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Detecting in vivo urokinase plasminogen activator activity with a catalyCEST MRI contrast agent.

ABSTRACT: Urokinase plasminogen activator (uPA) promotes tumor invasion and metastasis. The monitoring of uPA activity using molecular imaging may have prognostic value and be predictive for response to anti-cancer therapies. However, the detection of in vivo enzyme activity with molecular imaging remains a challenge. To address this problem, we designed a nonmetallic contrast agent, GR-4Am-SA, that can be detected with chemical exchange saturation transfer (CEST) MRI. This agent has a peptide that is cleaved by uPA, which causes a CEST signal at 5.0 ppm to decrease, and also has a salicylic acid moiety that can produce a CEST signal at 9.5 ppm, which is largely unresponsive to enzyme activity. The two CEST signals were used to determine a reaction coordinate, representing the extent of enzyme-catalyzed cleavage of the GR-4Am-SA agent during an experimental study. Initial biochemical studies showed that GR-4Am-SA could detect uPA activity in reducing conditions. Subsequently, we used our catalyCEST MRI protocol with the agent to detect the uPA catalysis of GR-4Am-SA in a flank xenograft model of Capan-2 pancreatic cancer. The results showed an average reaction coordinate of 80% ± 8%, which was strongly dependent on the CEST signal at 5.0 ppm. The relative independence of the reaction coordinate on the CEST signal at 9.5 ppm showed that the detection of enzyme activity was largely independent of the concentration of GR-4Am-SA within the tumor tissue. These results demonstrated the advantages of a single CEST agent with biomarker-responsive and unresponsive signals for reliably assessing enzyme activity during in vivo cancer studies.

SUBMITTER: Sinharay S

PROVIDER: S-EPMC5704996 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Detecting in vivo urokinase plasminogen activator activity with a catalyCEST MRI contrast agent.

Sinharay Sanhita S Howison Christine M CM Baker Amanda F AF Pagel Mark D MD

NMR in biomedicine 20170329 7

Urokinase plasminogen activator (uPA) promotes tumor invasion and metastasis. The monitoring of uPA activity using molecular imaging may have prognostic value and be predictive for response to anti-cancer therapies. However, the detection of in vivo enzyme activity with molecular imaging remains a challenge. To address this problem, we designed a nonmetallic contrast agent, GR-4Am-SA, that can be detected with chemical exchange saturation transfer (CEST) MRI. This agent has a peptide that is cle ...[more]

PMID: 28370884

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Project description:Data from clinical studies, cell culture, and animal models implicate the urokinase (uPA)/Plasminogen (Plg) system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/Plg stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression to clarify mechanisms of uPA/Plg-accelerated atherosclerosis and aneurysm formation. Microarray studies were performed to identify potential mediators of uPA-accelerated atherosclerosis. These studies identified S100A8 and S100A9 mRNA as the most highly upregulated transcripts in uPA-overexpressing macrophages; upregulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also upregulated in aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is upregulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Bioinformatics analysis of the microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm—in a second animal model—that macrophage-expressed uPA stimulates atherosclerosis and aortic dilation. They also implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease. Six independent biological replicate RNA samples were prepared from thioglycollate-elicited peritoneal macrophages from mice of two different genotypes: SR-uPA+/0 transgenic (overexpress uPA in macrophages) and nontransgenic, respectively), for a total of 12 independent RNA samples. Both transgenic and nontransgenic mice were Apoe-/- and were fed Western diet from 5-15 weeks before peritoneal macrophages were elicited. In addition, from the six samples of each genotype, a pooled sample was prepared by combining the six genotype-specific samples. Each of the two pooled samples was assayed on the BeadChip in two technical replicates, for a total of 16 hybridizations performed using two Illumina Mouse Ref-8 v1.1 chips.

Dataset Information

Detecting in vivo urokinase plasminogen activator activity with a catalyCEST MRI contrast agent.

Publications

Detecting in vivo urokinase plasminogen activator activity with a catalyCEST MRI contrast agent.

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