Viability Analysis and High-Content Live-Cell Imaging for Drug Testing in Prostate Cancer Xenograft-Derived Organoids.

Tumor organoids have been pushed forward as advanced model systems for in vitro oncology drug testing, with the eventual goal to direct personalized cancer treatments. However, drug testing efforts suffer from a large variation in experimental conditions for organoid culturing and organoid treatment. Moreover, most drug tests are restricted to whole-well viability as the sole read-out, thereby losing important information about key biological aspects that might be impacted due to the use of administered drugs. These bulk read-outs also discard potential inter-organoid heterogeneity in drug responses. To tackle these issues, we developed a systematic approach for processing organoids from prostate cancer (PCa) patient-derived xenografts (PDXs) for viability-based drug testing and identified essential conditions and quality checks for consistent results. In addition, we generated an imaging-based drug testing procedure using high-content fluorescence microscopy in living PCa organoids to detect various modalities of cell death. Individual organoids and cell nuclei in organoids were segmented and quantified using a dye combination of Hoechst 33342, propidium iodide and Caspase 3/7 Green, allowing the identification of cytostatic and cytotoxic treatment effects. Our procedures provide important insights into the mechanistic actions of tested drugs. Moreover, these methods can be adapted for tumor organoids originating from other cancer types to increase organoid-based drug test validity, and ultimately, accelerate clinical implementation.

Darolutamide added to docetaxel augments antitumor effect in models of prostate cancer through cell cycle arrest at the G1-S transition.

Resistance to taxane chemotherapy is frequently observed in metastatic prostate cancer. The androgen receptor (AR) is a major driver of prostate cancer and a key regulator of the G1-S cell cycle checkpoint, promoting cancer cell proliferation by irreversible passage to the S-phase. We hypothesized that AR signaling inhibitor (ARSi) darolutamide in combination with docetaxel could augment antitumor effect by impeding the proliferation of taxane-resistant cancer cells. We monitored cell viability in organoids, tumor volume and PSA secretion in patient-derived xenografts (PDXs) and analyzed cell cycle and signaling pathway alterations. Combination treatment increased anti-tumor effect in androgen-sensitive, AR-positive prostate cancer organoids and PDXs. Equally beneficial effects of darolutamide added to docetaxel were observed in a castration-resistant model, progressive on docetaxel, enzalutamide and cabazitaxel. In vitro studies showed that docetaxel treatment with simultaneous darolutamide resulted in a reduction of cells entering the S-phase in contrast to only docetaxel. Molecular analysis in the prostate cancer cell line LNCaP revealed an upregulation of Cyclin Dependent Kinase inhibitor p21, supporting blockade of S-phase entry and cell proliferation. Our results provide a preclinical support for combining taxanes and darolutamide as a multimodal treatment strategy in metastatic prostate cancer patients progressive on ARSi and taxane chemotherapy.

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

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.

Modeling Prostate Cancer Treatment Responses in the Organoid Era: 3D Environment Impacts Drug Testing.

Organoid-based studies have revolutionized in vitro preclinical research and hold great promise for the cancer research field, including prostate cancer (PCa). However, experimental variability in organoid drug testing complicates reproducibility. For example, we observed PCa organoids to be less affected by cabazitaxel, abiraterone and enzalutamide as compared to corresponding single cells prior to organoid assembly. We hypothesized that three-dimensional (3D) organoid organization and the use of various 3D scaffolds impact treatment efficacy. Live-cell imaging of androgen-induced androgen receptor (AR) nuclear translocation and taxane-induced tubulin stabilization was used to investigate the impact of 3D scaffolds, spatial organoid distribution and organoid size on treatment effect. Scaffolds delayed AR translocation and tubulin stabilization, with Matrigel causing a more pronounced delay than synthetic hydrogel as well as incomplete tubulin stabilization. Drug effect was further attenuated the more centrally organoids were located in the scaffold dome. Moreover, cells in the organoid core revealed a delayed treatment effect compared to cells in the organoid periphery, underscoring the impact of organoid size. These findings indicate that analysis of organoid drug responses needs careful interpretation and requires dedicated read-outs with consideration of underlying technical aspects.

The Outcome for Patients With Pathologic Node-Positive Prostate Cancer Treated With Intensity Modulated Radiation Therapy and Androgen Deprivation Therapy: A Case-Matched Analysis of pN1 and pN0 Patients.

PURPOSE: Improved outcome is reported after surgery or external beam radiation therapy (EBRT) plus androgen deprivation therapy (ADT) for patients with lymph node (LN) positive (N1) prostate cancer (PC). Surgical series have shown that pathologic (p)N1 PC does not behave the same in all patients. The aim of this study was to perform a matched-case analysis to compare the outcome of pN1 and pN0 PC after high-dose EBRT plus ADT. METHODS AND MATERIALS: Radiation therapy up to 80 Gy was delivered to the prostate with a minimal dose of 45 Gy to the pelvis for pN1 patients. After matching, Kaplan-Meier statistics were used to compare the 5-year biochemical and clinical relapse-free survival (bRFS and cRFS), prostate cancer-specific survival (PCSS), and overall survival (OS). Acute and late rectal and urinary toxicity was evaluated. RESULTS: Sixty-nine pN1 PC patients were matched 1:1 with pN0 PC patients. The median follow-up time was 60 months. The 5-year bRFS and cRFS for pN1 versus pN0 PC patients were 65% ± 7% versus 79% ± 5% (P=.08) and 70% ± 6% versus 83% ± 5% (P=.04) respectively. No significant difference was found in bRFS or cRFS rates between low volume pN1 (≤2 positive LNs) and pN0 patients. The 5-year PCSS and OS were comparable between pN1 and pN0 PC patients: PCSS: 92% ± 4% versus 93% ± 3% (P=.66); OS: 82% ± 5% versus 80% ± 5% (P=.58). Severe toxicity was rare for both groups, although pN1 patients experienced significantly more acute grade 2 rectal toxicity. CONCLUSION: Primary EBRT plus 2 to 3 years of ADT is a legitimate treatment option for pN1 PC patients, especially those with ≤2 positive LNs, and this with bRFS and cRFS rates comparable to those in pN0 PC patients. For pN1 PC patients with >2 positive LNs, bRFS and cRFS are worse than in pN0 patients, but even in this subgroup, long-term disease control is obtained.