Peter Rickhaus
email: peter.rickhaus@unibas.ch

Ming-Hao Liu

Péter Makk

Romain Maurand

Samuel Hess

Simon Zihlmann

Markus Weiss

Klaus Richter

Christian Schönenberger

Guiding of Electrons in a Few Mode Ballistic Graphene Channel

In graphene, the extremely fast charge carriers can be controlled by electron-optical elements, such as waveguides, in which the transmissivity is tuned by the wavelength. In this work charge carriers are guided in a suspended ballistic few-mode graphene channel, defined by electrostatic gating. By depleting the channel, a reduction of mode number and steps in the conductance are observed, until the channel is completely emptied. The measurements are supported by tight-binding transport calculations including the full electrostatics of the sample.

The wavelength of electrons at the Fermi energy in graphene [1] can be tuned locally by changing the carrier density n with gate-electrodes [2,3]. By forming regions of different wavelengths λ = 4π/|n|, elements known from optics such as lenses [4], filters [5] or Fabry-Pérot interferometers [6][7][8] can be mimicked. The reflection, refraction and transmission behavior of graphene's massless charge carriers in a spatially varying potential is analogous to the propagation of an electromagnetic wave in media with varying refractive index. An assembly of such media is used to guide photons in optical fibers (OFs). These consist of materials that are assembled in a way that the refractive index in the light-carrying core is larger than the refractive index in the coating. Similar conditions can be achieved in graphene using local gates. The possibility to form electronic waveguides in graphene has attracted a lot of theoretical interest. [9][10][11][12][13][14][15][16] However, up to now experiments were performed only in diffusive samples [17], making the comparison to optics difficult. In addition, a graphene waveguide is expected to show a distinct behavior once a p-n interface is involved. At a p-n interface the polarity of charge carriers is inverting. Due to the additional confinement of such an interface it is possible to transport one or few modes in a waveguide. In optics, single mode fibers are used for long distant communication since they are not limited by modal dispersion. In a similar way single or few-mode electron-optic fibers can be beneficial for quantum information communication.

So far, confinement of charge carriers has been mainly achieved with hard-wall potentials, as provided by the edges in graphene-nanoribbons [18][19][20] or the induced gap in bilayer graphene [21,22]. Whereas the former suffers from irregular edges, the latter is performed in gapped bilayer graphene which does not host relativistic Dirac particles. Even though electrons were successfully guided in the bulk of single-layer graphene by making use of snake states [23][24][25] their transport properties is significantly altered by the involved perpendicular magnetic field. It is a different and very challenging task to confine the charge carriers in an electrostatic waveguide [14,16]. In this letter we report on the formation of a narrow tunable ballistic electrostatic channel in graphene, that can be operated as an optical fiber and features controllable single mode filling.

In single layer graphene, the local Fermi energy E = hv F |n|π, with v F ≈ 10 6 ms -1 the Fermi velocity in graphene, takes the role of the refractive index. By changing this energy using local gates, a waveguide can be formed. This guiding principle and a possible geometry is shown in Figure 1a. The refraction at an interface is described by Snell's law in graphene: The small value for k x will lead to leakage as sketched in e. f, By increasing the Fermi-energy E in the second mode becomes available and k x of the first mode is increased. The first mode can be guided in the channel, as illustrated in g. h, Due to this mechanism, the expected conductance plateaus will be smoothened and shifted with respect to M compared to the situation of a hard-wall channel (dashed line).

E in • sin(θ in ) = E out • sin(θ out
They are still expected to occur at values of 4 e 2 /h.

In addition to this OF guiding, charge carriers in graphene can also propagate in a channel that has interfaces at which the polarity of the charge carrier is inverted. Such a channel can be formed by tuning the channel region to electron-like (n) and the outer region to hole-like (p) doping, or vice versa as indicated by the hatched quadrants in Figure 1b [17]. Since at a p-n interface the density is zero, it is naturally reflective. If the transition from p-to n-doping is gradual, the low-density region is increased in size leading to an even more reflective interface. In other words, a smooth p-n interface can guide electrons more efficiently than a sharp one. Losses out of a p-n channel are in both cases mostly caused by trajectories perpendicular to the interface that are transmitted with probability one (Klein tunneling) [5,26,27] as sketched in Figure 1c.

At low densities n in , the wavelength in the channel is such that λ/2 becomes larger than the channel width W . This situation of a depleted channel is sketched in the dark blue region in Figure 1b. By increasing n in , the local Fermi-energy E in increases such that the first mode M = 1 in the channel can be populated, as shown in the sketch of the band-diagram in Figure 1d. Yet, due to the low value of the wavevector k x in propagation direction x, the angle of incidence at the p-n interface is close to perpendicular and the mode will leak out of the channel (Figure 1e) and cannot be observed in transport along the channel. Nevertheless, by increasing E in , k x increases and the angle of incidence towards the p-n interface becomes larger. A corresponding band-diagram is sketched in Figure 1f, where already the second mode (M = 2) is available. In this situation, the first mode can be guided and the second mode leaks out of the channel since the Fermi-energy crosses this band at low k x . Compared to a channel where the outer regions are forbidden for electrons, the expected conductance plateaus for transport along the channel will be smoothened due to the above discussed mechanism. We still expect conductance steps at 4e 2 /h, as sketched in Figure 1h. A more detailed analysis is given in the supplementary material.

We realized an electrostatic channel in a four-terminal suspended graphene device. A 3dimensional design is shown in Figure 2a. By applying a voltage on the backgate (blue) and bottomgate (gold), the density can be tuned locally to guide electrons between the source (S) and collector (C) contact. The guiding losses can be measured at the side drain contacts (D1, D2). A scanning electron micrograph of the measured device is given in Figure 2b. The graphene flake is roughly 4 × 2 µm 2 in size. The fabrication follows the recipe of Refs. 28, 29. Graphene is transferred onto a sacrificial polymer layer (LOR) using a dry-transfer technique, and after defining the contacts, suspended. It is cleaned by in-situ current annealing between the D1 and D2 contact.

As a first characterization the conductance G D1D2 between D1 and D2 is measured while the S and C contacts are floating. We correct for the capacitive cross talk between backgate and bottomgate voltage and plot the data as a function of channel densities n in and n out . The conversion from voltage to density is given by the gating efficiency which we extracted from the electrostatic simulation (for details, see supplementary material). Throughout this paper negative densities correspond to holes, positive to electron charge carriers. In the colorscale-map of Figure 3a, the conductance drops drastically once a p-n junction is formed (i.e. the blue regions). Figure 3b shows the field effect along uniform doping which reveals a pronounced Dirac point. The very sharp transition from the unipolar to the bipolar region occurs within n in ≈ 10 9 cm -2 . This demonstrates the very high quality of the graphene. In addition, the regular Fabry-Pérot pattern (marked with arrows and sketched in the inset of Figure 3a) indicates ballistic transport in the channel [7,8].

We now discuss the guiding efficiency measurement (fig. 3c). An AC voltage is applied to the S contact and current is measured at C, D1 and D2 using an IV-converter and a Lock-In detector on each terminal. The guiding efficiency γ = I SC /I tot is then given by the current I SC measured at C divided by the total current I tot = I SD1 + I SD2 + I SC . It is important to notice that γ includes the injection efficiency into the channel which is limiting the overall maximum efficiency to 42% in this device. In this complex geometry it is not possible to discriminate the losses of the channel itself from losses due to poor coupling of the charge carriers into the channel. The colorscale map γ(n in , n out ) shows high guiding efficiency in the expected regions: A large γ is observed in the OF triangles, and the efficiency is even increased once a p-n-junction is formed. This is best seen by taking a cut at n in = 6 • 10 10 cm -2 shown in Figure 3d and indicated by a red arrow in Figure 3c. Charge carriers cannot be guided for n out > n in and for uniform doping (n out = n in ) roughly 29% of the charge carriers reach the C contact. In the OF regime, the efficiency increases drastically and reaches its maximum if the outer region is depleted, i.e. n out ≈ 0. The formation of a p-n-interface leads to an increased γ compared to the regime where only OF guiding is present.

Even though OF and p-n guiding mechanisms have been discussed for a diffusive sample in Ref.

[17] here we demonstrate their occurrence in the expected regions in a density-density map of a ballistic sample.

The lowest efficiency in the colorscale map (fig. 3c) is observed in the empty-channel region (dark blue). A step-wise increase of γ starting from the depleted channel towards larger n in is seen in the colorscale map. By taking cuts around n out = 6 • 10 10 cm -2 plateaus corresponding to a change in mode number M become apparent (Figure 3e). For this cut we averaged the curves between n out = 5...7 • 10 10 cm -2 to wash out features that are changing with n out . More details on these features are given later in the manuscript. The plateaus are also present at the opposite polarity, i.e. an n-doped channel and a p-doped outer region. They can be seen for example in cuts around n out = -5 • 10 10 cm -2 as given in Figure 3f.

For a comprehensive understanding, we compare the experiment to transport calculations based on an ideal electrostatic model obtained by finite-element simulations following the gating and graphene flake geometry of our device. Using the scalable tight-binding model for graphene [30], the full 4 × 2 µm 2 flake with realistic on-site energy profiles V (x, y) from the electrostatics can be considered and the main features of the guiding efficiency map can be reproduced by applying the real-space Green's function method to compute the conductance. The results of the simulations are shown in Figure 4a. The main features of the measurement are well captured: There is a high guiding efficiency in the OF triangles which is even increased in the p-n-p-regime. We see a very low efficiency at low n in where the channel is empty.

The emerging modes confined in the channel are observed along lines indicated by gray dashed arrows in Figure 4a. Additional resonances (indicated by black arrows) in the p-n-p regions appear and will be referred to as two-cavity resonances. They are related to states spatially extending over both the inner channel and the cavity regions confined by D1 and D2. They are therefore influenced by both n in and n out and are roughly parallel to the anti-diagonal in the density map.

Further analysis is given in the supplementary material.

From the simulations we further extract the space-resolved local current density J = J x e x + J y e y in the different guiding situations by applying a small DC voltage difference between the S and C contact with D1 and D2 grounded. In Figure 4b we plot J(x, y) = [J 2

x (x, y) + J 2 y (x, y)] right contact. The loss (i.e. current towards D1 or D2) is given by trajectories injected at S that reach the interface close to normal incidence. For the given densities we obtain θ c = 52 • and we sketch a corresponding dashed line in the Figure . By the formation of a p-n-interface this loss is reduced drastically as shown in Figure 4c. Outside the channel, the current is almost completely suppressed. Only close to the injector contact a loss current is observed, corresponding to trajectories aligned perpendicular to the channel border. For smooth p-n-junctions, this is exactly what is expected [27] due to strong Klein collimation. Inside the p-n-channel, a complex interference pattern forms. By reducing the density in the channel only the first mode is filled (Figure 4d) and as a consequence the complex interference pattern of Figure 4c disappears. Even though the channel appears to be lossless, the injection into the single-mode channel is rather inefficient, i.e.

there is a number of modes outside the channel that can be populated. For this reason the current density in the outer cavity does not disappear completely. By further reducing n in , the channel can be emptied completely, as it becomes apparent in Figure 4e.

We now show in figure 5a-c the conductance between source and collector in the interesting region of biploar doping where the channel is close to full depletion and plateaus in the conductance can be expected due to quantized filling. In Figure 5a e 2 /h here. Compared to the two-cavity resonances they do not disappear if the side contacts (D1 and D2) are reflective, as we reveal in the supporting material, where we also show steps in the conductance for a second device. The number of modes M in the middle of the channel (x = 0) can be read off from the number of peaks in J(x = 0, y) in Figures 5d-g, where the potential landscape V (x = 0, y) from the electrostatics is also shown. On top the J(x, y) profiles for

x close to 0 are given. The positions where the profiles were imaged are labeled with stars in Figure 5c, and their shape is representative for the whole respective conductance range marked by colored lines in Figure 5c. The V (x = 0, y) profiles reveal that the density is gradually changing from n outside to p inside of the channel. The smoothness in the potential landscape is due to the large distance of ≈ 400 nm between the graphene and gate electrodes. The channel formed by the potential in Figure 5d can transport only one mode, but since k x is small and since the channel is very leaky for small angles as explained in Figure 1d-e., only a vanishing current is observed in the middle. By increasing the voltage on the inner gate, the minimum in the potential corresponding local current density J(x = 0, y). Top panel: J(x, y) for x close to 0. For the given density profile the channel could carry one single mode M = 1, which however is leaking out drastically such that almost no current is transported. The plots d-g are taken at the density marked by a star in c but are representative for the respective conductance regimes marked by colors in the outer two curves in c. e, As the channel gets deeper and wider, the first mode can be transported in the channel. f,g, The second and third mode appear when |n in | increases further. h,i, A model explaining the occurrence of the conductance plateaus at 2.2, 2.6 and 2.9e 2 /h in a. Regions that are doped by absorbates are colored in light blue. The channel resistance R ch is then not only given by 1/(N • 4e 2 /h) but modified by a transmission probability t into and out of the channel. Additionally, current can flow from S to C through the outer cavities, which is modeled by a parallel resistor R p . The measured resistance between S and C is then given by R = (1/(t 2 • N • 4e 2 /h) + R p ) -1 . The loss resistance from S to D, R l , is modified by a contact resistance in series R c . If we calculate G SC = 1/R ch at n out = 5 • 10 10 cm -2 for R p = 13.4kΩ and t = 0.32 the plateaus occur at 0, 4, 8 and 12e 2 /h. profile V (x = 0, y) decreases and the channel grows wider such that electrons gain momentum in x-direction and the first mode can be transported along the waveguide (Figure 5e). By further increasing the inner gate voltage, the second mode, M = 2 (Figure 5f), and third mode, M = 3 (Figure 5g), also become available for charge transport along the channel.

Having confirmed the origin of the non-dispersing features in the experiment we now compare the conductance values. In the experiment, the plateaus do not occur at G SC = N • 4e 2 /h (with N = 0, 1, 2, ...) but rather at G SC = 1.9, 2.2, 2.6 and 2.9e 2 /h in Figure 5a. A simple model, depicted in Figure 5h-i, explains this deviation by taking into account non-uniform cleanliness of the graphene. In Figure 5h regions that presumably are doped by absorbents on the surface are shaded with light-blue color. Such a distribution of dopants is likely to occur after current annealing since the contacts act as heat sinks and therefore attract residual absorbents when the graphene is hot [31]. In a simple resistor network (Figure 5i) we therefore model an additional resistor R p , due to trajectoires from S to C that do not penetrate the channel, in parallel to the channel resistance R ch . In addition the injection into the channel and the detection at the collector will be modified by a transmission probability t which is the transmission from the contact into and out of the channel. Therefore, R ch = 1/(t 2 • N • 4e 2 /h) and the measured resistance between S and C will be R = (1/R ch + 1/R p ) -1 = 1/G SC . For the G SC (n in ) curve at n out = 5 • 10 10 cm -2 plateaus at G SC = 0, 4, 8 and 12e 2 /h are obtained for R p = 13.4kΩ and t = 0.32. By subtracting a contact resistance R c = 2.9kΩ from the loss conductance G SD1D2 , we find guiding efficiencies γ = G SC /(G SC + G SD1D2 ) of 26%, 41% and 51% for the first, second and third plateau. R c is extracted for high, unipolar densities in the G SD1D2 map (shown in the supporting material). The values compare well to the simulation (Figure 4a) where 28%, 39% and 46% are obtained.

Apparently the main limitation in the experiment is the injection and detection efficiency, which is parametrized in t. It is with t = 0.32 a factor of 3 smaller than in the simulation, where t = 0.96 is found by applying the same resistor model. Improvements would focus on creating excellent contact to the graphene. This on one hand, increase t both at the source and collector contact. On the other hand, a reduction of R p could be expected since the D1, D2 contacts would be less reflective, i.e. trajectories from S that get reflected at D1 and enter C could be suppressed.

A strategy to increase t by shaping the graphene is discussed in the supplementary material.

In the simulation, however, t is large meaning that the ideal channel very efficiently transports modes once k x is sufficiently large. This efficient guiding is only possible due to the smooth p-n interface in our device [14] which is very reflective for larger angles of incidence θ. For comparison, a sharp p-n interface transmits trajectories with θ = 45 • with 50% probability [27] such that the mode sketched in Figure 1g could not be guided. For our smooth interface, on the other hand, 50% transmission is obtained for only θ ≈ 20 • , and at 45 • the transmission is close to zero. These values are taken for (n in , n out ) = (5, -10) • 10 10 cm -2 . Details are given in the supplementary material.

In conclusion, we have realized electrostatically defined electron waveguides in ballistic graphene with an aspect ratio >10 using a global and a channel gate. Typical channel parameters are ≈ 300 nm in width and 2.7 µm in length. The wavelengths of the charge carriers inand outside of the channel can be tuned by the gates independently. Using this tunability, we can distinguish and control the regions of optical fiber-and p-n guiding. We observe an increased guiding efficiency if the two mechanisms coexist. Using p-n guiding, clear steps in the channel conductance appear whenever the mode number changes by one. All experimental results are supported by self-consistent theoretical simulations which also reveal that a smooth p-n interface and a sufficiently large momentum k x along the channel is required to transport single modes.

The simulation provides also a powerful guide for future device improvements. It shows, for example, that by using an even smoother p-n interface to confine the electrons in the channel, modes could be guided for k x k y . In current devices the coupling from the injector contact to the channel and similarly from the channel to the detector contact is far from ideal. The injector and collector efficiencies of currently ≈ 35% can be increased either by using a p-n interface as a collimator or by etching the graphene flake. With such improvements guiding efficiencies > 80%, as demonstrated by the theory, are in reach. This then allows to explore one-dimensional transport in graphene without the need to etch nanoribbons. The difficulty of the required precise control over the sample edges at the atomic level in nanoribbons can then be circumvented. An electrostatically confined one-dimensional channel within the bulk of graphene could even be oriented intentionally into any preferred crystallographic direction allowing to study the symmetry of the one-dimensional bands. A high degree of confinement can further be used for fast switches with a potential visibility in excess of 80%. Supporting Figure 2. Original data and density mapping. a-b, 3D and top-view of the measured device with its dimensions used also for the simulations. c, The measured guiding conductance G SC between left and right contact, d, and the simultaneously measured loss G SD1D2 between S and D1 and D2 contact. e, The guiding efficiency γ is calculated from these two maps as described in the main text, i.e. γ = G SC /(G SC + G SD1D2 ). f, The Fabry-Pérot map is measured in a different configuration. g, The simulated guiding efficiency and the simulated Fabry-Pérot pattern h show the same crosstalk between the inner and outer gate. The voltage scale for the outer voltage V out differs from the experimental data, but not the V in scale. This is due to a problem with the backgate, i.e. the experimental V out scale is not trustworthy. the conductance G SD1D2 = (I D1 + I D2 )/V S between the source and side drain contacts. The guiding efficiency in Supporting Figure 2e is then given by γ = I SC /(I SD1 + I SD2 + I SC ) =

G SC /(G SC + G SD1D2 ).
Apparently the V out scale for γ and for the Fabry-Pérot map G D1D2 (Supporting Figure 2f) is shifted to high voltages. Whereas the global Dirac point is very close to V in = 0V, it occurs at roughly V out = 30V for the outer gate. The small shift in V in and the regular Fabry-Pérot pattern clearly demonstrate that the shift in V out is not due to doping of the graphene but rather due to experimental problems with the backgate, that also lead to gate-jumps in the Fabry-Pérot map (Supporting Figure 2f). The backgate was connected with a silver-paint and this connection might have become bad at low temperatures, leading to charging of the gates and to instabilities. We therefore discard the V out -scale since the values are apparently not trustworthy, while keeping the V in -scale.

By comparing to the simulated guiding efficiency (Supporting Figure 2g) and Fabry-Pérot map (Supporting Figure 2h) the correct density values for the experimental data can be extracted. This is possible since there are no free fitting parameters in the model and all geometrical parameters which do influence the capacitive crosstalk between the gates are fixed. In addition, the V in voltage scale can be compared. Both experimental and theoretical data are mapped from (V in , V out ) to (n in , n out ) by using the local density profiles n(x = 0, y) (Supporting Figure 2i). The maximal absolute density in the channel n in and of the maximal absolute density in the outer region n out are obtained for all voltages (V in , V out ). Such a mapping is done for the theoretical Fabry-Pérot map in Supporting Figure 2j which is not shown in the main text. The data are sheared since the capacitive crosstalk between backgate and bottomgate is corrected.

The spacing of the Fabry-Pérot resonances represents a good control for the voltage to density mapping. Since the potential is smooth and the cavity size is changing with applied gate voltage, the experimental Fabry-Pérot resonances are best compared to the oscillations obtained in the tight-binding simulation, as it is done in Supporting Figure 2k for the density in the channel and in Supporting Figure 2l for n out . As the periodicity of the measured and calculated oscillation is matching rather well, we conclude that the density is properly mapped. The density of two resonance maxima n i and n i+1 are spaced √ n i+1 -√ n i = √ π/W , where W is the width of the channel. This can be obtained using the interference condition k F W = N π with N an integer. From the two first maxima in Supporting Figure 2k (experimental or theoretical) a cavity size of 730 nm is obtained, which corresponds well to the cavity size obtained from electrostatic simulations (see Supporting Figure 2i). To highlight the contrast between the smooth p-n junction of our suspended graphene and the widely discussed abrupt junction, we show in Supporting Figure 3 the angle-resolved transmission T (φ) across a p-n junction, considering the realistic smooth potential V (y) obtained from the electrostatic simulation, as well as the abrupt case for comparison. The red curves correspond to a smooth potential step, the gray one to a sharp p-n interface. The T (φ) curves shown in Supporting Figure 3a and 3c consider symmetric and asymmetric pn junctions, respectively, and are calculated in the same way as ref. 32. In the symmetric case of Supporting Figure 3a,b with n in = -n out = 10 11 cm -2 , the transmission curve for the abrupt case corresponds to the wellknown expression [27], T (φ) = cos 2 (φ), so that T drops to one half at ±45 • , while for the smooth In Figure 1f in the main text we sketched a very much simplified band structure for the modes in order to illustrate the leakage of the guide modes with short k x . A more evolved picture is presented in Supporting Figure 5a-c, where we show band structures E(k x ) calculated by taking a unit cell laterally cut (along y) from the simulated graphene lattice at around x = 0. The flake is oriented such that the armchair edge is parallel to the p-n interface. The band structures are given for different densities n in , n out and the corresponding position is labeled in the guiding efficiency map, shown in Supporting Figure 5d. We observe zero guided mode in transport along the channel in Supporting Figure 5a, one mode in b, and two in c. The band structures consist of mainly two Dirac-like-cones. The one for hole-like transport at the Fermi energy corresponds to the outer cavities. Since n out is changing only little from Supporting Figure 5a-c, this part of the band-structure remains unchanged. Due to the rather high density, there is a large number of modes available. More interesting are the modes that appear in the inner cavity, corresponding picture shown in Figure 1f and Supporting Figure 5e by neglecting the fine-structure at low k x . We would expect to observe two modes in transport along the channel for Supporting Figure 5b due to the orange and the blue branch. However, by calculating k y = k 2 -k 2

Smooth versus sharp p-n interface

x for k = √ n in π = 0.022 nm -1 we obtain an angle of incidence of 60 • for the blue and only 25 • for the orange mode.

Comparing to the numbers given in Supporting Figure 3a and c for transmission at a smooth p-n interface it becomes apparent why in transport along the channel only one of the modes is seen:

the smaller angle of incidence for the second (orange) mode leads to leakage out of the channel, whereas the first (blue) mode can be guided efficiently.

FIG. 1 .

 1 FIG. 1. Guiding mechanisms related to densities in n in and outside n out of an electrostatic channel. a, Possible design of a guiding channel where the gray areas are electrical contacts. If the electron density inside a channel is larger than outside n in > n out , electron trajectories reaching the interface under an angle θ in larger than the angle of total internal reflection θ c stay in the channel, similar to an optical fiber (OF). b, The different guiding mechanisms are sketched depending on n in and n out . In the red shaded triangles OF guiding is present. The shading visualized θ c (n in , n out ). The hatched quadrants indicate the regions of pnp-guiding and the blue steps correspond to different mode number M in the channel. c, The formation of a p-n junction helps to keep charge carriers in the channel; loss is due to trajectories with (almost) zero angle of incidence. d, Sketch of a band diagram in the channel for the situation, when the first mode (M = 1) is populated. The small value for k x will lead to leakage as sketched in e. f, By increasing the Fermi-energy

FIG. 2 .

 2 FIG. 2. Realization of a guiding channel in a suspended graphene device. a, 3-dimensional design of the device. Tuning the density in the channel (n in ) with the bottomgate (gold) and the density outside of the channel (n out ) with the backgate (blue), the electrons can be guided between the source (S) and the collector (C) contacts. The losses are recorded at side drain contacts D1 and D2. b, False-colored SEM image of the measured device; scale-bar: 1 µm.

FIG. 3 .

 3 FIG. 3. Experimental results: Fabry-Pérot interferences across the channel and guiding efficiency along the channel. a, Two-terminal conductance G D1D2 between D1 and D2 as a function of the densities n in and n out (S and C are floating). The resonances marked with black arrows are due to Fabry-Pérot interferences in the channel as sketched in the inset. b, Field-effect as a function of uniform doping (cut along white arrow in a). c, Guiding efficiency I SC /I tot as a function of n in and n out . Here, an AC voltage is applied to the S contact and current is measured simultaneously at C and D1, D2. d, Cut in c at fixed n in = 6 • 10 10 cm -2 . The guiding efficiency increases drastically in the OF regime and is highest when pn and OF guiding coexist. e, The cut in c around n out = 6 • 10 10 cm -2 reveals steps in the guiding efficiency that are due to mode filling of the channel. Shown is an averaged curve of n out = 5...7 • 10 10 cm -2 .The steps are seen in the 2D plot in c and are marked there with gray vertical arrows. f, A similar cut at n out = -4... -6 • 10 10 cm -2 reveals steps also for inverted polarity.

+FIG. 4 .

 4 FIG. 4. Simulated guiding efficiency and local current density profiles based on tight-binding model. a, Calculated guiding efficiency following the same definition as the experiment, γ = I SC /I tot , using 3dimensional electrostatics given by the geometry of the device. Sequential mode filling of the channel is indicated by gray dashed lines. Black arrows mark a resonance pattern involving the inner cavity formed by the narrow channel and an outer cavity given by one outer contact D1,D2 and the channel. b, Local current density distribution for a small applied voltage on the source contact S. The densities are tuned to the optical fiber regime. The red dashed line shows the angle of total internal reflection. c, In the p-n-regime, the electrons are kept inside the channel very efficiently and complex resonance patterns are observed. Close to the S contact current is lost towards D1 and D2 due to Klein tunneling. d, Local current distribution for the first mode. e, At vanishing n in , the channel can be emptied, i.e., the local current density is zero inside the channel.

  and b several curves G SC (n in ) are given for values of n out = 5...7 • 10 10 cm -2 . They are offset from the red curve by multiples of n in = 0.4 • 10 10 cm -2 . The plateaus due to mode-filling are indicated by dashed lines and we mark the additional two-cavity resonances by the black arrows. A similar structure is observed for opposite polarity (Figure 5b). Although Fabry-Pérot resonances and mode filling occur on similar length and energy scales they can be distinguished by their different gate behaviour. The observed pattern is reproduced clearly in the tight binding simulation for G SC (n in ) shown in Figure 5c. Again the flat plateaus are marked with dashed lines and the dispersing resonances are indicated by arrows. Comparing to Figure 4a we confirm that the resonances indicated by the black arrows correspond to the above mentioned two-cavity resonances whereas the plateaus indicated by horizontal dashed lines are due to an increase of the channel mode-number. These plateaus occur at roughly 4, 8 and 12

FIG. 5 .

 5 FIG. 5. Discrete change of the number of modes in experimental and theoretical conductance data. a, Steps corresponding to mode-filling are seen in the experimental G SC (n in ) data at fixed n out = 5...7 • 10 10 cm -2 . The curves are offset from the red line by n in = 0.4 • 10 10 cm -2 . Mode filling (see Figure 1h) occurs along the dashed lines and an additional two-cavity resonance pattern is indicated by the black arrows. b, A comparable pattern, yet less clear, is observed for inverted polarity. c, The calculated G SC (n in ) curves reveal the plateaus, indicated by black dashed lines, and the two-cavity resonance pattern (black arrows). d, Local band offset V (y) (orange curve) at the middle of the channel x = 0. Blue curve:

i,

  The density mapping is done according to calculated density profiles as shown in this Figure. j, The G D1D2 (V in , V out ) to G D1D2 (n in , n out ) mapping shears the data since the capacitive crosstalk is corrected. k,l, The comparison of the Fabry-Pérot oscillation period between experiment and theory reveals a successful voltage to density mapping. Plotted are the oscillations as a function of n in in k and as a function of n out in l along the directions marked with arrows in j. The curves were normalized after subtraction of the background conductance.

Supporting Figure 3 .

 3 Angle-resolved transmission function (red curves) T (φ) across the pn junction between the guiding channel center and the outer cavity at carrier densities (n in , n out ) = (10, -10) × 10 10 cm -2 in a and (n in , n out ) = (2, -10) × 10 10 cm -2 in c, taking into account the smooth junction profiles V (y) obtained from the electrostatics and sketched (red curves) in b and d, respectively. Gray curves in a and c are T (φ) when the smooth V (y)'s are replaced with ideally abrupt profiles as sketched (gray steps) in b and d, where V in/out = -sgn(n in/out )hv F π|n in/out | is the band offset applied on the incoming/outgoing lead (shaded in light yellow).

2 - 5 .

 25 ) (meV)n in ,n out =(1.5,-6.3)x10 10 cm-2 n in ,n out =(2.7,-5.9)x10 10 cm -2 ) (meV) n in ,n out =(0.9,-6.5)x10 10 cm -Calculated band structures for translational invariance along x. a-c, Band structures E(k x ) in the empty-channel regime (a), when the first mode appears in transport (b) and for two modes (c). d, The corresponding positions are labeled on the calculated γ(n in , n out ) map. e, A simplified picture of b, also shown in Figure 1f of the main text. f, The maximum angles of incidence to the p-n interface are sketched for the two different modes.

Acknowledgments M.-H.L. and K.R. acknowledge financial support by the Deutsche Forschungsgemeinschaft (SFB 689). This work was further funded by the Swiss National Science Foundation, the Swiss Nanoscience Institute, the Swiss NCCR QSIT, the ESF programme Eurographene, the EU FP7 project SE2ND, the ERC Advanced Investigator Grant QUEST, and the EU flagship project graphene.

Author contributions P.R. and S.H. and S.Z. fabricated the devices. Measurements were performed by P.R., S.H., S.Z., P.M., R.M. and M.W. Simulations were performed by M.-H.L. C.S. and K.R. guided the work. All authors worked on the manuscript.

SUPPORTING INFORMATION Measurements on further devices

In an effort to reduce the injection losses into the channel we also fabricated samples that were etched at the sides, such as the one shown in Supporting Figure 1a. Here the injection into the channel is better defined, but unfortunately after current annealing, the device is not uniformly clean. In the Fabry-Pérot map G D1D2 (Supporting Figure 1b, the outer region exhibits a much stronger depletion of carrier densities (lower conductance) at the Dirac point, i.e. the lowest conductance is found at V out = 0.

Compared to the device shown in the main text, the one shown here exhibits a slightly higher absolute guiding efficiency of 49% compared to 42% before (Supporting Figure 1c). This is due to the optimized injection into the channel. The relative increase from the unipolar value of 27% to 49% in the p-n-guiding regime is with 81% much higher than the 50% relative increase in the older sample. The device was destructed by further attempts of current annealing. On the other hand, the OF guiding is much weaker than for the device shown in the main text. In addition, steps due to mode filling can not be clearly observed in this device. This is also due to a different geometry:

In this case, the channel was formed by three bottomgate electrodes instead of one electrode and a global backgate as in the main text. The designed channel width was with 600 nm much larger than in the main text, which further complicates the observation of single mode-filling. We conclude that, even though the device was in the channel region not as clean as the device shown in the main text, the injection properties were improved due to the optimized design.

The measured guiding efficiency of a third device is shown in Supporting Figure 1d. For geometrical reasons, this device exhibits less crosstalk between the gates, i.e. the white dashed lines are less tilted in this case. Even though the overall guiding efficiency is slightly lower, OF guiding and steps in the conductance G SC (V in ) are visible. The features appear to be similar to the mode filling steps described in the main text, i.

Voltage to density mapping

The detailed device geometry as used for the simulations is shown in Supporting Figure2a and b. The measurement setup for the guiding efficiency is explained in the main text: An AC voltage is applied to the source (S) contact and an AC current is simultaneously measured at the collector (C) and the side drain contacts (D1 and D2). In Supporting Figure 2c Additionally we also show a test where the S and C contacts are 500 nm apart (blue curve).


Lara Bellini

Thomas Strub

Nadia Habel

Charlotte Pandiani

Sandrine Marchetti

Arnaud Martel

Stéphanie Baillif

Béatrice Bailly-Maitre

Philippe Gual

Robert Ballotti

Corine Bertolotto
email: bertolot@unice.fr

Endoplasmic reticulum stress mediates resistance to BCL-2 inhibitor in uveal melanoma cells

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Uveal melanoma is the most common primary intraocular malignancy in adult population [START_REF] Krantz | Uveal melanoma: epidemiology, etiology, and treatment of primary disease[END_REF][START_REF] Singh | Uveal melanoma: epidemiologic aspects[END_REF] . Despite enucleation or radiotherapy of the primary lesion, metastases develop in 50% of patients, mainly to the liver. These metastases are remarkably refractory to conventional chemotherapies, immunotherapy with checkpoint inhibitors and external radiotherapy [START_REF] Rietschel | Variates of survival in metastatic uveal melanoma[END_REF][START_REF] Carvajal | Metastatic disease from uveal melanoma: treatment options and future prospects[END_REF] . The median survival of patients who develop liver metastasis is reported to be 4 to 15 months, and the one-year survival rate is estimated to be 10-15% [START_REF] Carvajal | Metastatic disease from uveal melanoma: treatment options and future prospects[END_REF] . This highlights an urgent need for an efficient treatment.

Defective apoptosis, which contributes to sustained cell survival, is a major causative factor in the development and progression of cancer. The ability of a cell to undergo apoptosis is governed by members of the BCL-2 protein family that are grouped into three sub-families based on the number of BH (BCL-2 Homology) domains they share (BH1-4). They can be anti-apoptotic (e.g., BCL-2, BCL-XL, MCL-1) or proapoptotic (e.g., BAX, BID, BIM, NOXA) [START_REF] Hussein | Analysis of Bcl-2 protein expression in choroidal melanomas[END_REF] , among which some of them only contain the BH3 domain [START_REF] Omonosova | BH3-only proteins in apoptosis and beyond: An overview[END_REF] . BCL-2 can exert its anti-apoptotic function by sequestering the activator BH3-only proteins or through direct interaction with apoptosis-activating factors such as BAX (BCL-2 associated-X-protein) and BAK (BCL-2 homologous antagonist/killer), thereby modulating mitochondrial cytochrome c release. The release of cytochrome c subsequently leads to caspase 9 activation and to apoptosome formation, which activates the other caspases (caspases 3-7), ending in cell apoptosis.

Overexpression of the pro-survival BCL-2 family members is commonly associated with cancer [START_REF] Adams | The BCL-2 arbiters of apoptosis and their growing role as cancer targets[END_REF] . Such deregulations can be exploited by chemotherapeutic strategies, such as the BH3-mimetic drugs, which inhibit the antiapoptotic proteins by occupying their BH3-binding groove, to counteract the apoptotic blocks, and halt tumor progression [START_REF] Harb | Bcl-xL anti-apoptotic network is dispensable for development and maintenance of CML but is required for disease progression where it represents a new therapeutic target[END_REF][START_REF] Tse | ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor[END_REF] . Several BH3 mimetics, including ABT-737, ABT-263 (Navitoclax) and ABT-199 (Venetoclax) have been developed as cancer therapeutics [START_REF] Merino | BH3-Mimetic Drugs: Blazing the Trail for New Cancer Medicines[END_REF] . ABT-263 (Navitoclax), an orally available derivative of ABT-737, was tested as a single agent in phase I/II for the treatment of different solid and haematological malignancies, yet side effects such as thrombocytopenia have been reported [START_REF] Tse | ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor[END_REF][START_REF] Cleary | A phase I clinical trial of navitoclax, a targeted high-affinity Bcl-2 family inhibitor, in combination with gemcitabine in patients with solid tumors[END_REF][START_REF] Vlahovic | A phase I safety and pharmacokinetic study of ABT-263 in combination with carboplatin/paclitaxel in the treatment of patients with solid tumors[END_REF] . ABT-199 is an oral second-generation BH3 mimetic that inhibits BCL-2, with much less activity against BCL-xL. It is the first BH3 mimetic drug approved by the US Food and Drug Administration for the treatment of some leukemias and lymphomas [START_REF] Greenman | Patterns of somatic mutation in human cancer genomes[END_REF] .

Alternatively, BCL-2 family members are contained in other multiprotein complexes at the endoplasmic reticulum (ER) that are involved in the control of diverse cellular processes including calcium homeostasis and autophagy to regulate the switch between adaptive and proapoptotic phases under stress. Increasing evidence indicates that a functional activity of BCL-2 on ER protects mitochondria under diverse circumstances [START_REF] Thomenius | Bcl-2 on the endoplasmic reticulum: Protecting the mitochondria from a distance[END_REF] .

In primary uveal melanoma, expression of BCL-2 is significantly higher compared to normal ocular structures, or choroidal melanocytes [START_REF] Hussein | Analysis of Bcl-2 protein expression in choroidal melanomas[END_REF][START_REF] Jay | Expression of bcl-2 in uveal malignant melanoma[END_REF] , suggesting that it may be involved in the development and progression of these lesions. However, effects of ABT drugs have not been assessed.

In this study, we showed in vitro and in vivo that ABT-263 has antiproliferative and proapoptotic activities in uveal melanoma cells derived from primary tumors and metastases. We demonstrated that the effect of ABT-263 in some cells is accompanied with the activation of the ER stress response pathway that exerted a cytoprotective effect. Blocking ER stress enhanced ABT-263 efficacy.

Material and methods

Cell cultures and reagents

Human uveal melanoma cell lines and short-term cultures derived from different patients with metastatic malignant melanoma cells were grown in DMEM supplemented with 7% FBS at 37°C in a humidified atmosphere containing 5% CO 2 .

Lipofectamine TM RNAiMAX and opti-MEM medium were purchased from Invitrogen (San Diego, CA, USA). ABT-263 was obtained from Euromedex and qVD from Clinisciences.

Western blot assays

Western blotting was performed as previously described [START_REF] Hilmi | IGF1 promotes resistance to apoptosis in melanoma cells through an increased expression of BCL2, BCL-X(L), and survivin[END_REF][START_REF] Bertolotto | A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma[END_REF] . Briefly, cell lysates (30 µg) were separated using SDS-PAGE, transferred onto a PVDF membrane and subsequently exposed to the appropriate antibodies. Antibodies to BCL-2 (ms-123-P0) was from neomarker, to caspase 3 (#610323) was from BD, to BAX (#8429) was from sigma, to PARP (#9542), BCL-XL (#2762), CASPASE 9 (#9502), PUMA (#4976), CHOP (#2895), PERK (#5683), Phospho-PERK (#3179), IRE1α (#3294), BIP (#3177), were from Cell Signaling Technology Inc, to phospho-IRE1α (#NB100-2323) was from Novus, to MCL1 (#sc-819), to NOXA (#sc-56169), and HSP90 (#sc-13119) were from Santa Cruz biotechnology. The proteins were visualized using the ECL system (Amersham). The western blots shown are representative of at least 3 independent experiments.

Cell death analysis by flow cytometry

Cells were seeded at a density of 100 000 cells/well, in 6-well plate and treated with ABT-263 for indicated time. Cells were harvested using accutase enzyme, washed twice with ice-cold phosphate-buffered saline, resuspended in a buffer (Hepes 250mM, NaCl 150mM,KCl 5mM, MgCl 2 2mM, CaCl 2 2mM) with DAPI (1µg/ml) and Annexin V-Alexa Fluor 647 conjugate (1/100) and incubated for 15 minutes at room temperature (25°C) in the dark. Samples were immediately analyzed by a flow cytometer (MACS QUANT) using a laser at 405 nm excitation with a bandpass filter at 425 nm and 475 nm for DAPI detection and a laser at 635 nm excitation with a bandpass filter at 650 nm and 665 nm for Alexa Fluor 647 dye. Annexin and DAPI mono-or double positive cells were counted as dead cells.

Colony formation assay

Human uveal melanoma cells were seeded onto 6-well plates. The cells were subsequently placed in a 37°C, 5% CO 2 incubator. Colonies of cells were grown before being stained with 0.04% crystal violet/2% ethanol in PBS for 30 min.

Photographs of the stained colonies were captured. The colony formation assay was performed in duplicate.

mRNA preparation and real-time/quantitative PCR

The mRNA was isolated using TRIzol (Invitrogen) according to a standard procedure.

QRT-PCR was performed using SYBR® Green I (Eurogentec, Seraing, Belgium) and Multiscribe Reverse Transcriptase (Applied Biosystems) and subsequently monitored using the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). The detection of the SB34 gene was used to normalize the results. Spliced Xbp1 primers were previously reported [START_REF] Van Schadewijk | A quantitative method for detection of spliced X-box binding protein-1 (XBP1) mRNA as a measure of endoplasmic reticulum (ER) stress[END_REF] . Primer sequences for each cDNA were designed using either Primer Express Software (Applied Biosystems) or qPrimer depot (http://primerdepot.nci.nih.gov), and these sequences are available upon request.

Animal experimentation

Animal experiments were performed in accordance with French law and approved by a local institutional ethical committee. The animals were maintained in a temperaturecontrolled facility (22°C) on a 12-h light/dark cycle and provided free access to food (standard laboratory chow diet from UAR, Epinay-S/Orge, France). Human Mel270 uvealmelanoma cells were subcutaneously inoculated into 8-week-old female, immune-deficient, athymic, nude FOXN1 nu mice (Harlan Laboratory). When the tumors became palpable (0.1-0.2 cm 3 ), the mice received an intraperitoneal injection of ABT-263 (50 mg/kg), dissolved in 10% DMSO 6 times per week. Control mice were injected with DMSO alone. The growth tumor curves were determined after measuring the tumor volume using the equation V=(L × W2)/2. At the end of the experiment, the mice were euthanized by cervical dislocation.

Statistical analysis

The data are presented as the means + SD and analyzed using a two-way ANOVA or two-sided t-test with Graph Pad Prism. The difference between both conditions was statistically significant at p-value <0.05.

Results

ABT-263 triggers apoptotic cell death of uveal melanoma cells.

We first conducted experiments to assess the effect of three ABT drugs, ABT-199, ABT-737 and ABT-263 on uveal melanoma cell proliferation using four human uveal melanoma cell lines. Mel270 [START_REF] Chen | Expression of MAGE genes in ocular melanoma during progression from primary to metastatic disease[END_REF] , 92.1 [START_REF] Waard-Siebinga | Establishment and characterization of an uveal-melanoma cell line[END_REF] and OMM2.5 [START_REF] Chen | Expression of MAGE genes in ocular melanoma during progression from primary to metastatic disease[END_REF] were not sensitive to ABT-199, and OMM1 [START_REF] Luyten | Establishment and characterization of primary and metastatic uveal melanoma cell lines[END_REF] showed a moderate proliferation inhibition (about 15%) (Fig. 1a).

Whereas Mel270 and 92.1 were also highly resistant to ABT-737 and ABT-263, both drugs strongly reduced proliferation of OMM1 and OMM2.5 cells. Accordingly, cell death was higher in OMM1 and OMM2.5 cells compared to Mel270 and 92.1 cells (Fig. 1b). Thus, ABT-263 and the related ABT-737 gave similar and superior results to ABT-199. Since ABT-263 is used in clinical trials, we decided to pursue with ABT-263 for subsequent studies.

In agreement with the previous observations, in a colony formation assay, the OMM1 and OMM2.5 cell lines were more sensitive than Mel270 and 92.1 cell lines to the long-term growth inhibitory activity of ABT-263 (Fig. 1c).

Western blot indicated that the effect of ABT-263 was associated with cell cycle arrest in Mel270 cells, as shown by a reduced CDK4 expression and an increase in the cell cycle inhibitors p27 and p21 (Supplementary Fig. 1).

We next assessed the ability of ABT-263 to induce apoptosis in the cell line panel.

ABT-263 cytotoxicity as measured by Annexin V/DAPI staining revealed that OMM1 and OMM2.5 uveal melanoma cells were much more sensitive than Mel270 and 92.1 uveal melanoma cell lines, inducing 80% versus 20-35% cell death, respectively (Fig. 2a). The pan-caspase inhibitor qVD triggered a protective effect in all cell lines. Additionally, ABT-263 induced a time-dependent decrease of the full-length PARP protein, and caspase 3 zymogen (Fig. 2b) in the different cell lines. The disappearance of total poly ADP-ribose polymerase (PARP) and of the zymogenic form of caspase 3 appeared weaker in Mel270 and 92.1 cells compared to OMM1 and OMM2.5 cells.

A dose-dependent cleavage of PARP, caspase 9 and caspase 3 was also observed in all cell lines (Fig. 2c). Cleavage of caspase 3 was detected earlier in the OMM1 metastatic cells than in Mel270 cells. As confirmation that cell death was due to apoptosis, pretreatment with the pan caspase inhibitor qVD prevented both PARP and caspase cleavage (Fig. 2c).

Protein immunoblot revealed that the expression level of the main anti-apoptotic BCL-2 subfamily members (BCL-2, MCL1, BCL-xL) was higher in OMM1 cell line compared to Mel270 cells (Fig. 2d). After 24h of treatment, the only noticeable change in primary cells was the increase in pro-apoptotic NOXA level (Fig. 2d). In OMM1 cells, but not in Mel270 cells, due to significant cell death, ABT-263 effect was accompanied by a reduced expression of pro-survival (BCL-xL, MCL1), and proapoptotic (PUMA, BAX) members (Fig. 2d). BCL-2 exhibited no change in response to ABT-263.

BH3-only proteins control the mitochondrial pathway to apoptosis. We therefore measured the mitochondrial membrane potential by tetramethylrhodamine ethyl ester (TMRE) staining. ABT-263 dose-dependently reduced the mitochondrial membrane potential in both Mel270 and OMM1 cells (Fig. 2e). Interestingly, while qVD treatment did not prevent mitochondrial membrane depolarization in Mel270 cells (Supplementary fig. 2a), it blocked this process following ABT-263 exposure in OMM1 cells (Supplementary fig. 2b), suggesting that different mechanisms of cell death induction operate in these the two cell lines.

Altogether, these data demonstrated that ABT-263-induced cell death via apoptosis in uveal melanoma cells. Notably, OMM1 and OMM2.5 melanoma cells appeared more sensitive to ABT-263 effects than Mel270 and 92.1 cells.

ABT-263 Prevents Growth of Human Uveal Melanoma Tumor Xenografts

Experiments were next conducted in vivo to investigate the antineoplastic effect of ABT-263. For this purpose, the Mel270 and OMM1 human uveal melanoma cells were engrafted subcutaneously on the flank of nude mouse, which were subsequently treated with ABT-263 or with its vehicle.

ABT-263 was found to be partially effective in the group of established Mel270 xenografts (Fig. 3a) whereas it induced marked tumor growth inhibition of metastatic OMM1 xenografts, leading in mice to complete tumor shrinkage (Fig. 3b). Excised tumors in the ABT-263 group weighted significantly less than those in the control group (Fig. 3c-d). There was no significant difference in body weight between mice treated with vehicle or ABT-263, indicating no toxicity issues caused by ABT-263 in vivo. Thus, ABT-263 proved to be highly efficient in killing metastatic uveal melanoma cells and not only prevented tumor growth in vitro but also in vivo.

ABT-263 induces endoplasmic reticulum (ER) stress

BCL-2 protein family is mainly known for its anti-apoptotic role operating at the mitochondria level, yet it is also recognized for its role in the endoplasmic reticulum (ER) [START_REF] Bonneau | Non-apoptotic roles of Bcl-2 family: The calcium connection[END_REF] . Since our different cell lines showed variable sensitivity to ABT-263, we decided to investigate whether these cell lines also differed in the effect of ABT-263 on the ER pathway.

ER stress leads to unfolded protein response (UPR) which is sensed by the ER chaperone protein glucose regulated protein 78 (GRP78/also called BIP) and activation of three known ER resident proteins: inositol requiring protein-1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor-6 (ATF6), and then converge to drive the expression of C/EBP homologous protein (CHOP) [START_REF] Bahar | Chemotherapy resistance explained through endoplasmic reticulum stress-dependent signaling[END_REF] .

ABT-263-mediated ER stress was analyzed in the cell lines panel using detection of BIP, ATF4 (PERK signaling) and CHOP as surrogates for an ER stress response. In both Mel270 and 92.1 cells, ABT-263 enhanced ATF4 and CHOP as well as BIP level (Fig. 4a). In contrast, induction of ATF4 and CHOP by ABT-263 was hardly detectable in OMM1 and OMM2.5 human uveal melanoma cells, and BIP was not detected in OMM1 cells. We also did not detect PERK and IRE1α phosphorylation (Thy980 and Ser724 respectively) in OMM1 and OMM2.5 cell lines treated with ABT-

(data not shown).

Of note, in contrast to ABT-263, tunicamycin, another ER stress inducer, was able to stimulate CHOP in OMM1, thus ruling out that the ER stress pathway is deficient in OMM1 and OMM2.5 cell lines (Supplementary Fig. 3). Furthermore, XBP1 splicing was significantly higher following ABT-263 exposure (Fig. 4b).

Kinetics analysis in Mel270 primary uveal melanoma cells revealed that the protein level of BIP was enhanced following treatment with ABT-263 (Fig. 4c). PERK and EIF2α were phosphorylated (Thy980 and Ser51 respectively) and ATF4 increased.

We also observed IRE1α phosphorylation and an increase in ATF6 expression and cleavage. There was also a significant upregulation of the very downstream ER stress effector CHOP (Fig. 4c). Treatment with the pan caspase inhibitor qVD did not impact the ABT-263 mediated change in ER molecules. These data indicate that in Mel270 uveal melanoma cells, ABT-263 activated all branches of UPR, and that UPR operated upstream of the apoptotic pathway.

Altogether, our data indicate that ABT-263 elicits activation of the ER stress pathway in Mel270 and 92.1 uveal melanoma cells, while there are signs of defective activation of this pathway in OMM1 and OMM2.5 cells.

ER stress protects from ABT-263-induced apoptosis

Chemotherapy regimens were previously found to cause ER stress and to activate UPR, which paradoxically is a process that prevents apoptosis of the cancer cells and promotes their survival.

To delineate the role of ER stress pathway activation in ABT-263 effects, we used genetic or pharmacological intervention to target these pathways. Although ATF6 increases XBP1 expression, the spliced form of XBP1 that activates the UPR efficiently is mediated by IRE1 [START_REF] Yoshida | XBP1 mRNA Is Induced by ATF6 and Spliced by IRE1 in Response to ER Stress to Produce a Highly Active Transcription Factor phorylation, the activated Ire1p specifically cleaves HAC1 precursor mRNA to remove an intron of 252 nucle-otides. The cleaved 5 and[END_REF] . We were therefore focusing on PERK and IRE1α, which are the two main branches.

We first analysed siRNA against IRE1. Whereas it efficiently reduced IRE1 expression in Mel270 and 92.1 cells, it did not enhance cell death mediated by ABT-263 treatment (Supplementary Fig. 4a-b). Further, although OMM1 and OMM2.5 cells were more sensitive to ABT-263, no additive effect was observed when ABT-263 was combined with siRNA against IRE1 (Supplementary Fig. 4b). siRNA against PERK efficiently inhibited PERK mRNA level as shown by QPCR in both 92.1 and Mel270 cells (Figure 5A and Supplementary Figure 5A). Inhibition of total PERK that translated into inhibition of its phosphorylation was also confirmed by western blot analysis in 92.1 cells (Fig. 5b). PERK inhibition also impaired EIF2α activation as revealed by the loss of EIF2α phosphorylation in both basal and ABT-263-treated 92.1 cells (Fig. 5c). To reveal the effect of combined ABT-263 and PERK downregulation, we decreased the ABT-263 concentration to 3 M, which did not induce cell death in 92.1 cells (Fig. 1a-b). Whereas ABT-263 treatment or PERK inhibition exhibited a relatively low effect on PARP cleavage and cell death when used alone, the combination significantly increased the effect of ABT-263 and its killing efficacy, illustrated by PARP cleavage and an increase in cell death (Fig. 5b-c). This same holds true for Mel270 cells (Supplementary Fig. 5b-c). We also tested the effect of GSK2606414, a cell-permeable PERK inhibitor. GSK2606414 treatment reduced CHOP and ATF4 accumulation, demonstrating its efficacy in Mel270 (Supplementary Fig. 5d). Whereas GSK2606414 alone displayed no effect on cell viability, the effect of ABT-263 was improved when combined with GSK2606414 (Fig. 5e and Supplementary Fig. 5e), consistent with data obtained with PERK siRNA. Altogether, inhibition of anti-apoptotic BCL-2 proteins by ABT-263 induces a protective feedback response in 92.1 and Mel270 uveal melanoma cells, via induction of the ER stress response that can be prevented with PERK inhibitor.

Discussion

Metastatic uveal melanoma represents a major clinical challenge since, thus far, there is no efficient systemic treatment. Uveal melanomas are genetically different from cutaneous melanomas and are highly resistant to the therapeutic options used to treat cutaneous melanomas. One of the main mechanisms for cancer cells escaping apoptosis induction is through over-expression or anti-apoptotic proteins, such as the BCL-2-protein family, of which BCL-2 is the prototype.

Certain malignancies, mostly leukemias and lymphomas, appear addicted to a single pro-survival protein (mainly BCL-2). In line with that, BCL-2 transgenic mice exclusively develop hematopoietic malignancies [START_REF] Monni | BCL2 Overexpression Associated With Chromosomal Amplification in Diffuse Large B-Cell Lymphoma[END_REF] .

BCL-2 was also previously shown to be overexpressed in uveal melanoma compared to normal tissue [START_REF] Sulkowska | Bcl-2 Expression in primary uveal melanoma[END_REF][START_REF] Nemati | Targeting Bcl-2/Bcl-XL induces antitumor activity in uveal melanoma patient-derived xenografts[END_REF] . Previous studies showed that inhibition of BCL-2 alone by antisense oligonucleotides caused cell death of uveal melanoma cells and that reduction in BCL-2 through the use of miR-182 suppressed the in vitro and in vivo growth of uveal melanoma cells [START_REF] Yan | Role of microRNA-182 in posterior uveal melanoma: regulation of tumor development through MITF, BCL2 and cyclin D2[END_REF] . Zeaxanthin, a carotenoid pigment, has also been shown to decrease uveal melanoma cell viability through BCL-2 inhibition [START_REF] Song | Zeaxanthin Induces Apoptosis in Human Uveal Melanoma Cells through Bcl-2 Family Proteins and Intrinsic Apoptosis Pathway. Evidence-Based Complement[END_REF] . However, the survival of solid tumors, is often safeguarded by multiple pro-survival BCL-2 family members [START_REF] Merino | BH3-Mimetic Drugs: Blazing the Trail for New Cancer Medicines[END_REF] . Thus, these tumors require drugs targeting both BCL-2 and BCL-xL [START_REF] Merino | BH3-Mimetic Drugs: Blazing the Trail for New Cancer Medicines[END_REF] . Consistent with this notion, our results showed that ABT-199 (mainly directed against BCL-2) lacked efficiency in killing uveal melanoma cells. In contrast, we showed that ABT-263 which targets BCL-2 and BCL-xL has anti-proliferative and pro-apoptotic activities in uveal melanoma cell lines. Noteworthy, in another study, monotherapy with S44563-2, another BCL-2/BCL-xL inhibitor, exhibited a limited effect on uveal melanoma cells; however, the killing efficacy of S44563-2 was improved when combined with fotemustine [START_REF] Nemati | Targeting Bcl-2/Bcl-XL induces antitumor activity in uveal melanoma patient-derived xenografts[END_REF] . Differences in the mechanisms of drug action may explained the discrepancies in ABT-263 and S44563-2 effects. Another explanation might be that the level of BCL-2 and BCL-xL expression varied in the cells selected in these two different studies and thereby influenced their response to ABT-263 and S44563-2.

The BCL-2 protein family has extensively been studied for their role in mitochondrial apoptosis. However, the BCL-2 family seems to play a crucial role also in the endoplasmic reticulum and on the crosstalk with the mitochondria, operating as a stress rheostat [START_REF] Rodriguez | Integrating stress signals at the endoplasmic reticulum: The BCL-2 protein family rheostat[END_REF] . We observed that ABT-263 caused endoplasmic reticulum (ER) stress and activated the unfolded protein response (UPR) in primary melanoma cells, whereas in metastatic cells, ABT-263 elicited only a partial activation of the UPR.

Although BIP increase following ABT-263 treatment was easily seen in Mel270, 92.1 and OMM2.5 uveal melanoma cells, BIP was hardly detected in OMM1 cells.

Increased BIP expression reflects the intensity of the UPR activation. Furthermore, CHOP and ATF4 elevation were not detected in OMM1 and OMM2.5 cell lines upon ABT-263 treatment. However, tunicamycin (a known ER stress inducer), enhanced CHOP levels (Supplementary figure 3), thereby indicating that this pathway is functional in the metastatic cells and that the lack of ER stress stimulation by ABT-263 is specific. Thus, most likely, OMM1 and OMM2.5 cells are more sensitive because of partial UPR activation that is not able to counteract the ABT-263 killing effect. It will be interesting to assess whether activation of UPR was employed by uveal melanoma cells used in the previous studies [START_REF] Yan | Role of microRNA-182 in posterior uveal melanoma: regulation of tumor development through MITF, BCL2 and cyclin D2[END_REF][START_REF] Song | Zeaxanthin Induces Apoptosis in Human Uveal Melanoma Cells through Bcl-2 Family Proteins and Intrinsic Apoptosis Pathway. Evidence-Based Complement[END_REF][START_REF] Némati | Targeting Bcl-2/Bcl-X L induces antitumor activity in uveal melanoma patient-derived xenografts[END_REF] . In this context, the UPR pathway may be a powerful novel target to improve drug efficacy in the treatment of these uveal melanoma tumors. Among the three UPR branches, two converge towards IRE1. Indeed, ATF6 increases XBP1 mRNA expression while IRE1α mediates its splicing, resulting in the translation of a spliced active form of XBP1 (XBP1s). The PERK-EIF2 axis enhances ATF4. Both XBP1s and ATF4 function as transcription factors that regulate a wide range of genes, which plays a crucial role in cell adaptation to stress conditions [START_REF] Rozpedek | The Role of the PERK/eIF2α/ATF4/CHOP Signaling Pathway in Tumor Progression During Endoplasmic Reticulum Stress[END_REF][START_REF] Shi | Unravel the molecular mechanism of XBP1 in regulating the biology of cancer cells[END_REF] .

Our results indicate that the protective effect mounted by Mel270 and 92.1 uveal melanoma cells in response to ABT-263 specifically involved the PERK/EIF2/ATF4 signaling cascade. Indeed, in contrast to IRE1 inhibition that did not change the effect of ABT-263, the combination of ABT-263 with PERK inhibition synergistically reduced the survival rate of primary uveal melanoma cells. Mel270 and 92.1 which are primary cells appeared more resistant to ABT-263 killing activity than OMM1 and OMM2.5 that are metastatic cells. Interestingly, following ABT-263 treatment, which targets both BCL-2 and BCL-xL, we did not observe in Mel270 and OMM1 cells a compensatory increase in the other anti-apoptotic proteins, ruling out the possibility that a change in the anti-apoptotic protein level causes the different sensitivity of the cell lines to ABT-263.

The difference in sensitivity of primary and metastatic cells may also reflect the addiction of the selected cell lines to pro-survival BCL-2 family members.

Another explanation could be that the uveal melanoma cell lines did not retain the major features of the original tissue. Indeed, we showed that ABT-263 was able to efficiently kill primary uveal melanoma cells that we freshly isolated from a human biopsy (Supplementary Figure 6). We are aware that a higher number of cell lines should be tested to firmly conclude on the response of primary versus metastatic

LegendsFigure 1 :

 1 Figure 1: Effect of ABT drugs on uveal melanoma cells. a. Viability test (XTT) after exposure of a panel of uveal melanoma cell lines to BH3 mimetics drugs for 48h. Results are represented as means +SD of a minimum of three independent experiments, **P-value<0.01, ***P-value<0.001. b. FACS analyses of Annexin V/DAPI double staining in a panel of uveal melanoma cells indicate dead (early, late apoptosis and necrosis in light grey) cells after exposure to BH3 mimetics drugs for 48h. Results are represented as means +SD of minimum three independent experiments, *P-value<0.05, **P-value<0.01, ***Pvalue<0.001. c. Colony formation assay of primary and metastatic uveal melanoma cells exposed to ABT-263 3M and 5 M. Colonies were stained with crystal violet after 7 days (representative micrographs are shown).

Figure 2 :

 2 Figure 2: ABT-263 induces apoptosis of uveal melanoma cells. a. FACS analyses of Annexin V/DAPI double staining in a panel of uveal melanoma cells indicate alive (white) or dead (early, late apoptosis and necrosis in light grey) cells after exposure to ABT-263 5μM for 48h in the absence or presence of qVD-OPh 20 μM. Results are represented as means +SD of three independent experiments, *P-value<0.05, ***P-value<0.001. b. Western blot analysis of PARP and caspase 3 in Mel270, 92.1, OMM2.5 and OMM1 uveal melanoma cells exposed to ABT-263 5μM for 24h and 48h. Detection of HSP90 serves as a loading control. Representative immunoblots are shown. c. Western blot analysis of PARP, caspase 9 and caspase 3 human primary Mel270 and metastatic OMM1 uveal melanoma cells exposed to ABT-263 3μM or 5μM for 48h in the absence or presence of qVD-OPh 20μM. Detection of HSP90 serves as a loading control. Representative immunoblots are shown. d. Western blot analysis of pro-and anti-apoptotic members of the BCL-2 family proteins in Mel270 and OMM1 uveal melanoma cells that were left untreated or treated with ABT-263 3 μM or 5μM for 24 hrs. HSP90 was used as a loading control. e. Detection of the mitochondrial membrane potential using TMRE staining in Mel270 and OMM1 uveal melanoma cells exposed to ABT-263 3μM or 5μM for 48h. Percent of TMRE negative cells is shown. ***P-value<0.001.

Figure 3 .

 3 Figure 3. ABT-263 impairs tumor growth in vivo. a. Mel270 and b. OMM1 uveal melanoma cells were subcutaneously engrafted into athymic nude mice (n=8 per group). Once the tumours reached 100mm3, mice were treated daily with ABT-263. The red arrow indicates the start of the treatment. The growth tumor curves were determined by measuring the tumor volume using the equation V= (L x W x W)/2, where V is tumor volume, W is tumor width, L is tumor length. Results are presented as mean (±SEM) tumor volumes (mm 3 ). **P-value< 0.01, ***P-value<0.001 are from 2-way anova test in ABT-263 treated versus vehicle at each point. c-d. The weight of the sub-cutaneous tumor from control (Ct) and ABT-263-treated mice is shown. *P-value<0.05, **P-value< 0.01.

Figure 4 :

 4 Figure 4: ABT-263 induces ER stress and activation of the unfolded protein response. a. Western blot of ER stress molecules in uveal melanoma cells treated with ABT-263 5μM for 24 h and 48 h. HSP90 is used as a loading control. Representative western blots are shown. b. XBP1 splicing in uveal melanoma cells exposed to ABT-263 5μM for 15h. *P-value<0.05, **P-value< 0.01. c. Kinetic analysis of ER stress proteins in Mel270 cells treated with ABT-263 5μM in the absence or presence of qVD-OPh 20 μM. HSP90 is used as a loading control. Representative western blots are shown.

Figure 5 :

 5 Figure 5: PERK dampens ABT-263 killing activity. a. Q-PCR analysis of PERK level in 92.1 uveal melanoma cells treated with control (siCtl) or PERK (siPERK) siRNA for 48 h. ***P-value< 0.001. b. Western blot analysis to PERK and P-PERK in 92.1 cells treated with control (siCtl) or PERP (siPERK) siRNA. HSP90 is used as a loading control. c. 92.1 uveal melanoma cells treated with control (siCtl) or PERK (siPERK) siRNA for 48 h before being exposed to ABT-263 3 M for 48 h. Representative western blot to P-EIF2, total EIF2 and PARP are shown. HSP90 serves as a loading control. d. 92.1 cells were treated as in (B). FACS analyses of Annexin V/DAPI double staining in uveal melanoma cells indicate alive (white) or dead (early, late apoptosis and necrosis in light grey) cells. ***P-value< 0.001. e. 92.1 cells were treated as in (B). FACS analyses of Annexin V/DAPI double staining in uveal melanoma cells indicate alive (white) or dead (early, late apoptosis and necrosis in light grey) cells. ***P-value< 0.001.

Acknowledgments

The authors thank Dr M.J. Jager for the critical reading and editing of this manuscript. This work was funded by La Ville de Nice, UCAJEDI "ANR-15-IDEX-01", ARC grant #20171206312 to C.B, ARC grant #20171206287 to BB-M and cancéropôle PACA..CP is a fellowship from la Ligue Nationale Contre le Cancer. The authors thanks Karine Bille and Marjorie Heim for their technical help. 92.1 uveal melanoma cells were provided by Dr. M.J. Jager (Leiden, The Netherlands), Mel202 and Mel270 uveal melanoma cells by Dr. B. Ksander (Boston, USA) and OMM1 uveal melanoma cells from Prof. G.P.M. Luyten, (Rotterdam, The Netherlands).

Competing interests

The authors declare no conflicts of interest.