Utility of FRET substrates in bladder cancer diagnosis

Bladder cancer (BC) is one of the most commonly diagnosed cancers worldwide and is characterized by a high recurrence rate. In this study, we present the use of fluorogenic substrates based on Fluorescence Resonance Energy Transfer (FRET) to differentiate cancer samples from healthy ones. The substrates used in this work are synthetic peptide-based probes designed according to known substrate specificities of proteases associated with cancer. In particular, the "single substrate" refers to a substrate selective for human neutrophil elastase (HNE), incorporating a peptide sequence specifically cleaved by this enzyme and labeled with a FRET donor-acceptor pair. In the multi-substrate approach, two or more distinct synthetic peptide substrates were employed, each targeting different proteases relevant to bladder cancer biology. Enzymatic activity was determined in urine samples collected from patients diagnosed with BC, while urine from individuals without epithelial neoplasms served as a control group. Due to the exploratory nature of this study, the cohort size was limited (12 BCE patients and 24 controls), and the results should be interpreted as preliminary. Enzymes present in cancer samples induced hydrolysis of the applied substrates, resulting in measurable fluorescence signals. Interestingly, when mixed urine samples from cancer patients and healthy individuals were tested, activity was detected only in the cancer-containing samples, whereas the control group remained inactive. However, pooling was used only as an initial screening step and does not reflect inter-individual variability. In contrast, when individual samples were analyzed, enzymatic activity was observed in both healthy and diseased samples, depending on the substrate used. We found that measuring a single HNE-specific substrate yielded a preliminary sensitivity of 83% with a specificity of 100% in this limited cohort. Combining two or more substrates increased sensitivity to 100%, while reducing specificity to 92%. These estimates are based on non-optimized thresholds and should be interpreted cautiously due to the lack of ROC curve analysis and external validation. Importantly, the observed enzymatic activity-particularly for HNE-may be influenced by inflammatory processes, including urinary tract infections, and therefore may not be fully cancer-specific. These findings suggest that a multi-substrate, multiplexed approach may improve the performance of urine-based screening for bladder cancer. Further validation in larger, well-controlled cohorts with normalization of urine concentration (e.g., to creatinine) is required.