Patient-Derived Xenografts and Organoids Recapitulate Castration-Resistant Prostate Cancer with Sustained Androgen Receptor Signaling

Abstract

Castration-resistant prostate cancer (CRPC) remains an incurable and lethal malignancy. The development of new CRPC treatment strategies is strongly impeded by the scarcity of representative, scalable and transferable preclinical models of advanced, androgen receptor (AR)-driven CRPC. Here, we present contemporary patient-derived xenografts (PDXs) and matching PDX-derived organoids (PDXOs) from CRPC patients who had undergone multiple lines of treatment. These models were comprehensively profiled at the morphologic, genomic (n = 8) and transcriptomic levels (n = 81). All are high-grade adenocarcinomas that exhibit copy number alterations and transcriptomic features representative of CRPC patient cohorts. We identified losses of PTEN and RB1, MYC amplifications, as well as genomic alterations in TP53 and in members of clinically actionable pathways such as AR, PI3K and DNA repair pathways. Importantly, the clinically observed continued reliance of CRPC tumors on AR signaling is preserved across the entire set of models, with AR amplification identified in four PDXs. We demonstrate that PDXs and PDXOs faithfully reflect donor tumors and mimic matching patient drug responses. In particular, our models predicted patient responses to subsequent treatments and captured sensitivities to previously received therapies. Collectively, these PDX-PDXO pairs constitute a reliable new resource for in-depth studies of treatment-induced, AR-driven resistance mechanisms. Moreover, PDXOs can be leveraged for large-scale tumor-specific drug response profiling critical for accelerating therapeutic advances in CRPC.

Keywords: androgen receptor; castration-resistant prostate cancer; drug testing; organoids; patient-derived xenografts.

Figure 1

Development of CRPC PDXs. (A) Pie chart of PCa samples subcutaneously implanted in intact male nude mice to generate PDXs. No take (yellow) indicates absence of tumor outgrowth during a monitoring period of six months. Tumors take (green) represents successful engraftment followed by one to two mouse passages. PDX line (blue) indicates established PDXs through continuous mouse-to-mouse propagation. (B) Clinical and pathological patient characteristics for established PDXs. Treatment history prior to sampling is provided, including local treatments (radical prostatectomy (RP) and radiation therapy (RTx)), and systemic treatments (luteinizing hormone-releasing hormone (LHRH) agonists, chemotherapy, and AR signaling inhibitors). * Indicates identical patient origin. (C–F) Treatment timelines of the patients that gave rise to PC2400 (C), PC2412 (D), PC2416 and PC2416-DEC (E), and PC2459 (F) PDXs, depicted as PSA response from time of diagnosis (day 0) to death. Time point of tissue sampling for PDX engraftment is indicated by a red arrow.

Figure 2

Characteristics of CRPC xenografts. (A) Growth trajectories of PDXs in the first 600 days after engraftment. Each step marks a mouse-to-mouse transplantation. Time from tumor engraftment to first mouse-to-mouse transplantation is indicated by an arrow. (B) Histological architecture of PDXs (top panel) and original patient tumors (bottom panel), examined by hematoxylin and eosin staining. Scale bars equal 100 µm. (C) CNA profiles of CRPC PDXs (PC2400, PC2412, PC2416, PC2416-DEC and PC2459), HSPC PDXs (PC82, PC295 and PC310) and a normal prostate tissue control from an unmatched source, across a subset of genes and genomic regions recurrently altered in CRPC. Grouping per biological pathway. Amplifications (blue), homozygous deletions (red), partial losses (yellow) and unaltered regions (grey) are indicated based on ASCAT analysis of SNP array data (n = 1 for each PDX). ASCAT analysis failed for PC2412 PDX due to sample complexity. All PC2412 CNA calls were determined manually except for the region 10q with complex rearrangements (white). CNA frequency bar plots are provided for each PDX (top panel) and each genomic region (right panel). Additional alterations present in PC2416-DEC PDX, as compared to PC2416 PDX are marked with a white asterisk. (D) t-SNE plot of pathway activity in CRPC PDXs (PC2400 (n = 5), PC2412 (n = 5), PC2416 (n = 5), PC2416-DEC (n = 5) and PC2459 (n = 3); red) and HSPC PDXs (PC82 (n = 2), PC295 (n = 2), PC310 (n = 7); blue), based on GSVA scores for 50 MSigDB cancer hallmark gene sets, using RNA sequencing data. (E) Nuclear ERG staining in PC2459 PDX. Scale bar indicates 100 µm. (F) Copy number profile of chromosome 21 in PC2459 PDX, displaying the ERG and TMPRSS2 genes on 21q22.2-3. BAF: B allele frequency; LRR: log R ratio; CN: total copy number.

Figure 3

Establishment of PDXOs that reflect phenotypic and transcriptomic profiles of matching PDX and patient tumors. (A) Schematic workflow for creating PCa PDXs and PDXOs. Figure adapted from Van Hemelryk et al. [47]. (B) Top panel: Representative bright field images of PDXOs cultured in their optimal growth medium. Bottom panel: histological morphology of equivalent PDXOs, assessed by hematoxylin and eosin staining. Scale bars equal 50 µm. (C) Swimmer’s plot visualizing the number of days PDXOs could be viably maintained in each of the three distinct organoid culture media: PCOM (blue), APCOM (yellow) and PGM (orange). Organoid passages are marked with a red dot. Arrows indicate vital organoid culture beyond 180 days of in vitro propagation. (D) PDXO growth curves during long-term culturing, plotted as the cumulative number of wells over time and visualized up until six months after culture initiation. Each red dot represents a passage. (E) t-SNE plot based on gene expression profiles of original patient samples (n = 4), derived PDXs (n = 34) and PDXOs (n = 39), as quantified by RNA sequencing.

Figure 4

AR signaling status of CRPC PDXs and PDXOs. (A) B-allele frequency (BAF) and log R ratio (LRR) for the region of interest on chromosome Xq containing the AR gene and upstream AR enhancer, in PDXs PC2400 and PC2412. Dashed box on the chromosome X ideogram indicates the genomic region displayed above. (B) Nuclear AR staining in CRPC PDXs (left panel, scale bars 100 µm) and matching PDXOs (right panel, scale bars 50 µm). (C) Landscape plot of AR activity and NE signature scores in PDX (n = 34) and patient (n = 4) tumors. Thresholds separating samples with low versus high scores are marked with dashed lines. (D) Gene expression heatmap of a curated set of AR and NE marker genes shown across the entire dataset comprising original patient tumors (blue, n = 4), PDXs (white, n = 34) and PDXOs (yellow, n = 39) with different culture medium conditions (green: APCOM; red: PCOM; purple: PGM; white: not applicable (NA)). Expression levels are visualized as vst counts, based on RNA sequencing data. (E) Tumor growth responses after surgical castration (Cx) and testosterone supplementation (+T) in CRPC PDXs. When tumors passed a volume of 300 mm³ (day 0), mice underwent either Cx (blue and green; PC2400 n = 10, PC2412 n = 12, PC2416 n = 11, PC2416-DEC n = 10) or sham Cx (yellow; n = 6 for each PDX). Thirty days after Cx, mice were implanted with a testosterone pellet (blue; PC2400 n = 5, PC2412 n = 6, PC2416 n = 5, PC2416-DEC n = 5) or placebo pellet (green; PC2400 n = 5, PC2412 n = 6, PC2416 n = 6, PC2416-DEC n = 5). (F) Dose-response curves of PC2412 and PC2416-DEC PDXOs exposed to enzalutamide treatment. Dots mark mean and error bars SEM of three and four individual experiments (six technical replicates per condition) for PC2412 and PC2416-DEC PDXOs, respectively. Cell viability was normalized to vehicle (DMSO) controls.

Figure 5

Responses of PDXs and PDXOs to taxane treatment. (A) Kaplan Meier plots depicting disease-specific survival of the tumor-bearing mice treated with placebo, docetaxel or cabazitaxel for PDXs PC2400 (placebo n = 6, docetaxel n = 7, cabazitaxel n = 8), PC2412 (placebo n = 7, docetaxel n = 8, cabazitaxel n = 7), PC2416 (placebo n = 8, docetaxel n = 7, cabazitaxel n = 7), and PC2416-DEC (placebo n = 7, docetaxel n = 8, cabazitaxel n = 8). Disease-specific survival was determined from the start of treatment (day 0) until tumor volumes exceeded 1500 mm³. Censored mice did not develop tumors of >1500 mm³ during a maximum follow-up of 90 days or died from other causes. Tables display restricted mean survival benefit of docetaxel/cabazitaxel over placebo. (B) Dose-response curves of PC2412 PDXOs (upper panel) and PC2416-DEC PDXOs (lower panel) exposed to cabazitaxel (blue) or docetaxel (green). Dots indicate mean and error bars SEM of three and four individual experiments, respectively (six technical replicates per condition). Cell viability was normalized to vehicle (ethanol) controls. Half-maximal inhibitory concentration (IC50) was calculated by sigmoidal-curve fitting.