In a second experiment, mice (n = 5 for anti-hCD44 mAb, n = 6 for anti-hCD45 mAb) received one injection per week for three weeks of the minimal dose of the fluorescent mAb or PBS (control group) to determine the effects of the mAb on leukemia growth. Leukemia progression was monitored using BLI and life span was recorded. Survival distribution of treated and control groups of HL60-Luc tumor-bearing mice were statistically compared using the Log-rank test. Leukemia growth inhibition was calculated from BLI data, as the ratio of the median bioluminescent signal of mAb- treated versus control groups: T/C (%) = (median bioluminescent signal of mAb-treated group on day X / median bioluminescent signal of control group on day X) x 100. Use of the fluorescent AF750 anti-hCD45 monoclonal antibody to detect leukemic foci in an experimental model of patient acute myeloid leukemia sample 5 mg AF750 anti-hCD45 mAb have been intravenously injected to patient AML cells-bearing mice. After 24h, mice were imaged using FRI. Fluorescent bones were removed to assess the number of leukemic cells present in mouse bone marrow with flow cytometry and an immunohistochemical analysis was performed to detect human CD45+ cells in order to confirm that the fluorescent signals correspond to tumor foci. The fluorescent organs were also removed to perform immunohistochemical analysis. Immunohistochemical and flow cytometry analyses Human CD45+ cells were detected by immunohistochemistry in formalin/paraffin-embedded sections of bone or organs. Sections were stained using an automated system DAKO Autostainer. Bone marrow cells were flushed from the tibia and the femur and made into single cell suspensions for analysis by flow cytometry to determine the percentage of CD45+ cells over the total number of blasts. Results In vitro validation The degree of labeling calculated from the absorptions at 280nm and 752nm were 1.96 and 2.1 for the AF750 labeled anti- hCD44 mAb and anti-hCD45 mAb, respectively. Measurement of fluorescent AF750 mAb binding to cells was done to confirm the receptor-specific targeting of the mAb labeled with AF750. The cell binding was highest with HL60-Luc (leukemia) and negligible with HCT116-Luc (colon cancer). The majority of the binding could be blocked by first incubating the cells with an excess of non-fluorescent mAb. The percent of total AF750-mAb bound to HL60-Luc cells after 2h was 61611 and 3567 for the AF750 labeled anti-hCD44 mAb and anti-hCD45 mAb, respectively. When the cells were first incubated with an excess of mAb to saturate the receptors, the percent of total AF750-mAb bound fell to ,3% for each mAb. Determination of the minimal fluorescent monoclonal antibody dose zSeveral doses of mAbs, between 1 and 10 mg were intravenously injected to leukemia-bearing mice to determine the minimal fluorescent mAb dose to detect all the tumor foci. In the case of anti-hCD44 mAb, a dose of 1 mg was sufficient to detect all bioluminescent leukemic foci in the mice with a good co-localization between fluorescent and bioluminescent signals with an average Pearson correlation coefficient of 0.71 (Figure 1) and a maximal intensity obtained 48h post-injection of the fluorescent mAb. Optical Imaging of Leukemia Figure 1. Bioluminescent (A) and fluorescent (B) of HL60-Luc tumor-bearing mice 48h post-injection of 1 mg fluorescent CD44 mAb. doi:10.1371/journal.pone.0030690.g001 For anti-hCD45 mAb, a dose of 5 mg was required to detect all the foci with a good co-localization between fluorescent and biolumines- cent signals with an average Pearson correlation coefficient of 0.65 (Figure 2) with an optimal imaging point 24h post-injection of the fluorescent mAb. Effects of the fluorescent monoclonal antibodies on HL60-Luc leukemia development in mice One week after the first injection of 1 mg fluorescent anti-hCD44 mAb, BLI showed a decrease of HL60-Luc leukemia progression and the maximal leukemia growth inhibition achieved was 98.8% (T/ C = 1.2%) on day 49 (Figure 3a). Furthermore, the bioluminescent signal of all the foci decreased whatever their location in the mice. This indicated a satisfactory biodistribution of this mAb allowing an exposure of all the tumor foci. In addition, treatment with anti-hCD44 mAb resulted in a significant increase of survival of leukemia-bearing mice, as assessed by the Log-rank test (p = 0.009) (Figure 3b). The anti- hCD44 mAb had a very strong impact on this leukemia model. In contrast, repeated injections of 5 mg fluorescent anti-hCD45 mAb did not modify in vivo leukemia progression, as assessed using BLI. Indeed, there was no significant difference on the bioluminescent signal progression between the control group and the mAb treated group (Figure 4a). Furthermore, treatment with anti-hCD45 mAb did not result in any modification of the survival of HL60-Luc-bearing mice, as assessed by the Log-rank test (p = 0.411) (Figure 4b). So, it would seem that the anti-hCD45 mAb has no impact on the leukemia progression in this model. Detection of patient acute myeloid leukemia cells in NOD/SCID mice using the fluorescent AF750 anti-hCD45 monoclonal antibody The FRI performed 24h post-injection of the fluorescent anti- hCD45 antibody to AML-bearing mice showed several fluorescent PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e30690