(64)Cu-Labeled Ubiquitin for PET Imaging of CXCR4 Expression in Mouse Breast Tumor

[Image: see text] Ubiquitin has been recently identified as a chemokine receptor 4 (CXCR4) natural ligand, offering great potential for positron emission computed tomography (PET) imaging of CXCR4 expression. This study reports the preparation and evaluation of ((64)Cu)-radiolabeled ubiquitin for CXCR4 imaging. The ubiquitin was first fused with a C-terminal GGCGG sequence, and the resulting recombinant ubiquitin derivative UbCG4 was then functionalized with the trans-cyclooctene (TCO) moiety via thiol–maleimide click reaction, followed by (64)Cu-radiolabeling through the TCO/Tz (tetrazine)-based Diels–Alder click reaction. In the prepared in vitro studies, the prepared ((64)Cu)-UbCG4 showed significantly higher specific uptakes in the 4T1 breast cancer cells compared with the uptakes in the CXCR4-knockdown 4T1 cells. In the in vivo evaluation in the 4T1-xenograft mouse model, ((64)Cu)-UbCG4 demonstrated a similar tumor uptake but much lower backgrounds compared with (64)Cu-labeled AMD3465. These results suggested that ((64)Cu)-UbCG4 could serve as a potent PET tracer for the noninvasive imaging of CXCR4 expression in tumors.