Detection of Intestinal Cancer by Local, Topical Application of a Quenched Fluorescence Probe for Cysteine Cathepsins

Segal, Ehud; Prestwood, Tyler R.; Linden, Wouter A. van der; Carmi, Yaron; Bhattacharya, Nupur; Withana, Nimali P.; Verdoes, Martijn; Habtezion, Aida; Engleman, Edgar G.; Bogyo, Matthew

doi:10.1016/j.chembiol.2014.11.008

Cited by 71 publications

(60 citation statements)

References 48 publications

(53 reference statements)

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“…We and others have previously demonstrated that small-molecule ABPs can be used to topically label active cathepsins in excised tissues (37,38). Two fresh human carotid endarterectomy specimens were split into sections to perform labeling using the optical probe BMV109 (Fig.…”

Section: Topical Application Of Activity-based Probe On Human Carotidmentioning

confidence: 99%

Dual-Modality Activity-Based Probes as Molecular Imaging Agents for Vascular Inflammation

Withana

Saito

et al. 2016

J Nucl Med

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Macrophages are cellular mediators of vascular inflammation and are involved in the formation of atherosclerotic plaques. These immune cells secrete proteases such as matrix metalloproteinases and cathepsins that contribute to disease formation and progression. Here, we demonstrate that activity-based probes (ABPs) targeting cysteine cathepsins can be used in murine models of atherosclerosis to noninvasively image activated macrophage populations using both optical and PET/CT methods. The probes can also be used to topically label human carotid plaques demonstrating similar specific labeling of activated macrophage populations. Methods: Macrophage-rich carotid lesions were induced in FVB mice fed on a high-fat diet by streptozotocin injection followed by ligation of the left common carotid artery. Mice with carotid atherosclerotic plaques were injected with the optical or dual-modality probes BMV109 and BMV101, respectively, via the tail vein and noninvasively imaged by optical and small-animal PET/CT at different time points. After noninvasive imaging, the murine carotid arteries were imaged in situ and ex vivo, followed by immunofluorescence staining to confirm target labeling. Additionally, human carotid plaques were topically labeled with the probe and analyzed by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunofluorescence staining to confirm the primary targets of the probe. Results: Quantitative analysis of the signal intensity from both optical and PET/CT imaging showed significantly higher levels of accumulation of BMV109 and BMV101 (P , 0.005 and P , 0.05, respectively) in the ligated left carotid arteries than the right carotid or healthy arteries. Immunofluorescence staining for macrophages in cross-sectional slices of the murine artery demonstrated substantial infiltration of macrophages in the neointima and adventitia of the ligated left carotid arteries compared with the right. Analysis of the human plaque tissues by sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that the primary targets of the probe were cathepsins X, B, S, and L. Immunofluorescence labeling of the human tissue with the probe demonstrated colocalization of the probe with CD68, elastin, and cathepsin S, similar to that observed in the experimental carotid inflammation murine model. Conclusion: We demonstrate that ABPs targeting the cysteine cathepsins can be used in murine models of atherosclerosis to noninvasively image activated macrophage populations using both optical and PET/CT methods. The probes could also be used to topically label human carotid plaques demonstrating similar specific labeling of activated macrophage populations. Therefore, ABPs targeting the cysteine cathepsins are potentially valuable new reagents for rapid and noninvasive imaging of atherosclerotic disease progression and plaque vulnerability.

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Section: Topical Application Of Activity-based Probe On Human Carotidmentioning

confidence: 99%

Dual-Modality Activity-Based Probes as Molecular Imaging Agents for Vascular Inflammation

Withana

Saito

et al. 2016

J Nucl Med

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“…The use of fluorescently labeled markers that target tumors allows clear detection of margins, making it possible to achieve complete tumor resection while preserving healthy surrounding structures and tissue. Investigators have recently developed molecular imaging probes to image disease-associated proteases [14][15][16][17][18] . In addition to being viable targets for the treatment of disease, proteases are also potential biomarkers for diagnostic purposes 19 .…”

Section: Existing Methods For Visualization Of Tumor Marginsmentioning

confidence: 99%

“…BMV109 contains a Cy5 fluorophore, a sulfo-QSY21 quencher and a phenoxymethyl ketone electrophile that labels cysteine cathepsins B, S, L and X 17,18 (Fig. 1a).…”

Section: Experimental Designmentioning

confidence: 99%

“…The probe rapidly and selectively labeled tumor cells, distinguishing cancerous tissue from surrounding healthy tissue with a positive predictive value of 90.32% (95% CI of 74.22 to 97.85%) 17 . This protocol details the original published method, and it includes additional data obtained using a specific inhibitor capable of blocking the probe signal in order to demonstrate the selectivity of the method for active cathepsins.…”

Section: Advantages and Applications Of Using Abps To Assess Tumor Bumentioning

confidence: 99%

“…The benefit of using these covalent quenched probes over other probes, such as substrate probes, is that they have higher protease selectivity, they rapidly penetrate cells, and they provide a durable signal owing to the fact that they remain bound to the active protease 25 . We have recently demonstrated that qABPs can be topically applied to colon tissues to detect intestinal cancer 17 .…”

Section: Advantages and Applications Of Using Abps To Assess Tumor Bumentioning

confidence: 99%

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Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes

et al. 2015

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Active enzymes, such as proteases, often serve as valuable biomarkers for various disease pathologies. Therefore, methods to detect specific enzyme activities in biological samples can provide information to guide disease detection and diagnosis and to increase our understanding of the biological roles of specific enzyme targets. In this protocol, we outline methods for the topical application of fluorescently quenched activity-based probes (qABPs) to fresh-frozen tissue samples. This technique enables rapid imaging of enzyme activity at cellular resolution, and it can be combined with antibody labeling for immunodiagnosis. In this method, fresh-frozen tissue sections are fixed, incubated with the probe and imaged using fluorescence microscopy. This provides an advance over classical immunohistochemistry (IHC) in that it is rapid (4-8 h) and inexpensive, and it provides information on enzyme activity. Furthermore, it can be used with any of the growing number of fluorescent ABPs to provide data for more effective disease monitoring and diagnosis.

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Chemical Probes Targeting Proteases for Imaging and Diagnostics in Cancer

Gonçalves

Verhelst

2018

Extracellular Targeting of Cell Signaling in Cancer

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Detection of Intestinal Cancer by Local, Topical Application of a Quenched Fluorescence Probe for Cysteine Cathepsins

Cited by 71 publications

References 48 publications

Dual-Modality Activity-Based Probes as Molecular Imaging Agents for Vascular Inflammation

Dual-Modality Activity-Based Probes as Molecular Imaging Agents for Vascular Inflammation

Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes

Chemical Probes Targeting Proteases for Imaging and Diagnostics in Cancer

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