The neuroendocrine transition in prostate cancer is dynamic and dependent on ASCL1

Abstract

Lineage plasticity is a hallmark of cancer progression that impacts therapy outcomes, yet the mechanisms mediating this process remain unclear. Here, we introduce a versatile in vivo platform to interrogate neuroendocrine lineage transformation throughout prostate cancer progression. Transplanted mouse prostate organoids with human-relevant driver mutations (Rb1^-/-; Trp53^-/-; cMyc⁺ or Pten^-/-; Trp53^-/-; cMyc⁺) develop adenocarcinomas, but only those with Rb1 deletion advance to aggressive, ASCL1⁺ neuroendocrine prostate cancer (NEPC) resistant to androgen receptor signaling inhibitors. Notably, this transition requires an in vivo microenvironment not replicated by conventional organoid culture. Using multiplexed immunofluorescence and spatial transcriptomics, we reveal that ASCL1⁺ cells arise from KRT8⁺ luminal cells, progressing into transcriptionally heterogeneous ASCL1⁺;KRT8^- NEPC. Ascl1 loss in established NEPC causes transient regression followed by recurrence, but its deletion before transplantation abrogates lineage plasticity, resulting in castration-sensitive adenocarcinomas. This dynamic model highlights the importance of therapy timing and offers a platform to identify additional lineage plasticity drivers.

Fig. 1. Rapid establishment of genetically defined prostate cancer with prostate organoids transplanted into immunocompetent syngeneic hosts.

a, Schematic of timeline required to establish, propagate, edit and select for organoids harboring mutations in cancer-associated genes before transplantation into immunocompetent hosts for tumor establishment. b, Representative microscopy of Pten^−/−; Trp53^−/−; cMyc^T58A (PtPM) organoids, and stereoscopic and fluorescence images of OT prostate tumors with PRAD histology. Tumor images are representative of n = 22 mice. c, Representative microscopy of Rb1^−/−; Trp53^−/−; cMyc^T58A (RPM) organoids and stereoscopic and fluorescence images of OT prostate tumors and lung metastases (mets) (top). Representative histological assessment of RPM-PRAD and RPM-NEPC primary tumor or metastases histology at varying magnifications (bottom). Primary and metastatic microscopy and histology are representative of n = 25 mice. LN, lymph node (iliac). d, Phospho-histone H3 (Ser10; pHH3)-positive tumor cells per total tumor area (µm²). Each data point represents the average number of pHH3⁺ cells per individual tumor subset by tumor histology (PRAD versus NEPC) and experimental end point. PtPM-Early (<4 weeks), n = 14; PtPM-Late (>6 weeks), n = 8; RPM-PRAD, n = 11; and RPM-NEPC, n = 14 tumors. Statistics were derived using a one-way ANOVA with Tukey’s multiple comparisons correction. Error bars denote mean and s.d. e, Survival of mice transplanted with the indicated cell numbers of PtPM, RPM and Pten^−/−; Rb1^−/−; cMyc^T58A (PtRM) ex vivo edited organoids. PtPM 1k, n = 5; PtPM 250k, n = 5; RPM 250k, n = 14; and PtRM 250k, n = 8 mice. Statistics derived from the log-rank (Mantel–Cox) test for each pairwise comparison. f, Metastatic disease penetrance of the indicated organoid transplants. Regional metastases include dissemination into the iliac LNs. Distal metastases include dissemination to kidney, pancreas, liver or lungs. Statistics were derived from a two-sided Fisher’s exact test. The number of biological replicates is indicated within the figure. Scale bars are indicated within each panel. Source data

Fig. 2. Molecular characterization of PtPM and RPM primary prostate tumor transplants demonstrates emergence of neuroendocrine carcinoma marker expression.

a, Representative histological analysis of PtPM (top) and RPM (bottom) tumors isolated at 4 weeks after transplantation. Serial sections depict immunohistochemical staining of the indicated markers. Data are representative of n = 22 tumors. b, Representative histological analysis of RPM tumors isolated at 10 weeks after transplantation. Serial sections depict immunohistochemical staining of the indicated markers. Data are representative of n = 25 tumors. c, Volcano plot depiction of the log₂ fold change in RNA expression of primary (OT) RPM tumors relative to primary (OT) PtPM tumors. Genes that meet or surpass the indicated thresholds of significance (two-sided Wald test with Benjamini–Hochberg multiple comparisons correction) and FC in expression are color coded as depicted in the figure legend. Data are derived from the comparison of PtPM (n = 10) and RPM (n = 8) tumors. d, Heatmap depicting the z-score normalized differential expression of select genes in PtPM versus RPM tumors. Time points of isolation are color coded in the figure as they are in a. Genes are grouped by the listed class or pathway. Early PtPM, 4 weeks; early RPM, ≤6 weeks. Late PtPM, 5 weeks; late RPM, 10 weeks. Data are related to samples used in c. e, Enrichment plots (GSEA) of established expression signatures of a GEMM of NEPC harboring conditional deletion of Pten, Rb1 and Trp53 (PtRP) (left) and histologically verified human NEPC within RPM primary tumors (right). FDR and normalized enrichment score (NES) are indicated in the figure. Analysis derived from the transcriptional profiles of multiple independent RPM tumors (n = 8) relative to PtPM tumors (n = 10). Data are related to samples used in d. All scale bars are noted in each panel and are of equivalent magnification across each marker. NS, not significant; FC, fold change. Source data

Fig. 3. Multiplexed immunofluorescence identifies unique spatial distribution of immune cells within RPM prostate tumors, with local depletion of immune cell types in NEPC areas.

a, Schematic representation of the methods used to process RPM tumors for 20-plex mIF. b, Representative H&E (top) and serial section (bottom) depicting a 3-marker pseudo-colored 10-week RPM tumor. Histological regions (PRAD versus NEPC) are denoted in the H&E and demarcated by dotted yellow line. c, Representative enhanced magnification of lymphoid (left) and myeloid cell markers (middle) and serially sectioned H&E (right). Staining was repeated independently twice with similar results. d, Representative segmented field of view (FoV) for the indicated general lymphoid cell types in a 10-week RPM tumor. e, Representative mIF of the indicated pseudo-colored lymphocyte markers within NEPC (left) or PRAD (middle). Data are presented as a segmented FoV indicating the localization of each lymphoid and tumor cell type in space (right). f, Representative segmented FoV for the indicated general myeloid cell types in a 10-week RPM tumor. g, Representative mIF of the indicated pseudo-colored myeloid and tumor histotype markers. Segmented FoV indicating the localization of each myeloid and tumor cell type in space (right). For b–g, images are representative of n = 3 tumors. For d–g, stains are representative of n = 2 experiments repeated with similar results. h, Frequency distribution of CD8⁺ T cells within binned distance outside or inside the defined interface region (NEPC or PRAD). i, Mean distance of the indicated cell types to the nearest histotype boundary. Statistics are derived from a two-sided Student’s t-test. Error bars denote mean and s.d. j, Frequency distribution of Mac2 cells (CD11b^lo; CD11c⁺; F4/80⁺) within each binned distance outside or inside of the defined interface region (NEPC or PRAD). Data are calculated as in h. For h,j error bar represents mean and s.e.m. of the cell counts per bin. Shaded regions in h,j were approximated through the Loess method. The dotted line in h–j represents the boundary of the tumor histotype or tumor edge. Scale bars are denoted within each panel. Data derived from n = 3 independent tumor samples. Infiltration analyses are representative of n = 3 distinct NEPC and PRAD boundaries. Source data

Fig. 4. NEPC metastatic lesions are T cell excluded but retain macrophage infiltrates.

a, Representative segmented FoV for the indicated cell types within n = 4 draining lymph node (LN) metastases derived from n = 2 mice (OT) with RPM tumors. b, Representative segmented FoV of macrophages (IBA-1⁺) within liver or lung sections (n = 3 mice each) obtained from mice (OT) with RPM tumors. Note, liver-resident macrophages (Kupffer cells) are IBA-1⁺. c, Representative segmented FoV of T cells (CD4⁺ or CD8⁺) within liver or lung sections (n = 3 mice each) obtained from mice (OT) with RPM tumors. d, Representative zoomed in segmented FoV for all cell types listed within a draining LN metastasis. Scale denotes relative cell size. Image representative of n = 4 lymph nodes with similar results. e, Representative zoomed in segmented FoV across serial lung sections obtained from mice (OT) with RPM tumors, identifying NEPC metastatic nodules infiltrated with (left) macrophage subsets or (right) T cell subsets. Images representative of n = 3 lung sections stained for myeloid or lymphoid panel with similar results. f, Representative mIF of the indicated cell type markers across distinct metastatic sites obtained from mice OT transplanted with RPM organoids. Images are representative of n = 3 mouse samples for indicated each tissue. Staining was repeated independently twice with similar results. g, Neighborhood composition heatmap of cell types found within RPM draining LN metastases demonstrating the proximity of the source cell relative to a neighboring cell (20-pixel distance). Data are derived from n = 4 independent metastatic LN samples isolated from n = 2 mice. h, Frequency distribution for macrophages (IBA1⁺) or T cells (CD4⁺ or CD8⁺) within each binned distance outside or inside of RPM lung metastatic samples. i, Frequency distribution for macrophages (IBA1⁺) or T cells (CD4⁺ or CD8⁺) within each binned distance outside or inside of RPM liver metastatic samples quantified as in h. Shaded region in panels h,i approximated through the Loess method. Error bar in h,i represents mean and s.e.m. of the cell counts per bin. Dotted line in h,i represents the boundary of a tumor histotype or tumor edge. All metastatic tumors per section within an individual mouse were combined for infiltration analysis and subsequently averaged between replicates (n = 3 mice). Source data

Fig. 5. PrismSpot reveals spatial transcriptomic heterogeneity within NEPC marked by Ascl1 coexpressed with distinct NE-related TFs.

a, Representative confocal images of 7-plex mIF of the indicated markers. Second and fourth images are digitally magnified versions of the first and third panel from the left. Data are representative of n = 29 RPM tumors at varying time points post OT. b, Percentage of all ASCL1⁺ cells coexpressing KRT5, KRT8 or KRT-negative within an individual RPM OT tumor. Data are derived from the average percentage of cells within each tumor across n = 10 tumors 6 weeks after OT. c, H&E stains of 10-week RPM tumors (n = 2) (left). Tumors A and B are outlined in red and blue dotted lines, respectively. NEPC regions are highlighted in black dotted lines. BayesPrism inferred cell type fraction for NEPC (middle). The log₂ fold expression of Ascl1 overlayed on the tumor histology (right). d, Workflow of PrismSpot method. BayesPrism infers the posterior of cell-type-specific gene expression (U) and cell type fraction (μ) of each spot. The expression profile of the cell type of interest (NEPC) was selected as the input for Hotspot analysis. e, Heatmap shows PrismSpot output of the pairwise local correlation z-scores of n = 71 TFs of high consensus scores (>0.8) and significant spatial autocorrelation (FDR < 0.01). TFs are clustered into n = 3 modules based on pairwise local correlations between all TFs of significant spatial autocorrelation. f, Spatial expression patterns of TFs within each module are visualized using smoothed summary module scores. Images are representative of n = 2 RPM OT tumors. Spatial analyses were repeated on technical replicates with similar results across n = 2 tumors. g, Beeswarm plot shows the log₂ FC in expression of TFs in each module between bulk RNA-seq of human NEPC (n = 9) and PRAD (n = 50) samples. Dot size shows the two-sided P values based on a Wilcoxon test. All scale bars are indicated within each figure panel.

Fig. 6. Loss of Ascl1 results in abrogated NEPC establishment and castration-sensitivity.

a, Schematic for the generation of RPM-Ascl1^wt and RPM-Ascl1^KO tumors transplanted into the flanks or prostates of immunocompetent C57BL/6J hosts. b, Longitudinal SQ tumor volumes of the indicated tumor genotypes and host backgrounds. Statistics derived using two-way ANOVA with Tukey’s multiple comparisons correction for data collected between days 0–56 to ensure equal sample size comparisons. Error bars denote mean and s.e.m. n = 6 tumors across each group. Castration or sham surgery performed 14 days post SQ transplantation. c, Longitudinal SQ tumor volumes of the indicated tumor genotypes and host backgrounds. Statistics were derived using two-way ANOVA with Tukey’s multiple comparisons correction for data collected between 0–16 days post treatment start to ensure equal sample size comparisons. Error bars denote mean and s.d. Ascl1^wt-vehicle, Ascl1^wt-vehicle and Ascl1^KO-degarelix, n = 8 tumors; Ascl1^KO-vehicle, n = 9 tumors. Vehicle or degarelix treatment was initiated upon tumor establishment (≥150 mm³). d, Representative H&E of SQ (top) and OT (bottom) tumors isolated at end point. Genotype and treatment groups are listed within the figure panel. Scale bars are denoted within the figure panel. Data are related to samples in b,c and Extended Data Fig. 8c,d. e, Stacked bar charts representing percentage of OT tumor area composed of the histological categories depicted in the figure legend. Data are quantified tumor histology compared in b and represent average tumor area. f, Stacked bar charts representing percentage of SQ tumor area composed of the histological categories depicted in the figure legend. Data are quantified tumor histology compared in c and represent the average tumor area. For e,f, error bars indicate mean and s.d. g, Pie charts representing percentage of mice with metastatic disease (regional and distal) in intact or castrated hosts of the indicated genotypes. Statistics are derived from a two-sided Fisher’s exact test, P = 0.0137. The number of biological replicates is indicated in the figure panel. Scale bars are denoted in the figure panels. Source data

Fig. 7. Loss of Ascl1 results in enhanced AR expression and proportion of KRT8+ tumor cells.

a, Representative confocal images of the tumors isolated from mice in Fig. 6d. Scale bars and pseudocolor legend indicated within the figure. Stains were repeated independently twice with similar results. b, Density plots of the log₂(x + 1) transformed ASCL1 mean fluorescence intensity from all (OT and SQ) tumor cells. Tumor cells subset by all cells staining negatively for vimentin. Tumor genotype and treatment indicated in the figure panel. c, Density plots of the log₂(x + 1) transformed AR mean fluorescence intensity from all OT tumor cells within the indicated genotypes and treatment groups. Tumor cells subset by all cells staining negatively for vimentin and positively for KRT8 and AR. d, Area under the curve (AUC) for all KRT8⁺:AR⁺ tumor cells (VIM⁻) across both SQ and OT tumors, containing a log₂-transformed nuclear AR intensity score ≥3. Statistics derived using two-way ANOVA with Tukey’s multiple comparisons correction. Error bars indicate mean and s.d. Combined OT and SQ tumor sample sizes for all quantification and analysis performed in Fig. 7: n = 11 (Ascl1^wt and Ascl1^KO intact groups), n = 12 (Ascl1^wt castrate group), n = 9 (Ascl1^KO castrate group). FAU, fluorescence arbitrary unit. Source data

Fig. 8. Loss of Ascl1 in established NEPC results in modest tumor control and increased tumor heterogeneity.

a, Schematic of Ascl1 Dox-inducible in vivo platform. RPM-Ascl1^KO organoids infected with inducible mScarlet (Ctrl) or Ascl1-P2A-mScarlet (Ascl1) vectors were transplanted OT into mice fed Dox chow (primary recipient host, 1^o). Tumor volume was monitored by ultrasound. Upon primary tumor establishment, mice were randomized into Dox ON (maintained) or Dox OFF (withdrawal) groups. b, Survival curves of Ctrl or Ascl1 induced OT tumors following Dox maintenance (ON groups) or withdrawal groups (OFF groups). Statistics were derived from a log-rank (Mantel–Cox) test comparing primary Ascl1 ON to primary Ascl1 OFF groups. Ctrl ON n = 7, Ctrl OFF n = 8, Ascl1 ON n = 11, Ascl1 OFF n = 13 mice. c, Schematic of SQ Ascl1 Dox-inducible in vivo platform (secondary recipient host, 2^o). Ascl1 ON primary tumors were dissociated for flow cytometry to enrich for RPM-NEPC cells used for transplantation assays into the flanks of secondary recipient mice fed Dox chow. Tumor volume was monitored by caliper. Upon tumor establishment, mice were randomized into Dox ON (maintained) or Dox OFF (withdrawal) groups. d, Survival curves of Ctrl or Ascl1 induced secondary tumors following Dox maintenance (ON groups) or withdrawal groups (OFF). Statistics were derived from a log-rank (Mantel–Cox) test. Ascl1 ON n = 5 and Ascl1 OFF n = 7 mice per group. e, Serial sections from secondary transplanted mice (SQ) stained for the indicated markers by H&E and IHC. f, Representative NEUROD1 IHC within a secondary transplant containing mostly NEPC histology. Data in e are representative of n = 5 tumors. Stains were repeated independently twice with similar results. g, Average percent marker positive nuclei (left) or cells (right) across 2^o SQ Ascl1 ON (n = 5) or OFF (n = 4) tumors. Statistics are derived from a two-sided Student’s t-test. Error bars indicate mean and s.d. Scale bars are depicted in the figure panels. Source data

Extended Data Fig. 1. Engineering of clinically relevant mutations within an allelic mouse prostate organoid series.

a. Oncoprint of the indicated genes frequently mutated in human primary prostate cancer. Data (n = 1633 patient samples) obtained from the studies indicated within the figure legend. b. Schematic of the lentiviral vector used within this study to overexpress cMyc^T58A transcriptionally linked to EGFP in organoids. c. Representative brightfield (left), GFP fluorescence (center), and hematoxylin & eosin (H&E) stains (right) of organoids harboring mutations in indicated tumor suppressors and oncogenes. Images are representative of n = 3 technical replicate organoid samples per genotype. H&E and GFP imaging were repeated independently twice with similar results. d. Representative confocal images of 7-plex mIF stains of organoids of the indicated proteins. Data are representative of n = 3 technical replicates per genotype. e. Percentage of unique cell types expressing the indicated markers in organoid culture. Data representative of n = 3 technical replicates and related to images in panel d. Error bars denote mean and s.d. All scale bars and pseudocolor legends indicated within the figure panel. Source data

Extended Data Fig. 2. Transplantation of 3D, but not 2D, cultured mouse organoids maintain PRAD histology and marker profiles.

a. Schematic representation of steps taken to establish OT prostate tumors in mice from edited organoids grown in matrigel (3D) or monolayer culture (2D) conditions. b. Representative brightfield images of the indicated organoids seeded in monolayer growth. Images taken 5 days post seeding. Images representative of n = 2 independent experiments with similar results. c. Western blot validation of knockout efficiency 5 days post electroporation with Cas9 in complex with purified sgRNA. As in panel a, edited organoids were seeded in matrigel or monolayer culture prior to lysis and western validation. Data representative of n = 2 independent experiments with similar results. RB1, cMYC, HSP90, and P53 were blotted on a single gel. EGFP, AR, and PTEN were blotted on a second gel with an independent HSP90 blot shown in Source Extended Data. d. Representative H&E stains (low and high magnification images) of established mouse models (Hi-MYC), human prostate cancer, and organoid OT-derived prostate tumors and are representative of n = 5 tumor samples. Data related to panels a-c. OT prostate tumors derived from transplantation of organoids grown in (top) monolayer or (bottom) traditional 3D matrigel conditions. e. Representative Synaptophysin (SYP) or ASCL1 IHC of tumors isolated from mice transplanted with RP organoids grown in (top) monolayer or (bottom) traditional 3D matrigel conditions. Data representative of n = 2 tumors per stain. f. Percentage of mice with tumors per genotype and organoid growth conditions. Sample size (mice) per genotype indicated within the figure panel. g. Survival of mice OT transplanted with 250k dissociated organoids grown in matrigel. Sample size (mice) per cohort indicated within the figure legend. h. Representative H&E stains of n = 1 mouse that developed OT tumors following transplantation of PtRM organoids grown in matrigel. i. Representative phospho-histone H3 IHC stains of PtPM or RPM prostate tumors isolated 4 weeks post OT. Histological classification performed using serial sectioned H&E. Dotted line represents the boundary of PRAD and NEPC. Images related to quantification shown in Fig. 1d. j. Average percentage of KRT8+ or KRT5+ tumor cells relative to total detected cells (DAPI+ nuclei) within RPM tumors 5 weeks post OT. Data represent the average positive cell number per tumor. Data derived from n = 5 RPM OT tumors. k. Average percentage of total ASCL1+ cells relative to total detected cells (DAPI+ nuclei) within RPM OT tumors at the indicated time points. Each data point represents the average marker positive cell per mouse tumor. 2–3 weeks, n = 4; 4–5 weeks, n = 6; 6–8 weeks, n = 8; and 10 weeks, n = 11 tumors. Statistics derived using one-way ANOVA with Tukey’s multiple comparisons correction. l. Percentage of cells staining positively for nuclear ASCL1 from tumors harvest 4 to 5 weeks post OT. Data represent the average positive cell number per tumor. Data derived from n = 4 (PtPM) and n = 5 (RPM) OT tumors per group. For panels j-l, error bars indicate mean and s.d. For panels j, l statistics derived using two-sided Student's t-test. All scale bars denoted within the figure panel. Source data

Extended Data Fig. 3. Metastatic tumors in RPM model establish with high penetrance NEPC histology and marker profile.

a. Representative ASCL1 IHC stains from iliac lymph node metastases isolated from RPM OT mice. Dotted line represents the boundary of tumor and normal tissue. b. Representative serially sectioned immunohistochemical stains from metastases isolated across (top) liver and (bottom) lung tissue in RPM OT mice. Scale bars denoted in figure legend. VIM = VIMENTIN, SYP = SYNAPTOPHYSIN. c. Representative H&E stains and histological grade of metastases isolated from regional lymph nodes, kidney, liver, and lung tissue from RPM OT mice. Data related to panels a-b. Scale bars denoted within the figure panel. d. Pie charts demonstrating percentage of mice harboring distinct histotypes of prostate cancer in the indicated metastatic regions. Sample size denoted in figure panel. e. Representative mIF staining for lineage markers in metastastic tumors isolated from regional lymph nodes, liver, and lung tissue from RPM OT mice. Data representative of n = 3 mice. f. Percentage of unique cell types expressing the indicated lineage markers in RPM metastatic samples. For panels a-d, images and data are derived from n = 25 lymph node, n = 5 liver, and n = 9 lung metastatic samples from RPM OT mice. For panel e-f, images and data are representative of n = 3 lymph node, n = 3 liver, and n = 3 lung metastatic samples from RPM OT mice. Error bars denote mean and s.d. All scale bars and pseudo-coloring denoted in the figure panel. Source data

Extended Data Fig. 4. Bulk gene expression profiling of mouse tumors highlights the requirement for Rb1 loss and the TME to induce and maintain NEPC fate.

a. (Top) Principal component analysis of bulk RNA-seq data isolated from RPM or PtPM OT tumors. (Bottom) SYP immunohistochemical stains from the RPM tumors ordered by increasing percentage of SYP+ cells/tumor. RPM, n = 8 and PtPM, n = 10 tumors. Data related to Fig. 2a–d. b. Unsupervised hierarchical clustering of variant stabilized transcript normalized expression of the top 100 differentially expressed genes (columns) within tumors (rows). Data related Fig. 2c. c. GSEA enrichment plots of established expression signatures of (top) a GEMM of AR and Ascl1-co-expressing NEPC harboring conditional deletion of Pten, Rb1, and Trp53 (PtRP), and (right) histologically verified human NEPC expressing NEUROD1 within RPM primary tumors. FDR and NES indicated in the figure. Analysis derived from the transcriptional profiles of RPM tumors (n = 8) relative to PtPM tumors (n = 10). Data related to samples used in Fig. 2c, d. d. Quantitative PCR of Ascl1 transcripts across n = 2 RPM organoids (org), tumors, and n = 2 tumor-derived organoids (tumoroids) at passage 1 (P1) or passage 4 (P4) post isolation. Each data point indicates technical quadruplicate values and bars represent mean and s.d. Data representative of n = 2 independent experiments with similar results. Source data

Extended Data Fig. 5. RPM tumors dynamically transition into NEPC and establish strong T cell exclusion.

a. Representative (top) H&E, (middle) EGFP, and mIF (bottom) of RPM tumors isolated at the indicated time points. EGFP and mIF images are matched sections. Data representative of n = 3 tumors stained by COMET. Scale bar for all images indicates 20 μm. Pseudo-coloring listed within the figure panel. b. Cell number of each indicated stromal and vascular cell types within each binned distance outside or inside the defined interface region (NEPC or PRAD). c. Frequency distribution of each indicated stromal and vascular cell types within each binned distance outside or inside the defined interface region (NEPC or PRAD). d. Dot plot depicting mean cell density outside or inside NEPC or PRAD tumor regions for the indicated non-immune stromal cell types. VIM+, mesenchymal cells: LYVE1+, lymphatic endothelial cells; CD31+, endothelial cells. Statistics for panels c-d derived from two-sided Student's t-test. e. Spatial cell type density (cell density within 25 μm radius) for the indicated tumor cell types and lymphocytes across 10-week RPM tumors (n = 2). Data representative of n = 3 RPM tumors with similar results. Heatmap represents average cell density (cell number/μm²). f. Mean lymphocyte cell count relative to nearest PRAD or NEPC boundary. g. Data as in panel f but normalized to the binned tumor area. h. Percentage of TCF1-negative (neg), intermediate (int), or high (hi) CD8 T cells within RPM tumors. Data points represent the mean number of indicated CD8 T cells across TCF1 expression groups and error bars denote standard deviation. i. (Top) Representative mIF stains from RPM 10-week tumors of the indicated lymphocyte markers. (Bottom) Segmented FoV where each dot represents a CD8 T cell coordinates within a 10-week RPM tumor section. Each dot is color coded based on predetermined thresholds for TCF1 expression, FAU = fluorescence arbitrary units. Data related to panel h. Dotted line in panels b, d, f-g represents the boundary of the histotype to a different histotype or the edge of a tumor. Positive values indicate cells found outside the histotype boundary; negative values indicate cells found inside the histotype boundary. Data and images in panels h-i derived from n = 7 tumors. Data in panels b-d, f-g derived from n = 3 tumors. Error bars denote mean and s.e.m. in panels b, f, and g and mean and s.d. in panels c-d, h. Dotted line in panels b, d, f-g indicate the boundary of a PRAD or NEPC tumor region. Smoothened data curves in panels b, f, and g fit by Loess method. Source data

Extended Data Fig. 6. A subpopulation of tumor-associated macrophages is retained in mouse and human NEPC.

a. Frequency distribution of each indicated cell type within each binned distance outside or inside the defined interface region (NEPC or PRAD). b. Frequency distribution of each indicated cell type density within each binned distance from defined interface region (NEPC or PRAD). c. Dot plot depicting mean cell density away from the boundary of NEPC or PRAD tumor regions for the indicated macrophage subtypes. d. Dot plot depicting mean cell distance (μm) away from the boundary of NEPC or PRAD tumor regions for the indicated macrophage subtypes. e. Spatial cell type density (cell density within 25-μm radius) for the indicated myeloid cell types across 10-week RPM tumors (n = 2). Heatmap represents average cell density (cell number/μm²) and are representative of n = 3 RPM tumors with similar results. f. ITGAX (CD11c) expression across previously published tumor-associated macrophage populations identified within human PRAD (n = 9) and NEPC (n = 3) tumors sequenced by scRNA-seq. Violin and box plots are median centered and depict the 25th and 75th percentiles of ITGAX expression, respectively. g. Gene expression modules of TAM subsets identified within human PRAD and NEPC samples displayed in UMAP space, related to panel f. Scale bar represents module score. h. ITGAX (CD11c) expression across all myeloid cell types identified within human PRAD and NEPC samples displayed in UMAP space, related to panels f-g. Scale bar represents raw expression counts. i. Representative mIF of a human prostatectomy verified to contain mixed PRAD and NEPC pathology. Left zoomed out panels contain white dotted line indicating zoomed in regions on the right. Dotted yellow line represents the boundary of a pan-cytokeratin+ (panCK) PRAD and ASCL1+ NEPC. For panels h-i, images are representative of n = 1 human mixed histology tumor sample imaged across multiple distinct tumor regions of interest. Staining was repeated n = 2 times with similar results. j. Representative mIF of two distinct regions within the human NEPC sample shown in panel i. Dotted square indicates magnified inset shown adjacent to lower magnification view. Error bars in panels a-b represent mean and s.e.m. across n = 3 RPM tumors. Error bars in panels c-d represent mean and s.d. across n = 3 RPM tumors. Statistics in panel c-d derived by two-sided Student's t-test. Dotted line in panels a-b, d represents the boundary of the histotype to a different histotype or the edge of a tumor. Positive values indicate cells found outside the histotype boundary; negative values indicate cells found inside the histotype boundary. Smoothened data curves in panels a-b fit by Loess method. Source data

Extended Data Fig. 7. RPM tumors establish NEPC with heterogeneous expression of distinct NE TFs.

a. Scatter plot shows the mean cell type fraction inferred by BayesPrism across n = 2 technical replicates for each cell type in each region, colored by regions defined by histology. b. Same as panel a. but colored by cell type. NE = neuroendocrine, EMT = epithelial-to-mesenchymal transition tumor cells, Tumor L/B = tumor cells with gene expression associated with luminal and basal lineages. c. Spatial expression (10X Visium) across the indicated RPM tumor (n = 2) spatial modules identified by PrismSpot (related to Fig. 5e, f). Scale bar represents raw expression counts. d. Heatmap of the observed gene overlap normalized to expected gene overlap between PrismSpot modules and several tumor clusters derived by previously published GEMM models of NEPC. Robust PrismSpot modules identified in red font.

Extended Data Fig. 8. Ascl1 loss prior to NE transition confers androgen dependence.

a. Individual longitudinal SQ tumor volumes as determined by caliper. Castration or sham surgery was performed 14 days post organoid transplantation. n = 6 tumors across each group. Data related to Fig. 6b. b. Final SQ tumor mass at experimental end point. n = 6 tumors across each group. Statistics derived by one-way ANOVA with Sidak’s multiple comparisons correction. c. Individual longitudinal OT tumor volumes determined by ultrasound. n = 6 tumors per group. Castration or sham surgery was performed 14 days post organoid transplantation. d. Final OT prostate tumor volumes determined by ultrasound. Measurements derived from Ascl1^wt intact n = 5; Ascl1^wt castrate n = 3; Ascl1^KO intact n = 5; and Ascl1^KO castrate n = 6 mice. Statistics derived by one-way ANOVA with Sidak’s multiple comparisons correction. e. Bar charts representing percentage of SQ tumor area composed of the histological categories depicted in the figure legend. Data related to Fig. 6e. Each dot represents the average area per tumor. Statistics derived by two-way ANOVA with Tukey’s multiple comparisons correction. Error bar denotes mean and standard deviation. n = 6 tumors per group, except for the Ascl1^KO castrate group (n = 3 tumors). f. Bar charts representing percentage of OT tumor area composed of the histological categories depicted in the figure legend. Data related to Fig. 6f. Each dot represents the average area per tumor. Statistics derived by two-way ANOVA with Tukey’s multiple comparisons correction. Error bar denotes mean and s.d. n = 6 tumors per group. g. Final OT tumor mass at experimental end point. n = 6 tumors per group. Statistics derived by one-way ANOVA. Data related to panel c; however, after cessation of ultrasound measurements, tumors were isolated and weighed once palpable or distress was observed. h. (Left) schematic of SQ transplantation assay by which mice are randomized into vehicle or degarelix treatment arms after tumors reach predetermined volume as measured by caliper. (Middle) Validation of serum testosterone depletion assessed by ELISA collected 2 weeks post single dose of degarelix (15 mg/kg) or vehicle (5% mannitol). Box and whisker plot denotes minima and maxima data points, are mean centered, and display the top (90th) and bottom (10th) percentiles. Statistics derived using two-sided Student’s t-test. Data derived from serum measurements isolated from vehicle, n = 5; and degarelix, n = 5 mice. (Right) Longitudinal SQ caliper measurements of RPM-Ascl1^WT vehicle and degarelix treated n = 8, RPM-Ascl1^KO vehicle n = 9, RPM-Ascl1^KO degarelix n = 8 mice per group. i. Survival of vehicle or degarelix treated mice with SQ transplants of the indicated RPM organoid genotypes. Statistics derived from the Log-rank (Mantel-Cox) test for each pairwise comparison. Data related to Fig. 6c and panel h. RPM-Ascl1^WT vehicle n = 9, RPM-Ascl1^WT degarelix n = 6, RPM-Ascl1^KO vehicle n = 9, and RPM-Ascl1^KO degarelix n = 8 mice. j. Representative (left) H&E stain and (right) IHC of EGFP of RPM-Ascl1^KO tumor (n = 1) growth in a castrated host demonstrating chondrosarcomatoid histopathology. Scale bars denoted within the figure. k. (Left) Stacked bar charts representing percentage of SQ tumor area composed of the histological categories depicted in the figure legend. Data are quantified histology of RPM tumors treated with vehicle (n = 5 mice) or degarelix (n = 4 mice) for 4 weeks after tumor establishment (≥150 mm³) and represent average tumor area. (Right) Stacked bar charts of the percentage of AR- and ASCL1-pos (positive) or neg (negative) tumor cells (defined as EGFP+; CD45-; VIMENTIN-) within vehicle or degarelix treated RPM SQ tumors. Vehicle n = 5; and Degarelix n = 4 tumors. Error bars in panels b, d, e-f, g, and k indicate mean and s.d. Source data

Extended Data Fig. 9. Ascl1 loss prior to the NE transition promotes development of PRAD with increased KRT8 and AR expression.

a. Number of ASCL1+ nuclei (left), KRT8+ cells (middle) and KRT5+ cells (right) in the indicated genotypes (legend on right hand-side) and treatment groups. b. Percentage of ASCL1+ nuclei (left), KRT8+ cells (middle) and KRT5+ cells (right) relative to total cells (DAPI+ nuclei) in the indicated genotypes (legend on right hand-side) and treatment groups. c. Percentage of ASCL1+ cells (DAPI+ nuclei) co-expressing either KRT8 or KRT5 within RPM-Ascl1^WT tumors in either intact or castrated hosts. Statistics in panels a-c derived from two-way ANOVA with Tukey’s multiple comparisons correction. Error bars in panels a-c denote mean and standard deviation. For a-c, data represents the average number of cells positive for the indicated marker across biologically independent tumors of the defined genotype and treatment cohort. Ascl1^WT intact, n = 11; Ascl1^WT castrate, n = 11; Ascl1^KO intact, n = 12; Ascl1^KO castrate, n = 9 tumors. d. Field of view images depicting maximum intensity score for all segmented cells within representative RPM tumors of the indicated genotype and treatment group. FAU = fluorescence arbitrary units. Data representative of n = 10 tumors. e. Density plots of the log₂(x + 1) transformed mean fluorescence intensity for each nuclear protein. Each density plot represents signal intensity of tumor cells in tumor sections. f. Density plots of the log₂(x + 1) transformed mean fluorescence intensity for each cytoplasmic protein. Each density plot represents signal intensity of tumor cells across n = 12 tumors. Source data

Extended Data Fig. 10. Ascl1 loss in established NEPC promotes tumor heterogeneity.

a. Schematic of the dox-inducible lentiviral vector used within this conditionally overexpress (left) Ascl1-P2A-mScarlet or (right) mScarlet in RPM-Ascl1^KO organoids. b. Representative stereoscopic images (brightfield and fluorescent) of OT tumors isolated from the indicated dox-maintained or withdrawn conditions. Scale bar represents 1 mm. Images are representative of Ctrl ON n = 7, Ctrl OFF n = 8, Ascl1 ON n = 11, Ascl1 OFF n = 13 tumors. c. Tumor volumes determined by ultrasound 4 weeks post OT of the indicated groups. All mice were maintained on dox chow. Statistics derived from two-sided Student's t-test and error bars denote mean and s.d. Ctrl ON n = 15; and Ascl1 ON n = 24 tumors. d. (Left) Representative stereoscopic images (brightfield and fluorescence) of the draining lymph nodes and lungs of mice bearing OT Ascl1 ON tumors. (Right) Pie charts indicating frequency of regional or distal micro-metastatic dissemination. Mouse sample size indicated in the figure legend. e. Percent change in primary recipient (1^o) OT tumor volume measurements between 4–5 weeks of Ascl1 ON, as determined by ultrasound. Ascl1 OFF mice have been withdrawn from dox chow for 1 week. f. Longitudinal primary OT tumor volumes determined by ultrasound for the indicated groups. For panels e-f, Ascl1 ON n = 11; and Ascl1 OFF n = 13 mice. g. Longitudinal secondary recipient (2^o) SQ tumor volumes determined by caliper for the indicated groups. Ascl1 ON and OFF n = 10 tumors. h. Representative H&E images of the indicated groups spanning both primary recipient (OT) and secondary recipient (SQ) transplanted mice. i. Representative serially sectioned tumors stained for H&E and IHC of the indicated markers across Ascl1 ON and Ascl1 OFF secondary transplant recipient mice (SQ). Histology and IHC representative of tumors quantified in panels a-g. Stains were repeated n = 2 times with similar results. Images displayed represent regions maintaining NEPC histology and high fraction of NCAM-1 marker expression. All scale bars depicted in the figure panels. Source data