D95-01 Spatial Immunochemistry and Integrated Proteomics Reveal New Insights Into Airway Disease in Bronchopulmonary Dysplasia

Abstract Rationale Bronchopulmonary Dysplasia (BPD) is a disease of prematurity related to an arrest of normal lung development characterized by both airway and parenchymal disease. The objective of this study was to apply proteomics and multiplex immunohistochemistry on airway and alveolar sections isolated by laser capture microscopy (LCM) to identify region-specific proteins and pathways in BPD on a near single-cell level. Methods Pediatric human donors aged 1.3 to 3 years were classified based on history of prematurity and histopathologic severity of BPD (N = 3 “Healed” BPD hBPD and N = 3 “Established” BPD eBPD) with respective full-term (N = 6) age-matched controls. Four distinct tissue regions were isolated by LCM: large airway (AW), membranous bronchiole (MB), respiratory bronchiole (RB), and alveolar (ALV). Proteins were identified by 2 unique peptide fragments by tandem mass spectrometry, comparing abundances by ANOVA, paired t-tests, and presence-absence glm-binomial tests. MxIF microscopy (30-40 target antibodies) was performed on lung tissue microarrays enriched for the four regions targeting proteins differentially expressed in proteomics. Novel segmentation of MxIF images was performed using DeepCell, scipy, scanpy, and Napari to achieve near single-cell resolution, with manual thresholds applied to each channel. UMAPs were generated to identify cell clusters, and neighborhood analysis was performed to determine cell niches among BPD classifications for each LCM region. Results Proteomics detected 3261 unique proteins, 2149 differentially quantifiable in the alveolar region, and 2396 in the combined airway regions, with strong validation by region for canonical proteins (Figure 1A). We observed increased HEXA and HEXB and decreased ICAM1 in eBPD compared to hBPD or controls. Pathway enrichment in eBPD airways included inner mitochondrial membrane (GO) and oxidative phosphorylation (KEGG), which included PRDX1 and SFXN1. In addition, several ECM annexins (ANXA1, ANXA4, and ANXA5) were upregulated in eBPD airways. MxIF identified LYVE1, SMA, and COL6A1 as the proteins most significantly modulated in airway eBPD regions (Figure 1E), where calmodulin was found in lower abundance in multiple clusters. Single-cell clustering of spatial data identified 29 clusters (Figure 1B), which were mapped to cell types based on their marker profiles (Figure 1C) and remapped onto the tissue cores (Figure 1D). Preliminary neighborhood analyses demonstrated airway-specific localization of Macrophages and PROX1+ cells in eBPD. Conclusions eBPD is associated with dysregulation of the airway epithelium, immune cells, and extracellular matrix turnover. Region-specific alterations in protein abundance, cellular niches and neighborhoods, and pathway dysregulation can be identified in human BPD lung tissues at near single-cell resolution. This abstract is funded by: NIH U54HL165443 and U01HL148861