Using lung organoids to recapitulate HRas/NRas-double knockout alveolar maturation defects in mice

We have previously demonstrated that HRas/NRas-double null mutant (DKO) exhibited delayed lung maturation as revealed by increased retention of alveolar progenitors and bi-potent progenitors in distal lung epithelium (Fuentes-Mateos et al., 2019). Primary lung fibroblasts from newborn pups devoid of HRas and/or NRas were used to analyse whether the lung mesenchyme was responsible for the observed lung defects. Our results showed that lung fibroblasts lacking HRas had a dysregulation of the Hox5-Wnt2-FGF7/10 pathway. To investigate the relevance of epithelium-mesenchymal interactions in these processes, we generated lung organoids by co-culturing lung fibroblasts from HRas and/or NRas KO lungs with epithelial cells from the lungs of wildtype mice. The organoids lacking HRas and NRas recapitulated the alterations observed in the lung fibroblasts cultures and in newborn pups, with fewer alveolar organoids after 14-days of culture. This defect was corrected obtaining increased alveolar differentiation levels after NAC administration and, even more strongly, after MEK inhibition with UO126 inhibitor. Additionally, activation of epithelial KRas and downstream ERK was higher in lungs from DKO pups, and these signalling defects were corrected by antenatal dexamethasone, and more robustly with NAC. Our data show that combined absence of HRas and NRas leads to aberrant alveolar maturation that is possibly mechanistically linked to epithelial KRas gain-of-function. This phenotype is partially mitigated after NAC treatment and completely reversed with MEK inhibitor. This study suggests that a physiological modulation of KRas signalling is critical for control of murine late lung maturation.