title: Biodistribution of Evans blue in an orthotopic AY-27 rat bladder urothelial cell carcinoma model: implication for the improved diagnosis of non-muscle-invasive bladder cancer (NMIBC) using dye-guided white-light cystoscopy.
abstract: OBJECTIVE
  To investigate the possibility of using Evans blue (EB) as a novel diagnostic tool to detect bladder tumours with white-light (WL) cystoscopy, in this preclinical study we examine the biodistribution of EB in the different layers (urothelium, submucosa, muscle) of a normal rat bladder and a rat bladder bearing a malignant urothelium composed of syngeneic AY-27 tumour cells.
  
  
  METHODS
  EB was instilled into both normal as well as tumour-bearing rat bladders. After instillation, bladders were removed and snap frozen in liquid nitrogen. The distribution of EB in the different layers was quantified using fluorescence microscopy. To gain more insight into the mechanism underlying the selective accumulation of EB in tumour tissue, bladder sections were prepared for ultrastructural investigations by means of transmission electron microscopy (TEM). In addition, we also examined the expression of E-cadherin, claudin-1 and desmoglein-1 by immunohistochemistry to study the integrity of the bladder wall, as these molecules are key constituents of adherens junctions, tight junctions and desmosomes, respectively.
  
  
  RESULTS
  In most cases, the accumulation of EB in malignant bladders was substantially higher than in healthy bladders, at least when 1 mm EB instillations were used. In case of a 1 mm EB instillation for 2 h, the EB-associated fluorescence in malignant urothelial tissue was 55-times higher than the fluorescence found in normal urothelium. Ultrastructurally, malignant tissue displayed wider intercellular spaces and a decreased number of cell junction components compared with normal tissue, pointing to defects in the urothelial barrier. There were no differences in the expression of E-cadherin, whereas desmoglein-1 staining was stronger in the membranes of healthy bladder urothelium compared with tumour tissue. Claudin-1 expression was negative in all samples tested.
  
  
  CONCLUSIONS
  EB is selectively taken up by tumour tissue after intravesical instillations in rats bearing bladder tumours. The lower expression of desmoglein-1 in tumour samples, together with the reduced presence of desmosomes seen with TEM, likely imply that desmosomes play an important role in the ultrastructural differences between healthy rat urothelium and tumour tissue, and secondary to that, to the differential uptake of EB in both tissues. We consider that our findings could be useful for future clinical developments in the field of diagnostics for bladder cancer.
authors.affiliation: Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, University of Leuven, Leuven, Belgium.
authors.forename: Sanne
authors.initials: S
authors.lastname: Elsen
authors.affiliation: Laboratory of Translational Cell and Tissue Research, Department of Imaging and Pathology, University of Leuven, Leuven, Belgium.
authors.forename: Evelyne
authors.initials: E
authors.lastname: Lerut
authors.affiliation: Department of Urology, University Hospital Leuven, Leuven, Belgium.
authors.forename: Ben
authors.initials: B
authors.lastname: Van Cleynenbreugel
authors.affiliation: Department of Urology, University Hospital Leuven, Leuven, Belgium.
authors.forename: Frank
authors.initials: F
authors.lastname: van der Aa
authors.affiliation: Department of Urology, University Hospital Leuven, Leuven, Belgium.
authors.forename: Hein
authors.initials: H
authors.lastname: van Poppel
authors.affiliation: Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, University of Leuven, Leuven, Belgium.
authors.forename: Peter A
authors.initials: PA
authors.lastname: de Witte
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003558:Cystoscopy / Q000379:methods*; D005070:Evans Blue / Q000493:pharmacokinetics*; D005260:Female; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D051381:Rats; D014018:Tissue Distribution; D001743:Urinary Bladder / Q000378:metabolism / Q000473:pathology*; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis / Q000378:metabolism / Q000473:pathology*
chemical_list: D005456:Fluorescent Dyes; D005070:Evans Blue
keywords: Evans blue; non-muscle-invasive bladder cancer; orthotopic animal model; white-light cystoscopy