Abstract
Background: The interleukin-1-receptor antagonist IL1RA (encoded by the IL1RN 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. Objective: Based on these observations, here we investigated the potential roles of IL1RA, IL1A, and IL1B in bladder cancer cell invasion in vitro. Methods: Cell culture, real-time impedance sensing, invasion assays (Boyden chamber, pig bladder model), qPCR, Western blot, ELISA, gene overexpression. Results: 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. Conclusions: 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.
Highlights
Urothelial carcinoma of the urinary bladder (UCB) can be grossly subdivided into non-muscle invasive (NMIBC) and muscle-invasive tumors (MIBC)
We previously showed that IL1RA is highly expressed in the bladder urothelium, while its levels are downregulated in MIBC and low IL1RA levels are associated with aggressive disease [20]
IL1RN could be detected, though at lower levels compared to the primary tissue samples, in the non-invasive RT4 cells and the immortalized benign cell line UROtsa, while
Summary
Urothelial carcinoma of the urinary bladder (UCB) can be grossly subdivided into non-muscle invasive (NMIBC) and muscle-invasive tumors (MIBC). A scoring system, based on clinical and pathologic criteria, aims to predict progression by subdividing NMIBC into low-, intermediate-, and high-risk tumors [2]. While low-risk tumors have a small likelihood of recurrence and progression, high-risk tumors, such as pT1 or carcinoma in situ (CIS), are known to become muscle-invasive in 50% of patients, if left untreated [3]. The most accurate important prognostic index is the current WHO 2016 histologic grading system, where a high tumor grade strongly correlates with progression to a muscle-invasive stage [4]. High throughput sequencing analyses provided a comprehensive molecular characterization of bladder cancer [5]. FGFR3 mutations are commonly found in papillary, non-invasive, low-grade carcinomas [4]
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