Related Experiment Video
Updated: Feb 28, 2026
Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models
Published on: March 13, 2018
Low-Background Cancer Imaging with a Bioorthogonal Fluorescence Probe and Engineered Reporter Enzyme Bearing a
Ziyi Wang1, Ryosuke Kojima1, Rikuki Kiji2
1Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
Abstract:
Combinatorial use of an antibody-reporter enzyme conjugate and a fluorescence probe activated by the enzyme is a powerful strategy for fluorescence-guided cancer surgery. However, conventional probes for typical reporter enzymes lack sufficient bioorthogonality, leading to high background signals in nontarget tissues. We screened a library of HMRef (rhodol derivative)-based fluorescence probes with various sugar moieties and found that HMRef-β-d-Fucose is bioorthogonal in mammalian systems but is activated by a metagenomic glycosidase, Td2F2. Directed evolution generated a mutant with a kcat/Km of 3.3 × 105/M/sec, 7.3 times higher than wild-type Td2F2 and comparable to β-galactosidase (LacZ) with its corresponding probe. Theoretical calculation suggested the E296G mutation facilitates probe access to the enzyme's active site. In a proof-of-concept study, SKOV-3 cells, which endogenously express HER2, were visualized with minimal background in the mesentery of a mouse model using HMRef-β-d-Fucose and engineered Td2F2 conjugated or fused to a HER2-binding antibody or nanobody.
Insights
This study developed a novel bioorthogonal fluorescence probe (HMRef-β-d-Fucose) and an engineered enzyme (Td2F2) for precise cancer surgery. This combination minimizes background signals, enabling clearer visualization of cancer cells in vivo.
Area of Science:
- Biochemistry
- Molecular Biology
- Medical Imaging
Background:
- Fluorescence-guided surgery enhances precision but is limited by probe bioorthogonality and high background signals.
- Conventional probes often lack specificity, leading to inaccurate tumor margin detection.
Purpose of the Study:
- To develop a highly bioorthogonal fluorescence probe and enzyme system for improved cancer cell visualization.
- To engineer a reporter enzyme with enhanced catalytic activity for probe activation.
Main Methods:
- Screening of rhodol derivative-based probes, identification of HMRef-β-d-Fucose.
- Directed evolution of a metagenomic glycosidase (Td2F2) for improved probe activation.
- In vivo proof-of-concept study using HER2-expressing SKOV-3 cells in a mouse model.
Main Results:
- HMRef-β-d-Fucose demonstrated bioorthogonality in mammalian systems.
- Engineered Td2F2 showed a 7.3-fold increase in catalytic efficiency (kcat/Km) compared to wild-type.
- Successful visualization of HER2-expressing cells with minimal background in a mouse model.
Conclusions:
- The HMRef-β-d-Fucose and engineered Td2F2 system offers a promising strategy for fluorescence-guided cancer surgery.
- This approach significantly reduces background noise, improving the accuracy of tumor detection.
Related Concept Videos
03:31Labeling DNA Probes
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
02:11Reporter Genes