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Logo 22nd ISBC & 20th ISLS


Analytical, clinical and medical applications of luminescence

Bioluminescence for the Screening of Colon Cancer: the role of Notch signaling and the gut microbiota

Cristiana Caliceti1, Angela Punzo1, Alessia Silla2, Vanessa Bevilacqua3, Laura Mezzanotte4, Giada Moroni5, Antimo Gioiello5, Vadim Viviani6, Sylvia Daunert7, Aldo Roda8

1Department of Biomedical and Neuromotor Sciences, University of Bologna, 40138 Bologna, Italy, 2Department for Life Quality Sciences, University of Bologna, 47921 Rimini, Italy, 3Faculty of Medical and Health Sciences, Pontifical Catholic University of São Paulo (PUC), Sorocaba 05014-901, Brazil, 4Department of Molecular Genetics, Erasmus MC, 3015 CN Rotterdam, the Netherlands, 5Department of Pharmaceutical Sciences, University of Perugia, 06123 Perugia, Italy, 6Department of Physics, Chemistry and Mathematics, Federal University of São Carlos (UFSCar), Sorocaba 18052-780, Brazil, 7Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Miami FL 33136, US, 8INBB, National Institute of Biostructures and Biosystems, 00136 Rome, Italy

E-mail: cristiana.caliceti@unibo.it

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.

Keywords: Bioluminescence; Recombinant protein; Notch pathway; Colorectal cancer; Imaging, “caged” amino luciferin, bile salt hydrolase, bile acids, gut microbiota

Acknowledgments: This work was supported by Fondazione Cassa di Risparmio di Bologna (ID 20010 COD SME 2022.0071)

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