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Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry.

ABSTRACT: Vertebrate glycans constitute a large, important, and dynamic set of post-translational modifications that are notoriously difficult to manipulate and image. Although the chemical reporter strategy has been used in conjunction with bioorthogonal chemistry to image the external glycosylation state of live zebrafish and detect tumor-associated glycans in mice, the ability to image glycans systemically within a live organism has remained elusive. Here, we report a method that combines the metabolic incorporation of a cyclooctyne-functionalized sialic acid derivative with a ligation reaction of a fluorogenic tetrazine, allowing for the imaging of sialylated glycoconjugates within live zebrafish embryos.

SUBMITTER: Agarwal P

PROVIDER: S-EPMC4694582 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry.

Agarwal Paresh P Beahm Brendan J BJ Shieh Peyton P Bertozzi Carolyn R CR

Angewandte Chemie (International ed. in English) 20150731 39

Vertebrate glycans constitute a large, important, and dynamic set of post-translational modifications that are notoriously difficult to manipulate and image. Although the chemical reporter strategy has been used in conjunction with bioorthogonal chemistry to image the external glycosylation state of live zebrafish and detect tumor-associated glycans in mice, the ability to image glycans systemically within a live organism has remained elusive. Here, we report a method that combines the metabolic ...[more]

PMID: 26230529

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Project description:UnlabelledThe specificity of antibodies have made immunoconjugates promising vectors for the delivery of radioisotopes to cancer cells; however, their long pharmacologic half-lives necessitate the use of radioisotopes with long physical half-lives, a combination that leads to high radiation doses to patients. Therefore, the development of targeting modalities that harness the advantages of antibodies without their pharmacokinetic limitations is desirable. To this end, we report the development of a methodology for pretargeted PET imaging based on the bioorthogonal Diels-Alder click reaction between tetrazine and transcyclooctene.MethodsA proof-of-concept system based on the A33 antibody, SW1222 colorectal cancer cells, and (64)Cu was used. The huA33 antibody was covalently modified with transcyclooctene, and a NOTA-modified tetrazine was synthesized and radiolabeled with (64)Cu. Pretargeted in vivo biodistribution and PET imaging experiments were performed with athymic nude mice bearing A33 antigen-expressing, SW1222 colorectal cancer xenografts.ResultsThe huA33 antibody was modified with transcyclooctene to produce a conjugate with high immunoreactivity, and the (64)Cu-NOTA-labeled tetrazine ligand was synthesized with greater than 99% purity and a specific activity of 9-10 MBq/μg. For in vivo experiments, mice bearing SW1222 xenografts were injected with transcyclooctene-modified A33; after allowing 24 h for accumulation of the antibody in the tumor, the mice were injected with (64)Cu-NOTA-labeled tetrazine for PET imaging and biodistribution experiments. At 12 h after injection, the retention of uptake in the tumor (4.1 ± 0.3 percent injected dose per gram), coupled with the fecal excretion of excess radioligand, produced images with high tumor-to-background ratios. PET imaging and biodistribution experiments performed using A33 directly labeled with either (64)Cu or (89)Zr revealed that although absolute tumor uptake was higher with the directly radiolabeled antibodies, the pretargeted system yielded comparable images and tumor-to-muscle ratios at 12 and 24 h after injection. Further, dosimetry calculations revealed that the (64)Cu pretargeting system resulted in only a fraction of the absorbed background dose of A33 directly labeled with (89)Zr (0.0124 mSv/MBq vs. 0.4162 mSv/MBq, respectively).ConclusionThe high quality of the images produced by this pretargeting approach, combined with the ability of the methodology to dramatically reduce nontarget radiation doses to patients, marks this system as a strong candidate for clinical translation.

Dataset Information

Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry.

Publications

Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry.

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