Spatially discrete signalling niches regulate fibroblast heterogeneity in human lung cancer

Fibroblasts are functionally heterogeneous cells, capable of promoting and suppressing tumour progression. Across cancer types, the extent and cause of this phenotypic diversity remains unknown. We used single-cell RNA sequencing and multiplexed immunohistochemistry to examine fibroblast heterogeneity in human lung and non-small cell lung cancer (NSCLC) samples. This identified seven fibroblast subpopulations: including inflammatory fibroblasts and myofibroblasts (representing terminal differentiation states), quiescent fibroblasts, proto-myofibroblasts (x2) and proto-inflammatory fibroblasts (x2). Fibroblast subpopulations were variably distributed throughout tissues but accumulated at discrete niches associated with differentiation status. Bioinformatics analyses suggested TGF-ß1 and IL-1 as key regulators of myofibroblastic and inflammatory differentiation respectively. However, in vitro analyses showed that whilst TGF-ß1 stimulation in combination with increased tissue tension could induce myofibroblast marker expression, it failed to fully re-capitulate ex-vivo phenotypes. Similarly, IL-1ß treatment only induced upregulation of a subset of inflammatory fibroblast marker genes. In silico modelling of ligand-receptor signalling identified additional pathways and cell interactions likely to be involved in fibroblast activation, This highlighted a potential role for IL-11 and IL-6 (among other ligands) in myofibroblast and inflammatory fibroblast activation respectively. This analysis provides valuable insight into fibroblast subtypes and differentiation mechanisms in NSCLC. Overall design: mRNA profiles of cultured primary human lung fibroblasts