Modelling adaptation of PDAC to microenvironmental pressures

Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal disease with the lowest survival rate of all cancers. It is characterised by a dense stroma, few neoplastic cells, and extreme heterogeneity in both the stromal and epithelial compartments. As PDAC is characterised by therapy resistance and high relapse rates, we first focused on the effects of neoadjuvant therapy on cancer cells. We established a library of patient-derived organoids (PDOs) from treated and non-treated tumours. The integration of DNA and RNA-sequencing revealed few differences between the two cohorts, with transcriptomic signatures of metabolic and epigenetic reprogramming enriched in post-treatment tumours. Next, we explored the importance of microenvironmental cues, present in the culture medium, on the propagation of PDOs. We found that the WNT pathway activator, RSPO3, is an essential cue for organoids’ survival, and its expression is mostly confined to the stromal compartment of PDAC tissues (i.e., fibroblasts and endothelial cells). In keeping with this finding, endothelial cells could rescue PDOs survival in the absence of RSPO. Leveraging this known dependency of PDOs on the exogenous supplementation of stromal cues, we modelled the dynamics of oncogene amplifications on extrachromosomal DNA (ecDNAs). EcDNAs are circular elements containing oncogenes that are inherited through a non-Mendelian pattern and can supercharge oncogene expression due to increased chromatin accessibility. We found that PDOs can adapt to the withdrawal of Wnt ligands in the culture medium also through increased activity of MYC and that this occurs more rapidly when MYC is on ecDNAs. ecDNAs drove exceptionally high dosage of MYC and allowed cancer cells rapid adaptation to microenvironmental changes. However, the continued maintenance of extrachromosomal MYC was uniquely ensured by the presence of selective pressure. MYC dosage affected cell morphology and dependence of cancer cells on stromal niche factors, with the highest MYC levels correlating with squamous-like phenotypes. Collectively, this work provides the first omics analysis of PDOs from treated tumours, evidence that endothelial cells can support PDAC cells by providing niche factors and the first detailed analysis of ecDNAs in PDAC as a new genetic mechanism driving MYC heterogeneity.