NanoSIMS analysis of an isotopically labelled organometallic ruthenium(II) drug to probe its distribution and state in vitro

Abstract

The in vitro inter- and intra-cellular distribution of an isotopically labelled ruthenium(II)–arene (RAPTA) anti-metastatic compound in human ovarian cancer cells was imaged using nano-scale secondary ion mass spectrometry (NanoSIMS). Ultra-high resolution isotopic images of ¹³C, ¹⁵N, and Ru indicate that the phosphine ligand remains coordinated to the ruthenium(II) ion whereas the arene detaches. The complex localizes mainly on the membrane or at the interface between cells which correlates with its anti-metastatic effects.

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Ruthenium based anti-tumor compounds are under development as alternatives to classical platinum based drugs, the latter being widely used in the clinic, but are limited by resistance and severe side-effects.^1–4 Among the various types of ruthenium compounds under investigation are the bifunctional ruthenium(II)–arene (RAPTA) complexes, a class of organometallic ruthenium(II) complexes with a monodentate 1,2,5-triaza-7-phosphatricyclo[3.3.1.1.]decane (PTA) ligand (Fig. 1). The lead RAPTA compounds possess attractive anti-metastatic⁵ and anti-angiogenic⁶ properties. Although a number of intracellular targets have been identified for this class of compounds,^7–9 the full mechanism of action remains to be elucidated and, understanding the relative magnitude of extra- and intracellular accumulation and distribution would be useful in this respect. Previous attempts to elucidate the distribution of RAPTA complexes utilizing an anthracene florophore¹⁰ or an acetal functionalized arene¹¹ were only moderately successful owing partly to the need for significant structural modifications to the parent molecule. Here, we describe the use of NanoSIMS to detect ruthenium and isotopically labelled ligands bound to the ruthenium center in Ru(η⁶-toluene)(PTA)Cl₂ (RAPTA-T, Fig. 1).

Fig. 1 Examples of RAPTA complexes.

NanoSIMS has previously been used to detect Au^12,13 and Pt^14,15 metal-based drugs in cells, demonstrating its potential in applications of determining the distribution of these class of compounds in vitro. NanoSIMS provides a spatial resolution of up to 50 nm and is able to detect up to 7 masses simultaneously at high mass-resolution. For example, in this study, we simultaneously detected the ions ¹²C₂⁻, ¹³C¹²C⁻, ¹⁴N¹²C⁻, ¹⁵N¹²C⁻, ³¹P⁻, ³²S⁻ and ¹⁰²Ru⁻. This capability allows the cellular distribution of non-essential and/or isotopically labelled elements to be mapped in vitro. The major obstacles to imaging heavy metal elements in cells with NanoSIMS are the often relatively low ionization yield and the loss of compounds of interest from cells during the sample preparation procedures required for NanoSIMS analysis.¹⁶

In this study, a cisplatin resistant ovarian cancer cell line (A2780CR) was exposed to 500 µM of isotopically labelled RAPTA-T (enriched with 6 atoms of ¹³C at the η⁶-toluene, and 3 atoms of ¹⁵N at the PTA) for 24 hours. Subsequently the cells were fixed and prepared for NanoSIMS imaging as semi-thin sections (see ESI† for more details).

In order to image the distribution of ¹³C, ¹⁵N, and Ru and thus to visualize where in cells the RAPTA-T molecules located, and if they stay intact, using the NanoSIMS we sputtered semi-thin sections with a primary Cs⁺-beam current around 4 pA and a probe size of ∼150 µm (see ESI† for details) for a scanning time of ca. 22 hours, corresponding to 120 consecutive images with 256 × 256 pixels over an area of 30 × 30 µm². ¹⁰²Ru⁻ counts steadily increased with time, plateauing around 8 hours (∼40 planes) into the analysis (Fig. S2, ESI†), demonstrating that a large dose of Cs⁺ implantation is required before efficient ionization of ¹⁰²Ru⁻ is achieved. Such long analysis times represent a severe challenge with respect to machine stability. For example, even small thermal perturbations can cause the instrument, and hence the images, to drift. By minimizing any thermal perturbation to the instrument for over one week (including not entering the lab-space around the instrument), we obtained an image drift totaling only 6 pixels during the 22 hours acquisition period, corresponding to 0.7 microns. Such stable instrument conditions make it possible to add all images together with a minimum of drift correction, and thus obtain clear total images of even very weak signals.

Fig. 2 shows the elemental distribution maps obtained from a resin-embedded section of A2780CR cells after 24 hours exposure to 500 µM of isotopically labelled RAPTA-T. Highly resolved images of ¹⁴N¹²C⁻, ³²S⁻, and ³¹P⁻ allow clear visualization of the cellular compartments of the cell (labelled in Fig. 3B). The ¹³C/¹²C map shows faint variations in the ¹³C/¹²C between cell interiors and the adjacent epoxy resin (also observed in untreated controls, Fig. S1, ESI†), but no clear enrichments that can be ascribed to the presence of isotopically labeled RAPTA-T molecules, or its subcomponents. This absence of discernable ¹³C enrichment in regions clearly enriched in ¹⁵N and Ru (Fig. 2 and 4) could indicate that sample preparation (which includes epoxy embedding) dilutes the ¹³C-isotopic enrichment from the ¹³C-enriched η⁶-toluene ligands to below detection limit of the NanoSIMS.¹⁷ However, given the strength of the ¹⁵N enrichment observed (Fig. 2 and 4), the presence of the corresponding ¹³C-enriched η⁶-toluene ligands should be visible in these NanoSIMS ¹³C/¹²C images, which would reveal ¹³C-enrichment anomalies down to about 30‰. Thus, it is not unreasonable to hypothesize that the ¹³C-enriched η⁶-toluene ligands have partially detached from the complex and have been diluted over the sample. Indeed, dissociation of the arene has been previously observed in binding studies to isolated oligonucleotides.¹⁸ In humans, this detached toluene would undergo detoxifaction in the liver to hippuric acid which would then be excreted in the kidneys.¹⁹

Fig. 2 Secondary ion maps of ³¹P⁻/¹²C₂⁻, ³²S⁻/¹²C₂⁻, ¹⁴N¹²C⁻/¹²C₂⁻, ¹⁵N¹²C⁻/¹⁴N¹²C⁻, ¹⁰²Ru⁻/¹²C₂⁻ and ¹³C¹²C⁻/¹²C₂⁻ (figure labels have been simplified) in A2780CR cells treated with ¹⁵N and ¹³C-labelled RAPTA-T (500 µM, 24 hours). White line (AB) represents the line profile shown in Fig. 4. The ³¹P⁻/¹²C₂⁻, ³²S⁻/¹²C₂⁻, ¹⁴N¹²C⁻/¹²C₂⁻ maps allows identification of cellular compartments. Clear ¹⁵N and Ru enrichments are observed corresponding to RAPTA-T. No significant ¹³C enrichment (above the ¹³C/¹²C ratio of the epoxy resin) is observed.

Fig. 3 Composite RBG images of semi thin sections of A2780CR cells treated with ¹⁵N and ¹³C labelled RAPTA-T (500 µM, 24 hours). ¹⁵N¹²C⁻/¹⁴N¹²C⁻ is coloured green and ³¹P⁻/¹²C₂⁻, ³²S⁻/¹²C₂⁻, ¹⁴N¹²C⁻/¹²C₂⁻ and ¹⁰²Ru⁻/¹²C₂⁻ is coloured red (figure labels have been simplified). Subcellular compartments N (nucleus), Nu (nucleolus), C (cytoplasm), NM (nuclear membrane) and CM (cell membrane) have been labelled in image B. The ¹⁰²Ru⁻/¹²C₂⁻ and ¹⁵N¹²C⁻/¹⁴N¹²C⁻ images show clear co-accumulation of both Ru & ¹⁵N. RAPTA-T seems to localize on the cell membrane and between cells and its accumulation does not appear correlated with that of P, S or N.

Fig. 4 Line profiles of ¹³C¹²C⁻/¹²C₂⁻ (A) and overlaid line profile of ¹⁵N¹²C⁻/¹⁴N¹²C⁻ and ¹⁰²Ru⁻/¹²C₂⁻ (B) across line AB (shown in Fig. 2). Four clear peaks of ¹⁵N¹²C⁻/¹⁴N¹²C⁻ and ¹⁰²Ru⁻/¹²C₂⁻ are seen correlating perfectly, demonstrating the co-enrichment of ¹⁵N and Ru. In contrast, the ¹³C¹²C⁻/¹²C₂⁻ ratio show no enrichment above the statistical noise level.

On the other hand, overlaid images of ¹⁵N/¹⁴N and Ru/C (Fig. 3D) and line profiles (Fig. 4B), reveal co-accumulation of ¹⁵N and Ru indicating that the PTA ligand remains coordinated to the metal center after 24 hours. Enrichment in ¹⁵N (hereby used as a marker for RAPTA-T enrichment) is mainly seen on the cell membrane or interphase between cells (Fig. 3A–C). The observed localization of RAPTA-T indicates that interactions with membrane receptors or extracellular proteins are likely to be critical to its mode of action. In this respect, it has been previously shown that RAPTA-T interacts with cell adhesion proteins such as fibronectin and collagen IV preventing detachment and re-adhesion of metastatic tumor cells.^20,21

Only small pockets of ¹⁰²Ru⁻ were observed inside the cells, (Fig. 2, arrows). From the cellular compartments identified (Fig. 3B), these pockets seem to lie generally within the nuclear region of the cells which is likely as RAPTA complexes have been shown to bind to histones.^7,8 However, the exact subcellular localization cannot be determined accurately without correlated electron microscope imaging. The lack of correlation between these ¹⁰²Ru⁻ hotspots and ³²S⁻ shows that RAPTA-T distributes differently to cisplatin, which was found to accumulate in the nucleolus and S-rich regions of the cells.¹⁴ This difference is not unexpected considering the contrasting in vitro and in vivo anti-tumor effects of cisplatin and RAPTA-T, respectively. The observed distribution pattern is in reasonable agreement with cell uptake studies of RAPTA-T in A2780 CR cells,²² where ruthenium was found in the membrane as well as the particulate, cytoskeletal and nuclear fractions upon similar treatment conditions.

From our data, the observed co-accumulation of ¹⁵N and Ru shows that the PTA ligand remains coordinated to the ruthenium ion. This result highlights one of the key strengths of NanoSIMS for the detection of metal-based drugs, i.e. that the stability/liability of the ligands coordinated to the metal center can be probed via isotopic labelling. The ability to differentiate between the accumulation of a compound on the membrane versus intracellular accumulation in specific organelles illustrates the utility of the NanoSIMS relative to other techniques used to probe metallodrug distribution, such as inductively coupled plasma mass spectrometry and atomic absorption spectroscopy,^22–25 where such a spatial distinction cannot be made without cell fractionation, a process likely to introduce other distribution artifacts.

With recent developments in NanoSIMS, the technique has emerged as a powerful tool for exploring the distribution of metal-based drugs in cells, combining high sensitivity of detection with the ability to study the state of metal-bound ligands following appropriate isotopic labelling. In the case of RAPTA-T, the liability of the η⁶-toluene ligand presumably acts, in addition to aquation, as an activation mechanism of the compound, although from the data the extent of detachment is difficult to assess. Moreover, the observed accumulation of Ru on the membrane or at the interface between cells is in agreement with a number of in vitro and in vivo properties of RAPTA-T^22,26 and provides further insight into the drugs mechanism of action.

This research was supported by the NCCR Chemical Biology, funded by the Swiss National Science Foundation.

Notes and references

1. M. A. Jakupec, M. Galanski, V. B. Arion, C. G. Hartinger and B. K. Keppler, Dalton Trans., 2008, 183–194.
2. E. Reisner, V. B. Arion, B. K. Keppler and A. J. L. Pombeiro, Inorg. Chim. Acta, 2008, 361, 1569–1583.
3. A. Bergamo, C. Gaiddon, J. H. M. Schellens, J. H. Beijnen and G. Sava, J. Inorg. Biochem., 2012, 106, 90–99.
4. M. Groessl and C. G. Hartinger, Anal. Bioanal. Chem., 2012, 405, 1791–1808.
5. C. Scolaro, A. Bergamo, L. Brescacin, R. Delfino, M. Cocchietto, G. Laurenczy, T. J. Geldbach, G. Sava and P. J. Dyson, J. Med. Chem., 2005, 48, 4161–4171.
6. A. Weiss, R. H. Berndsen, M. Dubois, C. Müller, R. Schibli, A. W. Griffioen, P. J. Dyson and P. Nowak-Sliwinska, Chem. Sci., 2014, 5, 4742–4748.
7. Z. Adhireksan, G. E. Davey, P. Campomanes, M. Groessl, C. M. Clavel, H. Yu, A. A. Nazarov, C. H. F. Yeo, W. H. Ang, P. Dröge, U. Rothlisberger, P. J. Dyson and C. A. Davey, Nat. Commun., 2014, 5, 3462.
8. B. Wu, M. S. Ong, M. Groessl, Z. Adhireksan, C. G. Hartinger, P. J. Dyson and C. A. Davey, Chem. – Eur. J., 2011, 17, 3562–3566.
9. M. V. Babak, S. M. Meier, K. V. M. Huber, J. Reynisson, A. A. Legin, M. A. Jakupec, A. Roller, A. Stukalov, M. Gridling, K. L. Bennett, J. Colinge, W. Berger, P. J. Dyson, G. Superti-Furga, B. K. Keppler and C. G. Hartinger, Chem. Sci., 2015, 6, 2449–2456.
10. A. A. Nazarov, J. Risse, W. H. Ang, F. Schmitt, O. Zava, A. Ruggi, M. Groessl, R. Scopelitti, L. Juillerat-Jeanneret, C. G. Hartinger and P. J. Dyson, Inorg. Chem., 2012, 51, 3633–3639.
11. Y. Q. Tan, P. J. Dyson and W. H. Ang, Organometallics, 2011, 30, 5965–5971.
12. L. E. Wedlock, M. R. Kilburn, J. B. Cliff, L. Filgueira, M. Saunders and S. J. Berners-Price, Metallomics, 2011, 3, 917–925.
13. L. E. Wedlock and S. J. Berners-Price, Aust. J. Chem., 2011, 64, 692–704.
14. A. A. Legin, A. Schintlmeister, M. A. Jakupec, M. Galanski, I. Lichtscheidl, M. Wagner and B. K. Keppler, Chem. Sci., 2014, 5, 3135–3143.
15. L. E. Wedlock, M. R. Kilburn, R. Liu, J. A. Shaw, S. J. Berners-Price and N. P. Farrell, Chem. Commun., 2013, 49, 6944–6946.
16. P. Hoppe, S. Cohen and A. Meibom, Geostand. Geoanal. Res., 2013, 37, 111–154.
17. S. H. Kopf, S. E. McGlynn, A. Green-Saxena, Y. Guan, D. K. Newman and V. J. Orphan, Environ. Microbiol., 2015, 17, 2542–2556.
18. A. Dorcier, P. J. Dyson, C. Gossens, U. Rothlisberger, R. Scopelliti and I. Tavernelli, Organometallics, 2005, 24, 2114–2123.
19. L. Flowers, W. Boyes, S. Foster, M. Gehlhaus, K. Hogan, A. Marcus, P. McClure and M. Osier, U. S. Environ. Prot. Agency, 2005, 1–107.
20. A. Bergamo, A. Masi, P. J. Dyson and G. Sava, Int. J. Oncol., 2008, 1281–1289.
21. P. Nowak-Sliwinska, J. R. van Beijnum, A. Casini, A. A. Nazarov, G. Wagnières, H. van den Bergh, P. J. Dyson and A. W. Griffioen, J. Med. Chem., 2011, 54, 3895–3902.
22. D. A. Wolters, M. Stefanopoulou, P. J. Dyson and M. Groessl, Metallomics, 2012, 4, 1185.
23. M. Groessl, O. Zava and P. J. Dyson, Metallomics, 2011, 3, 591–599.
24. A. R. Timerbaev, J. Anal. At. Spectrom., 2014, 29, 1058–1072.
25. A. Zayed, T. Shoeib, S. E. Taylor, G. D. D. Jones, A. L. Thomas, J. P. Wood, H. J. Reid and B. L. Sharp, Int. J. Mass Spectrom., 2011, 307, 70–78.
26. A. Bergamo, A. Masi, P. J. Dyson and G. Sava, Int. J. Oncol., 2008, 33, 1281–1289.

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Footnote

† Electronic supplementary information (ESI) available: Nano-SIMS data, cell culture and sample preparation, and nano-SIMS parameter settings. See DOI: 10.1039/c5cc06983a

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