General Strategy for Bioluminescence Sensing of Peptidase Activity In Vivo Based on Tumor-Targeting Probiotic

Abstract

The abnormally expressed peptidases in human tissues are associated with many kinds of cancers. Monitoring of endogenous peptidase activity could allow us for pathophysiology elucidation and early clinical diagnosis. Herein, we developed a general strategy for bioluminescence (BL) sensing of peptidase activity in vivo based on tumor-targeting probiotics. The probiotic that harbored a luciferase-encoding plasmid was used to target and colonize tumor and provide luciferase for BL imaging. The peptide-based probes Lc and GPc were applied to track leucine aminopeptidase (LAP) and dipeptidyl peptidase IV (DPPIV) activity, respectively, by simply adding l-leucine and Gly-Pro dipeptides at the N-terminus of d-cysteine, which were specifically controlled by peptidase cleavage and released free d-cysteine to conduct a subsequent click condensation reaction with 2-cyano-6-hydroxybenzothiazole (HCBT) to produce firefly luciferin in situ, giving rise to a strong BL signal. Neither gene modification of cells of interest nor complicated synthesis was required in this BL system. Encouraged by these advantages, we successfully used our probes to monitor LAP and DPPIV activities in vitro and in vivo, respectively. A good linearity between BL and peptidase was obtained in the concentration range of 2.5–40.0 mU/mL with a limit of detection (LOD) of 1.1 mU/mL (55 ng/mL) for LAP and 2.0–40.0 mU/mL with a LOD of 0.78 mU/mL (1.15 ng/mL) for DPPIV, respectively. Additionally, approximately 5-fold (LAP) and 10-fold (DPPIV) differences in the BL signal before and after treatment with a specific inhibitor were also obtained for in vivo BL imaging. All these results reflected the potential application value of our probes in BL sensing of peptidase activity. We envision that our strategy may be a useful approach for monitoring a wide range of peptidases in tumors, especially in primary tumors.

Chemically Enhanced Live Probiotic for In Vivo Tumor Targeting and Inhibition

“…Necrosis is commonly found in the advanced stages of a solid tumor when the size of the tumor was relatively large . Therefore, EcN localized well in large tumors . To verify the colonization ability of PCB, relatively smaller tumors were used in this experiment.…”

“…39 Therefore, EcN localized well in large tumors. 40 To verify the colonization ability of PCB, relatively smaller tumors were used in this experiment. In vivo colonization of PCB was imaged by using D-luciferin.…”

Atg4B and Cathepsin B-Triggered in Situ Luciferin Formation for Precise Cancer Autophagy Bioluminescence Imaging

“…During tumor autophagy, these two compounds are cleaved by their corresponding autophagy enzymes to yield d -Cys and CBT, respectively, which undergo a CBT-Cys click reaction to yield d -aminoluciferin inside cancer cells, − turning tumor BL “on”. In fact, this in situ luciferin formation approach has been successfully applied for dual-analyte imaging in inflammation and tumor mice models. , And in this study, we found it was also well-performed in precise cancer autophagy BL imaging.…”

Enhancing the Contrast of Tumor Imaging for Image-Guided Surgery Using a Tumor-Targeting Probiotic with the Continuous Expression of a Biomarker

“…The existence of bacteria in solid tumors has been well-documented for a long time. Bacteria have gained unprecedented attention due to their unique capabilities, such as intratumoral selective colonization, and as vectors for delivering prodrug-converting enzymes. − Anaerobic bacteria can penetrate and proliferate in the hypoxic regions of tumors. − Recently, many studies have reported that the number of bacteria accumulated and proliferated in tumor cells might be 1000× higher than that in reticuloendothelial organs . More importantly, bacteria are easily eliminated by antibiotics in normal tissues, allowing them to play important roles in cancer diagnosis and treatment.…”

“…40−42 Recently, many studies have reported that the number of bacteria accumulated and proliferated in tumor cells might be 1000× higher than that in reticuloendothelial organs. 43 More importantly, bacteria are easily eliminated by antibiotics in normal tissues, allowing them to play important roles in cancer diagnosis and treatment. Compared with other attenuated tumor-targeting pathogenic bacteria, the facultative anaerobic probiotic Escherichia coli Nissle 1917 (EcN) has a superior ability to target and colonize in tumor tissues without pathogenicity.…”