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Targeting Phosphatidylserine for Positron Emission Tomography Imaging of Cell Death: Annexin V versus Phosphatidylserine-Binding Peptides Open Access


Other title
gallium-68 (68Ga)
phosphatidylserine (PS)
EL4 lymphoma
annexin V
fluorine-18 (18F)
molecular imaging
positron emission tomography (PET)
copper-64 (64Cu)
cell death
Type of item
Degree grantor
University of Alberta
Author or creator
Perreault, Amanda C
Supervisor and department
Wuest, Frank (Oncology)
Examining committee member and department
Hitt, Mary (Oncology)
Goping, Ing Swie (Biochemistry)
Wuest, Melinda (Oncology)
Makis, Villiam (Radiology & Diagnostic Imaging)
Department of Oncology
Experimental Oncology
Date accepted
Graduation date
Master of Science
Degree level
There is currently no clinically approved, widely used molecular imaging agent for in vivo detection of cell death. Such an agent would be highly useful for early assessment of treatment efficacy in cancer patients, as well as for monitoring many other diseases in which cell death plays an important role. Phosphatidylserine (PS), a phospholipid that is normally confined to the inner leaflet of the cell membrane, becomes externalized on cells undergoing cell death. The accessibility of PS on dying cells makes it an attractive target for a cell death imaging probe. In this study, we have radiolabelled and evaluated several PS-targeting biomolecules as potential candidates for positron emission tomography (PET) imaging of chemotherapy-induced tumour cell death, including the well-known PS-targeting protein annexin V and three literature-reported PS-binding peptides. Wild-type annexin V was labelled with fluorine-18 (18F) using two techniques: a random labelling approach that targets any of the 23 primary amines present on the protein, and a site-selective labelling approach that targets the protein’s single cysteine residue. Using an EL4 mouse lymphoma cell binding assay, we determined that both labelling techniques produced an 18F-labelled product that could detect cell death induced by chemotherapeutic agent camptothecin, and that labelling technique did not have a significant effect on the ability of 18F-labelled annexin V to bind to PS. Using a well-established mouse EL4 tumour model of cell death induced by combination treatment with etoposide and cyclophosphamide, we were unable to demonstrate the ability of 18F-annexin V to image EL4 tumour cell death in vivo. This is in contrast to previous reports that have shown successful preclinical imaging of chemotherapy-induced cell death using 18F-labelled wild-type annexin V. Possible reasons for our negative result include poor penetration of 18F-annexin V into EL4 tumours, lack of sufficient Ca2+ concentrations required for 18F-annexin V binding to PS, and lack of imaging during the time of greatest PS exposure. We also investigated three literature-reported PS-binding peptides as potential candidates for development of a PET agent for imaging cell death: LIKKPF, PGDLSR, and PS-binding peptide 6 (PSBP-6). We developed a novel competitive radiometric PS binding assay that revealed very poor PS-binding affinities of LIKKPF- and PGDLSR-based peptide conjugates, while PSBP-6-based conjugates showed more favourable affinities. We radiolabelled each of the PS-binding peptides with positron-emitting radionuclides to develop them for PET imaging. Hexapeptides PGDLSR and LIKKPF were successfully labelled with 18F, but neither of these radiolabelled peptides showed suitable stability in vivo. PSBP-6 was conjugated with metal chelator 1,4,7-triazacyclononane-triacetic acid (NOTA) to be labelled with radiometals gallium-68 (68Ga) and copper-64 (64Cu). Both 68Ga-NOTA-PSBP-6 and 64Cu-NOTA-PSBP-6 showed much better in vivo stability than the radiolabelled hexapeptides. Our cell binding assay showed that 64Cu-NOTA-PSBP-6 could detect camptothecin-induced cell death in the absence of Ca2+. However, supplementation with Ca2+ seemed to enhance the ability of 64Cu-NOTA-PSBP-6 to detect cells undergoing cell death. Using our EL4 lymphoma tumour model, we found that 64Cu-NOTA-PSBP-6 uptake was significantly higher in tumours receiving chemotherapeutic treatment compared to controls, which was apparent 5 minutes after injection with the radiopeptide. However, tumour retention of 64Cu-NOTA-PSBP-6 was poor, likely due to a combination of low affinity for PS, rapid proteolytic breakdown in the plasma, and fast blood clearance. We also labelled PSBP-6 with fluorescein isothiocyanate (FITC), and flow cytometry studies showed significantly higher binding of this fluorescent peptide to EL4 cells treated with camptothecin compared to untreated cells. Fluorescence microscopy studies revealed that FITC-PSBP-6 is not internalized while the cell membrane is still intact, but remains bound to the cell membrane. Further evaluation of the ability of 18F-labelled wild-type annexin V to detect tumour cell death in vivo will require optimization of the current EL4 model or the use of other preclinical tumour models of therapy-induced cell death. On the other hand, 64Cu-labelled PS-binding peptide PSBP-6 shows promise as an alternative probe for imaging cell death in vivo using PET. Modifications to further improve the PS-binding affinity, metabolic stability, blood clearance profile and tumour retention of 64Cu-NOTA-PSBP-6 are needed in order to “fine-tune” this radiopeptide for optimal imaging.
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
Citation for previous publication
Perreault A, Knight J, Wang M, Way J, Wuest F. 18F-labeled wild-type annexin V: Comparison of random and site-selective radiolabeling methods. Amino Acids. 2016;48(1):65-74. doi: 10.1007/s00726-015-2068-0.Wuest M, Perreault A, Kapty J, Richter S, Foerster C, Bergman C, Way J, Mercer J, Wuest F. Radiopharmacological evaluation of 18F-labeled phosphatidylserine-binding peptides for molecular imaging of apoptosis. Nucl Med Biol. 2015;42(11):864-74. doi: 10.1016/j.nucmedbio.2015.06.011.Perreault A, Richter S, Foerster C, Bergman C, Wuest M, Wuest F. 68Ga-labeled peptides for targeting phosphatidylserine (PS) in apoptosis. J Labelled Comp Radiopharm. 2015;58(1):S131.Foerster C, Perreault A, Wuest M, Bergman C, Wuest F. A novel radiometric phosphatidylserine (PS) binding assay based on [64Cu]Cu-NOTA-annexin-V. Nucl Med Biol. 2014;41(7):642–3.Knight J, Perreault A, Wang M, Way J, Wuest F. Radiolabeling annexin-V with fluorine-18: random and site-selective approaches. J Labelled Comp Radiopharm. 2013;56(1):S159.

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