In prostate cancer, the zinc levels in the gland are significantly reduced compared to healthy tissue and benign prostatic hyperplasia (BPH)¹. In this work we distinguish between the healthy mouse prostate and malignant prostate in a transgenic prostate cancer model (TRAMP) on the basis of Zn(II) content with MRI. Synthesis: Gd-CP027 was synthesized and characterized using published protocols². In vitro fluorescence: Normal human prostate epithelial cells (ATCC,VA, USA) were cultured using vendor protocols. Cells were incubated with 75µM ZnSO₄ in the medium for 72 hours at 37°C prior to study. The fluorescent zinc probe, ZIMIR, provided by Dr. Li at UT Southwestern was added to the cells to form a final concentration of 1 µM. Cells were then incubated at room temperature for 20 minutes and washed with SAB before glucose challenge. Zn(II) secretion in response to glucose was observed after adding 18mM D-glucose along with 10µM EDTA and 2µM DPAS. Epifluorescence images post-stimulation were obtained with an inverted Nikon wide-field fluorescence microscope. In vivo MRI: All animal experiments were performed in accordance to approved guidelines by the UT Southwestern IACUC committee. Ten TRAMP mice of the strain C57BL/6-Tg(TRAMP)8247Ng/J were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were fasted for at least 12 hours; the animals were anaesthetized with isofluorane and catheterized via the tail vein. Two pre-injection 3D T₁-weighted gradient echo scans were obtained (TE/TR 1.69/3.34ms, NEX4, Matrix 128x128x128) using a 9.4T Varian MRI scanner. After 50µl of 20% w/v D-glucose IP, 0.07mmol/Kg Gd-CP027 was injected IV using a syringe pump at a rate of 70µl/min. Immediately after injection, a series of 3D T₁-weighted scans were obtained to observe the dynamic glucose response and Zn(II) release in the prostate. The imaging protocol was repeated every two weeks until large tumors were found.The zinc sensor displays an increase in T₁ relaxivity at 0.5 T of almost 8-fold upon binding to Zn(II) and human serum albumin (HSA) as seen in the schematic in Figure 1. Although this is dramatically affected at higher magnetic fields the “on” 9.4 T relaxivity of 9.4mM-1s^-1 is still sufficiently sensitive to obtain an indirect measurement of prostatic zinc in vivo. Prostate epithelial intracellular zinc is mainly stored in zinc-rich granules and bound to a series of metalloproteins and citrate³, in this work we discovered that the use of D-glucose as an external secretagogue stimulates Zn(II) secretion in the prostate and thus make Zn(II) readily available to our Gd-based sensor. In figure 2 we see this phenomenon in RWPE-1 cells where the cells were cultured in low glucose conditions and provided with a zinc supplement available for intracellular storage. The zinc fluorescent probe, ZIMIR, anchors its lipophilic side-chains to cellular membranes and therefore is ideal to observe the movement of Zn(II) ions from inside the cell to its surroundings as a response to D-glucose. Figure 2 shows that approximately 5 minutes after addition of D-glucose to the medium we observe clear fluorescence emitted by the probe and thus validating the concept that a sudden increase in D-glucose stimulates the Zn(II) secreting mechanisms in prostate epithelial cells. The use of Zn(II) to differentiate between healthy prostate cells and aberrant cells was tested here by monitoring the spontaneous tumor formation in a TRAMP model. The mice were scanned upon arrival at 7-11 weeks old and subsequently every 1-2 weeks. Figure 3 shows three sets of representative mice, each showing distinctive stages of tumor formation. The bottom panel displays ~50% prostate enhancement as a result of D-glucose IP and Gd-CP027 IV injection, this enhancement may indicate that the levels of Zn(II) are not yet depleted and thus characteristic of healthy prostate. Middle panel, mouse at 11 weeks of age, show a hypointense region in the dorsolateral lobe of the prostate, indicative of cells deficient in Zn(II), top panel, mouse at 19 weeks of age, illustrate a large tumor stemming from the dorsolateral lobe of the prostate, it is evident that our Zn(II) sensitive contrast agent still enhances the minimal healthy prostate left but fails to enhance the tumor theoretically due to the lack of Zn(II) present in the tissue.We conclude that our Gd-based zinc sensor successfully differentiates healthy prostate tissue from prostate cancer tissue in the TRAMP model in vivo. Capitalizing on the known differences in Zn(II) content and that there is no current technique to differentiate between normal, malignant, and benign hyperplastic prostate tissue non-invasively, this lays the foundation for accurate differential diagnosis using glucose-stimulated release of Zn(II) as an imaging biomarker of prostatic disease.