Establishment of Mouse Orthotopic Urinary Bladder Tumor Model and Its Analysis by Light and Electron Microscopy.

Mouse tumor models are an important tool in cancer research, and the orthotopic cancer cell transplantation model is the most widely used among them. Methods for establishing tumor models may differ in many ways, including the selection of cancer cell lines and the type of urinary bladder pretreatment. Here, we describe our mouse orthotopic bladder tumor model using a labeled MB49 urothelial cancer cell line and chemical pretreatment with the cationic polypeptide poly-L-lysine to traumatize the bladder epithelium. Double labeling of MB49 cancer cells by their transduction with GFP and internalization of metal nanoparticles allows the study of their implantation process from the first hours to several days after intravesical injection, as well as the analysis of developed tumors after 3 weeks. Thus, our model provides a comprehensive analysis of the early and late stages of tumor development in the bladder at the light and electron microscopic level.

Sex-Dependent Differences in Blood-Urine Barrier Are Subtle but Significant in Healthy and Chronically Inflamed Mouse Bladders.

The urothelium is a vital permeability barrier that prevents the uncontrolled flow of urinary components into and out of the bladder interstitium. Our study addressed the question of possible sex-specific variations in the urothelium of healthy mice and their impact on chronic bladder inflammation. We found that healthy female bladders have a less robust barrier function than male bladders, as indicated by significant differences in transepithelial electrical resistance (TEER) values. These differences could be attributed to detected higher claudin 2 mRNA expression and a less pronounced glycocalyx in females than in males. In addition, TEER measurements showed delayed barrier recovery in chronically inflamed female bladders. We found subtle differences in the expressions of genes involved in the regulation of the actin cytoskeleton between the sexes, as well as pronounced urothelial hyperplasia in females compensating for attenuated barrier function. The identified genetic variations in glycosylation pathways may also contribute to this divergence. Our findings add to the growing body of literature on the intricate sex-specific nuances of urothelial permeability function and their implications for chronic bladder inflammation. Understanding these differences could lead to tailored diagnostic and therapeutic approaches in the treatment of bladder disorders in the future.

Molecular Profiling of Inflammatory Processes in a Mouse Model of IC/BPS: From the Complete Transcriptome to Major Sex-Related Histological Features of the Urinary Bladder.

Animal models are invaluable in the research of the pathophysiology of interstitial cystitis/bladder pain syndrome (IC/BPS), a chronic aseptic urinary bladder disease of unknown etiology that primarily affects women. Here, a mouse model of IC/BPS was induced with multiple low-dose cyclophosphamide (CYP) applications and thoroughly characterized by RNA sequencing, qPCR, Western blot, and immunolabeling to elucidate key inflammatory processes and sex-dependent differences in the bladder inflammatory response. CYP treatment resulted in the upregulation of inflammatory transcripts such as Ccl8, Eda2r, and Vegfd, which are predominantly involved in innate immunity pathways, recapitulating the crucial findings in the bladder transcriptome of IC/BPS patients. The JAK/STAT signaling pathway was analyzed in detail, and the JAK3/STAT3 interaction was found to be most activated in cells of the bladder urothelium and lamina propria. Sex-based data analysis revealed that cell proliferation was more pronounced in male bladders, while innate immunity and tissue remodeling processes were the most distinctive responses of female bladders to CYP treatment. These processes were also reflected in prominent histological changes in the bladder. The study provides an invaluable reference dataset for preclinical research on IC/BPS and an insight into the sex-specific mechanisms involved in the development of IC/BPS pathology, which may explain the more frequent occurrence of this disease in women.

Molecular and Cellular Markers in Chlorhexidine-Induced Peritoneal Fibrosis in Mice.

Understanding the tissue changes and molecular mechanisms of preclinical models is essential for creating an optimal experimental design for credible translation into clinics. In our study, a chlorhexidine (CHX)-induced mouse model of peritoneal fibrosis was used to analyze histological and molecular/cellular alterations induced by 1 and 3 weeks of intraperitoneal CHX application. CHX treatment for 1 week already caused injury, degradation, and loss of mesothelial cells, resulting in local inflammation, with the most severe structural changes occurring in the peritoneum around the ventral parts of the abdominal wall. The local inflammatory response in the abdominal wall showed no prominent differences between 1 and 3 weeks. We observed an increase in polymorphonuclear cells in the blood but no evidence of systemic inflammation as measured by serum levels of serum amyloid A and interleukin-6. CHX-induced fibrosis in the abdominal wall was more pronounced after 3 weeks, but the gene expression of fibrotic markers did not change over time. Complement system molecules were strongly expressed in the abdominal wall of CHX-treated mice. To conclude, both histological and molecular changes were already present in week 1, allowing examination at the onset of fibrosis. This is crucial information for refining further experiments and limiting the amount of unnecessary animal suffering.

Comprehensive transcriptome profiling of urothelial cells following TNFα stimulation in an in vitro interstitial cystitis/bladder pain syndrome model.

Urothelial cells of the urinary bladder play a critical role in the development and progression of interstitial cystitis/bladder pain syndrome (IC/BPS), a chronic and debilitating inflammatory disease. Given the lack of data on the exact phenotype and function of urothelial cells in an inflammatory setting (as in IC/BPS), we performed the first in-depth characterization of these cells using RNA sequencing, qPCR, ELISA, Western blot, and immunofluorescence. After TNFα stimulation, urothelial cells in the in vitro model of IC/BPS showed marked upregulation of several proinflammatory mediators, such as SAA, C3, IFNGR1, IL1α, IL1ß, IL8, IL23A, IL32, CXCL1, CXCL5, CXCL10, CXCL11, TNFAIPR, TNFRSF1B, and BIRC3, involved in processes and pathways of innate immunity, including granulocyte migration and chemotaxis, inflammatory response, and complement activation, as well as TLR-, NOD-like receptor- and NFkB-signaling pathways, suggesting their active role in shaping the local immune response of the bladder. Our study demonstrates that the TNFα-stimulated urothelial cells recapitulate key observations found in the bladders of patients with IC/BPS, underpinning their utility as a suitable in vitro model for understanding IC/BPS mechanisms and confirming the role of TNFα signaling as an important component of the associated pathology. The present study also identifies novel upregulated gene targets of TNFα in urothelial cells, including genes encoding the acute phase protein SAA, complement component C3, and the cytokine receptor IFNGR1, which could be exploited as therapeutic targets of IC/BPS. Altogether, our study provides a reference database of the phenotype of urothelial cells in an inflammatory environment that will not only increase our knowledge of their role in IC/BPS, but also advance our understanding of how urothelial cells shape tissue immunity in the bladder.

Cobalt Ferrite Magnetic Nanoparticles for Tracing Mesenchymal Stem Cells in Tissue: A Preliminary Study.

Therapy with mesenchymal stem cells (MSCs) is promising in many diseases. Evaluation of their efficacy depends on adequate follow-up of MSCs after transplantation. Several studies have shown that MSCs can be labeled and subsequently visualized with magnetic nanoparticles (NPs). We investigated the homing of MSCs labeled with magnetic cobalt ferrite NPs in experimentally induced acute kidney injury in mice. To explore the homing of MSCs after systemic infusion into mice, we developed a pre-infusion strategy for optimal tracing and identification of MSCs with polyacrylic acid-coated cobalt ferrite (CoFe2O4) NPs by light and transmission electron microscopy (TEM) in various organs of mice with cisplatin-induced acute kidney injury and control mice. By correlative microscopy, we detected MSCs labeled with NPs in the lungs, spleen, kidney, and intestine of cisplatin-treated mice and in the lungs and spleen of control mice. Our results confirm that labeling MSCs with metal NPs did not affect the ultrastructure of MSCs and their ability to settle in various organs. This study demonstrates the usefulness of cobalt ferrite NPs in ex vivo visualization of MSCs and offers correlative microscopy as a useful method in routine histopathology laboratories for tracing MSCs in paraffin-embedded tissue.

Oleic Acid Protects Endothelial Cells from Silica-Coated Superparamagnetic Iron Oxide Nanoparticles (SPIONs)-Induced Oxidative Stress and Cell Death.

Superparamagnetic iron oxide nanoparticles (SPIONs) have great potential for use in medicine, but they may cause side effects due to oxidative stress. In our study, we investigated the effects of silica-coated SPIONs on endothelial cells and whether oleic acid (OA) can protect the cells from their harmful effects. We used viability assays, flow cytometry, infrared spectroscopy, fluorescence microscopy, and transmission electron microscopy. Our results show that silica-coated SPIONs are internalized by endothelial cells, where they increase the amount of reactive oxygen species (ROS) and cause cell death. Exposure to silica-coated SPIONs induced accumulation of lipid droplets (LD) that was not dependent on diacylglycerol acyltransferase (DGAT)-mediated LD biogenesis, suggesting that silica-coated SPIONs suppress LD degradation. Addition of exogenous OA promoted LD biogenesis and reduced SPION-dependent increases in oxidative stress and cell death. However, exogenous OA protected cells from SPION-induced cell damage even in the presence of DGAT inhibitors, implying that LDs are not required for the protective effect of exogenous OA. The molecular phenotype of the cells determined by Fourier transform infrared spectroscopy confirmed the destructive effect of silica-coated SPIONs and the ameliorative role of OA in the case of oxidative stress. Thus, exogenous OA protects endothelial cells from SPION-induced oxidative stress and cell death independent of its incorporation into triglycerides.

Lectins as Biomarkers of IC/BPS Disease: A Comparative Study of Glycosylation Patterns in Human Pathologic Urothelium and IC/BPS Experimental Models.

Pathophysiology of interstitial cystitis/bladder pain syndrome (IC/BPS) remains poorly understood, as well as its effective diagnosis and therapy. Studying changes in tissue glycosylation patterns under pathological conditions is a promising way of discovering novel biomarkers and therapeutic targets. The glycobiology of IC/BPS is largely understudied, therefore we compared glycosylation patterns of normal human urothelium with the urothelium of IC/BPS patients using a selection of 10 plant-based lectins with different monosaccharide preferences. We also compared lectin binding to human urothelium with the two most cited experimental models of IC/BPS, specifically, TNFα-treated human urothelial cell line RT4 and cyclophosphamide-induced chronic cystitis in C57BL6/J mice. Furthermore, binding of four of the selected lectins (ConA, DSL, Jacalin and WGA) was evaluated qualitatively by means of fluorescence microscopy, and quantitatively by fluorescence intensity (F.I.) measurements. Our results reveal a significant reduction in F.I. of Jacalin, as well as a prominent change in the WGA labeling pattern in the urothelium of IC/BPS patients, suggesting their potential use as promising additional biomarkers for histopathological diagnosis of IC/BPS. We have also shown that urothelial glycosylation patterns between selected experimental models and patients with IC/BPS are similar enough to offer an adequate platform for preclinical study of IC/BPS glycobiology.

Just Seeing Is Not Enough for Believing: Immunolabelling as Indisputable Proof of SARS-CoV-2 Virions in Infected Tissue.

BACKGROUND: There is increasing evidence that identification of SARS-CoV-2 virions by transmission electron microscopy could be misleading due to the similar morphology of virions and ubiquitous cell structures. This study thus aimed to establish methods for indisputable proof of the presence of SARS-CoV-2 virions in the observed tissue. METHODS: We developed a variant of the correlative microscopy approach for SARS-CoV-2 protein identification using immunohistochemical labelling of SARS-CoV-2 proteins on light and electron microscopy levels. We also performed immunogold labelling of SARS-CoV-2 virions. RESULTS: Immunohistochemistry (IHC) of SARS-CoV-2 nucleocapsid proteins and subsequent correlative microscopy undoubtedly proved the presence of SARS-CoV-2 virions in the analysed human nasopharyngeal tissue. The presence of SARS-CoV-2 virions was also confirmed by immunogold labelling for the first time. CONCLUSIONS: Immunoelectron microscopy is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Furthermore, we developed a variant of correlative microscopy that allows pathologists to check the results of IHC performed first on routinely used paraffin-embedded samples, followed by semithin, and finally by ultrathin sections. Both methodological approaches indisputably proved the presence of SARS-CoV-2 virions in cells.

A Systematic Review of Therapeutic Approaches Used in Experimental Models of Interstitial Cystitis/Bladder Pain Syndrome.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a multifactorial, chronic bladder disorder with limited therapeutic options currently available. The present review provides an extensive overview of therapeutic approaches used in in vitro, ex vivo, and in vivo experimental models of IC/BPS. Publications were identified by electronic search of three online databases. Data were extracted for study design, type of treatment, main findings, and outcome, as well as for methodological quality and the reporting of measures to avoid bias. A total of 100 full-text articles were included. The majority of identified articles evaluated therapeutic agents currently recommended to treat IC/BPS by the American Urological Association guidelines (21%) and therapeutic agents currently approved to treat other diseases (11%). More recently published articles assessed therapeutic approaches using stem cells (11%) and plant-derived agents (10%), while novel potential drug targets identified were proteinase-activated (6%) and purinergic (4%) receptors, transient receptor potential channels (3%), microRNAs (2%), and activation of the cannabinoid system (7%). Our results show that the reported methodological quality of animal studies could be substantially improved, and measures to avoid bias should be more consistently reported in order to increase the value of preclinical research in IC/BPS for potential translation to a clinical setting.

How Cancer Cells Invade Bladder Epithelium and Form Tumors: The Mouse Bladder Tumor Model as a Model of Tumor Recurrence in Patients.

Non-muscle-invasive bladder cancer is the most common form of bladder cancer. The main problem in managing bladder tumors is the high recurrence after the transurethral resection of bladder tumors (TURBT). Our study aimed to examine the fate of intravesically applied cancer cells as the implantation of cancer cells after TURBT is thought to be a cause of tumor recurrence. We established an orthotopic mouse bladder tumor model with MB49-GFP cancer cells and traced them during the first three days to define their location and contacts with normal urothelial cells. Data were obtained by Western blot, immunolabeling, and light and electron microscopy. We showed that within the first two hours, applied cancer cells adhered to the traumatized epithelium by cell projections containing α3ß1 integrin on their tips. Cancer cells then migrated through the epithelium and on day 3, they reached the basal lamina or even penetrated it. In established bladder tumors, E-cadherin and desmoplakin 1/2 were shown as feasible immunohistochemical markers of tumor margins based on the immunolabeling of various junctional proteins. Altogether, these results for the first time illustrate cancer cell implantation in vivo mimicking cellular events of tumor recurrence in bladder cancer patients.

Human mesenchymal stromal cells from different tissues exhibit unique responses to different inflammatory stimuli.

BACKGROUND: Mesenchymal stromal cell (MSC) - based therapies are emerging as promising treatment of various autoimmune diseases, however the utility of different MSC tissue sources remains elusive. We aimed to characterize MSC from different origins, namely bone marrow (BM), adipose tissue (AT) and umbilical cord (UC) and determine their functional effects on normal human lung fibroblasts (NHLF). METHODS: BM-, AT- or UC-MSC were isolated each from 3 different healthy donors. The gene expression and protein secretion were analyzed at basal level, along with TNFα-, IL-1ß- and SAA- stimulated cells using real-time PCR and Luminex technology. Effect of conditioned medium (CM) from different MSC sources on migration was determined with wound scratch assay, while mitotic and apoptotic rates were studied using immunofluorescence microscopy. RESULTS: BM-MSC expressed highest basal mRNA levels of SDF1 and VCAM-1, while other genes were similarly expressed between MSC origins. TNFα priming of AT-MSC gained a prominent increase in IDO1 and CCL5 gene expression, with 928-fold and 4396-fold changes, respectively. Among all tissue sources, basal UC-MSC released highest protein levels of most measured analytes, including IL-6, IL-8, MCP-1, ICAM1, HGF, MMP1 and CH3L1. BM- and AT-MSC derived CM enhanced wound closure in NHLF, while an opposite effect was observed with UC-MSC derived CM. Our data also suggests that MSC-CM could contribute to decreased mitotic potential and increased apoptotic rate in lung fibroblasts. CONCLUSIONS: Our study highlights origin-specific MSC profile differences and emphasizes a heterogenic response of different MSC to inflammatory stimuli.

Does the Urothelium of Old Mice Regenerate after Chitosan Injury as Quickly as the Urothelium of Young Mice?

The aging of organisms leads to a decreased ability of tissue to regenerate after injury. The regeneration of the bladder urothelium after induced desquamation with biopolymer chitosan has been studied in young mice but not in old mice. Chitosan is a suitable inducer of urothelial desquamation because it is known to be non-toxic. We used chitosan for desquamation of urothelial cells in order to compare the dynamics of urothelial regeneration after injury between young and old mice. Our aim was to determine whether the urothelial function and structure of old mice is restored as fast as in young mice, and to evaluate the inflammatory response due to chitosan treatment. We discovered that the urothelial function restored comparably fast in both age groups and that the urothelium of young and old mice recovered within 5 days after injury, although the onset of proliferation and differentiation appeared later in old mice. Acute inflammation markers showed some differences in the inflammatory response in young versus old mice, but in both age groups, chitosan caused short-term acute inflammation. In conclusion, the restoration of urothelial function is not impaired in old mice, but the regeneration of the urothelial structure in old mice slightly lags behind the regeneration in young mice.

Characterization of Plasma-Derived Small Extracellular Vesicles Indicates Ongoing Endothelial and Platelet Activation in Patients with Thrombotic Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is a systemic autoimmune disease, characterized by thrombosis, obstetric complications and the presence of antiphospholipid antibodies (aPL), which drive endothelial injury and thrombophilia. Extracellular vesicles (EVs) have been implicated in endothelial and thrombotic pathologies. Here, we characterized the quantity, cellular origin and the surface expression of biologically active molecules in small EVs (sEVs) isolated from the plasma of thrombotic APS patients (n = 14), aPL-negative patients with idiopathic thrombosis (aPL-neg IT, n = 5) and healthy blood donors (HBD, n = 7). Nanoparticle tracking analysis showed similar sEV sizes (110-170 nm) between the groups, with an increased quantity of sEVs in patients with APS and aPL-neg IT compared to HBD. MACSPlex analysis of 37 different sEV surface markers showed endothelial (CD31), platelet (CD41b and CD42a), leukocyte (CD45), CD8 lymphocyte and APC (HLA-ABC) cell-derived sEVs. Except for CD8, these molecules were comparably expressed in all study groups. sEVs from APS patients were specifically enriched in surface expression of CD62P, suggesting endothelial and platelet activation in APS. Additionally, APS patients exhibited increased CD133/1 expression compared to aPL-neg IT, suggesting endothelial damage in APS patients. These findings demonstrate enhanced shedding, and distinct biological properties of sEVs in thrombotic APS.

Poly-L-lysine as an Effective and Safe Desquamation Inducer of Urinary Bladder Epithelium.

Induced desquamation of urinary bladder epithelial cells, also called urothelial cells, is frequently used in studies of bladder epithelial regeneration and also in treating recurrent bacterial cystitis. Positively charged polymer chitosan is known to cause large-scale desquamation of terminally differentiated urothelial cells called umbrella cells. Aiming to compare the desquamation ability of another polycation poly-L-lysine, we studied the effect of this polymer on the functional and structural integrity of the urothelium in ex vivo and in vivo experiments. The urothelium was analyzed by measuring transepithelial electrical resistance, and the structural changes of its luminal surface were analyzed with scanning electron microscopy. The results revealed a selective and concentration-dependent desquamation effect of poly-L-lysine on superficial urothelial cells followed by quick regeneration of the urothelium, which functionally and structurally recovers in 2 to 3 h after poly-L-lysine-induced injury. Poly-L-lysine was thus proven to be a promising polymer to be used when desquamation of urothelial cells is required in basic and potentially clinical studies.

Triple labelling of actin filaments, intermediate filaments and microtubules for broad application in cell biology: uncovering the cytoskeletal composition in tunneling nanotubes.

We report a protocol for simultaneous triple labelling of intermediate filaments, microtubules and actin filaments. The described procedure offers an optimal preservation of the structure and antigenicity of individual representatives of cytoskeletal elements and is applicable for labelling of tissue samples and cultured cells. Namely, we demonstrate that using this protocol the cytoskeletal elements are well-preserved and detectable in the whole mount urinary bladder tissue pieces, cryosections of the urinary bladder, and in cultured normal and cancer urothelial cells including their delicate intercellular connections such as tunneling nanotubes (TnTs). The protocol uncovers for the first time the co-distribution of actin filaments, intermediate filaments and microtubules in TnTs, which were up to now known as mono- or bi-cytoskeletal structures. Presented triple labelling protocol provides an efficient tool for studying co-distribution of actin filaments, intermediate filaments, and microtubules and therefore offers new insights into their cellular and tissue distribution.

Interleukin-1ß Induces Intracellular Serum Amyloid A1 Expression in Human Coronary Artery Endothelial Cells and Promotes its Intercellular Exchange.

Serum amyloid A (SAA) is an acute-phase protein with important, pathogenic role in the development of atherosclerosis. Since dysfunctional endothelium represents a key early step in atherogenesis, we aimed to determine whether induced human coronary artery endothelial cells (HCAEC) modulate SAA1/2/4 expression and influence intracellular location and intercellular transport of SAA1. HCAEC were stimulated with 1 ng/ml IL-1ß, 10 ng/ml IL-6, and/or 1 µM dexamethasone for 24 h. QPCR, Western blots, ELISA, and immunofluorescent labeling were performed for detection of SAA1/2/4 mRNA and protein levels, respectively. In SAA1 transport experiments, FITC- or Cy3-labeled SAA1 were added to HCAEC separately, for 24 h, followed by a combined incubation of SAA1-FITC and SAA1-Cy3 positive cells, with IL-1ß and analysis by flow cytometry. IL-1ß upregulated SAA1 (119.9-fold, p < 0.01) and SAA2 (9.3-fold; p < 0.05) mRNA expression levels, while mRNA expression of SAA4 was not affected. Intracellular SAA1 was found mainly as a monomer, while SAA2 and SAA4 formed octamers as analyzed by Western blots. Within HCAEC, SAA1/2/4 located mostly to the perinuclear area and tunneling membrane nanotubes. Co-culturing of SAA1-FITC and SAA1-Cy3 positive cells for 48 h showed a significantly higher percentage of double positive cells in IL-1ß-stimulated (mean ± SD; 60 ± 4%) vs. non-stimulated cells (48 ± 2%; p < 0.05). IL-1ß induces SAA1 expression in HCAEC and promotes its intercellular exchange, suggesting that direct communication between cells in inflammatory conditions could ultimately lead to faster development of atherosclerosis in coronary arteries.

How cancer cells attach to urinary bladder epithelium in vivo: study of the early stages of tumorigenesis in an orthotopic mouse bladder tumor model.

The majority of bladder cancers in humans are non-muscle-invasive cancers that recur frequently after standard treatment procedures. Mouse models are widely used to develop anti-tumor treatments. The purpose of our work was to establish an orthotopic mouse bladder tumor model and to explore early stages of implantation of cancerous MB49 cells in vivo using various labeling and microscopic techniques. To distinguish cancer cells from normal urothelial cells in mouse urinary bladders, we performed molecular characterization of MB49 cells before intravesical injection experiments. In this new approach we applied internalized metal nanoparticles to unequivocally discriminate cancer cells from normal cells. This method revealed that cancer cells attached to the urothelium or basal lamina within just 1 hour of intravesical injection, whereas small tumors and localized hyperplastic urothelial regions developed within two days. We found that cancer cells initially adhere to normal urothelial cells through filopodia and by focal contacts with basal lamina. This is the first in vivo characterization of intercellular contacts between cancerous and normal urothelial cells in the bladder. Our study yields new data about poorly known early events of tumorigenesis in vivo, which could be helpful for the translation into clinic.

The Use of Polymer Chitosan in Intravesical Treatment of Urinary Bladder Cancer and Infections.

The most frequent diseases of the urinary bladder are bacterial infections and bladder cancers. For both diseases, very high recurrence rates are characteristic: 50⁻80% for bladder cancer and more than 50% for bladder infections, causing loss of millions of dollars per year for medical treatment and sick leave. Despite years of searching for better treatment, the prevalence of bladder infections and bladder cancer remains unchanged and is even increasing in recent years. Very encouraging results in treatment of both diseases recently culminated from studies combining biopolymer chitosan with immunotherapy, and chitosan with antibiotics for treatment of bladder cancer and cystitis, respectably. In both pathways of research, the discoveries involving chitosan reached a successful long-lasting cure. The property of chitosan that boosted the effectivity of illness-specific drugs is its ability to enhance the accessibility of these drugs to the very sources of both pathologies that individual treatments without chitosan failed to achieve. Chitosan can thus be recognised as a very promising co-player in treatment of bladder cancer and bacterial cystitis.