The Bladder Tumor Microenvironment Components That Modulate the Tumor and Impact Therapy.

The tumor microenvironment (TME) is complex and involves many different cell types that seemingly work together in helping cancer cells evade immune monitoring and survive therapy. The advent of single-cell sequencing has greatly increased our knowledge of the cell types present in the tumor microenvironment and their role in the developing cancer. This, coupled with clinical data showing that cancer development and the response to therapy may be influenced by drugs that indirectly influence the tumor environment, highlights the need to better understand how the cells present in the TME work together. This review looks at the different cell types (cancer cells, cancer stem cells, endothelial cells, pericytes, adipose cells, cancer-associated fibroblasts, and neuronal cells) in the bladder tumor microenvironment. Their impact on immune activation and on shaping the microenvironment are discussed as well as the effects of hypertensive drugs and anesthetics on bladder cancer.

Tumor-targeting albumin nanoparticles as an efficacious drug delivery system and potential diagnostic tool in non-muscle-invasive bladder cancer therapy.

Current intravesical chemotherapy for non-muscle invasive bladder cancer (NMIBC) has limited efficacy due to loss of the instilled agent from urine voiding and the agent's lack of specificity for the tumors. We developed a nanocarrier (txCD47-HNP, ∼100 nm) based on human serum albumin conjugated with a peptide that targets the cluster of differentiation 47 receptor overexpressed on bladder cancer (BC) cells. The IC50 of gemcitabine elaidate (GEM) loaded in the txCD47-HNP was almost an order of magnitude lower than that of free GEM. In a mouse orthotopic BC model, GEM loaded in txCD47-HNP effectively reduced the tumor burden. Tumor cells in BC patients' urine can also be targeted by fluorescence-labeled txCD47-HNP resulting in >83 % of the cells exhibiting fluorescence. Thus, txCD47-HNP can potentially be a theranostic agent in NMIBC management by serving as a targeted drug delivery vehicle as well as an alternative to urine cytology.

Predictive biomarkers of response to bacillus Calmette-Guérin immunotherapy and bacillus Calmette-Guérin failure for non-muscle invasive bladder cancer.

Within the heterogeneous population of patients with bacillus Calmette-Guérin failure, there are clear differences in prognosis and therapy with regard to the timeline when bacillus Calmette-Guérin failure occurred. There are a variety of classifications which include bacillus Calmette-Guérin refractory disease, relapsing, unresponsive, and intolerant. Further profiling of these patients may help to shed light on other forms of therapy that are less radical. We hereby summarize the different biomarkers that predicts for response to bacillus Calmette-Guérin immunotherapy and bacillus Calmette-Guérin failure for non-muscle invasive bladder cancer.

Tryptophan-kynurenine ratio as a biomarker of bladder cancer.

OBJECTIVES: To investigate plasma and urinary kynurenine (KYN)-tryptophan (TRP) ratios in bladder cancer, expression of indoleamine 2,3-dioxygenase 1 (IDO1) in relation to tryptophan 2,3-dioxygenase (TDO2) in bladder tumour, and the correlation of KYN-TRP ratio with bladder tumour burden. METHODS: Metabotyping of the TRP-KYN metabolic axis was performed via a clinical case-control study. Expression of IDO1 and TDO2 was measured in human biopsied tissues. Correlational experiments between KYN-TRP ratio and bladder tumour were performed using a murine orthotopic prostate-specific antigen (PSA)-secreting MB49 bladder cancer model. RESULTS: We established for the first time that plasma TRP level was significantly decreased, while both plasma and urinary KYN-TRP ratios were significantly higher in bladder cancer patients, and expression level of IDO1 but not TDO2 was increased in human bladder tumour. We reported the positive correlation between IDO1 expression, KYN-TRP ratio, normalized PSA to creatinine, and bladder tumour burden in the murine model. CONCLUSION: Kynurenine-tryptophan ratio is a promising surveillance biomarker for bladder cancer, but would require further validation before clinical translation.

Gmcsf and Ifnα gene therapy improves the response to BCG immunotherapy in a murine model of bladder cancer.

Aim: To develop a strategy to improve response to bacillus Calmette-Gueri (BCG) using cytokine gene therapy (Gmcsf + Ifnα). Materials & methods: MB49-PSA tumor-bearing C57BL/6N mice were assigned into four groups: control; Gmcsf + Ifnα therapy; BCG therapy or combined therapy (Gmcsf + Ifnα and BCG). In schedule 1, cytokine gene therapy was delivered before BCG therapy (eight instillations). In schedule 2, cytokine gene and BCG therapy were instilled alternatively (eight instillations). Tumors were analyzed by immunohistochemistry and mRNA analysis and urinary immune cells by flow cytometry. Results: Combined therapy in schedule 2 reduced tumor growth, increased immune cell recruitment and was associated with reduced inflammation when compared with BCG therapy. Conclusion: Alternating cytokine gene delivery with BCG therapy modulates the tumor environment increasing receptivity to BCG.

The liver-gut microbiota axis modulates hepatotoxicity of tacrine in the rat.

The gut microbiota possesses diverse metabolic activities, but its contribution toward heterogeneous toxicological responses is poorly understood. In this study, we investigated the role of the liver-gut microbiota axis in underpinning the hepatotoxicity of tacrine. We employed an integrated strategy combining pharmacokinetics, toxicology, metabonomics, genomics, and metagenomics to elucidate and validate the mechanism of tacrine-induced hepatotoxicity in Lister hooded rats. Pharmacokinetic studies in rats demonstrated 3.3-fold higher systemic exposure to tacrine in strong responders that experienced transaminitis, revealing enhanced enterohepatic recycling of deglucuronidated tacrine in this subgroup, not attributable to variation in hepatic disposition gene expression. Metabonomic studies implicated variations in gut microbial activities that mapped onto tacrine-induced transaminitis. Metagenomics delineated greater deglucuronidation capabilities in strong responders, based on differential gut microbial composition (e.g., Lactobacillus, Bacteroides, and Enterobacteriaceae) and approximately 9% higher ß-glucuronidase gene abundance compared with nonresponders. In the validation study, coadministration with oral ß-glucuronidase derived from Escherichia coli and pretreatment with vancomycin and imipenem significantly modulated the susceptibility to tacrine-induced transaminitis in vivo. CONCLUSION: This study establishes pertinent gut microbial influences in modifying the hepatotoxicity of tacrine, providing insights for personalized medicine initiatives. (Hepatology 2018;67:282-295).

The Role of Vitamin D Receptor Polymorphisms in Predicting the Response to Therapy for Nonmuscle Invasive Bladder Carcinoma.

PURPOSE: Clinical and pathological predictors of bladder carcinoma recurrence and progression are relatively well defined. However, there is a paucity of genetic data specifically on the association of single nucleotide polymorphisms in specific genes for predicting recurrence and progression following immunotherapy. The VDR gene was found to regulate the immunomodulatory effects of vitamin D and it enhances the innate immunity system. We evaluated 3 VDR single nucleotide polymorphisms and their predictive role on the response to immunotherapy. MATERIALS AND METHODS: Patients with bladder cancer at intermediate-high risk who underwent post-transurethral resection intravesical bacillus Calmette-Guérin in Singapore and Hong Kong from 1995 to 2014 were recruited for analysis. We evaluated 3 VDR single nucleotide polymorphisms using polymerase chain reaction. Kaplan-Meier survival curves and relationships with outcomes were analyzed by multivariable Cox regression. RESULTS: A total of 338 predominantly Chinese patients were included in study. Individuals carrying the VDR genotype Bsm A/G were significantly associated with lower time to recurrence after bacillus Calmette-Guérin therapy (p <0.001). On multivariable analysis the HR of recurrence in patients with the Bsm A allele was 3.95 times that in patients without the allele (p = 0.037). Patients with the VDR GATC subhaplotype were 3.05 times more likely than patients with other subhaplotypes to experience recurrences (p = 0.003). Study limitations include the small sample size and the lack of information on previous bacillus Calmette-Guérin vaccine exposure and on vitamin D levels. CONCLUSIONS: Our findings in this study suggest that various VDR single nucleotide polymorphisms are associated with recurrences after bacillus Calmette-Guérin immunotherapy. Further functional studies should be performed to elucidate the significance of the VDR gene in the management of bladder cancer and the potential therapy implications.

Metabonomic profiling of bladder cancer.

Early diagnosis and life-long surveillance are clinically important to improve the long-term survival of bladder cancer patients. Currently, a noninvasive biomarker that is as sensitive and specific as cystoscopy in detecting bladder tumors is lacking. Metabonomics is a complementary approach for identifying perturbed metabolic pathways in bladder cancer. Significant progress has been made using modern metabonomic techniques to characterize and distinguish bladder cancer patients from control subjects, identify marker metabolites, and shed insights on the disease biology and potential therapeutic targets. With its rapid development, metabonomics has the potential to impact the clinical management of bladder cancer patients in the future by revolutionizing the diagnosis and life-long surveillance strategies and stratifying patients for diagnostic, surgical, and therapeutic clinical trials. An introduction to metabonomics, typical metabonomic workflow, and critical evaluation of metabonomic investigations in identifying biomarkers for the diagnosis of bladder cancer are presented.

Functionalized mesoporous silica nanoparticles with mucoadhesive and sustained drug release properties for potential bladder cancer therapy.

The synthesis of a series of ß-cyclodextrin modified mesoporous silica nanoparticles with hydroxyl, amino, and thiol groups was described. A comparison of their mucoadhesive properties and potential as a drug delivery system for superficial bladder cancer therapy was made. The thiol-functionalized nanoparticles exhibit significantly higher mucoadhesivity on the urothelium as compared to the hydroxyl- and amino-functionalized nanoparticles. This is attributed to the formation of disulfide bonds between the thiol-functionalized nanoparticles and cysteine-rich subdomains of mucus glycoproteins. An anticancer drug, doxorubicin, was loaded into the mesopores of the thiol-functionalized nanoparticles, and sustained drug release triggered by acidic pH was achieved. The present study demonstrates that thiol-functionalized mesoporous silica nanoparticles are promising as a mucoadhesive and sustained drug delivery system for superficial bladder cancer therapy.

Urinary metabotyping of bladder cancer using two-dimensional gas chromatography time-of-flight mass spectrometry.

Cystoscopy is the gold standard clinical diagnosis of human bladder cancer (BC). As cystoscopy is expensive and invasive, it compromises patients' compliance toward surveillance screening and challenges the detection of recurrent BC. Therefore, the development of a noninvasive method for the diagnosis and surveillance of BC and the elucidation of BC progression become pertinent. In this study, urine samples from 38 BC patients and 61 non-BC controls were subjected to urinary metabotyping using two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS). Subsequent to data preprocessing and chemometric analysis, the orthogonal partial least-squares discriminant analysis (OPLS-DA, R2X=0.278, R2Y=0.904 and Q2Y (cumulative)=0.398) model was validated using permutation tests and receiver operating characteristic (ROC) analysis. Marker metabolites were further screened from the OPLS-DA model using statistical tests. GC×GC-TOFMS urinary metabotyping demonstrated 100% specificity and 71% sensitivity in detecting BC, while 100% specificity and 46% sensitivity were observed via cytology. In addition, the model revealed 46 metabolites that characterize human BC. Among the perturbed metabolic pathways, our clinical finding on the alteration of the tryptophan-quinolinic metabolic axis in BC suggested the potential roles of kynurenine in the malignancy and therapy of BC. In conclusion, global urinary metabotyping holds potential for the noninvasive diagnosis and surveillance of BC in clinics. In addition, perturbed metabolic pathways gleaned from urinary metabotyping shed new and established insights on the biology of human BC.

Wirelessly Activated Nanotherapeutics for In Vivo Programmable Photodynamic-Chemotherapy of Orthotopic Bladder Cancer.

Photochemical internalization (PCI) is a promising intervention using photodynamic therapy (PDT) to enhance the activity of chemotherapeutic drugs. However, current bladder cancer treatments involve high-dose chemotherapy and high-irradiance PDT which cause debilitating side effects. Moreover, low penetration of light and drugs in target tissues and cumbersome light delivery procedures hinder the clinical utility of PDT and chemotherapy combination for PCI. To circumvent these challenges, a photodynamic-chemotherapy approach is developed comprising tumor-targeting glycosylated nanocarriers, coloaded with chlorin e6 (Ce6) and gemcitabine elaidate (GemE), and a miniaturized implantable wirelessly powered light-emitting diode (LED) as a light source. The device successfully delivers four weekly light doses to the bladder while the nanocarrier promoted the specific accumulation of drugs in tumors. This approach facilitates the combination of low-irradiance PDT (1 mW cm-2 ) and low-dose chemotherapy (≈1500× lower than clinical dose) which significantly cures and controls orthotopic disease burden (90% treated vs control, 35%) in mice, demonstrating a potential new bladder cancer treatment option.

Beyond diabetes mellitus: role of metformin in non-muscle-invasive bladder cancer.

INTRODUCTION: Usage of metformin is associated with improved survival in lung, breast and prostate cancer, and metformin has been shown to inhibit cancer cell growth and proliferation in in vitro studies. Given the lack of clinical data on metformin use in patients with bladder cancer, we aimed to evaluate the role of metformin in their oncological outcomes. METHODS: Medication use data from a prospectively maintained database of 122 patients with non-muscle-invasive bladder cancer treated with intravesical Bacille Calmette-Guerin (BCG), who were recruited under a randomised, double-blinded, controlled clinical trial, was collected and analysed. Kaplan-Meier curves were used to assess overall survival (OS) and disease-specific survival (DSS). RESULTS: At a median follow-up duration of 102 (range 3-357) months, 53 (43.4%) patients experienced disease recurrence and 21 (17.2%) experienced disease progression. There was no significant difference in mortality between patients with and without diabetes mellitus. There was significant difference in OS between patients without diabetes mellitus, patients with diabetes mellitus on metformin and patients with diabetes mellitus but not on metformin (p = 0.033); patients with diabetes mellitus on metformin had the best prognosis. Metformin use was associated with significantly lower DSS (p = 0.042). Other oral hypoglycaemic agents, insulin or statins were not associated with disease recurrence or progression. CONCLUSION: Metformin use was associated with improved oncological outcomes in patients with non-muscle-invasive bladder cancer treated with intravesical BCG. Prospective studies with larger patient populations are needed to validate the role of metformin as potential therapy for bladder cancer.

Lactobacilli secreting a tumor antigen and IL15 activates neutrophils and dendritic cells and generates cytotoxic T lymphocytes against cancer cells.

Lactobacillus rhamnosus GG (LGG) has been used to successfully induce tumor regression in an orthotopic model of bladder cancer. Increased infiltration of neutrophils and macrophages into the tumor mass was observed after therapy. This study evaluates the potential of LGG to induce a directed anti-tumor response. Lactobacilli were modified to secrete the prostate specific antigen (PSA) or IL15 and PSA (IL-15-PSA). Neutrophils and DC were exposed to LGG for 2 h as in clinical therapy for bladder cancer. Recombinant LGG activated neutrophils (elevated MHC class I expression) induced DC maturation (increased expression of CD86, CD80, CD40, MHC II and CD83), T cell proliferation and PSA specific cytotoxic T lymphocytes (CTL) activity. IL15 enhanced direct DC activation of CTL. Thus LGG secreting tumor antigens may activate antigen specific immune responses when instilled intravesically and IL15 could enhance this response.

Lactobacillus rhamnosus GG secreting an antigen and Interleukin-2 translocates across the gastrointestinal tract and induces an antigen specific immune response.

Lactobacillus rhamnosus strain GG (LGG) is a probiotic organism. In this present study, LGG that express the green fluorescence protein (LGG-GFP) and IL-2 and GFP as a fusion protein (LGG-IL-2-GFP) were used to examine bacterial uptake and the immune response induced by oral immunization. Using TEM to examine the intestinal tissue, the Lactobacilli were localized in M cells and in venules. After oral immunization, most of the bacteria were excreted in feces only a small fraction (0.15%) was retained in the intestine at 48 hr. However, more LGG-IL-2-GFP was found in the MLN and spleen than LGG-GFP. The loop ligation method was used to evaluate LGG uptake and both LGG-GFP and LGG-IL-2-GFP were found to translocate at the same rate. Analysis of LGG internalization in J774 macrophage cells indicated that IL-2 increased survival of LGG and this may explain the increased presence of these bacteria in the MLN for a longer period. After oral immunization, specific mucosal antibody production as well as GFP specific CTL activity was demonstrated. IL-2 co-expression with GFP further enhanced antibody production and CTL activity. In conclusion, Lactobacillus rhamnosus GG expressing an antigen could generate an effective immune response to the antigen and IL-2 improved the response generated probably by increasing LGG expressing antigen survival in immune cells.

Tumor and microenvironment modification during progression of murine orthotopic bladder cancer.

The aim of this study was to monitor changes in the expression of immune-related genes in the bladder after tumor implantation. Mice were orthotopically implanted with MB49-PSA cells (C57BL/6 mice) on day 1 and terminated on days 7, 14, 21, and 28. Another mouse model (MBT-2/C3H mice) was examined at day 7. Gene expression analysis was performed using a TaqMan Low Density Mouse Immune Panel (Applied Biosystems, USA) on RNA extracted from the bladders. Selected genes were reconfirmed by real-time PCR analysis and RT-PCR on the mRNA from other animals. Immune suppressive (IL13, IL1ß, PTGS2, NOS2, IL10, CTLA4, and CCL22) and immune stimulatory genes (CSF2, GZMB, IFNγ, CXCL10, TNFα, CD80, IL12a, and IL6) and AGTR2 were increased by day 7. By day 28, IL10, CCL2, CCL5, CXCL11, CTLA4, GZMB, IFNγ, CSF2, and IL6 were significantly increased. Therapeutic strategies involving TH1 induction and TH2 dampening may improve responses to immunotherapy.

Intravesical High Dose BCG Tokyo and Low Dose BCG Tokyo with GMCSF+IFN α Induce Systemic Immunity in a Murine Orthotopic Bladder Cancer Model.

This study evaluates a short therapy schedule for bladder cancer using BCG Tokyo. BCG Tokyo was evaluated in vitro using bone marrow derived dendritic cells, neutrophils, RAW macrophages and the murine bladder cancer cell line, MB49PSA, and compared to other BCG strains. BCG Tokyo > BCG TICE at inducing cytokine production. In vivo, high dose (1 × 107 colony forming units (cfu)) and low dose (1 × 106 cfu) BCG Tokyo with and without cytokine genes (GMCSF + IFNα) were evaluated in C57BL/6J mice (n = 12-16 per group) with orthotopically implanted MB49PSA cells. Mice were treated with four instillations of cytokine gene therapy and BCG therapy. Both high dose BCG alone and low dose BCG combined with cytokine gene therapy were similarly effective. In the second part the responsive groups, mice (n = 27) were monitored by urinary PSA analysis for a further 7 weeks after therapy cessation. More mice were cured at day 84 than at day 42 confirming activation of the immune system. Cured mice resisted the re-challenge with subcutaneous tumors unlike naïve, age matched mice. Antigen specific T cells recognizing BCG, HY and PSA were identified. Thus, fewer intravesical instillations, with high dose BCG Tokyo or low dose BCG Tokyo with GMCSF + IFNα gene therapy, can induce effective systemic immunity.

Polymorphism in autophagy gene ATG2B is not associated with bladder cancer recurrence after intravesical Bacillus Calmette-Guerin (BCG) immunotherapy in Asian patients.

OBJECTIVES: Optimal patient stratification is critical in the era of personalized medicine. Germline polymorphisms play an important role in the treatment response of various human diseases, including bladder cancer. Intravesical BCG therapy is widely-used for bladder cancer. However, tumor recurrence and progression are very common. Stratification based on germline polymorphisms may contribute to circumvent this clinical challenge. Autophagy pathway plays an important role in the nonspecific protective effects of BCG. Patients that carry C allele of rs3759601 in autophagy gene ATG2B showed increased risk of recurrence and progression in European population. We thus sought to analyze rs3759601 and its relevance in BCG response in Asian NMIBC patients. METHOD: Functional impact of rs3759601 ATG2B (p.Gln1383Glu) was analyzed by bioinformatics programs including NCBI Conserved Domain Search, Clustal Omega, Polyphen and SIFT. NMIBC patients who received intravesical BCG at multiple hospitals in Singapore from 1995 to 2016 were included. These patients were genotyped for rs3759601 using high resolution melt analysis. The rs3759601 polymorphism was studied in correlation with the bladder cancer recurrence rate and disease progression rate in our cohort. Statistical analysis was conducted using Kaplan-Meier plots and the Chi-squared analysis. RESULTS: In total, 307 individules were included in the study including 161 NMIBC patients and 146 healthy controls, predominately Chinese. The rs3759601 genotype distributions in our NMIBC patients were (GG 72.1%; GC 27.9%; CC 0%), which were distinct from the Dutch report (GG 32.8%; GC 47.4% and CC 19.8%, Buffen K et al, 2014). Consistently, the C allele frequencies of rs3759601 are 0.171 in our controls and 0.177 in East Asians from 1,000 Genome, but 0.406 in Europeans from 1,000 Genome. In silico analysis suggested rs3759601 ATG2B (p.Gln1383Glu) alteration is unlikely to be functionally deleterious. Statistical analysis revealed no significant association between ATG2B rs3759601 C allele and risk of bladder cancer recurrence (P= 0.353, GC vs. GG: hazard ratio [HR]= 1.324), or cancer progression (P= 0.454, GC vs. GG: HR = 0.658). CONCLUSION: In contrast to European NMIBC patients, ATG2B rs3759601 C allele is much less common in Asians and it not associated with BCG response in Asian NMIBC patients.

Mindfulness Awareness Practice (MAP) to Prevent Dementia in Older Adults with Mild Cognitive Impairment: Protocol of a Randomized Controlled Trial and Implementation Outcomes.

BACKGROUND: With an aging population, developing non-pharmacological interventions (NPIs) to delay dementia has become critical. Apart from cognitive decline, dementia is associated with multiple pathophysiology, including increased oxidative stress, dysregulated gene expressions, cytokine, neurotrophin, and stress markers, telomere shortening, and deteriorations in brain connectivity. Although mindfulness practices have been proposed to ameliorate these biological changes, no empirical studies were conducted. We thus aimed to investigate the effects of mindfulness awareness practice (MAP) to prevent cognitive decline and improve peripheral biomarkers in community-dwelling older adults diagnosed with mild cognitive impairment (MCI). METHODS/DESIGN: This was a single-blinded and parallel-group randomized controlled trial with two arms (intervention and active control arms), conducted over nine months. A total of 60 consenting community-dwelling older adults diagnosed with MCI were planned to be randomized in a 1:1 ratio to either the MAP or the Health Education Program (HEP). Interventions were performed weekly for the initial 12 weeks, and monthly for the subsequent six months. Outcome measures were assessed at baseline, 3-month, and 9-month post-intervention by blinded assessors. Primary outcomes were neurocognitive tests, comprehensive peripheral biomarkers, and brain imaging scans. Secondary outcomes included basic health screening measures, affective symptoms, and measures of physical functions. Linear-mixed models were used to examine the effects of MAP on these outcome measures. SIGNIFICANCE: This is the first randomized controlled trial to systematically investigate the effects of a mindfulness intervention in improving cognitive functions and various biomarkers in community-dwelling older adults diagnosed with MCI. Our findings have the potential to inform mindfulness intervention as a novel approach to delay dementia.

Noninvasive urinary metabonomic diagnosis of human bladder cancer.

Cystoscopy is considered the gold standard for the clinical diagnosis of human bladder cancer (BC). As cystoscopy is expensive and invasive, it may compromise patients' compliance and account for the failure in detecting recurrent BC in some patients. In this paper, we investigated the role of urinary metabonomics in the diagnosis of human BC. Gas chromatography/time-of-flight mass spectrometry was applied for the urinary metabolic profiling of 24 BC patients and 51 non-BC controls. The acquired data were analyzed using multivariate principal component analysis followed by orthogonal partial least-squares discriminant analysis (OPLS-DA). Model validity was verified using permutation tests and receiver operating characteristic (ROC) analysis. BC patients were clearly distinguished from non-BC subjects based on their global urinary metabolic profiles (OPLS-DA, 4 latent variables, R(2)X = 0.420, R(2)Y = 0.912 and Q(2) (cumulative) = 0.245; ROC AUC of 0.90; 15 marker metabolites). One-hundred percent sensitivity in detecting BC was observed using urinary metabonomics versus 33% sensitivity achieved by urinary cytology. Additionally, urinary metabonomics exhibited potential in the staging and grading of bladder tumors. In summary, urinary metabonomics is amenable for the noninvasive diagnosis of human BC.