Purpose

The healthy prostate contains the highest concentration of mobile zinc of all soft tissues in the body. This level decreases dramatically during the development of prostate cancer^1,2. Therefore, non-invasive detection of prostate zinc content may potentially be applied for an early diagnosis of malignant transformation. A new approach is to use ¹⁹F MRI with fluorinated probes, termed iCEST, which is capable to detect sub-millimolar Zn²⁺ concentrations³. Here, we show that the iCEST MRI is able to differentiate between normal and malignant prostate cells (as outlined in Figure 1) and to detect zinc deficiency in vivo in an orthotopic prostate cancer mouse model.

Methods

Normal (RWPE1), ZIP1 zinc transporter-downregulated (RWPE2), and malignant (DU145 and LNCaP) prostate cells were incubated with 75 μM ZnSO₄ for 72 h and then washed with secretion assay buffer for 3 times. Induction of zinc secretion by glucose stimulation was carried out according to a previous report². After centrifugation, cells were divided equally into three wells, and co-incubated with 2.5 mM 5,5’,6,6’-tetrafluoro-1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (TF-BAPTA) and 0, 3, or 18 mM glucose for 1 h, respectively. The supernatant of cells was collected in 5 mm NMR tubes. MRI experiments were performed on a Bruker 17.6 T scanner with vertical bore. For ¹⁹F iCEST MRI, the center frequency was set at the frequency of the ¹⁹F atom at the 6 position (0.0 ppm) of TF-BAPTA. A modified RARE sequence (TR/TE=3,053/2.87 ms, RARE factor=16, 8 mm slice, FOV=1.6×1.6 cm, matrix size=32×32, resolution=0.5×0.5 mm, average number=96 and a saturation pulse B₁=2.4 µT/3 s) was used. 1 x 10⁶ cancer cells were injected into the prostate of 6-8 weeks old immunodeficient NSG mice and allowed to grow for 21 days. All normal NSG mice and orthotopic tumor-bearing NSG mice were fasted for 12 h before the iCEST MRI procedure. Immediately after intraperitoneal (i.p.) injection of 80 μL of 20% w/v D-glucose and injection of 0.15 g/kg bw TF-BAPTA into the anterior prostate through a catheter, iCEST MRI images were collected. A modified RARE sequence was used with TR/TE=3,053/2.87 ms, RARE factor=16, 3 mm slice thickness, FOV=2.6×2.6 cm, matrix size=32×32, resolution=0.8×0.8 mm, average number=8 and a saturation pulse B₁=2.4 µT/3 s.

Results

In vitro, the strongest iCEST signal was observed for glucose-stimulated RWPE1 cells with a normal zinc transporter level (Figure 2d). In normal prostate cells with a downregulated ZIP1 zinc transporter (RWPE2), a weaker iCEST signal was observed. No signal could be observed for DU145 and LNCaP prostate cancer cells. The concentration of released zinc from RWPE1 cells stimulated by 3 mM and 18 mM glucose was calculated to be approximately 20 μM and 40 μM, respectively. In vivo, the strongest iCEST signal was observed for the normal prostate following i.p. glucose stimulation (Figure 2f). Both the normal prostate without glucose stimulation and the two orthotopic tumor models with glucose stimulation showed much weaker iCEST signal.

Conclusions

Using iCEST MRI, differences in glucose-induced zinc secretion between normal and malignant prostate cells can be readily detected, both in vitro and in vivo. The feasibility of serially monitoring the conversion of normal prostate cells into malignant cells is now further being evaluated in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model.

Acknowledgements

This project was supported by R03 EB018882 and the Pearl and Yueh-Heng Yang Foundation.

References

¹Ghosh, S. K. et al. Cancer Res. 2010, 70, 6119. ²Clavijo Jordan, V. et al. Proc. Natl. Acad. Sci. 2016, 113, E5464. ³Bar-Shir, A. et al. J. Am. Chem. Soc. 2015, 137, 78.