Bolenz Christian, Ikinger Eva-Maria, Ströbel Philipp, Trojan Lutz, Steidler Annette, Fernández Mario I, Honeck Patrick, Gabriel Ute, Weiss Christel, Grobholz Rainer, Alken Peter, Michel Maurice Stephan
Department of Urology, Mannheim Medical Centre, University of Heidelberg, Germany.
Eur Urol. 2009 Sep;56(3):504-11. doi: 10.1016/j.eururo.2008.07.042. Epub 2008 Jul 30.
Urothelial carcinoma (UC) is associated with a high local recurrence rate despite intravesical therapy. There is a lack of representative preclinical models for standardized testing of novel experimental therapies.
To develop an ex vivo model for human UC and to evaluate its ability to generate reproducible and reliable results when testing cytotoxic agents.
DESIGN, SETTING, AND PARTICIPANTS: Normal human urothelium (NHU) and bladder UC explants were collected from patients treated at our institution. A total of 195 surgical explants were cultured on a gelatine matrix. Tissue viability was regularly assessed using nicotinamide adenine dinucleotide (NADH) diaphorase enzymehistochemistry. Topical paclitaxel (PTX) or mitomycin C (MMC) chemotherapy was performed in a subset of 45 UC specimens.
All patients underwent radical cystectomy (RC) or primary transurethral resection (TUR) of a bladder UC.
Triple immunofluorescence (pan-cytokeratin [pan-CK]; 4',6-diamidin-2'-phenylindol-dihydrochloride [DAPI]; terminal deoxynucleotidyl transferase biotin-dUTP nick-end labelling [TUNEL]) and caspase-3 staining of paraffin sections was performed. Proliferation rates were assessed using Ki-67 labelling indices. Apoptosis (percent) was quantified in representative tissue areas to characterize culture stability and to assess antineoplastic effects.
No signs of necrosis and no significant changes in apoptosis were observed during the first 12 d of culture. Of all explants, 88.5% were vital after 20 d. In a highly reproducible fashion, topical chemotherapy resulted in significantly increased apoptosis (37.4% [19.0-75.0%] for PTX and 36.2% [18.8-46.7%] for MMC) compared with controls (7.5% [3.0-26.8%]; p<0.001]). No statistically significant difference was observed regarding the effects of the two chemotherapeutic agents (p=0.119).
The presented human ex vivo model takes UC heterogeneity into account and serves as a valuable translational tool. It offers an attractive alternative to preclinical cell line experiments or animal models and may even be used for prospective toxicity and drug efficacy tests in individual patients.
尽管进行了膀胱内治疗，但尿路上皮癌（UC）的局部复发率仍然很高。目前缺乏用于新型实验性治疗标准化测试的代表性临床前模型。
建立一种人UC的体外模型，并评估其在测试细胞毒性药物时产生可重复且可靠结果的能力。
设计、场所和参与者：从我们机构接受治疗的患者中收集正常人尿路上皮（NHU）和膀胱UC组织块。总共195个手术组织块在明胶基质上培养。使用烟酰胺腺嘌呤二核苷酸（NADH）黄递酶酶组织化学定期评估组织活力。在45个UC标本的子集中进行局部紫杉醇（PTX）或丝裂霉素C（MMC）化疗。
所有患者均接受膀胱UC的根治性膀胱切除术（RC）或初次经尿道切除术（TUR）。
对石蜡切片进行三重免疫荧光（泛细胞角蛋白[pan-CK]；4',6-二脒基-2'-苯基吲哚二盐酸盐[DAPI]；末端脱氧核苷酸转移酶生物素-dUTP缺口末端标记[TUNEL]）和半胱天冬酶-3染色。使用Ki-67标记指数评估增殖率。在代表性组织区域中对凋亡（百分比）进行定量，以表征培养稳定性并评估抗肿瘤效果。
在培养的前12天内未观察到坏死迹象，凋亡也无明显变化。在所有组织块中，20天后88.5%仍具有活力。与对照组（7.5%[3.0-26.8%]；p<0.001）相比，局部化疗以高度可重复的方式导致凋亡显著增加（PTX为37.4%[19.0-75.0%]，MMC为36.2%[18.8-至46.7%]）。两种化疗药物的效果之间未观察到统计学上显著差异（p=0.119）。
所提出的人体外模型考虑了UC的异质性，是一种有价值的转化工具。它为临床前细胞系实验或动物模型提供了有吸引力的替代方案，甚至可用于个体患者的前瞻性毒性和药物疗效测试。
