Jag1-regulated hybrid-EMT state identifies a subset of CD9hi pancreatic tumour-initiating cells with enriched stemness

We previously identified a pancreatic ductal adenocarcinoma (PDAC) subpopulation capable of initiating PDAC and giving rise to PDAC heterogeneity, marked by the tetraspanin CD9. PDAC shows great cellular heterogeneity, with pronounced epithelial and mesenchymal cancer cell populations, and it has been suggested that cancer cells with hybrid epithelial-mesenchymal transition (EMT) features peculiarly contribute to tumour aggressiveness. Here, we identify a stable hybrid-EMT subpopulation that is highly enriched for tumour initiating cell (TIC) properties which constituted a subpopulation of CD9hi TICs1. Hybrid-EMT CD9hi TICs showed increased capacity to form organoids and generate epithelial and mesenchymal tumour cell progeny. Interestingly, in contrast to the majority of PDAC organoids with cystic morphology, hybrid-EMT organoids exhibited a solid morphology and were able to give rise to both cystic and solid organoids, implying a relationship between stemness and solid morphology. Cell depletion of hybrid-EMT cells led to gradual collapse of organoids formation capability, suggesting hybrid-EMT cells are required for long-term organoid formation. Mechanistically, hybrid-EMT cells upregulated Jag1, which promoted their self-renewal by activating the Notch signalling pathway. Thus, the hybrid EMT state is a defining feature of stem cells in PDAC. To evaluate the role of Jag1 in the regulation of hybrid-EMT CD9hi tumour initiating cells (TICs), we generated Jag1 knocked-down mouse PDAC organoids by lentiviral shRNA, and then performed transcriptomic profiling on control (shCtrl) and Jag1 knocked-down (shJag1_3) organoids by RNA-sequencing (RNA-seq). Three biological replicates for each group were used and comparative gene expression analysis was conducted.