Yvan Capowiez
email: yvan.capowiez@inrae.fr

Stéphane Sammartino

Thomas Keller

Nicolas Bottinelli

Decreased burrowing activity of endogeic earthworms and effects on water infiltration in response to an increase in soil bulk density

Keywords: compaction, burrow system, Aporrectodea caliginosa, Allolobophora chlorotica, behaviour

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

Introduction

Compaction, partly due to an increase of engine loads, is a major threat to agricultural and forest soils and their resilience [START_REF] Hansen | Effects of manure treatment and soil compaction on plant production of a dairy farm system converting to organic farming practice[END_REF]. Earthworms are particularly sensitive to soil compaction as they burrow their way in the soil either by pushing the soil aside or ingesting it [START_REF] Beylich | Evaluation of soil compaction effects on soil biota and soil biological processes in soils[END_REF][START_REF] Keller | Long-term soil structure observatory for monitoring post-compaction evolution of soil structure[END_REF]. Indeed, ingestion is the only possible way of moving in highly compacted soils [START_REF] Dexter | Tunnelling in soil by earthworms[END_REF] and often results in higher quantities of excreted surface casts [START_REF] Joschko | Assessment of earthworm burrowing efficiency in compacted soil with a combination of morphological and soil physical measurements[END_REF].

A high BD also means that more energy is spent burrowing [START_REF] Ruiz | Experimental evaluation of earthworm and plant root soil penetration-cavity expansion models using cone penetrometer analogs[END_REF]. Although earthworms generally avoid highly compacted zones (Stovold et al., 1994), epi-anecic such as Lumbricus terrestris, earthworms have been observed to burrow in them under semi-field conditions in the presence of a plough pan [START_REF] Capowiez | The influence of tillage type and compaction on earthworm communities and the consequences for macroporosity and water infiltration in crop fields[END_REF]. Epi-anecics and other species have also been found to burrow in compacted zones below wheels tracks under field conditions [START_REF] Capowiez | Role of earthworm in regenerating soil structure after compaction in reduced tillage systems[END_REF].

Thus bulk density (BD) is one important factor indirectly governing earthworm abundance in croplands [START_REF] Chan | Soil compaction controls the abundance, biomass and distribution of earthworms in a single dairy farm in south-eastern Australia[END_REF][START_REF] Ouellet | A heuristic model to predict earthworm biomass in agroecosystems based on selected management and soil properties[END_REF][START_REF] Capowiez | The influence of tillage type and compaction on earthworm communities and the consequences for macroporosity and water infiltration in crop fields[END_REF], forests [START_REF] Jordan | Effect of soil compaction and organic matter removal on two earthworm populations and some soil properties in a hardwood forest[END_REF][START_REF] Bottinelli | Slow recovery of earthworm populations after heavy traffic in two forest soils in northern France[END_REF], tallgrass prairies [START_REF] Althoff | Response of soil microbial and invertebrate communities to tracked vehicle disturbance in tallgrass prairie[END_REF] and urban soils [START_REF] Smetak | Earthworm population density and diversity in different-aged urban systems[END_REF]. Among earthworms, anecic and epi-anecic earthworms (sensu [START_REF] Bouché | Lombriciens de France. Ecologie et systématique[END_REF]) are thought to be more powerful axially [START_REF] Keudel | Axial and radial pressure exerted by earthworms of different ecological groups[END_REF] and thus less sensitive to compaction [START_REF] Beylich | Evaluation of soil compaction effects on soil biota and soil biological processes in soils[END_REF]. Paradoxically, much more information has been reported on the effects of soil compaction on anecic or epi-anecic earthworm behavior than on other ecological categories. [START_REF] Rushton | The effects of soil compaction on Lumbricus terrestris and its possible implications for populations on land reclaimed from open-cast coal mining[END_REF] and [START_REF] Joschko | Assessment of earthworm burrowing efficiency in compacted soil with a combination of morphological and soil physical measurements[END_REF] demonstrated that burrow length was decreased when L. terrestris was challenged with increased BD. Stovold et al. (1994) made a similar observation for Aporrectodea nocturna in a choice experiment. [START_REF] Kretzschmar | Burrowing ability of the earthworm Aporrectodea longa limited by soil compaction and water potential[END_REF] estimated that Aporrectodea longa burrowing activity, as assessed by surface cast production, decreased for higher BD. This bias towards the anecic or epi-anecic species is explained by the fact that these species have a greater influence on water infiltration since their burrows are more vertical, more continuous (because less refilled) and have surface openings [START_REF] Capowiez | Morphological and functional characterisation of the burrow systems of six earthworm species (Lumbricidae)[END_REF]. In fact, compaction impacts not only burrow numbers but also burrow continuity [START_REF] Schäffer | Changes in the macro-pore structure of restored soil caused by compaction beneath heavy agricultural machinery: a morphometric study[END_REF][START_REF] Capowiez | Role of earthworm in regenerating soil structure after compaction in reduced tillage systems[END_REF] and this in turn can drastically reduce water infiltration [START_REF] Kim | Compaction effects on soil macropore geometry and related parameters for an arable field[END_REF]. In comparison, information regarding the effects of compaction on endogeic earthworms is scarce. [START_REF] Langmaack | Quantitative analysis of earthworm burrow systems with respect to biological soil-structure regeneration after soil compaction[END_REF] studied A. caliginosa burrowing behavior in compacted vs non-compacted soils and concluded that in response to compaction this species increased burrow continuity but no clear trend was observed for burrow length. [START_REF] Dexter | Tunnelling in soil by earthworms[END_REF] also found that high BD did not have a negative impact on A. caliginosa burrowing. In contrast, [START_REF] Söchtig | Effect of soil compaction on activity and biomass of endogeic lumbricids in arable soil[END_REF] determined that A. caliginosa burrow length decreased linearly when pore volume decreased (and thus BD increased).

In most of these studies, the effects of high BD on earthworms were measured indirectly, i.e. either by assessing surface cast production (assuming most of the casts were produced at the soil surface) or the burrows on the outer borders of soil cores (assuming these burrows were representative of burrows in the cores). This latter case is due to the absence of non-invasive techniques for estimating burrows in soil cores in past decades. Nevertheless the first application of X-ray tomography to analyze earthworm systems was in the early 90s [START_REF] Joschko | A nondestructive method for the morphological assessment of earthworm burrow systems in 3 dimensions by X-ray computed-tomography[END_REF] and medical scanners are now readily available to solve this problem. Microtomography with an higher resolution can now also be used (Balseiro-Romano et al., 2020). Another point that sometimes limits the relevance of past studies is the fact that they were observed using choice experiments: earthworms were placed in mesocosms containing soils with different BD and their burrowing activity was estimated in the different situations (Stovold et al., 1994;[START_REF] Söchtig | Effect of soil compaction on activity and biomass of endogeic lumbricids in arable soil[END_REF]. This kind of study provides information on earthworm preferences but not really on their ability to burrow and live in compacted soils. Furthermore, in most studies only two to three BD were tested [START_REF] Joschko | Assessment of earthworm burrowing efficiency in compacted soil with a combination of morphological and soil physical measurements[END_REF][START_REF] Joschko | Earthworm burrow system development assessed by means of X-ray computed tomography[END_REF][START_REF] Langmaack | Quantitative analysis of earthworm burrow systems with respect to biological soil-structure regeneration after soil compaction[END_REF]Stovold et al., 1994;[START_REF] Jégou | Impact of soil compaction on earthworm burrow systems using X-ray computed tomography. Preliminary study[END_REF][START_REF] Capowiez | Role of earthworm in regenerating soil structure after compaction in reduced tillage systems[END_REF] whereas recent studies suggested that depending on the soil texture, earthworm responses in terms of burrowing are not always monotonic (see Figure 5 in [START_REF] Pöhlitz | Computed tomography as an extension of classical methods in the analysis of soil compaction, exemplified on samples from two tillage treatments and at two moisture tensions[END_REF].

Overall, there is an increasing demand for accurate estimates of the effects of BD on earthworm burrowing [START_REF] Roeben | Towards a spatiotemporally explicit toxicokinetic-toxicodynamic model for earthworm toxicity[END_REF] since this kind of information is required, for example, to simulate earthworm behavior and better assess their real exposure to pesticides. However, there is a scarcity of useable data in the literature, especially for endogeic species.

Thus, in the present study, we carried out a lab experiment with soil cores compacted at five different BD currently observed in orchard soils close to Avignon, France. We then studied the resulting effects on the burrow system characteristics of two very common endogeic species (Aporrectodea caliginosa and Allolobophora chlorotica). We also determined how BD and earthworm species modified water infiltration. Furthermore, it is known that earthworms can compact the soil when they burrow [START_REF] Rogasik | Micro-scale dry bulk density variation around earthworm (Lumbricus terrestris L.) burrows based on X-ray computed tomography[END_REF][START_REF] Capowiez | Using X-ray tomography to quantify earthworm bioturbation non-destructively in repacked soil cores[END_REF][START_REF] Milleret | Impact of roots, mycorrhizas and earthworms on soil physical properties as assessed by shrinkage analysis[END_REF] and thus we assessed whether the lateral compaction around the burrows was influenced by soil BD for these species.

Material and Methods

Soil and earthworms

Soil (0-20 cm depth) and earthworms were sampled in an orchard abandoned since at least ten years in Avignon, in the south east of France. The soil has the following characteristics: pH = 8.2, clay = 19.7%, silt = 50.8%, sand = 29.5%, OM = 3.4%, CEC = 8.48 cmol C kg -1 . The soil was sieved at 3 mm and kept at constant humidity (20% on a mass basis, i.e. about 80% of its water holding capacity). In this orchard, we found high densities of earthworms (> 300 individuals m -2 ) and the most dominant species were two endogeic species: Allolobophora chlorotica (leucotypic form according to [START_REF] Bouché | Lombriciens de France. Ecologie et systématique[END_REF] but often called pink morph) and Aporrectodea caliginosa. However, these two species presumably do not occupy exactly the same niche as these species were indeed described as an 'intermediate' and 'endogeic' species by [START_REF] Bouché | Lombriciens de France. Ecologie et systématique[END_REF], respectively. Recently, [START_REF] Bottinelli | An explicit definition of earthworm ecological categories -Marcel Bouché's triangle revisited[END_REF] demonstrated that each lumbricid earthworm species can be categorized by three percentages into the three main ecological categories defined by [START_REF] Bouché | Soil organisms as components of ecosystems[END_REF]. According to that study, A. chlorotica (leucotypic form) is 97% endogeic and 3% anecic, whereas A. caliginosa is 80% endogeic, 16% epigeic and 4% anecic.

Mesocosms and experimental design

Mesocosms were PVC cores (height: 30 cm, diameter:16 cm) filled with sieved soil. For each soil layer (600 g of soil at 80% WHC), the soil was pressed to the desired bulk density using a hydraulic press with increasing pressures. For this experiment, 50 cores were prepared with ten cores for each soil bulk density (BD): four with A. caliginosa, four with A. chlorotica and two control cores without earthworms. The five levels of BD were 1.18, 1.23, 1.29, 1.34 and 1.38 g cm -3 . This range was chosen to encompass the BD observed in orchards, from tilled zones under the row to compacted zones below wheel tracks (personal observation). In each core, we introduced three individuals of either A. caliginosa or A. chlorotica (thus a density of 150 individuals m -2 ) or no earthworms. These endogeic densities can be found in most agricultural soils (excepted vineyards). Only adults were used in the experiment and the mean (+/-SD) biomass for each species was 0.57 (+/-0.04) and 0.44 (+/-0.04) g for A. caliginosa and A. chlorotica, respectively. At the end of the experiment (5 weeks), 5 ml of chloroform was poured into each core to kill earthworms and prevent further burrowing. In each core, surface casts were sampled, dried for 48 h at 110°C and weighed.

X-ray computed tomography and burrow system characterization

Macroporosity within the soil cores was analyzed by X-ray computed tomography using a medical scanner (BrightSpeed Exel 4, General Electric) in the INRA Nancy research centre. The scanner settings were 130 kV and 50 mA. DICOM images were transformed into 8-bit images using the fol-lowing parameters WL = -1000 and WW = 2000 HU. The greylevel histograms of the images showed two well separated peaks, one for the soil matrix and one for the porosity. We decided to choose a binary threshold value set at 1/3 of the distance between the two peaks. Since the peak value is dependent on the density of the soil matrix, this ensures that differences in density between BD treatments did not influence the total macroporosity volume. Images were filtered using a mean filter of radius 2 to remove some noise. Burrow system 3D reconstructions and skeletonizations were obtained using Avizo software (ThermoScientific). Other computations were carried out using ImageJ [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] and the 'SoilJ' and 'BoneJ' plugins. We thus estimated burrow system volume. Mean burrow diameter was estimated by selecting the most circular pores ('circularity' > 0.8 in ImageJ) on 2D images, assessing their area and computing an equivalent diameter. To assess burrow system continuity, as no standardized measure exists, we compute the number of burrows whose vertical extension was larger than 30% of the core length (i.e. 9 cm) was counted. We also computed the degree of anisotropy of the burrow system using BoneJ ; this algorithm assesses whether objects are oriented in the same direction (degree of anisotropy close to 0) or randomly in 3D (degree of anisotropy close to 1). To assess whether the endogeic species compacted the soil around their respective burrows, we selected the 8 mm spatial zones around the burrows (as assessed visually on the images) and simply computed the mean greylevel in these zones. For cores without earthworms, we randomly selected zones of similar size in the images to compute the mean greylevel value.

Water infiltration

To study the effects of the presence of an earthworm burrow system and its characteristics on water infiltration, we used the same protocols in [START_REF] Capowiez | Morphological and functional characterisation of the burrow systems of six earthworm species (Lumbricidae)[END_REF]. Soil cores were placed on a funnel to be able to observe water drainage below the cores. In brief, 150 ml of water was poured onto a soil core, the time was recorded until the water was completely absorbed and then a new volume was added. We also noted the time at which water began to drain ('breakthrough time'). This procedure was repeated until a linear trend was observed (i.e. same time intervals between applied water volumes), which was typically after around eight volumes. This single-ring protocol allowed the steady-state infiltration rate to be estimated by linear regression for each core. The first data points of the relationship between time and the number of water volumes poured were removed.

Statistical analysis

Our experimental design was balanced for comparing the responses of the two species (if we excluded cores without earthworms). For count data (burrow continuity), we used a GLM with a quasi-poisson family. For other burrow system characteristics and cast production, we used ANOVA since no large deviations from normality (Shapiro-Wilk test: all p-values > 0.061) or homoscedasticity (Levene test: all p-values > 0.047) were detected. In both cases, we first ran the analyses with two factors (BD and earthworm species) and then if the interaction was not significant (the most common case), we ran two tests (GLM or ANOVA) for each species separately. For infiltration data (infiltration rate and breakthrough time), we applied the same analysis but we also computed the infiltration gain, i.e. the difference between infiltration of each species compared to the cores at the same BD but without earthworms. These gains were also analyzed using a two-way ANOVA and then two one-way ANOVA. Linear regressions were carried out for burrow volume with the 'lm' function. All analyses were made in R (R core team, 2019).

Results

Effects of bulk density on the earthworm burrow system

Examples of the 3D burrow systems are shown in Figure 1. The two-way ANOVA indicated that BD had a highly significant effect on burrow volume whereas species and the interaction between BD and species were not significant (Table S1). One-way ANOVA for each species separately showed that burrow volumes were significantly larger at 1.18 g cm -3 than at other BD for both species (Fig. 2). At 1.38 g cm -3 , the burrow volume was significantly lower than at 1.18 and 1.23 g cm -3 for both species. The decrease in burrow volume between the minimal (1.18 g cm -3 ) and maximal (1.38 g cm -3 ) tested BD was high with -75% and -80% for A. caliginosa and A. chlorotica, respectively. The linear regressions between BD and burrow volume were significant (p = 0.027 and 0.0071) and the R 2 values were 0.79 and 0.91 and for A. caliginosa and A. chlorotica respectively, even if the linear trend was more obvious for the latter species. We observed very similar decreases and significant differences in burrow lengths (Table 1).

BD and species (p < 0.001) also had a significant influence on burrow diameter, but no interaction was observed between these factors (p = 0.21). For. A. caliginosa, burrow diameter was only significantly different at the lowest BD compared to the highest BD. For A. chlorotica diameter values at the two lowest BD were significantly different from those at the other BD (Table 1).

BD also has a significant effect on burrow continuity, assessed as the number of burrows with a vertical elongation larger than 9 cm (Table S1). At the lowest BD, a mean of 6 and 7 such burrows were observed for A. caliginosa and A. chlorotica respectively. At the highest BD, only one such burrow was observed on average for both species (Fig. 3). Thus, BD had a highly significant effect on burrow continuity (p < 0.001), whereas species and interaction did not (p = 0.17 and 0.16, respectively). For A. caliginosa, only the continuity for the lowest and the highest BD were different, whereas for A. chlorotica, continuity at the three highest BD was significantly different from that at the lowest BD.

The degree of anisotropy of the burrow systems increased with increasing BD whereas species and the interaction were not significant (Table S1 and Fig. 4). For both species, the degree of anisotropy for the two lowest BD was significantly lower than those observed at the highest BD which suggests that increased BD leads to less randomly orientated burrow systems. Finally BD or species (results not shown) did not affect burrow system verticality (mean deviation from the vertical of the segments of the skeleton).

Surface cast production was low (less than 10 g of dry soil) and not influenced by BD for A. chlorotica (Table 1). In contrast, for A. caliginosa, cast production was higher (15-32 g of dry soil; Table 1) and was the highest at a BD of 1.34 g cm -3 and significantly different from the amount produced at the two lowest tested BD.

Effects on water infiltration

BD (p < 0.001) had a significant impact on infiltration rates and breakthrough times, but the earthworm factor (with three modalities: A. caliginosa, A. chlorotica and no earthworm) and the interaction were not significant (p = 0.36 and 0.55 respectively for infiltration rate ; p = 0.30 and 0.21 respectively for breakthrough time). When BD increased, infiltration rates decreased and breakthrough times increased (Table 2).

When infiltration was expressed relative to the control without earthworms, BD has a significant impact (p < 0.001) but species (with two modalities: A. caliginosa and A. chlorotica) and the interaction were not significant (p = 0.26 and 0.17 respectively). Significant differences due to earthworm's presence were only observed at the lowest BD for A. caliginosa and at the two lowest BD for A. chlorotica (Fig. 5). No significant effect was observed for breakthrough times expressed relative to the control (data not shown).

Effects on soil bulk density around the burrows

The mean soil BD around the burrows increased when BD of the soil core increased (Fig. S2). Soil BD had a highly significant effect (p < 0.001) as did the presence of earthworms (p < 0.001), but there was no significant interaction between these two factors (p = 0.83). For each BD, the differ-ence in BD around the burrows appeared to be similar (around five greylevels corresponding to 59 HU) in the presence or absence of earthworms.

Discussion

Effects of bulk density on earthworm burrowing and casting behavior BD strongly and negatively influenced all the geometrical characteristics of the burrow systems of both endogeic species. First of all in terms of magnitude, the volumes (and lengths) of the burrow systems decreased almost linearly with increasing BD. Thus with our soil which lies between loam and silt loam soil, we did not observe a non-monotonic relationship between BD and soil macroporosity as reported by [START_REF] Pöhlitz | Computed tomography as an extension of classical methods in the analysis of soil compaction, exemplified on samples from two tillage treatments and at two moisture tensions[END_REF] using the epi-anecic earthworm L. terrestris. This earthworm species has a mono-typical burrowing behavior with the creation of an often single vertical burrow, eventually with a few lateral branches close to the surface resulting in a Y-shaped burrow system [START_REF] Shipitalo | Occupancy and geometrical properties of Lumbricus terrestris L. burrows affecting infiltration[END_REF]. These features make this species less prone to drastically decreasing burrow length since their burrow is also a vital shelter.

Burrow diameter gradually decreased when BD increased for both species, but this change was more marked for A. chlorotica for which we already observed a significant difference between 1.18 and 1.29 g cm -3 . As the earthworms were of similar weight at the beginning of the experiment, this suggests that they may have created thinner burrows in denser soil, probably to save energy.

Other burrow system characteristics were also modified. The most drastic change was burrow system continuity. Its decrease with increasing BD was almost linear for A. caliginosa, but for A. chlorotica the most important changes occurred between 1.18 and 1.29 g cm -3 with a sharp decrease of -60%. This suggests that the burrows of this species were less vertically continuous as soon as the BD increases. Moreover, the degree of anisotropy of the burrow systems increased significantly with BD for both species. This showed that the burrows had no preferential orientation at low BD but then a trend for a non-preferential orientation appeared for higher BD. Taken together, these ob-higher BD values, even if we did not observe significant differences for verticality.

Overall, we did not observe large differences between the responses of the two species to the increase in BD. However, for most of the burrow system characteristics (except the degree of anisotropy) the decreases observed at 1.34 compared to 1.18 g cm -3 were less marked for A. caliginosa than for A. chlorotica, even if the values observed for a BD of 1.38 g cm -3 were similar for both species. Thus although a BD of 1.38 g cm -3 was a strongly limiting factor for both species, A.caliginosa appeared to have a better resistance to lower but still high BD (1.34 g cm -3 ).

Another striking difference between the two species was the production of casts at the surface with A. chlorotica producing very few casts regardless of the BD. In contrast, A. caliginosa produced more casts and showed a tendency to cast more at the soil surface when BD increased (except for the highest BD but in that case the burrow volume was very limited). This corroborates previous findings [START_REF] Rushton | The effects of soil compaction on Lumbricus terrestris and its possible implications for populations on land reclaimed from open-cast coal mining[END_REF][START_REF] Kretzschmar | Burrowing ability of the earthworm Aporrectodea longa limited by soil compaction and water potential[END_REF] showing that when the soil is compacted the earthworms tend to produce more surface casts in order to increase the air-filled porosity and aeration within the soil. If we assume that the casts had the same BD than the soil from which they were created, we could compute the ratio of the volume of macroporosity (burrows) that was deposited at the soil surface as casts. In this case, the proportion of burrow volume increased with SBD for both species (Fig. S3) ; this increase was however higher for A. caliginosa with casts representing 100% of the burrow volume at the highest BD tested. The difference between the two species may partly be explained by the fact that A. caliginosa is epigeic at 16% (A. chlorotica at 0%) and thus is more prone to defecate at the soil surface.

Consequences for water infiltration

Breakthrough time characterizes the transient regime of infiltration while the infiltration rate is a measure of the stationary flow regime. In control samples without earthworms, infiltration rates decreased by a factor of 4.4 and and breakthrough time increased by a factor of 7.6 when BD increases from 1.18 to 1.38 g cm -3 . The occurence of macropores (earthworm burrows) in the homogeneous porous sample tended to mitigate the effect but only for the lowest BD. Infiltration rates and breakthrough times increased when BD increased. This finding was expected since an increased BD means lower macroporosity and generally a decreased continuity of such pores as illustrated in the present study with the decreased burrow continuity.

Endogeic burrows are often discontinuous, since they can be refilled by casts, and often less connected to the surface [START_REF] Capowiez | Quantitative estimates of burrow construction and destruction by anecic and endogeic earthworms in repacked soil cores[END_REF]. Thus their involvement in increased water transfer efficiency is still open for debate. In the present study, we demonstrated that the effect of the activity of endogeic species on infiltration was only significant for low BD (1.18 g cm -3 for A. caliginosa and 1.18 and 1.23 g cm -3 for A. chlorotica). This may appear to be counterintuitive: when the soil is compacted the burrows could have been preferential pathways for air and water transfer [START_REF] Mossadeghi-Björklund | Effects of subsoil compaction on hydraulic properties and preferential flow in a Swedish clay soil[END_REF]. However these pathways are less vertical and only partial since the burrow continuity rapidly decreased when BD increased. In addition, the burrow systems were less extensive at moderate and high BD and thus their functional role was limited.

We observed a small but significant increase in the soil matrix greylevels surrounding earthworm burrows (for both species). Thus, regardless of the initial BD, earthworm burrowing appears to cause a constant increase in the BD around the burrows. This also limit the roles of earthworm burrows in water transfer [START_REF] Pelisek | Investigation of soil water infiltration at a scale of individual earthworm channels[END_REF] since the two species produced discontinuous burrows and thus water would need to enter and go out of several burrows to reach the bottom of the cores. [START_REF] Rogasik | Micro-scale dry bulk density variation around earthworm (Lumbricus terrestris L.) burrows based on X-ray computed tomography[END_REF] previously showed that L. terrestris burrowing caused a lateral compaction of the soil around the burrows. We further demonstrate that this is true for the two studied endogeic species. However, we were unable to detect an influence of BD on the intensity of this lateral compaction even though the burrow diameter significantly decreased at the highest tested BD.

Conclusions

We demonstrated that increasing BD impaired the burrowing behavior of two common endogeic species, and that total burrow volume and length decreased linearly with increasing BD. Although the earthworms promoted the soil infiltration capacity compared to control samples without earthworms, their effect was limited and only significant at low BD. These results were obtained using a single soil and only two species. The main findings could be compared with previous results obtained in other soils by computing an effective BD [START_REF] Beylich | Evaluation of soil compaction effects on soil biota and soil biological processes in soils[END_REF] or by using a reference BD (Keller and Hakansson, 2010). Our results could be used in simulation models of earthworm behaviour, such as the model recently presented by [START_REF] Roeben | Towards a spatiotemporally explicit toxicokinetic-toxicodynamic model for earthworm toxicity[END_REF]. In this model, an energy budget is applied for each earthworm and our data could help parameterize the energy costs of burrowing as a function of bulk density.

Fig. 1

 1 Figure legends:

Fig. 2

 2 Fig. 2 Volume of burrows (means + SE) created by three individuals of either A. chlorotica or A.

Fig. 3

 3 Fig. 3 Continuity of the burrow systems (means + SE) made by three individuals of either A. chlo-

Fig. 4

 4 Fig. 4 Degree of anisotropy of the burrow systems (means + SE) made by three individuals of either

Fig. 5

 5 Fig. 5 Gain in water infiltration (means + SE) compared to control without earthworms, when three

)

  g cm -3 1.18 1.23 1.29 1.34 1.38 1.18 1.23 1.29 1.34 1.38 Bulk density (g cm -3 ) Volume of burrows (cm 3 1.23 1.29 1.34 1.38 1.18 1.23 1.29 1.34 1.38 Bulk density (g cm -3 ) Continuity (Nb of burrows > 9 cm vertically) 1.23 1.29 1.34 1.38 1.18 1.23 1.29 1.34 1.38 Bulk density (g cm -3 )

Table 1 .

 1 Effects of an increase of soil bulk density on mean (SE) burrow diameter, total length and surface cast production in soil cores incubated with three individuals of either Aporrectodea caliginosa or Allolobophora chlorotica. ANOVA were carried out for each species separately.Values bearing different letters are different at the 5% significance threshold. (5.66) 38.52 ab (3.52) 34.74 b (6.73) 35.02 b (5.93) 15.73 c (3.95) Surface cast production (g) 17.17 b (3.65) 15.17 b (1.84) 24.36 ab (4.97) 32.11 a (5.43) 24.85 ab (6.33)

	A. caliginosa		A. chlorotica
	***
	)
	Gain in water infiltration (Lh -1	***	*

.23 1.29 1.34 1.38 1.18 1.23 1.29 1.34 1.38

Bulk density (g cm -3 ) a

Table 2 .

 2 Mean (SE) infiltration rates and breakthrough times in the soil cores incubated with three individuals of either A. caliginosa or A. chlorotica or no earthworm (control) for six weeks. Values bearing different letters are statistically different (each species tested separately).

			Soil bulk density (g cm -3 )
		1.18x	1.23 x	1.29 x	1.34 x	1.38 x
	Infiltration rate (L h -1 )
	A. caliginosa	9.95 a (1.58)	3.70 b (0.96)	4.00 b (1.00)	1.66 b (0.64)	1.12 b (0.52)
	A. chlorotica	8.72 a (1.48)	4.91 ab (1.11)	2.48 b (0.79)	1.15 b (0.54)	0.84 b (0.46)
	Control	4.61 (1.07)	2.50 (0.79)	2.45 (0.78)	1.71 (0.65)	1.04 (0.51)
	Breakthrough times (mn)
	A. caliginosa	0.55 b (0.27)	6.89 a (1.31)	6.73 a (1.29)	9.08 a (1.51)	14.28 a (1.89)
	A. chlorotica	1.93 b (0.69)	2.58 b (0.80)	9.60 ab (1.55) 18.80 a (2.17) 12.63 a (1.78)
	Control	2.23 (0.75)	6.93 (1.32)	11.35 (1.68)	15.72 (1.98)	16.96 (2.06)

Abbreviations

BD: soil bulk density


Jacques Rouanet

Mercedes Quintana

Philippe Auzeloux

Florent Cachin

Dr Françoise Degoul
email: francoise.degoul@inserm.fr

Benzamide derivative radiotracers targeting melanin for melanoma imaging and therapy: Preclinical/clinical development and combination with other treatments

Keywords: Metastatic melanoma, Melanin, Radiopharmaceuticals, Benzamides, Targeted radionuclide therapy, Imaging

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

Introduction

Cutaneous melanoma is the malignant skin tumor caused by melanocytes [START_REF] Liu | Developmental pathways activated in melanocytes and melanoma[END_REF][START_REF] Miller | Melanoma[END_REF][START_REF] Paluncic | Roads to melanoma: Key pathways and emerging players in melanoma progression and oncogenic signaling[END_REF][START_REF] Shain | The Genetic Evolution of Melanoma from Precursor Lesions[END_REF]. These cells, located in the basal layer of the epidermis and the hair follicle, produce melanin, the pigment that protects the skin from DNA damage due to ultraviolet (UV) radiation and is responsible for tanning. Another hypothesis for the origin of primary tumor formation suggests that melanoma could arise from melanocyte stem cells residing in the bulge of the hair follicle, rather than differentiated melanocytes in the basal layer [START_REF] Diener | Reemergence of neural crest stem cell-like states in melanoma during disease progression and treatment[END_REF][START_REF] Moon | Melanocyte Stem Cell Activation and Translocation Initiate Cutaneous Melanoma in Response to UV Exposure[END_REF][START_REF] Sun | A novel mouse model demonstrates that oncogenic melanocyte stem cells engender melanoma resembling human disease[END_REF]. Cutaneous melanomas account for 10% of skin cancers with an incidence of 3.1 per 100,000 worldwide in 2018 and causing 47,200 deaths [START_REF] Ferlay | Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods[END_REF]. They can appear on healthy skin (70-80% of cases) or as a result of the malignant transformation of a naevus [START_REF] Liu | Developmental pathways activated in melanocytes and melanoma[END_REF][START_REF] Miller | Melanoma[END_REF][START_REF] Paluncic | Roads to melanoma: Key pathways and emerging players in melanoma progression and oncogenic signaling[END_REF][START_REF] Shain | The Genetic Evolution of Melanoma from Precursor Lesions[END_REF]. Cutaneous melanoma is a term that covers different histological subtypes: superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma and lentigo melanoma. Around eight percent of cutaneous melanomas are amelanotic [START_REF] Thomas | Comparison of clinicopathologic features and survival of histopathologically amelanotic and pigmented melanomas: a population-based study[END_REF].

Acral melanomas, usually located in the plantar and palmar areas and in sublingual zones, represent 2-3% of all melanomas [START_REF] Saida | Characteristic distribution of melanin columns in the cornified layer of acquired acral nevus: an important clue for histopathologic differentiation from early acral melanoma[END_REF]. Their formation and progression are independent of UV exposure and have been associated with c-kit mutations [START_REF] Rawson | Unexpected UVR and non-UVR mutation burden in some acral and cutaneous melanomas[END_REF]. Acral melanoma is often dramatic because diagnosed after the occurrence of metastases that did not respond well to current targeted therapies.

Other types of malignant melanocytic proliferation and pigmented tumors also exist in the mucosa (lips, oral and nasal cavities, vulva, vagina, uterus, etc.) and eyes, leading to mucosal and ocular (or uveal) melanomas, respectively. These cancers, characterized by specific oncogenic mechanisms, are less frequent than cutaneous melanoma [START_REF] Goldemberg | An Update on Mucosal Melanoma: Future Directions[END_REF][START_REF] Wu | Clinicopathological and prognostic significance and molecular mechanisms governing uveal melanoma[END_REF] and respond differently to treatments [START_REF] Rossi | Efficacy of immune checkpoint inhibitors in different types of melanoma[END_REF]. The prognosis is good for cutaneous melanoma detected early enough, i.e. with a thickness of less than 1 mm (Breslow index) and no identified metastases, allowing surgical removal.

Delayed diagnosis reduces the survival rate considerably because of the high metastatic potential of this type of cancer.

A wide range of treatments is now available, metastasis surgery, radiation therapy and chemotherapy being the oldest. Indeed, until the early 2010s, the only systemic treatments available for metastatic melanoma were chemotherapy [START_REF] Hodi | Improved survival with ipilimumab in patients with metastatic melanoma[END_REF] (dacarbazine) or immunotherapy (IL-2), with poor efficacy (5-10% survival at 5 years). Since then, the management of metastatic melanoma has seen significant therapeutic progress with therapies targeting the MAPK pathway, including BRAF V600E [START_REF] Flaherty | Inhibition of mutated, activated BRAF in metastatic melanoma[END_REF] and MEK-activated proteins [START_REF] Long | Dabrafenib and trametinib versus dabrafenib and placebo for Val600 BRAF-mutant melanoma: a multicentre, double-blind, phase 3 randomised controlled trial[END_REF], as well as immunotherapy (Larkin et al., 2015) to eliminate the inhibition and escape mechanisms of the antitumor immune response. These antibodies are directed against immune checkpoint inhibitors, such as CTLA-4 [START_REF] Hodi | Improved survival with ipilimumab in patients with metastatic melanoma[END_REF], PD-1 and its ligand PD-L1 [START_REF] Robert | Nivolumab in previously untreated melanoma without BRAF mutation[END_REF]. For more details, see reviews [START_REF] Mason | Current and emerging systemic therapies for cutaneous metastatic melanoma[END_REF]. Median overall survival has thus increased to 2 years [START_REF] Luke | Targeted agents and immunotherapies: optimizing outcomes in melanoma[END_REF], although more time is required to be able to confirm with any accuracy the encouraging increase in the number of long-term survivors (> 2 years).

Furthermore, various protocols combining immunotherapy [START_REF] Postow | Nivolumab and ipilimumab versus ipilimumab in untreated melanoma[END_REF], targeted therapies [START_REF] Long | Dabrafenib and trametinib versus dabrafenib and placebo for Val600 BRAF-mutant melanoma: a multicentre, double-blind, phase 3 randomised controlled trial[END_REF] and stereotactic radiotherapy [START_REF] Williams | Phase 1 Study of Ipilimumab Combined With Whole Brain Radiation Therapy or Radiosurgery for Melanoma Patients With Brain Metastases[END_REF] are in routine use or in the first or second phases of clinical trials. Although these new treatments have significantly improved the prognosis of metastatic melanoma, some melanomas do not respond or become resistant to treatment after just a few months.

Nuclear medicine can benefit cancer therapeutics through theranostics, i.e. the combined use of radiolabeled drugs for target identification (via imaging) and treatment delivery using dedicated radionuclides [START_REF] Langbein | Future of Theranostics: An Outlook on Precision Oncology in Nuclear Medicine[END_REF]. Another advantage of nuclear therapy relies on off-target effects, i.e. bystander and abscopal effects [START_REF] Pouget | Introduction to radiobiology of targeted radionuclide therapy[END_REF][START_REF] Pouget | Targeted and Off-Target (Bystander and Abscopal) Effects of Radiation Therapy: Redox Mechanisms and Risk/Benefit Analysis[END_REF]. Both nuclear imaging and targeted radionuclide therapy (TRT) involve the delivery of radiopharmaceuticals to melanomas using a specific vector coupled with a dedicated radionuclide. In addition to the small melanin radiotracers reviewed herein, there are a number of other vectors available for melanoma nuclear imaging and TRT, briefly discussed below. A recent review presents research on additional imaging tracers, such as the peptide ligands of integrin, [START_REF] Wei | PET and SPECT Imaging of Melanoma: State of the Art[END_REF]. Peptide analogs of the α-melanocyte-stimulating hormone (α-MSH) targeting melanocortin receptor-1 (MC1-R) [START_REF] Miao | 99mTc-and 111In-labeled alpha-melanocyte-stimulating hormone peptides as imaging probes for primary and pulmonary metastatic melanoma detection[END_REF]Miao & Quinn, 2007) allowed imaging in preclinical models of murine melanomas and provided promising preclinical results in systemic TRT [START_REF] Miao | Melanoma therapy via peptide-targeted {alpha}-radiation[END_REF][START_REF] Miao | Therapeutic efficacy of a 188Relabeled alpha-melanocyte-stimulating hormone peptide analog in murine and human melanomabearing mouse models[END_REF][START_REF] Miao | Therapeutic efficacy of a 177Lu-labeled DOTA conjugated alphamelanocyte-stimulating hormone peptide in a murine melanoma-bearing mouse model[END_REF]. However, their high renal concentration will result in renal toxicity, thus impairing their clinical use. Integrin targeting has also been proposed in a phase 1 clinical trial for melanoma imaging [START_REF] Phillips | Clinical translation of an ultrasmall inorganic optical-PET imaging nanoparticle probe[END_REF] with RGD-functionalized nanoparticles, radiolabeled with Iodine-124. These nanoparticles were functionalized with α-MSH peptides in a Lutetium-177 TRT preclinical study with promising results for a future translational clinical study [START_REF] Zhang | Targeted melanoma radiotherapy using ultrasmall 177Lu-labeled α-melanocyte stimulating hormone-functionalized core-shell silica nanoparticles[END_REF].

The melanin pigment itself may also be a specific target for antibodies or peptides, as demonstrated in melanomas [START_REF] Norain | Targeted Radionuclide Therapy of Melanoma[END_REF] [START_REF] Dadachova | Pre-clinical evaluation and efficacy studies of a melanin-binding IgM antibody labeled with 188Re against experimental human metastatic melanoma in nude mice[END_REF][START_REF] Klein | Safety and efficacy of 188rhenium-labeled antibody to melanin in patients with metastatic melanoma[END_REF][START_REF] Revskaya | Radioimmunotherapy of experimental human metastatic melanoma with melanin-binding antibodies and in combination with dacarbazine[END_REF][START_REF] Thompson | 166Ho and 90Y labeled 6D2 monoclonal antibody for targeted radiotherapy of melanoma: comparison with 188Re radiolabel[END_REF] [START_REF] Auzeloux | Technetium-99m radiolabelling of an N-amino-alkyl-benzamide nitrido-and oxo-technetium bis(aminoethanethiol) derivative synthesis and biological results. Potential melanoma tracer agents[END_REF][START_REF] Dadachova | Radiolabeled melanin-binding peptides are safe and effective in treatment of human pigmented melanoma in a mouse model of disease[END_REF]. [ 188 Re]6D2 mAb was also tested in one phase I clinical trial [START_REF] Klein | Safety and efficacy of 188rhenium-labeled antibody to melanin in patients with metastatic melanoma[END_REF] and has shown good tolerance, localization in melanoma metastases and antitumor activity. Despite these promising results, this work did not lead to phase II trials.

Back in 1972, the first molecules reported to bind melanin were chlorpromazine (an anti-psychotic derivative of phenothiazine) and chloroquine (anti-malaria drug) [START_REF] Lindquist | The melanin affinity of chloroquine and chlorpromazine studied by whole body autoradiography[END_REF]. Our group came into the melanin ligand research field with a study on adiphenin, an inhibitor of nicotinic receptors showing high retention in pigmented tissues (eyes and skin) [START_REF] Meyniel | Perspectives of scintigraphic diagnosis of malignant lymphoma using a new radiopharmaceutical[END_REF]. Other compounds are also known to bind with melanin, such as methylene blue (MB) or acridine orange drugs [START_REF] Mårs | Pheomelanin as a binding site for drugs and chemicals[END_REF]. Comparison of these molecule structures reveals a common pattern of aromatic rings and a "tertiary amino group" (Figure 1). The next step was to synthesize and select the best structures exhibiting these characteristics and a radiolabeling site, usually an iodine atom. This review starts by describing the key features of melanin and then recapitulates the benzamides and derivatives that have been tested in clinical trials, focusing on their radiosynthesis. The last part will be dedicated to therapeutic approaches in preclinical experiments and clinical trials with a specific focus on some promising combination strategies.

Melanin pigment

Localization and melanogenesis

Melanin is synthesized inside specialized melanocyte organelles called melanosomes under genetic and biochemical regulation (for reviews on pigmentation genes, see [START_REF] Pavan | The Genetics of Human Skin and Hair Pigmentation[END_REF][START_REF] Rocha | The Evolutionary History of Human Skin Pigmentation[END_REF][START_REF] Sturm | Molecular genetics of human pigmentation diversity[END_REF]). After maturation, the melanosomes are transferred to other cells, mainly to surrounding keratinocytes (Figure 2A). In the epidermis, melanocytes and keratinocytes form a melanin unit, by associating one melanocyte and around 36 keratinocytes [START_REF] Haass | Adhesion, migration and communication in melanocytes and melanoma[END_REF]. This pigment transfer is responsible for skin pigmentation and plays a central role in protecting the cells against ultraviolet (UV) radiation damage. Melanin is also responsible for the pigmentation of the hair, eyes, and mucosa. Melanin itself is a complex biopolymer made up of two chemically different melanins, called eumelanin (EM) and pheomelanin (PM). Eumelanin is a brown-black pigment, while PM has a yellow to reddish-brown color, ascribed to the cysteine. EM is constituted of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2carboxylic acid (DHICA) monomers, while benzothiazine and benzothiazole are the PM monomers produced by the melanogenesis pathway (Figure 2B). Considering the diversity of the DHI/DHICA ratio in EM, the degree of polymerization, and the availability of cysteine that governs PM synthesis, natural melanin constitutes a complex, heterogeneous, high molecular weight structure.

Functions: photo-protection vs photo-toxicity

The close association between UV exposure and its genotoxic effects that lead to the development of skin cancer, notably melanoma, is well established [START_REF] Erdmann | International trends in the incidence of malignant melanoma 1953-2008-are recent generations at higher or lower risk[END_REF]. Melanin photo-protection against UVB (290-320 nm) and UVA (320-400 nm) radiation has been demonstrated in vitro (human melanocytes) [START_REF] Barker | Comparison of the responses of human melanocytes with different melanin contents to ultraviolet B irradiation[END_REF][START_REF] Smit | Melanin offers protection against induction of cyclobutane pyrimidine dimers and 6-4 photoproducts by UVB in cultured human melanocytes[END_REF] and in situ (skin biopsies of healthy donors) [START_REF] Tadokoro | UV-induced DNA damage and melanin content in human skin differing in racial/ethnic origin[END_REF][START_REF] Yamaguchi | Human skin responses to UV radiation: pigment in the upper epidermis protects against DNA damage in the lower epidermis and facilitates apoptosis[END_REF][START_REF] Mouret | Cyclobutane pyrimidine dimers are predominant DNA lesions in whole human skin exposed to UVA radiation[END_REF]. The most studied pigment, EM, shows an exceptionally broad spectrum of UV-Vis absorption, with a decreasing exponential profile (Figure 2C) as well as an absorbed radiation dissipation capacity that supports its natural photo-protection action (for a review, see [START_REF] Meredith | The physical and chemical properties of eumelanin[END_REF]. Melanin radioprotection is also mediated by radical scavenging and oxygen depletion throughout melanogenesis. Notwithstanding its photo-protective actions, melanin exhibits potential toxicity. In the presence of UVA irradiation, both pigments undergo oxidative degradation (Wakamatsu et al., 2012). However, the antagonistic effect of melanin on radiation protection is mainly attributed to PM. Among the main causes of cell oxidative stress mediated by PM are the antioxidant depletion (i.e. glutathione (GSH) and NADH) and the production of reactive oxygen species (ROS) and the formation of cyclobutane pyrimidine dimers (CPD) leading to DNA damage [START_REF] Chedekel | Photodestruction of pheomelanin: role of oxygen[END_REF][START_REF] Hill | Comparative action spectrum for ultraviolet light killing of mouse melanocytes from different genetic coat color backgrounds[END_REF][START_REF] Hill | UVA, Pheomelanin and the Carcinogenesis of Melanoma[END_REF][START_REF] Panzella | Red human hair pheomelanin is a potent pro-oxidant mediating UV-independent contributory mechanisms of melanomagenesis[END_REF][START_REF] Premi | Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure[END_REF]. In vivo demonstration of the spontaneous development of melanomas by BRAF v600E mutant mice that produce PM when not exposed to UV provides a link with the pro-oxidant potential of PM [START_REF] Mitra | An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background[END_REF].

Melanin and melanoma

Melanoma incidence is lower in dark-skinned than in light-skinned individuals [START_REF] Bertrand | Melanoma Risk and Melanocyte Biology[END_REF][START_REF] Erdmann | International trends in the incidence of malignant melanoma 1953-2008-are recent generations at higher or lower risk[END_REF][START_REF] Mackie | Epidemiology of invasive cutaneous melanoma[END_REF]. Melanin is present in most primary melanoma tumors since only 1.8 to 8.1% are amelanotic [START_REF] Koch | Amelanotic melanoma: the great masquerader[END_REF]. Amelanotic melanomas usually present a thicker Breslow index, a higher mitotic rate, and are associated with a lower survival rate than pigmented melanomas [START_REF] Wee | Clinically amelanotic or hypomelanotic melanoma: Anatomic distribution, risk factors, and survival[END_REF]. It has been reported that melanoma cells lose their melanogenesis ability during metastatic dissemination, and it is assumed that about 50% of melanoma-derived metastases are pigmented [START_REF] Cachin | 123)I-BZA2 as a melanin-targeted radiotracer for the identification of melanoma metastases: results and perspectives of a multicenter phase III clinical trial[END_REF]. The loss of pigmentation is usually considered as a loss of differentiation and a marker of disease progression [START_REF] Miller | Melanoma[END_REF].

However, the presence of melanin in tumors can be deleterious to patients undergoing chemotherapy [START_REF] Chen | Melanosomal sequestration of cytotoxic drugs contributes to the intractability of malignant melanomas[END_REF][START_REF] Chen | Influence of Melanosome Dynamics on Melanoma Drug Sensitivity[END_REF] or radiotherapy [START_REF] Brożyna | Melanogenesis Affects Overall and Disease-Free Survival in Patients with Stage III and IV Melanoma[END_REF][START_REF] Brożyna | Melanin content in melanoma metastases affects the outcome of radiotherapy[END_REF], related to intrinsic melanin trapping and radiation-protection abilities. Conversely, a beneficial role of melanin on melanomas has been also demonstrated in vitro [START_REF] Sarna | Nanomechanical analysis of pigmented human melanoma cells[END_REF][START_REF] Sarna | Cell elasticity is an important indicator of the metastatic phenotype of melanoma cells[END_REF][START_REF] Sarna | Nanomechanical Phenotype of Melanoma Cells Depends Solely on the Amount of Endogenous Pigment in the Cells[END_REF] and in vivo [START_REF] Sarna | Melanin presence inhibits melanoma cell spread in mice in a unique mechanical fashion[END_REF] due to reduced cell elasticity. Interestingly, near-infrared pump-probe imaging showed structural changes to the pigments in melanoma biopsies and pointed to DHICA EM fragmentation as a marker of melanoma metabolic activity and metastatic potential [START_REF] Matthews | Pump-Probe Imaging Differentiates Melanoma from Melanocytic Nevi[END_REF][START_REF] Ju | Unraveling the molecular nature of melanin changes in metastatic cancer[END_REF]. The relationship between structure, melanin function and changes depending on the melanoma stage deserves further investigation to understand its significance in biological terms.

As described above, melanin is an obstacle to external radiotherapy and chemotherapy for melanoma because of its photo-protection and scavenging properties. However, its presence in melanomas and the discovery of melanin drug affinities have promoted studies on radionuclide-labeled compounds to enable specific tumor targeting.

Radiotracers binding to melanin

Discovery of strong affinities between certain drugs and melanin

Ocular toxicology cases associated with a drug affinity for melanin were first reported in the middle of the 20 th century, with a phenothiazine compound [START_REF] Verrey | Dégénérescence pigmentaire de la rétine d'origine médicamenteuse[END_REF] and chloroquine [START_REF] Hobbs | RETINOPATHY FOLLOWING CHLOROQUINE THERAPY[END_REF]. In 1972, a biodistribution study on mice and monkeys confirmed the accumulation of radiolabeled chloroquine and chlorpromazine in pigmented tissues [START_REF] Lindquist | The melanin affinity of chloroquine and chlorpromazine studied by whole body autoradiography[END_REF]. These observations led to studies of the drug-melanin interaction mechanisms, mainly for the toxicological studies required for drug development (for a review, see [START_REF] Ings | The melanin binding of drugs and its implications[END_REF]. The compounds with the highest affinity for melanin have two chemical structural similarities: firstly, an aromatic or heteroaromatic ring and, secondly, a protonated amine function at physiological pH (Figure 1). Early on, an analogue of chloroquine radiolabeled with iodine-125 was investigated for visualizing malignant melanomas and metastases in humans [START_REF] Beierwaltes | Visualizing Human Malignant Melanoma and Metastases: Use of Chloroquine Analog Tagged With Iodine 125[END_REF]. Unfortunately, the physical properties of iodine-125 (half-life 59 days, main gamma emission energy 27 keV) are not suitable for low patient dosimetry and do not enable resolution sufficient for nuclear medicine imaging applications. In the late 1980s, our team worked on aromatic iodinated amino compounds, including adiphenine, to explore the central nervous system. Unexpectedly, an accumulation of radioactivity was observed in richly pigmented tissues, leading to the first radiotracer N-(2diethylaminoethyl)-4-iodobenzamide (BZA), specifically designed to diagnose malignant melanoma [START_REF] Michelot | Synthesis and evaluation of new iodine-125 radiopharmaceuticals as potential tracers for malignant melanoma[END_REF].

3.2. BZA and benzamide derivatives with a high affinity for melanin BZA, like many benzamide structures, is known for its affinity for σ-receptors [START_REF] Schmidt | The Molecular Function of σ Receptors: Past, Present, and Future[END_REF]. σ-Receptors are cellular receptors found in various organs and expressed by melanoma cells [START_REF] Georgiadis | Sigma Receptor (σR) Ligands with Antiproliferative and Anticancer Activity[END_REF][START_REF] John | A malignant melanoma imaging agent: synthesis, characterization, in vitro binding and biodistribution of iodine-125-(2-piperidinylaminoethyl)4-iodobenzamide[END_REF]. They were first identified as targets of benzamides. However, it is now assumed that BZA directly binds with melanin for three main reasons: 1) BZA fixation is independent of its molar activity, suggesting a non-saturable mechanism different from the receptor-ligand interaction [START_REF] Dittmann | In vitro studies on the cellular uptake of melanoma imaging aminoalkyl-iodobenzamide derivatives (ABA)[END_REF]. 2) In vitro BZA uptake in melanoma is not correlated with the presence of σreceptors (M. [START_REF] Eisenhut | Radioiodinated N-(2-diethylaminoethyl)benzamide derivatives with high melanoma uptake: structure-affinity relationships, metabolic fate, and intracellular localization[END_REF][START_REF] Oltmanns | Benzamides as melanotropic carriers for radioisotopes, metals, cytotoxic agents and as enzyme inhibitors[END_REF], but correlation was found between melanin and benzamide uptake [START_REF] Labarre | Melanin affinity of N-(2-diethylaminoethyl)-4-iodobenzamide, an effective melanoma imaging agent[END_REF]. 3) Secondary ion mass spectrometry showed that BZA co-localized with intracytoplasmic melanin [START_REF] Guerquin-Kern | Ultra-structural cell distribution of the melanoma marker iodobenzamide: improved potentiality of SIMS imaging in life sciences[END_REF][START_REF] Chéhadé | Secondary ion mass spectrometry as a tool for investigating radiopharmaceutical distribution at the cellular level: the example of I-BZA and (14)C-I-BZA[END_REF]. However, the reversible interaction between BZA and melanin excludes a covalent bond [START_REF] Labarre | Melanin affinity of N-(2-diethylaminoethyl)-4-iodobenzamide, an effective melanoma imaging agent[END_REF]. The presence of the protonated cation on tertiary amine favors ionic interaction with carboxylates on one side, and a πinteraction between the aromatic ring and the heteroaromatic ring (indole) on the other (Figure 3). This specific non-saturable interaction leads to significant pharmacomodulation for nuclear medicine.

In the 1990s, various teams performed pharmacomodulation, mainly to investigate the position of iodine [START_REF] Brandau | Structure Distribution Relationship of Iodine-123-Iodobenzamides as Tracers for the Detection of Melanotic Melanoma[END_REF][START_REF] Nicholl | Pharmacokinetics of iodine-123-IMBA for melanoma imaging[END_REF], the length of the carbon chain between the two nitrogen atoms, and the terminal amine function [START_REF] Dittmann | In vitro studies on the cellular uptake of melanoma imaging aminoalkyl-iodobenzamide derivatives (ABA)[END_REF][START_REF] John | A malignant melanoma imaging agent: synthesis, characterization, in vitro binding and biodistribution of iodine-125-(2-piperidinylaminoethyl)4-iodobenzamide[END_REF][START_REF] Labarre | Evaluation in mice of some iodinated melanoma imaging agents using cryosectioning and multi-wire proportional counting[END_REF][START_REF] Moins | Synthesis, characterization and comparative biodistribution study of a new series of p-iodine-125 benzamides as potential melanoma imaging agents[END_REF][START_REF] Moreau | Synthesis, radiolabeling, and preliminary evaluation in mice of some (N-Diethylaminoethyl)-4-iodobenzamide derivatives as melanoma imaging agents[END_REF][START_REF] Nicholl | Pharmacokinetics of iodine-123-IMBA for melanoma imaging[END_REF]. Almost all these molecules maintained strong affinity for melanoma in preclinical studies. More recently, the aromatic ring has been modified by adding phenyl substituents (M. [START_REF] Eisenhut | Radioiodinated N-(2-diethylaminoethyl)benzamide derivatives with high melanoma uptake: structure-affinity relationships, metabolic fate, and intracellular localization[END_REF][START_REF] Nicholl | Pharmacokinetics of iodine-123-IMBA for melanoma imaging[END_REF], and using new heteroaromatic analogs [START_REF] Chezal | Evaluation of radiolabeled (hetero)aromatic analogues of N-(2-diethylaminoethyl)-4-iodobenzamide for imaging and targeted radionuclide therapy of melanoma[END_REF][START_REF] Greguric | Discovery of[END_REF].

Many of these structures have shown good potential as melanoma radiotracers in preclinical studies and have been reviewed elsewhere [START_REF] Oltmanns | Benzamides as melanotropic carriers for radioisotopes, metals, cytotoxic agents and as enzyme inhibitors[END_REF][START_REF] Wei | PET and SPECT Imaging of Melanoma: State of the Art[END_REF].

Radiotracers binding with melanin polymers for clinical applications

Radioisotope selection

In parallel to chemical BZA pharmacomodulation, a number of attempts has been made to find a radionuclide replacement for iodine-123/131. For SPECT diagnosis, the more available technetium-99m has been extensively investigated with moderate success [START_REF] Auzeloux | Technetium-99m radiolabelling of an N-amino-alkyl-benzamide nitrido-and oxo-technetium bis(aminoethanethiol) derivative synthesis and biological results. Potential melanoma tracer agents[END_REF][START_REF] Auzeloux | A potential melanoma tracer: synthesis, radiolabeling, and biodistribution in mice of a new nitridotechnetium bis(aminothiol) derivative pharmacomodulated by a N-(diethylaminoethyl)benzamide[END_REF][START_REF] Auzeloux | Technetium-99m radiolabelling of an N-amino-alkyl-benzamide nitrido-and oxo-technetium bis(aminoethanethiol) derivative synthesis and biological results. Potential melanoma tracer agents[END_REF][START_REF] Auzeloux | Synthesis and biodistribution of a new oxo-technetium-99m bis(aminothiol) complex as a potential melanoma tracer[END_REF][START_REF] Friebe | 3+1" mixed-ligand oxotechnetium(V) complexes with affinity for melanoma: synthesis and evaluation in vitro and in vivo[END_REF][START_REF] Friebe | 99mTc]oxotechnetium(V) complexes amine-amide-dithiol chelates with dialkylaminoalkyl substituents as potential diagnostic probes for malignant melanoma[END_REF][START_REF] Eisenhut | Melanoma uptake of (99m)Tc complexes containing the N-(2diethylaminoethyl)benzamide structural element[END_REF][START_REF] N'dongo | Preparation and biological evaluation of cyclopentadienyl-based 99mTc-complexes [(Cp-R)99mTc(CO)3] mimicking benzamides for malignant melanoma targeting[END_REF][START_REF] Moura | 99mTc(I)/Re(I) tricarbonyl complexes for in vivo targeting of melanotic melanoma: Synthesis and biological evaluation[END_REF]. The metallic nature of technetium requires the presence of a chelator group, resulting in partial loss of the melanin affinity. For PET imaging, fluorine-18 (S.-Y. [START_REF] Wu | Synthesis and preclinical characterization of [18F]FPBZA: a novel PET probe for melanoma[END_REF]Garg et al., 2017;[START_REF] Greguric | Discovery of[END_REF][START_REF] Greguric | Discovery of[END_REF][START_REF] Pyo | Ultrasensitive detection of malignant melanoma using PET molecular imaging probes[END_REF][START_REF] Ren | Melanin-targeted preclinical PET imaging of melanoma metastasis[END_REF], carbon-11 [START_REF] Garg | Selective targeting of melanoma using N-(2diethylaminoethyl) 4-[18F]fluoroethoxy benzamide (4-[18F]FEBZA): a novel PET imaging probe[END_REF][START_REF] Igawa | Development of a Novel Carbon-11 Labeled PET Radioligand for Melanin-Concentrating Hormone Receptor 1[END_REF] and gallium-68 complex (Kim et al., 2012b(Kim et al., , 2012a) ) have provided interesting results in preclinical studies. For radionuclide therapy, the alpha emitter, astatine-211, was considered in early studies, with a radio-analog of methylene blue in preclinical models [START_REF] Link | Uptake and therapeutic effectiveness of 125I-and 211At-methylene blue for pigmented melanoma in an animal model system[END_REF][START_REF] Link | 211At-methylene blue for targeted radiotherapy of disseminated melanoma: microscopic analysis of tumour versus normal tissue damage[END_REF][START_REF] Link | Targeting melanoma with 211At/131I-methylene blue: preclinical and clinical experience[END_REF].

Our team investigated both iodinated and fluorinated derivatives of quinoxaline. The same molecule can be radiolabeled with fluorine-18 for PET imaging or with iodine-131 for therapeutic applications [START_REF] Billaud | Synthesis, radiofluorination, and in vivo evaluation of novel fluorinated and iodinated radiotracers for PET imaging and targeted radionuclide therapy of melanoma[END_REF][START_REF] Billaud | Synthesis, radiolabeling and preliminary in vivo evaluation of multimodal radiotracers for PET imaging and targeted radionuclide therapy of pigmented melanoma[END_REF][START_REF] Maisonial | Synthesis, radioiodination and in vivo screening of novel potent iodinated and fluorinated radiotracers as melanoma imaging and therapeutic probes[END_REF]. This multimodal approach has shown promising preclinical results in theranostic approaches for pigmented metastatic melanoma: the best compound, injection, tumor doubling time increased from 1.8 ± 0.3 to 2.9 ± 0.5 days (controls vs treated), associated with higher median survival in the B16-BL6 murine model [START_REF] Rbah-Vidal | Theranostic Approach for Metastatic Pigmented Melanoma Using ICF15002, a Multimodal Radiotracer for Both PET Imaging and Targeted Radionuclide Therapy[END_REF]. Until now, only radiohalogenated benzamide derivatives have been injected into humans, radiolabeled with iodine-123/131 or fluorine-18. The radionuclide properties are summarized in Table 1.

The iodine radioisotope pair offers a strategic advantage in terms of development as the same molecule can be used for both SPECT with iodine-123 or therapy with iodine-131 [START_REF] Mier | Radiopharmaceutical Therapy of Patients with Metastasized Melanoma with the Melanin-Binding Benzamide 131I-BA52[END_REF].

However, for diagnosis applications, more studies are being conducted on fluorine-18 because of its availability ( 18 F is the radioisotope present in FDG, the 'gold standard' in nuclear medicine for tumor imaging) and because of benefits of PET vs SPECT (PET presents higher resolution and a better signal-tonoise ratio).

Radiopharmaceutical production

There are many chemical methods to incorporate a radio-halogen onto a small molecule (for radioiodination see [START_REF] Dubost | Recent Advances in Synthetic Methods for Radioiodination[END_REF] and for radiofluorination see [START_REF] Preshlock | 18 F-Labeling of Arenes and Heteroarenes for Applications in Positron Emission Tomography[END_REF]. These protocols involve two strong constraints: radiochemistry takes place at the trace scale (concentrations µM to pM) and the final product must comply with injectable drug regulations. Thus, for a radiopharmaceutical application, four main points must be respected: 1) the labeling time must be as short as possible; this parameter, while important for radioiodination, becomes crucial for the fluor-18 radiolabeling process; 2) radiochemical yields (RCY) must be as high as possible to reduce the activity, cost and radiation exposure involved; 3) the purification method must prefer single-use cartridges instead of a column; 4) the process must limit the formation of radioactive by-products.

For all these reasons, for the 10 molecules studied in clinical trials for malignant melanoma (Table 2) ). This is a nucleophilic substitution reaction, requiring the presence of a strong oxidant. This regiospecific method is most often used for radioiodation because of its very soft conditions (18-25°C) and fast kinetics (only a few minutes). The limit of this method for clinical application is that it eliminates stannane compounds in the final formulation; a validated method for final purification (HPLC or cartridge) is therefore required.

Preclinical and clinical results

Biodistribution, imaging, dosimetry and potential adverse effects

The main concern with radiopharmaceuticals is dosimetry. Radiotoxicity must be minimized in imaging, non-targeted organs must be spared in therapeutic protocols, but enough activity must be delivered to ensure anti-tumor efficiency. The dose in internal radiotherapy is closely related to the amount of radiolabeled ligand in tissues, causing prolonged exposure to different radiations for a given radionuclide, inducing a low absorbed dose rate. This is quite different from EBRT, which involves a homogeneous high dose rate and short exposure [START_REF] Pouget | Introduction to radiobiology of targeted radionuclide therapy[END_REF]. Studies on melanin ligands, as for other radiopharmaceuticals, usually refer to the ratio of tumor to blood or muscle for pharmacokinetics and the percentage of injected activity per gram of tissues (Table 3). This percentage varied from 9 to 41% in preclinical model tumors for the most recent studies on structures transferred to clinical trials (Table 3).

Dosimetry was calculated with the MIRD formalism, taking into account activity in the source and the Sfactor derived from computed values [START_REF] Bolch | MIRD pamphlet No. 21: a generalized schema for radiopharmaceutical dosimetry--standardization of nomenclature[END_REF]. S-factors are defined as the mean absorbed dose in the target organ per decay in the source organ [START_REF] Stabin | Developments in the internal dosimetry of radiopharmaceuticals[END_REF]. These S-factors were calculated by MONTE-CARLO simulation to increase accuracy and to provide a closed fit with reality [START_REF] Perrot | Internal dosimetry through GATE simulations of preclinical radiotherapy using a melanin-targeting ligand[END_REF]; they are used for specific personal dosimetry in the ongoing MELRIV1 trial (NCT03784625).

Various melanoma cell lines were used in the preclinical models but most investigations were carried out on highly pigmented murine B16 cell lines grafted subcutaneously into C57Bl/6 mice [START_REF] Poste | In vitro selection of murine B16 melanoma variants with enhanced tissue-invasive properties[END_REF]. This syngeneic model demonstrates high fixation of radiolabeled benzamides in the eyes, particularly in the pigmented retinal epithelium, and choroid, both structures being close to the retina [START_REF] Degoul | In vivo efficacy of melanoma internal radionuclide therapy with a 131Ilabelled melanin-targeting heteroarylcarboxamide molecule[END_REF]. This specific targeting is not expected to have an impact in humans as dosimetry extrapolations gave values below the maximum tolerated doses [START_REF] Jouberton | Radiation dosimetry of [131 I]ICF01012 in rabbits: Application to targeted radionuclide therapy for human melanoma treatment[END_REF]. Furthermore, the imaging and therapy protocols already performed on humans did not assess accumulation in the eyes, except in the case of uveal melanoma [START_REF] Everaert | Visualizing Ocular Melanoma Using Iodine-123-N-(2-Diethylaminoethyl)4-Iodobenzamide SPECT[END_REF][START_REF] Sillaire-Houtmann | Phase 2 clinical study of 123I-N-(2-diethylaminoethyl)-2-iodobenzamide in the diagnostic of primary and metastatic ocular melanoma[END_REF]. As mentioned previously, the melanin-binding of these molecules is not covalent and non-saturable, which allows high and low specific activities for these radiotracers. The kinetics of the radiolabeled ligands in pigmented tissues (mainly the eyes and melanomas) follow a curve that plateaus, indicating their affinity for melanin with the highest percentage of injected activity in pigmented tissues [START_REF] Perrot | Internal dosimetry through GATE simulations of preclinical radiotherapy using a melanin-targeting ligand[END_REF]. Radioiodinated benzamides can also affect skin and hair follicle melanocytes, and melanized neurons. Our results, and those from others obtained in preclinical models, did not evidence such toxicities. In the B16 preclinical model, cutaneous melanocytes only present in follicles were not modified qualitatively or quantitatively by [START_REF] Degoul | In vivo efficacy of melanoma internal radionuclide therapy with a 131Ilabelled melanin-targeting heteroarylcarboxamide molecule[END_REF]. Concerning neurons and the blood-brain barrier, we have already shown that the [ 125 I]BZA2 radiotracer can evidence brain metastases [START_REF] Cachin | 123)I-BZA2 as a melanin-targeted radiotracer for the identification of melanoma metastases: results and perspectives of a multicenter phase III clinical trial[END_REF], proving that the radiotracer can pass through the blood-brain barrier in patients. However, dosimetry for the whole brain was not critical in preclinical models (monkeys and mice) [START_REF] Joyal | Preclinical evaluation of an 131I-labeled benzamide for targeted radiotherapy of metastatic melanoma[END_REF][START_REF] Viallard | 123 I]ICF01012 melanoma imaging and [ 131 I]ICF01012 dosimetry allow adapted internal targeted radiotherapy in preclinical melanoma models[END_REF] or also for extrapolated human doses [START_REF] Jouberton | Radiation dosimetry of [131 I]ICF01012 in rabbits: Application to targeted radionuclide therapy for human melanoma treatment[END_REF]. Finally, the most convincing argument for TRT safety is that the first TRT on humans did not cause adverse effects in patients receiving [ 131 I]BA52 [START_REF] Mier | Radiopharmaceutical Therapy of Patients with Metastasized Melanoma with the Melanin-Binding Benzamide 131I-BA52[END_REF].

[ 131 I]ICF01012
As far as SPECT imaging is concerned, the high specificity of the radiolabeled benzamides has been clearly demonstrated by various trials, with values above 86% (Table 3). However, when these tracers were tested for PET imaging with [ 18 F]FDG, sensitivity was lower [START_REF] Cachin | 123)I-BZA2 as a melanin-targeted radiotracer for the identification of melanoma metastases: results and perspectives of a multicenter phase III clinical trial[END_REF], resulting in the interruption of benzamide development for SPECT imaging. Nevertheless, the size of these molecules is compatible with fluorine radiolabeling for PET imaging, as exemplified with [ 18 F]P3BZA with 17% IA post injection, leading to good imaging quality in mice and subsequently in human PET scans [START_REF] Ma | Biodistribution, Radiation Dosimetry, and Clinical Application of a Melanin-Targeted PET Probe, 18F-P3BZA, in Patients[END_REF].

This tracer should be compared with [ 18 F]FDG to evaluate its benefits.

Antitumoral effects of benzamide-based TRT

TRT efficacy has been assessed in preclinical studies in both syngeneic (B16-F0, B16-F10 and B16-BL6) and xenograft (M4Beu, SK-MEL-3) models. We used different clones of B16 melanoma, selected for their high metastazing capacities, for serial IV injection of cell lines from lung metastases, i.e. B16-F10 to B16-F0.

B16-BL6 differs from its parent B16-F10 in that it is able to cross the bladder membrane [START_REF] Poste | In vitro selection of murine B16 melanoma variants with enhanced tissue-invasive properties[END_REF].

We observed that B16-BL6 exhibited higher melanin than B16-F0 and B16-F10 [START_REF] Rondepierre | Proteomic studies of B16 lines: involvement of annexin A1 in melanoma dissemination[END_REF].

For instance, we showed that two injections of 18.5 MBq of [ 131 I]ICF01012 significantly inhibited the growth of B16-F0 and B16-BL6 tumors [START_REF] Bonnet | Anti-melanoma efficacy of internal radionuclide therapy in relation to melanin target distribution[END_REF][START_REF] Bonnet-Duquennoy | Targeted radionuclide therapy of melanoma: anti-tumoural efficacy studies of a new 131I labelled potential agent[END_REF][START_REF] Viallard | 123 I]ICF01012 melanoma imaging and [ 131 I]ICF01012 dosimetry allow adapted internal targeted radiotherapy in preclinical melanoma models[END_REF].

This therapeutic effect was also found with [ 131 I]5-IPN367 on B16-F10 tumors with an identical therapeutic protocol [START_REF] Xu | Targeted radiotherapy of pigmented melanoma with 131I-5-IPN[END_REF]. Similarly, in xenograft models, the injection of

[ 131 I]ICF01012 or [ 131 I]MIP-1145
led to a significant slow-down of tumor growth in human pigmented tumors and increased survival [START_REF] Bonnet-Duquennoy | Targeted radionuclide therapy of melanoma: anti-tumoural efficacy studies of a new 131I labelled potential agent[END_REF][START_REF] Bonnet | Anti-melanoma efficacy of internal radionuclide therapy in relation to melanin target distribution[END_REF][START_REF] Joyal | Preclinical evaluation of an 131I-labeled benzamide for targeted radiotherapy of metastatic melanoma[END_REF][START_REF] Degoul | In vivo efficacy of melanoma internal radionuclide therapy with a 131Ilabelled melanin-targeting heteroarylcarboxamide molecule[END_REF]. TRT efficacy on tumor growth and survival was dependent upon the number and scheduling of radiopharmaceutical injections in both B16 and SK-MEL-3 models [START_REF] Bonnet | Anti-melanoma efficacy of internal radionuclide therapy in relation to melanin target distribution[END_REF][START_REF] Degoul | In vivo efficacy of melanoma internal radionuclide therapy with a 131Ilabelled melanin-targeting heteroarylcarboxamide molecule[END_REF][START_REF] Joyal | Preclinical evaluation of an 131I-labeled benzamide for targeted radiotherapy of metastatic melanoma[END_REF][START_REF] Xu | Targeted radiotherapy of pigmented melanoma with 131I-5-IPN[END_REF] and was obviously related to the amount of melanin [START_REF] Viallard | 123 I]ICF01012 melanoma imaging and [ 131 I]ICF01012 dosimetry allow adapted internal targeted radiotherapy in preclinical melanoma models[END_REF].

[ 131 I]BA52, a
molecule derived from [ 131 I]MIP-1145, was tested in metastatic melanoma patients. This unique TRT clinical trial showed that of the five patients, two were still alive 2 years after treatment [START_REF] Mier | Radiopharmaceutical Therapy of Patients with Metastasized Melanoma with the Melanin-Binding Benzamide 131I-BA52[END_REF]. These patients received the higher activity, emphasizing the importance of dosimetry. Furthermore, a phase I clinical trial is currently assessing the safety and efficacy of the [ 131 I]ICF01012 molecule (NCT03784625) (Table 3).

Biological effects of benzamide-based TRT

Compared with EBRT, the cellular and molecular effects of TRT are not so well documented and researchers are still working to decipher the TRT mechanisms, particularly with respect to dosimetry [START_REF] Pouget | Introduction to radiobiology of targeted radionuclide therapy[END_REF]. However, by analogy with EBRT, different non-mutually exclusive biological mechanisms are likely to be induced after TRT: 1) the DNA double-strand breaks and the cell cycle are altered, related to p53 mutational status [START_REF] Baskar | Biological response of cancer cells to radiation treatment[END_REF][START_REF] Pawlik | Role of cell cycle in mediating sensitivity to radiotherapy[END_REF]; 2) metastatic mechanisms involving VEGF expression are altered, and epithelial-mesenchymal transition (EMT) is altered and inhibited [START_REF] Fujita | Irradiation induces diverse changes in invasive potential in cancer cell lines[END_REF][START_REF] Moncharmont | Radiation-enhanced cell migration/invasion process: a review[END_REF]; 3) the antitumor immune response is stimulated [START_REF] Rodríguez-Ruiz | Immunological Mechanisms Responsible for Radiation-Induced Abscopal Effect[END_REF]. The biological effects of TRT described below are summarized in Figure 4.

Effects on tumor cells

In B16-F0 and B16-BL6 tumors, TRT induces striking cellular abnormalities with anisocytosis and giant cells [START_REF] Bonnet | Anti-melanoma efficacy of internal radionuclide therapy in relation to melanin target distribution[END_REF][START_REF] Degoul | In vivo efficacy of melanoma internal radionuclide therapy with a 131Ilabelled melanin-targeting heteroarylcarboxamide molecule[END_REF]. Both [ 131 I]ICF01012 and [ 131 I]5-IPN induce a higher proportion of G2-phase blocked cells, suggesting that the main mechanism of TRT toxicity is the mitotic catastrophe [START_REF] Degoul | In vivo efficacy of melanoma internal radionuclide therapy with a 131Ilabelled melanin-targeting heteroarylcarboxamide molecule[END_REF][START_REF] Xu | Targeted radiotherapy of pigmented melanoma with 131I-5-IPN[END_REF]. This hypothesis is supported by morphological nuclear abnormalities, cytoskeleton alterations (tubulin-13 reduced) and consumption of metabolites (amino acids and glucose). In tumors following [ 131 I]ICF01012 injection, DNA damage due to irradiation is indicated by the significant increase in SubG1 phase cells and Ser-15 phosphorylation induction of the p53 protein [START_REF] Akil | Targeted Radionuclide Therapy Decreases Melanoma Lung Invasion by Modifying Epithelial-Mesenchymal Transition-Like Mechanisms[END_REF][START_REF] Degoul | In vivo efficacy of melanoma internal radionuclide therapy with a 131Ilabelled melanin-targeting heteroarylcarboxamide molecule[END_REF]. However, apoptosis is not the main mechanism of [ 131 I]ICF01012 efficacy in this model [START_REF] Degoul | In vivo efficacy of melanoma internal radionuclide therapy with a 131Ilabelled melanin-targeting heteroarylcarboxamide molecule[END_REF]. With [ 131 I]ICF01012 and [ 131 I]5-IPN, tumor cell proliferation was reduced [START_REF] Bonnet-Duquennoy | Targeted radionuclide therapy of melanoma: anti-tumoural efficacy studies of a new 131I labelled potential agent[END_REF][START_REF] Degoul | In vivo efficacy of melanoma internal radionuclide therapy with a 131Ilabelled melanin-targeting heteroarylcarboxamide molecule[END_REF][START_REF] Xu | Targeted radiotherapy of pigmented melanoma with 131I-5-IPN[END_REF]. Reduced clonogenicity [START_REF] Akil | Targeted Radionuclide Therapy Decreases Melanoma Lung Invasion by Modifying Epithelial-Mesenchymal Transition-Like Mechanisms[END_REF] and a decrease in the phosphorylated forms of ERK and AKT (proteins involved in cell survival) were observed after [ 131 I]ICF01012 injection [START_REF] Degoul | In vivo efficacy of melanoma internal radionuclide therapy with a 131Ilabelled melanin-targeting heteroarylcarboxamide molecule[END_REF].

Effect on metastases: angiogenesis and epithelial-mesenchymal transition (EMT)

Although the invasion modifications induced by EBRT have been described [START_REF] Sundahl | Effects of radiation on the metastatic process[END_REF], few data are available on the impact of TRT on metastasis and particularly on EMT-like mechanisms. TRT with [ 131 I]ICF01012 reduces the number of spontaneous lung metastases from subcutaneous primary B16-BL6 tumors [START_REF] Bonnet-Duquennoy | Targeted radionuclide therapy of melanoma: anti-tumoural efficacy studies of a new 131I labelled potential agent[END_REF] and significantly decreases the number and size of B16-BL6 colonies in the lungs [START_REF] Akil | Targeted Radionuclide Therapy Decreases Melanoma Lung Invasion by Modifying Epithelial-Mesenchymal Transition-Like Mechanisms[END_REF]. This might be due to an anti-angiogenic effect, by VEGF downregulation and up-regulation of an anti-angiogenic protein, IP10 [START_REF] Degoul | In vivo efficacy of melanoma internal radionuclide therapy with a 131Ilabelled melanin-targeting heteroarylcarboxamide molecule[END_REF][START_REF] Xu | Targeted radiotherapy of pigmented melanoma with 131I-5-IPN[END_REF]. A decrease in blood vessels has also been found in [ 131 I]5-IPN tumors [START_REF] Xu | Targeted radiotherapy of pigmented melanoma with 131I-5-IPN[END_REF]. Alternatively,

[ 131 I]ICF01012
reduces the acquisition of the EMT-like phenotype by decreasing the expression of EMT-likerelated genes (Sparc,Brn2,Zeb2,Mitf) and by reducing the protein expression of two EMT-like markers, Ncadherin and vimentine [START_REF] Akil | Targeted Radionuclide Therapy Decreases Melanoma Lung Invasion by Modifying Epithelial-Mesenchymal Transition-Like Mechanisms[END_REF]. Further investigations are required to improve understanding of the precise mechanisms underlying the effects of TRT on metastatic processes.

Effect on antitumor immune response

Antitumor immune response has been widely studied in melanoma, which is a tumor able to spontaneously regress due to a high intrinsic mutational burden (for a review, see [START_REF] Passarelli | Immune system and melanoma biology: a balance between immunosurveillance and immune escape[END_REF]. In the same way, EBRT, which leads to DNA alterations and immunogenic cell death, has been shown to modify the antitumor immune response (for a review, see [START_REF] Rodríguez-Ruiz | Immunological Mechanisms Responsible for Radiation-Induced Abscopal Effect[END_REF]. A few studies report changes in the antitumor immune response after TRT (recruitment of specific antigen-presenting cells (Y. [START_REF] Wu | Induction of Anti-Tumor Immune Responses by Peptide Receptor Radionuclide Therapy with (177)Lu-DOTATATE in a Murine Model of a Human Neuroendocrine Tumor[END_REF], induction of a specific T cell immune response [START_REF] Chakraborty | Use of radiolabeled monoclonal antibody to enhance vaccine-mediated antitumor effects[END_REF][START_REF] Keisari | Activation of local and systemic antitumor immune responses by ablation of solid tumors with intratumoral electrochemical or alpha radiation treatments[END_REF] and a memory T cell immune response [START_REF] Hernandez | 90Y-NM600 targeted radionuclide therapy induces immunologic memory in syngeneic models of T-cell Non-Hodgkin's Lymphoma[END_REF]. However, all these data were obtained in non-melanoma tumors and with different vectorization strategies.

In a B16-F10 murine model, [ 131 I]ICF01012 efficacy has been found to depend on the immunocompetence of the mice [START_REF] Rouanet | Immune checkpoint inhibitors reverse tolerogenic mechanisms induced by melanoma targeted radionuclide therapy[END_REF]. Furthermore, [ 131 I]ICF01012 modifies the antitumor immune response by inducing immunogenic cell death (exposure of annexin A1 and calreticulin at the cell membrane). This stimulates the recruitment of cytotoxic T cells, responsible for tumor immune destruction, regulatory T cells involved in immune tolerance, and innate immune cells (NK cells and macrophages) in the tumor environment [START_REF] Rouanet | Immune checkpoint inhibitors reverse tolerogenic mechanisms induced by melanoma targeted radionuclide therapy[END_REF].

TRT combined with other treatments

In spite of the promise of the antitumoral effects of TRT, the advent of targeted therapies and immunotherapy in the early 2010s cast doubt upon this strategy. Combining TRT with ICI and DNA repair inhibitors makes sense as TRT induces DSB and immune tolerance (see paragraph 4.3).

Immune checkpoint inhibitors

Combining TRT with ICI has been assessed using different approaches involving antibodies targeting melanin [START_REF] Jiao | Evaluating the Combination of Radioimmunotherapy and Immunotherapy in a Melanoma Mouse Model[END_REF][START_REF] Nosanchuk | Structure-function analysis and therapeutic efficacy of antibodies to fungal melanin for melanoma radioimmunotherapy[END_REF], a peptide targeting the integrin VLA-4 [START_REF] Choi | Combined VLA-4-Targeted Radionuclide Therapy and Immunotherapy in a Mouse Model of Melanoma[END_REF] and melanin ligands [START_REF] Rouanet | Immune checkpoint inhibitors reverse tolerogenic mechanisms induced by melanoma targeted radionuclide therapy[END_REF]. In this last study, [ 131 I]ICF01012 combined with an anti-CTLA-4 antibody resulted in a significant increase in survival, confirming the importance of immune tolerance after

[ 131 I]ICF01012.
The association of [ 131 I]ICF01012 with anti-PD-1 or anti-PD-L1 antibodies did not bring any significant benefit in terms of survival, showing that T cell exhaustion is not a major phenomenon after TRT.

Further analyses, using the combination of [ 131 I]ICF01012 with an anti-CTLA-4 antibody associated with an anti-PD-1 or an anti-PD-L1 antibody, suggest that the [ 131 I]ICF01012 + anti-CTLA-4 combination limits TRTinduced immune tolerance but induces T cell exhaustion. It is interesting to note that a similar observation was made on a B16 model following combined EBRT and ICI treatment (Twyman-Saint Victor et al., 2015).

Finally, these results confirm that the combination of TRT and ICI is a promising approach for the treatment of metastatic melanoma and opens the prospect of clinical trials for this type of combination.

coDbait

coDbait is a small DNA molecule which mimics a DNA double-strand break and disorganizes the DNA repair mechanisms by trapping repair enzymes [START_REF] Biau | A preclinical study combining the DNA repair inhibitor Dbait with radiotherapy for the treatment of melanoma[END_REF][START_REF] Croset | Inhibition of DNA damage repair by artificial activation of PARP with siDNA[END_REF][START_REF] Quanz | Hyperactivation of DNA-PK by double-strand break mimicking molecules disorganizes DNA damage response[END_REF]. It has been shown that combining [ 131 I]ICF01012 with coDbait results in a synergistic effect or additive effect on tumor growth and median survival, without increasing toxicity, in human SK-Mel 3 melanoma xenografts and in murine syngeneic B16-BL6 models [START_REF] Viallard | Targeting DNA repair by coDbait enhances melanoma targeted radionuclide therapy[END_REF]. However, there are no current plans for clinical trials on a DNA repair trap combined with TRT, even though Dbait associated with EBRT has proven its safety in melanoma patients (Le [START_REF] Tourneau | First-in-human phase I study of the DNA-repair inhibitor DT01 in combination with radiotherapy in patients with skin metastases from melanoma[END_REF].

Summary and perspectives

Benzamide-based radioligands targeting melanin were first developed for imaging. Although their specificity is well established, these structures are more suitable for therapeutic purposes because of the high capacity of [ 18 F]FDG to detect melanomas. In terms of TRT, we believe that, although many questions remain, the use of radiolabeled benzamides may constitute a therapeutic solution for melanomas resistant to therapy. TRT is an original strategy for the systemic targeting of not only pigmented lesions, but also unpigmented metastases through the abscopal effect. Furthermore, immunogenic modifications of treated TRT tumors can enhance the efficacy of ICI. Based on this observation, we are planning a future trial combining TRT with ICI inhibitors. [START_REF] Ito | Chemistry of Mixed Melanogenesis-Pivotal Roles of Dopaquinone[END_REF][START_REF] Ozeki | Chemical characterization of pheomelanogenesis starting from dihydroxyphenylalanine or tyrosine and cysteine. Effects of tyrosinase and cysteine concentrations and reaction time[END_REF]. After PM production, the benzothiazine is gradually converted to benzothiazole [START_REF] Wakamatsu | Chemical analysis of late stages of pheomelanogenesis: conversion of dihydrobenzothiazine to a benzothiazole structure[END_REF]. The depletion of Cys therefore promotes the spontaneous cyclization of DQ, giving rise to DOPAchrome (DC) [START_REF] Del Marmol | Cysteine deprivation promotes eumelanogenesis in human melanoma cells[END_REF][START_REF] Ozeki | Chemical characterization of pheomelanogenesis starting from dihydroxyphenylalanine or tyrosine and cysteine. Effects of tyrosinase and cysteine concentrations and reaction time[END_REF]. This is rearranged to produce the constituent monomers of EM: 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The enzyme DOPAchrome tautomerase (DCT), also called tyrosinase-related protein 2 (Tyrp2), catalyses the production of DHICA [START_REF] Aroca | Regulation of the final phase of mammalian melanogenesis. The role of dopachrome tautomerase and the ratio between 5,6dihydroxyindole-2-carboxylic acid and 5,6-dihydroxyindole[END_REF]. Subsequent oxidation of both dihydroxyindoles forms the EM polymer. C) Overall view of the functions of melanin and its role on melanoma. Table 3. Summary of radiolabeled benzamide derivatives injected into humans with corresponding preclinical studies on mice. The parameters given are for imaging the tumor-to-blood or tumor-to-muscle activity ratios (T/B, T/M) and the percentage of activity injected into the tumors allowing dosimetry to be calculated. For patient studies, the number of the clinical trial is indicated when available. For imaging, sensitivity and specificity calculations are based on lesion analysis. T/M 60 minutes 18.5+4.8 [START_REF] Ma | Biodistribution, Radiation Dosimetry, and Clinical Application of a Melanin-Targeted PET Probe, 18F-P3BZA, in Patients[END_REF]

Acronym

  [ 18 F]-N-(12-ethyl-1fluoro-3,6,9-trioxa-12-azatetradecan14-yl)-6-iodoquinoxaline-2-carboxamide ([ 18 F]ICF15002), demonstrated high and early tumor uptake (14.33%ID/g, 1 hour after injection). After [ 131 I]ICF15002

  , only four different radiolabeling methods were used. The oldest, and simplest one is the isotopic exchange used for [ 123 I]BZA and N-(2-diethylaminoethyl)-2-iodobenzamide ([ 123 I]BZA2). The chemical precursor corresponds to the last radiolabeled compound studied. However, final molecular activity levels are low (always < GBq/µmol). Nevertheless, this disadvantage is not a problem because the BZA derivative-melanin binding process is not saturable. Direct electrophilic aromatic substitution has been used with molecules involving an activating group. This method was successfully employed for the radioiodination of MB, N-((1ethylpyrrolidin-2-yl)methyl)-2-hydroxy-3-iodo-6-methoxybenzamide (IBZM), N-(2-diethylaminoethyl)-3iodo-4-methoxybenzamide (IMBA) and N-(2-diethylaminoethyl)-4-(4-fluorobenzamido)-5-iodo-2methoxybenzamide (MIP-1145). Using a chloro-or bromo-chemical precursor, it is also possible to carry out a halogen exchange involving nucleophilic aromatic substitution. BZA and BZA2 were thus radiolabeled with iodine-123 from the bromo-precursor, N-(2-diethylaminoethyl)-5-fluoropicolinamide (P3BZA) was radiolabeled with fluorine-18 from the bromo-precursor and N-(2-diethylaminoethyl)-6-fluoronicotinamide (Mel050) with fluorine-18 from the chloro-precursor. Note that the last two methods, direct substitution and halogen exchange, require a purification step to separate the chemical precursor from the final radiopharmaceutical. The most recently published method uses Iodo-destannylation of the butyltin precursor for both N-(4-((2-diethylamino-ethylcarbamoyl)-2-iodo-5-methoxyphenyl)benzo[1,3]dioxole-5carboxamide ([ 123/131 I]BA52) and N-(2-diethylaminoethyl)-6-iodoquinoxaline-2-carboxamide ([ 131 I]ICF01012

Figure 1 Figure 1 .

 11 Figure 1

Figure 2 Figure 2 .

 22 Figure 2

Figure 3 Figure 3 .

 33 Figure 3

  34

Figure 4 Figure 4 .

 44 Figure 4

188 Re]6D2, [ 166 Ho]6D2, [ 90 Y]6D2, [ 188 Re]8C3, [ 213 Bi]8C3, [ 177 Lu]8C3)

  . Dadachova et al. successfully tested some TRT strategies in preclinical models with different radiolabeled melanin-binding antibodies ([

188 Re]4B4,[ 177 Lu]4B4, [ 166 Ho]4B4, [ 153 Sm]4B4)

  and a melanin-binding decapeptide ([

131 I]MIP-1145 [ 131 I]BA52

		Preclinical models		Clinical trials
		Imaging T/M or T/B ratio or % of	Therapy Dosimetry	Number of patients	Imaging Sensitivity	Therapy Dosimetry	References
		IA/gr		/ NCT	Specificity	PFS
	[ 123 I]IBZM			1	Case report		(L. S. Maffioli et al., 1993)
				11	87%, nd		(L. Maffioli et al., 1994)
				21	80%, 100%		(Larisch et al., 1998)
	[ 123 I]BZA	B16: 3.5% IA/gr (6 hrs p.i.		110 (phase II)	81%, 100%		(Michelot et al., 1991)
		18 (6 hrs p.i.)					(Michelot et al., 1993)
		19 (6 hrs p.i.)
	[ 123 I]IMBA	B16: 3.5% IA/gr (6 hrs p.i).		2	nd		(Nicholl et al., 1997)
		88 (6 hrs p.i.)
		123 (6 hrs p.i.)
	[ 123 I]MB	HX118: 3% (3 hrs p.i.)					(Link et al., 1996)
	[ 131 I]MB	20 (3 hrs p.i.)		11	80%, 100%		(Link et al., 1998)
		14 (3 hrs p.i.)
	[ 123 I]BZA2	B16:					(Moreau et al., 1995)
		5 (6 hrs p.i.)		25	100%, 95%		(Nicole Moins et al., 2002)
		10 (6 hrs p.i.)		40	75%		(Sillaire-Houtmann et al., 2004)
				87	23%, 86%		(Cachin et al., 2014)
	[ 18 F]Mel050	B16: 9±0.6 (3 hrs p.i.)		NCT01620749			(Denoyer et al., 2010)
	[ SK-Mel-3: 9% (4 hrs p.i.)					(Joyal et al., 2010)
		25 (3 hrs p.i.)				12.2 Gy per GBq	(Mier et al., 2014)
				9		<2 years in 3/5
						receiving >4.5 GBq
	[ 131 I]ICF01012	B16-F0: 41% (6 hrs p.i.)	108 Gy/37 MBq	NCT03784625			(Chezal et al., 2008; Bonnet-
			(B16-BL6)	Recruiting			Duquennoy et al., 2009)
		SK-Mel-3: 27% (6 hrs p.i.)	52 Gy/75 MBq				(Bonnet et al., 2010; Degoul et al.,
			(SK-Mel-3)				2013; Viallard et al., 2015)
	[ 18 F]P3BZA	B16-F10: 17% (2 hrs p.i.)		NCT03033485
		88 (2 hrs p.i.)		6 controls
		37 (2 hrs p.i.)		5 patients

Conflict of Interest Statement:

The authors declare that they have no conflicts of interest. Table 1. Main properties of the three radionuclides used in clinical studies involving benzamide derivatives.

Tables

Radio-isotope

Half Sep-pak cartridge 60-80% >98% 24-84 GBq/µmol [START_REF] Chezal | Evaluation of radiolabeled (hetero)aromatic analogues of N-(2-diethylaminoethyl)-4-iodobenzamide for imaging and targeted radionuclide therapy of melanoma[END_REF] This article n.p.: not published