Progress in the Research and Targeted Therapy of ErbB/HER Receptors in Urothelial Bladder Cancer

Bladder cancer is a lethal malignancy and a majority of bladder cancer arise from urothelial cells. Infiltration and metastasis are barriers for the radical cystectomy to achieve favored outcome and are the main cause of death. Systemic therapy, including chemotherapy, targeted therapy, and immunotherapy, is fundamental for these patients. erbB/HER receptors are found to be overexpressed in a subgroup of urothelial carcinoma, targeting erbB/HER receptors in these patients was found to be an efficient way in the era of genetic testing. To evaluate the role of erbB/HER receptors in bladder cancer, we reviewed the literature and ongoing clinical trials as regards to this topic to unveil the context of erbB/HER receptors in bladder cancer, which probably help to solidate the theoretical basis and might instruct further research.

Supplementation of Probiotic Butyricicoccus pullicaecorum Mediates Anticancer Effect on Bladder Urothelial Cells by Regulating Butyrate-Responsive Molecular Signatures

In bladder cancer, urothelial carcinoma is the most common histologic subtype, accounting for more than 90% of cases. Pathogenic effects due to the dysbiosis of gut microbiota are localized not only in the colon, but also in regulating bladder cancer distally. Butyrate, a short-chain fatty acid produced by gut microbial metabolism, is mainly studied in colon diseases. Therefore, the resolution of the anti-cancer effects of butyrate-producing microbes on bladder urothelial cells and knowledge of the butyrate-responsive molecules must have clinical significance. Here, we demonstrate a correlation between urothelial cancer of the bladder and Butyricicoccus pullicaecorum. This butyrate-producing microbe or their metabolite, butyrate, mediated anti-cancer effects on bladder urothelial cells by regulating cell cycle, cell growth, apoptosis, and gene expression. For example, a tumor suppressor against urothelial cancer of the bladder, bladder cancer-associated protein, was induced in butyrate-treated HT1376 cells, a human urinary bladder cancer cell line. In conclusion, urothelial cancer of the bladder is a significant health problem. To improve the health of bladder urothelial cells, supplementation of B. pullicaecorum may be necessary and can further regulate butyrate-responsive molecular signatures.

Evasion of Toll-like Receptor Recognition by Escherichia coli is mediated via Population Level Regulation of Flagellin Production

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infections. Analysis of the innate immune response in immortalised urothelial cells suggests that the bacterial flagellar subunit, flagellin, is key in inducing host defences. A panel of 39 clinical uro-associated Escherichia coli isolates recovered from either asymptomatic bacteruria (ASB), cystitis or pyelonephritis patients, were characterised for motility and their ability to induce an innate response in urothelial cells stably transfected with a NFκB luciferase reporter. Twenty-four isolates (60%) were identified as motile with strains recovered from cystitis patients exhibiting a bipolar motility distribution pattern (P < 0.005) and associated with a 2-5 fold increase in NFκB signalling. Although two isolates were associated with swarm sizes of >7 cm and NFκB activities of >30 fold (P = 0.029), data overall suggested bacterial motility and the NFκB signalling response were not directly correlated. To explore whether the signalling response reflected antigenic variation flagellin was purified from 11 different isolates and the urothelial cell challenges repeated. Purified flagellin filaments generated comparable (30.4±1.8 to 46.1±2.5 fold, P = NS) NFκB signalling responses, irrespective of either the source of the isolate or H-serotype. These data argued against any variability between isolates being related to flagellin itself. To determine the roles, if any, of flagellar abundance in inducing these responses flagellar hook numbers of a range of cystitis and ABU isolates were quantified using a plasmid encoded flagellar hook gene flgEA240C. Foci data suggested isolates were averaging between 1 and 2 flagella per cell, while only 10 to 60% each isolates population exhibited foci. These data suggested selective pressures exist in the urinary tract that allow uro-associated E. coli strains to maintain motility exploiting population heterogeneity to prevent host TLR5 recognition.

Single-cell RNA sequencing of urinary cells reveals distinct cellular diversity in COVID-19-associated AKI

Background: Acute kidney injury (AKI) is a common sequela of infection with SARS-CoV-2 and contributes to the severity and mortality from COVID-19. Here, we tested the hypothesis that kidney alterations induced by COVID-19-associated AKI could be detected in cells collected from urine. Methods: We performed single-cell RNA sequencing (scRNAseq) on cells recovered from the urine of eight hospitalized COVID-19 patients with (n=5) or without AKI (n=3) as well as four non-COVID-19 AKI patients (n=4) to assess differences in cellular composition and gene expression during AKI. Results: Analysis of 30,076 cells revealed a diverse array of cell types, most of which were kidney, urothelial, and immune cells. Pathway analysis of tubular cells from patients with AKI showed enrichment of transcripts associated with damage-related pathways compared to those without AKI. ACE2 and TMPRSS2 expression were highest in urothelial cells amongst cell types recovered. Notably, in one patient we detected SARS-CoV-2 viral RNA in urothelial cells. These same cells were enriched for transcripts associated with anti-viral and anti-inflammatory pathways. Conclusions: We successfully performed scRNAseq on urinary sediment from hospitalized patients with COVID-19 to noninvasively study cellular alterations associated with AKI and established a dataset that includes both injured and uninjured kidney cells. Additionally, we provide preliminary evidence of direct infection of urinary bladder cells by SARS-CoV-2. The urinary sediment contains a wealth of information and is a useful resource for studying the pathophysiology and cellular alterations that occur in kidney diseases.

The Dynamics of Pathology Dataset Creation Using Urine Cytology as an Example

<b><i>Introduction:</i></b> Dataset creation is one of the first tasks required for training AI algorithms but is underestimated in pathology. High-quality data are essential for training algorithms and data should be labelled accurately and include sufficient morphological diversity. The dynamics and challenges of labelling a urine cytology dataset using The Paris System (TPS) criteria are presented. <b><i>Methods:</i></b> 2,454 images were labelled by pathologist consensus via video conferencing over a 14-day period. During the labelling sessions, the dynamics of the labelling process were recorded. Quality assurance images were randomly selected from images labelled in previous sessions within this study and randomly distributed throughout new labelling sessions. To assess the effect of time on the labelling process, the labelled set of images was split into 2 groups according to the median relative label time and the time taken to label images and intersession agreement were assessed. <b><i>Results:</i></b> Labelling sessions ranged from 24 m 11 s to 41 m 06 s in length, with a median of 33 m 47 s. The majority of the 2,454 images were labelled as benign urothelial cells, with atypical and malignant urothelial cells more sparsely represented. The time taken to label individual images ranged from 1 s to 42 s with a median of 2.9 s. Labelling times differed significantly among categories, with the median label time for the atypical urothelial category being 7.2 s, followed by the malignant urothelial category at 3.8 s and the benign urothelial category at 2.9 s. The overall intersession agreement for quality assurance images was substantial. The level of agreement differed among classes of urothelial cells – benign and malignant urothelial cell classes showed almost perfect agreement and the atypical urothelial cell class showed moderate agreement. Image labelling times seemed to speed up, and there was no evidence of worsening of intersession agreement with session time. <b><i>Discussion/Conclusion:</i></b> Important aspects of pathology dataset creation are presented, illustrating the significant resources required for labelling a large dataset. We present evidence that the time taken to categorise urine cytology images varies by diagnosis/class. The known challenges relating to the reproducibility of the AUC (atypical) category in TPS when compared to the NHGUC (benign) or HGUC (malignant) categories is also confirmed.

The importance of morphometric parameters in differentiating benign/reactive urothelial cells from low-grade urothelial carcinoma: computer-assisted study on urine specimens

Background. Urine cytology is deemed a sensitive method in detection of high-grade urothelial carcinoma. In contrast, detection of low-grade urothelial carcinoma (LGUC) and its differentiation from reactive lesions is difficult with urinary cytology. Objective. Our study aims to determine the effectiveness of morphometric parameters in differentiating reactive urothelial cells from LGUC by cytological examination of urine specimens. Methods. Voided urine samples were used for the study, while the cases were randomized into two groups: those diagnosed with LGUC (first group; N=10) and those which were not diagnosed with LGUC (second group; N=10). The morphometric parameters of major nuclear diameter (MaND), minor nuclear diameter (MiND), mean nuclear area (MNA), cell diameter (CD), mean cell area (MCA), as well as MaND/CD, MiND/CD, MiND/MaND and MNA/MCA ratios were measured on 100 urothelial cells for each case through ScopeImage® 9.0 software. Results. A statistically significant difference was found between the mean values of MiND/CD (p=0.017) and MNA/MCA (p=0.002) ratios of groups. The mean value of both parameters in the first group constituted 0.2 and higher, and below 0.2 in the second group. Conclusion. The ratios of MiND/CD and MNA/MCA in urothelial cells proved significantly higher in patients with LGUC than benign/reactive cases. The reliability of these findings in differentiating LGUC from benign/reactive lesions needs to be verified through studies examining a large number of cases. These parameters can be assessed much faster through a special software enabling an automatic measurement and thus can be used in routine cytological examination.

Isolation and \(\textit{in vitro}\) cultivation of human urothelial cells from urine of patients

In clinical practice the number of urothelial cells collected by biopsy are limited and the procedure requires general anaesthesia. Therefore, in order to acquire enough urothelial cells for in vitro engineering of the urothelium, in this research we aim to isolate urothelial cells from human urine by an alternative, effective, low-cost and safe technique rather than using the indicated method. Sixty human urine samples had been collected from patients and volunteers, cells then were precipitated by centrifugation and cultured. Following the isolation process, these cells were characterized by the immunocytochemical method using some specific antibodies. There are 2 types of cells were successfully isolated from with different shape and morphology, one of them grew randomly while the others formed smooth-edge contours and cobblestone-like cell morphology. These cells were characterized by immunostaining with a specific marker, both of these cells were positive for urothelial marker cytokeratin 7. All these results were taken into consideration, the isolated cells were urothelial cells observed in the urine-derived cell population. These results will be used for in vitro studies in toxicological and clinical research, and it will be the premised research to determine the cell mechanical properties and then develop a promising method for early diagnosis of bladder cancer.