Schneider Lisa, Liu Junnan, Zhang Cheng, Azoitei Anca, Meessen Sabine, Zheng Xi, Cremer Catharina, Gorzelanny Christian, Kempe-Gonzales Sybille, Brunner Cornelia, Wezel Felix, Bolenz Christian, Gunes Cagatay, John Axel
Department of Urology, Ulm University Hospital, 89081 Ulm, Germany.
Department of Dermatology und Venerology, UKE, 20246 Hamburg, Germany.
Int J Mol Sci. 2021 May 30;22(11):5875. doi: 10.3390/ijms22115875.
The interleukin-1-receptor antagonist IL1RA (encoded by the gene) is a potent competitive antagonist to interleukin-1 (IL1) and thereby is mainly involved in the regulation of inflammation. Previous data indicated a role of IL1RA in muscle-invasive urothelial carcinoma of the bladder (UCB) as well as an IL1-dependent decrease in tissue barrier function, potentially contributing to cancer cell invasion.
Based on these observations, here we investigated the potential roles of IL1RA, IL1A, and IL1B in bladder cancer cell invasion in vitro.
Cell culture, real-time impedance sensing, invasion assays (Boyden chamber, pig bladder model), qPCR, Western blot, ELISA, gene overexpression.
We observed a loss of IL1RA expression in invasive, high-grade bladder cancer cell lines T24, UMUC-3, and HT1197 while IL1RA expression was readily detectable in the immortalized UROtsa cells, the non-invasive bladder cancer cell line RT4, and in benign patient urothelium. Thus, we modified the invasive human bladder cancer cell line T24 to ectopically express IL1RA, and measured changes in cell migration/invasion using the xCELLigence Real-Time-Cell-Analysis (RTCA) system and the Boyden chamber assay. The real-time observation data showed a significant decrease of cell migration and invasion in T24 cells overexpressing IL1RA (T24-IL1RA), compared to cells harboring an empty vector (T24-EV). Concurrently, tumor cytokines, e.g., IL1B, attenuated the vascular endothelial barrier, which resulted in a reduction of the Cell Index (CI), an impedance-based dimensionless unit. This reduction could be reverted by the simultaneous incubation with IL1RA. Moreover, we used an ex vivo porcine organ culture system to evaluate cell invasion capacity and showed that T24-IL1RA cells showed significantly less invasive capacity compared to parental T24 cells or T24-EV.
Taken together, our results indicate an inverse correlation between IL1RA expression and tumor cell invasive capacity and migration, suggesting that IL1RA plays a role in bladder carcinogenesis, while the exact mechanisms by which IL1RA influences tumor cells migration/invasion remain to be clarified in future studies. Furthermore, we confirmed that real-time impedance sensing and the porcine ex vivo organ culture methods are powerful tools to discover differences in cancer cell migration and invasion.
白细胞介素-1受体拮抗剂IL1RA（由该基因编码）是白细胞介素-1（IL1）的强效竞争性拮抗剂，因此主要参与炎症调节。先前的数据表明IL1RA在膀胱肌肉浸润性尿路上皮癌（UCB）中发挥作用，以及IL1依赖性的组织屏障功能降低，这可能有助于癌细胞侵袭。
基于这些观察结果，我们在此研究了IL1RA、IL1A和IL1B在体外膀胱癌细胞侵袭中的潜在作用。
细胞培养、实时阻抗传感、侵袭试验（Boyden小室、猪膀胱模型）、qPCR、蛋白质免疫印迹法、酶联免疫吸附测定、基因过表达。
我们观察到侵袭性高级别膀胱癌细胞系T24、UMUC-3和HT1197中IL1RA表达缺失，而在永生化的UROtsa细胞、非侵袭性膀胱癌细胞系RT4和良性患者尿路上皮中可轻易检测到IL1RA表达。因此，我们对侵袭性人膀胱癌细胞系T24进行改造，使其异位表达IL1RA，并使用xCELLigence实时细胞分析（RTCA）系统和Boyden小室试验测量细胞迁移/侵袭的变化。实时观察数据显示，与携带空载体的细胞（T24-EV）相比，过表达IL1RA的T24细胞（T24-IL1RA）的细胞迁移和侵袭显著减少。同时，肿瘤细胞因子，如IL1B，削弱了血管内皮屏障，导致细胞指数（CI）降低，CI是一个基于阻抗的无量纲单位。通过与IL1RA同时孵育可逆转这种降低。此外，我们使用体外猪器官培养系统评估细胞侵袭能力，结果显示与亲本T24细胞或T24-EV相比，T24-IL1RA细胞的侵袭能力明显降低。
综上所述，我们的结果表明IL1RA表达与肿瘤细胞侵袭能力和迁移呈负相关，提示IL1RA在膀胱癌发生中起作用，而IL1RA影响肿瘤细胞迁移/侵袭的确切机制仍有待在未来研究中阐明。此外，我们证实实时阻抗传感和猪体外器官培养方法是发现癌细胞迁移和侵袭差异的有力工具。
