Defining the challenges and opportunities for using patient-derived models in prostate cancer research.

BACKGROUND: There are relatively few widely used models of prostate cancer compared to other common malignancies. This impedes translational prostate cancer research because the range of models does not reflect the diversity of disease seen in clinical practice. In response to this challenge, research laboratories around the world have been developing new patient-derived models of prostate cancer, including xenografts, organoids, and tumor explants. METHODS: In May 2023, we held a workshop at the Monash University Prato Campus for researchers with expertise in establishing and using a variety of patient-derived models of prostate cancer. This review summarizes our collective ideas on how patient-derived models are currently being used, the common challenges, and future opportunities for maximizing their usefulness in prostate cancer research. RESULTS: An increasing number of patient-derived models for prostate cancer are being developed. Despite their individual limitations and varying success rates, these models are valuable resources for exploring new concepts in prostate cancer biology and for preclinical testing of potential treatments. Here we focus on the need for larger collections of models that represent the changing treatment landscape of prostate cancer, robust readouts for preclinical testing, improved in vitro culture conditions, and integration of the tumor microenvironment. Additional priorities include ensuring model reproducibility, standardization, and replication, and streamlining the exchange of models and data sets among research groups. CONCLUSIONS: There are several opportunities to maximize the impact of patient-derived models on prostate cancer research. We must develop large, diverse and accessible cohorts of models and more sophisticated methods for emulating the intricacy of patient tumors. In this way, we can use the samples that are generously donated by patients to advance the outcomes of patients in the future.

The future of patient-derived xenografts in prostate cancer research.

Patient-derived xenografts (PDXs) are generated by engrafting human tumours into mice. Serially transplantable PDXs are used to study tumour biology and test therapeutics, linking the laboratory to the clinic. Although few prostate cancer PDXs are available in large repositories, over 330 prostate cancer PDXs have been established, spanning broad clinical stages, genotypes and phenotypes. Nevertheless, more PDXs are needed to reflect patient diversity, and to study new treatments and emerging mechanisms of resistance. We can maximize the use of PDXs by exchanging models and datasets, and by depositing PDXs into biorepositories, but we must address the impediments to accessing PDXs, such as institutional, ethical and legal agreements. Through collaboration, researchers will gain greater access to PDXs representing diverse features of prostate cancer.

Potent Stimulation of the Androgen Receptor Instigates a Viral Mimicry Response in Prostate Cancer.

Inhibiting the androgen receptor (AR), a ligand-activated transcription factor, with androgen deprivation therapy is a standard-of-care treatment for metastatic prostate cancer. Paradoxically, activation of AR can also inhibit the growth of prostate cancer in some patients and experimental systems, but the mechanisms underlying this phenomenon are poorly understood. This study exploited a potent synthetic androgen, methyltestosterone (MeT), to investigate AR agonist-induced growth inhibition. MeT strongly inhibited growth of prostate cancer cells expressing AR, but not AR-negative models. Genes and pathways regulated by MeT were highly analogous to those regulated by DHT, although MeT induced a quantitatively greater androgenic response in prostate cancer cells. MeT potently downregulated DNA methyltransferases, leading to global DNA hypomethylation. These epigenomic changes were associated with dysregulation of transposable element expression, including upregulation of endogenous retrovirus (ERV) transcripts after sustained MeT treatment. Increased ERV expression led to accumulation of double-stranded RNA and a "viral mimicry" response characterized by activation of IFN signaling, upregulation of MHC class I molecules, and enhanced recognition of murine prostate cancer cells by CD8+ T cells. Positive associations between AR activity and ERVs/antiviral pathways were evident in patient transcriptomic data, supporting the clinical relevance of our findings. Collectively, our study reveals that the potent androgen MeT can increase the immunogenicity of prostate cancer cells via a viral mimicry response, a finding that has potential implications for the development of strategies to sensitize this cancer type to immunotherapies. Significance: Our study demonstrates that potent androgen stimulation of prostate cancer cells can elicit a viral mimicry response, resulting in enhanced IFN signaling. This finding may have implications for the development of strategies to sensitize prostate cancer to immunotherapies.

The MURAL collection of prostate cancer patient-derived xenografts enables discovery through preclinical models of uro-oncology.

Preclinical testing is a crucial step in evaluating cancer therapeutics. We aimed to establish a significant resource of patient-derived xenografts (PDXs) of prostate cancer for rapid and systematic evaluation of candidate therapies. The PDX collection comprises 59 tumors collected from 30 patients between 2012-2020, coinciding with availability of abiraterone and enzalutamide. The PDXs represent the clinico-pathological and genomic spectrum of prostate cancer, from treatment-naïve primary tumors to castration-resistant metastases. Inter- and intra-tumor heterogeneity in adenocarcinoma and neuroendocrine phenotypes is evident from bulk and single-cell RNA sequencing data. Organoids can be cultured from PDXs, providing further capabilities for preclinical studies. Using a 1 x 1 x 1 design, we rapidly identify tumors with exceptional responses to combination treatments. To govern the distribution of PDXs, we formed the Melbourne Urological Research Alliance (MURAL). This PDX collection is a substantial resource, expanding the capacity to test and prioritize effective treatments for prospective clinical trials in prostate cancer.

Androgen receptor enhancer amplification in matched patient-derived xenografts of primary and castrate-resistant prostate cancer.

Amplifications of the androgen receptor (AR) occur in up to 80% of men with castration-resistant prostate cancer (CRPC). Recent studies highlighted that these amplifications not only span the AR gene but usually encompass a distal enhancer. This represents a newly recognised, non-coding mechanism of resistance to AR-directed therapies, including enzalutamide. To study disease progression before and after AR amplification, we used tumour samples from a castrate-sensitive primary tumour and castrate-resistant metastasis of the same patient. For subsequent functional and genomic studies, we established serially transplantable patient-derived xenografts (PDXs). Whole genome sequencing showed that alterations associated with poor prognosis, such as TP53 and PTEN loss, existed before androgen deprivation therapy, followed by co-amplification of the AR gene and enhancer after the development of metastatic CRPC. The PDX of the primary tumour, without the AR amplification, was sensitive to AR-directed treatments, including castration, enzalutamide, and apalutamide. The PDX of the metastasis, with the AR amplification, had higher AR and AR-V7 expression in castrate conditions, and was resistant to castration, apalutamide, and enzalutamide in vivo. Treatment with a BET inhibitor outperformed the AR-directed therapies for the metastasis, resulting in tumour regression for some, but not all, grafts. Therefore, this study provides novel matched PDXs to test potential treatments that target the overabundance of AR in tumours with AR enhancer amplifications. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Early intervention exercise training does not delay prostate cancer progression in Pten-/- mice.

BACKGROUND: There is convincing evidence that men with advanced prostate cancer experience improved quality of life as a result of exercise therapy, although there is limited preclinical, and no clinical, data to directly support the notion that exercise training improves prostate cancer prognosis or outcome. The aim of this study was to investigate the effect of regular exercise training on the early stages of prostate cancer progression, as well as assessing whether alterations to prostate cancer metabolism are induced by exercise. METHODS: Mice with prostate-specific deletion of Pten (Pten-/- ) remained sedentary or underwent 6 weeks of endurance exercise training or high-intensity exercise training involving treadmill running. At the conclusion of the training period, the prostate lobes were excised. A portion of fresh tissue was used to assess glucose, glutamine, and fatty acid metabolism by radiometric techniques and a second portion was fixed for histopathology. RESULTS: Despite the implementation of an effective exercise regime, as confirmed by improvements in running capacity, neither prostate mass, cell proliferation or the incidence of high-grade prostate intraepithelial hyperplasia or noninvasive carcinoma in situ were significantly different between groups. Similarly, neither glucose uptake, oxidation and de novo lipogenesis, glutamine oxidation, or fatty acid uptake, oxidation and storage into various lipids were significantly different in prostate tissue obtained from untrained and exercise trained mice. CONCLUSIONS: These results show that 6 weeks of moderate or high-intensity exercise training does not alter substrate metabolism in the prostate or slow the progression of Pten-null prostate cancer. These results question whether exercise is a useful therapy to prevent or delay prostate cancer progression.

Establishing a cryopreservation protocol for patient-derived xenografts of prostate cancer.

BACKGROUND: Serially transplantable patient-derived xenografts (PDXs) are invaluable preclinical models for studying tumor biology and evaluating therapeutic agents. As these models are challenging to establish from prostate cancer specimens, the ability to preserve them through cryopreservation has several advantages for ongoing research. Despite this, there is still uncertainty about the ability to cryopreserve PDXs of prostate cancer. This study compared three different cryopreservation protocols to identify a method that can be used to reproducibly cryopreserve a diverse cohort of prostate cancer PDX models. METHODS: One serially transplantable prostate cancer PDX from the Melbourne Urological Research Alliance cohort was used to compare three cryopreservation protocols: slow freezing in fetal calf serum (FCS) with 10% dimethyl sulfoxide (DMSO), FCS with 10% DMSO supplemented with the Rho-associated kinase (ROCK) inhibitor Y-27632 and vitrification. The efficiency of the slow freezing protocols was then assessed in 17 additional prostate cancer PDXs. Following cryopreservation, PDXs were re-established in host mice that were either intact and supplemented with testosterone or castrated. Graft take rate, tumor growth, histological features, and transcriptome profiles before and after cryopreservation were compared. RESULTS: Slow freezing maintained the viability and histological features of prostate cancer PDXs, and the addition of a ROCK inhibitor increased their growth following cryopreservation. Using the slow freezing method, we re-established 100% of PDXs grown in either testosterone-supplemented or castrated host mice. Importantly, the long-term tumor growth rate and transcriptome profile were maintained following cryopreservation. CONCLUSION: This study has identified a protocol to reliably cryopreserve and re-establish a diverse cohort of serially transplantable PDXs of prostate cancer. This study has the potential to significantly improve the practicality of maintaining PDX models. Cryopreservation may also increase the accessibility of these important resources and provide new opportunities for preclinical studies on a broader spectrum of prostate tumors.

Identification of Metabolically Distinct Adipocyte Progenitor Cells in Human Adipose Tissues.

Adipocyte progenitor cells (APCs) provide the reservoir of regenerative cells to produce new adipocytes, although their identity in humans remains elusive. Using FACS analysis, gene expression profiling, and metabolic and proteomic analyses, we identified three APC subtypes in human white adipose tissues. The APC subtypes are molecularly distinct but possess similar proliferative and adipogenic capacities. Adipocytes derived from APCs with high CD34 expression exhibit exceedingly high rates of lipid flux compared with APCs with low or no CD34 expression, while adipocytes produced from CD34- APCs display beige-like adipocyte properties and a unique endocrine profile. APCs were more abundant in gluteofemoral compared with abdominal subcutaneous and omental adipose tissues, and the distribution of APC subtypes varies between depots and in patients with type 2 diabetes. These findings provide a mechanistic explanation for the heterogeneity of human white adipose tissue and a potential basis for dysregulated adipocyte function in type 2 diabetes.

Suppressing fatty acid uptake has therapeutic effects in preclinical models of prostate cancer.

Metabolism alterations are hallmarks of cancer, but the involvement of lipid metabolism in disease progression is unclear. We investigated the role of lipid metabolism in prostate cancer using tissue from patients with prostate cancer and patient-derived xenograft mouse models. We showed that fatty acid uptake was increased in human prostate cancer and that these fatty acids were directed toward biomass production. These changes were mediated, at least partly, by the fatty acid transporter CD36, which was associated with aggressive disease. Deleting Cd36 in the prostate of cancer-susceptible Pten-/- mice reduced fatty acid uptake and the abundance of oncogenic signaling lipids and slowed cancer progression. Moreover, CD36 antibody therapy reduced cancer severity in patient-derived xenografts. We further demonstrated cross-talk between fatty acid uptake and de novo lipogenesis and found that dual targeting of these pathways more potently inhibited proliferation of human cancer-derived organoids compared to the single treatments. These findings identify a critical role for CD36-mediated fatty acid uptake in prostate cancer and suggest that targeting fatty acid uptake might be an effective strategy for treating prostate cancer.

Patient-derived Models of Abiraterone- and Enzalutamide-resistant Prostate Cancer Reveal Sensitivity to Ribosome-directed Therapy.

BACKGROUND: The intractability of castration-resistant prostate cancer (CRPC) is exacerbated by tumour heterogeneity, including diverse alterations to the androgen receptor (AR) axis and AR-independent phenotypes. The availability of additional models encompassing this heterogeneity would facilitate the identification of more effective therapies for CRPC. OBJECTIVE: To discover therapeutic strategies by exploiting patient-derived models that exemplify the heterogeneity of CRPC. DESIGN, SETTING, AND PARTICIPANTS: Four new patient-derived xenografts (PDXs) were established from independent metastases of two patients and characterised using integrative genomics. A panel of rationally selected drugs was tested using an innovative ex vivo PDX culture system. INTERVENTION: The following drugs were evaluated: AR signalling inhibitors (enzalutamide and galeterone), a PARP inhibitor (talazoparib), a chemotherapeutic (cisplatin), a CDK4/6 inhibitor (ribociclib), bromodomain and extraterminal (BET) protein inhibitors (iBET151 and JQ1), and inhibitors of ribosome biogenesis/function (RNA polymerase I inhibitor CX-5461 and pan-PIM kinase inhibitor CX-6258). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Drug efficacy in ex vivo cultures of PDX tissues was evaluated using immunohistochemistry for Ki67 and cleaved caspase-3 levels. Candidate drugs were also tested for antitumour efficacy in vivo, with tumour volume being the primary endpoint. Two-tailed t tests were used to compare drug and control treatments. RESULTS AND LIMITATIONS: Integrative genomics revealed that the new PDXs exhibited heterogeneous mechanisms of resistance, including known and novel AR mutations, genomic structural rearrangements of the AR gene, and a neuroendocrine-like AR-null phenotype. Despite their heterogeneity, all models were sensitive to the combination of ribosome-targeting agents CX-5461 and CX-6258. CONCLUSIONS: This study demonstrates that ribosome-targeting drugs may be effective against diverse CRPC subtypes including AR-null disease, and highlights the potential of contemporary patient-derived models to prioritise treatment strategies for clinical translation. PATIENT SUMMARY: Diverse types of therapy-resistant prostate cancers are sensitive to a new combination of drugs that inhibit protein synthesis pathways in cancer cells.

A rare castration-resistant progenitor cell population is highly enriched in Pten-null prostate tumours.

Castration-resistant prostate cancer is a lethal disease. The cell type(s) that survive androgen deprivation remain poorly described, despite global efforts to understand the various mechanisms of therapy resistance. We recently identified in wild-type (WT) mouse prostates a rare population of luminal progenitor cells that we called LSCmed according to their FACS profile (Lin- /Sca-1+ /CD49fmed ). Here, we investigated the prevalence and castration resistance of LSCmed in various mouse models of prostate tumourigenesis (Pb-PRL, Ptenpc-/- , and Hi-Myc mice). LSCmed prevalence is low (∼8%, similar to WT) in Hi-Myc mice, where prostatic androgen receptor signalling is unaltered, but is significantly higher in the two other models, where androgen receptor signalling is decreased, rising up to more than 80% in Ptenpc-/- prostates. LSCmed tolerate androgen deprivation and persist or are enriched 2-3 weeks after castration. The tumour-initiating properties of LSCmed from Ptenpc-/- mice were demonstrated by regeneration of tumours in vivo. Transcriptomic analysis revealed that LSCmed represent a unique cell entity as their gene expression profile is different from luminal and basal/stem cells, but shares markers of each. Their intrinsic androgen signalling is markedly decreased, explaining why LSCmed tolerate androgen deprivation. This also illuminates why Ptenpc-/- tumours are castration-resistant since LSCmed represent the most prevalent cell type in this model. We validated CK4 as a specific marker for LSCmed on sorted cells and prostate tissues by immunostaining, allowing for the detection of LSCmed in various mouse prostate specimens. In castrated Ptenpc-/- prostates, there was significant proliferation of CK4+ cells, further demonstrating their key role in castration-resistant prostate cancer progression. Taken together, this study identifies LSCmed as a probable source of prostate cancer relapse after androgen deprivation and as a new therapeutic target for the prevention of castrate-resistant prostate cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Dual Inhibition of RNA Pol I Transcription and PIM Kinase as a New Therapeutic Approach to Treat Advanced Prostate Cancer.

PURPOSE: The MYC oncogene is frequently overexpressed in prostate cancer. Upregulation of ribosome biogenesis and function is characteristic of MYC-driven tumors. In addition, PIM kinases activate MYC signaling and mRNA translation in prostate cancer and cooperate with MYC to accelerate tumorigenesis. Here, we investigate the efficacy of a single and dual approach targeting ribosome biogenesis and function to treat prostate cancer. EXPERIMENTAL DESIGN: The inhibition of ribosomal RNA (rRNA) synthesis with CX-5461, a potent, selective, and orally bioavailable inhibitor of RNA polymerase I (Pol I) transcription, has been successfully exploited therapeutically but only in models of hematologic malignancy. CX-5461 and CX-6258, a pan-PIM kinase inhibitor, were tested alone and in combination in prostate cancer cell lines, in Hi-MYC- and PTEN-deficient mouse models and in patient-derived xenografts (PDX) of metastatic tissue obtained from a patient with castration-resistant prostate cancer. RESULTS: CX-5461 inhibited anchorage-independent growth and induced cell-cycle arrest in prostate cancer cell lines at nanomolar concentrations. Oral administration of 50 mg/kg CX-5461 induced TP53 expression and activity and reduced proliferation (MKI67) and invasion (loss of ductal actin) in Hi-MYC tumors, but not in PTEN-null (low MYC) tumors. While 100 mg/kg CX-6258 showed limited effect alone, its combination with CX-5461 further suppressed proliferation and dramatically reduced large invasive lesions in both models. This rational combination strategy significantly inhibited proliferation and induced cell death in PDX of prostate cancer. CONCLUSIONS: Our results demonstrate preclinical efficacy of targeting the ribosome at multiple levels and provide a new approach for the treatment of prostate cancer. Clin Cancer Res; 22(22); 5539-52. ©2016 AACR.

Obesity does not promote tumorigenesis of localized patient-derived prostate cancer xenografts.

There are established epidemiological links between obesity and the severity of prostate cancer. We directly tested this relationship by assessing tumorigenicity of patient-derived xenografts (PDXs) of moderate-grade localized prostate cancer in lean and obese severe combined immunodeficiency (SCID) mice. Mice were rendered obese and insulin resistant by high-fat feeding for 6 weeks prior to transplantation, and PDXs were assessed 10 weeks thereafter. Histological analysis of PDX grafts showed no differences in tumor pathology, prostate-specific antigen, androgen receptor and homeobox protein Nkx-3.1 expression, or proliferation index in lean versus obese mice. Whilst systemic obesity per se did not promote prostate tumorigenicity, we next asked whether the peri-prostatic adipose tissue (PPAT), which covers the prostate anteriorly, plays a role in prostate tumorigenesis. In vitro studies in a cellularized co-culture model of stromal and epithelial cells demonstrated that factors secreted from human PPAT are pro-tumorigenic. Accordingly, we recapitulated the prostate-PPAT spatial relationship by co-grafting human PPAT with prostate cancer in PDX grafts. PDX tissues were harvested 10 weeks after grafting, and histological analysis revealed no evidence of enhanced tumorigenesis with PPAT compared to prostate cancer grafts alone. Altogether, these data demonstrate that prostate cancer tumorigenicity is not accelerated in the setting of diet-induced obesity or in the presence of human PPAT, prompting the need for further work to define the at-risk populations of obesity-driven tumorigenesis and the biological factors linking obesity, adipose tissue and prostate cancer pathogenesis.

Long term sertraline effects on neural structures in depressed and nondepressed adult female nonhuman primates.

BACKGROUND: Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed for mood and other disorders. However, their neural effects are difficult to study due to patient compliance and drug history variability, and rarely studied in those prescribed SSRIs for non-mood disorders. Here we evaluated SSRI effects on neural volumetrics in depressed and nondepressed monkeys. METHODS: 42 socially-housed cynomolgus monkeys were randomized to treatment balanced on pretreatment depressive behavior and body weight. Monkeys were trained for oral administration of placebo or 20 mg/kg sertraline HCl daily for 18 months and depressive and anxious behavior recorded. Volumes of neural regions of interest in depression were measured in magnetic resonance images and analyzed by 2 (depressed, nondepressed)×2 (placebo, sertraline) ANOVA. RESULTS: Sertraline reduced anxiety (p=0.04) but not depressive behavior (p=0.43). Left Brodmann's Area (BA) 32 was smaller in depressed than nondepressed monkeys (main effect of depression: p<0.05). Sertraline and depression status interacted to affect volumes of left Anterior Cingulate Cortex (ACC), left BA24, right hippocampus (HC), and right anterior HC (sertraline×depression interactions: all p's<0.05). In the Placebo group, depressed monkeys had smaller right anterior HC and left ACC than nondepressed monkeys. In nondepressed monkeys, sertraline reduced right HC volume, especially right anterior HC volume. In depressed monkeys sertraline increased left ACC volume. In nondepressed monkeys, sertraline reduced left BA24 volumes resulting in smaller BA24 volumes in nondepressed than sertraline-treated depressed monkeys. CONCLUSIONS: These observations suggest that SSRIs may differentially affect neural structures in depressed and nondepressed individuals.

A bioengineered microenvironment to quantitatively measure the tumorigenic properties of cancer-associated fibroblasts in human prostate cancer.

Stromal-epithelial cell interactions play an important role in cancer and the tumor stroma is regarded as a therapeutic target. In vivo xenografting is commonly used to study cellular interactions not mimicked or quantified in conventional 2D culture systems. To interrogate the effects of tumor stroma (cancer-associated fibroblasts or CAFs) on epithelia, we created a bioengineered microenvironment using human prostatic tissues. Patient-matched CAFs and non-malignant prostatic fibroblasts (NPFs) from men with moderate (Gleason 7) and aggressive (Gleason 8-9 or castrate-resistant) prostate cancer were cultured with non-tumorigenic BPH-1 epithelial cells. Changes in the morphology, motility and phenotype of BPH-1 cells in response to CAFs and NPFs were analyzed using immunofluorescence and quantitative cell morphometric analyses. The matrix protein gene expression of CAFs, with proven tumorigenicity in vivo, had a significantly different gene expression profile of matrix proteins compared to patient matched NPFs. In co-culture with CAFs (but not NPFs), BPH-1 cells had a more invasive, elongated phenotype with increased motility and a more directed pattern of cell migration. CAFs from more aggressive tumors (Gleason 8-9 or CRPC) were not quantitatively different to moderate grade CAFs. Overall, our bioengineered microenvironment provides a novel 3D in vitro platform to systematically investigate the effects of tumor stroma on prostate cancer progression.