Spatial Transcriptomics Reveals Region-specific Transcriptional Profiles in Human Lungs
Anne Marie Cawley
Mentor: Dr. Kenichi Okuda, Department of Medicine, Division of Pulmonary Diseases and Critical Care Medicine, The University of North Carolina at Chapel Hill Marsico Lung Institute/CF Research Center.
Date/Time: August 22nd, 2025 at 10:45 AM.
Abstract: The Human Biomolecular Atlas Program (HuBMAP) is a collaborative research initiative that aims to map the entire healthy human body at the cellular level (Jain et al., 2023). As part of this program, we conducted spatial whole transcriptome sequencing analysis on human lung sections from nine distinct regions along the proximal-to-distal axis (N=6 donors). The GeoMX spatial whole transcriptome assay allows for whole transcriptome analysis within selected regions of interest (ROIs) in formalin-fixed paraffin-embedded (FFPE) sections. We used tissue microarray (TMA) approach, allowing systematic evaluation of regional and structural transcriptomic differences in non-diseased human lung tissues.
Thirty-micrometer cores were extracted and assembled into TMAs (15 cores/slide, four slides). ROIs were assigned to specific lung structures based on H&E morphology. Gene counts per ROI were generated from GeoMX data, Q3 normalized, and analyzed using linear mixed-effect models to identify differential expression and enriched pathways across lung regions. Lung structures and regions were used as fixed-effects and random-effects included TMA cores and donor codes to account for technical and inter-individual variations. Filtering and data normalization were performed, and differential expression was conducted using the Bioconductor package, variancePartition, specifically the dream function. Gene set enrichment analysis was conducted using the Bioconductor R package fgsea, and heatmaps and volcano plots were generated from Q3 normalized data.
Our findings highlight transcriptomic differences between lung regions and structures. Differential expression analysis comparing proximal airway epithelium to terminal bronchiolar (distal airway) epithelium demonstrated enrichment of SCGB3A2 and SFTPB, established distal airway markers, in terminal bronchiolar epithelium. In contrast, proximal airway epithelium showed enrichment of secretory mucins (MUC5AC, MUC5B), and SERPINB3, known proximal airway markers. Pathway analysis revealed significant upregulation of cilia-associated genes in proximal airway epithelium and surfactant metabolism pathways in terminal bronchiolar epithelium, reflecting region-specific functional specialization in non-diseased airway epithelia.
These findings provide a spatially resolved transcriptomic roadmap of the human lung, offering a foundation for understanding how region-specific epithelial identities are altered in lung diseases. Such knowledge is essential for developing novel therapeutic strategies for lung diseases.
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