Fig. 1: Multiregional sampling of radical prostatectomy patient tumours.

a Five locally advanced prostate cancer patients with radical prostatectomy: PCAL37, PCAL34, PCAL25, PCAL10 and PCAL03. Created in BioRender. Weischenfeldt, J. (2025) https://BioRender.com/jglk9y9. Prostate organs were sliced according to the schematic. Prostate zones are coloured. PZ., Peripheral Zone (light orange); TZ., Transition Zone (purple); CZ., Central Zone (red); AFS, Anterior Fibromuscular Stroma (green); Tumour (grey); LNM, lymph node metastasis (brown). b Representative example of pathologist 3D prostate slices and areas selected for analysis (red dots). Prostate zones are marked in dashed lines, coloured as in (a), and tumour-containing areas in grey. c Single nuclei were extracted from fresh-frozen tissue punches and used for both snRNA-seq and bulk low-pass WGS. Right, spatial 3D representation of selected areas relative to the centroid of the prostate. Created in BioRender. Weischenfeldt, J. (2025) https://BioRender.com/5tar688. d UMAP showing the integrated data from all five patients. Semi-supervised integration was used to integrate datasets based on cell-type identity. Four major cell type clusters were present with the following cell type groupings: immune compartment (mast cells (light pink), MNPs (dark purple), T cells (dark pink) and B cells (coral)); stromal compartment (fibroblasts (light blue), endothelial cells (dark blue)); normal epithelial cells (luminal epithelial (LE, light green), club epithelial (CE, olive green), hillock epithelial (HE, yellow), basal (amber) and glial cells (mint green)); malignant epithelial cells (malignant (teal green), proliferating (light purple) and ciliated (orange)). e Barplot showing cell-type composition across areas for each patient, coloured by cell type as in (d). Tiles above show the number of cells captured per area, tumour content, TME entropy, distance from centroid, prostate anatomical zones, and Gleason grade. f Dot plot of cell-type specific marker genes. Dot size represents the percentage of cells within a group expressing the marker, and colour indicates scaled average expression within the cell group. Avg.exp., average expression. g Cell type variation. Left - bar plot of the Gini coefficient by cell type across all samples with cell types and number of samples for LE (43), Fibroblasts (35), Basal (10), Endothelial (34), MNPs (37), T cells (24), CE (6), Mast (6), B cells (13), HE (2), Glial (1), Malignant (43), Proliferating (22), Ciliated (10). Middle - box plot of cell type proportions, aggregated across all samples. Coloured by cell type as in (d). Right - tile plot of the percentage of samples in which each cell type is present. Panels (d–g) represent the integrated data of 356,860 single nuclei from 43 samples from all five patients. Box plots show the median (line), interquartile range (box), whiskers extending to the most extreme data points within 1.5× the interquartile range, and individual points beyond the whiskers representing outliers.