Deregulated Expression of IL-37 in Patients with Bladder Urothelial Cancer: The Diagnostic Potential of the IL-37e Isoform

Abstract

Cellular and molecular immune components play a crucial role in the development and perpetuation of human malignancies, shaping anti-tumor responses. A novel immune regulator is interleukin-37 (IL-37), already shown to be involved in the inflammation associated with the pathophysiology of many human disorders, including cancer. The interplay between tumor and immune cells is of great importance, especially for highly immunogenic tumors such as bladder urothelial carcinoma (BLCA). This study aimed to investigate the potential of IL-37 and its receptor SIGIRR (single immunoglobulin IL-1-related receptor) to serve as prognostic and/or diagnostic markers in patients with BLCA. To this end, a series of bioinformatics tools processing -omics datasets and specifically designed qPCR assays on human BLCA tumors and cancer cell lines were utilized. Bioinformatics analysis revealed that IL-37 levels correlate with BLCA tumor development and are higher in patients with longer overall survival. Furthermore, mutations on SIGIRR are associated with enhanced infiltration of the tumor by regulatory T cells and dendritic cells. Based on the qPCR validation experiments, BLCA epithelial cells express the IL-37c and IL-37e isoforms, while the latter is the predominant variant detected in tumor biopsies, also associated with higher grade and the non-muscle-invasive type. This is the first time, to the best of our knowledge, that IL-37 and SIGIRR levels have been assessed in BLCA tumor lesions, and associations with pathological and survival parameters are described, while a transcript variant-specific signature is indicated to have a diagnostic potential. These data strongly indicate the need for further investigation of the involvement of this cytokine and interconnected molecules in the pathophysiology of the disease and its prospective as a therapeutic target and biomarker for BLCA.

Keywords: advanced stage; biomarker; bladder cancer; interleukin (IL-)37; survival.

Figure 1

(A) Violin plots depicting the differential expression levels of IL-37 and SIGIRR in bladder tissues from BLCA (orange; n = 411) vs. non-BLCA (light green; n = 30) individuals or paired tumor versus adjacent normal tissues from BLCA patients (n = 19 pairs). Median expression levels, Mann–Whitney p-values and fold changes between medians are reported. Data were obtained from www.tnmplot.com [36] (accessed on 1 October 2022). (B) Kaplan–Meier plots depicting the probability of OS in months in BLCA patients exhibiting high (red) or low (black) expression levels of IL-37 and SIGIRR. Hazard ratio (HR), logrank p-values and number of patients with either high or low gene expression, categorized also into those who survived for 50, 100 and 150 months, are reported. Graphs were exported from www.kmplot.com [37] (accessed on 1 December 2022).

Figure 2

Box and Tukey whisker diagrams showing the differential distribution of IL-37 and SIGIRR expression levels (FPKMs) as analyzed through RNA sequencing in BLCA samples of different histological types, stages, nodal metastases and mutation statuses. Data were obtained from http://ualcan.path.uab.edu/ [38] (accessed on 20 December 2022). Asterisks designate statistically significant differences compared to normal samples, between groups (where accompanied by brackets) as analyzed using the unpaired t-test with Welch’s correction or statistically significant linear trends between group means and left-to-right order (where accompanied by an arrow) as analyzed using the one-way ANOVA test; *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 3

(A) Violin plots depicting the distribution of infiltrating B cells in BLCA tumors without versus with mutation on IL-37 and the distribution of infiltrating CD4⁺ T cells, regulatory T cells (Tregs), B cells, dendritic cells (DCs) and endothelial cells in BLCA tumors without versus with mutation on SIGIRR. Wilcoxon p-values and log₂ (fold changes, FC) are reported. (Β) Scatter plot diagrams depicting the linear association between levels of IL-37 gene expression (log₂TPM; y-axis) and infiltration of BLCA tumors by CD8⁺ T cells, CD4⁺ T cells, regulatory T cells (Tregs) and neutrophils (x-axis). Spearman’s rho and p-values are reported. Data were filtered for tumor purity. Graphs were exported from http://timer.cistrome.org/ [38] (accessed on 20 December 2022).

Figure 4

(A) Bar diagrams depicting the relative quantification (RQ) mRNA levels of IL-37 (a-e) isoforms in human T24 and RT4 BLCA cell lines (left) and indicative BLCA tumor samples (OK238, OK239, OK249) (right), using specifically developed qPCR assays. Data are represented as mean ± standard error (SE) of three independent experiments. (B) Dot plot diagrams portraying the differential distribution of IL-37e in BLCA tumor biopsies of different grades. Analysis was performed by processing total BLCA samples and non-muscle-invasive samples separately, applying Mann–Whitney t-test; *: p < 0.05.