Perspective Patel, Cole, Bradshaw et al.
radiolabel could possibly affect the pharmacokinetic (PK)/PD properties of the compound and, in turn, the in vivo behavior of the drug [23,24]. In imaging experi- ments it is the label that is followed rather than the parent compound it is bound to; thus, the patent drug cannot be distinguished from any biotransformation products formed.
PD/TD responses in tumors studied by MALDI-MSI One of the major application areas of MALDI-MSI has been the study of cancerous tissue. In order to study PD/TD responses in tumors, the unique characteristics of the tumor vasculature needs consideration. Owing to the blood capillary network being notoriously sinu- soidal, leaky and malformed, drug delivery in itself can present great challenges [25]. Areas of heterogeneous hypoxia within the tumor microenvironment are inevi- table with inadequate oxygenation as a result of the substandard tumor vasculature. One example of the complexities involved in the
analysis of drug compounds within hypoxic biological microenvironments was described in a study by Atkin- son et al. [26]. Here, the distribution of the bioreduc- tive alkylaminoanthraquinone drug AQ4N (banoxan- trone) was examined in treated H460 human tumor xenograph tissue using MALDI-MSI. The presence of hypoxic regions within the sample resulted in a phar- macokinetic profile where the inactive prodrug AQ4N, intermediate metabolite AQ4M and active compound AQ4 were all present. The nature of MALDI-MSI not only allowed the simultaneous observation of each spe- cies, but also the correlation of metabolic responses via coregistration of endogenous metabolites (i.e., ATP as a marker of hypoxia colocalized with cytotoxic AQ4) [26]. To support data reproducibility and the generation
of clean interpretable spectra, scrupulous sample prepa- ration is essential in order to minimize contaminating factors. Optimization of appropriate solvent wash steps aid tumor tissue fixation and/or eliminate unwanted molecular classes, without
removing compounds of
interest. A major concern for MALDI-MSI analysis within the small molecule mass range (m/z 50–1000) is the issue of ion suppression by matrix ions, mainly owing to the high proton affinity [27]. Conversely, sup- pression of peptides of interest, which are low in abun- dance, can also be subjected to similar issues merely by the pharmacological action of the antitumor agent administered. Vascular disrupting agents have been
Key term
Ion mobility separation (IMS): A method for separating gas phase ions according to their collisional cross-section, hence providing another dimension to MS data.
found to present this challenge during dose response relationship studies [25]. MALDI-MSI was employed to observe the effect of treatment with combretastatin A-4-phosphate (fosbretabulin), a vascular disrupting agent, on mouse VEGF120 fibrosarcoma tissue. A strat- egy incorporating ‘on-tissue’ tryptic digestion, MS/MS identification of peptides and ion mobility separa- tion to improve specificity was employed. By taking tumor samples at a number of time points after treat- ment, gross effects were clearly visible through changes in the expression of certain peptides. These were identi- fied as originating from hemoglobin and were indica- tive of the disruption of the tumor vasculature. Multi- variate statistical analysis of the MALDI-MSI data set was not, in this case, able to reveal more subtle changes taking place in the tumor samples. In this PD study, signals of low abundance were masked by the dominant hemoglobin signals. As observed in this case, suscepti- bility of the experimental model to the proposed drug treatment is a crucial factor. Interference and suppres- sion caused by high abundant proteins/lipids could also prohibit analysis of key molecules relevant to a disease pathway in other studies. Munteanu et al. [28] used the monitoring of histone
deacetylase drug target engagement to assess tumor- selective PD signatures by imaging drug-induced mass shifts. Protein fingerprints of post-histone deacetylase inhibitors generated by MALDI-MS provided label-free quantification of hyperacetylated histones and responses from this method were then used to study the spatial distribution of acetylated histones using MALDI-MSI. Histones are highly abundant and readily observable in tumor tissue using MALDI-MSI and this paper is a tes- timony to the usefulness of exploiting highly abundant proteins when analyzing pharmacological responses in cancerous tissue. MALDI-MSI has perhaps been disregarded as a high-
throughput technique for the purpose of patient biopsy analysis within the clinical setting. Djidja and cowork- ers [29] challenged this view by demonstrating the great potential of the technique for multiplex imaging using formalin-fixed paraffin-embedded tissue microarrays (TMA). TMAs had, up until this point, been predomi- nantly analyzed by immunohistochemistry teamed with a statistical methodology (i.e., hierarchical clus- tering) [30–33]. Employing MALDI as an imaging tech- nique demonstrated that tumor classification of patient control/treated tumor biopsy cores was possible. This could in the future enable multiple PD/TD distribu- tion profiles with corresponding relative ion intensities throughout the TMA sample cohort to be generated. In order to gain acceptance in routine oncological
clinical diagnostics, MALDI-MSI would have to meet various criteria. These include validation of the data
94 Bioanalysis (2015) 7(1) future science group
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