Extracellular matrix phenotyping by imaging mass cytometry defines distinct cellular matrix environments associated...

The extracellular matrix (ECM) forms the scaffold in which cells reside and interact. The composition of this scaffold guides the development of local immune responses and tissue function. With the advent of multiplexed spatial imaging methodologies, investigating the intricacies of cellular spatial organisation is more accessible than ever. However, the relationship between cellular organisation and ECM composition has been broadly overlooked. Using imaging mass cytometry, we investigated the association between cellular niches and their surrounding matrix environment during allergic airway inflammation in two commonly used in-bred mouse strains. We have integrated both intracellular and extracellular analysis by first classifying cells according to their canonical marker expression and then utilising a novel pipeline to independently characterise their ECM environment. Applying this methodology across three distinct lung tissue regions we reveal region-specific and spatially constrained allergic inflammatory responses. In our model of allergic airway inflammation, recruited neutrophils were dispersed within the alveolar parenchyma, alongside a loss of alveolar type-I cells and an expansion of alveolar type-II cells. This activated parenchyma was associated with increased proximity to hyaluronan and chondroitin sulphate. In contrast, infiltrating CD11b⁺ and MHCII⁺ cells accumulated in the adventitial cuff specifically in BALB/c mice. This region was around the airway-adjacent pulmonary artery and aligned with an expansion of the subepithelial airway region. This expanded subepithelial region was enriched for closely interacting stromal and CD11b⁺ immune cells which overlaid regions enriched for type-I and type-III collagen. The detailed map of cell and matrix interactions generated here provides a critical resource for understanding ECM contribution to specific lung niches during allergic disease. It reveals strain-dependent differences in inflammation and remodelling and generates novel hypotheses for targeting aspects of remodelling during allergic airway pathology.