Development of Zinc-Specific iCEST MRI as an Imaging Biomarker for Prostate Cancer

Abstract

The healthy prostate contains the highest concentration of mobile zinc in the body. As this level decreases dramatically during the initial development of prostate cancer, in vivo detection of prostate zinc content may be applied for diagnosis of prostate cancer. Using ¹⁹ F ion chemical exchange saturation transfer magnetic resonance imaging (iCEST MRI) and TF-BAPTA as a fluorinated Zn-binding probe with micromolar sensitivity, we show that iCEST MRI is able to differentiate between normal and malignant prostate cells with a 10-fold difference in contrast following glucose-stimulated zinc secretion in vitro. The iCEST signal decreased in normal prostate cells upon downregulation of the ZIP1 zinc transporter. In vivo, using an orthotopic prostate cancer mouse model and a transgenic adenocarcinoma of the mouse prostate (TRAMP) model, a gradual decrease of >300 % in iCEST contrast following the transition of normal prostate epithelial cells to cancer cells was detected.

Keywords: MRI; imaging agents; prostate cancer; zinc.

Figure 1.

ICEST MRI only detects zinc at the Zn-TF-BAPTA-specific offset frequency. a) Orientation of sample phantoms containing 10 mm TF-BAPTA and 100 μM Ion in SAB. b) ¹H MRI, c) ¹⁹F MRI, and d) ICEST signal (Δω = −2.8 ppm, B₁ = 2.4 μT) overlaid on ¹⁹F MRI. e) ICEST spectra of 10 mm TF-BAPTA + 100 μM Zn²⁺ for three saturation pulses with different powers. f) ICEST contrast (−2.8 ppm) of 10 mm TF-BAPTA + 100 μM Zn²⁺ as a function of pH.

Figure 2.

ICEST MRI can distinguish between normal and cancerous prostate cells In vitro. a) ¹⁹F ICEST spectra, b) ¹⁹F MRI and ICEST signal overlaid on ¹⁹F MRI. c) ICEST signal of supernatants of cells treated with 5 mM TF-BAPTA and 18 mM, 3, or 0 mM D-glucose. d) Calibration curve of χ_Zn (Zn²⁺/TF-BAPTA, μM mM⁻¹) vs. ICEST contrast. Various zinc concentrations were added to 5 mM TF-BAPTA (R² = 0.994). e) Amount of cellular glucose-induced zinc secretion calculated from the equation shown in (d). All measurements were performed at Δω = −2.8 ppm.

Figure 3.

a) Zinc-activated TFLZn (green) fluorescent images of RWPE1, RWPE2, LNCaP, and DU145 cells, counterstained with propidium iodide (PI, red). Scale bar = 50 μm. b) Calibration curve of zinc fluorescence in SAB for 500 μm TFLZn (λ_ex/λ_em = 380/510 nm). c) Calculated secreted zinc levels for D-glucose at different concentrations.

Figure 4.

a) ¹⁹F MRI, iCEST MRI, and iCEST spectra of control NSG mice (normal prostate) before and after glucose injection, and of LNCaP and DU145-bearing NSG mice after glucose injection. All mice were injected with 0.15 g kg⁻¹ of TF-BAPTA, GSZS was induced by i.p. injection of 80 μL of 20% (w/v) D-glucose. All measurements were performed at Δω = −2.8 ppm. b) H&E staining of normal and tumor-bearing NSG mice.

Figures 5.

a) ¹⁹F MRI, iCEST MRI, and iCEST spectra of 10-week old TRAMP mice before and after glucose injection, and of 17-week and 24-week old TRAMP mice after glucose injection. b) Quantification of iCEST signal after glucose stimulation. Data are shown as mean ± SEM (n = 4). All mice were injected with 0.15 gkg⁻¹ of TF-BAPTA, GSZS was induced by i.p. injection of 80 μL of 20% (w/v) D-glucose. All measurements were performed at Δω = −2.8 ppm. c) H&E and PCNA staining (green = PCNA, blue = DAPI) of AP in 10-week, 17-week, and 24-week old TRAMP mice.

Scheme 1.

Conceptual design of Zn-specific cellular ICEST MRI. a) Chemical structure of free and zinc-bound TF-BAPTA. b) Schematic of glucose-stimulated extracellular zinc secretion with subsequent chelation by TF-BAPTA, which generates an ICEST signal In normal but not In cancerous prostate cells.