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Detection of Alternative Splicing During Epithelial-Mesenchymal Transition
Published on: October 9, 2014
In vivo imaging of mesenchymal-epithelial transition factor (c-Met) expression using an optical imaging system
Eun-Mi Kim1, Eun-Hye Park, Su-Jin Cheong
1Department of Nuclear Medicine, Research Institute of Clinical Medicine, Cyclotron Research Center, Chonbuk National University Medical School and Hospital, Jeonju, Jeonbuk, Republic of Korea.
Abstract:
Mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase that has been shown to be overexpressed and mutated in a variety of malignancies, such as glioma. We have recently found that an (125)I-radiolabeled Gly-Gly-Gly (GGG)- or 8-aminooctanoic acid (AOC)-containing c-Met binding peptide (cMBP) specifically targets c-Met receptor in vivo and in vitro. In this report, cyanine dye 5.5 (Cy5.5)-conjugated GGG- or AOC-containing cMBPs were evaluated in human cancer cell xenografts in order to investigate the possibility of c-Met receptor targeting using an optical imaging system. The receptor binding affinity of Cy5.5-conjugated peptides was tested in 96-well plates coated with a c-Met/Fc chimeric protein. Optical imaging studies were performed in U87MG and Ramos bearing athymic mice. The binding affinities of Cy5.5-conjugated GGG- or AOC-containing cMBPs were determined to be 0.318 and 0.342 microM, respectively. Confocal images show that Cy5.5-conjugated peptides bound mainly to the cell surface and that peptide binding was clearly inhibited by free cMBP. Subcutaneous U87MG tumors were clearly visualized with each of the two fluorescent probes. Of the two, cMBP-AOC-Cy5.5 displayed higher tumor uptake and tumor-to-normal tissue ratios at 10 min to 24 h postinjection in the U87MG tumor model. For the in vivo blocking study, cMBP-AOC-Cy5.5 (4 nmol) was co-injected with cold cMBP (0.13 micromol) into the U87MG xenograft mice. Image-based tumoral uptake decreased up to approximately 35%. These results suggest that Cy5.5-conjugated cMBP could potentially be used to detect c-Met-positive cancers in vivo. However, additional modifications to this optical imaging agent are needed to further improve its efficacy.
Insights
New fluorescent peptide probes targeting the c-Met receptor show promise for optical imaging of cancer. These c-Met binding peptides (cMBPs) conjugated with Cy5.5 successfully visualized tumors in vivo, indicating potential for detecting c-Met-positive cancers.
Area of Science:
- Oncology
- Molecular Imaging
- Bioconjugation Chemistry
Background:
- The c-Met receptor tyrosine kinase is implicated in various cancers, including glioma.
- Previous studies developed radiolabeled c-Met binding peptides (cMBPs) for targeted imaging.
- Optical imaging offers a non-invasive method for cancer detection.
Purpose of the Study:
- To evaluate cyanine dye 5.5 (Cy5.5)-conjugated cMBPs for optical imaging of c-Met-positive tumors.
- To assess the in vitro and in vivo targeting capabilities of these novel probes.
Main Methods:
- Synthesis of Cy5.5-conjugated GGG- and AOC-containing cMBPs.
- In vitro binding affinity assays using c-Met/Fc chimeric protein.
- In vivo optical imaging in U87MG and Ramos tumor-bearing athymic mice.
- Tumor uptake and blocking studies to confirm target specificity.
Main Results:
- Cy5.5-conjugated cMBPs demonstrated specific binding to c-Met with binding affinities of 0.318 µM (GGG) and 0.342 µM (AOC).
- Confocal microscopy confirmed cell surface binding, inhibited by free cMBP.
- Subcutaneous U87MG tumors were visualized; cMBP-AOC-Cy5.5 showed higher tumor uptake and ratios.
- In vivo blocking studies reduced tumoral uptake by ~35%, confirming target specificity.
Conclusions:
- Cy5.5-conjugated cMBPs are potential optical imaging agents for detecting c-Met-positive cancers.
- The cMBP-AOC-Cy5.5 probe exhibited superior performance in tumor models.
- Further modifications are necessary to enhance the efficacy of these optical imaging agents.
