Combination Tetrathiomolybdate and Radiation Therapy in a Mouse Model of Head and Neck Squamous Cell Carcinoma

Abstract

To assess the effect of combining tetrathiomolybdate therapy and radiation treatment (RT) on tumor growth in the mouse head and neck squamous cell carcinoma (HNSCC) model. One million HNSCC cells were injected subcutaneously into the flanks of C3H/HeJ mice and the tumors grown to an average of 301 mm3 (day 0). Mice were randomized into 4 groups: (a) no therapy, (b) tetrathiomolybdate alone, (c) RT alone, or (d) tetrathiomolybdate + RT. Data from 3 experiments with these 4 groups were analyzed. A gaussian mixed model was fit to the initialized logarithm of the tumor size counts between days 7 and 16 (linear component), and growth rates were compared. Assays using 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazolium bromide (MTT) were conducted on HNSCC cells in culture with varying doses of tetrathiomolybdate. Treated mice were given tetrathiomolybdate in their water and observed for clinical evidence of toxic effects associated with copper depletion as measured by ceruloplasmin assay. When tumor sizes reached an average of 535 mm(3), mice receiving RT were given a single fraction of 750 rad (7.5 Gy), a dose determined in previous experiments to slow but not cure tumor growth, permitting an examination of interaction of radiation with tetrathiomolybdate. Data from 3 separate experiments were analyzed. There were a total of 37 mice in the untreated group, 32 mice in the tetrathiomolybdate alone group, 38 mice in the RT alone group, and 46 mice in the tetrathiomolybdate + RT group. Ceruloplasmin assays showed that we had obtained adequate copper reduction throughout the experiments to inhibit angiogenesis with minimal toxic effects. The tetrathiomolybdate + RT combined therapy group of mice showed a statistically significant decrease in tumor growth compared with both the tetrathiomolybdate alone (P = .001) and RT alone groups (P<.001). The combination of the anti-angiogenic copper chelating agent tetrathiomolybdate with RT improved local control of HNSCC in an isogenic mouse model compared with either therapy alone.