Targeting Phosphatidylserine with a64Cu-Labeled Peptide for Molecular Imaging of Apoptosis.
Molecular imaging of programmed cell death (apoptosis) in vivo is an innovative strategy for early assessment of treatment response and treatment efficacy in cancer patients. Externalization of phosphatidylserine (PS) to the cell membrane surface of dying cells makes this phospholipid an attractive molecular target for the development of apoptosis imaging probes. In this study, we have radiolabeled PS-binding 14-mer peptide FNFRLKAGAKIRFG (PSBP-6) with positron-emitter copper-64 (64Cu) for PET imaging of apoptosis. Peptide PSBP-6 was conjugated with radiometal chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) through an aminovaleric acid (Ava) linker for subsequent radiolabeling with64Cu to prepare radiotracer64Cu-NOTA-Ava-PSBP-6. PS-binding potencies of PSBP-6, NOTA-Ava-PSBP-6, andnatCu-NOTA-Ava-PSBP-6 were determined in a competitive radiometric PS-binding assay. Radiotracer64Cu-NOTA-Ava-PSBP-6 was studied in camptothecin-induced apoptotic EL4 mouse lymphoma cells and in a murine EL4 tumor model of apoptosis using dynamic PET imaging. Peptide PSBP-6 was also conjugated via an Ava linker with fluorescein isothiocyanate (FITC). FITC-Ava-PSBP-6 was evaluated in flow cytometry and fluorescence confocal microscopy experiments. Radiopeptide64Cu-NOTA-Ava-PSBP-6 was synthesized in high radiochemical yields of >95%. The IC50values for PS-binding potency of PSBP-6, NOTA-Ava-PSBP-6, andnatCu-NOTA-PSBP-6 were 600 μM, 30 μM, and 23 μM, respectively. A competitive radiometric cell binding assay confirmed binding of64Cu-NOTA-Ava-PSBP-6 to camptothecin-induced apoptotic EL4 cells in a Ca2+-independent manner. PET imaging studies demonstrated significantly higher uptake of64Cu-NOTA-Ava-PSBP-6 in apoptotic EL4 tumors (SUV5min0.95 ± 0.04) compared to control tumors (SUV5min0.74 ± 0.03). Flow cytometry studies showed significantly higher binding of FITC-Ava-PSBP-6 to EL4 cells treated with camptothecin compared to untreated cells. Fluorescence microscopy studies revealed that FITC-Ava-PSBP-6 was binding to cell membranes of early apoptotic cells, but was internalized in late apoptotic and necrotic cells. The present study showed that radiotracer64Cu-NOTA-Ava-PSBP-6 holds promise as a first peptide-based PET imaging agent for molecular imaging of apoptosis. However, additional "fine-tuning" of64Cu-NOTA-Ava-PSBP-6 is required to enhance PS-binding potency and in vivo stability to improve tumor uptake and retention.
Department of Oncology, Cross Cancer Institute, University of Alberta , 11560 University Avenue, Edmonton, Alberta T6G 2X4, Canada.
Amanda
A
Perreault
Department of Oncology, Cross Cancer Institute, University of Alberta , 11560 University Avenue, Edmonton, Alberta T6G 2X4, Canada.
Susan
S
Richter
Department of Oncology, Cross Cancer Institute, University of Alberta , 11560 University Avenue, Edmonton, Alberta T6G 2X4, Canada.
Cody
C
Bergman
Department of Oncology, Cross Cancer Institute, University of Alberta , 11560 University Avenue, Edmonton, Alberta T6G 2X4, Canada.
Melinda
M
Wuest
Department of Oncology, Cross Cancer Institute, University of Alberta , 11560 University Avenue, Edmonton, Alberta T6G 2X4, Canada.
Frank
F
Wuest
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D045744:Cell Line, Tumor; D003301:Copper Radioisotopes / Q000032:analysis*; D005434:Flow Cytometry; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D057054:Molecular Imaging / Q000379:methods*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D010718:Phosphatidylserines / Q000737:chemistry*; D049268:Positron-Emission Tomography / Q000379:methods
D003301:Copper Radioisotopes; D010455:Peptides; D010718:Phosphatidylserines
64Cu; apoptosis; peptide; phosphatidylserine; positron emission tomography (PET)