Alkaline Phosphatase and ATG4B Sequentially Activated Fluorescent Probe for Cancer Cell-Specific Live Imaging of...

Tracking autophagy in cancer cells is crucial for enhancing cancer therapies. Existing methods are often inefficient and cannot distinguish cancer from normal cells during autophagy. Herein, a sequentially activated peptide probe, NBD-1p-Dabcyl, was developed for achieving cancer cell-specific imaging of autophagy. The probe self-assembled and fluoresced brightly upon sequential processing by alkaline phosphatase (ALP) and autophagy-related protease (ATG4B), where NBD-1p-Dabcyl was dephosphorylated by ALP to give NBD-1-Dabcyl, which was then processed by ATG4B into nanofibers emitting strong fluorescence. Notably, the bright fluorescence of NBD was observed in cancer cells MDA-MB-231 and HeLa, while normal cells NIH3T3 exhibited weaker fluorescence, allowing differentiation between cancer and normal cells using a rapamycin (Rap)-induced autophagy cell model. The enhanced fluorescence in cancer cells was attributed to the higher activities of intracellular ALP and ATG4B. Next, NBD-1p-Dabcyl was used to assess the inhibition efficiency of an autophagy inhibitor NSC 185058 in MDA-MB-231 cells, where a strong correlation between fluorescence intensity and inhibitor concentration suggested that NBD-1p-Dabcyl could predict the activity of autophagy inhibitors. Finally, animal experiments revealed that NBD-1p-Dabcyl effectively facilitated in situ fluorescence imaging of autophagy in tumor tissues. The design of this sequentially activated peptide probe offers a practical approach for monitoring autophagy in cancer cells, enabling high-throughput screening of autophagy inhibitors for cancer therapy.