Dual-oxygen pancreatic cancer organoids recapitulate basal–classical heterogeneity validated by spatial transcriptomics

Abstract

Pancreatic ductal adenocarcinoma (PDAC) comprises heterogeneous classical and basal-like subtypes. Although pancreatic cancer organoids (PCOs) are widely used, normoxic cultures preferentially maintain the classical subtype, limiting their ability to represent intrinsic intratumoral heterogeneity. Because PDAC develops in profoundly hypoxic microenvironments, the influence of oxygen tension on organoid modelling remains unclear. We performed spatially resolved single-cell transcriptomics on 12 primary PDAC tumours to map the distributions of classical and basal-like cells. Paired organoids were generated under normoxic (20% O₂; NORM-PCOs) and hypoxic (1% O₂; HYPO-PCOs) conditions, and morphology, subtype scores, proliferation and drug responses were compared. Spatial profiling revealed gradients of basal-like–classical heterogeneity across tumours, with basal-like cells enriched in hypoxic regions. HYPO-PCOs tended to retain basal-like traits, including solid or multi-layered morphology, and increased proliferation, along with a tendency toward reduced sensitivity to chemotherapeutic agents. In contrast, NORM-PCOs more often exhibited mono-layered morphology and showed higher classical subtype scores. Hypoxic culture conditions promote the preservation of basal-like subpopulations in PDAC organoids, more accurately reflecting the basal–classical heterogeneity of primary tumours. Hypoxia-induced basal-like features remained stable after oxygen switching, indicating a fixed phenotype that informs organoid modelling and offers insight into PDAC progression.