Noninvasive imaging using radioactive
annexin V is an emerging strategy for the assessment of cell death in vivo (F. G. Blankenberg, and H. W. Strauss. Apoptosis, 6: 117-123, 2001.). Therefore, we investigated whether
annexin V labeled with the fluorophore
Cy5.5 (Cy) could serve as a probe for imaging of
tumor apoptosis using near infrared fluorescence (NIRF). We prepared active Cy-
annexin (an equimolar
dye:
protein ratio) that bound to apoptotic Jurkat T cells and an inactive Cy-
annexin probe (>2
dyes/mol
protein) that did not. Active Cy
annexin was used to image a 9L
gliosarcoma, constitutively expressing
green fluorescent protein marker, and the CR8 variant of
Lewis lung carcinoma, stably transfected to express DsRed2. The expression of transfected fluorescent
protein provided an indication of
tumor margins and a means of defining
tumor-associated NIRF signal intensity with both
tumor models.
Tumors were imaged with and without
cyclophosphamide treatment. In both
tumor models active Cy-
annexin V tumor NIRF signal increased two to three times after the treatment.
Tumor NIRF signal developed by 75 min after active Cy-
annexin injection and remained for a 20-h observation period. Inactive
annexin V was used as a control in the CR8
carcinoma experiments and resulted in a low nonspecific signal. With the 9L gliomosacrcoma model, active Cy-
annexin V bound to both
tumor cells (Cy-
annexin V staining only) and endothelial cells (costained with Cy-
annexin V and antibody to the endothelial marker CD31). Our results demonstrate that active Cy-
annexin can be used as a NIRF probe to image apoptosis from outside an intact living animal and may provide nonradioactive method of measuring the antiproliferative effects of
cancer chemotherapeutic regimens.