Rapid Copper Acquisition by Developing Murine Mesothelioma: Decreasing Bioavailable Copper Slows Tumor Growth, Normalizes Vessels and Promotes T Cell Infiltration

Abstract

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 anti-angiogenic 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.

Behind the Link between Copper and Angiogenesis: Established Mechanisms and an Overview on the Role of Vascular Copper Transport Systems

“…To this day, the anti-angiogenic potential of metal starvation represents a major research target in the field of clinical oncology. In fact, in vitro and in vivo studies on cancer models indicate that copper deprivation may not act directly on the tumour cells, but on the local vasculature, thereby cutting off nutrients to proliferating cancer cells [8,235]. However, it is not clear whether copper limitation prevents the growth of tumour blood vessels through the inhibition of endothelial cell proliferation or if it just affects the regularity of neo-formed structures.…”

“…However, it is not clear whether copper limitation prevents the growth of tumour blood vessels through the inhibition of endothelial cell proliferation or if it just affects the regularity of neo-formed structures. In this sense, it is noteworthy that HUVECs, an election model for in vitro angiogenic studies, continue to replicate in the presence of trientine and TM (tetrathiomolybdate) copper-chelating agents, and that passive copper reduction by penicillamine only induces slight, but not significant proliferation [8,235]. Accordingly, dietary regimens including copper-chelating agents (penicillamine, trientine or TM) in a mesothelioma mouse model demonstrated that the anti-angiogenic and tumour-suppressive action is due to the normalization of blood vessels, functional for sustaining CD4 + T cell infiltration into the tumour microenvironment, but not anti-proliferative effects [235].…”

“…In this sense, it is noteworthy that HUVECs, an election model for in vitro angiogenic studies, continue to replicate in the presence of trientine and TM (tetrathiomolybdate) copper-chelating agents, and that passive copper reduction by penicillamine only induces slight, but not significant proliferation [8,235]. Accordingly, dietary regimens including copper-chelating agents (penicillamine, trientine or TM) in a mesothelioma mouse model demonstrated that the anti-angiogenic and tumour-suppressive action is due to the normalization of blood vessels, functional for sustaining CD4 + T cell infiltration into the tumour microenvironment, but not anti-proliferative effects [235]. Several in vivo experiments demonstrated that vascular responses to copper starvation are heterogeneous and seem to be influenced by organ-specific endothelial phenotypes, previously discussed in [236].…”

Copper as the Target for Anticancer Nanomedicine

“…Given between meals, TM is absorbed into the blood, and forms complexes with serum copper and albumin, reducing the bioavailable copper. [76] A phase II trial examined a combination of D-pen and a low-copper diet in human glioblastoma multiforme (ClinicalTrials.gov Identifier: NCT00003751). [48,[67][68][69][70][71][72][73][74] A small phase II clinical trial showed that the event-free survival for 75 advanced breast cancer patients with TM-treatment was 75.6% at a median followup of 6.9 years (ClinicalTrials.gov Identifier: NCT00195091).…”

“…[75] D-Pen depletes copper by binding free copper in blood and tissues, forming a stable soluble complex that is excreted in urine. [76] A phase II trial examined a combination of D-pen and a low-copper diet in human glioblastoma multiforme (ClinicalTrials.gov Identifier: NCT00003751). The serum copper levels fell to the desired level of <50 µg dL −1 after 2 months of therapy.…”