Patient derived organoid established under hypoxic is preferrentially enriched for Moffitt, basal like subtype that corresponse to the dedifferentiate portion of the tumour section.
It is widely accepted that the desmoplastic feature of pancreatic ductal adenocarcinoma (PDAC) and the associated hypoxic microenvironment strongly correlate with a more malignant phenotype that is more resistance to conventional-, targeted- and immuno-therapies. Whether chronic hypoxia in the PDAC stromal gives rise to distinct populations driven by genetics diversity or adoption of distinct phenotypic states in response to low oxygen-tension is poorly understood.  To overcome potential selection bias during the establishment of primary 3D organoids lines, single cell dissociated from each surgically resected tumour were split and established directly in 20% O­2 (normoxia) and 1% O2 (hypoxia), yielding cystic and solid organoid morphology respectively. Organoid established under hypoxia (HYPO-PCO) displayed higher expression of EMT-related proteins, a Moffitt basil-like subtype transcriptomes and higher resistance to 5-FU than organoid established under normoxia (NORMO-PCO). Strikingly, while NORMO-PCO subjected to hypoxia displayed the expected gene signatures enriched including inflammatory response, mTORC1 signalling and glycolysis, these signatures were also enriched when compared to HYPO-PCO under hypoxia. This indicates that the two organoid lines captured distinct population from the same tumour which they were derived from.  Given recent appreciation for the better understanding of and the ability to target hypoxia to improve PDAC therapeutic responses, this study highlights the importance of our approach to study hypoxic population over the more conventional model which study hypoxic response in organoids established under normoxia. Overall design: Dissociated clinical specimens were split into half, and subjected to identical establishment protocol with the exception of oxygen level being at either 20% (normoxia) or 1% (hypoxia). A total of three passages were performed in medium without EGF for the purpose of enriching pancreatic cancer organoids. Following establishment respective RNA samples were collected before switching to the opposite oxygen concentrations and cultured for a total of three passages before further RNA samples were collected.