a better option for understanding cell-specific responses and post-injury regeneration capacity of the tissue. The present study evaluated the activated cellular pathways of a human lung organoid model containing six major proximal and distal epithelial cell types co cultured with macrophages (Issa et al., 2024), microinjected with three CBN at low (2, 2.6, and 6.6 ng/mm2) and high doses (10, 13, and 33 ng/mm2) for 12 h or 6 d. Our study combines bulk RNA-seq of all exposed conditions with single-cell RNA-seq from untreated organoids, using the latter as a reference to deconvolute cell-type-specific responses. With this approach, we systematically compare deconvolution approaches and identify the model that is applicable for CBN-specific cellular pathways. We identify cell types, cell abundance, and activated toxicity pathways across different time points and concentrations using bulk data integrating in vivo findings. We suggest that this methodology is useful to future studies, where high-throughput bulk RNA-seq coupled with single-cell–guided deconvolution would allow exploration of multiple time points and conditions.