Michael Dobosz

Drug-Based Optical Agents: Infiltrating Clinics at Lower Risk

Using drugs as optical imaging agents and “microdosing” amounts reduces risk for clinical translation of fluorescence molecular imaging. Fluorescent agents with specificity to cellular and subcellular moieties present promise for enhancing diagnostics and theranostics, yet challenges associated with regulatory approvals of experimental agents stifle the clinical translation. As a result, targeted fluorescent agents have remained predominantly as preclinical imaging tools. We discuss the potential of using optically labeled drugs to accelerate the clinical acceptance of optical and optoacoustic agents, in analogy to nuclear medicine approaches. This strategy, corroborated with microdosing studies, outlines a promising approach for overcoming bottlenecks and advancing photonic clinical imaging.

Deep tissue imaging: a review from a preclinical cancer research perspective

This review delves into the rapidly evolving field of deep tissue imaging at cellular resolution, reviewing popular tissue clearing and staining methods in combination with light-sheet fluorescence microscopy (LSFM) including quantification and three-dimensional visualization tools, the field of applications and perspective, particularly with the focus on preclinical cancer research and drug development. The LSFM technique presented here allows an extremely fast optical sectioning for three-dimensional reconstruction of centimeter-sized tissue samples at cellular resolution. However, optical clearing methods are required to receive optical transparent tissue. Application of either tissue autofluorescence, in vivo fluorescence labeling, endogenous fluorescence or ex vivo whole-mount immunolabeling enables three-dimensional in situ visualization of morphological and functional features of unsectioned whole-mount tissue samples. This powerful and innovative imaging technique opens up a new dimension of tissue analysis providing detailed and comprehensive insights into biology. It enables the investigation of normal and pathological tissue features and disease progression and allows precise monitoring of potential therapeutic interventions in intact biological tissue on a cellular level.

Dynamic contrast-enhanced micro-computed tomography correlates with 3-dimensional fluorescence ultramicroscopy in antiangiogenic therapy of breast cancer xenografts.

ObjectivesDynamic contrast-enhanced (DCE) micro–computed tomography (micro-CT) has emerged as a valuable imaging tool to noninvasively obtain quantitative physiological biomarkers of drug effect in preclinical studies of antiangiogenic compounds. In this study, we explored the ability of DCE micro-CT to assess the antiangiogenic treatment response in breast cancer xenografts and correlated the results to the structural vessel response obtained from 3-dimensional (3D) fluorescence ultramicroscopy (UM). Material and MethodsTwo groups of tumor-bearing mice (KPL-4) underwent DCE micro-CT imaging using a fast preclinical dual-source micro-CT system (TomoScope Synergy Twin, CT Imaging GmbH, Erlangen, Germany). Mice were treated with either a monoclonal antibody against the vascular endothelial growth factor or an unspecific control antibody. Changes in vascular physiology were assessed measuring the mean value of the relative blood volume (rBV) and the permeability-surface area product (PS) in different tumor regions of interest (tumor center, tumor periphery, and total tumor tissue). Parametric maps of rBV were calculated of the tumor volume to assess the intratumoral vascular heterogeneity. Isotropic 3D UM vessel scans were performed from excised tumor tissue, and automated 3D segmentation algorithms were used to determine the microvessel density (MVD), relative vessel volume, and vessel diameters. In addition, the accumulation of coinjected fluorescence-labeled trastuzumab was quantified in the UM tissue scans to obtain an indirect measure of vessel permeability. Results of the DCE micro-CT were compared with corresponding results obtained by ex vivo UM. For validation, DCE micro-CT and UM parameters were compared with conventional histology and tumor volume. ResultsExamination of the parametric rBV maps revealed significantly different patterns of intratumoral blood supply between treated and control tumors. Whereas control tumors showed a characteristic vascular rim pattern with considerably elevated rBV values in the tumor periphery, treated tumors showed a widely homogeneous blood supply. Compared with UM, the physiological rBV maps showed excellent agreement with the spatial morphology of the intratumoral vascular architecture. Regional assessment of mean physiological values exhibited a significant decrease in rBV (P < 0.01) and PS (P < 0.05) in the tumor periphery after anti–vascular endothelial growth factor treatment. Structural validation with UM showed a significant reduction in reduction of relative vessel volume (rVV) (P < 0.01) and MVD (P < 0.01) in the corresponding tumor region. The reduction in rBV correlated well with the rVV (R = 0.73 for single values and R = 0.95 for mean values). Spatial maps of antibody penetration showed a significantly reduced antibody accumulation (P < 0.01) in the tumor tissue after treatment and agreed well with the physiological change of PS. Examination of vessel diameters revealed a size-dependent antiangiogenic treatment effect, which showed a significant reduction in MVD (P < 0.001) for vessels with diameters smaller than 25 &mgr;m. No treatment effect was observed by tumor volume. ConclusionsNoninvasive DCE micro-CT provides valuable physiological information of antiangiogenic drug effect in the intact animal and correlates with ex vivo structural analysis of 3D UM. The combined use of DCE micro-CT with UM constitutes a complementary imaging toolset that can help to enhance our understanding of antiangiogenic drug mechanisms of action in preclinical drug research.

Noninvasive measurement of pharmacokinetics by near-infrared fluorescence imaging in the eye of mice.

Abstract. Purpose: For generating preclinical pharmacokinetics (PKs) of compounds, blood is drawn at different time points and levels are quantified by different analytical methods. In order to receive statistically meaningful data, 3 to 5 animals are used for each time point to get serum peak-level and half-life of the compound. Both characteristics are determined by data interpolation, which may influence the accuracy of these values. We provide a method that allows continuous monitoring of blood levels noninvasively by measuring the fluorescence intensity of labeled compounds in the eye and other body regions of anesthetized mice. Procedures: The method evaluation was performed with four different fluorescent compounds: (i) indocyanine green, a nontargeting dye; (ii) OsteoSense750, a bone targeting agent; (iii) tumor targeting Trastuzumab-Alexa750; and (iv) its F(ab′)2-alxea750 fragment. The latter was used for a direct comparison between fluorescence imaging and classical blood analysis using enzyme-linked immunosorbent assay (ELISA). Results: We found an excellent correlation between blood levels measured by noninvasive eye imaging with the results generated by classical methods. A strong correlation between eye imaging and ELISA was demonstrated for the F(ab′)2 fragment. Whole body imaging revealed a compound accumulation in the expected regions (e.g., liver, bone). Conclusions: The combination of eye and whole body fluorescence imaging enables the simultaneous measurement of blood PKs and biodistribution of fluorescent-labeled compounds.

Improved decision making for prioritizing tumor targeting antibodies in human xenografts: Utility of fluorescence imaging to verify tumor target expression, antibody binding and optimization of dosage and application schedule

ABSTRACT Preclinical efficacy studies of antibodies targeting a tumor-associated antigen are only justified when the expression of the relevant antigen has been demonstrated. Conventionally, antigen expression level is examined by immunohistochemistry of formalin-fixed paraffin-embedded tumor tissue section. This method represents the diagnostic “gold standard” for tumor target evaluation, but is affected by a number of factors, such as epitope masking and insufficient antigen retrieval. As a consequence, variances and discrepancies in histological staining results can occur, which may influence decision-making and therapeutic outcome. To overcome these problems, we have used different fluorescence-labeled therapeutic antibodies targeting human epidermal growth factor receptor (HER) family members and insulin-like growth factor-1 receptor (IGF1R) in combination with fluorescence imaging modalities to determine tumor antigen expression, drug-target interaction, and biodistribution and tumor saturation kinetics in non-small cell lung cancer xenografts. For this, whole-body fluorescence intensities of labeled antibodies, applied as a single compound or antibody mixture, were measured in Calu-1 and Calu-3 tumor-bearing mice, then ex vivo multispectral tumor tissue analysis at microscopic resolution was performed. With the aid of this simple and fast imaging method, we were able to analyze the tumor cell receptor status of HER1–3 and IGF1R, monitor the antibody-target interaction and evaluate the receptor binding sites of anti-HER2-targeting antibodies. Based on this, the most suitable tumor model, best therapeutic antibody, and optimal treatment dosage and application schedule was selected. Predictions drawn from obtained imaging data were in excellent concordance with outcome of conducted preclinical efficacy studies. Our results clearly demonstrate the great potential of combined in vivo and ex vivo fluorescence imaging for the preclinical development and characterization of monoclonal antibodies.

Abstract 2071A: Anti-VEGF treatment in orthotopic breast cancer xenografts: Dynamic contrast-enhanced micro-CT correlates with 3D multispectral fluorescence histology

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Aim: Here, we evaluate the ability of dynamic contrast-enhanced micro-computed tomography (DCE micro-CT) in combination with 3D fluorescence ultramicroscopy (UM) to assess the early physiological and morphological treatment response of orthotopic breast tumors to anti-angiogenic drug therapy. Methods: Non-invasive DCE micro-CT was performed on breast cancer xenografts (KPL-4) that were treated twice with an antibody against the vascular endothelial growth factor (VEGF) or a control antibody. Tumor physiology was assessed in different tumor regions of interests measuring the relative blood volume (rBV) and the permeability-surface area product (PS). Parametric maps were calculated on a voxel-by-voxel basis to evaluate intra-tumor vascular heterogeneity. Mean physiological measurements were compared with morphological measures of the tumor vascular architecture (microvessel density (MVD) and relative vessel volume (rVV)) as obtained by 3-dimensional fluorescence UM. Additionally, vessel leakage was assessed by UM quantifying the penetration strength of a co-injected fluorescence-labeled therapeutic antibody into the tumor tissue. Results: Examination of the parametric maps revealed significantly different spatial patterns of intra-tumoral blood supply (rBV) between anti-VEGF-treated tumors and control. While a characteristic rim vascularization (high rBV values in the tumor periphery) was observed in the control tumors, treated tumors showed a widely homogeneous blood supply. Compared with high-resolution UM, these physiological rBV maps showed excellent agreement with the morphological appearance of the 3D tumor vascular architecture. Assessment of the mean physiology showed a significant decrease of rBV (p<0.01) and PS (p<0.05) in the tumor periphery after anti-angiogenic treatment. The reduction of rBV correlated well with a significant reduction of rVV (p<0.01) in the corresponding tumor region. The decrease in PS was found consistent with a significantly reduced antibody uptake (p<0.01) in the tumor tissue after treatment. No treatment effect was observed by tumor volume. Conclusions: DCE micro-CT used along with UM provides comprehensive and complementary information of the physiological and morphological anti-angiogenic treatment response in breast cancer xenografts. This technology may help to improve current standard methods in the assessment of anti-angiogenic drug efficacy in preclinical drug development. Citation Format: Thomas Poschinger, Anja Renner, Fabian Eisa, Michael Dobosz, Robert Brauweiler, Willi A. Kalender, Werner Scheuer. Anti-VEGF treatment in orthotopic breast cancer xenografts: Dynamic contrast-enhanced micro-CT correlates with 3D multispectral fluorescence histology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2071A. doi:10.1158/1538-7445.AM2014-2071A