sup 18^F-FDG, ^sup 18^F-4FMFES, and ^sup 89^Zr-trastuzumab PET imaging protocol in a preclinical breast cancer model

Objectives: Hormone therapy is highly effective in estrogen receptor positive (ER+) breast cancers. However, if recurrence occurs, ER expression is often downregulated and tumors become increasingly difficult to detect and treat. Recently, aberrant signalling caused by HER2 overexpression on the surface of breast cancer cells was shown to compensate for the loss of ER signalling in hormone therapy resistance. Hence, PET imaging ER and HER2 target-specific tracers has potential for improved characterization of breast cancer. A PET imaging protocol was designed for whole-body monitoring of ER and HER2 status using purposely developed ER and HER2 target-specific probes. 4-fluoro-11β-methoxy-16a-[18F]-fluoroestradiol [18F-4FMFES] is able to detect ER-positive tumors with increased accuracy relative to 18F-fluoroestradiol. 89Zr-labeled trastuzumab is a recent tracer for imaging HER2+ breast cancer. 18F-FDG is a routine tracer used for breast cancer detection. Methods: Commercially available p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS) was conjugated to the lysine-NH2 groups of trastuzumab using known standard procedures. Labeling was achieved using 89Zr-chloride mixed with 1 mg DFO-trastuzumab in PBS at pH 7.0 and room temperature for 16 h. Purification of 89Zr-trastuzumab was performed by centrifugal filtration. The conjugate was radiochemically characterized using instant thin layer chromatography and SDS-PAGE. 18F-4FMFES and 18F-FDG were prepared as previously described. The MCF7 (ER+, HER2-) and ZR-75-1 (ER+, HER2+) breast cancer cell lines were implanted subcutaneously on the shoulders of NOD/SCID mice (n=5). Day 1 of the imaging protocol commenced when tumors were 4 mm in diameter. As a reference, a whole-body PET scan was performed in mice injected with 18F-FDG on day 1. On day 2, 2.0 ± 0.7 MBq of 18F-4FMFES was intravenously administered followed by PET imaging 1 h post-administration. Immediately after 18F-4FMFES imaging, mice were injected with ~50 μg (1.6 ± 0.2 MBq) 89Zr-trastuzumab. On day 6, mice were imaged again by PET for 30 min. A semi-quantitative analysis of the tracer uptake in tumors was carried out to obtain values expressed as %ID/g. Results: 89Zr-trastuzumab specific activity was 30 MBq/mg (4.5 TBq/mmol), with radiochemical purity ...95%. Tumor cell implantation timing demonstrated that both MCF7 and ZR-75-1 tumors could be developed simultaneously. MCF7 tumors had 18F-FDG and 18F-4FMFES uptake of 2.5 ± 0.2 %ID/g and 2.3 ± 1.2 %ID