Multiplexed mAbs: a new strategy in preclinical time-domain imaging of acute myeloid leukemia

Abstract

Key Points• Multiplexing antibodies against common human epitopes all labeled with the same fluorophore facilitates optical imaging of heterogenous AML.• Multiplexing fluorescently labeled monoclonal antibodies permits optical imaging of primary patient xenograft pathology/therapy response.Antibodies play a fundamental role in diagnostic immunophenotyping of leukemias and in cell-targeting therapy. However, this versatility is not reflected in imaging diagnostics. In the present study, we labeled anti-human mAbs monochromatically against selected human myeloid markers expressed on acute myeloid leukemia (AML) cells, all with the same near-infrared fluorochrome. In a novel "multiplexing" strategy, we then combined these mAbs to overcome the limiting target-to-background ratio to image multiple xenografts of AML. Time-domain imaging was used to discriminate autofluorescence from the distinct fluorophore-conjugated antibodies. Imaging with multiplexed mAbs demonstrated superior imaging of AML to green fluorescent protein or bioluminescence and permitted evaluation of therapeutic efficacy with the standard combination of anthracycline and cytarabine in primary patient xenografts. Multiplexing mAbs against CD11b and CD11c provided surrogate imaging biomarkers of differentiation therapy in an acute promyelocytic leukemia model treated with all-trans retinoic acid combined with the histone-deacetylase inhibitor valproic acid. We present herein an optimizedapplication of multiplexed immunolabeling in vivo for optical imaging of AML cellxenografts that provides reproducible, highly accurate disease staging and monitoring of therapeutic effects. (Blood. 2013;121(7):e34-e42) IntroductionDespite modern advances in therapeutics and improvement in the diagnosis of acute myeloid leukemia (AML) subtypes, the majority of patients die from their disease. 1,2 Thus, in the absence of definitive in vitro models of human AML and failure of significant numbers of new drugs late in clinical trials, 3,4 it is essential that murine AML models are further developed to exploit more specific, targeted therapeutics. Although preclinical development of therapeutics has previously exploited genetic and syngeneic models of AML, 5,6 subcutaneous xenografting of human cell lines in immunodeficient mice has been a popular screening tool. 7 However, the ease of measuring therapeutic efficacy with simple caliper measurements in subcutaneous tumors is offset by the recognition that these models do not typify clinical disease. 8,9 True evaluation of therapeutic potential in this disease necessitates preclinical screening in multiple, systemic, or orthotopic xenograft models of AML, increasingly with primary patient cells that reflect the heterogeneity of the disease. 10 To facilitate the use of humanized orthotopic AML models in therapeutic settings, investigators have increasingly turned to noninvasive preclinical imaging techniques to define efficacy. 11 Optical imaging of reporter gene expression 12,13 is the preferred modality owing to low cost an...

SIRT1 Activation by a c-MYC Oncogenic Network Promotes the Maintenance and Drug Resistance of Human FLT3-ITD Acute Myeloid Leukemia Stem Cells

“…Secondary transplantation of BM cells from mice receiving combination treatment resulted in significantly reduced engraftment in PB at 8 and 16 weeks (Figure 5D) and in BM at 16 weeks (Figures 5E and S5E) compared to AC220 treatment alone, indicating reduced LSC capacity of residual cells. We also studied another sample from a chemotherapy-refractory AML patient using molecular imaging of primary patient xenografts with fluorescently labeled monoclonal antibodies, as described previously (McCormack et al, 2013). We observed significant differences between combination and single-arm groups after 4 weeks of therapy (combination versus TV6, p = 0.0005; combination versus AC220, p = 0.015) (Figures 5F and 5G).…”

“…After engraftment was confirmed, mice were treated with AC220, TV6, the combination, or vehicle for 4 weeks or with chemotherapy as described elsewhere (McCormack et al, 2013). BM, PB, and spleen cells were analyzed for engraftment by flow cytometry analysis of human CD45 + cells.…”

Recent developments in multiplexing techniques for immunohistochemistry

“…For example, the simultaneous use of two antibodies that are specific for separate markers of leukemia, CD11b and CD11c, but conjugated with the same color fluorescent dye, more clearly demarcates leukemia-afflicted tissues. This double antibody staining for different cancer-specific antigens, combined with the use of time-domain imaging to minimize auto-fluorescence, significantly enhanced the signal-to-background ratio [12]. Because one cannot distinguish the relative positions or abundance of the antigen labeled by each antibody, this type of unicolor and multi-antibody method is not considered multiplexing for the purposes of this review.…”

Application of Theranostics to Measure and Treat Cell Heterogeneity in Cancer

“…Flow cytometry is routinely used to diagnose leukemias and lymphomas from blood samples and fresh tissues [69]. Multiplexed immunolabeling has been used to stage cancer cells and to monitor therapeutic effects, for example, in the case of acute myelogenous leukemia [70]. A flow cytometry protocol has been developed for the detection of ER, PR, HER2, epidermal growth factor receptor (EGFR), and E-cadherin in breast epithelial cells [71], and flow cytometry may have applications in the detection of breast cancer cells in peripheral blood [72].…”