In vivo and in vitro measurement of apoptosis in breast cancer cells using 99mTc-EC-annexin V

Objective: The purpose of this study was to develop an imaging technique to measure and monitor tumor cells undergoing programmed death caused by radiation and chemotherapy using Tc-99m-EC-annexin V. Annexin V has been used to measure programmed cell death both in vitro and in vivo. Assessment of apoptosis would be useful to evaluate the efficacy and mechanisms of therapy and disease progression or regression. Methods: Ethylenedicysteine (EC) was conjugated to annexin V using sulfo-N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl as coupling agents. The yield of EC-annexin V was 100%. In vitro cellular uptake, pre- and post-radiation (10-30 Gy) and paclitaxel treatment, was quantified using Tc-99m-EC-annexin V. Tissue distribution and planar imaging of Tc-99m-EC-annexin V were determined in breast tumor-bearing mts at 0.5, 2, and 4 hrs. To demonstrate in vivo cell apoptosis that occurred during chemotherapy, a group of mts was treated with paclitaxel and planar imaging studies were conducted at 0.5-4 hrs. Computer outlined region of interest (ROI) was used to quantify tumor uptake on day 3 and clay 5 post-treatment. Results: In vitro cellular uptake showed that there was significantly increased uptake of Tc-99m-EC-annexin V after irradiation (10-30 Gy) and paclitaxel treatment In vivo biodistribution of Tc-99m-EC-annexin in breast tumor-bearing rats showed increased tumor-to-blood, tumor-to-lung and tumor-to-muscle count density ratios as a function of time. Conversely, tumor-to-blood count density ratios showed a time-dependent decrease with Tc-99m-EC in the same time period Planar images confirmed that the tumors could be visualized clearly with 99mTc-EC-annexin. There was a significant difference of ROI ratios between pre- and post-paclitaxel treatment groups at 2 and 4 hrs post injection. Conclusion: The results indicate that apoptosis can be quantified using Tc-99m-EC-annexin and that it is feasible to use Tc-99m-EC-annexin to image tumor apoptosis.