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Updated: Mar 2, 2026
Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT
Published on: April 29, 2017
Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT
Sabrina H L Hoffmann1, Andreas Maurer1, Dorothea I Reck1
1Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Eberhard Karls University Tübingen.
Insights
This study presents a novel method for tracking cells in vivo using Copper-64 (⁶⁴Cu) radiolabeling of monoclonal antibodies. This technique allows for non-invasive PET/CT imaging of cell targeting and tracking in a mouse model.
Area of Science:
- Nuclear Medicine
- Immunology
- Cell Biology
Background:
- Positron Emission Tomography (PET)/Computed Tomography (CT) imaging is crucial for non-invasive cell tracking.
- Developing stable and specific radiolabeling methods for cell tracking is an ongoing challenge.
Purpose of the Study:
- To detail a protocol for producing ⁶⁴Cu and radiolabeling monoclonal antibodies (mAbs) for cell tracking.
- To evaluate the in vitro and in vivo performance of ⁶⁴Cu-labeled cells for PET/CT imaging.
- To demonstrate the utility of this method in an airway delayed-type hypersensitivity reaction (DTHR) mouse model.
Main Methods:
- Conjugation of mAbs with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator.
- Production of radioactive ⁶⁴Cu and subsequent radiolabeling of DOTA-conjugated mAbs.
- Expansion of T helper cells and their radiolabeling.
- In vitro assessment of cell viability, apoptosis, functionality (IFN-γ ELISA), radiolabel uptake, and labeling stability.
- Induction of airway DTHR in BALB/c mice for in vivo cell tracking.
- PET/CT imaging acquisition, reconstruction, and analysis.
Main Results:
- The ⁶⁴Cu-mAb targeting approach demonstrated high specificity and stability with reduced cellular toxicity and low efflux rates.
- In vitro assays confirmed the integrity and functionality of radiolabeled cells.
- Non-invasive in vivo cell tracking in the DTHR mouse model was achieved with PET/CT, showing an optimal signal-to-background ratio for up to 48 hours.
Conclusions:
- The developed ⁶⁴Cu-antibody receptor targeting protocol enables robust, non-invasive in vivo cell tracking via PET/CT.
- This method offers advantages over conventional PET tracers, including improved specificity, stability, and reduced toxicity.
- The protocol is adaptable for various cell types and animal models with membrane-bound receptors.
Abstract:
This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described. In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-γ-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-γ enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented. The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.
