Time course of apoptotic tumor response using ^99mTc-annexin V following a single dose of chemotherapy: Comparison with immunohistological findings in an experimental animal model

Background and purpose: Annexin V (AV), a human protein with a high affinity for membrane-bound phospholipid (phosphatidylserine, PS), has been labeled with ^99mTc and shown to be useful for detecting apoptosis in vivo. We assessed the potential of ^99mTc-AV for detecting apoptotic tumor response and examined the change of radioactivity to chemotherapy in an experimental model. Methods: HYNIC-annexin V was labeled with ^99mTc (^99mTc-AV). Rats were inoculated with allogenic hepatoma cells (KDH-8) into the left calf muscle. Eleven days after tumor inoculation, rats were randomized to receive a single dose of cyclophosphamide (CP; 150 mg/kg, ip) or to serve as control. ^99mTc-AV was injected 4, 12 and 20 h following the chemotherapy. Radioactivity in tissues was determined 6 h after injection of ^99mTc-AV by means of ex vivo tissue counting. TUNEL and caspase-3 staining was performed to detect apoptosis in the tumor. We measured tumor blood flow by ¹⁴C-iodoantipyrine (IAP) on both treated and control rats (n=4, respectively). Results: Cyclophosphamide treatment significantly increased the tumor uptake of ^99mTc-AV at 20 h but not at earlier phases, coinciding with an increased number of immunohistological positive cells. There was no significant change in tumor blood flow by a cyclophosphamide. Conclusions: In vivo detection of apoptosis with ^99mTc-Annexin V can be used as a non-invasive means to assess tumor response to chemotherapy without the influence of blood flow. Effective detection required approximately 20 h, not 4 h, for the best timing of annexin V imaging after the start of chemotherapy in a clinical setting.