Mutation Hotspots Found in Bladder Cancer Aid Prediction of Carcinogenic Risk in Normal Urothelium.

More than 80,000 new cases of bladder cancer are estimated to be diagnosed in 2023. However, the 5-year survival rate for bladder cancer has not changed in decades, highlighting the need for prevention. Numerous cancer-causing mutations are present in the urothelium long before signs of cancer arise. Mutation hotspots in cancer-driving genes were identified in non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) tumor samples. Mutation burden within the hotspot regions was measured in normal urothelium with a low and high risk of cancer. A significant correlation was found between the mutation burden in normal urothelium and bladder cancer tissue within the hotspot regions. A combination of measured hotspot burden and personal risk factors was used to fit machine learning classification models. The efficacy of each model to differentiate between adjacent benign urothelium from bladder cancer patients and normal urothelium from healthy donors was measured. A random forest model using a combination of personal risk factors and mutations within MIBC hotspots yielded the highest AUC of 0.9286 for the prediction of high- vs. low-risk normal urothelium. Currently, there are no effective biomarkers to assess subclinical field disease and early carcinogenic progression in the bladder. Our findings demonstrate novel differences in mutation hotspots in NMIBC and MIBC and provide the first evidence for mutation hotspots to aid in the assessment of cancer risk in the normal urothelium. Early risk assessment and identification of patients at high risk of bladder cancer before the clinical presentation of the disease can pave the way for targeted personalized preventative therapy.

Comparison of SureSelect and Nextera Exome Capture Performance in Single-Cell Sequencing.

BACKGROUND: Advances in single-cell sequencing provide unprecedented opportunities for clinical examination of circulating tumor cells, cancer stem cells, and other rare cells responsible for disease progression and drug resistance. On the genomic level, single-cell whole exome sequencing (scWES) started to gain popularity with its unique potentials in characterizing mutational landscapes at a single-cell level. Currently, there is little known about the performance of different exome capture kits in scWES. Nextera rapid capture (NXT; Illumina, Inc.) has been the only exome capture kit recommended for scWES by Fluidigm C1, a widely accessed system in single-cell preparation. RESULTS: In this study, we compared the performance of NXT following Fluidigm's protocol with Agilent SureSelectXT Target Enrichment System (AGL), another exome capture kit widely used for bulk sequencing. We created DNA libraries of 192 single cells isolated from spheres grown from a melanoma specimen using Fluidigm C1. Twelve high-yield cells were selected to perform dual-exome capture and sequencing using AGL and NXT in parallel. After mapping and coverage analysis, AGL outperformed NXT in coverage uniformity, mapping rates of reads, exome capture rates, and low PCR duplicate rates. For germline variant calling, AGL achieved better performance in overlap with known variants in dbSNP and transition-transversion ratios. Using calls from high coverage bulk sequencing from blood DNA as the golden standard, AGL-based scWES demonstrated high positive predictive values, and medium to high sensitivity. Lastly, we evaluated somatic mutation calling by comparing single-cell data with the matched blood sequence as control. On average, 300 mutations were identified in each cell. In 10 of 12 cells, higher numbers of mutations were identified using AGL than NXT, probably caused by coverage depth. When mutations are adequately covered in both AGL and NXT data, the two methods showed very high concordance (93-100% per cell). CONCLUSIONS: Our results suggest that AGL can also be used for scWES when there is sufficient DNA, and it yields better data quality than the current Fluidigm's protocol using NXT.

Current Update of Patient-Derived Xenograft Model for Translational Breast Cancer Research.

Despite recent advances in the treatment of patients with breast cancer (BrCa), BrCa remains the third leading cause of cancer death for women in the US due to intrinsic or acquired resistance to therapy. Continued understanding of gene expression profiling and genomic sequencing has clarified underlying intratumoral molecular heterogeneity. Recently, the patient-derived xenograft (PDX) models have emerged as a novel tool to address the issues of BrCa genomics and tumor heterogeneity, and to critically transform translational BrCa research in the preclinical setting. PDX models are generated by xenografting cancer tissue fragments obtained from patients to immune deficient mice, and can be passaged into next generations of mice. Generally, in contrast to conventional xenograft using cancer cell lines, PDXs are biologically more stable and recapitulate the individual tumor morphology, gene expression, and drug susceptibility of each patient. PDX may better model the original patient's tumor by retaining tumor heterogeneity, gene expression, and similar response to treatment. PDX models are thus thought to be more translationally relevant, especially as a drug development tool, because PDXs can capture the genetic character and heterogeneity that exists within a single patient's tumor and across a population of patients' tumors. PDX models also hold enormous potential for identifying predictive markers for therapeutic response. It has been repeatedly shown that PDX models demonstrate similar levels of activity as compared to the clinical response to therapeutic interventions. Therefore, this enables identification of therapeutic interventions that can most likely benefit a patient. This allows us to address the issues of BrCa genomics and tumor heterogeneity using PDXs in "pre-clinical" trials. Herein, we reviewed recent scientific development and future perspectives using PDX models in BrCa.

Metastatic phenotype in CWR22 prostate cancer xenograft following castration.

BACKGROUND: CWR22 is a human xenograft model of primary prostate cancer (PCa) that is often utilized to study castration recurrent (CR) PCa. CWR22 recapitulates clinical response to androgen deprivation therapy (ADT), in that tumors regress in response to castration, but can recur after a period of time. METHODS: Two cohorts of mice, totaling 117 mice were implanted with CWR22, allowed to develop tumors, castrated by pellet removal and followed for a period of 32 and 50 weeks. Mice presenting with tumors >2.0 cm(3) at the primary site, moribund appearance, or palpable masses other than the primary tumor were sacrificed prior to the endpoint of the study. Tumor tissue, serum, and abnormal lesions were collected upon necropsy and analyzed by IHC, H&E, and PCR for presence of metastatic lesions arising from CWR22. RESULTS: Herein, we report that CWR22 progresses after castration from a primary, hormonal therapy-naïve tumor to metastatic disease in 20% of castrated nude mice. Histological examination of CWR22 primary tumors revealed distinct pathologies that correlated with metastatic outcome after castration. CONCLUSION: This is the first report and characterization of spontaneous metastasis in the CWR22 model, thus, CWR22 is a bona-fide model of clinical PCa representing the full progression from androgen-sensitive, primary PCa to metastatic CR-PCa.

E2f binding-deficient Rb1 protein suppresses prostate tumor progression in vivo.

Mutational inactivation of the RB1 tumor suppressor gene initiates retinoblastoma and other human cancers. RB1 protein (pRb) restrains cell proliferation by binding E2f transcription factors and repressing the expression of cell cycle target genes. It is presumed that loss of pRb/E2f interaction accounts for tumor initiation, but this has not been directly tested. RB1 mutation is a late event in other human cancers, suggesting a role in tumor progression as well as initiation. It is currently unknown whether RB1 mutation drives tumor progression and, if so, whether loss of pRb/E2f interaction is responsible. We have characterized tumorigenesis in mice expressing a mutant pRb that is specifically deficient in binding E2f. In endocrine tissue, the mutant pRb has no detectable effect on tumorigenesis. In contrast, it significantly delays progression to invasive and lethal prostate cancer. Tumor delay is associated with induction of a senescence response. We conclude that the pRb/E2f interaction is critical for preventing tumor initiation, but that pRb can use additional context-dependent mechanisms to restrain tumor progression.

Enhancing PDT efficacy in NMIBC: Efflux inhibitor mediated improvement of PpIX levels and efficacy of the combination of PpIX-PDT and SO-cleavable prodrugs.

Protoporphyrin IX (PpIX)-based photodynamic therapy (PDT) has shown limited efficacy in nonmuscle-invasive bladder cancer (NMIBC). To improve PDT efficacy, we developed singlet oxygen-cleavable prodrugs. These prodrugs, when combined with PpIX-PDT, induce cancer cell death through both PDT and drug release mechanisms. Inhibition of PpIX efflux was reported to be an effective strategy to improve PpIX-PDT in certain cancer cells. Our main goal was to investigate whether adding an efflux inhibitor to the combination of PpIX and prodrugs can improve the PpIX levels in bladder cancer cells and the release of active drugs, thus improving the overall efficacy of the treatment. We treated bladder cancer cell lines with lapatinib and evaluated intracellular PpIX fluorescence, finding significantly increased accumulation. Combining lapatinib with prodrugs led to significantly reduced cell viability compared to prodrugs or PpIX-PDT alone. The effect of lapatinib depended on the expression level of the efflux pump in bladder cancer cells. Interestingly, lapatinib increased paclitaxel (PTX) prodrug uptake by threefold compared to prodrug alone. Adding an efflux inhibitor (e.g., lapatinib) into bladder instillation solutions could be a straightforward and effective strategy for NMIBC treatment, particularly in tumors expressing efflux pumps, with the potential for clinical translation.

Mitochondrial targeting improves the selectivity of singlet-oxygen cleavable prodrugs in NMIBC treatment.

Mitochondria play an essential role in cancer treatment by providing apoptotic signals. Hexyl aminolevulinate, an FDA-approved diagnosis for non-muscle invasive bladder cancer, induces the production of protoporphyrin IX (PpIX) preferentially by mitochondria in cancer cells. Photosensitizer PpIX upon illumination can release active chemotherapy drugs from singlet oxygen-activatable prodrugs. Prodrugs placed close enough to PpIX formed in mitochondria can improve the antitumor efficiency of PpIX-PDT. The preferred uptake of prodrugs by cancer cells and tumors can further enhance the selective damage of cancer cells over non-cancer cells and surrounding normal tissues. Mitochondriotropic prodrugs of anticancer drugs, such as paclitaxel and SN-38, were synthesized using rhodamine, a mitochondrial-targeting moiety. In vitro, the mitochondrial targeting helped achieve preferential cellular uptake in cancer cells. In RT112 cells (human bladder cancer cells), intracellular prodrug concentrations were 2-3 times higher than the intracellular prodrug concentrations in BdEC cells (human bladder epithelial cells), after 2 h incubation. In an orthotopic rat bladder tumor model, mitochondria-targeted prodrugs achieved as much as 34 times higher prodrug diffusion in the tumor area compared to the nontumor bladder area. Overall, mitochondria targeting made prodrugs more effective in targeting cancer cells and tumors over non-tumor areas, thereby reducing nonspecific toxicity.

Comparison of red and green light for treating non-muscle invasive bladder cancer in rats using singlet oxygen-cleavable prodrugs with PPIX-PDT.

It has been 30 years since Photofrin-PDT was approved for the treatment of bladder cancer in Canada. However, Photofrin-PDT failed to gain popularity due to bladder complications. The PDT with red light and IV-administered Photofrin could permanently damage the bladder muscle. We have been developing a new combination strategy of PpIX-PDT with singlet oxygen-cleavable prodrugs for NMIBC with minimal side effects, avoiding damage to the bladder muscle layer. PpIX can be excited by either green (532 nm) or red (635 nm) light. Red light could be more efficacious in vivo due to its deeper tissue penetration than green light. Since HAL preferentially produces PpIX in tumors, we hypothesized that illuminating PpIX with red light might spare the muscle layer. PpIX-PDT was used to compare green and red laser efficacy in vitro and in vivo. The IC50 of in vitro PpIX-PDT was 18 mW/cm2 with the red laser and 22 mW/cm2 with the green laser. The in vivo efficacy of the red laser with 50, 75, and 100 mW total dose was similar to the same dose of green laser in reducing tumor volume. Combining PpIX-PDT with prodrugs methyl-linked mitomycin C (Mt-L-MMC) and rhodamine-linked SN-38 (Rh-L-SN-38) significantly improved efficacy (tumor volume comparison). PpIX-PDT or PpIX-PDT + prodrug combination did not cause muscle damage in histological analysis. Overall, a combination of PpIX-PDT and prodrug with 635 nm laser is promising for non-muscle invasive bladder cancer treatment.

A synergistic two-drug therapy specifically targets a DNA repair dysregulation that occurs in p53-deficient colorectal and pancreatic cancers.

The tumor-suppressor p53 is commonly inactivated in colorectal cancer and pancreatic ductal adenocarcinoma, but existing treatment options for p53-mutant (p53Mut) cancer are largely ineffective. Here, we report a therapeutic strategy for p53Mut tumors based on abnormalities in the DNA repair response. Investigation of DNA repair upon challenge with thymidine analogs reveals a dysregulation in DNA repair response in p53Mut cells that leads to accumulation of DNA breaks. Thymidine analogs do not interrupt DNA synthesis but induce DNA repair that involves a p53-dependent checkpoint. Inhibitors of poly(ADP-ribose) polymerase (PARPis) markedly enhance DNA double-strand breaks and cell death induced by thymidine analogs in p53Mut cells, whereas p53 wild-type cells respond with p53-dependent inhibition of the cell cycle. Combinations of trifluorothymidine and PARPi agents demonstrate superior anti-neoplastic activity in p53Mut cancer models. These findings support a two-drug combination strategy to improve outcomes for patients with p53Mut cancer.

A regulatory feedback loop between Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and the androgen receptor in prostate cancer progression.

The androgen receptor (AR) plays a critical role in prostate cancer (PCa) progression, however, the molecular mechanisms by which the AR regulates cell proliferation in androgen-dependent and castration-resistant PCa are incompletely understood. We report that Ca(2+)/calmodulin-dependent kinase kinase 2 (CaMKK2) expression increases and becomes nuclear or perinuclear in advanced PCa. In the TRAMP (transgenic adenocarcinoma of mouse prostate) model of PCa, CaMKK2 expression increases with PCa progression with many cells exhibiting nuclear staining. CaMKK2 expression is higher in human castration-resistant tumor xenografts compared with androgen-responsive xenografts and is markedly higher in the AR-expressing, tumorigenic cell line LNCaP compared with cell lines that are AR-nonexpressing and/or nontumorigenic. In LNCaP cells, dihydrotestosterone induced CaMKK2 mRNA and protein expression and translocation of CaMKK2 to the nucleus. Conversely, androgen withdrawal suppressed CaMKK2 expression. Knockdown of CaMKK2 expression by RNAi reduced LNCaP cell proliferation and increased percentages of cells in G(1) phase, whereas correspondingly reducing percentages in S phase, of the cell cycle. CaMKK2 knockdown reduced expression of the AR target gene prostate-specific antigen at both mRNA and protein levels, AR transcriptional activity driven by androgen responsive elements from the prostate-specific probasin gene promoter and levels of the AR-regulated cell cycle proteins, cyclin D1 and hyperphosphorylated Rb. Our results suggest that in PCa progression, CaMKK2 and the AR are in a feedback loop in which CaMKK2 is induced by the AR to maintain AR activity, AR-dependent cell cycle control, and continued cell proliferation.

Preclinical evaluation of singlet oxygen-cleavable prodrugs in combination with protoporphyrin IX-photodynamic therapy in an orthotopic rat model of non-muscle-invasive bladder cancer†.

Photodynamic therapy (PDT) initially employed red light, which caused some patients to experience permanent bladder contractions. PDT using the FDA-approved drug hexaminolevulinate (HAL), which produces protoporphyrin IX (PpIX) in the tumor, showed some promise but has low efficacy in treating non-muscle-invasive bladder cancer (NMIBC). We developed singlet oxygen-activatable prodrugs of two anticancer drugs, paclitaxel and mitomycin C, to enhance the antitumor effect of PpIX-PDT without producing systemic side effects, by promoting only local release of the active chemotherapeutic agent. Orthotopic NMIBC model was used to compare the efficacy of prodrugs only, PpIX-PDT, and prodrugs + PpIX-PDT. 532 nm laser with a total power of 50 mW for 20 min (60 J, single treatment) was used with HAL and prodrugs. Histology and microscopic methods with image analysis were used to evaluate the tumor staging, antitumor efficacy, and local toxicity. Prodrug + PpIX-PDT produced superior antitumor efficacy than PpIX-PDT alone with statistical significance. Both PpIX-PDT alone and combination therapy resulted in mild damage to the bladder epithelium in the normal bladder area with no apparent damage to the muscle layer. Overall, SO-cleavable prodrugs improved the antitumor efficacy of PpIX-PDT without causing severe and permanent damage to the bladder muscle layer.

Photodynamic Therapy for Bladder Cancers, A Focused Review†.

Bladder cancer is the first cancer for which PDT was clinically approved in 1993. Unfortunately, it was unsuccessful due to side effects like bladder contraction. Here, we summarized the recent progress of PDT for bladder cancers, focusing on photosensitizers and formulations. General strategies to minimize side effects are intravesical administration of photosensitizers, use of targeting strategies for photosensitizers and better control of light. Non-muscle invasive bladder cancers are more suitable for PDT than muscle invasive and metastatic bladder cancers. In 2010, the FDA approved blue light cystoscopy, using PpIX fluorescence, for photodynamic diagnosis of non-muscle invasive bladder cancer. PpIX produced from HAL was also used in PDT but was not successful due to low therapeutic efficacy. To enhance the efficacy of PpIX-PDT, we have been working on combining it with singlet oxygen-activatable prodrugs. The use of these prodrugs increases the therapeutic efficacy of the PpIX-PDT. It also improves tumor selectivity of the prodrugs due to the preferential formation of PpIX in cancer cells resulting in decreased off-target toxicity. Future challenges include improving prodrugs and light delivery across the bladder barrier to deeper tumor tissue and generating an effective therapeutic response in an In vivo setting without causing collateral damage to bladder function.

Mechanistic review of sulforaphane as a chemoprotective agent in bladder cancer.

Regular consumption of cruciferous vegetables has numerous health benefits, including reduced cancer risk and improved patient outcomes. Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with a chemoprotective role against epithelial cancers, particularly of the bladder. Epithelial cells have several functions, including secretion, absorption, filtration, and protection from environmental insults. The specialized stratified epithelium of the bladder has direct and frequent contact with carcinogenic agents, increasing the likelihood of cancer initiation at this site. Carcinogen exposure, particularly from cigarette smoke or occupational exposure to aromatic amines, are the most significant risk factors for bladder cancer due to their ability to activate inflammatory pathways, induce free radicals, and damage DNA. SFN acts as an antioxidant by activating phase II enzymes involved in carcinogen detoxification to prevent DNA damage and inhibit tumor initiation, modulates multiple signaling pathways to inhibit tumor growth and progression, and has anti-inflammatory and immune-modulating properties to help protect against cancer. Due to these chemoprotective mechanisms, SFN has been studied as both mono- and adjuvant therapy in several bladder cancer models. Here we present a review of the effects of SFN on carcinogen-induced bladder cancer to support the inclusion of cruciferous vegetables as a chemoprotective strategy.

HotSPOT: A Computational Tool to Design Targeted Sequencing Panels to Assess Early Photocarcinogenesis.

Mutations found in skin are acquired in specific patterns, clustering around mutation-prone genomic locations. The most mutation-prone genomic areas, mutation hotspots, first induce the growth of small cell clones in healthy skin. Mutations accumulate over time, and clones with driver mutations may give rise to skin cancer. Early mutation accumulation is a crucial first step in photocarcinogenesis. Therefore, a sufficient understanding of the process may help predict disease onset and identify avenues for skin cancer prevention. Early epidermal mutation profiles are typically established using high-depth targeted next-generation sequencing. However, there is currently a lack of tools for designing custom panels to capture mutation-enriched genomic regions efficiently. To address this issue, we created a computational algorithm that implements a pseudo-exhaustive approach to identify the best genomic areas to target. We benchmarked the current algorithm in three independent mutation datasets of human epidermal samples. Compared to the sequencing panel designs originally used in these publications, the mutation capture efficacy (number of mutations/base pairs sequenced) of our designed panel improved 9.6-12.1-fold. Mutation burden in the chronically sun-exposed and intermittently sun-exposed normal epidermis was measured within genomic regions identified by hotSPOT based on cutaneous squamous cell carcinoma (cSCC) mutation patterns. We found a significant increase in mutation capture efficacy and mutation burden in cSCC hotspots in chronically sun-exposed vs. intermittently sun-exposed epidermis (p < 0.0001). Our results show that our hotSPOT web application provides a publicly available resource for researchers to design custom panels, enabling efficient detection of somatic mutations in clinically normal tissues and other similar targeted sequencing studies. Moreover, hotSPOT also enables the comparison of mutation burden between normal tissues and cancer.

Syngeneic model of carcinogen-induced tumor mimics basal/squamous, stromal-rich, and neuroendocrine molecular and immunological features of muscle-invasive bladder cancer.

Introduction: Bladder cancer is a heterogenous disease and the emerging knowledge on molecular classification of bladder tumors may impact treatment decisions based on molecular subtype. Pre-clinical models representing each subtype are needed to test novel therapies. Carcinogen-induced bladder cancer models represent heterogeneous, immune-competent, pre-clinical testing options with many features found in the human disease. Methods: Invasive bladder tumors were induced in C57BL/6 mice when continuously exposed to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in the drinking water. Tumors were excised and serially passed by subcutaneous implantation into sex-matched syngeneic C57BL/6 hosts. Eight lines were named BBN-induced Urothelium Roswell Park (BURP) tumor lines. BURP lines were characterized by applying consensus molecular classification to RNA expression, histopathology, and immune profiles by CIBERSORT. Two lines were further characterized for cisplatin response. Results: Eight BURP tumor lines were established with 3 male and 3 female BURP tumor lines, having the basal/squamous (BaSq) molecular phenotype and morphology. BURP-16SR was established from a male mouse and has a stromal-rich (SR) molecular phenotype and a sarcomatoid carcinoma morphology. BURP-19NE was established from a male mouse and has a neuroendocrine (NE)-like molecular phenotype and poorly differentiated morphology. The established BURP tumor lines have unique immune profiles with fewer immune infiltrates compared to their originating BBN-induced tumors. The immune profiles of the BURP tumor lines capture some of the features observed in the molecular classifications of human bladder cancer. BURP-16SR growth was inhibited by cisplatin treatment, while BURP-24BaSq did not respond to cisplatin. Discussion: The BURP lines represent several molecular classifications, including basal/squamous, stroma-rich, and NE-like. The stroma-rich (BURP-16SR) and NE-like (BURP-19NE) represent unique immunocompetent models that can be used to test novel treatments in these less common bladder cancer subtypes. Six basal/squamous tumor lines were established from both male and female mice. Overall, the BURP tumor lines have less heterogeneity than the carcinogen-induced tumors and can be used to evaluate treatment response without the confounding mixed response often observed in heterogeneous tumors. Additionally, basal/squamous tumor lines were established and maintained in both male and female mice, thereby allowing these tumor lines to be used to compare differential treatment responses between sexes.

Altered corepressor SMRT expression and recruitment to target genes as a mechanism that change the response to androgens in prostate cancer progression.

Androgen receptor (AR) is required for the development and progression of prostate cancer (CaP) from androgen-dependence to androgen-resistance. Both corepressors and coactivators regulate AR-mediated transcriptional activity, and aberrant expression or activity due to mutation(s) contributes to changes in AR function in the progression to androgen resistance acquired during hormonal ablation therapies. Primary culture of epithelial cells from androgen-dependent CWR22 and androgen-resistant CWR22R xenograft tumors were used to evaluate the effect of androgens on AR function, and the association with coactivators (SRC-1 and TIF-2) and corepressors (SMRT and NCoR). Both androgen-dependent CWR22 and androgen-resistant CWR22R cells expressed functional AR as the receptor bind ligand with high affinity and increased trafficking to the nuclei in the presence of androgens. However, in the presence of androgens, AR-mediated transcriptional activity in androgen-sensitive CWR22 cells was limited to a 2-fold increase, as compared to a 6-fold increase in androgen-resistance CWR22R cells. In androgen-sensitive CWR22 cells, immunoblot, confocal microscopy, and chromatin immunoprecipitation assays indicated that the androgen bound AR transcriptional initiation complex in the PSA promoter contained corepressor SMRT, resulting in limited receptor transcriptional activity. In contrast, increased AR-mediated transcriptional activity in the CWR22R cells was consistent with decreased expression and recruitment of the corepressors SMRT/NCoR, as well as increased recruitment of the coactivator TIF-2 to the receptor complex. Similar changes in the response to androgens were observed in the LNCaP/C4-2 model of androgen resistance prostate cancer. Thus, altered recruitment and loss of corepressors SMRT/NCoR may provide a mechanism that changes the response of AR function to ligands and contributes to the progression of the advanced stages of human prostate cancer.

Role of prostate stem cells and treatment strategies in benign prostate hyperplasia.

Benign prostate hyperplasia (BPH) is a progressive disease with a direct correlation between incidence and age. Since the treatment and management of BPH involve harmful side effects and decreased quality of life for the patient, the primary focus of research should be to find better and longer-lasting therapeutic options. The mechanisms regulating prostate stem cells in development can be exploited to decrease prostate growth. BPH is defined as the overgrowth of the prostate, and BPH is often diagnosed when lower urinary tract symptoms (LUTS) of urine storage or voiding symptoms cause patients to seek treatment. While multiple factors are involved in the hyperplastic growth of the stromal and epithelial compartments of the prostate, the clonal proliferation of stem cells is considered one of the main reasons for BPH initiation and regrowth of the prostate after therapies for BPH fail. Several theories explain possible reasons for the involvement of stem cells in the development, progression, and pathogenesis of BPH. The aim of the current review is to discuss current literature on the fundamentals of prostate development and the role of stem cells in BPH. This review examines the rationale for the hypothesis that unregulated stem cell properties can lead to BPH and therapeutic targeting of stem cells may reduce treatment-related side effects and prevent the regrowth of the prostate.

Generation of a novel transgenic mouse model for bioluminescent monitoring of survivin gene activity in vivo at various pathophysiological processes: survivin expression overlaps with stem cell markers.

Survival has been implicated to play an important role in various pathophysiological processes. However, because of a lack of appropriate animal models, the role and dynamic expression of survivin during pathophysiology are not well defined. We generated a human survivin gene promoter-driven luciferase transgenic mouse model (SPlucTg) so that dynamic survivin gene activity can be monitored during various pathophysiological conditions using in vivo imaging. Our results show that, consistent with survivin positivity in testis, luciferase activity in normal SPlucTg mice was detected in the testis of male mice. Furthermore, similar to the known requirement of transient expression of survivin for pathophysiological responses, we observed a transient luciferase expression in castrated SPlucTg male mice after supplement of androgen. Significantly, it was reported that survivin expression turns on during mouse liver injury and regeneration; a transient and dose-dependent luciferase expression in the mouse liver was observed after administration of carbon tetrachloride into SPlucTg mice. We further demonstrated that luciferase activity closely correlates with endogenous survivin expression. We also demonstrated that only a subset of cells expresses survivin, and its expression overlaps with the expression of several stem cell markers tested. Thus, we have generated a unique animal model for analysis of diverse pathophysiological processes and possible stem cell distribution/activity in vivo.

A simple quantitative PCR assay to determine TRAMP transgene zygosity.

BACKGROUND: The TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model remains one of the most widely used transgenic mouse models of prostate cancer. This is due to its ability to recapitulate with ~100% penetrance multiple aspects of the human disease such as prostatic intraepithelial neoplasia lesions, invasive carcinoma, progression to castration-resistant prostate cancer including aggressive neuroendocrine prostate cancer and metastasis. Despite its popularity, the use of TRAMP mice is limited/slowed by the inability to distinguish the zygosity of the TRAMP transgene. This is especially true for breeding strategies implementing multiple crosses and alleles and when the rapid generation of large animal cohorts with the desired genotype is needed. METHODS: We developed a quantitative PCR (qPCR) approach to determine the relative TRAMP transgene copy number of mice. RESULTS: This method was validated by three independent laboratories across two institutions, which successfully identified the genotype of the mice 98.2% of the time (165/168) in the first attempt. The genotypes of the uncertain mice were correctly identified in the repeated experiments. CONCLUSIONS: We develop the first straightforward, qPCR approach to reliably determine the TRAMP transgene zygosity. The development of this qPCR-based genotyping method enables researchers to streamline breeding strategies when creating complex genetic mouse models involving TRAMP mice; thus, ultimately reducing the required animal numbers, cost, and investigator time.

Patient derived models of bladder cancer enrich the signal of the tumor cell transcriptome facilitating the analysis of the tumor cell compartment.

The evolving paradigm of the molecular classification of bladder cancer requires models that represent the classifications with less heterogeneity. Robust transcriptome based molecular classifications are essential to address tumor heterogeneity. Patient derived models (PDMs) are a powerful preclinical tool to study specific tumor compartments. We tested if the consensus molecular subtype analysis was applicable to PDMs and evaluated the tumor compartment each model represents. PDMs derived from surgical specimens were established as xenografts (PDX), organoids (PDO), and spheroids (PDS). The surgical specimens and PDMs were molecularly characterized by RNA sequencing. PDMs that were established in immune deficient mice or in vitro significantly downregulated transcripts related to the immune and stromal compartments compared to the surgical specimens. However, PDMs upregulate a patient-specific bladder cancer cell signal which allowed for analysis of cancer cell pathways independent of the tumor microenvironment. Based on transcriptomic signatures, PDMs are more similar to their surgical specimen than the model type; indicating that the PDMs retained unique features of the tumor from which the PDM was derived. When comparing models, PDX models were the most similar to the surgical specimen, while PDO and PDS models were most similar to each other. When the consensus molecular subtype classification system was applied to both the surgical samples and the three PDMs, good concordance was found between all samples indicating that this system of classification can be applied to PDO and PDS models. PDMs reduce tumor heterogeneity and allow analysis of tumor cells while maintaining the gene expression profile representative of the original tumor.