Rapid copper acquisition by developing murine mesothelioma: Decreasing bioavailable copper slows tumor growth, normalizes vessels and promotes T cell infiltration

Copper, an essential trace element acquired through nutrition, is an important co-factor for pro-angiogenic factors including vascular endothelial growth factor (VEGF). Decreasing bioavailable copper has been used as an antiangiogenic and anti-cancer strategy with promising results. However, the role of copper and its potential as a therapy in mesothelioma is not yet well understood. Therefore, we monitored copper levels in progressing murine mesothelioma tumors and analyzed the effects of lowering bioavailable copper. Copper levels in tumors and organs were assayed using atomic absorption spectrophotometry. Mesothelioma tumors rapidly sequestered copper at early stages of development, the copper was then dispersed throughout growing tumor tissues. These data imply that copper uptake may play an important role in early tumor development. Lowering bioavailable copper using the copper chelators, penicillamine, trientine or tetrathiomolybdate, slowed in vivo mesothelioma growth but did not provide any cures similar to using cisplatin chemotherapy or anti-VEGF receptor antibody therapy. The impact of copper lowering on tumor blood vessels and tumor infiltrating T cells was measured using flow cytometry and confocal microscopy. Copper lowering was associated with reduced tumor vessel diameter, reduced endothelial cell proliferation (reduced Ki67 expression) and lower surface ICAM/CD54 expression implying reduced endothelial cell activation, in a process similar to endothelial normalization. Copper lowering was also associated with a CD4+ T cell infiltrate. In conclusion, these data suggest copper lowering is a potentially useful anti-mesothelioma treatment strategy that slows tumor growth to provide a window of opportunity for inclusion of other treatment modalities to improve patient outcomes

Reducing bioavailable copper slows tumor growth rate.

<p>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.</p

Bioavailable copper levels can be decreased in vivo by copper chelating agents.

<p>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.</p

Copper lowering in vivo promotes CD4⁺ T cell infiltration.

<p>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.</p