B75-36 Spatiotemporal Analysis of Post-pneumonectomy Lung Regeneration

Rationale

Chronic lung diseases are a leading cause of morbidity and mortality worldwide, with no regenerative therapies currently available. Post-pneumonectomy lung regeneration (PPLR) represents a natural example of robust adult alveolar regrowth and provides a powerful in vivo system for studying the mechanisms by which lung tissue successfully remodels and regenerates. Yet, the spatial and temporal logic by which diverse lung cell populations coordinate this process remains poorly defined.

Methods

Adult rats of both sexes underwent pneumonectomy and tissue from the remaining lung was harvested at Days 0, 3, 7, and 14 post-surgery. A 3x8 tissue microarray was constructed from paraffin-embedded samples to capture all time points, sexes, and anatomical regions of interest, including subpleural and adventitial compartments. We combined single-cell RNA sequencing with high-resolution spatial transcriptomics (10x Genomics Xenium) to profile lung tissue. To study this multicellular coordination, we constructed spatial tissue graphs and applied NICHES-based spatial connectomics to identify ligand-receptor-mediated cell-cell interactions.

Results

We found that lung tissue followed a reproducible trajectory progressing from early inflammation, regenerative remodeling, and subsequent resolution. Early inflammation (Day 3) was characterized by mesothelial cell activation, increased macrophage infiltration, and widespread coupling of mechanotransducive, inflammatory, and developmental signaling programs. During peak remodeling (Day 7), localized remodeling regions emerged that were marked by co-localization of transitional epithelial (Krt17⁺), fibroblast (Dkk2⁺), and macrophage (Spp1⁺) populations, and exhibited loss of mature microvasculature. These regions also displayed high-dimensional connectivity signatures integrating fibrotic, developmental, and inflammatory pathways, suggesting coordinated tissue-level remodeling rather than individual cell state changes. During late resolution (Day 14), fibrotic-like signaling networks diminished, while angiogenic pathways resurfaced and secondary septation was observed, coincident with restoration of differentiation of alveolar epithelial and endothelial populations. Although not fully resolved, these findings indicate progression of the tissue towards a homeostatic state.

Conclusions

Our findings establish a tissue systems biology framework for understanding multicellular coordination during tissue remodeling. PPLR provides a reproducible model for understanding how similar biological pathways and networks can lead to regeneration in some contexts but persistent fibrosis in chronic lung disease. This work demonstrates the feasibility and power of spatial transcriptomics and connectomics to elucidate regenerative pathways that have been previously difficult to study through traditional approaches.

This abstract is funded by: NIH grants