Jeanne Kergomard

Frédéric Carrière

Gilles Paboeuf

Franck Artzner

Nathalie Barouh

Claire Bourlieu

Dr Véronique Vié
email: veronique.vie@univ-rennes1.fr

Interfacial organization and phase behavior of mixed galactolipid-DPPCphytosterol assemblies at the air-water interface and in hydrated mesophases

Keywords: AFM: atomic force microscopy digalactosyldiacylglycerol DPPC: 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine phytosterol PUFA: polyunsaturated fatty acids SAXS: X-ray scattering 4 galactolipid, phytosterol, natural surfactant, vegetal photosynthetic assemblies

Plant polar lipids formed stable monolayers at the air/water interface.

• Phytosterols reduced the lateral elasticity of galactolipid and DPPC monolayer.

• DPPC has affected MGDG-DGDG interactions.

INTRODUCTION

Galactolipids are the most abundant polar lipids found in higher plants. These glycolipids are actively involved in the structuration of the plant membranes and are particularly concentrated in the photosynthetic chloroplast membranes (80% wt. of total non-pigmented lipids), which contain large amounts of monogalactosyldiacylglycerol (MGDG, 53% wt.) and digalactosyldiacylglycerol (DGDG, 27% wt.) [START_REF] Gurevich | Poly-unsaturated phospholipids increase the hypolipidemic effect of Lovastatin[END_REF]. MGDG possess a small 1--galactose polar head bound to a diacylglycerol, giving it a conical shape, which can induce curvatures in lamellar phases [START_REF] Lee | Membrane lipids : It's only a phase[END_REF]. On the other hand, DGDG has a larger polar head with an additional -galactose, linked to -galactose [START_REF] Mizusawa | The role of lipids in photosystem II[END_REF] and adopts a cylindrical shape in solution. Both GL possess two esterified acyl chains at the sn-1 and sn-2 position of the glycerol backbone, whose molecular motion depends on the number of unsaturation.

Due to their different structure, MGDG and DGDG exhibit distinct phase behaviors, which govern the overall membrane architecture (Fig. 1). Indeed, studies on the phase behavior of GL have shown that DGDG forms lamellar phases (L) and induces bilayer formation, whereas unsaturated MGDG tends to form inverted hexagonal structures (HII) in aqueous solution [START_REF] Brentel | Phase equilibria of mixtures of plant galactolipids. The formation of a bicontinuous cubic phase[END_REF]. In addition to their biological functions in photosynthetic membranes, GL are of nutritional interest since they are rich in polyunsaturated fatty acids (PUFA), and their consumption provides essential 3 fatty acids [START_REF] Sahaka | The digestion of galactolipids and its ubiquitous function in Nature for the uptake of the essential αlinolenic acid[END_REF]. Phytosterols (pS) are also among the major compounds of photosynthetic plant membranes where they play crucial roles in regulating the physical properties of membranes [START_REF] Evans | Methods in plant biochemistry : 1. plant phenolics[END_REF]Ostlund, 2002). More than 250 species of phytosterols have been reported, the most abundant being -sitosterol (70% wt.), stigmasterol (20% wt.) and campesterol (5% wt.) [START_REF] Evans | Methods in plant biochemistry : 1. plant phenolics[END_REF]. These pS in their free form participate in the structuration of membranes by stabilizing polar lipid bilayers, although to a lesser extent than their cholesterol counterpart in animal cell membranes [START_REF] Moreau | Phytosterols, phytostanols, and their conjugates in foods : Structural diversity, quantitative analysis, and health-promoting uses[END_REF]. Beyond their structural properties, phytosterols are also compounds of nutritional and medical importance. Their consumption from fruits and vegetables has been shown to lower the total cholesterol and LDL levels in plasma in humans [START_REF] St-Onge | Phytosterols and human lipid metabolism : Efficacy, safety, and novel foods[END_REF]. The interesting structural and nutritional properties of GL and pS have raised interest in their use in various applications, which requires a deep understanding of the organization and interactions of these plant polar lipids.

The monolayer behavior of saturated GL has already been extensively studied at the air/water interface [START_REF] Hoyo | Monogalactosyldiacylglycerol and digalactosyldiacylglycerol role, physical states, applications and biomimetic monolayer films[END_REF][START_REF] Tomoaia-Cotişel | Hysteresis in compressionexpansion cycles of distearoylmonogalactosylglycerol monolayers[END_REF]. Nevertheless, it should be noted that GL acyl chains in higher plants are mostly polyunsaturated, and thus less stable since they are more susceptible to oxidation (Tomoala-Cotişel et al., 1983). Monolayer studies have also been performed on GL mixtures with unsaturation indices greater than 1, highlighting the differences in interfacial behavior between saturated and unsaturated GL. Using Langmuir films, [START_REF] Bottier | Galactosyl headgroup interactions control the molecular packing of wheat lipids in Langmuir films and in hydrated liquid-crystalline mesophases[END_REF] studied the interfacial behavior of GL from different wheat tissues and highlighted the miscibility of MGDG and DGDG in water but also their tendency to phase separation in mixture. Overall, interface organization and packing were governed by interactions between the sugar polar heads of GL [START_REF] Bishop | Monolayer properties of chloroplast lipids[END_REF], but also depended on their fatty acid composition and the number of unsaturations.

The impact of plant sterols in polar lipid bilayers has already been studied extensively, showing that they alter phase transitions and membrane fluidity [START_REF] Itzhaki | Age-Related Changes in Petal Membranes from Attached and Detached Rose Flowers[END_REF][START_REF] Mckersie | Influence of Plant Sterols on the Phase Properties of Phospholipid Bilayers 1[END_REF], depending on their chemical structure and concentration (Kamal & Raghunathan, 2012). The interactions between pS and dipalmitoylphosphatidylcholine (DPPC) have also been studied in model Langmuir films at the air/water interface, and the incorporation of -sitosterol and stigmasterol into PL monolayers has been shown to increase their packing [START_REF] Su | Condensation effect of cholesterol, stigmasterol, and sitosterol on dipalmitoylphosphatidylcholine in molecular monolayers[END_REF]. Nevertheless, to the best of our knowledge, few results are available today concerning the impact of pS and phospholipids on GL interactions and packing in monolayers.

The interfacial properties of MGDG and DGDG, as well as their modulation by DPPC and/or pS in mixed Langmuir monolayers at the air/water interface were studied by ellipsometry, tensiometry and atomic force microscopy (AFM). The phase behavior and physical properties of these mixtures in hydrated mesophases were also studied by small angle X-ray scattering (SAXS)

EXPERIMENTAL SECTION

Lipids

1,2-dipalmitoylphosphatidylcholine (DPPC), monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were purchased from Avanti Polar Lipids (see Table S1 in the supplementary material for the fatty acid repartition). Canola phytosterols were a gift from Cognis France (Estarac, France) obtained from desodorization distillates of canola oil. Typical molar composition (calculated from usual normalized GC procedures) was: β-sitosterol (50 mol%), campesterol (40 mol%) and brassicasterol (10 mol%).

Preparation of multicomponent lipid blends

Simple binary mixture of MGDG and DGDG (60:40, mol/mol) was prepared, namely GL. Ternary and quaternary mixtures of glycerophospholipids, galactolipids and phytosterols (see Table 1 for relative molar composition) were prepared to add some phase heterogeneity and to mimic the composition of natural plant photosynthetic membranes.

Ellipsometry and surface pressure measurements at the air/water interface

All reported experiments were performed at least in duplicate, using a computer controlled and user-programmable LB Teflon Langmuir trough (KSV Nima, Helsinki, Finland) of 77 cm², equipped with two mobile barriers, allowing to vary the surface. Before each experiment, the trough was carefully cleaned with ultrapure water and ethanol to get rid of the surface-active residual impurities. The surface pressure was measured using a Wilhelmy balance connected to a microelectronic feedback system (Nima Technology, Cambridge, UK). The values of surface pressure () were recorded every 15 s with a precision of 0.2 mN/m. A home-made automated ellipsometer in a "null ellipsometer" configuration was used to carry out the ellipsometric angle () measurement [START_REF] Berge | Ellipsometry Study of 2D Crystallization of 1-Alcohol Monolayers at the Water Surface[END_REF]. The laser beam probed a surface of 1 mm 2 and a depth of 1 µm and allowed the qualitative monitoring of the thickness of the lipid monolayer formed at the air/water interface. Values of  were recorded every 15 s with a precision of ± 0.5 °.

Formation of the lipid monolayers and Langmuir Blodgett transfer

For all the characterizations, the aqueous phase was composed of a 10 mM Tris HCl buffer solution (100 mM NaCl, 5 mM CaCl2, 10 mM Tris) at pH 7. The monolayers were formed by spreading a few microliters of 1 mM solution of lipids in CHCl3/MeOH (2:1, v/v) at the air/water interface using a Hamilton microliter syringe. The surface area of the Langmuir trough was set at 35 cm² and the lipids were deposited up to a surface pressure of 20 mN/m. The film was then equilibrated for 10 minutes before each measurement, allowing the evaporation of solvent and lipid organization at the air/water interface. After 10 minutes equilibrium, the interfacial film was transferred onto a freshly cleaved mica plate, using the Langmuir Blodgett method (lipid-transfer ratio ~ 1.0). All lipid layers were sampled at a very low speed (0.5 mm.min -1 ) by raising vertically the mica plate through the air/water interface. The surface pressure was kept constant during the sampling to maintain the lateral organization of lipids and thus avoid desorption phenomena and interfacial disorganization.

Compression isotherms and compressibility modulus C -1

The surface area of the Langmuir trough was set at 77 cm² before compression. Few microliters of 1 mM solution of lipids in CHCl3/MeOH (2:1, v/v) were spread at the interface until the pressure reaches 0.1 mN/m. The surface pressure as a function the surface area of the trough (-A isotherm) was then recorded upon a symmetrical compression of the lipid monolayer using the two barriers, from 77 cm² to 21 cm², until the collapse of the film. The barrier speed was set at 5 mm²/min.

Compression isotherms allow to obtain several parameters on the behavior of monolayers under compression. The minimum molecular area corresponds to the mean molecular area occupied by the molecules at the collapse of the film and was calculated as the intercept value between the pressure plateau at collapse and the tangent at the end of the -A isotherm. The lift-off area was determined as the mean molecular area occupied by the molecules when the pressure begins to increase under compression. Finally, the monolayer compressibility was obtained from the first derivate of -A isotherm and the mean molecular area of lipids deposited at the interface [START_REF] Gaines | Nanomechanics of Lipid Bilayers : Heads or Tails?[END_REF][START_REF] Smaby | Phosphatidylcholine acyl unsaturation modulates the decrease in interfacial elasticity induced by cholesterol[END_REF]. The equation (1) was used to determine the compressibility coefficient (Cs) of the monolayer.

(1

) 𝐶𝑠 = - 1 𝐴 × 𝑑𝐴 𝑑𝜋
where  is the surface pressure and A is the mean molecular area of the lipids forming the monolayer, estimated from the surface of the trough and the amounts of lipids deposited. The reciprocal of compressibility coefficient, the compressibility modulus, i.e., Cs -1 was represented as a function of the mean molecular area (Ų/molecules). Cs -1 values were obtained at equally spaced surface pressures (0.2 mN/m intervals).

Topographic visualization of the monolayer interface using atomic force microscopy

Imaging of the films transferred to mica plates was carried out using an AFM (Multimode Nanoscope 5, Bruker, France). The PeakForce Quantitative Nanomechanical Mapping (QNM in air, 20°C) was used in all the experiments. This mode provides high-resolution AFM images of the sample nanoscale surface topography, as well as the modulus and adhesion, at an acquisition speed comparable to the tapping mode. A standard silicon cantilever was used (0.06 N/m, SNL-10, Bruker, France), and the scan rate was set at 1 Hz. The force was minimized during all scans and the scanner size was 100×100 µm². The processed images analyzed by the open-source platform Gwyddion are representative of at least duplicated experiments. The differences of height of the LC and LE domains were assessed by the plot of height profile based on three cross-sections of the image using the software Gwyddion (2.55). For all AFM images, the color scale covers a height range of 15 nm, with the darkest color corresponding to the lowest domains and the lightest color to the highest domains, the fluid background corresponding to zero. A color scale is provided in Figure 3 to facilitate interpretation of the images.

Determination of the phase behavior of the model lipid mixture in aqueous dispersions by SAXS

The phase behavior of the model lipid mixture was investigated in aqueous dispersions using Xray scattering with a homemade set-up at the Rennes Physique Institute. The X-ray scattering results were collected with a Pilatus 300 K (Dectris, Switzerland), mounted on a microsource Xray generator GeniX 3D (Xenocs, France) operating at 30 W. The monochromatic CuKa radiation was of l = 1.541 Å. The X-ray diffraction patterns were recorded in a reciprocal space q = (4.

sin)/ from repetitive distances q=0.012 to 1.742 Å -1 . Small Angle X-ray Scattering (SAXS) and Wide-Angle X-ray Scattering (WAXS) regions were used to determine the supramolecular organization (long range order) of the lipid/water systems and the packing of acyl chains (short range order), respectively. The samples were prepared by evaporation of the solvent containing the solubilized lipids and then hydration at 5% wt of the lipid films in a Tris buffer solution pH 7 (0.5 mg of lipids hydrated in 10 µL of Tris buffer). The samples were then introduced in thin calibrated quartz capillaries (Ø 1.5 mm, GLAS W. Muller, Berlin, Germany) before being centrifuged and sealed with candle wax. For the analysis, they have been introduced in a capillary holder accommodating 19 capillaries at controlled temperature. The homogeneity of the sample was checked at two y-positions. The analyses were carried out following a heating and cooling temperature ramp from 12 °C to 42 °C and 42 °C to 12 °C, respectively, every 8°C with an exposure time per point of 10 minutes. The results were collected by a homemade program and the positions of Bragg reflections were determined by the Igor Pro 7.0 software (Wavemetrics, US).

RESULTS AND DISCUSSION

Study of the compression isotherms -A of the GLx/pSy systemsinfluence of the pS concentration

The absence of shoulder and/or inflection point on the compression isotherm of the GL mixture (Fig. 2.A) was characteristic of a homogeneous liquid-expanded (LE) single phase, which could be explained by the high unsaturation content of the mixture. Indeed, the unsaturations decreased the intensity of van der Walls interactions between the acyl chains, and could hinder the establishment of a tight packing between GL molecules in the monolayer. The isotherm of the GL film indicated a lift-off area at around 230.8 ± 1.8 Ų/molecule (2.3 nm²/molecule) and showed a regular increase of the surface pressure until the collapse of the film at =39.9 ± 0.2 mN/m and at a limiting area of 81.0 ± 0.6 Ų/molecule (0.8 nm²/molecule). The evolution of the -A isotherm of the GL film was consistent with the study of [START_REF] Bottier | Galactosyl headgroup interactions control the molecular packing of wheat lipids in Langmuir films and in hydrated liquid-crystalline mesophases[END_REF] on purified wheat MGDG and DGDG monolayers, pure or in mixture. Nevertheless, a higher collapse pressure of 47 mN/m had been obtained for the less unsaturated wheat MGDG/DGDG equimolar mixture, at a lower mean molecular area of 70 Ų/molecule (0.7 nm²/molecule). This result was not surprising, as unsaturated acyl chains cannot adopt a very tight packing, which explains a larger average molecular area and a lower surface pressure at collapse as the number of unsaturation increases. The presence of unsaturations in the alkyl chains could also explains the significantly lower Cs -1 max which was obtain for the GL monolayer in our study (Fig. 2.B) compared to the Cs -1 max reported by [START_REF] Hoyo | Monogalactosyldiacylglycerol and digalactosyldiacylglycerol role, physical states, applications and biomimetic monolayer films[END_REF] on the 2:1 mixture of saturated GL (48 versus 247 mN/m respectively). These values were determined for pressure values of interest, between 30 and 40 mN/m, =35 mN/m having been proposed as the equivalence surface pressure between lipid monolayers and cellular bilayers [START_REF] Marsh | Lateral pressure in membranes[END_REF][START_REF] Marsh | Lateral Pressure Profile, Spontaneous Curvature Frustration, and the Incorporation and Conformation of Proteins in Membranes[END_REF]. This decrease in Cs -1 max with increasing unsaturation number had also been reported by [START_REF] Gzyl-Malcher | Differences in surface behaviour of galactolipoids originating from different kind of wheat tissue cultivated in vitro[END_REF], reflecting the higher lateral elasticity of unsaturated monolayers compared to the saturated ones.

The addition of pS in the GL mixture did not significantly impact the surface pressure at the collapse of the film (Fig. 2.A). The addition of 5 and 10 mol% pS to the GL mixture did not significantly impact the limiting area. In contrast, the limiting area was affected by the addition of 30 mol% of pS and shifted from 81.0 ± 0.6 to 66.1 ± 0.5 Ų/molecule (0.8 to 0.7 nm²/molecule).

The inclusion of pS has also significantly altered the Cs -1 max of the monolayers (Fig. 2.B., logarithmic scale), and this effect was particularly marked in the presence of 30 mol% of pS in the GL mixture. Indeed, a Cs -1 max of 85.7 mN/m was reached for the GL70/pS30 blend, compared to 72.8 mN/m for the GL90/pS10 and GL95/pS5 blends and 48.0 mN/m for the pure GL system. The greater impact of phytosterols for a content of 0.3 mol% on the compressibility of the monolayer was consistent with the results presented in Figure 2.D. Indeed, the phase diagram showed a nonideal behavior of the GL and pS blends, and this effect was even more noticeable for a molar concentration of pS of 30% in the system. The reduction of the mean molecular area at collapse and the increase of Cs -1 max with the increase of pS concentration could be attributed either to the condensation of the unsaturated acyl chains or to the reorientation of the sugar polar heads, induced by the presence of the pS molecule, thus decreasing the lateral elasticity of the monolayers [START_REF] Li | Cholesterol Decreases the Interfacial Elasticity and Detergent Solubility of Sphingomyelins[END_REF]. Indeed, the presence of a largely hydrophobic small molecule such as pS will also impact the tilt of the polar heads, which will increase the possibility of packing of the molecules, explaining the non-ideal behavior observed in the case of unsaturated GL monolayers.

Nevertheless, the animal counterpart of pS, the cholesterol, was shown to establish weaker interactions with unsaturated lipids, in comparison with saturated lipids [START_REF] Silvius | Role of cholesterol in lipid raft formation : Lessons from lipid model systems[END_REF], so the addition of DPPC in the mixtures could drastically alter the phenomena observed in GL-pS monolayers [START_REF] Jurak | Thermodynamic Aspects of Cholesterol Effect on Properties of Phospholipid Monolayers : Langmuir and Langmuir-Blodgett Monolayer Study[END_REF]. 2.

Influence

The addition of 10 mol% of pS in the GL mixture has induced a clear increase in the film thickness at collapse (8.2 versus 7.3 ± 0.5° for the GL90/pS10 versus GL mixture, respectively), while there was no significant change in collapse pressure, neither in the limiting area. These results

tend to indicate that the inclusion of the pS only impacted the orientation of the GL sugar polar heads, and not the acyl chain organization.

The addition of 50 mol% DPPC in the GL mixture led to an increase of the collapse surface pressure to =44.1 ± 0.2 mN/m (versus 39.9 ± 0.2 mN/m for the GL monolayer) as well as a shift in the mean molecular area at collapse to 60.4 ± 1.0 Ų/molecules (0.6 nm²/molecule), versus 81.0 ± 0.6 Ų/molecule (0.8 nm²/molecule) for the GL monolayer. These results indicated a strong condensing effect of DPPC, which could be explained by the presence of saturated acyl chains and smaller polar head of DPPC, which decreased the steric hindrance. An increase in the film thickness at collapse induced by the presence of DPPC was also observed (=8.9° vs 7.30° for the GL film), suggesting a reorientation of the sugar polar heads of GL, as well as the formation of condensed domains upon compression (Vié et al., 1998).

The addition of phytosterols at 10 mol% in the GL/DPPC film did not induce a significative variation of the collapse pressure (44.4 ± 0.2 mN/m versus 44.1 mN/m for the GL/DPPC) but triggered a shift of the limiting molecular area from 60.4 ± 1.0 to 55.0 ± 1.7 Ų/molecule (0.6 to 0.5 nm²/molecule), indicating an increase of the packing in the mixture in presence of pS. This decrease in the limiting area was not expected, given that pS probably interacts preferentially with DPPC than with PUFA-containing GL. Indeed, Botet-Carreras et al. ( 2019) have studied the impact of cholesterol on homo-and hetero-acids phospholipid monolayers by AFM and AFM-FS, confirming that the effect of cholesterol on unsaturated PL was weaker than the one exerted on saturated PL. Additionally, a variety of biophysical measurements have revealed that sterols have an aversion to PUFA (Huster et al., 1998;Niu & Litman, 2002;Pitman et al., 2004;Wassall et al., 2004), explaining the lower impact of pS in the presence of GL in the GL90/pS10 mixture, which contain a significant amount of PUFA. One possible explanation for such aversion of sterols for PUFA is the high disorder of PUFA acyl chains, which is incompatible with the usual orientation of cholesterol vertically with respect to interface plane. Thus, one can expect a change in the orientation of the sterol backbone (Harroun et al., 2006). Additionally, cholesterol has already been shown to induce order in lipids in fluid phase, whereas it has the opposite effect on lipids present in gel phase [START_REF] Gaines | Nanomechanics of Lipid Bilayers : Heads or Tails?[END_REF]. Given these general effects of sterols and their aversion to PUFA, we were expected a more pronounced disorganization of DPPC molecules than a condensation effect of GL polyunsaturated lipid chains, and thus a larger limiting area for the GL45/DPPC45/pS10 mixture compared to the GL50/DPPC50 mixture. Nevertheless, given the decrease in the limiting area of the mixture in the presence of pS, it is likely that the addition of pS induced GL organization in the presence of DPPC, raising the question of a potential miscibility between DPPC and MGDG and/or DGDG in the presence of pS, reflecting a more complex mixture.

Characterization by ellipsometry of interfacial films at 20 mN/m

The thickness of the three model systems was determined by ellipsometry at a surface pressure of 20 mN/m. The detailed molar compositions are given in Table 1. The values of ellipsometric angles obtained at 20 mN/m are presented in Table 3. Eight sub-model systems were also prepared to give a better understanding of the organization and interactions between polar lipids at the air/water interface (see supplementary data S1 for the detailed molar compositions and S2 for the ellipsometric angle values at 20 mN/m).

An ellipsometric angle of 7.5° was obtained for the mixed GL monolayer at 20 mN/m., which was identical to the value obtained with DGDG alone and higher than the value for MGDG (7.5 and 6.3 ± 0.5° respectively). This result was expected as the DGDG lipid film has already been shown to be thicker than the MGDG monolayer [START_REF] Bottier | Caractérisation des puroindolines, des galactolipides du blé et de leurs interactions : Mesures physiques aux interfaces[END_REF].

The addition of DPPC to GL at a molar fraction of 0.5 did not induce a significant variation in the thickness at 20 mN/m. This result was consistent with the data obtained with the sub-model systems MGDG50/DPPC50 and DGDG50/DPPC50 (6.3 and 7.4 ± 0.5°, respectively), in which the addition of DPPC did not induce a significant variation of the ellipsometric angle at 20 mN/m.

The addition of pS to the mixed model system of GL and DPPC, at a molar content of 10%, led to a slight decrease of the ellipsometric angle at 20 mN/m, but it was not significant. By comparison, pS induced a clear decrease in the thickness of the MGDG45/DPPC45/pS10 and DGDG45/DPPC45/pS10 sub-model systems (5.5 and 6.0 ± 0.5°, respectively), compared to the MGDG50/DPPC50 and DGDG50/DPPC50 systems (6.3 and 7.4 ± 0.5°, respectively). However, no significant variation in thickness was obtained for the sub-models MGDG90/pS10 and DGDG90/pS10, in comparison with the pure MGDG and DGDG systems. These results tend to indicate different interactions of pS with GL-DPPC mixed films and pure MGDG and DGDG.

In order to better understand the differences in the organization of the different models and sub-models, atomic force microscopy was used to visualize the interface of the films obtained by Langmuir Blodgett sampling.

Nanoscale topographic visualization of interfacial films at 20 mN/m using atomic force microscopy

Figure 3 shows AFM images of the interfacial organization of the three main model monolayers GL, GL50/DPPC50, and GL45/DPPC45/pS10 after their transfer by the Langmuir-Blodgett technique onto a mica plate and at a surface pressure of 20 ± 0.5 mN/m. The scale used was composed of different shades of brown, related to the differences in height in the monolayers.

These color variations were correlated with the different tilt values of the lipid molecules and thus provide information on the orientation of the molecules and their physical state.

The 5×5 µm² AFM image of the GL system (Fig. 3.A), was characteristic of a homogeneous liquid-expanded (LE) phase without phase separation, consistent with the presence of unsaturated acyl chains in the fluid phase. However, the 1×1 µm² image highlights the presence of surface roughness, with height differences between 2 to 8 Å (0.2 to 0.8 nm) with respect to the baseline. A similar segregation was previously observed by AFM on mixed films of MGDG:DGDG (68:32 mol/mol) by [START_REF] Sarkis | The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts[END_REF]. Additionally, [START_REF] Bottier | Galactosyl headgroup interactions control the molecular packing of wheat lipids in Langmuir films and in hydrated liquid-crystalline mesophases[END_REF] had observed the presence of irregular protrusions of 7 Å (0.7 nm) and 4 Å (0.4 nm) at surface pressures above 25 mN/m on monolayers of pure MGDG or in equimolar mixture with DGDG, respectively. The presence of these protrusions had been attributed to a specific organization of the MGDG polar heads between them or with those of DGDG, this roughness being absent on the AFM images of the DGDG monolayer. Indeed, a particular orientation of DGDG polar heads induced by the interactions between GL had been highlighted by PM-IRRAS data. The assumption was that the monogalactosyl polar head of MGDG could be responsible for the orientation of the digalactosyl group of DGDG parallel to the interface, with the formation of a network of hydrogen bridges between the polar heads and the water molecules at the interface [START_REF] Kanduc | Tight cohesion between glycolipid membranes results from balanced water-headgroup interactions[END_REF].

The addition of DPPC to the system (2) (Fig. 3.B) resulted in the appearance of 1 nm high condensed domains of varying shapes and sizes at the interface (5x5 µm² image), likely to be enriched in DPPC, given their degree of saturation. Additionally, a loss of the granularity on 1x1 µm² images suggests changes in the orientation of the galactosyl polar heads of GL, that could result from a modification by DPPC of the interactions between MGDG and DGDG. This would fit with the previous results obtained on the compressibility of the film.

The addition of pS to the GL50/DPPC50 mixture (Fig. 3.C) resulted in a subsequent dilution of the domains in the LC condensed phase, as well as a significant increase in the thickness of the domains (h=1.6 nm versus h=1.0 nm, with and without pS, respectively), in contrast to the unchanged value of the ellipsometric angle. The irregular domain boundaries also indicated a change in line tension induced upon addition of pS in the GL/DPPC system, and suggests a change in interactions between the different classes of lipids, with miscibility regions between the different components. Defects were also visible at the condensed phase domains, probably induced by the inclusion of pS and the local subsequent disorganization of the saturated acyl chains of DPPC.

In order to investigate the composition of the condensed phase domains and to better understand the interactions involved between the different classes of lipids, four sub-model systems were prepared, namely MGDG50/DPPC50, DGDG50/DPPC50, MGDG30/DPPC60/pS10 and the same trend as that observed in the GL45/DPPC45/pS10 model, for which a 45% increase in domain thickness was observed.

Overall, the results confirm the hypotheses of a different miscibility between DPPC and MGDG and between DPPC and DGDG. Moreover, it seems that the inclusion of pS in the systems induced, on the one hand, a transition from the DPPC-enriched gel phase to a less ordered phase, with the apparition of defects, and on the other hand, a transition from a fluid phase to a less ordered zone, with reduced height mismatch and a decrease of the line tension in the ternary systems. To validate these hypotheses and determine the miscibility of DPPC in mixture with MGDG or DGDG, as well as the impact of pS in GL/DPPC lipid systems, SAXS experiments were performed, in the presence of water in excess.

Phase behavior of mixed galactolipid mixtures in hydrated mesophase (SAXS)

The three-dimensional structures of unsaturated MGDG and DGDG, alone or in their equimolar mixture, have already been studied by analysis of the X-ray diffraction patterns and electron density profile at 20°C in excess of water [START_REF] Bottier | Galactosyl headgroup interactions control the molecular packing of wheat lipids in Langmuir films and in hydrated liquid-crystalline mesophases[END_REF][START_REF] Hoyo | Monogalactosyldiacylglycerol and digalactosyldiacylglycerol role, physical states, applications and biomimetic monolayer films[END_REF]. On one hand, the lamellar structure L of DGDG was evidenced by the presence of single peaks regularly spaced, with a lamellar periodicity of 54.9 Å. On the other hand, the X-ray diffraction profile obtained for MGDG showed six Bragg diffraction pics, with a hexagonal periodicity of 67.2 Å, that were attributed to an inverse hexagonal phase (HII) distribution, which has been observed in other studies over a wide temperature range (-15 to 80°C) [START_REF] Popova | Thermotropic phase behavior and headgroup interactions of the nonbilayer lipids phosphatidylethanolamine and monogalactosyldiacylglycerol in the dry state[END_REF]. The ability of unsaturated MGDG to form non-lamellar structures can be neutralized by mixing it with at least 50% of lipids forming lamellar phases. However, unsaturated MGDG can induce defects in the lamellar structure of MGDG/lipid mixtures forming bilayers when its proportion is between 20 and 50% [START_REF] Castro | NMR studies of membranes composed of glycolipids and phospholipids[END_REF]. Nevertheless, for the equimolar mixture of MGDG and DGDG in excess of water, the SAXS diffraction pattern obtained by Bottier et al. was the signature of a bicontinuous cubic phase (Im3m space group) with a cubic lattice parameter of 202 Å, showing the specific behavior of MGDG and DGDG in the equimolar mixture compared to the pure lipids. Indeed, the formation of an Im3m space group could result from specific interactions between the polar sugar heads of the MGDG and DGDG.

The three-dimensional organization of DPPC has also already been extensively studied by SAXS, showing a well-ordered multilayer structure in excess of water due to the so-called L gel phase at 20°C [START_REF] Et-Thakafy | Mechanical Properties of Membranes Composed of Gel-Phase or Fluid-Phase Phospholipids Probed on Liposomes by Atomic Force Spectroscopy[END_REF][START_REF] Varga | Localization of dihalogenated phenols in vesicle systems determined by contrast variation X-ray scattering[END_REF]. Nevertheless, the miscibility of the mixture of GL with DPPC forming lamellar phases has never been studied by SAXS.

First of all, the present study investigated the three-dimensional organization of heterogenous GLx/DPPCy mixtures in excess of water, and as a function of DPPC proportion.

Figure 5.A presented the X-Ray diffraction patterns of three mixtures of GLx/DPPCy in water, with DPPC at 66 (black curve), 50 (blue curve), and 33 mol% (green curve), respectively. The results showed that DPPC in large amount of 66 mol% induced a segregation between the components in water. Indeed, on the diffraction pattern (black line), the large diffraction peaks visible at 0.09 and 0.18 Å -1 were identified as the ones related to the L phase formed by pure DPPC at 20°C.

Additionally, a peak and a shoulder were visible on the diffraction pattern at 0.06 and 0.11 Å -1 , respectively, that could correspond to the diffraction pattern of the cubic phase of the MGDG-DGDG mixture in water, consistent with the results of [START_REF] Bottier | Galactosyl headgroup interactions control the molecular packing of wheat lipids in Langmuir films and in hydrated liquid-crystalline mesophases[END_REF]. These results tend to indicate a segregation between DPPC and the MGDG-DGDG mixture in water, leading to the existence of two non-miscible phases. Nevertheless, DPPC may also have disrupted the cubic phase by diluting the MGDG-DGDG interactions, coinciding with the distorted pattern of the MGDG-DGDG cubic phase. When DPPC was added at 50 mol% in the GL mixture, the diffraction pattern (blue line) showed a better miscibility between the three components of the system. Indeed, the intensity of the peaks presumably related to the cubic phase of the GL mixture was decreased and the pic 0.06 Å -1 was no longer visible, tending to show the existence of two miscible phases. These results were consistent with AFM images of the GL50DPPC50 mixture, where a fluid phase was visible, probably composed of a mixture of MGDG, DGDG and DPPC, as well as condensed domains, likely enriched in DPPC. At a ratio of 33 mol% DPPC, the diffraction pattern (green line)

indicates that the concentration of DPPC was not sufficient enough to segregate the three components of the system, and the slightly weaker MGDG-DGDG interactions have probably prevailed, leading to the existence of a single organized phase composed by MGDG, DGDG and DPPC. Overall, the SAXS data confirm the presence of significant segregation in the systems, depending on the DPPC proportion and implying the existence of very selective interactions between the different types of lipids.

To our knowledge, this study is the first on the impact of pS on heterogenous model plant membranes, while the impact of cholesterol on model of animal membranes made of phospholipid bilayers has been extensively studied. To better understand the impact of pS and the interactions between DPPC and MGDG/DGDG, the SAXS diffraction patterns of three other model systems were studied, i.e. MGDG45DPPC45pS10, GL45/DPPC45/pS10 and DGDG45/DPPC45/pS10 (fig. 5.B).

On one hand, results tend to show that MGDG and DGDG did not interact equivalently with the DPPC-pS mixture. First of all, the mixture of DGDG with DPPC-pS did not promote the presence of a single organized phase in water (see green line). Indeed, the diffraction pattern of the pure DPPC was clearly visible, with the L lamellar phase peaks at 0.09, 0.18, and 0.27 Å -1 . The diffraction pattern of the pure DGDG was also clearly evidenced, with the diffraction peaks reflecting the L phase at 0.11 and 0.22 Å -1 . These results are consistent with those obtained by [START_REF] Bottier | Galactosyl headgroup interactions control the molecular packing of wheat lipids in Langmuir films and in hydrated liquid-crystalline mesophases[END_REF] on the wheat DGDG/water system and they highlight the poor miscibility of DGDG with DPPC-pS system. On the contrary, in the case of the MGDG45/DPPC45/pS10 mixture (blue pattern), only one phase was clearly visible, with two peaks reflecting a lamellar phase at 0.09 and 0.18 Å -1 , but slightly shifted compared to pure DPPC in water. In the case of the GL45/DPPC45/pS10 mixture in water (black pattern), a shoulder was also visible at 0.11 Å -1 , compared to the diffraction pattern of the MGDG45/DPPC45/pS10 mixture, highlighting the formation of two phases: one phase enriched in MGDG-DPPC-pS and one phase containing a mixture of MGDG-DGDG. These results showed drastically different interactions between MGDG

CONCLUSION

In this study, tunable Langmuir model membrane of heterogenous assemblies of GL, DPPC and pS were used to study the structural behavior of the main polar lipids of vegetal photosynthetic membrane (MGDG and DGDG) at the air/water interface. The impact of pS and 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC) or both on the structural properties of GL membrane was studied.

The biophysical results obtained confirmed the good surfactant properties of these polar lipids. The addition of pS in the mixed GL systems up to 30 mol% induced a reorientation of the sugar polar heads, leading to a more important packing and thus to a reduction of the lateral elasticity of the monolayer. The study of the compressibility isotherms of the different films also allowed to highlight the complex miscibility of the heterogeneous ternary mixtures, and their singular behavior compared to the pure systems. Indeed, the results obtained on the GL45/DPPC45/pS10 mixture indicated preferential DPPC-pS interactions but a decrease in lateral elasticity despite the disorganization effect of pS on saturated lipids.

The inclusion of pS into the gel phase regions was visible on the AFM images, leading to a thickening of the domains and a local decrease of packing by the appearance of defects. This result highlighted phase miscibility between DPPC and GL, and in particular with MGDG, which was confirmed by AFM images and SAXS diffraction patterns. The favorable interaction between DPPC and pS may have caused the appearance of segregated zones, on one side, devoid of pS and enriched in MGDG-DGDG in the fluid phase, and on the other, enriched in pS and DPPC.

The pS-DPPC-rich areas could also have included the presence of MGDG, due to more favorable DPPC-MGDG interactions than DPPC-DGDG, raising the hypothesis of a heterogeneous MGDG-DPPC-pS composition of the gel phase domains. The characterization of these kinds of plant lipid mixtures is useful for the understanding of mechanisms such as their digestion by specific digestive enzymes.

FIGURES

Figure captions

.

  of GL, DPPC, and pS interfacial composition on collapse pressure and ellipsometric angle values Ellipsometry combined with tensiometry were used on Langmuir trough to determine the values of surface pressure (, mN/m), ellipsometric angle (, °) and limiting molecular area (Ų/molecule) at the collapse of mixed heterogenous films with various compositions in GL, DPPC and pS. The values of  and  at the collapse of the films are presented in Table

Figure 1

 1 Figure 1 Molecular structure and phase behaviour of A) monogalactodiacylglycerol (MGDG) and B) digalactosyldiacylglycerol (DGDG). L standing for lamellar phase and HII standing for hexagonal inverted phase.

Figure 2

 2 Figure2Compression isotherms of 1) GL (red), 2) GL95/pS5 (deep blue), 3) GL90/pS10 (green), 4) GL70/pS30 (black) monolayers at the air/water interface. A) The graph corresponds to the evolution of  (mN/m) as a function of the mean molecular area (Ų/molecules). B) The graph corresponds to the evolution of the surface compressibility moduli (Cs -1 , calculated following the equation (1)), as a function of  (mN/m) of 1), 2), 3) and 4). C) The graph corresponds to the evolution of  (mN/m) as a function of the mean molecular area (Ų/molecules) of the pure pS system. D) The graph corresponds to the mean molecular area (Ų/molecules) at 20 mN/m of each monolayer as a function of the percentage of pS in the systems (pure pS monolayer in light blue). The experiments were performed using Tris HCl buffer at pH 7 and were done in duplicate. Values of  were given with a standard deviation on duplicate measurements of ± 0.2 mN/m.

Figure 3

 3 Figure35x5 µm² and 1x1 µm² AFM topographic images of A) GL, B) GL50/DPPC50, and C) GL45/DPPC45/pS10 model monolayers. The height profiles were performed on a cross-section of the 1x1 µm² image. The Langmuir films were transfer at a surface pressure of 20 ± 0.5 mN/m on a mica plate using the Langmuir Blodgett method. Experiments were performed in a Tris HCl buffer at pH 7 and done in duplicate.

Figure 4

 4 Figure 4 5x5 µm² AFM topographic images of A) MGDG50/DPPC50, B) DGDG50/DPPC50, C) MGDG30/DPPC60/pS10, and D) DGDG30/DPPC60/pS10 monolayers. Zooms to 1x1 µm² were performed on samples C) and D) to allow visualization of defects in condensed areas. The height profiles were performed on three different cross-sections of the 5x5 µm² image. The Langmuir films were transfer at a surface pressure of 20 ± 0.5 mN/m on a mica plate using the Langmuir Blodgett method. The ellipsometric angle value at 20 mN/m was reported to compare the thickness between the four monolayers. Experiments were performed in a Tris HCl buffer at pH 7 and done in duplicate.

Figure 5

 5 Figure5SAXS patterns of the model systems recorded at 20 ± 0.5°C. A) GL/DPPC systems at different ratios: 33:67 mol/mol (black curve), 50:50 mol/mol (blue curve) and 67:33 mol/mol (green curve). B) DGDG/DPPC/pS (45:45:10 mol/mol/mol, green curve), GL/DPPC/pS (45:45:10 mol/mol/mol, black curve), MGDG/DPPC/pS (45:45:10 mol/mol/mol, blue curve)

Acknowledgments

The authors would like to acknowledge the BIOMIF platform ('Biological Molecules at fluid interfaces', IPR, Rennes, France) for allowing the biophysical characterization of samples presented in this article.

C. Bourlieu, V. Vié and J. Kergomard determined the outline and the content of the manuscript. J.

Kergomard wrote the manuscript and all the co-authors participated in the experimental design, the collection, the interpretation of data and the correction and implementation of the manuscript. All co-authors have approved the final article.

DGDG30/DPPC60/pS10 (mol%) mixtures. AFM images and height profiles of these sub-models are presented Figure 4.

In the absence of pS, the 5×5 µm² AFM image of the MGDG50/DPPC50 mixture (Fig. 4.A) showed the presence of LC phase domains (average domain area=0.1 µm²), of irregular shape and 1.7 nm height, as well as a background characteristic of a homogeneous fluid phase. The DGDG50/DPPC50 film (Fig. 4.B) presented a similar fluid background. However, the LC phase domains are more abundant but smaller (average domain area=0.0002 µm²), and present a greater height of 1.9 nm, in accordance with the higher ellipsometric angle for this mixture (6.3° versus 7.4° for the MGDG50/DPPC50 and DGDG50/DPPC50 mixtures, respectively). These observations confirm the different nature of MGDG and DGDG interactions with DPPC and question a possible segregation of MGDG-DGDG interactions in the presence of DPPC.

The addition of pS up to 10 mol% in these two previous sub-model systems changed the appearance of the interfacial films. First, the addition of pS in the MGDG/DPPC system (Fig. 4.C) induced a change in the line tension of the condensed phase domains, which showed irregular edges as well as the presence of defects at the larger domains, probably induced by the presence of pS, similar to the previous observations on the model systems. Nevertheless, a significant decrease of 18% in the height of these domains (1.7 nm vs. 1.4 nm in height, without and with pS, respectively) was observed, consistent with the evolution of the ellipsometric angle (6.3° vs. 5.5°, without and with pS, respectively). The addition of pS in the DGDG/DPPC mixture induced a similar evolution of the interface (Fig. 4.D): the line tension and the thickness of the condensed domains were also modified according to the same trend (16% decrease in thickness), and the presence of defects at the larger domains with irregular edges was still visible. This change in thickness did not showed and DGDG, respectively, and the DPPC-pS mixture, and highlight the preferential miscibility of MGDG with the DPPC-pS mixture.

On the other hand, the comparison between the GL50/DPPC50 (fig. 5. A, blue pattern) and the GL45/DPPC45/pS10 (fig. 5. B, black pattern) mixtures in water allowed us to highlight the impact of 10 mol% of pS in the system. As expected, pS did not formed a phase on its own but triggered a modification of the organization of the existing phases. Indeed, the inclusion of pS in the GL/DPPC system did not induce the appearance of new diffraction peaks, but has led to a contraction of the L diffraction peaks at 0.09 and 0.18 Å -1 , indication a homogenization in the miscibility of the two phases.

Overall, the results were consistent with the AFM images obtained on the GL45/DPPC45/pS10 monolayer, with the visualization of a fluid phase composing the background, probably enriched in MGDG-DGDG, and the presence of a gel phase enriched in MGDG-DPPC, in which the inclusion of pS has induced the formation of defects, leading to a thickening of the gel phase by decreasing the packing. Additionally, the observation of smaller domains in presence of pS on the AFM images was consistent with a lowering of the line tension at the edges of the gel domains, resulting from a better miscibility between the two phases and thus a reduced height mismatch.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. (1) GL 39.9 ± 0.2 7.3 ± 0.5 81.0 ± 0.6

(2) DPPC 55.3 ± 0.2 10.8 ± 0.5 44.1 ± 0.7

(3) GL50/DPPC50 44.1 ± 0.2 8.9 ± 0.5 60.4 ± 1.0 (4) GL90/pS10 41.1 ± 0.1 8.2 ± 0.5 79.5 ± 0.9

(5) GL45/DPPC45/pS10 44.4 ± 0.2 8.6 ± 0.5 55.0 ± 1.7

± stands for calculated standard deviation on duplicated measurement; for , since the system/instrumentation error is ± 0.5°, calculated SD ≤ 0.5 were minored by this instrumentation error. (1) GL 7.5 ± 0.5

(2) GL50/DPPC50 7.1 ± 0.5

(3) GL45/DPPC45/pS10 7.0 ± 0.5 ± stands for calculated standard deviation on duplicated measurement; the system/instrumentation error is ± 0.5°. Calculated SD ≤ 0.5 were minored by this instrumentation error.


Julien Bellec

Louis Rigal

Aurélien Hervouin

Raphaël Martins

Mathieu Lederlin

Nicolas Jaksic

Joël Castelli

Karim Benali

Renaud De Crevoisier

Antoine Simon

Cardiac radioablation for ventricular tachycardia: Which approach for incorporating cardiorespiratory motions into the planning target volume?

Keywords: Cardiac radioablation, ventricular tachycardia, motion management, ITV, CT-4D

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

Introduction

Cardiac radioablation (CR) has recently emerged as a therapeutic option to treat cardiac arrhythmias in patients with recurrent ventricular tachycardia (VT) after being treated using the standard workflow, including antiarrhythmic drugs and/or catheter radiofrequency ablation [START_REF] Cuculich | Noninvasive Cardiac Radiation for Ablation of Ventricular Tachycardia[END_REF]. CR involves the delivery of a single 25 Gy dose to the cardiac substrate identified as the source of abnormal electrical propagation. Both C-arm linear accelerators (linacs) (Elekta and Varian platforms) and the CyberKnife robotic system (Accuray), which are typically implemented for stereotactic body radiotherapy (SBRT), have been considered for CR of VT [START_REF] Van Der Ree | Cardiac radioablation-A systematic review[END_REF][START_REF] Lydiard | A Review of Cardiac Radioablation (CR) for Arrhythmias: Procedures, Technology, and Future Opportunities[END_REF].

One of the main technical challenges when implementing CR is the management of target motion induced by both heart beats and respiration. It has been shown in the literature that both cardiac and respiratory motions impact target position. Prusator et al. [START_REF] Prusator | Evaluation of motion compensation methods for non-invasive cardiac radioablation of ventricular tachycardia[END_REF] reported mean amplitudes of cardiac target displacements caused by heart contraction of 3.4 mm left/right, 4.3 mm anterior/posterior, and 4.1 mm superior/inferior. Mean target displacements induced by respiration have been reported as 3.9 mm left/right, 4.1 mm anterior/posterior, and 4.7 mm superior/inferior. Additionally, these motions are highly patient-specific, depending on the location of the target in the heart and the respiration amplitude [START_REF] Prusator | Evaluation of motion compensation methods for non-invasive cardiac radioablation of ventricular tachycardia[END_REF][START_REF] Knybel | Real-time measurement of ICD lead motion during stereotactic body radiotherapy of ventricular tachycardia[END_REF]. The main target motion management technique used in CR, especially when using a C-arm linear accelerator, is the generation of a cardiorespiratory internal target volume (ITV) based on cardiac-gated and/or respiration-gated four-dimensional computed-tomography (4D-CT) scans [START_REF] Lydiard | A Review of Cardiac Radioablation (CR) for Arrhythmias: Procedures, Technology, and Future Opportunities[END_REF]. A cardiorespiratory ITV is defined as encompassing all positions of the target during heartbeats and respiration. However, there is no consensus on the optimal approach for its generation, with the methods considered in the literature generally only weakly described. Therefore, a clinically feasible approach for defining the cardiorespiratory ITV must be defined and validated. It should answer several questions that remain open. For example, should cardiac motion be specifically integrated into the definition of cardiorespiratory ITV? What is the efficiency of a fixed-margin-based approach versus a personalized strategy? How can cardiac and respiratory 4D-CT scans be utilized and combined? This study sought to answer these questions. Hence, we evaluated different methods of defining the cardiorespiratory ITV based on cardiac-gated and respiration-gated 4D-CT scans, from the use of fixed margins to the utilization of the full dynamic sequences. To identify the most appropriate method, the proposed approaches were evaluated and compared using both geometric and dosimetric criteria.

Materials and methods

4D imaging description

CT images were acquired from four patients who were candidates to receive cardiac SBRT for VT.

For each patient, two CT sequences were acquired: a cardiac-gated 4D-CT scan [START_REF] Clayton | Assessment of the myocardium with cardiac computed tomography[END_REF] and a respiratorygated 4D-CT scan. Depending on the patient, other imaging modalities were used to delineate the clinical target volume (CTV) (TEP and/or electro-anatomical mapping).

Cardiac CT scan acquisition was performed with breath holding at submaximal inspiration, after thorough training, on a Somaton Force 384 slice CT scanner (Siemens, Munich, Germany). The reconstruction field of view was adjusted to include the entire heart, and intravenous contrast was administered to visualize the cardiac cavities. To demonstrate the entire cardiac cycle, 10 CT images sets were retrospectively reconstructed based on the electrocardiogram (ECG) with a slice thickness of 0.5 mm. Additionally, an average cardiac CT dataset was reconstructed from the 10 phases of the cardiac 4D-CT scan.

The respiratory-gated 4D-CT images were acquired under free breathing on a Confidence 24-slice CT scanner (Siemens, Munich, Germany). A sentinel surface-guided system (C-RAD, Stockholm, Sweden) was used to record respiratory signals during image acquisition and binning. A total of 10 respiratory phase-sorted CT datasets were retrospectively reconstructed with a slice thickness of 2 mm. Additionally, an average respiratory CT dataset was reconstructed from the 10 phases of the respiratory 4D-CT scan.

Cardiac 4D-CT datasets, respiratory 4D-CT datasets, and other useful imaging datasets, if available, were all imported into a MIM platform v7.0 (MIM Software Inc, Cleveland OH, USA), which was used for target volume definition.

Cardiorespiratory ITV creation

For each patient, five different methods were employed to generate the cardiorespiratory ITV. Figure 1 illustrates the concept of the methods. All were decomposed in two steps: first, the cardiac motions were integrated to define a cardiac ITV using the cardiac 4D-CT information, and then the respiratory motions were incorporated through respiratory 4D-CT images to generate the final cardiorespiratory ITV. The five evaluated methods differed in the number of phases incorporated from the 4D-CT scans to characterize cardiac and respiratory motions as defined below:

-Card_10ph/resp_10ph: It utilizes all the available images, i.e., combined all 10 cardiac phases with all 10 respiratory phases to generate the cardiorespiratory ITV.

-Card_2ph/resp_10ph: It generates cardiorespiratory ITV using the two extreme phases of the cardiac cycle combined with all 10 respiratory phases.

-Card_1ph/resp_10ph: It simulates a commonly used approach that consists of evaluating the combined cardiorespiratory target motions using only the respiratory 4D-CT scan [START_REF] Cuculich | Noninvasive Cardiac Radiation for Ablation of Ventricular Tachycardia[END_REF][START_REF] Robinson | Phase I/II Trial of Electrophysiology-Guided Noninvasive Cardiac Radioablation for Ventricular Tachycardia[END_REF][START_REF] Knutson | Radiation Therapy Workflow and Dosimetric Analysis from a Phase 1/2 Trial of Noninvasive Cardiac Radioablation for Ventricular Tachycardia[END_REF][START_REF] Lloyd | Clinical Experience of Stereotactic Body Radiation For Refractory Ventricular Tachycardia in Advanced Heart Failure Patients[END_REF][START_REF] Scholz | Risen from the dead: Cardiac stereotactic ablative radiotherapy as last rescue in a patient with refractory ventricular fibrillation storm[END_REF].

The assumption behind this approach is to consider that respiratory-phase-binned 4D-CT images are blurred by cardiac contraction and thus implicitly integrate cardiac motion information.

-Card_FM/resp_10ph: It simulates the use of fixed margins to compensate cardiac motions. An isotropic 3 mm margin has been used since it was reported in a study where patients were treated using a Cyberknife [START_REF] Gianni | Stereotactic arrhythmia radioablation for refractory scar-related ventricular tachycardia[END_REF]. In our study, the resulting volume was propagated over the 10 respiratory phases for the integration of respiratory motions.

-Card_2ph/resp_2ph: It integrates cardiorespiratory motions using only the two extreme phases of both the cardiac and respiratory cycles.

The practical flowchart for generating cardiorespiratory ITV with the card_10ph/resp_10ph method is described in detail in Figure 2. The creation of the cardiorespiratory ITV was divided into three main steps:

-First, the arrhythmogenic substrate (defined as the CTV in this study for consistency with conventional radiation oncology terminology) was delineated by a cardiologist on the end-ofdiastole CT dataset. Delineation of the end-of-diastole CTV was performed manually after aligning image planes with the three main axes of the heart [START_REF] Brownstein | Method and Atlas to Enable Targeting for Cardiac Radioablation Employing the American Heart Association Segmented Model[END_REF].

-Next, the end-of-diastole CTV was propagated from the end-of-diastole phase to the nine other cardiac phases through diffeomorphic demons deformable image registration (DIR) [START_REF] Vercauteren | Diffeomorphic demons: Efficient nonparametric image registration[END_REF].

A cardiac ITV was generated by merging the CTV from different numbers of phases, based on the method being considered. For the card_10ph/resp_10ph method, the cardiac ITV was created by the union of all 10 cardiac phase-related CTVs. For the card_2ph/resp_10ph and card_2ph_/resp_2ph methods, the cardiac ITV was defined as the union of the end-diastole CTV and the end-of-systole propagated CTV. For these three approaches, the resulting cardiac ITV was transferred to the average cardiac 4D-CT dataset. For the card_1ph/resp_10ph, no cardiac ITV was used, and only the end-of-diastole CTV was considered from the cardiac 4D-CT information. With the card_FM/resp_10ph method, a non-patient-specific cardiac ITV was defined as the end-of-diastole CTV, plus a fixed 3 mm isotropic margin expansion.

-Finally, the cardiorespiratory ITV with combined cardiac and respiratory target motions was generated by incorporating the respiratory 4D-CT scans. For this purpose, the cardiac ITV (or the end-of-diastole CTV for the card_1ph/resp_10ph method) was propagated on the 10 respiratory phases using a registration of the average cardiac 4D-CT image (or the end-ofdiastole phase CT for the card_1ph/resp_10ph and card_FM/resp_10ph methods) to each of the 10 phases of the respiratory 4D-CT scan. The registration process was performed as follows. Initially, automatic rigid registration based on intensity values was performed on the full volume of both images to roughly align the structures. Then, a second automatic rigid registration was performed on a volume of interest encompassing the whole heart only, to adjust the heart-to-heart registration while ignoring other high-intensity structures. Each registration result was visually inspected and validated by a radiation oncologist and a cardiologist. Using the resulting transformation matrices, cardiac ITV (or end-of-diastole CTV for the card_1ph/resp_10ph method) were propagated to all 10 phases of the respiratory 4D-CT scan and finally merged to generate the cardiorespiratory ITV.

The resulting cardiorespiratory ITVs from the five methods were finally associated with the average respiratory 4D-CT dataset, which is the commonly used 4D-CT dataset for treatment planning and dose calculation when implementing an ITV-based strategy [START_REF] Schwarz | Geometrical and dosimetrical uncertainties in hypofractionated radiotherapy of the lung: A review[END_REF][START_REF] Guckenberger | ESTRO ACROP consensus guideline on implementation and practice of stereotactic body radiotherapy for peripherally located early stage non-small cell lung cancer[END_REF].

Geometric evaluation

For evaluation purposes, the cardiorespiratory ITV from the card_10ph/resp_10ph method was considered as the reference as it incorporates all available information from the cardiac and respiratory 4D-CT scans. For each method, the geometric target coverage (Cg) and geometric spill (Sg) were quantified. The concepts of coverage and spill are illustrated in Figure 3. Here, Cg refers to the proportion of the evaluated cardiorespiratory ITV overlapping the reference cardiorespiratory ITV (i.e., generated with the card_10ph/resp_10ph method), and Sg refers to the proportion of the evaluated ITV that did not overlap with the reference cardiorespiratory ITV.

Dosimetric evaluation

For each patient and each method, a planning target volume (PTV) was generated using an isotropic 3 mm margin around the cardiorespiratory ITV. The 3 mm expansion was chosen based on the previous work of our group, which demonstrated that a 3 mm margin ensured correct target coverage for lung targets when using an ITV-based strategy in a Carm linac with advanced 4D-CBCT-based image guidance [START_REF] Bellec | ITV versus mid-ventilation for treatment planning in lung SBRT: a comparison of target coverage and PTV adequacy by using in-treatment 4D cone beam CT[END_REF]. A volumetric modulated arc therapy (VMAT) treatment plan was simulated using a v9.10 treatment planning system (Philips Radiation Oncology Systems, Milpitas, USA) with a VersaHD linac (Elekta, Crawley, UK). The treatment plans were created using two coplanar arcs of 358° with a 6 MV flattening filter free (FFF) photon beam. A dose of 25 Gy was prescribed to the PTV in a single fraction. For each treatment plan, the goal was to cover at least 95% of the PTV with at least 95% of the prescription dose (PTV D95% ≥ 23.75 Gy) while respecting target and organ-atrisk dose constraints from the RAVENTA trial [START_REF] Blanck | Radiosurgery for ventricular tachycardia: preclinical and clinical evidence and study design for a German multi-center multiplatform feasibility trial (RAVENTA)[END_REF]. Treatment plans were optimized to place hotspots within the ITV (with a maximum allowed dose of 35 Gy). Tuning structures (3, 15, and 40 mm rings) were used to increase the dose fall-off. Over all the patients and PTV approaches, treatment plans have been optimized to achieve a PTV coverage equal or higher than 95% (VPTV, 23.75 Gy ≥ 95%). To avoid any biases, a PTV coverage normalization was performed, for each patient, over the different PTV approaches. In practice, all patient-specific treatment plans were normalized after the optimization process in order to achieve the same PTV coverage with a coverage variation within +/-0.5% over all approaches.

Each method was evaluated dosimetrically by calculating the dosimetric coverage (Cd) and dosimetric spill (Sd) indices following the definitions described in Figure 4. Here, Cd refers to the proportion of the prescription isodose volume overlapping the reference PTV, and Sd refers to the proportion of the prescription isodose volume that did not overlap with the reference PTV. The prescription isodose volume was considered 95% of the prescribed dose (i.e. 23.75 Gy). The isodose value covering 95% of the reference PTV (D95%) and the median dose (D50%) to the heart volume minus the reference PTV were also reported for each method.

Results

The data of the four patients enrolled for CR for VT in our institution were processed using different approaches.

Geometric impact of cardiorespiratory motions on the target volume

Figure 5 illustrates the CTV, cardiac ITV, and cardiorespiratory ITV obtained using the card_10ph/resp_10ph method. The CTV differed among patients, both in terms of localization (septal, infero-basal, infero-lateral, and inferior, respectively, for patients 1-4) and volume (CTV ranging from Compared to the CTV, when incorporating all cardiac CT images, the cardiac ITV increased by a proportion of 39% to 75%. When incorporating respiratory motion using all respiratory CT images, the ITV was increased between 27% and 100%. In total, the generation of the cardiorespiratory ITV using all cardiac and respiratory 4D-CT images increased the target volume by between 76% (patient 1) and 249% (patient 4).

Geometric evaluation

Figure 6 presents the values of Cg and Sg obtained for the cardiorespiratory ITVs of the evaluated methods, with respect to the reference ITV (i.e., the ITV obtained by the card_10ph/resp_10ph method). The values obtained by this method are considered, by definition, as being optimal (Cg = 100%, Sg = 0%). The highest Cg obtained by another approach was the card_FM/resp_10ph method.

However, this method resulted in large Sg values, ranging from 34% (patient 3) to 52 % (Patient 1).

The card_2ph/resp_10ph method produced a Cg larger than 89% for all patients. The Cg obtained by the card_1ph/resp_10ph method ranged from 67% and 85%, and the values obtained by the card_2ph/resp_2ph method were between 72% and 88%. These methods produced a Sg value equal to 0%, except for card_1ph/resp_10ph of patient 2, with 4%.

Dosimetric evaluation

Figure 7 reports the volumes of the PTV generated using the five evaluated methods. As the card_10ph/resp_10ph method can be considered as the most robust approach for defining the cardiorespiratory ITV, the resulting PTV was considered as the reference PTV. Table 1 reports the variation of PTV volumes as a function of the cardiorespiratory ITV approach implemented. By comparison, the method using fixed margins for cardiac motion management (i.e., card_FM/resp_10ph method) systematically overestimated the PTV by 20% (patient 3) to 37 % (patient 1). Conversely, the exclusion of cardiac motion using the card_1ph/resp_10ph method produced the largest underestimation of PTV, with a mean underestimation of 18%. Meanwhile, the card_2ph/resp_10ph and card_2h/resp_2ph methods produced mean underestimations of 7% and 15%, respectively.

The PTV coverage (VPTV, 23.75 Gy) over all evaluated approaches ranged from 96.8% to 97.2%, from 95.4% to 95.6%, from 95.0% to 95.5% and from 96.0% to 96.2% for patients 1, 2, 3 and 4, respectively. The dose distribution optimized on the PTV created with the card_10ph/resp_10ph method is illustrated in Figure 8 for the four patients.

Figure 9 shows the Cd and Sd values obtained by the five methods with respect to the reference PTV.

Further, the D95% of the and D50% of the heart minus the PTV were reported.

The highest Cd (Fig. 9a) was obtained using the card_FM/resp_10ph method, with a mean Cd of 99%.

In decreasing order of performance, the methods card_10ph/resp_10ph, card_2ph/resp_10ph, card_2ph/resp_2ph, and card_1ph/resp_10ph produced mean Cd values of 96%, 93%, 91%, and 89%, respectively. This was confirmed using the D95% of the reference PTV, with the highest values for the card_FM/resp_10ph method producing a mean value of 26.3 Gy (higher than the prescribed dose of 25 Gy). The lowest values were obtained by using the card_1ph/resp_10ph method with 21.5 Gy for patient 4 (86% of the prescribed dose).

For dosimetric spill, the worst results (i.e., the highest Sd values) were obtained by the card_FM/resp_10ph method, with a mean value of 35%, and a maximum of 41%. In contrast, the lowest Sd was obtained by the card_2ph/resp_2ph and card_1ph/Resp_10ph methods, with mean Sd values of 12%, followed by the card_2ph/resp_10ph and card_10ph/resp_10ph methods, with mean Sd values of 15% and 19%, respectively. The D50% delivered to the heart minus PTV were all below the recommended 5 Gy value, except for the card_FM/resp_10ph method for patients 1 and 2.

Discussion

Due to the high delivered dose (25 Gy in a single fraction), CR of VT requires optimal definition of the PTV to obtain the expected therapeutic ratio, which involves the accurate incorporation of both cardiac-induced and respiratory-induced target motion. However, there is no consensus on the ideal management of these motions. In this study, we compared the geometric and dosimetric impacts of different methods used to generate a cardiorespiratory ITV by utilizing cardiac and respiratory 4D-CT scans. The variability among all considered indicators for these patients must be noted. Target volumes and localizations and the impact of cardiac and respiratory motions, such as coverage of target and organs at risk, varied significantly. To date, CTVs ranging from 3.5 to 89.0 mL have been reported [START_REF] Lydiard | A Review of Cardiac Radioablation (CR) for Arrhythmias: Procedures, Technology, and Future Opportunities[END_REF]. The volumes obtained in our study were included in this range (between 15 and 78 mL).

For patient 2, a larger CTV volume was observed compared to other patients since the CTV was delineated using only CT and PET imaging because of a contraindication to electro-anatomical exploration.

When using conventional C-arm linacs, different approaches have been reported to incorporate the cardiorespiratory motion uncertainties into the PTV [START_REF] Cuculich | Noninvasive Cardiac Radiation for Ablation of Ventricular Tachycardia[END_REF][START_REF] Lloyd | Clinical Experience of Stereotactic Body Radiation For Refractory Ventricular Tachycardia in Advanced Heart Failure Patients[END_REF][START_REF] Scholz | Risen from the dead: Cardiac stereotactic ablative radiotherapy as last rescue in a patient with refractory ventricular fibrillation storm[END_REF][START_REF] Krug | Stereotactic body radiotherapy for ventricular tachycardia (cardiac radiosurgery): First-in-patient treatment in Germany[END_REF]. Most approaches rely on a passive motion-encompassing technique using an ITV-based strategy. Some teams assessed combined cardiorespiratory motions on the sole respiration-correlated 4D-CT scan [START_REF] Cuculich | Noninvasive Cardiac Radiation for Ablation of Ventricular Tachycardia[END_REF][START_REF] Robinson | Phase I/II Trial of Electrophysiology-Guided Noninvasive Cardiac Radioablation for Ventricular Tachycardia[END_REF][START_REF] Knutson | Radiation Therapy Workflow and Dosimetric Analysis from a Phase 1/2 Trial of Noninvasive Cardiac Radioablation for Ventricular Tachycardia[END_REF][START_REF] Lloyd | Clinical Experience of Stereotactic Body Radiation For Refractory Ventricular Tachycardia in Advanced Heart Failure Patients[END_REF][START_REF] Scholz | Risen from the dead: Cardiac stereotactic ablative radiotherapy as last rescue in a patient with refractory ventricular fibrillation storm[END_REF], while others employed ECG-gated cardiac 4D-CT scans to isolate cardiac motion [START_REF] Krug | Stereotactic body radiotherapy for ventricular tachycardia (cardiac radiosurgery): First-in-patient treatment in Germany[END_REF][START_REF] Neuwirth | Stereotactic radiosurgery for ablation of ventricular tachycardia[END_REF][START_REF] Cvek | Cardiac Radiosurgery for Malignant Ventricular Tachycardia[END_REF]. When using a CyberKnife system, an active respiratory compensation technique was systematically implemented [START_REF] Adler | The Cyberknife: A Frameless Robotic System for Radiosurgery[END_REF]; thus, only cardiac ITV was considered [START_REF] Neuwirth | Stereotactic radiosurgery for ablation of ventricular tachycardia[END_REF][START_REF] Cvek | Cardiac Radiosurgery for Malignant Ventricular Tachycardia[END_REF][START_REF] Loo | Stereotactic Ablative Radiotherapy for the Treatment of Refractory Cardiac Ventricular Arrhythmia[END_REF][START_REF] Zeng | Stereotactic body radiation therapy for refractory ventricular tachycardia secondary to cardiac lipoma: A case report[END_REF]. Gianni et al. [START_REF] Gianni | Stereotactic arrhythmia radioablation for refractory scar-related ventricular tachycardia[END_REF] used fixed margins to account for cardiac motion. Other teams did not explicitly take into account the cardiac target motion when defining the planning target volume [START_REF] Peichl | A case report of successful elimination of recurrent ventricular tachycardia by repeated stereotactic radiotherapy: the importance of accurate target volume delineation[END_REF][START_REF] Mayinger | First magnetic resonance imaging-guided cardiac radioablation of sustained ventricular tachycardia[END_REF].

By considering all phases of both CT scans, we first demonstrated that both cardiac and respiratory motions have an impact on the target volume. Cardiac motion alone produced an increase in target volume up to 75%, while respiratory motion alone could double the target volume. When combining both motions, the increase in the target volume reached up to 249%. When implementing an ITVbased strategy, the integration of cardiorespiratory motion into the PTV during treatment planning is thus considered essential to ensure optimal treatment delivery.

In this study, we propose a workflow (card_10ph/resp_10ph) to generate a patient-specific cardiorespiratory ITV using all phases of both cardiac and respiratory CT scans. The workflow is based on the definition of a cardiac ITV from all phases of the cardiac 4D-CT scan using deformable image registration, and its propagation to all phases of the respiratory 4D-CT scan. However, its implementation is complex. In the absence of validated deformable registration tools enabling the propagation of the CTV from one phase of the cardiac CT scan to the others, the delineation of each phase may be required, which is time consuming and prone to variability. Thus, simplified approaches based on a subset of images would be clinically useful.

To define the cardiorespiratory ITV, methods based on the incorporation of a unique cardiac phase, with or without margins (card_FM/resp_10ph and card_1ph/resp_10ph), were not able to generate adequate target volumes for all patients. For example, the card_1ph/resp_10ph method resulted in geometric coverage Cg and dosimetric coverage Cd values for patient 1 of only 75% and 84%, respectively, which cannot be considered acceptable. The addition of a fixed 3 mm margin to the CTV improved target coverage for all patients, although it also increased the size of the PTV, and consequently the prescription isodose volume that spilled outside the target volume. Thus, the surrounding organs were unnecessarily exposed to risk. Other margin values may be considered, however, since the dynamics are highly patient specific, no unique value would be optimal for all the patients. The high spill phenomenon observed while using fixed margins is amplified when the shape of the target is close to a "C", which happens frequently considering the conical shape of the left ventricle (e.g. for patient 2). In this study, we evaluated the utilization of the two extreme phases of both CT scans, that is, end-of-diastole and end-of-systole for the cardiac CT scan, and end-ofinspiration and end-of-expiration for the respiratory CT scan. The method based on the two extreme phases for both CT scans (card_2ph/resp_2ph) did not produce adequate geometric and dosimetric target coverage for all patients (e.g., patient 1 produced a geometric coverage Cg of 74% and a dosimetric coverage of 83%). In contrast, utilizing the two extreme cardiac phases and all respiratory phases (card_2ph/resp_10ph) correctly characterized the cardiorespiratory motion, with a geometric coverage Cg ranging from 89% to 94%, leading to a clinically acceptable dosimetric target coverage, Cd, for all patients (ranging from 92% to 95%). All these approaches except the card_FM/resp_10ph method were, by definition, smaller than the reference approach (card_10ph/resp_10ph), thus resulting in geometric spill values equal or close to 0. The non-zero geometrical spill values corresponded to a registration between cardiac and respiratory CTs based on another image than the reference approach. Indeed, if the methods based on a dynamic cardiac CT exploited the average CT image, the method card_1ph/resp_10ph exploited the registration of the end-of-diastole phase, thus potentially leading to a difference in terms of resulting geometrical transform. Concerning the dosimetric indices, the ranking of the different methods was confirmed by the D95% index, yet it was important to note that no method presented here resulted in a D95% that was below 20Gy. The median dose to heart confirms the spill induced by card_FM/resp_10ph. It should also be noted that, in the context of CR for VT, the balance between efficacy (represented by the coverage in this study) and safety (represented by the spill) is an open question, especially because CR is still considered a last resort treatment for patients for whom standard treatments have failed.

This study demonstrates that using a single static cardiac CT scan combined with a respirationcorrelated 4D-CT scan is not a sufficiently robust approach to define the cardiorespiratory ITV. The method card_2ph/resp_10ph may be considered an acceptable compromise in terms of accuracy and clinical feasibility for incorporating cardiorespiratory motions into the target volume. It may be reasonable to manually delineate the target on the two cardiac phases corresponding to the end-ofdiastole and end-of-systole. Moreover, the incorporation of all respiratory phases can be considered clinical routine as it is implemented in the context of lung SBRT [START_REF] Schwarz | Geometrical and dosimetrical uncertainties in hypofractionated radiotherapy of the lung: A review[END_REF][START_REF] Guckenberger | ESTRO ACROP consensus guideline on implementation and practice of stereotactic body radiotherapy for peripherally located early stage non-small cell lung cancer[END_REF].

The main limitation of this study is the small patient cohort, which is due to the treatment of VT by SBRT still being an emerging technique that is only applied to a quite limited number of patients fitting specific criteria. Some uncertainties were not considered in the study, especially those related to the delineation of the target and the registration process. However, as all methods that were compared used the same delineations and registration method, these uncertainties should not have an impact on the conclusions drawn. Other imaging modalities to integrate cardiac motion may have been considered, such as echography [START_REF] Loo | Stereotactic Ablative Radiotherapy for the Treatment of Refractory Cardiac Ventricular Arrhythmia[END_REF][START_REF] Jumeau | Rescue procedure for an electrical storm using robotic non-invasive cardiac radio-ablation[END_REF]. Other methods to incorporate cardiorespiratory motions into the PTV may also have been considered such as probabilistic approaches as the mid-ventilation concept [START_REF] Thomas | An evaluation of the mid-ventilation method for the planning of stereotactic lung plans[END_REF][START_REF] Vander Veken | Incorporation of tumor motion directionality in margin recipe: The directional MidP strategy[END_REF]. The evaluation of a probabilistic approach in the context of CR would deserve further studies as it would require specific metrics estimating the target coverage over time. The impact on target delineation of the breath holding method when acquiring the cardiac CT scan may also be studied, since the shape of the left ventricle may be influenced by the respiration phase [START_REF] Claessen | Interaction between respiration and right versus left ventricular volumes at rest and during exercise: a real-time cardiac magnetic resonance study[END_REF]. In addition, the development of a dynamic anthropomorphic phantom would be a useful tool to simulate and evaluate the different approaches used for the management of cardiorespiratory motion in CR [START_REF] Erdoğan | Patient-specific tumor and respiratory monitoring phantom design for quality controls of stereotactic ablative body radiotherapy in lung cancer cases[END_REF]. Finally, all treatment plans were generated by considering treatment on a C-arm linac; we did not consider the use of CyberKnife robotic systems, which is a promising modality for the treatment of tachycardia [START_REF] Neuwirth | Stereotactic radiosurgery for ablation of ventricular tachycardia[END_REF]. MRI-linac is another quite promising modality, owing to its ability to visualize the heart in detail during the entire treatment [START_REF] Mayinger | First magnetic resonance imaging-guided cardiac radioablation of sustained ventricular tachycardia[END_REF]. Methods to derive the PTV from the CTV must be designed specifically to take advantage of the target localization abilities of these treatment devices [START_REF] Dieterich | SBRT targets that move with respiration[END_REF].

This study demonstrates that the method implemented to integrate cardiac and respiratory motions into the target volume has an important impact on the geometry of the PTV and on the dose received by the target and surrounding organs at risk. Considering that the approaches used in the literature are heterogeneous, defining standardized approaches should be considered as a priority to safely deliver cardiac SBRT.

Conclusion

Cardiac and respiratory motions induce a large increase in target volume in CR when no active motion compensation technique is implemented. These patient-specific cardiorespiratory motions should be assessed for each patient by combining information from cardiac-gated and respiratory-gated 4D-CT scans to optimize the PTV. If the incorporation of the entire 4D-CT datasets may be challenging in clinical practice, we have shown that using only two extreme cardiac phases combined with the 10 respiratory phases is a good compromise between efficiency and clinical feasibility for the generation of a cardiorespiratory ITV. The use of only a single cardiac phase with or without a fixed margin is not be recommended for optimal delivery of CR. Further studies with larger patient cohorts are warranted to support this conclusion.