Introduction: Angiogenesis plays a central role in tumor growth and metastasis. Growing evidence indicates that tumor progression is dependant upon an “angiogenic phenotype” expressed through various positive and negative angiogenic cytokines. Copper is a mandatory cofactor for the function of many of these mediators; therefore, reducing copper levels in the cellular milieu limits the formation of new blood vessels. Previous studies from our lab and other labs have shown that TM, a potent chelator of copper, lowers body stores of copper and suppresses primary tumor growth. The objective of this study is to investigate if TM treatment could inhibit tumor angiogenesis and distant metastasis in head and neck cancer.
Methods: We have used two in vivo models to study the effects of TM on tumor angiogenesis and tumor metastasis. In the first study, tumor cells (OSCC-3, 1 x 106) and endothelial cells (HDMEC, 1 x 106) were mixed with 100 µl of Matrigel and were implanted subcutaneously in the flanks of SCID mice. Mice were randomized into two groups and received TM (0.7-1.0 mg/mouse) or water, respectively, by daily oral gavaging. The treatment was initiated one week prior to tumor cell inoculation and was continued until the end of the experiment. Blood samples from these animals were collected at regular intervals to monitor copper suppression. TM dose was adjusted to maintain serum ceruloplasmin (CP) 20% of baseline. At the time of sacrifice, tumors and lungs were harvested. Lungs from each mouse were divided into two parts. One half of each lung was paraffin embedded and processed for immunohistochemistry. The other half was processed for colony formation assay by treating with collagenase and culturing the cells with G418 (400 µg/ml) for 7 days. Tumor sections were stained with factor-VIII antibody and blood vessel density was analyzed. In the second animal model, luciferases labeled OSCC-3 cells (500,000 cells) were injected in the SCID mice via the tail vein. Similar to flank model, animals were divided into two groups and treated with TM or water. Tumor metastasis to lungs was monitored for 6 weeks. At the end of 6 weeks, animals were sacrificed and lungs were harvested and processed as described above.
Results: Animals treated with TM showed significant decrease in tumor angiogenesis (blood vessel density) as compared to control animals (treated with water). In addition, animals treated with TM showed significantly lower lung metastasis in both in vivo models as compared to control group. Tumors cells from TM treated animals developed significantly smaller, less aggressive colonies and these colonies had significantly less number of tumor cells.
Conclusion: With its low toxicity profile and good oral availability, TM may be a potential adjuvant treatment option in humans for preventing head and neck cancer metastasis.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA