The pulmonary extracellular matrix (ECM) provides structural integrity and essential mechanical properties such as elastic recoil, but also serves as an information-rich signaling template that instructs cell identity and activity in health and disease. The spatial distribution and specificity of ECM proteins to the lungs distinct tissue niches is mostly unknown.

In this study, we use spatially resolved mass spectrometry-based proteomics to define global differences in the proteomic composition of anatomically and histologically defined regions of the distal human lung. We characterized the distal airway tree and associated arteries and veins from 12 human donors*, using both 3D surgical microdissection (n=5) and 2D laser-capture microdissection techniques (n=7). Our analysis identified 7594 proteins (including 446 matrisome proteins) from 3D dissections and 4898 proteins (including 341 matrisome proteins) from 2D dissections.

We identified gradients of ECM composition along the proximal-distal axis of the airways and vessels and discovered a specific ECM and immune niche in the respiratory bronchioles (RB). Integrative analysis of the spatial proteomes with donor-matched snRNAseq data and the Human Lung Cell Atlas (HLCA) further suggests the presence of a novel RB-associated fibroblast state that can be found across donors also in public datasets. As proof of concept, we identify and validate a subset of fibroblasts characterized by an ECM expression program enriched in RB via interactive imaging method. Given the potential origin of chronic lung diseases in distal airways, our discovery warrants further investigation of this novel RB specific ECM niche.