Role of Forkhead Box P3 in IFNγ-Mediated PD-L1 Expression and Bladder Cancer Epithelial-to-Mesenchymal Transition.

Antagonism of the PD-1/PD-L1 axis is a critical therapeutic strategy for patients with advanced bladder cancer. IFNγ functions as a key regulator of PD-L1 in both immune as well as cancer cells. Forkhead box P3 (FOXP3) is a transcription factor synonymous in T regulatory cell function but with increasingly described functions in cancer cells. Here, we investigated the relationship between FOXP3 and PD-L1 in bladder cancer. We showed that FOXP3 is critical in the ability for IFNγ to activate PD-L1 in bladder cancer cells. FOXP3 can bind to the PD-L1 promoter and induces a gene program that leads to regulation of multiple immune-related genes and genes involved in epithelial-to-mesenchymal transition (EMT). Using in vitro and in vivo human and murine models, we showed that FOXP3 can influence bladder cancer EMT as well as promote cancer metastases. Furthermore, FOXP3 may be a convergent factor for multiple activators of PD-L1, including the chemotherapeutic drug cisplatin. SIGNIFICANCE: Historically a key transcription factor driving T regulatory cell function, FOXP3 has an increasingly recognized role in cancer cells. In bladder cancer, we defined a novel mechanism whereby FOXP3 mediates the activation of the immune checkpoint PD-L1 by the cytokine IFNγ. We also showed that FOXP3 induces other immune checkpoints as well as genes involved in EMT, promoting immune resistance and cancer metastases.

UCHL1 is a potential molecular indicator and therapeutic target for neuroendocrine carcinomas.

Neuroendocrine carcinomas, such as neuroendocrine prostate cancer and small-cell lung cancer, commonly have a poor prognosis and limited therapeutic options. We report that ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is elevated in tissues and plasma from patients with neuroendocrine carcinomas. Loss of UCHL1 decreases tumor growth and inhibits metastasis of these malignancies. UCHL1 maintains neuroendocrine differentiation and promotes cancer progression by regulating nucleoporin, POM121, and p53. UCHL1 binds, deubiquitinates, and stabilizes POM121 to regulate POM121-associated nuclear transport of E2F1 and c-MYC. Treatment with the UCHL1 inhibitor LDN-57444 slows tumor growth and metastasis across neuroendocrine carcinomas. The combination of UCHL1 inhibitors with cisplatin, the standard of care used for neuroendocrine carcinomas, significantly delays tumor growth in pre-clinical settings. Our study reveals mechanisms of UCHL1 function in regulating the progression of neuroendocrine carcinomas and identifies UCHL1 as a therapeutic target and potential molecular indicator for diagnosing and monitoring treatment responses in these malignancies.

Tumor heterogeneity in VHL drives metastasis in clear cell renal cell carcinoma.

Loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is a hallmark of clear cell renal cell carcinoma (ccRCC). The importance of heterogeneity in the loss of this tumor suppressor has been under reported. To study the impact of intratumoral VHL heterogeneity observed in human ccRCC, we engineered VHL gene deletion in four RCC models, including a new primary tumor cell line derived from an aggressive metastatic case. The VHL gene-deleted (VHL-KO) cells underwent epithelial-to-mesenchymal transition (EMT) and exhibited increased motility but diminished proliferation and tumorigenicity compared to the parental VHL-expressing (VHL+) cells. Renal tumors with either VHL+ or VHL-KO cells alone exhibit minimal metastatic potential. Combined tumors displayed rampant lung metastases, highlighting a novel cooperative metastatic mechanism. The poorly proliferative VHL-KO cells stimulated the proliferation, EMT, and motility of neighboring VHL+ cells. Periostin (POSTN), a soluble protein overexpressed and secreted by VHL non-expressing (VHL-) cells, promoted metastasis by enhancing the motility of VHL-WT cells and facilitating tumor cell vascular escape. Genetic deletion or antibody blockade of POSTN dramatically suppressed lung metastases in our preclinical models. This work supports a new strategy to halt the progression of ccRCC by disrupting the critical metastatic crosstalk between heterogeneous cell populations within a tumor.

Donor-Derived Urothelial Carcinoma in Renal Transplant Recipients.

Cancer is a significant cause of morbidity and mortality in recipients of renal transplantation. The vast majority develop from recipient origins, whereas donor-derived malignancies are exceedingly rare. We report 2 cases of poorly differentiated donor-derived urothelial carcinoma (UC) in renal transplantation recipients. The first patient underwent a living-related-donor renal transplantation 24 years prior and presented with back pain, hematuria, and rising creatinine and was found to have a 14 cm mass in the renal allograft with regional lymphadenopathy and liver metastases. Pathology showed UC with small-cell differentiation. The second patient presented with hematuria and rising creatinine and was initially found to have muscle invasive bladder cancer seven years after a deceased donor renal transplantation. Nine months after radical cystectomy, a large 9 cm mass was found on his allograft, for which radical nephrectomy and excision of prior ileal conduit was performed. Pathology showed UC with sarcomatoid differentiation. Short tandem repeat (STR) genotyping confirmed donor-derived origins. Both patient tumors expressed PD-L1 suggesting an additional therapeutic avenue for these rare tumors.

A genetically defined disease model reveals that urothelial cells can initiate divergent bladder cancer phenotypes.

Small cell carcinoma of the bladder (SCCB) is a rare and lethal phenotype of bladder cancer. The pathogenesis and molecular features are unknown. Here, we established a genetically engineered SCCB model and a cohort of patient SCCB and urothelial carcinoma samples to characterize molecular similarities and differences between bladder cancer phenotypes. We demonstrate that SCCB shares a urothelial origin with other bladder cancer phenotypes by showing that urothelial cells driven by a set of defined oncogenic factors give rise to a mixture of tumor phenotypes, including small cell carcinoma, urothelial carcinoma, and squamous cell carcinoma. Tumor-derived single-cell clones also give rise to both SCCB and urothelial carcinoma in xenografts. Despite this shared urothelial origin, clinical SCCB samples have a distinct transcriptional profile and a unique transcriptional regulatory network. Using the transcriptional profile from our cohort, we identified cell surface proteins (CSPs) associated with the SCCB phenotype. We found that the majority of SCCB samples have PD-L1 expression in both tumor cells and tumor-infiltrating lymphocytes, suggesting that immune checkpoint inhibitors could be a treatment option for SCCB. We further demonstrate that our genetically engineered tumor model is a representative tool for investigating CSPs in SCCB by showing that it shares a similar a CSP profile with clinical samples and expresses SCCB-up-regulated CSPs at both the mRNA and protein levels. Our findings reveal distinct molecular features of SCCB and provide a transcriptional dataset and a preclinical model for further investigating SCCB biology.

Establishment of xenografts of urological cancers on chicken chorioallantoic membrane (CAM) to study metastasis.

Cancer of the urological system commonly occurs in the kidney, bladder, and prostate gland. The clear cell subtype of renal cell carcinoma (ccRCC) constitutes the great majority of kidney cancer. Metastatic ccRCC portends a very poor outcome with no effective treatment available. Prostate cancer is the most common cancer in males in the US. Despite recent advances in selective kinase inhibitors and immunotherapies, the rate of developing new treatment from bench to bedside is slow. A time-consuming step is at the animal drug testing stage, in which the mouse model is the gold standard. In the pursuit to streamline the in vivo cancer biology research and drug development, we explored the feasibility of the chicken chorioallantoic membrane (CAM) model to establish xenografts. The CAM model greatly shortens the time of tumor growth and lowers the cost comparing to immunocompromised mice. We generated CAM xenografts from ccRCC, bladder and prostate cancer, with established cancer cell lines and freshly isolated patient-derived tissues, either as primary tumor cells or small pieces of tumors. The successful CAM engraftment rate from the different tumor sources is 70% or above. Using our previously established metastatic ccRCC mouse model, we showed that the CAM xenograft maintains the same tumor growth pattern and metastatic behavior as observed in mice. Taken together, CAM can serve as a valuable platform to establish new patient-derived xenografts (PDXs) to study tumor biology, thus accelerating the development of individualized treatment to halt the deadly metastatic stage of cancer.

Sequencing of cancer cell subpopulations identifies micrometastases in a bladder cancer patient.

PURPOSE: Pathologic staging of bladder cancer patients remains a challenge. Standard-of-care histology exhibits limited sensitivity in detection of micrometastases, which can increase risk of cancer progression and delay potential adjuvant therapies. Here, we sought to develop a proof of concept novel molecular approach to improve detection of cancer micrometastasis. EXPERIMENTAL DESIGN: We combined fluorescence activated cell sorting and next-generation sequencing and performed whole-exome sequencing of total cancer cells and cancer cell subpopulations in multiple tumor specimens and regional lymph nodes in a single patient with muscle-invasive urothelial carcinoma of the bladder following radical cystectomy. RESULTS: Mean allele frequency analysis demonstrated a significant correlation between primary tumor cancer cells and cancer cells isolated from the lymph nodes, confirming lymph node disease despite negative pathologic staging. RNA-sequencing revealed intratumoral heterogeneity as well as enrichment for immune system and lipid metabolism gene sets in the micrometastatic cancer cell subpopulations. CONCLUSIONS: Our analysis illustrates how next-generation sequencing of cancer cell subpopulations may be utilized to enrich for cancer cell markers and enhance detection of bladder cancer micrometastases to improve pathologic staging and provide insight into cancer cell biology.

Development and Validation of an Automated Method to Identify Patients Undergoing Radical Cystectomy for Bladder Cancer Using Natural Language Processing.

INTRODUCTION: Measurement for quality improvement relies on accurate case identification and characterization. With electronic health records now widely deployed, natural language processing, the use of software to transform text into structured data, may enrich quality measurement. Accordingly we evaluated the application of natural language processing to radical cystectomy procedures for patients with bladder cancer. METHODS: From a sample of 497 procedures performed from March 2013 to October 2014 we identified radical cystectomy for primary bladder cancer using the approaches of 1) a natural language processing enhanced algorithm, 2) an administrative claims based algorithm and 3) manual chart review. We also characterized treatment with robotic surgery and continent urinary diversion. Using chart review as the reference standard we calculated the observed agreement (kappa statistic), sensitivity, specificity, positive predictive value and negative predictive value for natural language processing and administrative claims. RESULTS: We confirmed 84 radical cystectomies were performed for bladder cancer, with 50.0% robotic and 38.6% continent diversions. The natural language processing enhanced and claims based algorithms demonstrated 99.8% (κ=0.993, 95% CI 0.979-1.000) and 98.6% (κ=0.951, 95% CI 0.915-0.987) agreement with manual review, respectively. Both approaches accurately characterized robotic vs open surgery, with natural language processing enhanced algorithms showing 98.8% (κ=0.976, 95% CI 0.930-1.000) and claims based 90.5% (κ=0.810, 95% CI 0.686-0.933) agreement. For urinary diversion natural language processing enhanced algorithms correctly specified 96.4% of cases (κ=0.924, 95% CI 0.839-1.000) compared with 83.3% (κ=0.655, 95% CI 0.491-0.819). CONCLUSIONS: Natural language processing enhanced and claims based algorithms accurately identified radical cystectomy cases at our institution. However, natural language processing appears to better classify specific aspects of cystectomy surgery, highlighting a potential advantage of this emerging methodology.

Biased Expression of the FOXP3Δ3 Isoform in Aggressive Bladder Cancer Mediates Differentiation and Cisplatin Chemotherapy Resistance.

PURPOSE: The transcriptional regulation mediating cancer cell differentiation into distinct molecular subtypes and modulating sensitivity to existing treatments is an enticing therapeutic target. Our objective was to characterize the ability of the forkhead/winged transcription factor FOXP3 to modulate the differentiation of bladder cancer. EXPERIMENTAL DESIGN: Expression of FOXP3 was analyzed by immunohistochemistry in a tumor microarray of 587 samples and overall survival in a subset of 187 patients following radical cystectomy. Functional assays were performed in SW780 and HT1376 cell lines in vitro and in vivo and gene expression profiling performed by RNA-Seq. Validation was undertaken using gene expression profiles of 131 patients from The Cancer Genome Atlas (TCGA) consortium in bladder cancer. RESULTS: FOXP3 expression correlates with bladder cancer stage and inversely with overall survival, with biased expression of the FOXP3Δ3 isoform. Functional assays of FOXP3Δ3 demonstrated resistance to chemotherapy in vitro, whereas subcutaneous xenografts overexpressing FOXP3Δ3 developed larger and more poorly differentiated bladder cancers. RNA expression profiling revealed a unique FOXP3Δ3 gene signature supporting a role in chemotherapy resistance. Accordingly, knockdown of Foxp3 by siRNA in HT1376 cells conferred sensitivity to cisplatin- and gemcitabine-induced cytotoxicity. Validation in TCGA dataset demonstrated increased expression of FOXP3 in subtypes II to IV and skewing of molecular subtypes based on FOXP3Δ3-specific gene expression. CONCLUSIONS: (i) Biased expression of the FOXP3Δ3 isoform in bladder cancer inversely correlates with overall survival, (ii) FOXP3Δ3 induces a unique gene program that mediates cancer differentiation, and (iii) FOXP3Δ3 may augment chemotherapy resistance. Clin Cancer Res; 22(21); 5349-61. ©2016 AACR.

Gleason 6 Prostate Cancer: Translating Biology into Population Health.

PURPOSE: Gleason 6 (3+3) is the most commonly diagnosed prostate cancer among men with prostate specific antigen screening, the most histologically well differentiated and is associated with the most favorable prognosis. Despite its prevalence, considerable debate exists regarding the genetic features, clinical significance, natural history, metastatic potential and optimal management. MATERIALS AND METHODS: Members of the Young Urologic Oncologists in the Society of Urologic Oncology cooperated in a comprehensive search of the peer reviewed English medical literature on Gleason 6 prostate cancer, specifically focusing on the history of the Gleason scoring system, histological features, clinical characteristics, practice patterns and outcomes. RESULTS: The Gleason scoring system was devised in the early 1960s, widely adopted by 1987 and revised in 2005 with a more restrictive definition of Gleason 6 disease. There is near consensus that Gleason 6 meets pathological definitions of cancer, but controversy about whether it meets commonly accepted molecular and genetic criteria of cancer. Multiple clinical series suggest that the metastatic potential of contemporary Gleason 6 disease is negligible but not zero. Population based studies in the U.S. suggest that more than 90% of men newly diagnosed with prostate cancer undergo treatment and are exposed to the risk of morbidity for a cancer unlikely to cause symptoms or decrease life expectancy. Efforts have been proposed to minimize the number of men diagnosed with or treated for Gleason 6 prostate cancer. These include modifications to prostate specific antigen based screening strategies such as targeting high risk populations, decreasing the frequency of screening, recommending screening cessation, incorporating remaining life expectancy estimates, using shared decision making and novel biomarkers, and eliminating prostate specific antigen screening entirely. Large nonrandomized and randomized studies have shown that active surveillance is an effective management strategy for men with Gleason 6 disease. Active surveillance dramatically reduces the number of men undergoing treatment without apparent compromise of cancer related outcomes. CONCLUSIONS: The definition and clinical relevance of Gleason 6 prostate cancer have changed substantially since its introduction nearly 50 years ago. A high proportion of screen detected cancers are Gleason 6 and the metastatic potential is negligible. Dramatically reducing the diagnosis and treatment of Gleason 6 disease is likely to have a favorable impact on the net benefit of prostate cancer screening.

Synergy of Histone-Deacetylase Inhibitor AR-42 with Cisplatin in Bladder Cancer.

PURPOSE: Cisplatin based chemotherapy regimens form the basis of systemic bladder cancer treatment, although they show limited response rates and efficacy. Recent molecular analysis of bladder cancer revealed a high incidence of mutations in chromatin regulatory genes, suggesting a therapeutic avenue for histone deacetylase inhibitors. We investigated the ability of the novel histone deacetylase inhibitor AR-42 to synergize with cisplatin in preclinical models of bladder cancer. MATERIALS AND METHODS: We assessed the ability of the pan-histone deacetylase inhibitor AR-42 with and without cisplatin to destroy bladder cancer cells by survival and apoptosis assays in vitro, and by growth and differentiation in an in vivo xenograft model. We also assessed the response to the bladder cancer stem cell population by examining the effect of AR-42 on the CD44(+)CD49f(+) population with and without cisplatin. Synergy was calculated using combination indexes. RESULTS: The AR-42 and cisplatin combination synergistically destroyed bladder cancer cells via apoptosis and it influenced tumor growth and differentiation in vivo. When tested in the CD44(+)CD49f(+) bladder cancer stem cell population, AR-42 showed greater efficacy with and without cisplatin. CONCLUSIONS: AR-42 may be an attractive novel histone deacetylase inhibitor with activity against bladder cancer. Its efficacy in bladder cancer stem cells and synergy with cisplatin warrant further clinical investigation. Our in vitro and animal model studies provide preclinical evidence that AR-42 may be administered in conjunction with cisplatin based chemotherapy to improve the treatment of bladder cancer in patients.

Loss of MyD88 leads to more aggressive TRAMP prostate cancer and influences tumor infiltrating lymphocytes.

BACKGROUND: The influence of pattern recognition receptor (PRR) signaling in the prostate tumor microenvironment remains unclear. Although there may be a role for PRR agonists as adjuvants to therapy, prior evidence suggests tumor promoting as well as tumor inhibiting mechanisms. The purpose of this study is to examine the role of the key Toll-like receptor (TLR) signaling adaptor protein myeloid differentiation primary response gene 88 (MyD88) in prostate cancer development. METHODS: MyD88(-/-) mice in a C57Bl6 background were crossed with transgenic adenocarcinomas of the mouse prostate (TRAMP) mice to create MyD88(-/-) TRAMP(Tg+/-) animals, which were compared to MyD88(+/+) TRAMP(Tg+/-) animals and their non-transgenic counterparts at 30 weeks. Prostates were examined histologically, by immunohistochemistry and immunofluorescence staining, and by qPCR, to characterize tumor-infiltrating immune populations as well as activation of the downstream NF-κB pathway and androgen receptor (AR) expression. Splenocytes were examined for development of distinct immune cell populations. RESULTS: Absence of MyD88 led to increased prostatic intraepithelial neoplasm (PIN) and areas of well-differentiated adenocarcinoma in TRAMP transgenic mice. Analysis of infiltrating immune populations revealed an increase in CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs), as evidenced by increased expression of prostatic arginase-1 and iNOS as well as the cytokine IL-10, and a deficiency in NK cells in prostates from MyD88(-/-) TRAMP(Tg+/-) compared to MyD88(+/+) TRAMP(Tg+/-) mice, whereas a decrease in splenocytic NK cell differentiation was observed in MyD88(-/-) mice. Prostate tumors revealed no significant differences in NF-κB or AR expression in MyD88(+/+) TRAMP(Tg+/-) compared to MyD88(-/-) TRAMP(Tg+/-) mice. CONCLUSIONS: During prostate cancer development in the TRAMP model, MyD88 may play a role in limiting prostate tumorigenesis by altering tumor-infiltrating immune populations. This suggests that in the context of specific cancers, distinct PRRs and signaling pathways of innate immune signaling may influence the tumor microenvironment and represent a novel therapeutic strategy.

RARP in high-risk prostate cancer: use of multi-parametric MRI and nerve sparing techniques.

To examine the outcomes of patients with high-risk prostate cancer (PCa) treated by robot-assisted radical prostatectomy (RARP) and evaluate the value of multi-parametric magnetic resonance imaging (MRI) in estimating tumor stage, extracapsular extension, and grade, and the application of nerve sparing (NS) techniques. Patient demographics, preoperative imaging, surgical parameters, pathological features, functional and recurrence outcomes were collected retrospectively in patients with high-risk PCa who underwent RARP between December 2009 and October 2013. Pathological whole mount slides to assess NS were compared with potency, recovery of continence, and surgical margins (SM). Forty-four cases of high-risk PCa were identified with a median followup of 24 months and positive surgical margins (PSM) rate of 14%. Continence returned in 86%, with potency rate of 58%. Of the 25 cases with a preoperative multi-parametric MRI, MRI improved clinical staging from 28% to 88%, respectively. Following risk stratification of NS by microscopic analysis of whole mount pathology, patients with Group A (bilateral NS), Group B (unilateral NS), Group C (partial NS), and Group D (non-NS) had 100%, 92%, 91%, and 50% continence rates, and 100%, 80%, 45%, and 0% potency rates, respectively, with an inverse correlation to PSM. RARP in men with high-risk PCa can achieve favorable oncologic and functional outcomes. Preoperative MRI may localize high-grade tumors and improve clinical staging. Extent of NS is influenced by clinical staging and may balance potency and continence with PSMs.

Role of Rip2 in development of tumor-infiltrating MDSCs and bladder cancer metastasis.

Tumor invasion and metastases represent a complex series of molecular events that portends a poor prognosis. The contribution of inflammatory pathways mediating this process is not well understood. Nod-like receptors (NLRs) of innate immunity function as intracellular sensors of pathogen motifs and danger molecules. We propose a role of NLRs in tumor surveillance and in programming tumor-infiltrating lymphocytes (TILs). In this study, we examined the downstream serine/threonine and tyrosine kinase Rip2 in a murine model of bladder cancer. In Rip2-deficient C57Bl6 mice, larger orthotopic MB49 tumors developed with more numerous and higher incidence of metastases compared to wild-type controls. As such, increased tumor infiltration of CD11b+ Gr1hi myeloid-derived suppressor cells (MDSCs) with concomitant decrease in T cells and NK cells were observed in Rip2-deficient tumor bearing animals using orthotopic and subcutaneous tumor models. Rip2-deficient tumors showed enhanced epithelial-to-mesenchymal transition, with elevated expression of zeb1, zeb2, twist, and snail in the tumor microenvironment. We found that the absence of Rip2 plays an intrinsic role in fostering the development of granulocytic MDSCs by an autocrine and paracrine effect of granulocytic colony stimulating factor (G-CSF) expression. Our findings suggest that NLR pathways may be a novel modality to program TILs and influence tumor metastases.

Karnofsky performance status predicts overall survival, cancer-specific survival, and progression-free survival following radical cystectomy for urothelial carcinoma.

OBJECTIVE: Radical cystectomy (RC) can provide a survival advantage in patients with urothelial carcinoma of the bladder, but not without significant morbidity rates. Whether the ability of preoperative comorbidity or performance status metrics can stratify patients to overall survival (OS), cancer-specific survival (CSS), and progression-free survival (PFS) following RC is unclear. We analyze our RC experience from 2005 to 2010 to assess the prognostic power of American Society of Anesthesiologists (ASA) score, Charlson Comorbidity Index (CCI), and Karnofsky Performance Status (KPS) index as they relate to OS, CSS, and PFS. MATERIALS AND METHODS: A retrospective analysis was performed of 234 patients who underwent RC between January 2005 and December 2010; of these, 148 patients had sufficient data for OS, CSS, and PFS analysis. Multivariate Cox proportional hazard modeling generated hazard ratios using as independent variables patient age at surgery, gender, ethnicity, preoperative KPS, CCI, and ASA values, pathologic T-staging, the presence of nodal disease, use of radiation therapy, neoadjuvant chemotherapy, and adjuvant chemotherapy. A recursive partition analysis tree divided the population into high- and low-performance groups, and 5-year survival outcomes were evaluated. OS, CSS, and PFS were employed as Kaplan-Meier dependent variables with similar populations comprising high- and low-performance subgroups. RESULTS: Mean CSS was 46.8 months (95 % CI 43.2-50.4) with a 5-year CSS of 75 % and OS of 69 %. Patient age, pathologic T-stage, and KPS were identified as independent predictors of OS and CSS. Analysis of PFS as the continuous dependent variable identified only KPS as a statistically significant predictor of freedom from radiologic progression. No statistically significant predictive value was identified for nodal disease, neoadjuvant chemotherapy, adjuvant chemotherapy, gender, ethnicity, CCI, or ASA in terms of OS, CCS, or PFS. Patients with a KPS ≤ 80 had a shorter survival than patients with a KPS ≥ 90 in terms of OS, CSS, and PFS (log-rank Mantel-Cox: p < 0.01). For patients with a KPS ≤ 80, ~5-year CSS was 42 %, while for patients with a KPS ≥ 90 the 5-year survival was 81 %. These survival curves can be further stratified based on T-stage where patients with a KPS ≥ 90 and T2 have a 5-year CSS of 80 %, whereas patients with a KPS ≤ 80 and >T2 have a ~5-year CSS of 43 % (p < 0.0001). CONCLUSIONS: Our study suggests the use of KPS to have predictive capacity in terms of OS, CSS, and PFS. This information can be used to inform patients' survival expectations prior to proceeding with radical cystectomy.

Stromal modulation of bladder cancer-initiating cells in a subcutaneous tumor model.

The development of new cancer therapeutics would benefit from incorporating efficient tumor models that mimic human disease. We have developed a subcutaneous bladder tumor regeneration system that recapitulates primary human bladder tumor architecture by recombining benign human fetal bladder stromal cells with SW780 bladder carcinoma cells. As a first step, SW780 cells were seeded in ultra low attachment cultures in order to select for sphere-forming cells, the putative cancer stem cell (CSC) phenotype. Spheroids were combined with primary human fetal stromal cells or vehicle control and injected subcutaneously with Matrigel into NSG mice. SW780 bladder tumors that formed in the presence of stroma showed accelerated growth, muscle invasion, epithelial to mesenchymal transition (EMT), decreased differentiation, and greater activation of growth pathways compared to tumors formed in the absence of fetal stroma. Tumors grown with stroma also demonstrated a greater similarity to typical malignant bladder architecture, including the formation of papillary structures. In an effort to determine if cancer cells from primary tumors could form similar structures in vivo using this recombinatorial approach, putative CSCs, sorted based on the CD44(+)CD49f(+) antigenic profile, were collected and recombined with fetal bladder stromal cells and Matrigel prior to subcutaneous implantation. Retrieved grafts contained tumors that exhibited the same structure as the original primary human tumor. Primary bladder tumor regeneration using human fetal bladder stroma may help elucidate the influences of stroma on tumor growth and development, as well as provide an efficient and accessible system for therapeutic testing.

Primary carcinoid tumor of urinary bladder discovered on pelvic magnetic resonance imaging.

Pure primary carcinoid tumor of the urinary bladder is an exceedingly rare lesion with less than twenty cases reported in the English language literature. These tumors are typically small and rarely invade past the lamina propria. Amenable to transurethral resection alone, they are associated with a favorable prognosis. We present a case of pure primary carcinoid tumor of the bladder discovered on T2-weighted pelvic magnetic resonance imaging.

Immunological therapy in urological malignancy: novel combination strategies.

At present, immunotherapy in urological malignancy is experiencing a renaissance, particularly with the emergence of a host of innovative cancer vaccines. Herein, we will review promising immunotherapeutic approaches and evaluate the data supporting their inclusion in novel combination strategies.

Toll-like receptor 3-mediated suppression of TRAMP prostate cancer shows the critical role of type I interferons in tumor immune surveillance.

Inflammation has increasingly been recognized as a critical component influencing tumor growth. Recent reports have revealed conflicting evidence for the role of Toll-like receptors (TLR) in modulating tumorigenesis. In our study, we implicate TLR3 in mediating immune surveillance with increased growth of implanted transgenic adenocarcinoma of the mouse prostate (TRAMP) tumors in TLR3(-/-) compared with TLR3(+/+) mice. Activation of TLR3 by polyinosinic-polycytidylic acid (polyI:C) leads to induction of multiple inflammatory pathways, including NF-kappaB, mitogen-activated protein kinases, and interferon (IFN) regulatory factors. We explored the potential of TLR3 stimulation in prostate cancer immunotherapy and showed that treatment with polyI:C can strongly suppress both s.c. implanted TRAMP tumors in syngenic mice as well as orthotopic prostate cancers in TRAMP C57Bl6 x FvB F1 Tg(+/-) transgenic mice. Treated tumors remained well differentiated to moderately differentiated with increased infiltration of T lymphocytes and natural killer (NK) cells compared with poorly differentiated adenocarcinoma observed in untreated tumors. Like TLR3(-/-) mice, IFN-alpha receptor 1 (IFNAR1)(-/-) mice exhibited reduced tumor surveillance and impaired tumor suppression following polyI:C treatment. We observed that type I IFN-dependent induction of cytokines was responsible for NK activation, with depletion of NK cells leading to increased tumor growth as well as expansion of CD4(+)CD25(+)Foxp3(+) T regulatory lymphocytes. Our study therefore delineates the importance of IFNAR-dependent functions in TLR3-mediated tumor suppression and supports the use of TLR3 agonists for prostate cancer immune-based therapies.