NanoSIMS/EM/CLSM Imaging of Platinum-based Anticancer Drugs

Platinum drugs play an important role in cancer therapy mainly due to their high curative potential in certain malignancies. The mechanisms underlying the efficacy, selectivity and side effects of platinum drugs are not sufficiently well understood, though. Interactions with different cell organelles may contribute to these mechanisms, but currently there is no imaging technique available allowing the visualization of both the drug distribution and the ultrastructure within the same cell. In the proposed project, the distribution of a clinically relevant platinum drug (oxaliplatin) and two drug candidates will be imaged in two- and three-dimensionally cultured malignant and non-malignant cells as well as in murine normal and tumor tissues to identify the subcellular structures accumulating the drugs. For this purpose, novel combinations of nano-scale secondary ion mass spectrometry (NanoSIMS) with transmission and scanning electron microscopy (TEM, SEM) and confocal laser scanning microscopy (CLSM) will be applied, providing unprecedented opportunities for subcellular drug imaging. Platinum complexes containing ligands labeled with stable isotopes will be synthesized and used to study their intracellular dissociation/activation and relations between localization and biological activity/resistance by subcellular analyses of both platinum and the label. The synopsis of high-resolution drug distribution patterns, cell morphology and ultrastructure holds much promise for resolving questions concerning activity and side effects of the therapy as well as resistance to it. The new imaging approach will be extended from conventional monolayer cell cultures to multicellular spheroids and animal tissue samples. Novel sample preparation techniques will be established and optimized to enable the combined application of three different imaging techniques on the very same regions of interest and to minimize analyte distribution artifacts (e. g., washout and redistribution of analyte by dehydration and embedding). Parallel analyses with inductively coupled plasma mass spectrometry (ICP-MS) and elemental analyzerisotope ratio mass spectrometry (EA-IRMS) will provide (as yet unavailable) matrix-matched standards for calibration of NanoSIMS signal intensities, which is a prerequisite for quantitative analysis with the (per se semi- quantitative) NanoSIMS technology. The application of these analytical techniques will also aid in quantifying the effect of sample preparation on analyte concentration and in assessing the detection limits of the new imaging method.

While the crucial role of DNA as the main target for clinically relevant platinum drugs is generally accepted, the variety of possible interactions with cellular targets other than DNA has scarcely been studied. To examine the subcellular distribution of selected stable isotope- labelled platinum(II)- and platinum(IV)-based anticancer drugs a novel combination of nanometer-scale secondary ion mass spectrometry (NanoSIMS) with transmission electron microscopy (TEM) was elaborated. The application of the combinatorial imaging technique spread from conventional two-dimensionally cultured cell lines to 3D (spheroid) cultures and organs from treated mice. One of the major challenging tasks associated with combinatorial (Nano)SIMS/TEM application for trace element and isotope analyses is sample preparation. To elaborate a technique preserving both the structural and chemical integrity of the living cell high-pressure freezing followed by rapid, agitation-assisted freeze substitution and resin embedding was employed on adherently grown 2D cell cultures. This approach allowed us to investigate the distribution of isotopically dual labeled oxaliplatin in three human colon cancer cell lines (SW480, HCT116 wt and isogenic oxaliplatin resistant HCT116 oxR). For the first time, to our knowledge, the ultrastructurally correlated subcellular distribution of the clinically established platinum-based drug oxaliplatin in tumor cells was studied (Legin et al., submitted). Multicellular tumor spheroid models serve as an important three-dimensional in vitro cell model system as they mimic the complex tumor microenvironment and thus form a basis for valuable assays in drug discovery studies. In close collaboration with the Department of Analytical Chemistry, University of Vienna, we employed a state-of-the-art laser ablation- inductively coupled plasma-mass spectrometry (LA-ICP-MS) setup for high spatial resolution elemental imaging of multicellular tumor spheroids (Theiner S. et al., 2017). To explore the general applicability of the approach to the in vivo setting correlative analyses were carried out on mouse kidney and tumor samples upon administration of platinum(IV) compounds (isotopically non-labeled KP2156 and KP1819). Spatial platinum accumulation patterns were quantitatively assessed by LA-ICP-MS and used to select regions of interest for subcellular-scale imaging with NanoSIMS and TEM. Altogether, we demonstrated that elemental imaging can be effectively combined with up-scale and down-scale imaging techniques (Legin et al., 2016).