Fig. 1: Organoids from NPp53 mice recapitulate heterogeneity and neuroendocrine transdifferentiation of CRPC-NE.

a, H&E and immunofluorescence staining of sections from parental tumours and matched NPPO organoid lines established from NPp53 mice at passage 2. NPPO-2 and NPPO-3 are relatively homogeneous neuroendocrine lines, NPPO-1 and NPPO-5 are heterogeneous lines that contain neuroendocrine and AR⁺ non-neuroendocrine cells, and NPPO-4 and NPPO-6 are amphicrine lines with cells that express both AR and neuroendocrine markers. b, Diffusion component (DC) projection of scRNA-seq data from individual NPPO organoid lines analysed by VIPER. Proportions of cells in the three clusters are indicated by bars on the left of each plot. c, DC projection of a composite dataset of all five NPPO organoid lines. d, Left, VIPER-inferred activity for a published NEPC gene signature²⁰ (CRPC-NE), with colour scale indicating normalized enrichment score (NES). Right, CytoTRACE analysis. e, Activity profiles inferred by VIPER for the indicated proteins in the composite dataset; colour scales indicate NES. f, Dot plot of inferred protein activity in the three clusters. g, Pathway analysis using VIPER-inferred activities, with ten selected enriched pathways shown in the NPPO dataset. h, Schematic of the co-culture assay for neuroendocrine transdifferentiation. i,j, Immunofluorescence analysis of RFP-expressing NPPO-1nonNE and NPPO-1NE organoids cultured separately (i) or together in organoid chimeras (j) for four passages. Arrows indicate cells co-expressing RFP and VIM together with the neuroendocrine markers CHGA and SYP. k, DC projection of scRNA-seq data and VIPER-inferred activity for the indicated proteins from NPPO-1nonNE (top) and co-cultured organoids (bottom) at passage 4. Colour scales correspond to normalized gene counts. Note the presence of RFP⁺ cells in clusters 2 and 3 in the co-cultured organoids. Scale bars, 50 µm (a,i,j).