Dataset Information

Antibody-based PET of uPA/uPAR signaling with broad applicability for cancer imaging.

ABSTRACT: Mounting evidence suggests that the urokinase plasminogen activator (uPA) and its receptor (uPAR) play a central role in tumor progression. The goal of this study was to develop an 89Zr-labeled, antibody-based positron emission tomography (PET) tracer for quantitative imaging of the uPA/uPAR system. An anti-uPA monoclonal antibody (ATN-291) was conjugated with a deferoxamine (Df) derivative and subsequently labeled with 89Zr. Flow cytometry, microscopy studies, and competitive binding assays were conducted to validate the binding specificity of Df-ATN-291 against uPA. PET imaging with 89Zr-Df-ATN-291 was carried out in different tumors with distinct expression levels of uPA. Biodistribution, histology examination, and Western blotting were performed to correlate tumor uptake with uPA or uPAR expression. ATN-291 retained uPA binding affinity and specificity after Df conjugation. 89Zr-labeling of ATN-291 was achieved in good radiochemical yield and high specific activity. Serial PET imaging demonstrated that, in most tumors studied (except uPA- LNCaP), the uptake of 89Zr-Df-ATN-291 was higher compared to major organs at 120 h post-injection, providing excellent tumor contrast. The tumor-to-muscle ratio of 89Zr-Df-ATN-291 in U87MG was as high as 45.2 ± 9.0 at 120 h p.i. In vivo uPA specificity of 89Zr-Df-ATN-291 was confirmed by successful pharmacological blocking of tumor uptake with ATN-291 in U87MG tumors. Although the detailed mechanisms behind in vivo 89Zr-Df-ATN-291 tumor uptake remained to be further elucidated, quantitative PET imaging with 89Zr-Df-ATN-291 in tumors can facilitate oncologists to adopt more relevant cancer treatment planning.

SUBMITTER: Yang D

PROVIDER: S-EPMC5342023 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Antibody-based PET of uPA/uPAR signaling with broad applicability for cancer imaging.

Yang Dongzhi D Severin Gregory W GW Dougherty Casey A CA Lombardi Rachel R Chen Daiqin D Van Dort Marcian E ME Barnhart Todd E TE Ross Brian D BD Mazar Andrew P AP Hong Hao H

Oncotarget 20161101 45

Mounting evidence suggests that the urokinase plasminogen activator (uPA) and its receptor (uPAR) play a central role in tumor progression. The goal of this study was to develop an 89Zr-labeled, antibody-based positron emission tomography (PET) tracer for quantitative imaging of the uPA/uPAR system. An anti-uPA monoclonal antibody (ATN-291) was conjugated with a deferoxamine (Df) derivative and subsequently labeled with 89Zr. Flow cytometry, microscopy studies, and competitive binding assays wer ...[more]

PMID: 27729618

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Project description:The repair of injured tissue is a highly complex process that involves cell proliferation, differentiation, and migration. Cell migration requires the dismantling of intercellular contacts in the injured zone and their subsequent reconstitution in the wounded area. Urokinase-type plasminogen activator (uPA) is a serine proteinase found in multiple cell types including endothelial cells, smooth muscle cells, monocytes, and macrophages. A substantial body of experimental evidence with different cell types outside the central nervous system indicates that the binding of uPA to its receptor (uPAR) on the cell surface prompts cell migration by inducing plasmin-mediated degradation of the extracellular matrix. In contrast, although uPA and uPAR are abundantly found in astrocytes and uPA binding to uPAR triggers astrocytic activation, it is unknown if uPA also plays a role in astrocytic migration. Neuronal cadherin is a member of cell adhesion proteins pivotal for the formation of cell-cell contacts between astrocytes. More specifically, while the extracellular domain of neuronal cadherin interacts with the extracellular domain of neuronal cadherin in neighboring cells, its intracellular domain binds to β-catenin, which in turn links the complex to the actin cytoskeleton. Glycogen synthase kinase 3β is a serine-threonine kinase that prevents the cytoplasmic accumulation of β-catenin by inducing its phosphorylation at Ser33, Ser37, and Ser41, thus activating a sequence of events that lead to its proteasomal degradation. The data discussed in this perspective indicate that astrocytes release uPA following a mechanical injury, and that binding of this uPA to uPAR on the cell membrane induces the detachment of β-catenin from the intracellular domain of neuronal cadherin by triggering its extracellular signal-regulated kinase 1/2-mediated phosphorylation at Tyr650. Remarkably, this is followed by the cytoplasmic accumulation of β-catenin because uPA-induced extracellular signal-regulated kinase 1/2 activation also phosphorylates lipoprotein receptor-related protein 6 at Ser1490, which in turn, by recruiting glycogen synthase kinase 3β to its intracellular domain abrogates its effect on β-catenin. The cytoplasmic accumulation of β-catenin is followed by its nuclear translocation, where it induces the expression of uPAR, which is required for the migration of astrocytes from the injured edge into the wounded area.

Project description:Urokinase-type plasminogen activator receptor (uPAR) is overexpressed in human prostate cancer and uPAR has been found to be associated with metastatic disease and poor prognosis. AE105 is a small linear peptide with high binding affinity to uPAR. We synthesized an N-terminal NOTA-conjugated version (NOTA-AE105) for development of the first (18)F-labeled uPAR positron-emission-tomography PET ligand using the Al(18)F radiolabeling method. In this study, the potential of (18)F-AlF-NOTA-AE105 to specifically target uPAR-positive prostate tumors was investigated.NOTA-conjugated AE105 was synthesized and radiolabeled with (18)F-AlF according to a recently published optimized protocol. The labeled product was purified by reverse phase high performance liquid chromatography RP-HPLC. The tumor targeting properties were evaluated in mice with subcutaneously inoculated PC-3 xenografts using small animal PET and ex vivo biodistribution studies. uPAR-binding specificity was studied by coinjection of an excess of a uPAR antagonist peptide AE105 analogue (AE152).NOTA-AE105 was labeled with (18)F-AlF in high radiochemical purity (>92%) and yield (92.7%) and resulted in a specific activity of greater than 20GBq/?mol. A high and specific tumor uptake was found. At 1h post injection, the uptake of (18)F-AlF-NOTA-AE105 in PC-3 tumors was 4.22 ± 0.13%ID/g. uPAR-binding specificity was demonstrated by a reduced uptake of (18)F-AlF-NOTA-AE105 after coinjection of a blocking dose of uPAR antagonist at all three time points investigated. Good tumor-to-background ratio was observed with small animal PET and confirmed in the biodistribution analysis. Ex vivo uPAR expression analysis on extracted tumors confirmed human uPAR expression that correlated close with tumor uptake of (18)F-AlF-NOTA-AE105.The first (18)F-labeled uPAR PET ligand, (18)F-AlF-NOTA-AE105, has successfully been prepared and effectively visualized noninvasively uPAR positive prostate cancer. The favorable in vivo kinetics and easy production method facilitate its future clinical translation for identification of prostate cancer patients with an invasive phenotype and poor prognosis.

Project description:BackgroundLoss of intestinal epithelial barrier integrity is a critical component of Inflammatory Bowel Disease (IBD) pathogenesis. Co-expression regulation of ligand-receptor pairs in IBD mucosa has not been systematically studied. Targeting ligand-receptor pairs which are induced in IBD mucosa and function in intestinal epithelial barrier integrity may provide novel therapeutics for IBD.MethodsWe performed transcriptomic meta-analysis on public IBD datasets combined with cell surface protein-protein-interaction (PPI) databases. We explored primary human/mouse intestinal organoids and Caco-2 cells for expression and function studies of uPA-uPAR (prime hits from the meta-analysis). Epithelial barrier integrity was measured by Trans-Epithelial Electrical Resistance (TEER), FITC-Dextran permeability and tight junction assessment. Genetic (CRISPR, siRNA and KO mice) and pharmacological (small molecules, neutralizing antibody and peptide inhibitors) approaches were applied. Mice deficient of uPAR were studied using the Dextran Sulfate Sodium (DSS)-induced colitis model.FindingsThe IBD ligand-receptor meta-analysis led to the discovery of a coordinated upregulation of uPA and uPAR in IBD mucosa. Both genes were significantly upregulated during epithelial barrier breakdown in primary intestinal organoids and decreased during barrier formation. Genetic inhibition of uPAR or uPA, or pharmacologically blocking uPA-uPAR interaction protects against cytokine-induced barrier breakdown. Deficiency of uPAR in epithelial cells leads to enhanced EGF/EGFR signalling, a known regulator of epithelial homeostasis and repair. Mice deficient of uPAR display improved intestinal barrier function in vitro and during DSS-induced colitis in vivo.InterpretationOur findings suggest that blocking uPA-uPAR interaction via pharmacological agents protects the epithelial barrier from inflammation-induced damage, indicating a potential therapeutic target for IBD.FundingThe study was funded by Boehringer Ingelheim.

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Antibody-based PET of uPA/uPAR signaling with broad applicability for cancer imaging.

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Antibody-based PET of uPA/uPAR signaling with broad applicability for cancer imaging.

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