Novel Approach of MALDI Drug Imaging, Immunohistochemistry, and Digital Image Analysis for Drug Distribution Studies in Tissues Katharina Huber, † Annette Feuchtinger, † Daniela M. Borgmann, † Zhoulei Li, ‡ Michaela Aichler, † Stefanie M. Hauck, § Horst Zitzelsberger, ∥ Markus Schwaiger, ‡ Ulrich Keller, ⊥,# and Axel Walch* ,† † Research Unit Analytical Pathology, Institute of Pathology, Helmholtz Zentrum Mü nchen, German Research Center for Environmental Health, 85764 Neuherberg, Germany ‡ Department of Nuclear Medicine, Technische Universitä t Mü nchen, 80333 Mü nchen, Germany § Research Unit Protein Science, Helmholtz Zentrum Mü nchen, German Research Center for Environmental Health, 85764 Neuherberg, Germany ∥ Research Unit Radiation Cytogenetics, Helmholtz Zentrum Mü nchen, German Research Center for Environmental Health, 85764 Neuherberg, Germany ⊥ Department of Internal Medicine III, Technische Universitä t Mü nchen, 80333 Mü nchen, Germany # German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany ABSTRACT: Drug efficacy strongly depends on the presence of the drug substance at the target site. As vascularization is an important factor for the distribution of drugs in tissues, we analyzed drug distribution as a function of blood vessel localization in tumor tissue. To explore distribution of the anticancer drugs afatinib, erlotinib, and sorafenib, a combined approach of matrix-assisted laser desorption/ionization (MALDI) drug imaging and immunohistochemical vessel staining was applied and examined by digital image analysis. The following two xenograft models were investigated: (1) mice carrying squamous cell carcinoma (FaDu) xenografts (n tumor = 13) were treated with afatinib or erlotinib, and (2) sarcoma (A673) xenograft bearing mice (n tumor = 8) received sorafenib treatment. MALDI drug imaging revealed a heterogeneous distribution of all anticancer drugs. The tumor regions containing high drug levels were associated with a higher degree of vascularization than the regions without drug signals (p < 0.05). When correlating the impact of blood vessel size to drug abundance in the sarcoma model, a higher amount of small vessels was detected in the tumor regions with high drug levels compared to the tumor regions with low drug levels (p < 0.05). With the analysis of coregistered MALDI imaging and CD31 immunohistochemical data by digital image analysis, we demonstrate for the first time the potential of correlating MALDI drug imaging and immunohistochemistry. Here we describe a specific and precise approach for correlating histological features and pharmacokinetic properties of drugs at microscopic level, which will provide information for the improvement of drug design, administration formula or treatment schemes. I n drug discovery and development, the examination of drug distribution in tissues is crucial to characterize the pharmacological profile of drug substances. The presence of effective drug doses at the target sites is an important precondition for its efficacy. 1 Current techniques for the detection and localization of drugs in tissues are autoradiography (ARG), positron emission tomography (PET), and mass spectrometry imaging (MSI). 2,3 Because ARG and PET determine the decay of radioactive molecules, these techniques lack molecular specificity as the detectors are not capable of distinguishing between parent drug and its metabolites. 4 Liquid-based methods, such as liquid chromatography-mass spectrometry (LC-MS), do not demand any labels and allow the specific detection of the parent drug and its metabolites. However, the disadvantage of liquid-based modalities is the loss of spatial information due to tissue homogenization. 5 These limitations are overcome in MSI by the specific and simultaneous detection of drug compounds, metabolites, and endogenous molecules with high spatial resolution, without the need for radioactive labeling. In addition, the overlaying of mass spectrometry (MS) datasets with histological hematoxylin and eosin (H&E) stainings of consecutive tissue sections after MSI measurement provides an accurate allocation of MS signals and morphological structures. 6 Most drug therapies are administered orally or as injections, with subsequent absorption and distribution to the target tissue Received: June 5, 2014 Accepted: September 27, 2014 Published: September 28, 2014 Article pubs.acs.org/ac © 2014 American Chemical Society 10568 dx.doi.org/10.1021/ac502177y | Anal. Chem. 2014, 86, 10568−10575