Elsen Sanne, Lerut Evelyne, Van Cleynenbreugel Ben, van der Aa Frank, van Poppel Hein, de Witte Peter A
Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, University of Leuven, Leuven, Belgium.
Laboratory of Translational Cell and Tissue Research, Department of Imaging and Pathology, University of Leuven, Leuven, Belgium.
BJU Int. 2015 Sep;116(3):468-77. doi: 10.1111/bju.13113. Epub 2015 Apr 30.
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.
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.
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.
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.
在这项临床前研究中，探讨使用伊文思蓝（EB）作为一种新型诊断工具，通过白光（WL）膀胱镜检查来检测膀胱肿瘤的可能性，我们研究了EB在正常大鼠膀胱以及由同基因AY - 27肿瘤细胞构成的恶性尿路上皮大鼠膀胱的不同层（尿路上皮、黏膜下层、肌肉层）中的生物分布。
将EB注入正常大鼠膀胱和荷瘤大鼠膀胱。注入后，取出膀胱并在液氮中速冻。使用荧光显微镜对EB在不同层中的分布进行定量。为了更深入了解EB在肿瘤组织中选择性积聚的潜在机制，通过透射电子显微镜（TEM）制备膀胱切片进行超微结构研究。此外，我们还通过免疫组织化学检测E - 钙黏蛋白、闭合蛋白 - 1和桥粒芯糖蛋白 - 1的表达，以研究膀胱壁的完整性，因为这些分子分别是黏附连接、紧密连接和桥粒的关键组成部分。
在大多数情况下，至少当注入1mm EB时，恶性膀胱中EB的积聚明显高于健康膀胱。在注入1mm EB持续2小时的情况下，恶性尿路上皮组织中与EB相关的荧光比正常尿路上皮中的荧光高55倍。超微结构上，与正常组织相比，恶性组织显示出更宽的细胞间隙和减少的细胞连接成分数量，表明尿路上皮屏障存在缺陷。E - 钙黏蛋白的表达没有差异，而与肿瘤组织相比，健康膀胱尿路上皮细胞膜中的桥粒芯糖蛋白 - 1染色更强。在所有测试样本中，闭合蛋白 - 1表达均为阴性。
在荷膀胱肿瘤的大鼠中，膀胱内注入EB后，肿瘤组织可选择性摄取EB。肿瘤样本中桥粒芯糖蛋白 - 1表达较低，以及TEM观察到桥粒数量减少，这可能意味着桥粒在健康大鼠尿路上皮和肿瘤组织的超微结构差异中起重要作用，进而导致两种组织中EB摄取的差异。我们认为我们的发现可能对膀胱癌诊断领域未来的临床发展有用。
