Pancreatic ductal adenocarcinoma (PDAC) is characterised by very poor outcomes and a uniquely hypoxic tumour microenvironment caused by limited vascularisation and extensive desmoplastic stroma. Reported intratumoral oxygen tensions can be extremely low (under 0.7% O2), and this hypoxia contributes to treatment resistance, invasion, metastasis and poorer prognosis. Molecular profiling has identified two principal expression subtypes in PDAC—classical and basal-like—which can coexist within the same tumour and appear to reflect progression from well-differentiated to poorly differentiated states. Prior single-cell studies revealed subtype co-existence but lacked spatial resolution to map where these states localise within tumours and how microenvironmental factors such as oxygen tension relate to subtype distribution.
Patient-derived organoids (PCOs) are widely used to model PDAC biology and drug response ex vivo. However, standard organoid culture under normoxic conditions (around 20% O2) preferentially maintains the classical subtype, limiting the model’s capacity to reflect the full heterogeneity of primary tumours. Given the central role of hypoxia in driving basal-like phenotypes, the influence of oxygen tension on organoid phenotype and stability was evaluated in this study.
The investigators performed spatially resolved single-cell transcriptomics on 12 primary PDAC tumours to map the spatial distributions of classical and basal-like cells. From matched tumours, organoids were established and cultured under two oxygen conditions: normoxic (20% O2; NORM-PCOs) and hypoxic (1% O2; HYPO-PCOs). The study compared organoid morphology, expression-based subtype scores, proliferation and responses to chemotherapeutic agents across the two culture conditions. Spatial profiling of tumours was used to relate local oxygenation and tissue architecture to subtype enrichment.
Spatial transcriptomic profiling revealed gradients of basal-like–classical heterogeneity across primary PDAC specimens. Within tumours, regions of lower oxygenation were enriched for basal-like cells, whereas classical cells predominated in relatively oxygenated areas. These spatial relationships support the concept that local microenvironmental hypoxia is associated with basal-like transcriptional programmes and the emergence of more poorly differentiated cancer cell states.
Organoids cultured under hypoxic conditions (1% O2) displayed morphological and transcriptional features aligning with the basal-like subtype. HYPO-PCOs more often exhibited solid or multi-layered architectures, while NORM-PCOs more frequently formed mono-layered structures typical of classical-like organoids. Subtype scoring showed higher classical scores in NORM-PCOs compared with HYPO-PCOs, consistent with a tendency of normoxic culture to favor classical identity.
Crucially, culturing organoids at physiologic hypoxia preserved basal-like subpopulations that are otherwise underrepresented in standard normoxic conditions. The hypoxic culture conditions therefore improved the organoid model’s ability to recapitulate the basal–classical heterogeneity observed in primary tumours.
HYPO-PCOs demonstrated increased proliferation relative to NORM-PCOs, aligning with aggressive basal-like behaviour described in clinical and genomic studies. In addition, HYPO-PCOs showed a tendency toward reduced sensitivity to chemotherapeutic agents compared with normoxic counterparts, reflecting clinical observations that hypoxia-adapted PDAC cells are more therapy-resistant. The article reports these comparative tendencies; detailed quantitative metrics and specific drug-response values are provided in the source manuscript.
When oxygen conditions were switched experimentally, basal-like features induced or preserved under hypoxia remained stable, indicating a fixed phenotype rather than a rapidly reversible transcriptional state. This stability suggests that hypoxic selection or reprogramming can lock tumour cells into a basal-like identity that persists even after restoration of higher oxygen levels, with implications for disease progression and treatment resistance.
The work demonstrates that oxygen tension is a critical determinant of organoid phenotype in PDAC. Culturing patient-derived organoids under hypoxia (1% O2) preserves basal-like subpopulations and better recapitulates the spatially resolved basal–classical heterogeneity found in primary tumours than standard normoxic culture. These findings recommend integrating physiologic hypoxia into organoid protocols when modelling PDAC biology, intratumoral heterogeneity and treatment response. The observation that hypoxia-induced basal-like traits are stable after oxygen switching suggests hypoxia can create persistent phenotypic states relevant to progression and therapy resistance.
Note: The source article contains detailed experimental results, quantitative subtype scores, proliferation indices and specific chemotherapeutic response data; those numerical details and figures are reported in the original publication and are not reproduced in full here.