Transcriptomic and functional profiling of human intestinal organoids identifies enhanced calcium signalling and...

Although gastrointestinal symptoms of cystic fibrosis (CF) have long been overlooked, patients with CF have an unexplained 5-fold increased risk of colorectal cancer. Much of our understanding of the disease has relied on two-dimensional cell lines and animal models. However, the primary changes occurring in human CF epithelial cells remain largely unclear.

Donor-matched wild-type (WT) and CFTR knockout (KO) human intestinal organoid (HIO) lines were employed to dissect epithelial cell-intrinsic responses to CFTR dysfunction. Knockout was confirmed at the molecular level by qPCR and immunostaining, and functionally by the forskolin-induced swelling assay. Bulk RNA sequencing was then performed to assess key transcriptional differences between WT and KO organoids under steady state conditions and after IFNγ stimulation, and to evaluate the therapeutic potential of calcium channel blockade. Relevant changes were functionally validated using organoid-derived monolayers.

Loss of CFTR expression alone was sufficient to induce profound changes in gene expression across all conditions. Notably, CFTR KO organoids showed higher baseline MUC2 expression and increased THBS2 in an inflamed context. Calcium channel blockade with verapamil reversed these disease-associated changes at the RNA and protein levels.

By utilising gene-edited HIOs derived from the same donor, we reveal epithelial cell-intrinsic mechanisms downstream of CFTR loss. These findings, enabled by our experimental approach, offer important insights into the role of the intestinal epithelium in CF, independent of immune cells, the microbiome, and inflammation. They also lend support for therapeutic targeting of calcium signalling to reverse disease-associated transcriptomic changes.