Engineered Peptide Hydrogel Interface for Coupling Rapid Stemness Induction, In Situ Pathway Monitoring, and Direct...

Cancer stem cell (CSC) models are essential for understanding tumor progression and evaluating targeted therapies. However, conventional approaches rely on sphere formation, destructive characterization, and re-culture steps, resulting in prolonged workflows and limited reproducibility (15-30 days). Herein, we report an engineered functional peptide hydrogel interface that seamlessly integrates rapid CSC induction with in situ pathway monitoring and drug evaluation. The self-assembled Fmoc-diphenylalanine (Fmoc-FF) hydrogel provides a 3D microenvironment, while co-assembly with Fmoc-RGD peptides activates integrin-mediated signaling, inducing highly efficient stemness reprogramming of MCF-7 cells within 3 days. By incorporating a manganese porphyrin as a catalytic recognition element, the hydrogel-electrode interface enables electrochemical detection of nitric oxide (NO) as a functional readout of pathway activation. This design allows real-time, in situ monitoring of dynamic signaling processes during CSC induction. Furthermore, the NO-responsive electrochemical sensing platform enables direct evaluation of CSC-targeting drugs within the same 3D system, eliminating the need for sphere transfer and lysis-based assays. Compared with conventional methods, the integrated strategy significantly shortens the experimental workflow to 6-8 days while improving operational simplicity and functional readout capability. This work establishes a materials-driven strategy for coupling cell state programming with electrochemical signal transduction, enabling integrated interrogation of CSC biology and therapeutic response.