Abstracts

Colorectal cancer (CRC) is the second cause of death due to cancer worldwide and its incidence is estimated to increase by 60% in 2030 in developing countries. The stage at diagnosis is the most important indicator of survival, so predictive biomarkers are highly demanded. Recent studies suggest a pivotal role of epigenetic factors, such as “aberrant” Notch signaling activation and secondary bile acids (BAs) formation. To evaluate Notch signaling activation, a recombinant protein that combines the extracellular domain (ECD) of the Notch high affinity mutated form of the selective Notch ligand Jagged 1 (Jag1) with a Red emitting firefly luciferase (FLuc) was developed to understand if Jag1-FLuc binding with Notch correlates with CRC progression. Once a good linear correlation between the BL signal and the concentration of Jag1-FLuc protein was obtained up to 10 µg/mL in a cell-free system, we moved on to human colorectal adenocarcinoma cells Caco-2. Under the optimized conditions, we observed that BL signal increase was proportional to the Notch expression, with a linear range from 0.1 to 50 µg/mL of Jag1-FLuc obtaining a LOD and LOQ of 0.8 ± 0.2 and 6.0 ± 0.2 µg /mL, respectively. Next, BLI experiments were performed to examine the light output of Jag1-FLuc construct on Caco-2 cells and ex vivo on human biopsies derived from patients with different pre-CRC stages. Secondary BAs derive from the hydrolysis of amide bonds in conjugated primary BAs as a first step, a reaction catalyzed by microbial bile salt hydrolases (BSH) enzymes in gut microbiota. To uncover BSH abundance and its activity in human stools, we developed an effect-based BL bioassay based on an amino luciferin” caged” with chenodeoxycholic acid (aLuc-CDCA), thus mimicking the conjugated primary BA with glycine. Since the reaction occurs after the BSH-catalyzed hydrolysis that allows the release of the “free” amino luciferin, we investigated in vitro the deconjugation level of aLuc-CDCA (50 µM) in the presence of pure BSH (range 0-10 µg/mL) using different exposure times at 37°C. Kinetic profiles showed a good linear correlation between BL emission intensity and increased amount of BSH, after 60 min of incubation (LOD:0.8 µg/mL; LOQ: 1.6 µg/mL). To overcome the decrease in light output due to interferents in fecal matrices, we then exploited the possibility of using a whole cell-based BL assay in which Caco2 cells were genetically encoded with the firefly luciferase Amydetes Vivianii. This approach enables the protection of the luciferase by cell membranes, obtaining a good correlation between BL signal intensity and BSH amount with a LOD of 2.6 µg/mL, and has been successfully employed for quantifying BSH in human stools. These effect-based BL bioassays can represent new tools for the screening of pre-neoplastic CRC lesions and possible therapeutic approaches.