Synchrotron Radiation X-ray Fluorescence Elemental Mapping in Healthy versus Malignant Prostate Tissues Provides New Insights into the Glucose-Stimulated Zinc Trafficking in the Prostate As Discovered by MRI

Abstract

Prostatic zinc content is a known biomarker for discriminating normal healthy tissue from benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Given that zinc content is not readily measured without a tissue biopsy, we have been exploring noninvasive imaging methods to detect these diagnostic differences using a zinc-responsive MRI contrast agent. During imaging studies in mice, we observed that a bolus of glucose stimulates secretion of zinc from the prostate of fasted mice. This discovery allowed the use of a Gd-based zinc sensor to detect differential zinc secretion in regions of healthy versus malignant prostate tissue in a transgenic adenocarcinoma mouse model of PCa. Here, we used a zinc-responsive MRI agent to detect zinc release across the prostate during development of malignancy and confirm the loss of total tissue zinc by synchrotron radiation X-ray fluorescence (μSR-XRF). Quantitative μSR-XRF results show that the lateral lobe of the mouse prostate uniquely accumulates high concentrations of zinc, 1.06 ± 0.08 mM, and that the known loss of zinc content in the prostate is only observed in the lateral lobe during development of PCa. Additionally, we confirm that lesions identified by a loss of zinc secretion indeed represent malignant neoplasia and that the relative zinc concentration in the lesion is reduced to 0.370 ± 0.001 mM. The μSR-XRF data also provided insights into the mechanism of zinc secretion by showing that glucose promotes movement of zinc pools (∼1 mM) from the glandular lumen of the lateral lobe of the mouse prostate into the stromal/smooth muscle surrounding the glands. Co-localization of zinc and gadolinium in the stromal/smooth muscle areas as detected by μSR-XRF confirm that glucose initiates secretion of zinc from intracellular compartments into the extracellular spaces of the gland where it binds to the Gd-based agent and albumin promoting MR image enhancement.

Figure 1.

(A) Schematic to illustrate the mechanism of MRI zinc detection. (B) Current study design and sample preparation. After resection of the organ, 50 and 10 μm sections were cut and mounted for μSR-XRF and hematoxylin and eosin (H&E) staining, respectively.

Figure 2.

In vivo GSZS MRI identification of malignant prostate lesions and μSR-XRF validation of zinc and gadolinium content. (A) T₁-weighted gradient echo images of healthy C57BL6 and 21-week-old TRAMP mice prior to and 7 min after receiving a bolus of 0.07 mmol/kg GdL2 plus 2.2 mmol/kg glucose. The inset in the TRAMP mouse image shows a hypo-intense region in the lateral/ventral lobe, consistent with a nascent tumor. (B) Image analysis of ROIs drawn along the prostates of healthy and TRAMP animals. The contrast-to-noise ratio of prostate versus muscle tissue indicates a loss of GSZS in the TRAMP animal (N = 4 healthy, N = 6 TRAMP, *p < 0.05). (C) Representative H&E stains and μSR-XRF images of prostate tissue samples show a poorly differentiated tumor in the lateral lobe and the distribution of zinc, gadolinium, and phosphorus in those same slices. Concentration bars for zinc and gadolinium correspond to absolute quantified values in millimolar; absolute concentrations for P were not obtained due to low energy of the fluorescent signal.

Figure 3.

Glucose-stimulated movement of zinc pools in the prostate. (A) μSR-XRF of WT C57Bl6 animals without any glucose or GdL2 illustrates the endogenous distribution of zinc. (B) μSR-XRF of WT animals only receiving 0.07 mmol/kg GdL2 shows the distribution of zinc and gadolinium in the prostate. High resolution μSR-XRF shows glands found in the lateral lobe full of zinc-rich secretions. Phosphorus membrane seen in blue delineates the separation between intraglandular zinc and stromal gadolinium. (C) μSR-XRF of WT animals receiving both glucose and GdL2 shows that the distribution of zinc continues to concentrate in the lateral lobe, but at the glandular level, zinc is now secreted from the glands into the stromal space where it comes in contact with gadolinium. (D) μSR-XRF of 20–23-week-old TRAMP receiving glucose and GdL2 illustrates the relative reduction of zinc in the entire gland; at the glandular level, zinc is found predominantly in the smooth muscle surrounding the gland, and not distributed in the stroma as seen in C.

Figure 4.

Quantification of element concentration in different prostate lobes. (A) Mouse prostate section illustrating the ventral (yellow), lateral (gray), dorsal (blue), and anterior (green) lobes. Close inspection of histological structures and the decision chart from ref allowed consistent identification of lobular structures. (B) Quantified concentrations of zinc, gadolinium, copper, and iron, categorized by prostate lobe for healthy naïve WT animals (N = 3), WT mice receiving GdL2 and no glucose (N = 3), WT mice receiving GdL2 and glucose (N = 3), and 20–23-week-old TRAMP receiving GdL2 and glucose (N = 10).

Figure 5.

Glandular and stromal distribution of elements. (A) H&E stained section of prostate glands illustrating the glandular lumen (yellow line) and smooth muscle surrounding the gland as stroma (blue dotted line). (B) The distribution of zinc and gadolinium in WT mice receiving GdL2 and either saline or glucose and in TRAMP mice receiving GdL2 and glucose. *p < 0.05, **p < 0.01.