Optical Imaging of Disseminated Leukemia Models in Mice with Near-Infrared Probe Conjugated to a Monoclonal Antibody Sabrina Pesnel1,2*, Arnaud Pillon1, Laurent Cre´ ancier1, Ste´ phanie Lerondel2, Alain Le Pape2,3, Christian Recher4,5, Ce´ cile Demur4,5, Nicolas Guilbaud 1 , Anna Kruczynski1 1 Centre de Recherche en Oncologie Expe´rimentale, Institut de Recherche Pierre Fabre, Toulouse, France, 2 UPS n 44 TAAM – CIPA, CNRS, Orle´ans, France, 3 INSERM U618, Tours, France, 4 Service d’He´matologie, H oˆpital Purpan, CHU de Toulouse, Toulouse, France, 5 INSERM U563, Centre de Physiopathologie, CHU Purpan, Toulouse, France Abstract Background: The assessment of anticancer agents to treat leukemia needs to have animal models closer to the human pathology such as implantation in immunodeficient mice of leukemic cells from patient samples. A sensitive and early detection of tumor cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Therefore, we developed a fluorescent antibody based strategy to detect leukemic foci in mice bearing patient-derived leukemic cells using fluorescence reflectance imaging (FRI) to determine when to start treatments with novel antitumor agents. Methods: Two mAbs against the CD44 human myeloid marker or the CD45 human leukocyte marker were labeled with Alexa Fluor 750 and administered to leukemia-bearing mice after having verified the immunoreactivity in vitro. Bioluminescent leukemic cells (HL60-Luc) were used to compare the colocalization of the fluorescent mAb with these cells. The impact of the labeled antibodies on disease progression was further determined. Finally, the fluorescent hCD45 mAb was tested in mice engrafted with human leukemic cells. Results: The probe labeling did not modify the immunoreactivity of the mAbs. There was a satisfactory correlation between bioluminescence imaging (BLI) and FRI and low doses of mAb were sufficient to detect leukemic foci. However, anti-hCD44 mAb had a strong impact on the tumor proliferation contrary to anti-hCD45 mAb. The use of anti-hCD45 mAb allowed the detection of leukemic patient cells engrafted onto NOD/SCID mice. Conclusions: A mAb labeled with a near infrared fluorochrome is useful to detect leukemic foci in disseminated models provided that its potential impact on tumor proliferation has been thoroughly documented. Citation: Pesnel S, Pillon A, Cre´ancier L, Lerondel S, Le Pape A, et al. (2012) Optical Imaging of Disseminated Leukemia Models in Mice with Near-Infrared Probe Conjugated to a Monoclonal Antibody. PLoS ONE 7(1): e30690. doi:10.1371/journal.pone.0030690 Editor: Pierre Bobe´, Institut Jacques Monod, France Received August 22, 2011; Accepted December 22, 2011; Published January 27, 2012 Copyright: 2012 Pesnel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: These authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: spesnel@cnrs-orleans.fr Introduction Acute myeloid leukemia (AML) is a heterogeneous disorder of hematopoietic progenitor cells involving a block in differentiation. This incomplete maturation of leukemic blasts is usually associated with extensive cell proliferation, resulting in cell accumulation in bone marrow, peripheral blood, and eventually at ectopic locations like in the liver, in the spleen and in the central nervous system. Preclinical models that effectively recapitulate human disease are critical for expanding our knowledge of cancer biology and response to therapeutic agents. In order to develop more relevant tumor models, we implanted leukemic cells issued directly from patient samples intravenously in immunodeficient mice. However, the sensitive and early detection of leukemic cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Two methods are currently used to detect human leukemia development in mouse models: flow cytometry analysis and human DNA assessment using Polymerase Chain Reaction (PCR) on blood samples. These methods allow the detection of circulating leukemic cells but they do not allow to access to the location of the tumor foci. One way to obtain this information is to use an imaging modality. Due to the low quantity of patient leukemic cells available and to avoid modifying tumor cell properties, cells could not be genetically modified to express luciferase gene or fluorescent protein gene which would enable monitoring the disease with direct in vivo imaging of bioluminescent [1] or fluorescent tumor cells [2]. So, an alternative method consisted of designing a monoclonal antibody (mAb) conjugated to a near-infrared probe with better tissue penetration and less autofluorescence than a visible fluorophore [3,4]. Leukemic cells were characterized by different identification markers, used in flow cytometry, such as the CD44 myeloid and the CD45 leukocyte markers. CD44 is expressed by leukocytes, erythrocytes, epithelial cells and weakly by platelets; it has a PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e30690