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 99mTc-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 99mTc-EC-annexin V. Tissue distribution and planar imaging of 99mTc-EC-annexin V were determined in breast tumor-bearing rats at 0.5, 2, and 4 hrs. To demonstrate in vivo cell apoptosis that occurred during chemotherapy, a group of rats 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 day 5 post-treatment.


RESULTS
In vitro cellular uptake showed that there was significantly increased uptake of 99mTc-EC-annexin V after irradiation (10-30 Gy) and paclitaxel treatment. In vivo biodistribution of 99mTc-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 99mTc-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.


CONCLUSIONS
The results indicate that apoptosis can be quantified using 99mTc-EC-annexin and that it is feasible to use 99mTc-EC-annexin to image tumor apoptosis.
Univ. of Texas M. D. Anderson Cancer Center, Dept. of Nuclear Medicine, Box 59, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. dyang@di.mdacc.tmc.edu
D J
DJ
Yang
A
A
Azhdarinia
P
P
Wu
D F
DF
Yu
W
W
Tansey
S K
SK
Kalimi
E E
EE
Kim
D A
DA
Podoloff
D000818:Animals; D017304:Annexin A5 / Q000493:pharmacokinetics; D000972:Antineoplastic Agents, Phytogenic / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D005260:Female; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000000981:diagnostic imaging* / Q000188:drug therapy / Q000473:pathology; D015609:Organotechnetium Compounds / Q000493:pharmacokinetics; D017239:Paclitaxel / Q000627:therapeutic use*; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D051381:Rats; D011916:Rats, Inbred F344; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects
D017304:Annexin A5; D000972:Antineoplastic Agents, Phytogenic; D015609:Organotechnetium Compounds; D019275:Radiopharmaceuticals; C433841:technetium Tc 99m ethylenedicysteine annexin V; D017239:Paclitaxel