Urothelial Research Articles

Urinary and household chemical exposures in pet dogs with urothelial cell carcinoma.

Urothelial cell carcinoma (UCC) has been linked to environmental chemical exposures in people, but these risk factors are not well understood in dogs with UCC. We hypothesised that household chemical exposures contribute to the risk of UCC in pet dogs. This prospective cross-sectional case-control study included 37 dogs with UCC and 37 unaffected breed-, sex-, and age-matched controls. Dog owners completed an environmental questionnaire and household samples were collected and analysed for arsenic (in tap water and room dust) and acrolein (in room air). Urine samples from UCC dogs, control dogs, and consenting owners were analysed for inorganic arsenic species, the acrolein metabolite 3-HPMA, and the phenoxy herbicide 2,4-D. Public data on chlorination byproducts (total trihalomethanes) in municipal drinking water were also compared between case and control households. Dogs with UCC were more likely to swim in a pool (15.2%) compared with control dogs (0%) (OR 1.69, 95% CI = 1.69-∞; p = .02). Dogs with UCC also had more than 4-fold higher reported municipal water concentrations of chlorination byproducts (median 28.0 ppb) compared with controls (median 6.9 ppb; p < .0001). Dust arsenic concentrations were unexpectedly lower in case households (median 0.277 ng/cm2) compared with control households (median 0.401 ng/cm2; p = .0002). Other outcomes were not significantly different between groups. These data suggest that dog owners, especially those of breeds known to be at higher risk for UCC, consider limiting access to swimming pools and installing water filtration units that remove total trihalomethanes.

Phase II study investigating the efficacy and safety of glesatinib (MGCD265) in patients with advanced NSCLC containing MET activating alterations

ObjectivesDysregulated signaling by mesenchymal epithelial transition factor (MET) and heightened AXL activation are implicated in the pathogenesis of non-small cell lung cancer (NSCLC). Glesatinib (MGCD265) is an investigational, oral inhibitor of MET and AXL. Materials and methodsThis open-label, Phase II study investigated glesatinib (free-base suspension [FBS] capsule 1050 mg BID or spray-dried dispersion [SDD] tablet 750 mg BID) in patients with advanced, previously treated NSCLC across four cohorts grouped according to presence of MET activating mutations or amplification in tumor or ctDNA. The primary endpoint was objective response rate (ORR). ResultsSixty-eight patients were enrolled: n = 28 and n = 8 with MET exon 14 skipping mutations in tumor tissue and ctDNA, respectively, and n = 20 and n = 12 with MET gene amplification in tumor tissue and ctDNA, respectively. Overall, ORR was 11.8 %, median progression-free survival was 4.0 months, and median overall survival was 7.0 months. Among patients with MET activating mutations, ORR was 10.7 % with tumor testing and 25.0 % with ctDNA testing. For MET amplification, responses were observed only in patients enrolled by tumor testing (ORR 15.0 %). Diarrhea (82.4 %), nausea (50.0 %), increased alanine aminotransferase (41.2 %), fatigue (38.2 %), and increased aspartate aminotransferase (36.8 %) were the most frequent adverse events assessed as related to study medication. Glesatinib exposure was similar with the SDD tablet and FBS capsule formulations. The study was terminated early by the sponsor due to modest clinical activity. ConclusionsGlesatinib had an acceptable safety profile in patients with advanced, pre-treated NSCLC with MET activating alterations. Modest clinical activity was observed, which likely reflects suboptimal drug bioavailability suggested by previously reported Phase I data, and pharmacodynamic findings of lower than anticipated increases in circulating soluble shed MET ectodomain (s-MET).

Tumorigenesis of basal muscle invasive bladder cancer was mediated by PTEN protein degradation resulting from SNHG1 upregulation

BackgroundPhosphatase and tensin homolog deleted on chromosome ten (PTEN) serves as a powerful tumor suppressor, and has been found to be downregulated in human bladder cancer (BC) tissues. Despite this observation, the mechanisms contributing to PTEN’s downregulation have remained elusive.MethodsWe established targeted genes’ knockdown or overexpressed cell lines to explore the mechanism how it drove the malignant transformation of urothelial cells or promoted anchorageindependent growth of human basal muscle invasive BC (BMIBC) cells. The mice model was used to validate the conclusion in vivo. The important findings were also extended to human studies.ResultsIn this study, we discovered that mice exposed to N-butyl-N-(4-hydroxybu-tyl)nitrosamine (BBN), a specific bladder chemical carcinogen, exhibited primary BMIBC accompanied by a pronounced reduction in PTEN protein expression in vivo. Utilizing a lncRNA deep sequencing high-throughput platform, along with gain- and loss-of-function analyses, we identified small nucleolar RNA host gene 1 (SNHG1) as a critical lncRNA that might drive the formation of primary BMIBCs in BBN-treated mice. Cell culture results further demonstrated that BBN exposure significantly induced SNHG1 in normal human bladder urothelial cell UROtsa. Notably, the ectopic expression of SNHG1 alone was sufficient to induce malignant transformation in human urothelial cells, while SNHG1 knockdown effectively inhibited anchorage-independent growth of human BMIBCs. Our detailed investigation revealed that SNHG1 overexpression led to PTEN protein degradation through its direct interaction with HUR. This interaction reduced HUR binding to ubiquitin-specific peptidase 8 (USP8) mRNA, causing degradation of USP8 mRNA and a subsequent decrease in USP8 protein expression. The downregulation of USP8, in turn, increased PTEN polyubiquitination and degradation, culminating in cell malignant transformation and BMIBC anchorageindependent growth. In vivo studies confirmed the downregulation of PTEN and USP8, as well as their positive correlations in both BBN-treated mouse bladder urothelium and tumor tissues of bladder cancer in nude mice.ConclusionsOur findings, for the first time, demonstrate that overexpressed SNHG1 competes with USP8 for binding to HUR. This competition attenuates USP8 mRNA stability and protein expression, leading to PTEN protein degradation, consequently, this process drives urothelial cell malignant transformation and fosters BMIBC growth and primary BMIBC formation.

Role of Cranberry in Urinary Tract Infections

Urinary tract infection (UTI), both symptomatic and asymptomatic, occurs very frequently in the population, especially amongpatients with diabetes. With the advent of newer medications such as sodium-glucose co-transporter 2 (SGLT2) inhibitors, thereis greater focus on this disease. Cranberry products have been used earlier in various forms as a natural remedy for this disease.Certain compounds like flavonoids, distinctive proanthocyanidins with prominent active ingredients like phenolic acid inhibitthe adherence of fibrillae of Escherichia coli to the urothelial cells lining the urinary bladder. This article takes a brief look atthe disease and the role of cranberry as a natural remedy for UTI.

Targeting cancer-derived extracellular vesicles by combining CD147 inhibition with tissue factor pathway inhibitor for the management of urothelial cancer cells

BackgroundExtracellular vesicles (EVs), including microvesicles, hold promise for the management of bladder urothelial carcinoma (BLCA), particularly because of their utility in identifying therapeutic targets and their diagnostic potential using easily accessible urine samples. Among the transmembrane glycoproteins highly enriched in cancer-derived EVs, tissue factor (TF) and CD147 have been implicated in promoting tumor progression. In this in vitro study, we explored a novel approach to impede cancer cell migration and metastasis by simultaneously targeting these molecules on urothelial cancer-derived EVs.MethodsCell culture supernatants from invasive and non-invasive bladder cancer cell lines and urine samples from patients with BLCA were collected. Large, microvesicle-like EVs were isolated using sequential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and flow cytometry. The impact of urinary or cell supernatant-derived EVs on cellular phenotypes was evaluated using cell-based assays following combined treatment with a specific CD147 inhibitor alone or in combination with a tissue factor pathway inhibitor (TFPI), an endogenous anticoagulant protein that can be released by low-molecular-weight heparins.ResultsWe observed that EVs obtained from the urine samples of patients with muscle-invasive BLCA and from the aggressive bladder cancer cell line J82 exhibited higher TF activity and CD147 expression levels than did their non-invasive counterparts. The shedding of GFP-tagged CD147 into isolated vesicles demonstrated that the vesicles originated from plasma cell membranes. EVs originating from invasive cancer cells were found to trigger migration, secretion of matrix metalloproteinases (MMPs), and invasion. The same induction of MMP activity was replicated using EVs obtained from urine samples of patients with invasive BLCA. EVs derived from cancer cell clones overexpressing TF and CD147 were produced in higher quantities and exhibited a higher invasive potential than those from control cancer cells. TFPI interfered with the effect when used in conjunction with the CD147 inhibitor, further suppressing homotypic EV-induced migration, MMP production, and invasion.ConclusionsOur findings suggest that combining a CD147 inhibitor with low molecular weight heparins to induce TFPI release may be a promising therapeutic approach for urothelial cancer management. This combination can potentially suppress the tumor-promoting actions of cancer-derived microvesicle-like EVs, including collective matrix invasion.Graphical

An in-depth analysis on the effects of body composition in patients receiving neoadjuvant chemotherapy for urothelial cell carcinoma.

Neoadjuvant chemotherapy (NAC) is the standard of care for patients undergoing radical cystectomy (RC) for muscle-invasive bladder cancer (MIBC); however, NAC can be associated with significant side effects and morbidity in some patients. NAC may contribute to sarcopenia, obesity, and the combination of the two. Our study examined the effects of NAC on body composition and the association between body composition and adverse events. We created a retrospective database of patients with non-metastatic MIBC receiving NAC prior to RC. The change in skeletal muscle index (SMI) and fat mass index (FMI) was calculated using computed tomography (CT) scans done within three months prior to NAC and after the first two cycles. The association between body composition (sarcopenia, obesity, and sarcopenic obesity) and preoperative adverse events was investigated using a multivariable logistic regression. Changes in body composition were calculated using a paired Student's t-test. A total of 70 patients were included in our study. There was a mean decrease in SMI of 2.2±3.2 cm2/m2. Adiposity and FMI were unchanged by NAC. Sarcopenic obesity was found to be associated with adverse events among patients receiving NAC in the multivariable analysis. There was a total of 637 preoperative complications with grades 1-2 and 33 complications with grades 3-5. Based on our retrospective cohort study, NAC did not affect obesity and FMI, but there was a significant decrease in SMI. Sarcopenic obesity was associated with increased severity of NAC adverse events. As such, the presence of this factor may help predict tolerance of NAC.

ETV5 Silencing Produces Mesenchymal to Epithelial Transition in INS-1 (832/13) Cell Line.

ETV5 has been described to be involved in the epithelial to mesenchymal transition (EMT) mainly in cancer. It is known that EMT provokes cytoskeleton remodeling, improving cellular migratory, and invasive capabilities. Moreover, overexpression of ETV5 has been correlated to cancer development and this gene has been implicated in cell proliferation. However, little is known about the downregulation of ETV5 expression in a pancreatic cell line and the inverse mesenchymal to epithelial transition (MET). Therefore, we studied the implications of ETV5 silencing over the phenotype of the insulinoma INS-1 (832/13) cell line and described the MET by partial ETV5 silencing in the INS-1 (832/13) cell line. The downregulation of ETV5 expression was obtained by using ETV5 siRNA in the insulinoma rat cell line, INS-1 (832/13). Then, ETV5 knockdown provoked a MET phenotype observed by crystal violet staining and verified by immunohistochemistry against E-cadherin. Wound healing assay showed no migration, and F-actin stain revealed rearrangement of actin microfilaments. In addition, TGFβ1 and TGFβ3 were downregulated in the absence of ETV5. ETV5 silencing induces epithelial phenotype by downregulating TGFβ1 and TGFβ3 in INS-1 (832/13) cell line.

Urine cytology in the detection of renal cell carcinomas - a territory-wide multi-institutional retrospective review of more than 2 decades.

Compared with urothelial lesions of the upper urinary tract, the diagnostic performance of urine cytology in detection of renal cell carcinomas is underreported. This study aims to establish the role of urine cytology in the assessment of renal carcinomas by a multi-institute review of urine cytology from nephrectomy confirmed renal cell carcinomas, referenced against renal urothelial and squamous cell carcinomas. Records of nephrectomy performed from the 1990s to 2020s at three hospitals were retrieved and matched to urine cytology specimens collected within 1year prior. Patient demographics, specimen descriptors, and histology and staging parameters were reviewed and compared against cytologic diagnoses. There were 1147 cases of urine cytology matched with renal cell carcinomas, with 666 renal urothelial/squamous carcinomas for comparison. The detection rate for urothelial/squamous (atypia or above [C3+]: 63.1%; suspicious or above [C4+]: 24.0%) were higher than renal cell carcinoma (C3+: 13.1%; C4+: 1.5%) (p<0.001). The positive rate for upper tract urine exceeded other collection methods at 45.0% (C3+) and 10.0% (C4+) (p<.01). Other factors associated with increased positive rates were male sex, collecting duct carcinoma histology, nuclear grade, and renal/sinus involvement (p<.05). Multivariate analysis revealed additional positive correlations with presence of sarcomatoid tumor cells, lymphovascular invasion, and perinephric fat involvement (p<.05). Larger lesion size and higher urine volume did not improve detection rates (p<.05). The detection rate of renal cell carcinomas is suboptimal compared with urothelial carcinomas, although urine samples collected from cystoscopy or percutaneous nephrostomy significantly outperformed voided urine specimens.

Uropathogenic Escherichia coli causes significant urothelial damage in an ex vivo porcine bladder model, with no protective effect observed from cranberry or d-mannose.

Urinary tract infections (UTIs), primarily caused by uropathogenic Escherichia coli (UPEC), have an unclear impact on bladder mucosal physiology. This study investigates UPEC's effects on the urothelium and lamina propria using an ex vivo porcine bladder model. Bladder mucosal strips were analysed for contractile responses to acetylcholine, serotonin, and neurokinin A. Given rising antibiotic resistance, non-antibiotic agents such as cranberry and d-mannose were also evaluated for their potential to prevent UPEC-induced damage. The findings of the current study revealed that UPEC significantly compromised urothelial integrity, barrier function, and permeability, with loss of urothelial cells, uroplakins, and tight junction protein ZO-1 expression. Additionally, infected bladders exhibited a markedly enhanced contractile response to serotonin compared to uninfected controls. Notably, neither cranberry nor d-mannose offered protection against UPEC-mediated damage or mitigated the heightened serotonin-induced contractility. This study provides novel insights into how UPEC disrupts bladder cell biology and highlights the possible involvement of serotonin in the pathophysiology of UTIs.

Hispidulin targets PTGS2 to improve cyclophosphamide-induced cystitis by suppressing NLRP3 inflammasome.

Interstitial cystitis (IC) is a chronic bladder inflammation. Inhibition of prostaglandin G/H synthase 2 (PTGS2) is the most common method for controlling inflammation-related diseases. This study aimed to analyze the effects of hispidulin on the PTGS2 and NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammation in experimental IC models. A binding activity between hispidulin and PTGS2 was measured using molecular docking. Human urothelial cells (SV-HUC-1) were stimulated by 2ng/mL of interleukin (IL)-1β for 24h and cultured in a medium with different concentrations of hispidulin (2.5, 5, 10, 20µM) for 24h to observe the expressions of PTGS2 and NLRP3 protein. Cells overexpressing PTGS2 were established by PTGS2 cDNA transfection. In the IL-1β-treated cells, the NLRP3 inflammasome was measured after 20µM hispidulin treatment. In rats, animals were performed with three injections of 40mg/kg cyclophosphamide (CYP) and orally treated with 50mg/kg/day hispidulin or ibuprofen for 3days. The bladder pain was measured using Von Frey filaments, and the bladder pathology was observed using hematoxylin and eosin (H&E) staining. The expressions of PTGS2 and NLRP3 inflammasome were also observed in the bladder tissues. A good binding activity was found between hispidulin and PTGS2 (score = - 8.9kcal/mol). The levels of PTGS2 and NLRP3 inflammasome were decreased with the hispidulin dose increase in the IL-1β-treated cells (p < 0.05). Cells overexpressing PTGS2 weakened the protective effects of hispidulin in the IL-1β-treated cells (p < 0.01). In the CYP-treated rats, hispidulin treatment improved the bladder pain through decreasing the nociceptive score (p < 0.01) and suppressed the bladder inflammation through suppressing the expressions of PTGS2 and NLRP3 inflammasome in bladder tissues (p < 0.01). Additionally, the results of ibuprofen treatment were similar to the effects of hispidulin in the CYP-treated rats. This study demonstrates that hispidulin may be a new alternative drug for the IC treatment that binds PTGS2 to perform its functions.

A biophysically comprehensive model of urothelial afferent neurons: implications for sensory signalling in urinary bladder.

The urothelium is the innermost layer of the bladder wall; it plays a pivotal role in bladder sensory transduction by responding to chemical and mechanical stimuli. The urothelium also acts as a physical barrier between urine and the outer layers of the bladder wall. There is intricate sensory communication between the layers of the bladder wall and the neurons that supply the bladder, which eventually translates into the regulation of mechanical activity. In response to natural stimuli, urothelial cells release substances such as ATP, nitric oxide (NO), substance P, acetylcholine (ACh), and adenosine. These act on adjacent urothelial cells, myofibroblasts, and urothelial afferent neurons (UAN), controlling the contractile activity of the bladder. There is rising evidence on the importance of urothelial sensory signalling, yet a comprehensive understanding of the functioning of the urothelium-afferent neurons and the factors that govern it remains elusive to date. Until now, the biophysical studies done on UAN have been unable to provide adequate information on the ion channel composition of the neuron, which is paramount to understanding the electrical functioning of the UAN and, by extension, afferent signalling. To this end, we have attempted to model UAN to decipher the ionic mechanisms underlying the excitability of the UAN. In contrast to previous models, our model was built and validated using morphological and biophysical properties consistent with experimental findings for the UAN. The model included all the channels thus far known to be expressed in UAN, including; voltage-gated sodium and potassium channels, N, L, T, P/Q, R-type calcium channels, large-conductance calcium-dependent potassium (BK) channels, small conductance calcium-dependent (SK) channels, Hyperpolarisation activated cation (HCN) channels, transient receptor potential melastatin (TRPM8), transient receptor potential vanilloid (TRPV1) channel, calcium-activated chloride(CaCC) channels, and internal calcium dynamics. Our UAN model a) was constrained as far as possible by experimental data from the literature for the channels and the spiking activity, b) was validated by reproducing the experimental responses to current-clamp and voltage-clamp protocols c) was used as a base for modelling the non-urothelial afferent neurons (NUAN). Using our models, we also gained insights into the variations in ion channels between UAN and NUAN neurons.

Clinical and molecular characterization of urothelial (UC) vs. small cell carcinoma (SCC) of the urinary tract.

Clinical and molecular characterization of urothelial (UC) vs. small cell carcinoma (SCC) of the urinary tract.

A phase 1 study of IO102-IO103 vaccine plus pembrolizumab in patients with BCG-intolerant or unresponsive non-muscle invasive bladder cancer (NMIBC).

TPS716 Background: Pembrolizumab, an anti-PD1 antibody, is a treatment option for high-risk BCG-unresponsive NMIBC, but has a modest complete response (CR) rate of 41% at 3 months, and a median duration of CR of 16.2 months. IO102-IO103 is a novel immune-modulating cancer vaccine that stimulates activation of T cells targeting indoleamine-2,3-dioxygenase 1 (IDO1) and PDL1-positive cells, resulting in potentially increased susceptibility to anti-PD-1 blockade. Combination IO102-IO103 with nivolumab showed encouraging clinical activity and tolerability in previously anti-PD-1 naïve metastatic melanoma patients (NCT03047928). The existing data for synergistic effects of IO102-IO103 in combination with anti-PD1 therapy and the known expression of IDO in urothelial carcinoma lend rationale to this phase I trial designed to assess the feasibility, safety, and toxicity of IO102-IO103 plus pembrolizumab in patients with BCG-intolerant or unresponsive NMIBC. Methods: This open-label, single arm, phase 1 study enrolls patients with BCG-unresponsive or intolerant high-risk NMIBC who are ineligible for or have declined cystectomy and have adequate ECOG performance status (0-2), hematologic function, and end organ function. High-risk NMIBC is defined as T1, high-grade Ta, or CIS/Tis, with predominantly urothelial cell histology. Exclusion criteria include known active autoimmune disorders requiring immunosuppressive therapy or prior checkpoint inhibitor therapy. Eligible patients will be treated with pembrolizumab 200 mg IV on Day (D) 1, and IO102-IO103 85 mcg SQ on D1 and 8 of a 21-day cycle for the first two cycles, after which IO102-IO103 dosing will be D1 only, with treatment for up to 2 years. The primary endpoints of feasibility, safety, and toxicity will be evaluated by CTCAE v5.0 criteria and will be met if ≥10 patients out of 12 are able to complete the first cycle without experiencing pre-specified treatment-limiting toxicities. Key secondary endpoints include CR rate at 3 months, median duration of response, and cystectomy-free survival at 18 months. Biopsy specimens and serial urine and blood samples will be collected to evaluate potential biomarkers of response. Clinical trial information: NCT05843448 .

Engineered chloride-conducting channels in urothelial cells for potential treatment of interstitial cystitis

Engineered chloride-conducting channels in urothelial cells for potential treatment of interstitial cystitis

INVEST: A phase Ib window of opportunity study of atezolizumab administered either intravesically or direct tumour injection in patients with bladder cancer prior to radical cystectomy.

TPS710 Background: High-risk non-muscle invasive bladder cancer is a common and difficult to manage disease. The treatment of choice is radical cystectomy (RC), as BCG-unresponsive tumours have a poor prognosis. RC negatively impacts health related quality of life and therefore clinicians have explored alternative treatments. The FDA approved systemic pembrolizumab for BCG-refractory carcinoma in situ, but uptake is low, reflecting concerns around efficacy and safety for a non-invasive cancer. We hypothesize that intravesical administration of a PDL1 inhibitor could be effective with less systemic toxicity. However, it is unknown whether antibodies delivered via this route can reach the tumour vasculature. INVEST is a phase Ib window of opportunity study aiming to investigate the safety and preliminary activity of both passive instillation and direct injection into the tumour/bladder wall of intravesical atezolizumab prior to RC. Methods: Eligible participants (ECOG performance status 0-2) are awaiting RC for urothelial cell carcinoma of the bladder (any stage). Those with muscle invasive cancer must be ineligible for or refuse cisplatin based neo-adjuvant chemotherapy. RC will not be delayed by study treatment. Each dose confirmation stage uses a conventional 3+3 design to establish safety based upon dose limiting toxicities. The recommended single dose will be determined first of either 600mg or 1200mg (n=3-12). This dose will then be evaluated further to determine the recommended multiple dose of either 600mg or 1200mg administered weekly for 3-6 weeks (n=3-12). A dose expansion stage will then treat 10 patients at the recommended multiple dose to further evaluate safety and toxicity. This schema will be followed independently for both the passive instillation and direct injection routes of intravesical atezolizumab administration. Efficacy signals will be explored via progression-free survival at two years and pathological complete response rate. INVEST will assess the pharmacokinetic profile of intravesical atezolizumab, anti-drug antibodies to atezolizumab. Samples are being prospectively collected for future translational research. Sampling (fresh, frozen, and FFPE blocks) of scar, tumour and background urothelium is being performed on cystectomy specimens. Tissue microarrays will be created from TURBT and cystectomy tissue. Translational samples include pre- and post-treatment blood cfDNA, PBMCs and serum and urine cfDNA. We will assess drug penetration into the bladder wall and the effects of treatment on circulating tumour cells, and local and systemic immune cell composition. Enrolment began in May 2023. This is a single-site UK-based study and we are not currently seeking additional recruiting centres. Clinical trial information: ISRCTN15842444 .

6]-Shogaol Induces Apoptosis of Murine Bladder Cancer Cells.

Bladder cancer is considered one of the most aggressive neoplasms due to its recurrence and progression profile, and even with the improvement in diagnosis and treatment methods, the mortality rate has not shown a declining trend in recent decades. From this perspective, the search and development of more effective and safer therapeutic alternatives are necessary. Phytochemicals are excellent sources of active principles with therapeutic potential. [6]-Shogaol is a phenolic compound extracted from the ginger rhizomes that has shown antitumor effects in a wide variety of cancer models. However, there is no record in the literature of studies reporting these effects in models of bladder cancer. Thus, this study aimed to investigate the in vitro cytotoxic and pro-apoptotic potential of [6]-Shogaol against murine bladder cancer urothelial cells (MB49). The cytotoxic effects of [6]-Shogaol on cell viability (MTT method), cell morphology (light microscopy), alteration of proliferative processes (clonogenic assay), oxidative stress pathway (levels of reactive oxygen species) and the induction of apoptotic events (flow cytometry and high-resolution epifluorescence imaging) were evaluated in murine urothelial bladder cancer cell lines (MB49), relative to non-tumor murine fibroblasts (L929). The results showed that [6]-Shogaol was able to induce concentration-dependent cytotoxic effects, which compromised cell viability, exhibiting an inhibitory concentration of 50% of cells (IC50) of 146.8 µM for MB49 tumor cells and 236.0 µM for L929 non-tumor fibroblasts. In addition to inhibiting and altering the proliferative processes if colony formation, it presented pro-apoptotic activity identified through a quantitative analysis and the observation of apoptotic phenotypes, events apparently mediated by the induction of nuclear fragmentation. The data presented suggest that [6]-Shogaol has a higher concentration-dependent cytotoxic and apoptosis-inducing potential in MB49 cells than in L929 fibroblasts. These results may contribute to the development of therapeutic alternatives for bladder cancer.

Implementation of The Paris System for Reporting Urine Cytology improves diagnostic accuracy of selective upper urinary tract cytology.

The Paris System for Reporting Urine Cytology (TPS) recommends diagnostic criteria for urinary tract cytology, focusing primarily on the detection of high-grade urothelial carcinoma (HGUC) in the lower urinary tract. The second edition of TPS (TPS 2.0), published in 2022, extends these recommendations to the upper urinary tract (UUT); however, there is a lack of comprehensive data on this subject. In total, 223 consecutive UUT cytology specimens from 137 patients were retrieved and reclassified according to TPS 2.0 criteria and were compared with the original diagnosis based on the conventional system (CS). Histologic follow-up within a 3-month period was conducted for 43 patients. Histologic follow-up revealed 30 HGUCs, five low-grade urothelial carcinomas (LGUCs), and eight nonneoplastic fibrotic tissues. The risk of high-grade malignancy for each TPS diagnostic category was 16.7% for nondiagnostic/unsatisfactory, 2.3% for negative for HGUC (NHGUC), 42.1% for atypical urothelial cells, 50.0% for suspicious for HGUC (SHGUC), and 81.8% for HGUC. In all five cases of histologically diagnosed LGUC, the cytologic diagnosis was NHGUC. When SHGUC/HGUC was considered positive, the diagnostic accuracy of TPS had 63% sensitivity, 95% specificity, a 90% negative predictive value, and a 79% positive predictive value, which were better than those of CS. In addition, the TPS indices did not differ significantly between the specimens obtained before and after the application of contrast reagents. TPS implementation improved the accuracy of UUT cytology in predicting histologic HGUC, which was unaffected by the application of contrast reagents. These data indicate the usefulness of TPS for UUT cytology in routine clinical settings.

Cryptotanshinone Inhibits Bladder Cancer Cell Malignant Progression in a Lipopolysaccharide-Induced Inflammatory Microenvironment through NLRP3 Inhibition.

Bladder cancer (BC) is one of the most common malignancies of the urogenital system. This study assessed the nucleotide-binding oligomerization domain and leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) in BC as well as the effects of cryptotanshinone on changes in BC malignant behaviors and NLRP3 expression under a lipopolysaccharide (LPS)-induced inflammatory microenvironment. BC tissue specimens from 62 patients were collected for immunohistochemical detection of NLRP3 protein. BC and normal urothelial cell lines were cultured for the detection of NLRP3 mRNA and protein. Then, BC cells were pretreated with LPS to mimic the inflammatory tumor microenvironment. Next, these cells were incubated with a low or high dose of cryptotanshinone to assess its effects on tumor cell malignant behaviors as well as transfected with NLRP3 cDNA to confirm the role of NLRP3 in BC cells in vitro. High NLRP3 expression was associated with larger tumor diameters (>2 cm), muscle invasion, and metastasis. The levels of NLRP3 mRNA and protein were greater in BC cells than in normal urothelial cells. LPS pretreatment significantly promoted NLRP3 and inflammatory cytokine expression in BC cells, and induced cell viability, migration, and invasion. However, cryptotanshinone was able to reduce the LPS-induced increase of NLRP3 and inflammatory cytokine expression as well as the BC cell malignant progression. NLRP3 overexpression using NLRP3 cDNA further promoted BC cell malignant progression after LPS stimulation and reversed cryptotanshinone-reduced LPS-induced BC cell malignant behaviors. NLRP3 might possess oncogenic activity in BC, and the antitumor activity of cryptotanshinone in BC in vitro might be related to its inhibition of NLRP3 expression.

M6A modification mediates SLC3A2/SLC7A5 translation in 3-methylcholanthrene-induced uroepithelial transformation

3-Methylcholanthracene (3-MC) is one of the most carcinogenic polycyclic aromatic hydrocarbons (PAHs). Long-term exposure to PAHs has been thought of as an important factor in urothelial tumorigenesis. N6-methyladenosine (m6A) exists widely in eukaryotic organisms and regulates the expression level of specific genes by regulating mRNA stability, translation efficiency, and nuclear export efficiency. Currently, the potential molecular mechanisms that regulate m6A modification for 3-MC carcinogenesis remain unclear. Here, we profiled mRNA, m6A, translation and protein level using “-omics” methodologies, including transcriptomes, m6A profile, translatomes, and proteomics in 3-MC-transformed urothelial cells and control cells. The key molecules SLC3A2/SLC7A5 were screened and identified in 3-MC-induced uroepithelial transformation. Moreover, SLC7A5/SLC3A2 promoted uroepithelial cells malignant phenotype in vitro and in vivo. Mechanically, METTL3 and ALKBH5 mediated m6A modification of SLC3A2/SLC7A5 mRNA in 3-MC-induced uroepithelial transformation by upregulating the translation of SLC3A2/SLC7A5. Furthermore, programmable m6A modification of SLC3A2/SLC7A5 mRNA affected the expression of its proteins. Taken together, our results revealed that the m6A modification-mediated SLC3A2/SLC7A5 translation promoted 3-MC-induced uroepithelial transformation, suggesting that targeting m6A modification of SLC3A2/SLC7A5 may be a potential therapeutic strategy for bladder cancer related to PAHs.

High and selective cytotoxicity of ex vivo expanded allogeneic human natural killer cells from peripheral blood against bladder cancer: implications for natural killer cell instillation after transurethral resection of bladder tumor

BackgroundNon-muscle-invasive bladder cancer (NMIBC) is treated with transurethral resection of bladder tumor (TURBT) followed by intravesical instillation of chemotherapy or Bacillus Calmette–Guérin therapy. However, these treatments have a high recurrence rate and side effects, emphasizing the need for alternative instillations. Previously, we revealed that expanded allogeneic human natural killer (NK) cells from peripheral blood are a promising cellular therapy for prostate cancer. However, whether NK cells exhibit a similar killing effect in bladder cancer (BCa) remains unknown.MethodsExpansion, activation, and cryopreservation of allogeneic human NK cells obtained from peripheral blood were performed as we previously described. In vitro cytotoxicity was evaluated using the cell counting kit-8. The levels of perforin, granzyme B, interferon-γ, tumor necrosis factor-α, and chemokines (C-C-motif ligand [CCL]1, CCL2, CCL20, CCL3L1, and CCL4; C-X-C-motif ligand [CXCL]1, CXCL16, CXCL2, CXCL3, and CXCL8; and X-motif ligand 1 and 2) were determined using enzyme-linked immunosorbent assay. The expression of CD107a, major histocompatibility complex class I (MHC-I), MHC-I polypeptide-related sequences A and B (MICA/B), cytomegalovirus UL16-binding protein-2/5/6 (ULBP-2/5/6), B7-H6, CD56, CD69, CD25, killer cell Ig-like receptors (KIR)2DL1, KIRD3DL1, NKG2D, NKp30, NKp46, and CD16 of NK cells or BCa and normal urothelial cells were detected using flow cytometry. Cytotoxicity was evaluated using lactate dehydrogenase assay in patient-derived organoid models. BCa growth was monitored in vivo using calipers in male NOD-scid IL2rg−/− mice subcutaneously injected with 5637 and NK cells. Differential gene expressions were investigated using RNA sequence analysis. The chemotaxis of T cells was evaluated using transwell migration assays.ResultsWe revealed that the NK cells possess higher cytotoxicity against BCa lines with more production of cytokines than normal urothelial cells counterparts in vitro, demonstrated by upregulation of degranulation marker CD107a and increased interferon-γ secretion, by MICA/B/NKG2D and B7H6/NKp30-mediated activation. Furthermore, NK cells demonstrated antitumor effects against BCa in patient-derived organoids and BCa xenograft mouse models. NK cells secreted chemokines, including CCL1/2/20, to induce T-cell chemotaxis when encountering BCa cells.ConclusionsThe expanded NK cells exhibit potent cytotoxicity against BCa cells, with few toxic side effects on normal urothelial cells. In addition, NK cells recruit T cells by secreting a panel of chemokines, which supports the translational application of NK cell intravesical instillation after TURBT from bench to bedside for NMIBC treatment.