Urothelial Research Articles

Loss of ORP3 induces aneuploidy and promotes bladder cancer cell invasion through deregulated microtubule and actin dynamics

We have recently shown that loss of ORP3 leads to aneuploidy induction and promotes tumor formation. However, the specific mechanisms by which ORP3 contributes to ploidy-control and cancer initiation and progression is still unknown. Here, we report that ORP3 is highly expressed in ureter and bladder epithelium while its expression is downregulated in invasive bladder cancer cell lines and during tumor progression, both in human and in mouse bladder cancer. Moreover, we observed an increase in the incidence of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced invasive bladder carcinoma in the tissue-specific Orp3 knockout mice. Experimental data demonstrate that ORP3 protein interacts with γ-tubulin at the centrosomes and with components of actin cytoskeleton. Altering the expression of ORP3 induces aneuploidy and genomic instability in telomerase-immortalized urothelial cells with a stable karyotype and influences the migration and invasive capacity of bladder cancer cell lines. These findings demonstrate a crucial role of ORP3 in ploidy-control and indicate that ORP3 is a bona fide tumor suppressor protein. Of note, the presented data indicate that ORP3 affects both cell invasion and migration as well as genome stability through interactions with cytoskeletal components, providing a molecular link between aneuploidy and cell invasion and migration, two crucial characteristics of metastatic cells.

The p38-MITOGEN-ACTIVATED PROTEIN KINASE Signaling Pathway Is Involved in Leonurus artemisia Extract-Induced Inhibition of the Proliferation of Human Bladder Cancer BFTC-905 Cells via G1/G0 Arrest and Causes Apoptosis In Vitro.

Bladder cancer is a urothelial malignancy. Bladder cancer starts in the urothelial cells lining the inside of the bladder. The 5-year recurrence rate for bladder cancer ranges from 31% to 78%, and the progression rate is approximately 45%. To treat bladder cancer, intravesical drug therapy is often used. Leonurus artemisia extract (LaE) was obtained from medicinal samples of Chinese motherwort Scientific Chinese Medicine; L. artemisia has various biological effects. This study investigated the impact of LaE on human bladder cancer cells (the BFTC-905 cell line) and the molecular mechanism underlying apoptosis resulting from the activation of cell signal transduction pathways in bladder cancer cells. A cell counting kit-8 (CCK-8) assay was used to determine the effect of LaE on cell growth. The effect of LaE on migration ability was observed using a wound healing assay. The effects of LaE on the cell cycle, reactive oxygen species production, and apoptosis were investigated. Western blot analysis detected apoptosis-related and mitogen-activated protein kinase signaling pathway-related protein concentrations. At non-toxic concentrations, LaE inhibited the proliferation of BFTC-905 cells in a concentration-dependent manner, and the half-maximal inhibitory concentration (IC50) was 24.08172 µg/µL. LaE impaired the migration ability of BFTC-905 cells. LaE arrested the cell cycle in the G1 and G0 phases, increased reactive oxygen species production, and induced apoptosis. LaE increased Bax and p-ERK concentrations and decreased Bcl-2, cleaved caspase-3, and p-p38 concentrations. No differences in PARP, C-PARP, vimentin, e-cadherin, p-JNK, or TNF-alpha concentrations were observed. These results suggest that LaE inhibits the proliferation of human bladder cancer cells. Moreover, the mitogen-activated protein kinase signaling pathway is involved in the inhibition of the proliferation of BFTC-905 cells.

A novel rapid detection method for a single-nucleotide substitution mutation derived from canine urothelial and prostatic carcinoma cells present in small amounts in urine sediments.

For early detection of canine urothelial and prostatic carcinoma, we intend to develop and commercialize a simple and rapid detection method for the BRAF V595E mutation, a known mutation in this cancer. Detection of the single-nucleotide substitution in cancer cells contained in urine sediments is effective for early cancer diagnosis. However, urine sediment also contains many normal cells, and when there is a small relative composition of cancer cells, the mutation is difficult to detect by conventional methods other than next-generation sequencing. Our new detection method enables reliable discrimination with the same labor and cost as the PCR method. We compared the results of our new method with the results of the conventional Sanger method for 38 canine urine sediment samples, and the results of 34 samples were consistent between both methods. The remaining four results were all determined to be negative by the Sanger method and positive by our new method. For these four samples, the ratio of the mutated gene to the wild-type gene was estimated using a third-generation sequencer, and the ratio of the mutated gene was 0.1%-1.4%. We postulate that the Sanger method gave a negative result because of the low abundance of the mutated gene in these samples, proving the high sensitivity of our new method.

A comprehensive pan-cancer analysis of CDH5 in immunological response.

Cadherin 5 (CDH5) functions critically in maintaining cell adhesion and integrity of endothelial and vascular cells. The expression of CDH5 is abnormal in tumor cells, which may have great potential to serve as a new immune checkpoint. The current pan-cancer analysis was performed to better understand the role of CDH5 in tumor. The clinical significance and immunological function of CDH5 in pan-cancers were comprehensively analyzed based on the correlations between CDH5 and clinicopathologic features, prognosis values, tumor mutation burden (TMB), microsatellite instability (MSI), immune cells infiltration and immune response genes using 33 datasets from The Cancer Genome Atlas (TCGA). We further confirmed the expression of CDH5 in bladder cancer (BCa) tissues and cell lines. The CD8+ T cells were screened from peripheral blood of healthy controls and activated. BCa cell-CD8+ T cell co-culture assay and ELISA assay were carried out to verify the immunological function of CDH5. The expression of CDH5 was down-regulated in 8 types of tumors including in BCa but up-regulated in 4 types of tumors. CDH5 was significantly correlated with tumor stage in 6 types of tumors. In addition, CDH5 was positively or negatively correlated with tumor prognosis. Furthermore, CDH5 was closely associated with TMB in 15 types of tumors and with MSI in 9 types of tumors. KEGG-GSEA and Hallmarks-GSEA analyses results indicated that CDH5 was positively related to immune response in most tumor types. In many tumors, CDH5 showed a positive correlation with immune cell infiltration. Enrichment analyses demonstrated that CDH5 was significantly related to the expression of many immunomodulators and chemokines. Further experiments showed that CDH5 was low-expressed in BCa tissues and cell lines in comparison to adjacent normal tissues and normal urothelial cell line, but it was positively associated with a better prognosis of BCa patients. The results of in vitro co-culture assay and ELISA assay demonstrated that CDH5 could promote the function of CD8+ T cells in TME of BCa. In summary, CDH5 was positively associated with a favorable prognosis and effective immune response in tumors, showing a great potential to serve as a novel tumor biomarker and immune checkpoint.

Drug Resistance Mechanism and Therapeutic Strategy of Targeted Therapy of Non-small Cell Lung Cancer with MET Alterations

Mesenchymal to epithelial transition factor (MET) gene alterations involve in the proliferation, invasion, and metastasis of non-small cell lung cancer. MET-tyrosine kinase inhibitors (TKIs) have been approved to treat non-small cell lung cancer with MET alterations, and resistance to these TKIs is inevitable. Molecular mechanisms of resistance to MET-TKIs are completely unclear. The review focused on potential mechanisms of MET-TKIs resistance and therapeutics strategies to delay and prevent resistance. .

Geospatial co-distribution of human and canine urothelial cell carcinoma and associated environmental risk factors in Queensland, Australia

Geospatial co-distribution of human and canine urothelial cell carcinoma and associated environmental risk factors in Queensland, Australia

Nadofaragene firadenovec: a breakthrough in the field of bladder oncology

Muscle-invasive bladder tumors pose a grave mortality risk due to their propensity for distant metastases. The therapeutic spectrum for such tumors encompasses surgery, chemotherapy, and radiation, tailored to the cancer’s severity. In the context of high-risk Bacillus Calmette-Guérin (BCG)-unresponsive non-muscle invasive bladder cancer (NMIBC), a novel treatment has emerged as a beacon of hope. Nadofaragene firadenovec, a pioneering gene therapy, has gained worldwide approval for combating this condition, marking a watershed moment in bladder cancer therapy. Nadofaragene firadenovec is ingeniously designed to address high-risk BCG-unresponsive NMIBC, particularly carcinoma in situ (CIS) with or without papillary tumors, in adult patients. Rooted in a vector DNA, this therapy encodes interferon (IFN)-2b, which imparts urothelial cells with the ability to generate IFN-2b. The resulting cascade of events triggers a multifaceted assault on cancer, characterized by its immunostimulatory, antiangiogenic, and apoptotic effects. The therapeutic efficacy of nadofaragene firadenovec rests on its capacity to exploit the transformed urothelial cells to deliver these targeted anticancer activities. The evolutionary trajectory of nadofaragene firadenovec culminated in its monumental approval in December 2022 by the United States, signifying a pivotal juncture in the field. Notably, a segment of patients, approximately 30%, prove refractory to BCG treatment. For these individuals, alternative therapeutic avenues are imperative. Presently, the landscape for patients with non-muscle invasive bladder cancer lacks a definitive, enduring solution. Against this backdrop, the introduction of nadofaragene firadenovec heralds a momentous stride toward the global availability of an authorized therapeutic intervention.

Intravesical injections of autologous platelet-rich plasma for the treatment of refractory interstitial cystitis.

The urothelium acts as a barrier for the urinary bladder that prevents the influx of urinary toxic substances, electrolytes, urea nitrogen, and pathogens into the circulation. Acute or chronic inflammation of the urinary bladder may impair the regenerative function of urothelial cells and thus urothelial cell differentiation. In an inflamed bladder wall, mature apical cells are defective, resulting in impaired barrier function and thus increased urothelial permeability. This is considered to be the potential mechanism of the symptom trigger in patients with interstitial cystitis/bladder pain syndrome (IC/BPS). Previous studies have revealed that increased bladder inflammation, impaired urothelial cell maturation, a defective umbrella cell barrier, and defective junction proteins are prominent in IC/BPS bladders. Platelet-rich plasma (PRP) contains many growth factors and cytokines that are essential proteins for modulating inflammation and promoting tissue regeneration and thus wound healing. As such, PRP has been used as a regenerative therapy in many medical fields. Our preliminary studies have demonstrated that multiple intravesical PRP injections could improve symptoms in 70% of IC/BPS patients. Repeated PRP treatments also improve junctional protein, increase cytoskeleton protein expression, and decrease urinary inflammatory proteins. These preliminary results suggest that PRP injections might reduce bladder inflammation and improve urothelial cell regeneration in IC/BPS patients. This article reviews recently published clinical and basic research on the treatment potential of PRP for IC/BPS patients.

Tumorigenic effects of human mesenchymal stromal cells and fibroblasts on bladder cancer cells.

Patients with muscle-invasive bladder cancer face a poor prognosis due to rapid disease progression and chemoresistance. Thus, there is an urgent need for a new therapeutic treatment. The tumor microenvironment (TME) has crucial roles in tumor development, growth, progression, and therapy resistance. TME cells may also survive standard treatment of care and fire up disease recurrence. However, whether specific TME components have tumor-promoting or tumor-inhibitory properties depends on cell type and cancer entity. Thus, a deeper understanding of the interaction mechanisms between the TME and cancer cells is needed to develop new cancer treatment approaches that overcome therapy resistance. Little is known about the function and interaction between mesenchymal stromal cells (MSC) or fibroblasts (FB) as TME components and bladder cancer cells. We investigated the functional impact of conditioned media (CM) from primary cultures of different donors of MSC or FB on urothelial carcinoma cell lines (UCC) representing advanced disease stages, namely, BFTC-905, VMCUB-1, and UMUC-3. Underlying mechanisms were identified by RNA sequencing and protein analyses of cancer cells and of conditioned media by oncoarrays. Both FB- and MSC-CM had tumor-promoting effects on UCC. In some experiments, the impact of MSC-CM was more pronounced. CM augmented the aggressive phenotype of UCC, particularly of those with epithelial phenotype. Proliferation and migratory and invasive capacity were significantly increased; cisplatin sensitivity was reduced. RNA sequencing identified underlying mechanisms and molecules contributing to the observed phenotype changes. NRF2 and NF-κB signaling was affected, contributing to improved cisplatin detoxification. Likewise, interferon type I signaling was downregulated and regulators of epithelial mesenchymal transition (EMT) were increased. Altered protein abundance of CXCR4, hyaluronan receptor CD44, or TGFβ-signaling was induced by CM in cancer cells and may contribute to phenotypical changes. CM contained high levels of CCL2/MCP-1, MMPs, and interleukins which are well known for their impact on other cancer entities. The CM of two different TME components had overlapping tumor-promoting effects and increased chemoresistance. We identified underlying mechanisms and molecules contributing to the aggressiveness of bladder cancer cells. These need to be further investigated for targeting the TME to improve cancer therapy.

Pathway from Acute Kidney Injury to Chronic Kidney Disease: Molecules Involved in Renal Fibrosis.

Acute kidney injury (AKI) is one of the main conditions responsible for chronic kidney disease (CKD), including end-stage renal disease (ESRD) as a long-term complication. Besides short-term complications, such as electrolyte and acid-base disorders, fluid overload, bleeding complications or immune dysfunctions, AKI can develop chronic injuries and subsequent CKD through renal fibrosis pathways. Kidney fibrosis is a pathological process defined by excessive extracellular matrix (ECM) deposition, evidenced in chronic kidney injuries with maladaptive architecture restoration. So far, cited maladaptive kidney processes responsible for AKI to CKD transition were epithelial, endothelial, pericyte, macrophage and fibroblast transition to myofibroblasts. These are responsible for smooth muscle actin (SMA) synthesis and abnormal renal architecture. Recently, AKI progress to CKD or ESRD gained a lot of interest, with impressive progression in discovering the mechanisms involved in renal fibrosis, including cellular and molecular pathways. Risk factors mentioned in AKI progression to CKD are frequency and severity of kidney injury, chronic diseases such as uncontrolled hypertension, diabetes mellitus, obesity and unmodifiable risk factors (i.e., genetics, older age or gender). To provide a better understanding of AKI transition to CKD, we have selected relevant and updated information regarding the risk factors responsible for AKIs unfavorable long-term evolution and mechanisms incriminated in the progression to a chronic state, along with possible therapeutic approaches in preventing or delaying CKD from AKI.

G protein-coupled receptor 55 activated by palmitoylethanolamide is associated with the development of nocturia associated with circadian rhythm disorders

AimsBladder function is regulated by clock genes and dysregulation of circadian bladder function can cause nocturia. The blood concentration of palmitoylethanolamide (PEA), a fatty acid metabolite, changes with circadian rhythm. Clock gene abnormalities demonstrate the highest PEA levels during the sleep phase. PEA is a GPR55 agonist that influences urination; therefore, increased PEA during the sleep phase may cause nocturia. Herein, we investigated the function of GPR55 to evaluate the relationship between GPR55 and nocturia that evoked higher PEA during the sleep phase in patients with circadian rhythm disorders. Main methodsMale C57BL/6 mice were used. GPR55 localization was evaluated by immunofluorescence staining, qRT-PCR, and western blotting. Variations in PEA-induced intracellular Ca2+ concentrations were measured in primary cultured mouse urothelial cells (UCs) using Ca2+ imaging. PEA-induced NGF and PGI2 release in UCs was measured by ELISA. The micturition reflex pathway after PEA administration was evaluated using immunofluorescence staining. Key findingsGPR55 was predominant in the UC layer. PEA induced release of Ca2+ from the endoplasmic reticulum into the UC cytoplasm. ELISA and immunofluorescence staining revealed that NGF and PGI2 were released from bladder UCs, stimulated the pontine micturition center in mice, and induced nocturia. SignificanceThe loss of regular circadian metabolizing rhythm in fatty acids causes higher blood PEA levels during the sleep phase. Binding of PEA to GPR55 in UC may activate the downstream processes of the micturition reflex, leading to nocturia. These findings suggest a new mechanism for nocturia and its potential as a therapeutic target.

Synthesis and secretion of Nerve Growth Factor is regulated by Nitric Oxide in bladder cells in vitro under a hyperglycemic environment

Urine samples of female patients with overactive bladder (OAB) are characterized by low levels of nerve growth factor (NGF) and elevated concentrations of nitric oxide (NO) compared to healthy controls. We therefore examined how NO might regulate NGF synthesis using rat bladder smooth muscle (SMCs) and urothelial (UROs) cells in culture. In UROs, incubation in hyperglycemic conditions to mimic insulin insensitivity present in the OAB cohort increased secretion of NO and concomitantly decreased NGF, except when the NO synthase inhibitor, l-NAME (1 mM) was present. Sodium nitroprusside (SNP) (300 μM, 24 h), a NO generator, decreased NGF levels and decreased cyclic GMP (cGMP) content, a process validated by the cGMP synthase inhibitor ODQ (100 μM). Alternatively, SNP increased mRNA of both NGF and matrix metalloproteinase-9 (MMP-9). MMP-9 knockout of UROs by Crispr-Cas9 potently decreased the effect of SNP on NGF, implying a dependent role of NO on MMP-9. On the other hand, matrix metalloproteinase-7 (MMP-7) activity was increased by SNP, which taken together with increase in NGF mRNA, suggests a compensatory mechanism. In SMCs, hyperglycemic conditions had the same effect on extracellular content of NO and NGF than in UROs. SNP also decreased NGF secretion but increased cGMP content. Stable permeable analogs of cGMP 8-(4-Chlorophenylthio)-cGMP (1 mM) and N2,2′-O-Dibutyryl-cGMP (3 mM) inhibited NGF release. NGF and MMP-9 mRNA expression was unchanged by SNP. Deletion of MMP-9 in SMCs by Crispr-Cas9 did not alter the effect of SNP. Finally, SNP decreased MMP-7 activity, diminishing the conversion of proNGF to NGF. These results demonstrate that enhanced NO secretion triggered by high glucose decreases NGF secretion through pathways unique for each cell type that involve cGMP and proteases MMP-7 and MMP-9. These results might help to explain our observations from the urine from patients with OAB associated with metabolic syndrome.

Change in Splenic Volume as a Surrogate Marker for Immunotherapy Response in Patients with Advanced Urothelial and Renal Cell Carcinoma-Evaluation of a Novel Approach of Fully Automated Artificial Intelligence Based Splenic Segmentation.

In the treatment of advanced urothelial (aUC) and renal cell carcinoma (aRCC), biomarkers such as PD-1 and PD-L1 are not robust prognostic markers for immunotherapy (IO) response. Previously, a significant association between IO and a change in splenic volume (SV) was described for several tumour entities. To the best of our knowledge, this study presents the first correlation of SV to IO in aUC and aRCC. All patients with aUC (05/2017-10/2021) and aRCC (01/2012-05/2022) treated with IO at our academic centre were included. SV was measured at baseline, 3 and 9 months after initiation of IO using an in-house developed convolutional neural network-based spleen segmentation method. Uni- and multivariate Cox regression models for overall survival (OS) and progression-free survival (PFS) were used. In total, 35 patients with aUC and 30 patients with aRCC were included in the analysis. Lower SV at the three-month follow-up was significantly associated with improved OS in the aRCC group. We describe a new, innovative artificial intelligence-based approach of a radiological surrogate marker for IO response in aUC and aRCC which presents a promising new predictive imaging marker. The data presented implicate improved OS with lower follow-up SV in patients with aRCC.

Diuron and its metabolites induce mitochondrial dysfunction-mediated cytotoxicity in urothelial cells

In the environment, or during mammalian metabolism, the diuron herbicide (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is transformed mainly into 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU) and 3,4-dichloroaniline (DCA). Previous research suggests that such substances are toxic to the urothelium of Wistar rats where, under specific exposure conditions, they may induce urothelial cell degeneration, necrosis, hyperplasia, and eventually tumors. However, the intimate mechanisms of action associated with such chemical toxicity are not fully understood. In this context, the purpose of the current in vitro study was to analyze the underlying mechanisms involved in the urothelial toxicity of those chemicals, addressing cell death and the possible role of mitochondrial dysfunction. Thus, human 1T1 urothelial cells were exposed to six different concentrations of diuron, DCA, and DCPMU, ranging from 0.5 to 500 µM. The results showed that tested chemicals induced oxidative stress and mitochondrial damage, cell cycle instability, and cell death, which were more expressive at the higher concentrations of the metabolites. These data corroborate previous studies from this laboratory and, collectively, suggest mitochondrial dysfunction as an initiating event triggering urothelial cell degeneration and death.

LP-33: A urothelial cell model for the new therapeutics efficacy assessment

LP-33: A urothelial cell model for the new therapeutics efficacy assessment

P53, CK20, and FGFR3 Overexpression is Associated with the Characteristics of Urothelial Cell Carcinoma of the Bladder

The aim of this study was to investigate the association between the overexpression of tumor protein (P53), cytokeratin 20 (CK20), fibroblast growth factor receptor 3 (FGFR3), biomarkers and the grading, prognosis, heterogeneity, and relapse tendency of urothelial cell carcinomas (UCCs) of the bladder. A cross-sectional study was conducted using 413 samples of Iranian patients diagnosed with UCC of the bladder. The tissue microarray technique was used to evaluate the patterns of tumor tissue. Two pathologists scored tissue staining using a semi-quantitative scoring system. The results showed that P53 was a predictor of a high-grade pattern (the area under the curve (AUC)=0.620) with a best cut-off value of 95.0 using the receiver operating characteristic (ROC) curve. CK20 was another predictor of a high-grade pattern (AUC=0.745) with a best cut-off value of 15. However, the overexpression of both biomarkers was not associated with a heterogeneous pattern and could not predict tumor-associated death or relapse. The heterogeneous (odds ratio (OR)=4.535, p-value=0.001) and non-papillary (OR= 6.363, p-value= 0.001) patterns were effective predictors of tumor recurrence among all baseline variables, including patient and tumor characteristics. FGFR3 was positive in all specimens and was not a valuable biomarker for differentiating patterns. None of the variables predicted tumor prognosis. The study findings indicate that the intensity and percentage of cell staining for P53 and CK20 in the UCC of the bladder can aid in differentiating the grading patterns. The tendency of tumor relapse can be predicted by demonstrating heterogeneous and non-papillary patterns.

Bladder malignancy as a cause of spontaneous bladder rupture: A systematic review.

To characterise cases of spontaneous rupture of the urinary bladder in the context of bladder cancer. A systematic review was performed to characterise cases of spontaneous bladder rupture in patients with bladder cancer. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) system was utilised, with databases being searched for relevant cases. Patient characteristics were extracted, including age, sex, presenting signs and symptoms, management modalities, tumour histology and mortality. Thirty cases were included. Seventeen (57%) were male, and the median age of presentation was 59. Abdominal pain and peritonism were the most common presenting symptoms, in 80% and 60% of patients, respectively. Most patients (n = 16, 53%) had urothelial cell carcinoma. Nine patients (30%) died during their initial hospitalisation. Spontaneous bladder perforation in the context of bladder cancer is a rare cause of acute abdomen. The diagnosis is associated withhigh mortality, highlighting the aggressive nature of the malignancies that cause spontaneous bladder rupture. This raises important questions about the role of emergency cystectomy, the timing of systemic therapy and the appropriate involvement of palliative care.

Differential functions of RhoGDIβ in malignant transformation and progression of urothelial cell following N-butyl-N-(4-hydmoxybutyl) nitrosamine exposure

BackgroundFunctional role of Rho GDP-dissociation inhibitor beta (RhoGDIβ) in tumor biology appears to be contradictory across various studies. Thus, the exploration of the molecular mechanisms underlying the differential functions of this protein in urinary bladder carcinogenesis is highly significant in the field. Here, RhoGDIβ expression patterns, biological functions, and mechanisms leading to transformation and progression of human urothelial cells (UROtsa cells) were evaluated following varying lengths of exposure to the bladder carcinogen N-butyl-N-(4-hydmoxybutyl) nitrosamine (BBN).ResultsIt was seen that compared to expression in vehicle-treated control cells, RhoGDIβ protein expression was downregulated after 2-month of BBN exposure, but upregulated after 6-month of exposure. Assessments of cell function showed that RhoGDIβ inhibited UROtsa cell growth in cells with BBN for 2-month exposure, whereas it promoted the invasion of cells treated with BBN for 6 months. Mechanistic studies revealed that 2-month of BBN exposure markedly attenuated DNMT3a abundance, and this led to reduced miR-219a promoter methylation, increased miR-219a binding to the RhoGDIβ mRNA 3’UTR, and reduced RhoGDIβ protein translation. While after 6-mo of BBN treatment, the cells showed increased PP2A/JNK/C-Jun axis phosphorylation and this in turn mediated overall RhoGDIβ mRNA transcription and protein expression as well as invasion.ConclusionsThese findings indicate that RhoGDIβ is likely to inhibit the transformation of human urothelial cells during the early phase of BBN exposure, whereas it promotes invasion of the transformed/progressed urothelial cells in the late stage of BBN exposure. The studies also suggest that RhoGDIβ may be a useful biomarker for evaluating the progression of human bladder cancers.

Real-World Data on Combined EGFR-TKI and Crizotinib Treatment for Acquired and De Novo MET Amplification in Patients with Metastatic EGFR-Mutated NSCLC.

Amplification of the mesenchymal epithelial transition (MET) gene is a mechanism of acquired resistance to epidermal growth factor receptor (EGFR)-tyrosine-kinase-inhibitors (TKIs) in over 20% of patients with advanced EGFR-mutated (EGFRm+) non-small lung cancer (NSCLC). However, it may also occur de novo in 2-8% of EGFRm+ NSCLC cases as a potential mechanism of intrinsic resistance. These patients represent a group with unmet needs, since there is no standard therapy currently approved. Several new MET inhibitors are being investigated in clinical trials, but the results are awaited. Meanwhile, as an alternative strategy, combinations of EGFR-TKIs with the MET/ALK/ROS1-TKI Crizotinib may be used in this setting, despite this use is principally off-label. Thus, we studied five of these MET amplified cases receiving EGFR-TKI and Crizotinib doublet after progression on EGFR-TKI treatment to assess the benefits and challenges related to this combination and the possible occurrence of genomic and phenotypic co-alterations. Furthermore, we compared our cases with other real-world reports on Crizotinib/EGFR-TKI combinations, which appeared effective, especially in patients with high-level MET amplification. Yet, we observed that the co-occurrence of other genomic and phenotypical alterations may affect the response to combined EGFR-TKI and Crizotinib. Finally, given the heterogeneity of MET amplification, the diagnostic methods for assessing it may be discrepant. In this respect, we observed that for optimal detection, immunohistochemistry, fluorescence in situ hybridization, and next-generation sequencing should be used together, as these methods possess different sensitivities and complement each other in characterizing MET amplification. Additionally, we addressed the issue of managing EGFR-mutated NSCLC patients with de novo MET amplification causing primary EGFR-TKI resistance. We conclude that, while data from clinical trials with new MET inhibitors are still pending, adding Crizotinib to EGFR-TKI in NSCLC patients acquiring MET amplification at progression on EGFR-TKI monotherapy is a reasonable approach, with a progression-free survival of 3-19 months.

Insights into cellular behavior and micromolecular communication in urothelial micrografts

Autologous micrografting is a technique currently applied within skin wound healing, however, the potential use for surgical correction of other organs with epithelial lining, including the urinary bladder, remains largely unexplored. Currently, little is known about the micrograft expansion potential and the micromolecular events that occur in micrografted urothelial cells. In this study, we aimed to evaluate the proliferative potential of different porcine urothelial micrograft sizes in vitro, and, furthermore, to explore how urothelial micrografts communicate and which microcellular events are triggered. We demonstrated that increased tissue fragmentation subsequently potentiated the yield of proliferative cells and the cellular expansion potential, which confirms, that the micrografting principles of skin epithelium also apply to uroepithelium. Furthermore, we targeted the expression of the extracellular signal-regulated kinase (ERK) pathway and demonstrated that ERK activation occurred predominately at the micrograft borders and that ERK inhibition led to decreased urothelial migration and proliferation. Finally, we successfully isolated extracellular vesicles from the micrograft culture medium and evaluated their contents and relevance within various enriched biological processes. Our findings substantiate the potential of applying urothelial micrografting in future tissue-engineering models for reconstructive urological surgery, and, furthermore, highlights certain mechanisms as potential targets for future wound healing treatments.