Figure 2. Bioluminescent (A) and fluorescent (B) images of HL60-Luc tumor-bearing mice 24h post-injection of 5 mg fluorescent CD45 mAb. doi:10.1371/journal.pone.0030690.g002 foci in the femur, tibia, sternum spleen and liver (Figure 5). To confirm the specific fixation of the mAb on the human leukemic cells, flow cytometry and immunohistochemical analysis were performed on the organs showing a fluorescent signal. The flow cytometry analysis revealed a massive invasion of the bone marrow by hCD45+ leukemic cells (Figure 6a); 90% and 70% of the blasts present in the tibia and the femur, respectively, were hCD45+. The same results were obtained with control mice (without anti-hCD45 mAb injections). The immunohistochem- ical analysis of the bones, spleen and liver confirmed the presence of hCD45+ leukemic cells (Figure 6b). It showed that cancer cells had extensively invaded the bone marrow. These results revealed a satisfactory correlation between the fluorescent signal emitted by the anti-hCD45 mAb and the presence of leukemic cells in the bone marrow and other organs (spleen, Optical Imaging of Leukemia liver) demonstrating efficient targeting of the fluorescent mAb in the mice. Discussion Our aim was to develop a fluorescent probe for detection and staging of human leukemic foci in mice bearing patient-derived leukemic cells in order to determine when to start treatments of the mice with novel antitumor agents. Three strategies can be envisaged to image tumor cells without genetically modifying them. The first class uses cell markers: it is possible to use lipophilic tracers such as 1,1-dioctadecyl-3,3,3,3- tetramethylindotricarbocyanine iodide (DiR) or 1,1’-dioctadecyl- 3,3,39,39-tetramethylindotricarbocyanine (DiD) [13]. This type of tracer cannot be used to follow cells for a long time because at each cell division the number of fluorochromes presents on the cells decreases. Another option consists in labeling cells with radioisotopes (Indium 111 [14] or Copper 64 [15]) but it is not possible to image the cells for long time due to the short half-life of the isotopes. The second class uses metabolic markers such as the 18F-FluoroDeoxyGlucose (FDG) [16,17] but they are not tumor specific. The last class uses molecular targeted probes that are imaging agents against tissue modifications associated with a pathology or a cellular type such a the CD45 which is a marker of leucocytes. This targeting can be realized via an antibody, a peptide (for example the RGD peptide which binds to integrins) or the ligand of a receptor. For our application, we have chosen to use an antibody specific of the human leukemic cells. Antibodies can be labeled with chelates that bind radioactive metals [18,19]. However, radiolabeling exposes mice to ionizing radiations that can impact tumor progression in the case of repeated exams of radiosensitive tumors. Furthermore, despite high resolution and sensitivity, nuclear imaging suffers from severe constraints due to requirements of dedicated facilities and regulations about radiation use. Optical labeling overcomes these disadvantages but suffers from poor tissue penetration. Among the fluorochromes, near- infrared dyes have the greatest tissue penetration and a low autofluorescence, and so are the most used for in vivo imaging. We decided to use the fluorochrome Alexa fluor 750 because its emission wavelength at 780nm is best suited to reveal deep foci and with the additional advantage over Alexa fluor 680 of an absence of autofluorescence in abdomen. In cancer research, the most routinely used imaging modality to measure tumor burden is Figure 3. Effects of fluorescent CD44 mAb intravenously administered over three weeks on the bioluminescence signal of HL60-Luc tumor-bearing mice (A) and on life span (B). doi:10.1371/journal.pone.0030690.g003 PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e30690