PROJECT SUMMARY The advent of single cell RNA-sequencing (scRNA-seq) revolutionized our ability to study the molecular mechanisms underlying pulmonary fibrosis (PF). Our group has leveraged this approach to identify novel cell types and cell states, as well as to characterize the genetic architecture of gene expression in advanced PF. While this work has made significant impact, it has also raised important questions about the timing and coordination of disease pathogenesis and pre-clinical progression. Combining advances in imagining based spatial transcriptomics and a unique set of formalin-fixed, paraffin-embedded transbronchial biopsy samples from subjects in our At-Risk for Familial PF (FPF) Cohort, we can now characerize and localize molecular and cellular dysregulation during the earliest phases of disease. In preliminary studies, we performed spatial transcriptomic analysis of FFPE tissue from a small subset of the At-Risk for FPF Cohort, as well as a larger cohort of samples from declined donors and late-stage disease. Using these data we quantified cell type composition across samples and found higher representation of cells that are often depleted in single cell assays – e.g. AT1, endothelial, and fibroblast cells. Importantly, taking full advantage of the in situ nature of these data we identified distinct molecular niches using both cell-cell and transcriptional neighborhoods. Importantly, these approaches were able to distinguish between histologically similar regions with differing underlying cellular and molecular profiles. Furthermore, we found gene expression differences between healthy and dysregulated alveolar niches between controls and late stage disease were similar to expression differences that preceded radiographic disease in the At-Risk for FPF cohort. We hypothesize that disruption of specialized niche-specific regulatory programs in the distal lung results in a spatially coordinated activation of multicellular disease amplification programs to mediate the initiation and progression of pulmonary fibrosis. Our Specific Aims are to: 1) develop a spatially-resolved molecular atlas of pre-clinical FPF, 2) define disease- stage-specific regulatory mechanisms throughout the natural history of PF, and 3) identify niche-perturbations which promote and ameliorate disease progression in ex vivo models. Together this study represents a comprehensive analysis of the molecular and cellular mechanisms underlying PF. We anticipate the results of these analyses will identify novel therapeutically relevant targets that have immense potential to impact patients with PF.