Figure 1.
Developing mesothelioma tumors rapidly acquire copper.
Mice were sacrificed when tumors reached 1-16 mm2, 20-50 mm2 and > 50 mm2. Tumor growth rate shown in A. Copper levels from the different sized tumors (B); pooled data from 4 experiments, with n = 19 mice with 1-16mm2 tumors; n = 10 mice with 20-50mm2 tumors; n = 16 mice with > 50mm2 tumors; and n = 10 control mice is shown as mean ± SEM. Copper levels are also shown as individual weighed tumor samples to better reflect tumor size (C). Cu levels in whole tumor tissue relative to individual tumor weights are also shown (D).
Figure 2.
Bioavailable copper levels can be decreased in vivo by copper chelating agents.
Low, medium and high doses of TM (A and B), penicillamine (C and D) and trientine (E and F) were administered i.p. to healthy mice over 5 days. Mice were sacrificed on days 0, 2 and 5 Plasma ceruloplasmin (Cp) was assayed (A, C and E), and liver Cu levels analyzed (B, D and F). For Cu levels n = 3 mice/group. For Cp assays there were 11 mice for TM at day 0, these mice were divided into groups and given different doses of TM; 1 mouse/TM dose was culled and sampled at day 2 (n = 1/group) for each of the three doses; 3 mice were sampled for each of the 200 µg and 1000µg TM doses (n = 3/group), and 2 mice sampled for the 500µg TM treatment at day 5 (n = 2/group). For penicillamine and trientine there were 4 mice per treatment dose at day 0 (n = 4) and 1 mouse sampled at each time point for each dose (n = 1/group). Where possible, data is shown as mean ± SEM.
Figure 3.
Reducing bioavailable copper slows tumor growth rate.
AE17-bearing mice were given daily PBS, TM (500 µg/dose/mouse), penicillamine (2000 µg/dose/mouse) and trientine (700 µg/dose/mouse) throughout tumor growth (A). Pooled data is from one experiment (n = 4 or 5 mice/group). In a separate experiment, AE17-bearing mice were given cisplatin, TM or PBS; n = 6 mice/group (B). In another experiment AE17-bearing mice were given i.p. injections (800 µl) of PBS or 800 µg/dose/mouse anti-VEGFr antibody (C) (n = 10 mice/group). Arrow indicates when treatment was commenced. All data are shown as mean ± SEM. * p < 0.05.
Figure 4.
Decreasing bioavailable copper in vivo modulates tumor vessel dimensions.
HUVECs (5 x 103) were treated in vitro with PBS (control is shown as the black line) or log fold concentrations of tetrathiomolybdate (A), penicillamine (B) and trientine (C) and MTT assays performed 24 hrs later. Data from n = 2 experiments each with its own triplicates is shown as mean ± range. Mice treated with PBS, TM, penicillamine or trientine were sacrificed when tumors reached 100 mm2 and tumor-associated vessels visualized and measured on frozen tumor sections using FITC-anti-CD31 antibody and confocal imaging. Representative photographs are from individual mice that were not treated (control: D), given TM (E) or penicillamine (F). Pooled data counting a minimum of 100 cells per mouse showing tumor blood vessel (BV) diameter (G) and length (H) from control (normal mice; n = 4), penicillamine (Pen; n = 2), trientine (TT; n = 2) or Tm-treated (n = 2) mice are shown as mean ± SEM. * p < 0.05, ** p < 0.005.
Figure 5.
Copper lowering in vivo reduces tumor endothelia proliferation and ICAM (CD54) expression.
Mice treated with PBS, TM, penicillamine or trientine were sacrificed when tumors reached 100 mm2 and tumor endothelia analyzed by flow cytometry. Single cells were identified by excluding doublets by gating on FSC-A versus FSC-H plots; A. Isotype controls and single stains were used to set up negative and positive regions (not shown). Lymphocytes were excluded by gating. Backgating on CD31+CD105+ revealed the FSC/SSC region associated with tumor blood vessels (BV:B). CD31+CD105+ angiogenic (C and D) or CD31+CD34+ normal (E) endothelial cells were identified, gated and quantified. The proportions of Ki67+, CD105+ and CD54+ cells within each gate were determined (F). Pooled data counting > 20,000 CD31+CD105+ or CD31+CD34+ cells per mouse from 2 mice/treatment group and 4 control mice is shown as mean ± SEM. * p < 0.05; *** p < 0.001.
Figure 6.
Copper lowering in vivo promotes CD4+ T cell infiltration.
Mice treated with PBS, TM, penicillamine or trientine were sacrificed when tumors reached 100 mm2 and immune cell infiltration analyzed by flow cytometry. The SSC/FSC-A plot revealed the lymphocyte population (A). CD3+ regions were determined using the CD3-APC-Cy7 single stain (B). Triple staining identified CD3+CD4+ or CD3+CD8+ cells; representative contour plot (C). Pooled data from 2 mice/group shown as mean ± SEM (D). * p < 0.05.