David Cockayne

Howie

Application of electron optical techniques to the study of amorphous materials

Keywords: amorphous solids, electron diffraction, electron microscopy ): coherence volume amorphous solids, electron diffraction, electron microscopy, coherence volume

Despite the sub-angstrom resolution capability of modern transmission electron microscopes, the elucidation of the structure of amorphous materials by direct imaging methods is still challenged by problems of projection and data presentation identified over twenty years ago. The classical diffraction analysis, leading to the radial distribution function not only retains its essential importance but has been extended to deal with small scattering volumes in inhomogeneous samples by the use of focused electron probe illumination but retaining orientation averaging. Fluctuation microscopy provides a significant advance in the detection and statistical representation of mediumscale spatial variations in amorphous structure. Issues of scattering coherence are crucial in both these two approaches. Further opportunities for use of focused probes include the study of angular correlations in nano-diffraction patterns as well as fine structure in energy loss spectra. Finally there may be a return to the direct imaging approach thanks to recent advances in electron tomography and in aberration-corrected depth sectioning.

Introduction

High resolution transmission electron microscopy (HRTEM) has made many decisive contributions to direct structure determination in materials science but, in the case of amorphous specimens, it has proved very difficult to rival let alone surpass the more modest, spatially averaged statistical test of short range order (SRO) offered by the radial distribution function (RDF) analysis deduced from classical diffraction data. Initial experience with HRTEM bright and dark-field images, summarized in §2 below, revealed two serious problems [1]. Firstly the images, being essentially 2-d projections of the 3-d structure, are usually dominated by the purely random statistical overlap of contributions from atoms too far separated in the beam direction to be structurally correlated. Secondly, in cases where significant structural effects were identified in HRTEM images, these were not usually readily appreciated by non-experts and it proved impossible to devise some simple statistical conclusion to supplement the classical RDF function. The use of hollow cone illumination or equivalently annular dark field (ADF) imaging in scanning transmission electron microscopy (STEM) was then proposed as a means of reducing the unwanted coherent interference effects between widely separated atoms [2] but not actively followed up at that time.

In this impasse, micro-diffraction and nowadays nano-diffraction provide attractive alternatives to direct imaging particularly in samples with obvious spatial variations and are discussed in §3. Detailed analysis of the spotty ring structure of these diffraction patterns has presented serious but interesting challenges. Much greater progress has however been made by azimuthal averaging of the patterns and extraction of local RDF functions making the usual assumption of isotropic structure [3]. Almost two decades later, as discussed in §4, the hollow cone imaging ideas were substantially reworked and refined in the development of the fluctuation microscopy imaging method [4] through which spatial variations in amorphous structure are now routinely and successfully presented in the ideal simple angular scattering or radial separation plots [5]. Here the degree of spatial resolution and consequent inter-atomic scattering coherence have emerged as crucial parameters.

With continuing improvements in electron optics and in electron microscopy techniques it is often profitable to revisit previously intractable or difficult problems. We therefore conclude in §5 with a brief note of the possible impact on amorphous structure studies as a result of aberration correction (with improved depth resolution as well as lateral resolution), of electron tomography (with the capability of full determination of 3-d structures).

Early work in HRTEM imaging

The bright spots visible in dark field images of amorphous materials [6,7] and later the prominent small patches of fringes found in bright field images taken with tilted illumination [8] were initially interpreted as arising from nm scale crystallites or other ordered regions. However image simulations soon demonstrated that the dark field structure could be an essentially random speckle phenomenon with bright spot sizes purely related to the spatial resolution of the microscope and with no systematic dependence on defocus for its overall appearance. Likewise, when random effects dominate, the fringy appearance of bright field images is mainly determined by the image power spectrum and could be enhanced by a chromatic aberration filtering effect in tilted illumination [9]. By using axial illumination [START_REF] Freeman | The Structure of Non-Crystalline Materials[END_REF] and in some cases through focal series reconstruction [START_REF] Saxton | [END_REF], bright field images free of dangerous filtering effects were obtained where extended patches of fringes were generally much less noticeable. The image contrast mainly arises from alignments of atoms in the beam direction and in all but the thinnest samples such alignments involve atoms separated by over 3nm and thus reflect purely random statistics unless some unusual long range order is present. In such cases where the scattered waves have random phases, the only information provided by the high resolution bright field image is the power spectrum which is equally available from the diffraction pattern.

Image simulations carried out for a 1.5nm diameter crystallite surrounded by a region of random network showed that the crystal remained easily visible only when the total sample thickness was less than about 4nm [12]. For widely separated crystallites or other ordered regions comprising just a very small fraction of the sample volume, direct imaging would none the less often be a much more sensitive detector of their presence than classical diffraction broad beam data. These early micrographs gave a strong impression of the great variability of amorphous materials even when the averaged properties indicated by diffraction data seemed closely similar [1]. Amorphous Si or Ge films often seemed indistinguishable from random structures whereas silica was much more variable. In cases, where the images showed some noticeably larger patches of fringes in bright field or dark field spots larger than the instrumental resolution and exhibiting an optimum focus condition, more rigorous tests were devised and applied to assess their significance. These tests have included checks that the appearance of order disappeared and was not simply rearranged to new regions after phase randomization of the images whilst preserving the power spectrum [13] and autocorrelation measurements [14,15].

Although it was appreciated that stereo microscopy might provide a solution to the imaging overlap problem just described, it seemed in these early days virtually impossible to apply this successfully at the very high spatial resolution involved, particularly in the absence of obvious image markers enabling a particular region to be identified. Hollow cone illumination however allows images from different viewing directions to be incoherently and accurately superimposed as illustrated in fig. 1(a). In conventional TEM this can be achieved for a fairly small range of different viewing directions either with an annular illuminating aperture or by rocking the incident beam direction. By the reciprocity principle, the same result can be obtained more easily and over a larger angular range in STEM ADF imaging as indicated in fig. 1(b). The application of hollow cone dark field imaging to amorphous structures was thus investigated both experimentally and theoretically [2].

At each image point r = (x,y,z), where z denotes the plane of focus, the contribution to the coherent dark field intensity I from a particular incident plane wave component, defined by the vector K in fig. 1, is given by the expression

( ) ( ) ( ) 2 . exp , , j j j j j i z z a I r K ρ ρ K r - - - Σ = (1)
Here the atomic image function for an atom j at position r j = (ρ ρ ρ ρ j, z j ) = (x j ,y j ,z j ), with z j its distance from the focal plane, is given by

( ) ( ) ( ) ( ) ( ) q ap q j j j j j d i i K M K f i z z a σ θ γ π λ ∫ - - -       = - - r r q ρ ρ . exp )) ( exp( 4 , 2 (2)
where for simplicity the variation of the atomic scattering amplitude f and Debye Waller factor exp(-M) with position q in the objective aperture have been ignored. For later convenience in §3 and §4 below, we may note that when the radius Q of the objective aperture (or in STEM the probe forming aperture) is not too large we can ignore both the lens aberration term γ and the z component of q. Eqn. (2) then reduces to an Airy disc form

( ) ( ) ( ) j j j j j Q Q J Q K M K f i a ρ ρ ρ ρ ρ ρ - - -       = - 1 2 2 2 )) ( exp( 4 π π λ (3)
For discussing speckle effects, it is convenient to split the coherent dark field intensity of eqn (1) into a sum of single atom and different atom contributions I 1 and I 2 respectively.

( ) ( ) ( ) ( ) φ ρ cos exp .( exp * , , , , 2 2 1 j l j l z l j j l j j j iK z z iK a a K z I a z I ρ ρ ρ ρ - - Σ = ∆ Σ = ∆ ≠ (4)
Referring to fig. 1(b) it can be seen that K has components K z =2ksin 2 (α/2) and K ρ = ksin(α). In general the speckle effect, arising from the superposition of atoms closely aligned along the optic axis, is dominated by the second term with say N(N-1) contributions compared with the N contributions from the first term. To include the spread of angular illumination (assumed incoherent) we have to integrate eqn. (4) over the angular ranges 0< φ <2π and α 1 < α < α 2 corresponding to points in the condenser aperture or to points in the annular detector in the case of STEM. After the azimuthal integration over φ, the expression for I 2 becomes

( ) α ρ α α sin 2 sin 2 sin 2 sin 2 cos 2 1 0 2 2 , 2 jl jl jl jl jl j l j k J kz B kz A I             +       Σ = ≠ (5)
where ( )

l j l j jl a a a a A * * + = ( ) l j l j jl a a a a i B * * - = (6) l j jl ρ ρ - = ρ j l lj z z z - = (7)
Integration over a sufficient range of α, will average out the oscillations of the Bessel function except for very small values of ρ jl . Noting further that the B jl terms will be close to zero when ρ jl is small, the interference effect will also be restricted to atoms separated in the beam direction by less than z jl ≈ λ/(8sin 2 (α m /2)) where α m is an angle typical of the annular detector. Gibson and Howie [2] pointed out that the effect of these integrations is to introduce a cigar-shaped coherence volume within which the two atoms j and l must both lie if interference to occur. For the values of α in excess of 50mrad readily accessible in ADF STEM, we might expect this second effect to be useful in reducing the random speckle in typical dark field images of amorphous materials. Preliminary experiments suggested in fact that such reductions in speckle did occur leaving residual bright regions which could be more reliably linked to genuine regions of local order [2].

The problem of finding a convenient quantitative means of presenting any signs of local order discovered in this way was however not addressed though the possibility of mapping I 2 and higher moments of the dark field intensity was mentioned.

This apparently promising line of incoherent dark field imaging of amorphous materials was regrettably not pursued further at the time. Indeed it was not realized until some years later [16,17,18] that, in providing a basis for the general phenomenon of incoherent ADF STEM imaging, the simple effect of annular geometry described by these equations is a significant factor in addition to the arguments about thermal diffuse scattering that had previously been advanced [19]. As already noted above, for atoms with appreciable lateral separation ρ jl , interference (i.e. transverse coherence) is suppressed by the annular detector geometry. For overlapping atoms such as those with separation distance z jl in the same column of a crystal, the role of phonons is more important in suppressing the longitudinal coherence [18].

Nano-diffraction methods

To discuss the classical diffraction method it will be adequate here to start with the expression for the diffracted intensity from a monatomic sample neglecting constant prefactors

( ) ( ) ( ) ( ) [ ] l j l j i K M K f I r r K K - Σ - = . exp ) ( 2 exp , 2 (8)
In this approach, the overlap problem of high resolution amorphous imaging, is greatly reduced by making the assumption of statistical isotropy. We then find the Debye [20] expression for the diffracted intensity I(K)

( ) ( ) ∫ Σ - + - = dV Kr Kr r g M f M Nf K I jl jl jl j ) sin( ) 2 exp( ) 2 exp( 2 2 (9)
Here g(r jl ) is the pair distribution function measuring the probability of finding an atom j at distance r jl from atom l. This equation already demonstrates some of the effects of projection for randomly oriented bonds since the first diffracted maximum occurs near Kr jl = 5π /2 rather than at 2π where it would be for atom pairs aligned with K. Still more of the problems experienced in direct imaging are avoided in the classical diffraction approach by constructing the reduced intensity function Φ(K) = [I(K) -Nf 2 (K)exp(-2M)]/ Nf 2 (K)exp(-2M) which measures the deviation from purely random behavior and can be directly related by a Fourier transform to the reduced density function G(r) = 4πr[g(r) -ρ] where ρ is the mean atomic density. A useful indication of the averaged significant structure then results in the form of simple plots of Φ(K) vs K or G(r) vs r. This diffraction technique is long established in the scattering of X-rays and neutrons but, for the study of thin films, intergrannular layers and other nanovolumes, electrons are potentially unrivalled because of their high scattering cross section and our ability to focus them to small spots. There were some pioneering applications of electron diffraction to amorphous or highly disordered solids in the 1960s when removal of inelastically scattered electrons by energy filtering became more straightforward but the whole field has become much more active following the work of David Cockayne and his colleagues (for a detailed review see [3]). Their determination of the bond lengths in the C 70 molecule proved to be an early and striking success [21]. The consistency of their results with the full molecular structure was ensured by the ingenious procedure of relaxing the molecule through a simulated annealing algorithm [22] using -the measured G(r) as an interatomic pair potential. A subsequent and impressive comparison of their bond length data with various theories is given in [23].

These C 70 results were obtained with broad beam illumination but spurred interest in nanodiffraction work making use of the massive developments in instrumentation that had taken place since the field was first pioneered [24,25]. The extraction of the RDF function from typically 1-2 nm diameter local areas in for example intergrannular phase regions conveniently selected in TEM images has proved to be very fruitful. Individual nanodiffraction patterns display azimuthally structured or spotty diffraction rings so that azimuthal averaging has to be carried out before making the traditional analysis. These developments and the results obtained have been fully reviewed in [3,26]. Here we concentrate on the effects on the simple plane wave illumination theory of using focused illumination and of possible departures from isotropy in such nano regions.

The effects on classical diffraction and RDF analysis of using an annular range of incident beam directions have been investigated experimentally and computationally for both incoherent illumination [27,28] and fully coherent illumination [29]. Completely incoherent illumination will not of course exhibit any spatial localization but the experimental spot sizes used have diameters typically about twice as great as could be produced with fully coherent illumination with the aperture used and so fall between these two extremes. The effect of an angular spread of incoherent illumination can be corrected by applying a deconvolution procedure to the measured diffracted intensity I(K). Although the need for a two-dimensional deconvolution was not clearly emphasized, the experimental as well as computational results indicated that the main low-order peaks in the RDF function were not seriously affected [27,28]. In the case of coherent focused illumination, the effect was studied in amorphous Si both by simulation and by observation with different probe sizes [29]. The conclusion again was that the positions of the low order peaks in the RDF function are preserved for probe diameters greater than 1.2nm.

A different insight into these effects can be got by looking directly at the effect in eqn. (8) of including a range of incident plane waves as was done in §2. For the fully incoherent case we have to integrate the diffracted intensity over a range of incident q vectors within the illumination aperture shown in fig 1(b) (dividing by the aperture area for normalization) to obtain the result

( ) ( ) ( ) ( ) [ ] ( ) { } ( ) ( ) ( ) jl jl jl jl inc jl inc l j j l j inc Q Q J Q qdqd iq K F K F i N M K f I ρ ρ π ϕ φ ρ ρ ρ 1 2 , 2 2 cos exp . exp 2 exp = = - Σ + - = ∫ ≠ r r K K (10)
The correction factor is just the Airy function of the probe forming aperture.

In the coherent illumination case the integration over q has to be done on the expression for the diffracted amplitude before calculating the intensity. The correction factor is then the product of two Airy functions. To be detected in incoherent illumination (where no probe position is defined), it is then clear that the projected bond length ρ jl must simply be less than the about 2a = 2π/Q, the diameter of the resolution cylinder indicated in fig. 2 (a). Longer bonds will contribute only if they are sufficiently aligned to the z axis. The corresponding peak in the I(K) plot will therefore move slightly outwards. For coherent illumination, both projected atom positions must lie within the resolution volume so many fewer atom pairs can contribute. Summing up the results of many different probe positions effectively scrambles the phases of the incident plane waves and therefore reproduces the incoherent correction factor. Apart from restricting the volume sampled, coherent illumination will produce further filtering effects on bond lengths and orientations. Consider a bond of projected length ρ jl < 2π/Q as indicated in fig. 2(b). The area available for its projected centre to occupy lies within an ellipse of major axis a = π/Q and minor axis b = π/Qρ jl /2. The filtering factor is therefore roughly 1-Qρ jl /2π.

( ) ( ) ( ) l l j j jl coh Q Q J Q Q J K F ρ ρ ρ ρ ρ ρ ρ ρ - - - - =
For reliable application of the classical diffraction method it is obviously important to be confident about the validity of the assumption of structural isotropy. Significant failures, potentially signaled by an azimuthal φ K dependence of the diffraction ring structure, could however be hard to detect in rather noisy nano diffraction patterns. Furthermore, any simple structural dependence only on θ, the angle with the optic axis would not result in any such obvious effect. In the case of thin film structures, a dependence of bond orientation relative to the film normal could easily be imagined and would not be obvious unless the film were tilted to large angles. Some idea about the likely importance of departures from isotropy can be obtained by reworking the Debye equation ( 9) for some specific angular bond angular distributions B(θ,φ). Instead of the isotropic function B 1 = 1/4π, we can consider for example a simple θ-dependent function B 2 = [(1-f) + 3f cos 2 (θ)]/4π or a similar function relative to the x axis B 3 = [(1-f) + 3f sin 2 (θ)cos 2 (φ)]/4π. This last function could be relevant near a grain boundary or other interface normal to the x direction. Analytical expressions for these two examples can then be found to replace in eqn ( 9) the Debye factor D 1 = sinZ/ Z where Z = Kr jl . We find

( ) ( ) ( ) [ ] ( ) [ ] [ ] K K Z Z Z Z Z Z f Z Z Z Z f Z Z f f D Z Z Z Z f Z Z f f D φ φ 2 cos 2 sin cos 3 sin 3 3 2 cos sin 3 sin 2 1 , cos sin 3 sin 1 3 2 3 3 3 2 - - - - -       + = - + - = (12)
In fig. 3, D 2 (0.5) and D 2 (-0.5) are compared with the Debye factor D 1 . As expected, the diffraction ring moves outwards for f > 0 corresponding to a preponderance of bonds aligned with the z-axis. In fig. 4, D 3 (0.5,0) and D 3 (0.5,π/2), are compared with D 1 . For f > 0 the distribution B3 describes an enhanced bond concentration aligned with the x-axis. The ring pattern exhibits as expected an azimuthal effect being moved inwards for K directions parallel to the x-axis (φ K = 0) and outwards for directions normal to this. Apart from the shift in ring positions, it is noticeable in all these cases that there can be considerable changes in peak heights as a result of departures from isotropy. An inward shift of the ring position seems to be correlated with an increase in height. For more realistic modeling of departures from isotropy, the parameter f would need to be a function of bond length r jl . Eqns. ( 12) remain valid but are now functions of both r jl and Kr jl and not just Kr jl .

Fluctuation microscopy

Almost two decades after the work described in §2, a much more fruitful way of using the hollow cone imaging approach for amorphous structure investigations emerged [16,30]. Fluctuation microscopy is still advancing [31,5] as so far the best means of investigating MRO. Rather than studying the dark field image intensity I(r) in full image detail, the fluctuations, i.e. spatial variations, of the intensity are measured via the second moment function V(K,Q) = {< I 2 (r) > / < I(r) > 2 } -1. The observed dependence of V(K,Q) on the reciprocal space quantities K and Q (denoting the radii of the annulus radius and objective aperture respectively) can then be compared with computations for any assumed structure.

A crucial step in the development of fluctuation microscopy, running counter to most deeply embedded opinion in electron microscopy, was to appreciate the importance of operating at Q corresponding to a spatial resolution of 1.5nm or even worse. Whether this was realized in advance of the development of the method or was simply a consequence of the limitations of the hollow cone TEM instrumentation initially used is immaterial. At such modest spatial resolution, we can ignore defocus and other aberration effects so that eqn. (

j j j j Q A Q K M K f i a ρ ρ ρ ρ - -       = - 2 2 ) ( exp( 4 π π λ 2) becomes ( ) ( ) ( )
where A(x) = 2 J 1 (x)/x is yet again the usual Airy function. The term a j a l * + a l a j * is then the main factor contributing to the coherent intensity in eqn (5) and requires that the two atoms concerned have projected positions in overlapping Airy discs. In the light of the reciprocity principle the situation is identical to that discussed in §3 for nanodiffraction work in the STEM. Fluctuation microscopy can indeed also be efficiently carried out in STEM mode [32,33].

As before, the final Bessel function factor in eqn. (5) gives the usual Debye factor when integrated over bond orientations. Since the annulus defines only a small range of K ρ for each K ρ value studied, there is no integration over α and the highly localized coherence volume introduced in §2 does not arise. The effective coherence volume is defined by the specimen thickness and the instrumental resolution as in fig. 2.

The importance of matching the instrumental resolution to the structure under investigation by fluctuation microscopy has been very convincingly argued quite recently [34] in response to some observations purporting to show that the dependence of V(K,Q) on K conveys broadly just the same information as the diffracted intensity [35]. It Applications of fluctuation microscopy technique have yielded interesting results about the structure of evaporated amorphous tetrahedral semiconductors and the effect of annealing [36], light soaking [37] or low energy ion bombardment [38]. These initially surprising annealing studies have been to some extent confirmed by autocorrelation studies of HREM bright field images [15]. Fluctuation microscopy seems to have useful sensitivity to MRO lying in the 1-4nm region through higher order pair-pair correlation functions. Although the computations can be somewhat demanding, different atomic models can thus be tested against the intensity fluctuation data. The precise structural features, e.g. para-crystallites or voids, giving rise to MRO may often however be rather subtle and elusive [39]. Very recently, in Ag/In-incorporated Sb 2 Te amorphous films, fluctuation microscopy has been successfully extended to detect sub-critical nuclei for crystallization and monitor their population changes following laser pulse annealing [40].

Conclusions and future prospects

The challenge of elucidating amorphous structures has certainly forced microscopists to consider more carefully issues of coherence in imaging and presentation of data. Spatially localized nano-diffraction, retaining the classical analysis but with due attention to some of the coherence issues orientation averaging noted here, may offer useful information about variations in SRO. The low-angle scattering region, which may be cut off by the need to use focused illumination in nano-diffraction, can nevertheless yield bright field images whose power spectra can also be used to construct the RDF function [41]. Nano diffraction may be less successful in dealing with MRO except perhaps when this is manifested in pair correlation effects giving rise for instance to the first sharp diffraction peak noticed in silica and some other amorphous materials [42]. For the more general investigation and characterization of MRO, fluctuation microscopy seems currently the best approach and has certainly enjoyed some successes in linking the variations detected in MRO to wider properties of the amorphous samples.

Following a preliminary attempt to find angular correlations in the short range order scattering from nano regions [43], angular correlation functions or "correllograms" were constructed from a large number of nano diffraction patterns [44]. Some rather weak evidence for a significant structural signal was found in amorphous indium oxide thin films. More recently it has been suggested that the fluctuation microscopy method may be usefully extended to examine the angular intensity variance function [45].

When several different elements are present, electron scattering may lack the ability of neutron scattering to use isotope effects but energy loss spectroscopy is a powerful alternative tool. Short range order in the vicinity of specific atomic species can be investigated through above-edge fine structure (for discussion see [5]). Spatial variations recently observed in the near edge structure obtained with a 50nm probe from a calcium- The enduring mind set of electron microscopists, now reinforced with aberrationcorrected equipment, will no doubt ensure that high resolution imaging of amorphous materials remains on the agenda despite the early somewhat disappointing history. The reduced depth of field available at higher imaging apertures has already provided some degree of depth discrimination by through-focal imaging in ADF STEM [47] which may be useful in reducing the overlap problem. At least amorphous materials will be free from the channeling effects which complicate these experiments in crystals. Combining the narrow coherence volume of ADF STEM with tomography is certainly something which should be tried with amorphous materials now that the tomography technique has been so enormously developed.

One can be confident that no single one of these approaches will provide all the answers needed. To make significant progress in this demanding field, electron microscopists will not only have to master several of them but also to arrange access to amorphous samples whose important structure-dependent properties are well characterized.

  these observations were carried out at too high spatial resolution (high Q) for the fluctuation microscopy technique to be effective.

  glass indicate long-range fluctuations in composition as well as atomic and electronic structure[46].

Figure 1 .

 1 Figure captions

Figure 2 .

 2 Figure 2. Influence on atomic scattering coherence of the angular spread of imaging or illumination (the angle θ in figs. 1(a) and 1(b) respectively. The cylinder in (a) denotes the resolution volume with height equal to the specimen thickness and radius a = π/Q for an aperture of radius Q. Solid arrows denote bonds which scatter coherently for both coherent and incoherent illumination. Dashed arrows denote bonds which scatter coherently only for incoherent illumination. Coherent scattering from the remaining dotted arrow bonds is destroyed by the angular spread of illumination whether it is coherent or incoherent.

Figure 3 .

 3 Figure 3. Dependence on Z = Kr jl of D 1 the Debye function (denoted by ♦) and of the functions D 2 (f) for f = +0.5 (■) and for f = -0.5 (▲).

Figure 4 .

 4 Figure 4. Dependence on Z = Kr jl of D 1 the Debye function (denoted by ♦) and of the functions D 3 (f, φ Κ ) for f = +0.5, φ Κ = 0 or π (■) and for f = +0.5, φ Κ = π/2 or 3π/2 (▲).

http://mc.manuscriptcentral.com/pm-pml Philosophical Magazine & Philosophical Magazine Letters


Dirk Heshe

Stephanie Hoogestraat

Christine Brauckmann

Uwe Karst

Joachim Boos

PhD Claudia Lanvers-Kaminsky

Dichloroacetate Metabolically Targeted Therapy Defeats Cytotoxicity of Standard Anticancer Drugs

Keywords: DCA, Warburg effect, cancer, cisplatin, cytotoxicity

Purpose: The observation that the orphan drug dichloroacetate (DCA) selectively promotes mitochondria-regulated apoptosis and inhibits tumour growth in preclinical models by shifting the glucose metabolism in cancer cells from anaerobic to aerobic glycolysis attracted scientist, clinicians' but also patients' interests and prompted us to further evaluate DCA effects against paediatric malignancies. Methods: The effects of DCA on mitochondrial membrane potential ( m), cell viability, and induction of apoptosis were evaluated in paediatric tumour cell lines and the non-malignant cell line HEK293. In addition, combinations of DCA with the standard anticancer drugs cisplatin, doxorubicin, and temozolomide were tested and intra-and extra-cellular platinum species analysed. Results: DCA selectively induced phosphatidylserine externalisation and reduced m in paediatric tumour cells compared to HEK293 cells, but DCA concentrations ≤10mmol/L only moderately inhibited the growth of 18 paediatric tumour cell lines. DCA neither influenced the invitro stability of cisplatin nor the cellular cisplatin uptake, but it abrogated the cytotoxicity of cisplatin in 7 out of 10 cell lines. DCA also affected the cytotoxicity of doxorubicin, but did not influence the cytotoxicity of temozolomide. Despite of phosphatidylserine externalisation, DCA failed to activate caspase 3/7 and, moreover, suppressed caspase 3/7 activation by cisplatin and doxorubicin. Conclusions: Our results indicate that apart from the intriguing effects of DCA on the glucose metabolism of cancer cells the use of DCA for cancer treatment has to be evaluated carefully.

Moreover, compassionate use of the orally available drug by cancer patients themselves without medical supervision is strongly discouraged at present.

Introduction

Anaerobic glycolysis (also known as Warburg Effect) is a common feature of cancer cells. In contrast to normal cells, which metabolise most of their pyruvate via acetyl-CoA and the Krebs cycle in their mitochondria, malignant cells shift their pyruvate catabolism from this oxygen-dependent pathway to cytoplasmatic, anaerobic glycolysis [START_REF] Warburg | On respiratory impairment in cancer cells[END_REF][START_REF] Gatenby | Why do cancers have high aerobic glycolysis?[END_REF][START_REF] Samudio | Mitochondrial uncoupling and the Warburg effect: molecular basis for the reprogramming of cancer cell metabolism[END_REF]. The generic drug dichloroacetate (DCA) stimulates the activity of the enzyme pyruvate dehydrogenase (PDH) through inhibition of the enzyme pyruvate dehydrogenase kinase (PDK) [START_REF] Stacpoole | The pharmacology of dichloroacetate[END_REF][START_REF] Bowker-Kinley | Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex[END_REF]. Activation of PDH shifts the catabolism of pyruvate from oxygen-independent lactate formation to oxygendependent acetyl-CoA formation. The irreversible conversion of pyruvate into acetyl-CoA results in a sustained activation of the Krebs cycle, which in turn increases the production of reactive oxygen species (ROS) in the mitochondria.

The increased concentrations of ROS damage the redox sensitive complex I, inhibit H+ efflux and decrease the mitochondrial membrane potential ( m). This decrease of m again facilitates the opening of the m-sensitive mitochondrial transition pores (MTP) and promotes the efflux of cytochrome c (cyt c) and apoptosis-inducing factor (AIF). In addition, ROS translocated to the cytoplasm reduce the tonic inhibition of caspases by dilating Kv1.5 potassium ion channels andin a positive feedback loop -by increasing the expression of Kv1.5

potassium channels [START_REF] Bonnet | A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth[END_REF]. DCA was shown to inhibit cell growth over a wide range of tumour cells. In preclinical studies, DCA reduced m, induced apoptosis, and inhibited the proliferation of breast cancer, endometrial cancer, prostate cancer, non-small cell lung cancer, and glioblastoma cell lines [START_REF] Bonnet | A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth[END_REF][START_REF] Wong | Dichloroacetate induces apoptosis in endometrial cancer cells[END_REF][START_REF] Cao | Dichloroacetate (DCA) sensitizes both wild-type and over expressing Bcl-2 prostate cancer cells in vitro to radiation[END_REF]. In preclinical in vivo studies DCA reduced the growth of non-small cell lung cancer xenografts and significantly diminished the number of lung metastasis in a rat mammary adenocarcinoma metastasis model [START_REF] Bonnet | A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth[END_REF][START_REF] Sun | Reversal of the glycolytic phenotype by dichloroacetate inhibits metastatic breast cancer cell growth in vitro and in vivo[END_REF]. In addition, reversal of the glycolytic phenotype reduced the growth of pancreas tumour xenografts [START_REF] Chen | Oxygen consumption can regulate the growth of tumors, a new perspective on the Warburg effect[END_REF].

Since non-malignant cells usually do not use anaerobic glycolysis for energy production, DCA had no effect on proliferation and induction of apoptosis in nonmalignant cells [START_REF] Bonnet | A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth[END_REF][START_REF] Wong | Dichloroacetate induces apoptosis in endometrial cancer cells[END_REF]. Thus, selective modulation of the glycolytic phenotype of cancer cells by DCA seems to be a promising approach for an effective and tolerable treatment of cancer [START_REF] Xu | Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia[END_REF][START_REF] Michelakis | Dichloroacetate (DCA) as a potential metabolic-targeting therapy for cancer[END_REF]. For its ability to favour glucose metabolism by aerobic glycolysis, DCA has been used for more than 30 years for the treatment of lactate acidosis and inherited mitochondrial diseases in adults as well as in children [START_REF] Stacpoole | Efficacy of dichloroacetate as a lactatelowering drug[END_REF][START_REF] Stacpoole | Evaluation of long-term treatment of children with congenital lactic acidosis with dichloroacetate[END_REF][START_REF] Mori | Dichloroacetate treatment for mitochondrial cytopathy: long-term effects in MELAS[END_REF]. Though clinical trials in humans with cancer had yet to be performed, DCA was celebrated as the magic bullet against cancer by the public press further stimulating the attraction of DCA for cancer patients. Cancer patients opt for this orally available drug and can purchase it from several distributors [START_REF] Pearson | Cancer patients opt for unapproved drug[END_REF]. The promising preclinical results against adult malignancies along with the availability of safety data in adults and children provided a strong rationale for the evaluation of DCA against paediatric malignancies. Here we report on the effects of DCA against a panel of 18 immortal tumour cell lines representing six of the most common and aggressive paediatric tumour types.

Since DCA by decreasing m is supposed to promote mitochondria mediated apoptosis, we also analysed the combination of DCA with standard anticancer drugs.

Methods

Reagents

DCA and temozolomide were purchased from TOCRIS Bioscience (TOCRIS Cookson Ltd., Bristol, UK). Both drugs were dissolved in DMSO (Sigma-Aldrich, Deisenhofen, Germany) and further diluted with complete cell culture medium.

Cisplatin was purchased from Gry-Pharma GmbH (Kirchzarten, Germany) and doxorubicin was obtained from Pharmacia GmbH (Karlsruhe, Germany).

Cell culture

The cytotoxic effect of DCA was tested on a panel of 18 permanent human tumour cell lines including four Ewing sarcoma cell lines (CADO-ES-1, STA-ET-

Cell viability assays

Screening of cell viability was determined by the well acknowledged MTT assay and the CellTiter-Glo™ cell viability assay (Promega, Mannheim, Germany).

While the MTT assay detects viable cells via reduction

Evaluation of caspase 3/7 activation

Caspase activation was determined using the caspase-Glo® 3/7 assay supplied by Promega (Mannheim, Germany) according to the manufacturer's instructions.

Cells (5 × 10 4 cells/well) were seeded in white-walled 96-well plates (Greiner Bio-One GmbH, Solingen, Germany) treated with DCA, cisplatin, and doxorubicin as indicated. Luminescence was measured with the Fluoroskan Ascent FL (Thermo Scientific, Langenselbold, Germany).

Quantification of cellular platinum concentrations

The total platinum concentration in cells and cell culture medium was determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES) using a Spectro CIROS instrument (Spectro Analytical Instruments, Kleve, Germany).

Platinum emission was detected simultaneously at two different emission lines (214.40 nm and 299.97 nm). Cells and cell culture medium were analysed separately. The cells were dissolved in nitric/hydrochloric acid (1/3, v/v) and diluted with bidistilled water. The cell culture medium solutions were diluted with 1% nitric acid prior to analysis.

Quantification of cisplatin in cell culture medium

Cisplatin in cell culture medium was determined by application of liquid chromatography coupled to inductively coupled plasma-mass spectrometry (LC/ICP-MS) using an Agilent model 1200 HPLC and an Agilent 7500 ICP-MS (both from Agilent Technologies, Waldbronn, Germany). Cisplatin was identified based on its characteristic retention time in hydrophilic interaction liquid chromatography (HILIC) using a ZIC®-HILIC column (dimensions 150 x 2.1 mm, particle size 3.5 µm) from SeQuant (Marl, Germany) and a mobile phase containing aqueous ammonium formate buffer (20 mM, Sigma Aldrich Chemie GmbH, Steinheim, Germany) with a pH of 7.4 and methanol (Merck, Darmstadt, Germany). An isocratic flow of 60% methanol was used throughout. Proteins from the cell culture medium were precipitated with acetonitrile (Merck, Darmstadt, Germany) and separated by centrifugation prior to analysis.

Results

Since DCA was reported to decrease m in adult tumour cell lines already at concentrations of 0.5 mmol/L, we exposed 18 cell lines to seven 10-fold drug dilutions of DCA ranging from 10 nmol/L to 10 mmol/L and only observed a moderate reduction of cell viability at the highest test concentration (Fig. 1). In

Discussion

The outstanding experiments of Bonnet et al. identifying DCA as selective anticancer drug attracted oncologists' and also patients' attention [START_REF] Bonnet | A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth[END_REF][START_REF] Wong | Dichloroacetate induces apoptosis in endometrial cancer cells[END_REF][START_REF] Cao | Dichloroacetate (DCA) sensitizes both wild-type and over expressing Bcl-2 prostate cancer cells in vitro to radiation[END_REF][START_REF] Sun | Reversal of the glycolytic phenotype by dichloroacetate inhibits metastatic breast cancer cell growth in vitro and in vivo[END_REF][START_REF] Chen | Oxygen consumption can regulate the growth of tumors, a new perspective on the Warburg effect[END_REF][START_REF] Xu | Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia[END_REF][START_REF] Michelakis | Dichloroacetate (DCA) as a potential metabolic-targeting therapy for cancer[END_REF]. Though clinical trials in humans with cancer had yet to be performed and though accordingly DCA had not been approved for the treatment of cancer by FDA and EMA, it was celebrated as the magic bullet against cancer by the public press further stimulating the attraction of DCA for cancer patients. Cancer patients opt for this orally available drug and can purchase it from several distributors [START_REF] Pearson | Cancer patients opt for unapproved drug[END_REF].

We evaluated the effects of DCA against paediatric malignancies in vitro and only found moderate effects of DCA. In line with the observations of Bonnet et al.,

DCA was able to selectively initiate apoptosis in paediatric tumour cell lines compared to non malignant cells. However, high rates of phosphatidylserine externalisation and significant effects on m reduction were only measurable at concentrations of 25 mmol/L, while DCA concentrations of 10 mmol/L had little or no effect at all. This again was in line with the poor cytotoxic effects of DCA against paediatric tumour cell lines, which we observed at DCA concentrations up to 10 mmol/L. In contrast to some endometrial cancer cell lines no significant increase of cell viability at low DCA concentrations was observed for the cell lines analysed in this study [START_REF] Wong | Dichloroacetate induces apoptosis in endometrial cancer cells[END_REF]. Some factors might be responsible for the poor effects of DCA observed in this study. First of all anaerobic glycolysis might be less important for the metabolism of the paediatric tumours types studied [START_REF] Chen | Mitochondrial membrane potential in living cells[END_REF].

However, there are several lines of evidence (i.e. from fluorine-18fluorodeoxyglucose (FDG) positron emission tomography (PET), gene and protein expression profiling) that paediatric like adult tumours have a high glucose turnover and that anaerobic glycolysis might also be relevant for paediatric malignancies [START_REF] Völker | Positron emission tomography for staging of pediatric sarcoma patients: results of a prospective multicenter trial[END_REF][START_REF] Chen | Global genomic and proteomic analysis identifies biological pathways related to high-risk neuroblastoma[END_REF][START_REF] Wellmann | Activation of the HIF pathway in childhood ALL, prognostic implications of VEGF[END_REF][START_REF] Hulleman | Inhibition of glycolysis modulates prednisolone resistance in acute lymphoblastic leukemia cells[END_REF][START_REF] Lu | Induction of pyruvate dehydrogenase kinase-3 by hypoxia-inducible factor-1 promotes metabolic switch and drug resistance[END_REF]. Furthermore, the expression of the four PDK isoforms and/or of antiapoptotic proteins, like Bcl-2, PUMA or survivin might have influenced the sensitivity to DCA [START_REF] Bowker-Kinley | Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex[END_REF][START_REF] Wong | Dichloroacetate induces apoptosis in endometrial cancer cells[END_REF]. Especially the lack of caspase activation observed by us as well as Sun et al. might be attributed to over expression of antiapoptotic proteins [START_REF] Sun | Reversal of the glycolytic phenotype by dichloroacetate inhibits metastatic breast cancer cell growth in vitro and in vivo[END_REF]. Differences in the expression of Kv1.5 channels might also have discriminated between DCA-sensitive and -resistant cell lines, since activation of Kv1.5 channels by ROS results in a positive feedback loop, which promotes caspase activation [START_REF] Bonnet | A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth[END_REF]. Ongoing studies on the cell line specific glycolytic phenotype along with the cell line specific expression of the different PDK isoforms, Kv1.5 channels, and antiapoptotic proteins will further clarify these issues. Tolerable DCA dosages of 25 mg/kg/d given twice daily, which were clinically applied for the treatment of mitochondrial disorders, resulted in peak plasma concentrations of 100-250 µmol/L after the first dose and 200-550 µmol/L after 6 month of therapy [START_REF] Stacpoole | Role of dichloroacetate in the treatment of genetic mitochondrial diseases[END_REF]. Plasma concentrations of about 1 mmol/L were observed after the first application of 50 mg/kg DCA as well as after chronic exposure to 25 mg/kg DCA [START_REF] Mori | Dichloroacetate treatment for mitochondrial cytopathy: long-term effects in MELAS[END_REF][START_REF] Barshop | Chronic treatment of mitochondrial disease patients with dichloroacetate[END_REF]. Higher plasma concentrations would require higher dosages, whose tolerability has not been evaluated so far.

This already argues against an unguarded use of DCA against cancer. Even more important is the fact that DCA was able to fade out the cytotoxicity of standard anticancer drugs. Making cancer cells more susceptible to apoptosis by selectively switching their glucose metabolism from anaerobic to aerobic glycolysis is challenging, since it promises better response of cancer cells to chemotherapy. (VDAC). The VDAC plays an important role in the inhibition of apoptosis, but is also part of the mitochondrial transition pore (MTP) as described above, a downstream target of DCA [START_REF] Vyssokikh | The function of complexes between the outer mitochondrial membrane pore (VDAC) and the adenine nucleotide translocase in regulation of energy metabolism and apoptosis[END_REF]. Interestingly, VDACs seem to be one main target of cellular proteins for cisplatin [START_REF] Yang | Cisplatin preferentially binds mitochondrial DNA and voltage-dependent anion channel protein in the mitochondrial membrane of head and neck squamous cell carcinoma: possible role in apoptosis[END_REF]. Convenient to this hypothesis is the fact that one of the main interaction sites in cells for doxorubicin is the mitochondrial membrane, including VDACs [START_REF] Tokarska-Schlattner | Alterations in myocardial energy metabolism induced by the anti-cancer drug doxorubicin[END_REF]. The finding that there was no effect of DCA to temozolomide induced cell death adds further support to this hypothesis. To date, temozolomide is not known to interact with the mitochondria or the mitochondrial membrane. Further investigations on these interactions could help to decipher drug interactions between DCA and anticancer drugs, which in turn will help to develop optimal strategies for targeting the Warburg effect.

The Warburg effect is an interesting selective target for the treatment of cancer, however, our studies underline that targeting the Warburg effect for cancer treatment is far more complex than anticipated and more pre-clinical and clinical work is absolutely necessary to exclude any disadvantage for cancer patients.

Moreover, our observations strongly argue against compassionate use of DCA by cancer patients themselves without medical supervision.

Legends:

  of the yellow 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma, Deisenhofen, Germany) to a purple formazan dye by mitochondrial aldehyde dehydrogenases, CellTiterGlo™ identifies viable cells via their ATP content. The formation of the purple formazan dye correlates with the amount of viable cells and was quantified spectrophotometrically at a wavelength of 560 nm with an Ascent Multiscan® microplate reader (Thermo Fisher Scientific, Langenselbold, Germany). The ATP released from viable cells after lysis was detected via a luminescent dye, which was quantified with the Fluoroskan Ascent FL (Thermo Fisher Scientific, Langenselbold, Germany).Cells were exposed to serial drug dilutions as indicated in the text. The measured absorptions/ luminescence were used to calculate the rate of cell viability compared to untreated controls. In each experiment each concentration was tested in quadruplets and all experiments were repeated at least three times.Assessment of apoptosis by phosphatidylserine externalisation and mCells were seeded at 2 x 10 5 cells per well into six-well plates and left overnight to adhere and resume exponential cell growth. Then DCA was added and cells were incubated for another 24h to 72h. At the indicated time points, cells were harvested by trypsinization and resuspended in RPMI 1640. Alexa Fluor™ 488 Annexin V and MitoTracker™ Red were determined using the Vybrant™ Apoptosis Assay Kit #11 (Molecular Probes™). m was analysed by use of the MitoProbe™ DiIC 1 (5) Assay Kit for Flow Cytometry (Molecular Probes™). Fluorescence-activated cells were analysed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) and data were analyzed using the CellQuest software (Becton Dickinson).

  the medulloblastoma cell lines DAOY and UW228.2, the rhabdomyosarcoma cell lines RD and RH-30, and the Ewing sarcoma cell line CADO-ES-1 DCA concentrations of 10 mmol/L attenuated cell viability by 30% to 50% compared to untreated controls. The DCA effects on cell viability were not related to the rate of cell growth during the time of drug exposure as estimated from the increase of cell viability of untreated controls. Since stimulation of the PDH and the Krebs cycle by PDK inhibition through DCA might at least in part counteract measurements of cell viability, which are based on the activity of mitochondrial aldehyde dehydrogenases and cellular ATP content, we also analysed induction of apoptosis by annexin V staining with comparable results. Just like in the cytotoxicity screens, DCA concentrations of 10 mmol/L only sparsely increased the rate of apoptotic cells. Increasing the dose to 50 mmol/L DCA raised the rate of apoptotic cells up to 80% (Fig 2). In accordance with the report of Bonnet et al., this effect was selective for the tumour cell lines, as DCA concentrations of 50 mmol/L failed to induce significant levels of phosphatidylserine externalisation in the non-malignant human embryonic kidney 293 cells (Fig 2). In addition, DCA reduced m in a dose dependent manner. However, again exposure to DCA concentrations of 10 mmol/L for 72h only reduced m in a small portion of cells. About 50% of cells were affected after an exposure to 25 mmol/L DCA for 72h and m of all cells was decreased after exposure to 50 mmol/L DCA for 72h (Fig 3 a-c). At DCA concentrations of 50 mmol/L, a reduction of m was measurable already within 5 min in a small portion of cells. The rate of cells with reduced m increased after 24h and after 48h m of the whole cell population was reduced (Fig 3d-f). The reduction of m facilitates the release of pro apoptotic factors like cytochrome c and AIF, which in turn should facilitate apoptosis induced by standard anticancer drugs like cisplatin or doxorubicin. However, when combined with DCA, the cytotoxicity of cisplatin was reduced or even abolished in 7 out of 10 cell lines (Fig 4). The efficacy of cisplatin is based on the formation of an aquo-complex, which is responsible for the platination of DNA and proteins by nucleophiles. In order to exclude alleviation of cisplatin toxicity simply by chemical artefacts due to the high DCA concentrations used, we determined cisplatin concentrations in cell culture medium in the presence and the absence of DCA and found no differences between incubations with and without DCA (Fig 5a). In addition, the cellular uptake of platinum was not affected by DCA (Fig 5b). To determine whether DCA also alleviated the toxicity of other standard anticancer drugs, the cytotoxicity of doxorubicin and temozolomide alone and combined with DCA was analysed on the leukaemia cell lines MOLT-4 and CCRF-CEM. In these cell lines, DCA completely abolished the cytotoxicity of doxorubicin comparable to the DCA effect on cisplatin toxicity (Fig 6a). However, the cytotoxicity of temozolomide was neither reduced nor increased by combination with DCA (Fig 6b). In addition, DCA suppressed caspase 3/7 activation induced by cisplatin (Fig 6c) and doxorubicin (Fig 6d). Interestingly, despite induction of phosphatidylserine externalisation as an early event in apoptosis and despite of reduction of m, DCA treatment alone failed to activate caspase 3/7.

  Bonnet et al. observed a reduction of tumour size by DCA treatment in non-smallcell lung cancer xenografts but no complete remissions[START_REF] Bonnet | A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth[END_REF], which, in addition, argues for the combination of DCA with other anticancer drugs. Dhar and Lippard addressed this issue and designed a new platinum drug, mitaplatin, which consists of one molecule cisplatin with two molecules DCA[START_REF] Dhar | Mitaplatin, a potent fusion of cisplatin and the orphan drug dichloroacetate[END_REF]. Within the cell mitaplatin is reduced and cisplatin and DCA are released. Mitaplatin was toxic against a panel of five mainly adult tumour cell lines, but failed to increase the cytotoxicity of cisplatin. On the contrary against some cell lines (U2OS or HeLa) the cytotoxicity of mitaplatin was decreased compared to cisplatin alone. We tested DCA and cisplatin at a molar ration of 1:100. This excess of DCA might have obliterated the toxicity of cisplatin due to chemical artefacts, however neither intracellular platinum concentrations nor extracellular cisplatin concentrations were influenced by these high DCA concentrations. In addition, the cytotoxicity of doxorubicin, which in contrast to cisplatin exerts its toxicity against tumour cells by inhibition of topoisomerase II and not by DNA modifications, was also attenuated by DCA. The toxicity of temozolomide, another DNA modifying agent, however, was not compromised by DCA at the drug concentrations tested. Standard anticancer drugs, like cisplatin, doxorubicin, and temozolomide exert their effects mainly on proliferating cells and concomitant inhibition of proliferation reduces the cytotoxicity of standard anticancer drugs. DCA was reported to inhibit cell proliferation[START_REF] Bonnet | A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth[END_REF][START_REF] Wong | Dichloroacetate induces apoptosis in endometrial cancer cells[END_REF][START_REF] Cao | Dichloroacetate (DCA) sensitizes both wild-type and over expressing Bcl-2 prostate cancer cells in vitro to radiation[END_REF][START_REF] Sun | Reversal of the glycolytic phenotype by dichloroacetate inhibits metastatic breast cancer cell growth in vitro and in vivo[END_REF][START_REF] Chen | Oxygen consumption can regulate the growth of tumors, a new perspective on the Warburg effect[END_REF]. The reduction of cell viability, which we observed in 5 out 18 cell lines, might well be attributed to reduced cell proliferation. However, DCA reduced the cytotoxicity of cisplatin in DCA-sensitive as well as DCA-resistant cell lines suggesting that inhibition of proliferation by DCA was not responsible for the observed reduction of cisplatin toxicity by DCA. Apart from DNA, cisplatin and doxorubicin both impair mitochondrial function. One of the DCA-cisplatin and DCA-doxorubicin interaction sites could be the mitochondrial voltage-dependent anion channel

Figure 1 :)

 1 Figure 1: Cell viability of 18 paediatric tumour cell lines from six different tumour types (Ewing sarcoma (EWS), medulloblastoma (MB), Leukaemia, Osteosarcoma (OS), Neuroblastoma (NB), and Rhabdomyosarcoma (RMS) after exposure to 0.1 mmol/L (

Figure 2 :

 2 Figure 2: Annexin V-and Mitotracker™ -staining of CCRF-CEM and HEK293 cells after exposure to DCA concentrations of 10 mmol/L and 50 mmol/L for 48h. Cells were seeded at 2 x 10 5 cells per well into six-well plates and left overnight before DCA was added.

Figure 3 :

 3 Figure 3: Dose-and time-dependent reduction of m. CCRF-CEM cells were exposed to 10 mmol/L DCA (a), 25 mmol/L DCA (b), and 50 mmol/L DCA (c) for 72h and to 50 mmol/L DCA for 5 min (d), 24h (e), and 48h (f). Cells were seeded at 2 x 10 5 cells per well into six-well plates and left overnight before DCA was added. The grey line represents the m of untreated controls and the black line the m of DCA-treated cells.

Figure 4 :Figure 5 :

 45 Figure 4: Cell viability of paediatric tumour cell lines exposed to DCA, cisplatin, and a combination of both for 72h. Cisplatin and DCA were combined in a constant ratio of 1:100 ( 1 mmol/L DCA, 10 µmol/L Cisplatin, 1 mmol/L DCA + 10 µmol/L Cisplatin; 10 mmol/L DCA, 100 µmol/L Cisplatin, 10 mmol/L DCA + 100 µmol/L Cisplatin) Figure 5: To exclude chemical artefacts for the alleviation of cisplatin toxicity cisplatin concentrations were determined in cell culture medium after 4h and 24h in the presence and absence of DCA. CCRF-CEM cells were incubated with 100 µmol/L cisplatin alone ( ) or in along with 10 mmol/L DCA ( ).

Figure 6 :

 6 Figure 6: a. Cell viability of CCRF-CEM and MOLT-4 cells exposed for 72h to 1 mmol/L DCA ( ), 10 µmol/L doxorubicin ( ) or a combination of both ( ). b. Cell viability of CCRF-CEM and MOLT-4 cells exposed for 72h to 10 mmol/L DCA ( ), 100 µmol/L temozolomide ( ) or a combination of both ( ). The solid lines represent the viability of untreated controls. c and d. Caspase 3 activation in CCRF-CEM cells exposed to increasing DCA concentrations either alone ( ) or combined ( ) with 100 µmol/L cisplatin or 10 µmol/L doxorubicin. Caspase 3 activation was analysed after 24h. Cells (5 × 10 4 cells/well) were seeded and treated with DCA, cisplatin, and doxorubicin as indicated. Caspase activation was analysed 24h after drug exposure.

Acknowledgements:

This work was supported by the Karl Bröcker Stiftung Weseke.