Integrated single-nucleus and spatial transcriptomics define tumour cell metaprograms and tumour microenvironment organisation in choroid plexus tumours

Background Choroid plexus tumours (CPTs) are rare intraventricular brain neoplasms derived from choroid plexus epithelium, predominantly affecting young children. They include benign choroid plexus papilloma (CPP), atypical CPP (aCPP), and malignant choroid plexus carcinoma (CPC) and segregate into three DNA-methylation subgroups: paediatric A (pedA), paediatric B (pedB), and adult. Methods To resolve subtype-specific biology, we performed single-nucleus RNA sequencing on 43 CPTs and integrated 12 non-neoplastic foetal, paediatric, and adult choroid plexus references, complemented by spatial transcriptomics in a subset of 16 tumour sections and 2 normal choroid plexus samples. Results Tumour embeddings and correlation analyses demonstrated that CPTs cluster by methylation subtype rather than WHO grade, revealing epigenetically driven relationships despite divergent histology. We identified 10 tumour metaprograms spanning epithelial differentiation, ciliogenesis, proliferation, extra cellular matrix remodelling, and stress responses. PedA and adult tumours showed relatively homogeneous program distribution dominated by differentiation or ciliation states, whereas pedB tumours exhibited marked heterogeneity with selective enrichment of hypoxia, interferon signalling, early stress response, and TGF-β signalling. Spatial mapping revealed consistent anticorrelation of these programs with ciliogenesis and differentiation programs, suggesting stress-associated dedifferentiation. Elevated stress and hypoxia scores correlated with poorer survival. PedB tumours showed enrichment of interferon- and lipid-associated myeloid populations, that carried pro-angiogenic signatures. They were spatially co-localised with hypoxic and interferon I expressing tumour areas, that were predicted to interact with myeloid cells via immunosuppressive cues, shaping a pro-angiogenic, pro-tumour environment. Conclusions This study identifies stress- and hypoxia-associated dedifferentiation programs that are selectively enriched in pedB tumours, correlate with poor clinical outcome, and antagonise multiciliogenesis and epithelial differentiation. Their spatial co-localisation with immunosuppressive, pro-angiogenic macrophage populations links developmental arrest to microenvironmental remodelling and aggressive behaviour. These findings provide a biologically grounded framework for risk stratification and highlight hypoxia, angiogenesis and macrophage polarisation as candidate therapeutic targets in high-risk CPTs.