Jagged1-Fluc: A Luminescent Recombinant Protein as a Sensitive Tool for High-Throughput Screening of Colorectal...

Colorectal cancer (CRC) onset is closely linked to dysregulated Notch signaling, particularly involving receptors like Notch1 and Notch3, and ligands such as Jagged1 (Jag1), which are overexpressed in aggressive subtypes. To address the need for a quantitative assessment of Notch expression in clinical settings, we developed a luminescent recombinant probe (Jag1-Fluc) by fusing the extracellular domain of a high-affinity Jag1 mutant with red-emitting firefly luciferase. The Jag1-Fluc probe can bind to Notch, thus correlating the intensity of emitted light with the concentration of Notch in the sample. In cell-free assays, Jag1-Fluc demonstrated a linear luminescent signal across different concentrations (detection limit: 0.20 ± 0.03 μg/mL). When applied to living and fixed human colorectal cancer cells, the probe showed a dose-dependent light emission (5-50 μg/mL), confirming its ability to bind endogenous Notch receptors. Competitive binding assays using soluble human Jag1 chimera demonstrated concentration-dependent signal inhibition (IC₅₀ = 0.55 ± 0.06 μg/mL), validating probe specificity. Notably, in a small cohort of human biopsies, Jag1-Fluc enabled stratification of Notch expression, with luminescence intensity progressively increasing from hyperplastic polyps to low- and high-grade adenomas. These findings reinforce prior evidence linking Jag1 overexpression to CRC progression. The assay's simplicity and sensitivity highlight its potential for early CRC screening, paving the way for personalized medicine by enabling tailored therapeutic monitoring and strategies based on Notch signaling profiles. This approach potentially enhances treatment efficacy and reduces side effects, representing a valuable tool for precision oncology.