D95-02 Detailing the Extracellular Matrix Shifts in Bronchopulmonary Dysplasia Using Spatial Multi-omics

Abstract Rationale Bronchopulmonary dysplasia (BPD) is a chronic lung disease caused by premature birth and assisted-breathing methods including supplemental oxygen and mechanical ventilation. Many patients recover as they grow and mature, while others develop lifelong lung complications or do not survive. A hallmark of BPD is abnormal extracellular matrix (ECM) organization, characterized by disordered collagen and elastic fiber deposition, impairing lung structure and function. Precise ECM compositional changes in specific lung compartments remain poorly understood. Methods We developed a spatial multi-omics workflow to investigate molecular changes in formalin-fixed, paraffin-embedded lung tissues (5-μm-thick sections). Tissue microarrays were constructed with samples from donors with established BPD, healed BPD, and controls. The workflow combined (i) matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) of N-glycans, (ii) MALDI-MSI of ECM proteins, and (iii) highly multiplexed immunofluorescence (MxIF). In addition, bulk ECM proteomics using collagenase 3 was performed to build a library of ECM peptides. Data analysis was conducted using RomicsProcessor, a custom-made open-source R package, enabling spatial registration of modalities and identification of distinct cellular and molecular neighborhoods. Results Bulk ECM proteomics identified over 16,000 peptides from 125 ECM proteins including collagens, laminins, elastic fibers, and fibronectins. These results revealed proteins with significant abundance changes in BPD samples. Using MxIF segmentation and co-registration, lung compartments such as alveolar parenchyma, vessels (arteries/veins), airways, and smooth muscle-containing regions were delineated. Linear mixed model statistics and pseudobulk Student’s T-tests revealed significant changes in different segmented ROIs. Examples of changes include, increased COL1 and COL14 in smooth muscle-containing regions; significant ECM remodeling in the alveolar parenchyma with decreased COL3A1 and COL4A1, alongside increased COL1A1, COL1A2, COL2A1, COL3A1, COL6A1, COL6A2, COL14A1, fibulin, ANXA2, and ANXA3. Spatial N-glycomics revealed an increase in tetraantenary and monofucosylated glycans in the alveolar parenchyma of diseased donors, suggesting altered glycosylation processes with potential impacts on cell-cell signaling in these tissues. Conclusion This study provides the first detailed spatial characterization of ECM remodeling in BPD. By mapping molecular changes to specific lung compartments, this research opens avenues for new therapeutic strategies. Future work, including spatial transcriptomics, will identify cellular drivers behind ECM changes, advancing understanding and providing avenues for the treatment of bronchopulmonary dysplasia. This abstract is funded by: NHLBI, NIGMS