Preclinical models of bladder cancer: BBN and beyond.

Preclinical modelling is a crucial component of advancing the understanding of cancer biology and therapeutic development. Several models exist for understanding the pathobiology of bladder cancer and evaluating therapeutics. N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced bladder cancer is a commonly used model that recapitulates many of the features of human disease. Particularly in mice, BBN is a preferred laboratory model owing to a high level of reproducibility, high genetic fidelity to the human condition, and its relative ease of use. However, important aspects of the model are often overlooked in laboratory studies. Moreover, the advent of new models has yielded a variety of methodologies that complement the use of BBN. Toxicokinetics, histopathology, molecular genetics and sex can differ between available models and are important factors to consider in bladder cancer modelling.

Twelve-month recovery following road trauma: Results from an inception cohort in Vancouver, Canada.

BACKGROUND: Injury-related disability following road trauma is a major public health concern. Unfortunately, outcome following road trauma and risk factors for poor recovery are inadequately studied, especially for road trauma survivors with minor injuries that do not require hospitalization. OBJECTIVES: This manuscript reports 12-month recovery outcomes for a large cohort of road trauma survivors. METHODS: This was a prospective, observational inception cohort study of 1,480 road trauma survivors recruited between July 2018 and March 2020 from 3 trauma centres in British Columbia, Canada. Participants were aged ≥16 years and arrived in a participating emergency department within 24 h of a motor vehicle collision. Data on baseline health and injury severity were collected from structured interviews and medical records. Outcome measures, including the SF-12, were collected during follow-up interviews at 2, 4, 6 and 12 months. Predictors of recovery outcomes were identified using Cox proportional hazards models and summarized using hazard ratios. RESULTS: Only 42 % of participants self-reported full recovery and only 66 % reported a return to usual daily activities. Females, older individuals, pedestrians, and those who required hospital admission had a poorer recovery than other groups. Similar patterns were observed for the SF-12 physical component. For the SF-12 mental component, no significant differences were observed between participants admitted to hospital and those discharged home from the ED. Return to work was reported by 77 % of participants who had a paying job at baseline, with no significant differences between sex and age groups. CONCLUSIONS: In a large cohort of road trauma survivors, under half self-reported full recovery one year after the injury. Poor mental health recovery was observed in both participants admitted to hospital and those discharged home from the ED. This finding may indicate a need for early intervention and continued mental health monitoring for all injured individuals, including for those with less serious injuries.

Aging induces changes in cancer formation and microbial content in a murine model of bladder cancer.

Bladder cancer (BCa) incidence is tightly linked to aging. Older patients with BCa present with higher grade tumors and have worse outcomes on Bacillus-Calmette-Guerin (BCG) immunotherapy. Aging is also known to result in changes in the gut microbiome over mammalian lifespan, with recent studies linking alterations in the gut microbiome to changes in tumor immunity. There is limited information on the microbiome in BCa models though, despite known links to aging and immunotherapy. The purpose of this study was to evaluate how aging impacts tumor formation, inflammation, and the microbiome of mice given the model BCa carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). We hypothesized old animals would have larger, more inflamed tumors and a shift in their fecal microbiome compared to their younger counterparts. Young (~8-week-old) or old (~78-week-old) C57Bl/6J animals were administered 0.05% BBN in drinking water for 16 weeks and then euthanized or allowed to progress for an additional 4 weeks. After 16 weeks of BBN, old mice had higher bladder to body weight ratio than young mice, and also muscle invasive tumors, which were not seen in their young counterparts. Old animals also had increased innate immune recruitment, but CD4+/CD8+ T cell recruitment did not appear different. BBN dramatically altered the microbiome in both sets of animals as measured by ß-diversity, including changes in multiple genera of bacteria. These data suggest old mice have a differential response to BBN-induced BCa. Given the median age of patients with BCa, understanding how the aged phenotype interacts with BCa is imperative.

Quantitative image analysis of intracellular protein translocation in 3-dimensional tissues for pharmacodynamic studies of immunogenic cell death.

A recent development in cancer chemotherapy is to use cytotoxics to induce tumor-specific immune response through immunogenic cell death (ICD). In ICD, calreticulin is translocated from endoplasmic reticulum to cell membrane (ecto-CRT) which serves as the 'eat-me-signal' to antigen-presenting cells. Ecto-CRT measurements, e.g., by ecto-CRT immunostaining plus flow cytometry, can be used to study the pharmacodynamics of ICD in single cells, whereas ICD studies in intact 3-dimensional tissues such as human tumors require different approaches. The present study described a method that used (a) immunostaining with fluorescent antibodies followed by confocal microscopy to obtain the spatial locations of two molecules-of-interest (CRT and a marker protein WGA), and (b) machine-learning (trainable WEKA segmentation) and additional image processing tools to locate the target molecules, remove the interfering signals in the nucleus, cytosol and extracellular space, enable the distinction of the inner and outer edges of the cell membrane and thereby identify the cells with ecto-CRT. This method, when applied to 3-dimensional human bladder cancer cell spheroids, yielded drug-induced ecto-CRT measurements that were qualitatively comparable to the flow cytometry results obtained with single cells disaggregated from spheroids. This new method was applied to study drug-induced ICD in short-term cultures of surgical specimens of human patient bladder tumors.

Demographic and clinical profile of an inception cohort of road trauma survivors.

BACKGROUND: Road trauma is a major public health concern, often resulting in reduced health-related quality of life and prolonged absenteeism from work even after so-called 'minor' injuries that do not result in hospitalization. This manuscript compares pre-injury health, sociodemographic characteristics and injury details between age, sex, and road user categories in a cohort of 1,480 road trauma survivors. METHODS: This was a prospective observational inception cohort study of road trauma survivors recruited between July 2018 and March 2020 from three trauma centres in British Columbia, Canada. Participants were aged ≥ 16 years and arrived in a participating emergency department within 24 h of involvement in a motor vehicle collision. Data were collected from structured interviews and review of medical records. RESULTS: The cohort of 1,480 road trauma survivors included 280 pedestrians, 174 cyclists, 118 motorcyclists, 683 motor vehicle drivers, and 225 passengers. Median age was 40 (IQR = [27, 57]) years; 680 (46%) were female. Males and younger patients were significantly more likely to report better pre-injury physical health. Motorcyclists and cyclists tended to report better physical health and less severe somatic symptoms, whereas pedestrians and motor vehicle drivers reported better mental health. Injury severity and hospital admission rates were higher in pedestrians and motorcyclists and lower in motorists. Upper and lower extremity injuries were most common in pedestrians, cyclists and motorcyclists, whereas neck injuries were most common in motor vehicle drivers and passengers. CONCLUSIONS: In a large cohort of road trauma survivors, overall injury severity was low. Motorcyclists and pedestrians, but not cyclists, had more severe injuries than motorists. Extremity injuries were more common in vulnerable road users. Future research will investigate one-year recovery outcomes and identify risk factors for poor recovery.

Pyruvate Dehydrogenase Kinase 4 Deficiency Increases Tumorigenesis in a Murine Model of Bladder Cancer.

Pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial isozyme in the PDK family (PDK1-4) partially responsible for phosphorylation of pyruvate dehydrogenase (PDH). Phosphorylation of PDH is thought to result in a pro-proliferative shift in metabolism that sustains growth of cancer cells. Previous data from our lab indicate the pan-PDK inhibitor dichloroacetate (DCA) or acute genetic knockdown of PDK4 blocks proliferation of bladder cancer (BCa) cells. The goal of this study was to determine the role of PDK4 in an in vivo BCa model, with the hypothesis that genetic depletion of PDK4 would impair formation of BCa. PDK4-/- or WT animals were exposed to N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) for 16 weeks, and tumors were allowed to develop for up to 7 additional weeks. PDK4-/- mice had significantly larger tumors at later time points. When animals were treated with cisplatin, PDK4-/- animals still had larger tumors than WT mice. PDK4 expression was assessed in human tissue and in mice. WT mice lost expression of PDK4 as tumors became muscle-invasive. Similar results were observed in human samples, wherein tumors had less expression of PDK4 than benign tissue. In summary, PDK4 has a complex, multifunctional role in BCa and may represent an underrecognized tumor suppressor.

Role of MIF1/MIF2/CD74 interactions in bladder cancer.

Macrophage migration inhibitory factor (MIF1) is a pleiotropic cytokine involved in inflammation and cancer. Genetic knockout of Mif1 in the validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) model of bladder cancer (BCa) resulted in stage arrest at non-muscle-invasive disease in prior studies. Small-molecule inhibition of MIF1 reduced cancer-associated outcomes, but it did not fully recapitulate genetic models. D-dopachrome tautomerase (gene symbol DDT), commonly referred to as MIF2, is a functional homolog of MIF1, and both MIF1 and MIF2 can bind the cell surface receptor CD74 on multiple cell types to initiate a signaling cascade. It has been proposed that this interaction mediates part of the protumorigenic effects of MIF1 and MIF2 and may explain the discordance in prior studies. We hypothesized that MIF2 functions redundantly with MIF1 in BCa development and progression. The Cancer Genome Atlas (TCGA) analysis indicated MIF and DDT expression were increased in BCa patients compared to control. 4-Iodopyridine (4-IPP), a combined MIF1/MIF2 inhibitor, was more efficacious than ISO-1, a MIF1-only inhibitor, in preventing cellular proliferation in BCa cell lines. To evaluate these findings in vivo, wild-type (WT) and Mif1-/- animals were exposed to 0.05% BBN in drinking water for 16 weeks to initiate tumorigenesis and then evaluated over the subsequent 4 weeks for tumor formation and progression in the presence or absence of 4-IPP. 4-IPP reduced bladder weights in WT animals and bladder weights/tumor stage in Mif1-/- animals. To determine whether MIF1/MIF2 functioned through CD74 in BCa, WT or Cd74-/- animals were used in the same BBN model. Although these animals were partially protected against BBN-induced BCa, 4-IPP did not enhance this effect. In conclusion, our data suggest that MIF2 mechanistically functions in a similar protumorigenic manner to MIF1, and this is at least partially through CD74. Dual inhibition of MIF homologs is more efficacious at reducing tumor burden in this model of BCa. © 2022 The Pathological Society of Great Britain and Ireland.

FOXA1 repression drives lineage plasticity and immune heterogeneity in bladder cancers with squamous differentiation.

Cancers arising from the bladder urothelium often exhibit lineage plasticity with regions of urothelial carcinoma adjacent to or admixed with regions of divergent histomorphology, most commonly squamous differentiation. To define the biologic basis for and clinical significance of this morphologic heterogeneity, here we perform integrated genomic analyses of mixed histology bladder cancers with separable regions of urothelial and squamous differentiation. We find that squamous differentiation is a marker of intratumoral genomic and immunologic heterogeneity in patients with bladder cancer and a biomarker of intrinsic immunotherapy resistance. Phylogenetic analysis confirms that in all cases the urothelial and squamous regions are derived from a common shared precursor. Despite the presence of marked genomic heterogeneity between co-existent urothelial and squamous differentiated regions, no recurrent genomic alteration exclusive to the urothelial or squamous morphologies is identified. Rather, lineage plasticity in bladder cancers with squamous differentiation is associated with loss of expression of FOXA1, GATA3, and PPARG, transcription factors critical for maintenance of urothelial cell identity. Of clinical significance, lineage plasticity and PD-L1 expression is coordinately dysregulated via FOXA1, with patients exhibiting morphologic heterogeneity pre-treatment significantly less likely to respond to immune checkpoint inhibitors.

Bladder cancer, inflammageing and microbiomes.

Ageing is correlated with elevated bladder cancer incidence, morbidity and mortality. Advanced age is also associated with elevated markers of chronic inflammation and perturbations in gut and urinary tract microbiota. One reason for the increased incidence and mortality of bladder cancer in the elderly might be that age-associated changes in multiple microbiomes induce systemic metabolic changes that contribute to immune dysregulation with potentially tumorigenic effects. The gut and urinary microbiomes could be dysregulated in bladder cancer, although the effect of these changes is poorly understood. Each of these domains - the immune system, gut microbiome and urinary microbiome - might also influence the response of patients with bladder cancer to treatment. Improved understanding of age-related alterations to the immune system and gut and urinary microbiomes could provide possible insight into the risk of bladder cancer development and progression in the elderly. In patients with bladder cancer, improved understanding of microbiota might also provide potential targets for therapeutic intervention.

Identifying the lungs as a susceptible site for allele-specific regulatory changes associated with type 1 diabetes risk.

Type 1 diabetes (T1D) etiology is complex. We developed a machine learning approach that ranked the tissue-specific transcription regulatory effects for T1D SNPs and estimated their relative contributions to conversion to T1D by integrating case and control genotypes (Wellcome Trust Case Control Consortium and UK Biobank) with tissue-specific expression quantitative trait loci (eQTL) data. Here we show an eQTL (rs6679677) associated with changes to AP4B1-AS1 transcript levels in lung tissue makes the largest gene regulatory contribution to the risk of T1D development. Luciferase reporter assays confirmed allele-specific enhancer activity for the rs6679677 tagged locus in lung epithelial cells (i.e. A549 cells; C > A reduces expression, p = 0.005). Our results identify tissue-specific eQTLs for SNPs associated with T1D. The strongest tissue-specific eQTL effects were in the lung and may help explain associations between respiratory infections and risk of islet autoantibody seroconversion in young children.

Fosciclopirox suppresses growth of high-grade urothelial cancer by targeting the γ-secretase complex.

Ciclopirox (CPX) is an FDA-approved topical antifungal agent that has demonstrated preclinical anticancer activity in a number of solid and hematologic malignancies. Its clinical utility as an oral anticancer agent, however, is limited by poor oral bioavailability and gastrointestinal toxicity. Fosciclopirox, the phosphoryloxymethyl ester of CPX (Ciclopirox Prodrug, CPX-POM), selectively delivers the active metabolite, CPX, to the entire urinary tract following parenteral administration. We characterized the activity of CPX-POM and its major metabolites in in vitro and in vivo preclinical models of high-grade urothelial cancer. CPX inhibited cell proliferation, clonogenicity and spheroid formation, and increased cell cycle arrest at S and G0/G1 phases. Mechanistically, CPX suppressed activation of Notch signaling. Molecular modeling and cellular thermal shift assays demonstrated CPX binding to γ-secretase complex proteins Presenilin 1 and Nicastrin, which are essential for Notch activation. To establish in vivo preclinical proof of principle, we tested fosciclopirox in the validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) mouse bladder cancer model. Once-daily intraperitoneal administration of CPX-POM for four weeks at doses of 235 mg/kg and 470 mg/kg significantly decreased bladder weight, a surrogate for tumor volume, and resulted in a migration to lower stage tumors in CPX-POM treated animals. This was coupled with a reduction in the proliferation index. Additionally, there was a reduction in Presenilin 1 and Hes-1 expression in the bladder tissues of CPX-POM treated animals. Following the completion of the first-in-human Phase 1 trial (NCT03348514), the pharmacologic activity of fosciclopirox is currently being characterized in a Phase 1 expansion cohort study of muscle-invasive bladder cancer patients scheduled for cystectomy (NCT04608045) as well as a Phase 2 trial of newly diagnosed and recurrent urothelial cancer patients scheduled for transurethral resection of bladder tumors (NCT04525131).

Cystic Hygroma in a Dental Hygienist Reporting With Carpal Tunnel Syndrome: A Case Report.

OBJECTIVE: This purpose of this case report is to describe the chiropractic management of a patient who presented with symptoms of hand neuropathy. CLINICAL FEATURES: A 35-year-old woman presented with a 6-month history of numbness and tingling in the first and second digits of the right hand. Visual inspection revealed a large golf ball-like mass in the patient's right lower neck region. Orthopedic assessment revealed a Tinel's sign at the right carpal tunnel, positive Allen's maneuver, present flick sign, and diminished right radial pulse strength. Advanced diagnostic imaging had been taken previously at the ages of 11 and 24 years, and showed the presence of cystic hygroma in the patient's right axilla and lower neck region. INTERVENTION AND OUTCOME: The patient was treated using manipulative therapy to the thoracic spine, myofascial release therapy, and therapeutic ultrasound over the right carpal tunnel. Active home care included postural relief exercises and education about work-related ergonomics. Several functional and subjective improvements were seen within the first 2 weeks of treatment. Symptoms of right-hand numbness resolved after 8 treatments. CONCLUSION: In this case, the chiropractor originally thought the patient's hand numbness was due to a cystic hygroma; however, this was later considered an incidental finding. The patient's symptoms seemed to respond to chiropractic management and reduced within 1 month.

The Development of Hsp90ß-Selective Inhibitors to Overcome Detriments Associated with pan-Hsp90 Inhibition.

The 90 kD heat shock proteins (Hsp90) are molecular chaperones that are responsible for the folding of select proteins, many of which are directly associated with cancer progression. Consequently, inhibition of the Hsp90 protein folding machinery results in a combinatorial attack on numerous oncogenic pathways. Seventeen small-molecule inhibitors of Hsp90 have entered clinical trials for the treatment of cancer, all of which bind the Hsp90 N-terminus and exhibit pan-inhibitory activity against all four Hsp90 isoforms, which may lead to adverse effects. The development of Hsp90 isoform-selective inhibitors represents an alternative approach toward the treatment of cancer and may limit some of these detriments. Described herein, is a structure-based approach to develop isoform-selective inhibitors of Hsp90ß, which induces the degradation of select Hsp90 clients without concomitant induction of Hsp90 levels. Together, these initial studies support the development of Hsp90ß-selective inhibitors as a method for overcoming the detriments associated with pan-inhibition.

Natural products as a means of overcoming cisplatin chemoresistance in bladder cancer.

Cisplatin remains an integral part of the treatment for muscle invasive bladder cancer. A large number of patients do not respond to cisplatin-based chemotherapy and efficacious salvage regimens are limited. Immunotherapy has offered a second line of treatment; however, only approximately 20% of patients respond, and molecular subtyping of tumors indicates there may be significant overlap in those patients that respond to cisplatin and those patients that respond to immunotherapy. As such, restoring sensitivity to cisplatin remains a major hurdle to improving patient care. One potential source of compounds for enhancing cisplatin is naturally derived bioactive products such as phytochemicals, flavonoids and others. These compounds can activate a diverse array of different pathways, many of which can directly promote or inhibit cisplatin sensitivity. The purpose of this review is to understand current drug development in the area of natural products and to assess how these compounds may enhance cisplatin treatment in bladder cancer patients.

Thiol-ene enabled preparation of S-lipidated anti-HBV peptides.

Despite significant efforts made towards treatments for Hepatitis B virus (HBV), a long-term curative treatment has thus far eluded scientists. Recently, the Sodium Taurocholate Co-Transporting Polypeptide (NTCP) receptor has been identified as the entry pathway of HBV into hepatocytes. Myrcludex B, an N-terminally myristoylated 47-mer peptide mimic of the preS1 domain of the Hepatitis B virion, was identified as a potent protein-protein interaction (PPI) inhibitor blocking HBV fusion (IC50 = 140 pM). Herein we report an optimised chemical synthesis of Myrcludex B and a series of novel analogues. Employing a small modification to the Cysteine Lipidation of a Peptide or Amino acid (CLipPA) thiol-ene reaction, a library of S-lipidated Myrcludex B and truncated (21-mer) analogues were prepared, providing novel chemical space to probe for the discovery of novel anti-HBV peptides. The S-lipidated analogues showed an equivalent or a slight decrease (∼2-fold) in binding effectiveness to NTCP expressing hepatocytes compared to Myrcludex B. Three S-lipidated analogues were highly potent HBV inhibitors (IC50 0.97-3.32 nM). These results demonstrate that incorporation of heteroatoms into the lipid 'anchor' is tolerated by this antiviral scaffold and to the best of our knowledge constitutes the first report of potent S-lipidated antiviral peptides. Interestingly, despite only moderate reductions in binding effectiveness, truncated analogues possessed dramatically reduced inhibitory activity thus providing new insights into the structure activity relationship of these hitherto unreported antiviral S-lipopeptides.

Lumbar spinal epidural lipomatosis: A case report and review of the literature.

INTRODUCTION: Lumbar spinal epidural lipomatosis (SEL) is a rare condition defined by an excessive deposition of adipose tissue in the lumbar spinal canal. The objective of this case report is to document a clinical case of SEL presenting within a multidisciplinary spine clinic and to compare our clinical findings and management with the available literature. CASE PRESENTATION: A 51-year-old female presented at a spine clinic with low back pain, bilateral leg pain and difficulty walking. Magnetic resonance imaging of the lumbar spine showed evidence of severe central canal stenosis due to extensive epidural lipomatosis. She was initially advised to lose weight and undergo a 3-month course of physiotherapy. However, because of lack of improvement, she was scheduled for and underwent L4-S1 posterior spinal decompression and L4-L5 posterior spinal instrumented fusion. At 12-month follow-up, the patient reported no pain and retained the ability to walk regular distances without experiencing discomfort. DISCUSSION: This case report describes the conservative and surgical management of a case of lumbar spinal stenosis due to SEL. The therapeutic approach of patients with this condition is not standardized. As such, a discussion of the literature with respect to the diagnosis, clinical presentation, epidemiology, imaging appearance, risk factors, etiology, and management of SEL is also presented.

Best Practices to Optimise Quality and Outcomes of Transurethral Resection of Bladder Tumours.

CONTEXT: Transurethral resection of bladder tumour (TURBT) for bladder cancer (BC) is an underappreciated common urological procedure. TURBT outcomes are highly variable, and results are dependent on judgement and surgical skill. OBJECTIVE: To perform a narrative review and identify optimal best practice in TURBT including preparation, choice of equipment, procedural steps, surgical technique, and management of difficult scenarios and complications. EVIDENCE ACQUISITION: Medline, Embase, and the Cochrane Central Register of Controlled Trials were searched. Important studies were identified and reviewed by an international panel of urologists representing major urological societies and guideline panels with a record of academic publication in this field. In areas where the group identified a lack of evidence or agreement, discussions took place until a consensus was reached. EVIDENCE SYNTHESIS: A total of 814 studies were identified and 43 were included. The majority were retrospective (level of evidence 3), with only two prospective randomised trials. Four broad themes were identified, which formed the basis for the review: (1) the role of TURBT within the overall management of BC, (2) TURBT techniques, (3) measurement of outcomes including quality control and checklists, and (4) postoperative management. Familiarity with all aspects of the procedure is necessary to minimise morbidity and improve oncological outcomes. Development of new instruments and techniques, and prospective audit of TURBT outcomes are important future goals. CONCLUSIONS: TURBT is a common and challenging operation with known variable outcomes. To reduce these variations and optimise outcomes, best practice based on evidence and expert opinion is recommended. PATIENT SUMMARY: Transurethral resection of bladder tumour (TURBT) is a common but deceptively difficult urological operation. Optimal outcomes depend on experience and surgical skill. An international group of experienced TURBT surgeons review critical aspects of the procedure and share best practice to stimulate further discussion.

Fracture of an os peroneum.

Os peroneum is an accessory ossicle located within the peroneus longus tendon, present in 26% of the population. Fractures of the os peroneum present as pain localized on the lateral aspect of the foot resulting from direct trauma, muscle contraction, inversion injuries or chronic overuse injuries. We document a case of a fractured os peroneum that resulted in the insidious onset of ongoing pain that was managed conservatively.

L'os péronier est un os sésamoïde du tendon du muscle long péronier latéral observé chez 26% de la population. La fracture de l'os péronier se manifeste par une douleur localisée à la face latérale du pied à la suite d'un traumatisme direct, d'une contraction musculaire, d'une blessure d'inversion ou d'une blessure de surutilisation. Nous documentons un cas de fracture de l'os péronier ayant causé l'apparition insidieuse d'une douleur continue soignée par un traitement conservateur.