Early fibrotic niches establish tumour-permissive microenvironments [Experiment 3]

Pathologic transformation represents a pivotal yet poorly defined window during which early alterations in epithelial stem cells remodel their surrounding niche to prime tumour initiation. Here, we integrate single-cell, spatial, and functional analyses to map the dynamics of this early multicellular reorganisation. We show that KrasG12D-mutant alveolar type II cells undergo an early reprogramming transition that transforms them into signalling hubs, with the Amphiregulin (Areg)-EGFR axis emerging as a central driver of cross-compartmental cooperation. Mutant epithelial cells secrete Areg to activate EGFR in adjacent fibroblasts, eliciting a regenerative-like fibrotic programme. These reprogrammed fibroblasts, in turn, reshape the immune landscape by expanding and reprogramming alveolar macrophages, amplifying inflammatory signalling, immune recruitment, and epithelial plasticity. Together, these interactions establish a self-reinforcing multicellular circuit that generates and maintains a tumour permissive niche. Disrupting that circuit through genetic and pharmacological perturbation of the Areg-EGFR axis prevents both early niche cell reprogramming and tumour formation. Findings from KRASG12D-inducible human alveolar organoids and early-stage lung adenocarcinoma tissues confirm conservation of these interactions, identifying early mutant epithelial-stromal crosstalk as a targetable step in pathologic transformation with potential to avert establishment of treatment-resistant disease. Overall design: At 2 weeks post two doses of tamoxifen induction (0.2 mg/gbw) every other day, lung tissues were collected from Sftpc-CreERT2; Red2Kras; Aregflox/+ and Sftpc-CreERT2; Red2Kras; Aregflox/flox animals. Lungs from three mice of the same genotype were dissociated and pooled into a single-cell suspension. Lineage-labelled epithelial cells (EpCAM+RFP+) and a mixed population of mesenchymal and immune cells (1:1 ratio of mesenchymal cells (CD45–CD31–EpCAM–) and immune cells (CD45+CD31–EpCAM–)) were then sorted. For each genotype, two separate libraries were generated, resulting in a total of four libraries (2 x Aregflox/+ and 2 x Aregflox/flox). Libraries were sequenced using Illumina Novaseq 6000.