HaloTag as a reporter gene for PET

Objectives To employ HaloTag technology for positron emission tomography (PET), which involves 2 components: HaloTag protein (an engineered haloalkane dehalogenase which covalently binds to synthetic ligands) and HaloTag ligand (HTL).

Methods 4T1 murine breast cancer cells were stably transfected to express HaloTag protein on the surface (i.e. 4T1-HaloTag-ECS). Microscopy studies were performed to determine the cellular distribution of HaloTag, using commercial AlexaFluor488-HTL. We synthesized a new HTL which contains 1,4,7-triazacyclononane-N,N’N’’-triacetic acid (NOTA, for ⁶⁴Cu-labeling) and polyethylene glycol (PEG, to improve pharmacokinetics), which was termed NOTA-PEG-HTL2G (2G is 2nd generation). Pulse-chase experiments were conducted to evaluate the HaloTag binding efficiency of NOTA-PEG-HTL2G in vitro. ⁶⁴Cu-NOTA-PEG-HTL2G was studied for PET imaging in mice bearing 4T1-HaloTag-ECS or 4T1 tumors. Autoradiography and histology studies were carried out to confirm the HaloTag specificity of ⁶⁴Cu-NOTA-PEG-HTL2G in vivo.

Results Microscopy confirmed surface expression of HaloTag in 4T1-HaloTag-ECS cells, which specifically binds NOTA-PEG-HTL2G. The radiochemical yield was >85% for ⁶⁴Cu-NOTA-PEG-HTL2G, with purity of >98%. Uptake of ⁶⁴Cu-NOTA-PEG-HTL2G in 4T1-HaloTag-ECS tumors (4.3±0.5, 4.0±0.2, 4.1±0.2, 2.3±0.1, and 2.2±0.1%ID/g at 0.5, 3, 6, 16, and 24 h post-injection; n=3) was significantly higher than that in 4T1 tumors (3.0±0.3, 3.0±0.1, 3.0±0.2, 2.0±0.4, and 2.4±0.3 %ID/g respectively; n=3) at early time points. Other organs with significant tracer uptake included kidneys and liver, which indicated renal/hepatobiliary clearance. Autoradiography of tumor homogenates confirmed that ⁶⁴Cu-NOTA-PEG-HTL2G binds specifically to HaloTag protein in the 4T1-HaloTag-ECS tumor, corroborated by histology.

Conclusions HaloTag protein-specific targeting and PET imaging in vivo was achieved with ⁶⁴Cu-NOTA-PEG-HTL2G, which serves as a proof-of-principle for future non-invasive and sensitive tracking of HaloTag-transfected (stem) cells with PET, as well as many other studies of gene/protein/cell function in vivo.