A. Progressive cancer burden in TRAMP mice monitored by the cumulative weight of the genitourinary (GU) tract (includes prostate, seminal vesicles, testicles and empty urinary bladder). GU tracts were weighed from both wild type and TRAMP mice at the indicated ages (6-22 weeks) and normalised to respective mouse body weights (n=5-15 at each age). Horizontal black lines represent the mean GU tract weight at the indicated age. B. Representative photographs of harvested GU tracts from wild type and TRAMP mice at 10, 18 and 22 weeks of age. C. Histological examination of prostate lobes [anterior prostate (AP), dorsolateral prostate (DLP) and ventral prostate (VP)] with hematoxylin and eosin (H&E) staining, verifying disease grade in TRAMP mice at the indicated ages (6-22 weeks) (n=5 at each age). The most advanced proliferative lesion in each lobe signified the grade of disease. D. Representative H&E-stained sections displaying grades of prostate disease in TRAMP mice from hyperplasia, low-grade PIN, high-grade PIN, adenocarcinoma and invasive adenocarcinoma. H&E-stained normal prostate was obtained from wild type mice. (***p < 0.001).

Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine whether copper, zinc or iron concentrations change in prostate lobes and sera of TRAMP mice throughout disease progression. A-D. Copper concentrations in anterior prostate (AP), dorso-lateral prostate (DLP), ventral prostate (VP) and serum from both wild type and TRAMP mice at the indicated ages (6-22 weeks). Zinc E-H. and iron I-K. concentrations are also shown. Results represent mean ± STDEV (bar) and are shown as either μg/g wet weight for tissues (n=5-10 at each age) or μM for serum (n=10-17 at each age). (*p < 0.05; ***p < 0.001).

A. TRAMP adenocarcinoma cells (TRAMP-C1) have normal intracellular copper levels. Total intracellular copper was measured in both TRAMP-C1 and mouse primary prostate epithelial cells (PrECs) cultured under basal conditions. Results are shown as copper (ng) per 10⁶ cells. B. TRAMP adenocarcinoma cells (TRAMP-C1) have elevated intracellular ROS levels. Intracellular ROS was measured using the cell permeable fluorogenic probe H₂DCF-DA and flow cytometry. Results represent mean fluorescence intensity (MFI) (geometric mean). C. Copper-ionophores potently kill TRAMP adenocarcinoma cells (TRAMP-C1). TRAMP-C1 cells were treated for 18 hours with Cu^II(gtsm), disulfiram (DSF) or clioquinol alone or in combination with 20 μM CuCl₂. Ionophore concentrations are shown and cell viability was determined by the propidium iodide exclusion assay and flow cytometry. D. Copper-ionophores selectively kill TRAMP adenocarcinoma cells while not affecting the viability of mouse primary prostate epithelial cells (PrECs). Both cell lines were treated for 18 hours with Cu^II(gtsm), disulfiram or clioquinol in combination with 20 μM CuCl₂. Ionophore concentrations are shown and cell viability was determined by the propidium iodide exclusion assay and flow cytometry. E. Copper-ionophores generate intracellular ROS in TRAMP adenocarcinoma cells (TRAMP-C1) (i), but not in mouse primary prostate epithelial cells (PrECs) (ii). Both cell lines were treated for 2, 4, 6 or 18 hours with Cu^II(gtsm), disulfiram or clioquinol in combination with 20 μM CuCl₂. Ionophore concentrations are shown and intracellular ROS was measured using the cell permeable fluorogenic probe H₂DCF-DA and flow cytometry. Results represent mean fluorescence intensity (MFI) (geometric mean) F. TRAMP adenocarcinoma cells (TRAMP-C1) have markedly reduced antioxidant capacity. Reduced (GSH) (i) and oxidised (GSSG) (ii) glutathione were measured in TRAMP adenocarcinoma cells (TRAMP-C1) and mouse primary prostate epithelial cells (PrECs) by HPLC. (iii) The GSH:GSSG ratio is compared between both cell lines. G. Differential GSH expression in TRAMP adenocarcinoma cells (TRAMP-C1) treated with copper-ionophores. Reduced glutathione (GSH) was measured in mouse primary prostate epithelial cells (PrECs) (i) and TRAMP adenocarcinoma cells (TRAMP-C1) (ii) following treatment for 18 hours with sublethal concentrations of Cu^II(gtsm) (20 nM), disulfiram (150 nM) or clioquinol (2 μM) (with 20 μM CuCl₂). Glutathione (GSH & GSSG) was measured by HPLC. Results represent mean ± STDEV (bar) of triplicate determinations for each measurement. (*p < 0.05; **p < 0.01).

A&B. TRAMP mice harbouring the tx mutation (tx/TRAMP) display elevated copper in their liver and serum. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine copper concentrations in the liver and serum of 22-week old wild type, tx, TRAMP and tx/TRAMP mice (n=5-20 for each strain). Results represent mean ± STDEV (whisker plot) and are shown as either μg/g wet weight for tissue or μM for serum. C. Copper incorporation into serum ceruloplasmin is perturbed in TRAMP mice harbouring the tx mutation (tx/TRAMP). Serum ceruloplasmin oxidase activity (copper-dependent) was measured from 22-week old wild type (n=10), tx (n=11), TRAMP (n=17) and tx/TRAMP (n=10) mice using the o-dianisidine dihydrochloride based assay. Results are expressed as unit/litre (U/L) and presented as mean ± STDEV (bar). D-H. TRAMP mice harbouring the tx mutation (tx/TRAMP) display elevated copper in extrahepatic tissues. ICP-MS was used to determine copper concentrations in brain, kidney, spleen, lung and prostate lobes [anterior prostate (AP), dorsolateral prostate (DLP) and ventral prostate (VP)] of 22-week old wild type, tx, TRAMP and tx/TRAMP mice (n=4-20 for each strain). Results represent mean ± STDEV (whisker plot) and are shown as μg/g wet weight. I. Mouse prostate has no detectable level of Atp7b expression. (i) Real-time PCR quantification of Atp7b mRNA levels in liver (LIV), kidney (KID) and prostate (PRO) of 22-week old wild type mice (n=3). The level of Atp7b mRNA is compared against the liver. (ii) Western blot analysis of Atp7b expression in liver (LIV), kidney (KID) and prostate (PRO) of 22-week old wild type mice (50 μg protein). The WND4B antibody detected Atp7b in the liver and kidney at ~170kDa. β-actin was detected as a loading control. (*p < 0.05; **p < 0.01; ***p < 0.001).

A. The tx mutation significantly reduces prostate cancer burden in TRAMP mice. Genitourinary (GU) tracts were weighed from 22-week old and 26-week old wild type, tx, TRAMP and tx/TRAMP mice and normalised to respective mouse body weights (n=5-15 for each strain). The mean ± STDEV (whisker plot) for each strain is shown. B. The tx mutation significantly reduces prostate cancer disease severity (grade) in TRAMP mice. Histological examination of prostate lobes [anterior prostate, dorsolateral prostate and ventral prostate] with hematoxylin and eosin (H&E) staining, establishing disease grade in 26-week old wild type, tx, TRAMP and tx/TRAMP mice (n=5 for each strain). The most advanced proliferative lesion in each lobe signified the grade of disease. C. Representative H&E-stained sections displaying prostate disease grade in the prostate lobes [anterior prostate (AP), dorsolateral prostate (DLP) and ventral prostate (VP)] of 26-week old TRAMP and tx/TRAMP mice. (*p < 0.05; **p < 0.01; ***p < 0.001)