R Dupuy

M Bertin

G Féraud

X Michaut

P Marie-Jeanne

P Jeseck

L Philippe

V Baglin

R Cimino

C Romanzin

J.-H Fillion

Mechanism of Indirect Photon-induced Desorption at the Water Ice Surface

Electronic excitations near the surface of water ice lead to the desorption of adsorbed molecules, through a so far debated mechanism. A systematic study of photon-induced indirect desorption, revealed by the spectral dependence of the desorption (7-13 eV), is conducted for Ar, Kr, N2 and CO adsorbed on H2O or D2O amorphous ices. The mass and isotopic dependence and the increase of intrinsic desorption efficiency with photon energy all point to a mechanism of desorption induced by collisions between adsorbates and energetic H/D atoms, produced by photodissociation of water. This constitutes a direct and unambiguous experimental demonstration of the mechanism of indirect desorption of weakly adsorbed species on water ice, and sheds new light on the possibility of this mechanism in other systems. It also has implications for the description of photon-induced desorption in astrochemical models.

Water ice is ubiquitous on Earth and in space, and often plays the role, among others, of an environment onto which less abundant molecules adsorb, desorb and react [1][2][3][4][5]. Energetic irradiation of such environments will result in energy deposition mostly in the form of electronic excitations of the water matrix, which can subsequently affect molecules adsorbed on the ice through energy transfer or reaction with radicals. Adsorbate desorption is one of the possible outcomes. Photodesorption by vacuum-UV (VUV) photons is, for instance, an important process taking place at the surface of interstellar dust grains, which are covered by an icy mantle mainly composed of water, along with smaller amounts of molecules such as CO, CO 2 , N 2 or CH 4 [6]. It affects the gas and ice molecular abundances in the interstellar medium, and explains how some molecules (such as water) are observed in the gas at temperatures where their thermal desorption from grains is not possible [7,8].

A molecular-scale description of the mechanism by which an electronic excitation eventually causes desorption has so far been achieved only for a handful of molecular or atomic solids, such as rare-gas solids [9,10]. This is despite a considerable amount of experimental measurements on photodesorption focusing on model "pure" molecular ices [START_REF] Caro | Photon-Induced Desorption Processes in Astrophysical Ices[END_REF][START_REF] Mccoustra | Exciton-Promoted Desorption From Solid Water Surfaces[END_REF][START_REF] Öberg | [END_REF][14], including water ice, as detailed below. On the other hand, there is currently a lack of detailed understanding of the desorption properties -both the desorption mechanism and an estimate of the yield of the process -of more realistic "mixed" systems. In such systems, indirect desorption processeswhere an excited matrix species transfers energy to a different species which desorbs -play an important role. Indirect photodesorption has been studied for the astrophysically relevant CO-ice environment [15][16][17][18][19] but the exact mechanism remains elusive. Water ice is a more complex but very important system for which indirect desorption has been little explored.

What has been the object of many studies is the fate of electronic excitations at the pure water ice surface [14,[START_REF] Johnson | Photolysis and radiolysis of water ice[END_REF][21][22][23][24][25], and how they may lead to desorption of intact water [26][27][28][29][30][START_REF] Fillion | Astronomy & Astrophysics[END_REF][START_REF] Westley | [END_REF][33][34][35][36]. The dominant underlying mechanism for this latter process remains nonetheless debated.

Theoretical works [26,27] suggested the co-existence of two different mechanisms, initiated by water photodissociation: one where an energetic H atom issued from the dissociation transfers momentum to another molecule and "kicks" it out into vacuum, and another where a dissociated water molecule immediately recombines (geminate recombination) with the exothermic energy of recombination leading to desorption. Experimental measures of the rotational and kinetic energies of desorbing H 2 O (v=0) were shown to be compatible with these mechanisms [28]. However, other experimental and theoretical investigations [29,30], in principle very similar, favoured instead a more direct mechanism, without dissociation but involving exciton localization at the surface and an electronic repulsion of the excited water molecule relative to its surroundings, causing its desorption, in line with the classic MGR (Menzel-Gomer-Redhead) model [37]. In addition, we showed in a recent study [START_REF] Fillion | Astronomy & Astrophysics[END_REF] that the observed isotopic and temperature effects in the desorption from water ice required consideration of additional mechanisms, such as desorption channels linked with photochemistry. From the available experimental evidence, it is therefore not possible to conclude definitively on the existence of any of these mechanisms and whether they can be extrapolated to other closely related systems. The desorption of other molecules adsorbed on water ice, for instance, remains largely unexplored, despite its importance for a more realistic description of electronic desorption on actual icy surfaces.

In this letter we report a study of indirect VUV photon-induced desorption of molecules weakly adsorbed (E b < 130 meV) on amorphous water. We systematically measured indirect photodesorption yields for four adsorbates (Ar, Kr, CO, N 2 ) on either H 2 O or D 2 O ices. Our results reveal unambiguously that the energetic H atom collision mechanism dominates indirect desorption for the systems investigated here. It is therefore now clear that there are systems where this mechanism does operate and even dominates, although it is not the case for water desorption from the pure ice [30,[START_REF] Fillion | Astronomy & Astrophysics[END_REF].

The experiments were performed in an ultra-high vacuum chamber coupled to the DESIRS undulator beamline [38] of the SOLEIL synchrotron which provides monochromatic photons in the VUV range (7-13 eV). Ices are grown on a polycrystalline copper substrate cooled down to 15-100 K by exposition to a partial pressure of gas using a dosing tube. First a 100 ML eq (monolayer equivalent) water ice is grown at 100 K, yielding compact amorphous solid water [39] (c-ASW). 1 ML eq of the adsorbate is then deposited on top at a temperature of 15 K. The thickness of the deposited molecular ice is controlled and calibrated using the temperature programmed desorption (TPD) technique [40]. Photodesorption induced by irradiation of the ice is monitored in the gas phase by quadrupole mass spectrometry (QMS). We have only monitored desorption of the adsorbates, as intact water, OH or H fragments cannot be detected under these experimental conditions. The QMS is calibrated to derive absolute photodesorption yields, in molecules/incident photon. More experimental details are described in previous papers [17,41] and in the Supplemental Material, which includes ref. [START_REF] Doronin | Adsorption on interstellar analog surfaces: from atoms to organic molecules[END_REF][START_REF] Fayolle | [END_REF][44][45][46][47].

The photodesorption spectrum of Ar from the Ar/H 2 O system is presented on fig. 1. Also shown for comparison are the photodesorption spectrum of Ar for a pure 20 ML Ar ice, and the absorption spectrum of amorphous water ice taken from Lu et al. [48]. Spectral information provides here a direct evidence of indirect desorption. The pure Ar photodesorption spectrum is very different from the spectrum of Ar desorbing from Ar/H 2 O: the threshold for Ar absorption, and desorption from the pure ice, is 11.55 eV. Desorption of Ar below that photon energy in the Ar/H 2 O spectrum is therefore due to water ice excitation, as is seen from the resemblance between that part of the desorption spectrum and the absorption spectrum of water ice. We will focus here in particular on the first electronic excited state of condensed water, which is dissociative in the gas phase (where it corresponds to the 4a 1 1b 1 transition) and in condensed phase [24]. An apparent shift of the maximum of this state, observed at 8.65 eV in water ice absorption and at 8.95 eV in the Ar from Ar/H 2 O desorption spectrum, can be observed. This apparent shift actually corresponds to a deformation of the spectrum caused by an increase of desorption efficiency throughout the first electronic state of H 2 O ice. This is visible in the lower panel of fig. 1 where the Ar photodesorption spectrum was divided by the H 2 O absorption spectrum, yielding the intrinsic desorption efficiency. The efficiency increases almost linearly from 8 to 9 eV, then remains constant at higher photon energy. Similar experiments performed on 1 ML eq Kr, N 2 and CO adsorbed on compact amorphous H 2 O are displayed in fig. 2. The absolute photodesorption yields differ but the spectral shape for Kr and N 2 , whose first dipole-allowed electronic state are respectively at 9.9 eV and 12.4 eV in the condensed phase, is similar to Ar/H 2 O. For both these adsorbates, as for Ar, it is therefore straightforward to measure indirect desorption through the spectral signature of the first water ice excited state around 9 eV. This state appears again blue-shifted compared to the absorption spectrum in the desorption spectra. The excitation threshold of CO on the other hand is at 7.9 eV, and its first excited state overlaps with the water ice excited state. Still, the structure due to the vibronic bands of that CO excited state makes it so that one can still easily see the presence, and estimate the quantitative contribution, of indirect water-induced desorption to the overall CO desorption spectrum.

The experiments were repeated for the same adsorbates on D 2 O ice (grown under the same conditions) for comparison. The results are shown in fig. S2 and are quite similar to those obtained for H 2 O (including the increase of desorption efficiency throughout the first water ice electronic state), to the exception of the absolute photodesorption yield which is different.

The experimental results are summarized in table I, indicating the indirect desorption yields (as measured at the maximum of the first electronic state of water in the desorption spectrum, around 9 eV) for all investigated systems as well as the binding energy of each adsorbate to water ice and their mass. First we can observe that the indirect desorption yield goes in the order CO > N 2 > Ar > Kr, and second that for a given adsorbate, the indirect desorption yield is systematically higher on D 2 O ice than on H 2 O ice. These facts, along with the observed linear increase of desorption efficiency through the first water ice electronic state in the desorption spectra, allow us to discuss the possible mechanisms of indirect desorption. Indirect desorption on water ice has been demonstrated before in the case of adsorbed benzene, using electron-stimulated desorption [23,49]. In that case the authors concluded that an electronic excitation transfer occurred between water and benzene. In our case, we can exclude any mechanism of that kind, i.e. that would involve energy transfer to the internal modes of the adsorbates. We observe indirect desorption occurring in the case of Ar and Kr below their excitation threshold: an electronic energy transfer is energetically impossible for these two adsorbates, and there is no notion of transfer to other modes (vibrational, librational) for these atoms as there could be for molecules. Let us now turn to the mechanisms that have been proposed for intact water desorption from the pure ice. Interestingly, these mechanisms predict different isotopic effects. The mechanisms of excited state repulsion and geminate recombination mentioned earlier predict no or close to no isotopic effect, while the H/D atom collision mechanism implies a higher efficiency of desorption for D 2 O than for H 2 O, because D atoms transfer momentum to heavier species more efficiently than H atoms. In the most recent experiments on pure water ice [START_REF] Fillion | Astronomy & Astrophysics[END_REF], we observed for intact water desorption a higher desorption yield for H 2 O than for D 2 O, in contradiction with several of these mechanisms. However the reverse isotopic effect is observed here: indirect desorption is more efficient on D 2 O ices, implying a different dominant mechanism than for intact water desorption. The most fitting mechanism for indirect desorption is therefore energetic H/D atom collision, also called kick-out mechanism.

In the present case, where our adsorbates are atoms or diatomic molecules, this mechanism can be modelled as a pure kinetic energy transfer in a simple hard sphere collision. The fraction of transferred energy therefore simply depends on the masses of the colliding atom (H or D) and the adsorbate. It can be written as:

E kin,transf E kin,H/D = 4m H/D m ads (m H/D + m ads ) 2 (1)
Where E kin,transf is the kinetic energy transferred to the adsorbate upon collision, E kin,H/D the initial kinetic energy of the H/D atom, and m H/D and m ads the respective masses of the H/D atom and the adsorbate. Let us now estimate the kinetic energy of the H/D atom upon water dissociation. At a photon energy of 9 eV, considering a dissociation energy of 5.4 eV for H 2 O and ∼ 0.7 eV going into OH translational and internal energy [51], the H atom ends up with approximately 2.9 eV of kinetic energy. A D atom would have slightly less. We can calculate that the absolute amount of kinetic energy transferred to the adsorbate therefore varies between 100 and 600 meV for our systems.

Fig. 3 shows the indirect desorption yields of all investigated systems plotted against the estimated amount of kinetic energy that would be transferred to the adsorbate in a collision with an H/D atom (E kin,transf ). This figure shows a clear positive correlation. To confirm further the hypothesis of the collision mechanism, we need to know how the desorption probability of the adsorbate relates to the amount of transferred kinetic energy. This has been explored by Fredon et al. [52,53] in molecular dynamics simulations. In these works the authors look at the desorption and energy dissipation of molecules at the surface of water ice which have been given translational energy. They derive an empirical function for the desorption probability as a function of initially imparted translational energy:

P des (E kin ) = 1 -exp -ω E kin -E bind E bind (2)
with P des the desorption probability, E kin the kinetic energy imparted initially to the adsorbate, E bind the binding energy of the adsorbate, and ω a factor set to 0.3 which fits best their simulation results.

The desorption probability calculated from equation 2, is plotted on fig. 3 for E bind = 70, 100 and 130 meV and ω = 0.3. A good agreement with the experimental results is obtained, especially considering the error bars, relative calibration uncertainties and the fact that each species have slightly different binding energies. Some of the observed differences -e.g. the fact that the rare gases points tend to be closer to the lower curve and the diatomic points to the upper one, contrary to what would be expected from their binding energy differences -could indicate differences in the behaviour of atoms and molecules, as discussed below. The various sources of uncertainty however preclude firm conclusions.

The H/D collision mechanism also explains the linear increase of desorption efficiency observed between 8 and 9 eV (bottom panel of fig. 1). As photon energy increases throughout the first state, the amount of excess energy available increases as well and therefore the kinetic energy of the H/D atoms increases. This can (i) increase the amount of kinetic energy imparted by H/D atoms to the adsorbates, and (ii) increase the contribution of deeper layers of the ice to indirect desorption, as H/D atoms originating from deeper layers acquire sufficient kinetic energy to reach the surface and kick adsorbates. Both effects will lead to an increased desorption probability with photon energy. The changes in the desorption probabilities depend in principle on the different parameters in equation 2 and should therefore vary for the different adsorbates, leading to different spectrum deformations. The desorption efficiencies are shown in the Supplemental Material. While the changes are indeed not exactly similar, they remain too small to analyse further. Above 9 eV the increase in desorption efficiency is no longer observed, which can be explained by the loss of energy to other channels such as vibrational or electronic excitation of the fragments [26]. Higher electronic states of water ice can be excited that also have a dissociative character [START_REF] Schinke | Photodissociation dynamics: spectroscopy and fragmentation of small polyatomic molecules, Cambridge monographs on atomic, molecular and chemical physics[END_REF], and therefore they can also lead to production of energetic H atoms and collision desorption.

One possibility that needs to be discussed before concluding definitively in favor of the H/D collision, is a mechanism that would involve collisions with OH/OD fragments. The more favourable mass ratio with the adsorbates means that even though OH/OD fragments acquire much less kinetic energy than H/D fragments upon dissociation, they would still transfer enough kinetic en-ergy to induce desorption. The isotopic effect observed would then come not from the collision with the adsorbate, but from the photodissociation event, where OD fragments acquire almost twice more energy than OH fragment due to momentum conservation. Such a mechanism has however not been observed in the molecular dynamics simulations that observed the H/D collision mechanism. Furthermore, OH/OD fragments have a much lower mobility in ice [35], and from momentum conservation estimations [51] would transfer twice less energy to adsorbates than in the equivalent H/D collision. We therefore consider this mechanism as possible but non-dominant.

The full elucidation of the indirect desorption mechanism means that our results can be extrapolated to other systems. All weakly adsorbed atoms and diatomic molecules, at least, should follow a behaviour similar to what we observed. For these species the photodesorption yields can be derived from the desorption model of equations 1 and 2, scaled to the experimentally measured absolute values. As pointed out above, there could be slight differences in the behaviour of atoms and diatomic molecules considering that for the latter excitation of internal degrees of freedom are possible. We would for sure expect deviations for even more sizeable molecules, for which a hard sphere collision model will not be appropriate any more, and for cases where a reactive collision can compete. Already for the case of H 2 O desorption itself by the H collision mechanism, the theoretical calculations [26] point to the importance of the collision happening at the center of mass of the molecule (on the O atom) for the efficiency of the process. For adsorbates that form hydrogen bonds with water we may expect also different processes coming into play, such as electronic energy transfer as in the case of benzene on water ice [49]. Nonetheless, the present results could serve as a first basis for the modelling of indirect waterinduced desorption in e.g. astrochemical models.

As we outlined previously, the so-called "kick-out" mechanism, which was among the first mechanisms suggested to explain desorption from pure water ice [26], had not received a completely unambiguous experimental demonstration of its existence so far, due to the complexity of disentangling the different possible mechanisms from the available for pure water ice [30,[START_REF] Fillion | Astronomy & Astrophysics[END_REF]. We now provide such a demonstration in a slightly different system, weakly adsorbed molecules on water ice, for which all experimental facts converge. We thus shed light on a way indirect desorption can proceed. This is an important step towards a better comprehension of (indirect) desorption processes induced by electronic transitions and the fate of excitations at the water ice surface. This mechanism also deserves interest for any surface from which energetic H atoms could be produced (other hydride molecular ices, hydrogenated carbon surfaces...).

Figure 1 .

 1 Figure 1. Upper panel: photodesorption spectrum (desorption yield as a function of photon energy) of argon for 1 MLeq of Argon adsorbed on 100 ML compact amorphous solid water c-ASW (black line). Also shown for comparison are the absorption spectrum of amorphous H2O ice from Lu et al. [48] (green line, arbitrary units) and the photodesorption spectrum of Argon from 20 ML pure Argon ice (red line, scaled 1/200). Lower panel: photodesorption spectrum of argon 1 MLeq on c-ASW (black spectrum of the upper panel) divided by H2O absorption (green spectrum of the upper panel).

C O 1 M L e q o n H 2 ON 2 1 M L e q o n H 2 OK r 1 M L e q o n H 2 OFigure 2 .

 1212122 Figure 2. Photodesorption spectra (black lines) of 15 N2 from 15 N2 adsorbed on c-ASW (top panel), Kr from Kr adsorbed on c-ASW (middle panel) and 13 CO from 13 CO adsorbed on c-ASW (bottom panel). The absorption spectrum of c-ASW from Lu et al. (green line) is reproduced on the top panel. On the bottom panel, the photodesorption spectrum of 20 ML pure CO is also shown (scaled 1/19) and fitted under the CO/H2O photodesorption spectra to estimate the contribution of indirect desorption, indicated by the blue arrow.

Figure 3 .

 3 Figure 3. Indirect desorption yields for each system as a function of the fraction of kinetic energy transferred in the collision of an H/D atom with the adsorbate (see text for the formula). The indirect desorption yield is measured as the photodesorption yield at 9 eV at the maximum of the first electronic state of water ice. Error bars only reflect the signal to noise ratio of the desorption spectra, not errors in the relative calibration of the signals and coverage of the different species (see Supplemental Material). The green line is the desorption probability model from Fredon et al.[52] for a binding energy value of E bind = 70, 100 or 130 meV (upper, middle and lower curves) and an initial H/D kinetic energy of 2.9 eV.

Table I .

 I Summary of the investigated systems

	Adsorbate	Indirect yield a	Mass Binding energy b
		(mol/photon)	(a.m.u.)	(meV)
		on H2O	on D2O
	13 CO	3.4 ×10 -3 4.5 ×10 -3	29	122
	15 N2	3 ×10 -3 4 ×10 -3	30	99
	Ar	1.4 ×10 -3 3.8 ×10 -3	40	75
	Kr	5 ×10 -4 1.5 ×10 -3	84	118
	a H 2 O-induced indirect desorption yield of the molecule for
	single layer systems at 9 eV.
	b Binding energy on compact amorphous H 2 O for 1 ML, from
	Smith et al. [50].

We acknowledge SOLEIL for provision of synchrotron radiation facilities under the project 20180060 and we thank Laurent Nahon and the DESIRS beamline team for their help. This work was done in collaboration and with financial support by the European Organization for Nuclear Research (CERN) under the collaboration agreement KE3324/TE. The research was supported by the Programme National "Physique et Chimie du Milieu Interstellaire" (PCMI) of CNRS/INSU with INC/INP co-funded by CEA and CNES. Financial support from the LabEx MiChem, part of the French state funds managed by the ANR within the investissements d'avenir program under reference ANR-11-10EX-0004-02, and by the Ile-de-France region DIM ACAV program, is gratefully acknowledged.


Guillaume Hanouna

Ellie Tang

Joëlle Perez

Sophie Vandermeersch

Jean-Philippe Haymann

Laurent Baud

Emmanuel Letavernier
email: emmanuel.letavernier@aphp.fr

Prof Emmanuel Letavernier

Preventing Calpain Externalization by Reducing ABCA1 Activity with Probenecid Limits Melanoma Angiogenesis and Development

Keywords: calpain, calpastatin, angiogenesis, melanoma, ABCA1

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

Introduction

Calpains are calcium-activated neutral cysteine proteases [START_REF] Goll | The calpain system[END_REF][START_REF] Zatz | Calpains and disease[END_REF]. Two major isoforms, calpain µ and m are ubiquitously expressed, whereas the other isoforms are tissue-specific forms. Calpastatin, a specific endogenous inhibitor which contains four equivalent inhibitory domains, is responsible for the limited proteolysis of specific calpain substrates [START_REF] Goll | The calpain system[END_REF]. As they are expressed in the cytosol, these targets are thought to be intracellular. Structural determinants of calpain substrates are still not well defined. Several of them contain a sequence rich in proline, glutamic acid, serine, and threonine (PEST domain) enhancing both calpain binding and calpain-dependent proteolysis [START_REF] Shumway | The PEST domain of IkappaBalpha is necessary and sufficient for in vitro degradation by mu-calpain[END_REF][START_REF] Wang | A PEST sequence in ABCA1 regulates degradation by calpain protease and stabilization of ABCA1 by apoA-I[END_REF]. Using mice with calpastatin transgene overexpression (since calpain µ and m knock-out mice are not viable), we and others have demonstrated that calpains are involved in a great diversity of inflammatory diseases [START_REF] Howatt | Leukocyte Calpain Deficiency Reduces Angiotensin II-Induced Inflammation and Atherosclerosis But Not Abdominal Aortic Aneurysms in Mice[END_REF][START_REF] Letavernier | Calpains contribute to vascular repair in rapidly progressive form of glomerulonephritis: potential role of their externalization[END_REF][START_REF] Letavernier | Critical role of the calpain/calpastatin balance in acute allograft rejection[END_REF][START_REF] Letavernier | Targeting the calpain/calpastatin system as a new strategy to prevent cardiovascular remodeling in angiotensin II-induced hypertension[END_REF][START_REF] Wan | Calpastatin overexpression impairs postinfarct scar healing in mice by compromising reparative immune cell recruitment and activation[END_REF][START_REF] Zafrani | Calpastatin controls polymicrobial sepsis by limiting procoagulant microparticle release[END_REF].

The contribution of calpains in tumor processes has been also questioned [START_REF] Leloup | Calpains as potential anti-cancer targets[END_REF][START_REF] Moretti | Calpains and cancer: friends or enemies?[END_REF][START_REF] Ono | Calpain research for drug discovery: challenges and potential[END_REF][START_REF] Storr | The calpain system and cancer[END_REF]. Their expression and activity appear increased in numerous cancer types, in response to different oncoproteins and epidermal growth factor. As a consequence, calpains are involved in lessened adhesiveness and increased invasiveness of transformed cells, mainly through the cleavage of cytoskeletal proteins (amoeboid mechanism) and the MMP-dependent degradation of extracellular matrix components (mesenchymal mechanism). Tumor invasiveness and dissemination also depends on tumor neovascularization in response to angiogenic factors, including vascular endothelial growth factors (e.g. VEGF) which stimulate the endothelial cell migration through the mcalpain activity. The function of calpains in tumor cell death and survival appears ambiguous [START_REF] Leloup | Calpains as potential anti-cancer targets[END_REF][START_REF] Storr | The calpain system and cancer[END_REF]. In a recent study using a mouse model of melanoma, we observed that calpain inhibition restricted to melanoma cells amplifies their dissemination by accelerating migration process [START_REF] Raimbourg | The calpain/calpastatin system has opposing roles in growth and metastatic dissemination of melanoma[END_REF]. Meanwhile, calpain inhibition restricted to host cells blunts tumor infiltration by immune cells, allowing tumor cells to escape tumor niche and disseminate [START_REF] Raimbourg | The calpain/calpastatin system has opposing roles in growth and metastatic dissemination of melanoma[END_REF].

Recent studies demonstrated that calpains are also partially externalized. In extracellular localization, they limit paradoxically inflammatory processes and promote tissue repair [START_REF] Letavernier | Calpains: a double edge sword[END_REF][START_REF] Abe | Cell-associated activation of latent transforming growth factor-beta by calpain[END_REF][START_REF] Yoshimura | Chemerin reveals its chimeric nature[END_REF]. They decrease IL-17A expression as well, by causing Toll like receptor 2 (TLR2) cleavage and thus inhibition of TLR2-induced transcription of molecules essential for IL-17A induction [START_REF] Perez | Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression[END_REF]. In addition, we demonstrated that extracellular calpains participate in both epithelium regeneration in a model of acute kidney injury and angiogenesis in models of kidney inflammation [START_REF] Frangié | Extracellular calpains increase tubular epithelial cell mobility. Implications for kidney repair after ischemia[END_REF][START_REF] Letavernier | Calpains contribute to vascular repair in rapidly progressive form of glomerulonephritis: potential role of their externalization[END_REF]. Calpain exteriorization involves the ABCA1 transporter [START_REF] Perez | Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression[END_REF]. Given the involvement of intracellular calpains in cancer development and the opposite functions of intra-and extra-cellular calpains in inflammatory processes, we sought to assess the modulatory role of extracellular calpains in melanoma development. To this aim, we first used a model of transgenic mice expressing the entire coding sequence of the mouse calpastatin gene Cast-201 under the control of the human CRP promoter and a signal peptide (CRP/Calpast) [START_REF] Perez | Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression[END_REF]. This construction induces both liver expression and secretion of calpastatin and, hence, allows to blunt selectively the extracellular activity of calpains. We then used a pharmacological approach to limit calpain exteriorization through the ABCA1 transporter. Both studies demonstrate that blocking the extracellular activity of calpains reduces melanoma growth in mice mainly by limiting angiogenesis process.

Results

Limiting the activity of exteriorized calpains with extracellular calpastatin is protective against melanoma development in mouse.

To address the role of extracellular calpains in tumor development, we used transgenic mice expressing the mouse calpastatin gene under the control of the CRP gene promoter/signal peptide (CRP/Cast) [START_REF] Perez | Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression[END_REF]. Such a construct induces the liver expression of calpastatin which then reaches tissue extracellular medium via the blood stream. We subcutaneously implanted these CRP/Cast mice and wild type (WT) mice with melanoma cell line B16-F10 (10 6 cells/flank) [START_REF] Raimbourg | The calpain/calpastatin system has opposing roles in growth and metastatic dissemination of melanoma[END_REF]. Tumor growth monitored by caliper measurements was significantly reduced in CRP/Cast relative to WT mice (Figure 1a andb).

We next investigated the mechanisms whereby extracellular calpastatin limits melanoma development. The first possibility was that inhibition of the extracellular activity of calpains would slow down tumor cell proliferation but BrdU incorporation assays showed that the proliferation of B16-F10 cells was not affected by extracellular calpain activity (Figure 2a).

The second hypothesis was that a decrease of extracellular calpain activity would alter immune polarization or the production of specific cytokines by tumor cells and/or infiltrating immune cells. In particular, B16 melanoma expansion has been shown to be increased by IL-6/IL-17 (through an IL-6-Stat3 pathway involving ROR γt) and conversely decreased by IFNγ [START_REF] Wang | IL-17 can promote tumor growth through an IL-6-Stat3 signaling pathway[END_REF]. However, our previous study demonstrated clearly that inhibition of extracellular calpains increases IL-17 expression dramatically and IL-6 to a lesser extent while it does not affect IFN-γ production [START_REF] Perez | Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression[END_REF]. In CRP/Cast as compared with WT mice, gene expression analysis in melanoma tumors showed no significant change of IL-2, IL-6, ROR γt and Foxp3, (Figure 2b). Nevertheless, flow cytometry analysis of tumoral cells revealed a mild but significant increased number of CD8 positive cells infiltrating CRP/CAST tumors (Figure 2c-f)

The third hypothesis was that a decrease of extracellular calpain activity would limit melanoma angiogenesis, which plays a crucial role in melanoma growth [START_REF] Jour | Angiogenesis in melanoma: an update with a focus on current targeted therapies[END_REF][START_REF] Meierjohann | Hypoxia-independent drivers of melanoma angiogenesis[END_REF][START_REF] Pastushenko | Mechanisms of tumour vascularization in cutaneous malignant melanoma: clinical implications[END_REF]. To this aim, capillary density has been measured in whole melanoma harvested at day 10 and actually, extracellular calpastatin limited dramatically tumoral neoangiogenesis in CRP/CAST mice (Figure 2g-i).

Limiting the exteriorization of calpains by reducing ABCA1 activity is protective against melanoma development and tumoral neoangiogenesis in mouse

Earlier studies suggested that the ABCA1 transporter is involved in non-classical export of calpains [START_REF] Perez | Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression[END_REF]. Since drugs such as glyburide and probenecid are thought to be ABCA1 inhibitors, we first determined in vitro the effect of probenecid on calpain exteriorization from melanoma cell line B16-F10 [START_REF] Flieger | Regulated secretion of macrophage migration inhibitory factor is mediated by a non-classical pathway involving an ABC transporter[END_REF]. Extracellular calpain activity was reduced in a dose-dependent manner by probenecid (Figure 3a). Conversely, intracellular calpain activity increased when melanoma cells were exposed to probenecid, confirming that probenecid limits calpain efflux from melanoma cells (Figure 3b). Similar results were obtained with glyburide (Figure 3c-d). At last, ABCA1 silencing by SiRNA limited significantly calpain externalization (Figure 3e-f). SiRNA inhibited 52 to 65% of ABCA1 mRNA expression in each experiment.

Probenecid did not affect melanoma cell proliferation as shown by BrdU incorporation assays (Figure 4a). We next analyzed in vivo the effect of probenecid on melanoma development in WT mice. Tumor volume and weight were significantly reduced in mice given probenecid (50 mg per kg BW at days 0, 4 and 8) as compared with untreated mice (Figure 4b-c). Tumor growth and tumor weight at day 16 were significantly reduced by probenecid administration (Figure 4b-c). Another set of experiments was conducted to quantify angiogenesis at day 10: tumor volume was significantly decreased (101±28 µm 3 vs 314±83 µm 3 , p=0.02) and capillary density was reduced significantly in whole melanoma from mice treated with probenecid, evidencing the antiangiogenic role of probenecid in this model (Figure 4d).

Limiting the exteriorization of calpains by probenecid inhibits neo-angiogenesis ex-vivo and in vitro

To confirm that probenecid exerts antiangiogenic properties, the outgrowth of vessels from aortic rings exposed to probenecid has been measured at day 7. Probenecid decreased significantly ex vivo angiogenesis, even at low concentrations (Figure 5a). The exposure of aortic ring to calpains (4µM every 2 days) did not increase significantly the outgrowth of vessels, but the addition of calpains (4 µM every 2 days) to a medium containing 500 µM probenecid inhibited partly the antiangiogenic effect of probenecid, suggesting that probenecid inhibits angiogenesis by inhibiting calpain externalization from cells (Figure 5b-f).

To confirm that probenecid exerted antiangiogenic properties on endothelial cells through a decrease in calpain extracellular activity, the formation of capillaries in matrigel has been assessed in presence of probenecid and/or calpain and calpastatin. Probenecid (500 µM) reduced significantly capillary lenght (Figure 5g). Extracellular calpain inhibition by calpastatin inhibited capillary formation in a similar manner and the addition of probenecid (500 µM) to calpastatin did not limit further angiogenesis (Figure 5g). In a medium containing low amounts of fetal calf serum, extracellular calpain (4µg/mL) increased capillary formation (Figure 5h-j). Probenecid (500µM) decreased capillary formation significantly in similar conditions (Figure 5h,i,k). The addition of 4 µg/mL calpain to medium containing 500 µM probenecid restored capillary formation (Figure 5h,k,l).

Limiting the exteriorization of calpains by probenecid limits fibronectin cleavage

We investigated the mechanisms whereby limitation of calpain externalization by probenecid would limit (neo-)angiogenesis. Probenecid did not induce endothelial cell death and did not reduce endothelial cell proliferation in vitro (Figure 6a,b). It has been shown previously that externalized calpains can degrade specifically fibronectin, an extracellular matrix protein, and by this way increase epithelial cell migration properties [START_REF] Frangié | Extracellular calpains increase tubular epithelial cell mobility. Implications for kidney repair after ischemia[END_REF][START_REF] Letavernier | Calpains contribute to vascular repair in rapidly progressive form of glomerulonephritis: potential role of their externalization[END_REF]. In vivo, intact fibronectin was increased significantly in melanoma in the presence of probenecid, suggesting that probenecid actually limited significantly fibronectin cleavage due to calpain activity (Figure 6c-d).

Limiting the exteriorization of calpains by probenecid inhibits endothelial cell migration

Probenecid inhibited the migration of endothelial cells in a dose-dependent manner (Figure 6e). Conversely, extracellular calpain (4 µg/mL) increased endothelial cell migration (Figure 6f-h) and the addition of 4 µg/mL calpain to a medium containing 500 µM probenecid restored endothelial cell migration properties (Figure 6f,i,j).

Discussion

In this report, we show that calpains exteriorized via the ABCA1 transporter play a key role in melanoma neo-angiogenesis. The importance of neo-angiogenesis in melanoma maintenance, local growth and metastasis has been well demonstrated. Hypoxia-dependent processes lead to the activation of hypoxia-inducible factor-1 (HIF1), a potent inducer of pro-angiogenic factors, including VEGF, angiopoietin-2, matrix metalloprotease 14 (MMP 14), and angiogenin [START_REF] Meierjohann | Hypoxia-independent drivers of melanoma angiogenesis[END_REF]. Metalloproteinases expression by melanoma cells is responsible for the degradation of extracellular matrix proteins such as collagens and fibronectin which bind integrins, αvβ3 and α5β1, respectively, thereby amplifying tumor invasiveness [START_REF] Jour | Angiogenesis in melanoma: an update with a focus on current targeted therapies[END_REF]. Extracellular calpains also cleave matrix fibronectin, inducing a decrease in the linkage between endothelial cells and matrix with a switch from α5β1 to αvβ3 integrin-based adhesion. In turn, αvβ3 engagement promotes angiogenesis [START_REF] Cseh | Autocrine fibronectin directs matrix assembly and crosstalk between cell-matrix and cell-cell adhesion in vascular endothelial cells[END_REF]Lee et al. 2017). In addition, we have shown that truncated forms of fibronectin resulting from extracellular calpain activity contain both the cell binding domain III9 and the VEGF binding domain III13 [START_REF] Letavernier | Calpains contribute to vascular repair in rapidly progressive form of glomerulonephritis: potential role of their externalization[END_REF]. These fragments of fibronectin, when released by calpain activity, increase the angiogenic response to VEGF. Thus, limiting the extracellular activity of calpains in WT mice given probenecid or CRP/Cast mice could decrease angiogenic response by preventing those processes. A significant increase in CD8+ cells was observed in CRP/CAST melanoma. Interestingly, VEGF/VEGFR signalling reduces adhesion molecule expression in melanoma intratumoral vessels, reducing CD8+ cells recruitment [START_REF] Tan | Control of immune cell entry through the tumour vasculature: a missing link in optimising melanoma immunotherapy?[END_REF][START_REF] Georganaki | Vascular Targeting to Increase the Efficiency of Immune Checkpoint Blockade in Cancer[END_REF]. Whether extracellular calpain and calpastatin may influence tumor-induced endothelial cell anergy is unknown.

We have previously identified that calpains are exteriorized from lymphocytes through an ABCA1-dependent process involving the shedding of microvesicles [START_REF] Perez | Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression[END_REF][START_REF] Zafrani | Calpastatin controls polymicrobial sepsis by limiting procoagulant microparticle release[END_REF]. Interestingly, mouse B16-F10 melanoma cell lines generate microvesicles that support tumor neo-angiogenesis, invasion and metastasis [START_REF] Bland | Exosomes derived from B16F0 melanoma cells alter the transcriptome of cytotoxic T cells that impacts mitochondrial respiration[END_REF][START_REF] Peinado | Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET[END_REF]. The expression of ABCA1 appears high in melanoma cells, especially in pro-angiogenic stem cells, a specificity which could explain their sensitivity to the inhibitory effect of probenecid in neo-angiogenesis process [START_REF] Bachmeier | Overexpression of the ATP binding cassette gene ABCA1 determines resistance to Curcumin in M14 melanoma cells[END_REF][START_REF] Lin | Notch4+ cancer stem-like cells promote the metastatic and invasive ability of melanoma[END_REF]. ABCA1 could promote angiogenesis by increasing cholesterol efflux from the plasma membrane, what determines the formation of lipid rafts, positively affecting VEGFR2 dimerization and activation [START_REF] Zecchin | How Endothelial Cells Adapt Their Metabolism to Form Vessels in Tumors[END_REF]. It also facilitates the generation of HDL which carry sphingosine-1-phosphate (S1P), a bioactive lipid involved in neo-angiogenesis, including in murine melanoma (B16-F10) allograft model [START_REF] Visentin | Validation of an anti-sphingosine-1-phosphate antibody as a potential therapeutic in reducing growth, invasion, and angiogenesis in multiple tumor lineages[END_REF]). ABCA1 transporter involved in melanoma growth is also expressed by tumor infiltrating immune cells. Deletion of myeloid Abca1 limits melanoma growth, by decreasing the number of infiltrating myeloid derived suppressor cells (MDSCs), which promote tumor angiogenesis [START_REF] Sorrentino | Myeloid-derived suppressor cells contribute to A2B adenosine receptor-induced VEGF production and angiogenesis in a mouse melanoma model[END_REF][START_REF] Zamanian-Daryoush | Myeloid-specific genetic ablation of ATP-binding cassette transporter ABCA1 is protective against cancer[END_REF]). Thus, it appears that calpain exteriorization could involve the ABCA1 expression by both tumor cells and tumor infiltrating immune cells.

Our therapeutic approach to limit ABCA1 activity and, hence, melanoma neo-angiogenesis and growth has involved the use of probenecid, a uricosuric drug and an inhibitor of ABCA1 [START_REF] Ahmed | Repositioning of drugs using open-access data portal DTome: A test case with probenecid (Review)[END_REF][START_REF] Freeman | Inhibition of Pannexin 1 reduces tumorigenic properties of human melanoma[END_REF]. We have confirmed ABCA1 blunts calpain exteriorization from melanoma (Fig. 3), endothelial and myeloid cells (data not shown), while promoting its cell retention. Similar results were obtained by using glyburide, another inhibitor of ABCA1 and ABCA1 silencing by SiRNA (Fig. 3). This response is associated with a defect in capillary formation in vitro and neo-angiogenesis and melanoma growth in vivo. Interestingly, the addition of calpain to the extracellular milieu blunted totally the inhibition of angiogenesis induced by probenecid, supporting the hypothesis that probenecid anti-angiogenic properties imply calpain externalization. Nevertheless, it is possible that probenecid limits tumor angiogenesis also by additional mechanisms. For instance, this drug has been shown to block pannexin 1 (PANX1), a channel-forming glycoprotein responsible for the exteriorization of ATP [START_REF] Freeman | Inhibition of Pannexin 1 reduces tumorigenic properties of human melanoma[END_REF]). Its expression is upregulated in B16 melanoma cells as compared to normal melanocytes and its knockdown in B16 melanoma reverts tumor cells into a melanocytic-like phenotype and decreases tumor vascularization and growth [START_REF] Penuela | Loss of pannexin 1 attenuates melanoma progression by reversion to a melanocytic phenotype[END_REF][START_REF] Wu | A new perspective of mechanosensitive pannexin-1 channels in cancer metastasis: clues for the treatment of other stress-induced diseases[END_REF].

In conclusion, our present study suggests that ABCA1 pharmacological inhibition, using probenecid or any other specific inhibitory drug, is potentially a novel therapeutic approach in the prevention of melanoma growth. This protection is probably mainly explained by a slowing down of tumor neo-angiogenesis. Further studies exploring supplementary mechanisms involved in the response to ABCA1 inhibition and different tumor targets are warranted. Considering the complex and pleiotropic role of calpains in cell metabolism, synthetic calpain inhibitors may induce severe adverse events but a selective intervention on calpain externalization process could represent a reasonable therapeutic option.

Materials and methods

Mice and induction of melanoma. C57BL/6 male mice expressing the mouse calpastatin gene Cast-201 under the control of the human CRP promoter and a signal peptide (CRP/Calpast; European Mouse Mutant Archive ID EM/ 05917) and wild type C57BL/6 mice bred and housed in similar conditions (WT) were used [START_REF] Perez | Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression[END_REF]. Two-3 months WT and CRP/Calpast mice were inoculated subcutaneously under isoflurane anesthesia at the upper right part of the back with 10 6 B16-F10 cells (ATCC, USA). Tumor size was measured between day 9 and 15, 3 times a week, as described previously [START_REF] Raimbourg | The calpain/calpastatin system has opposing roles in growth and metastatic dissemination of melanoma[END_REF]. The sacrifice was performed at day 16 (tumor growth) or at day 10 (angiogenesis) under sodium pentobarbital anesthesia and tumor volume was calculated from radius of the 3 axis: Volume = 4/3x(π)x(r1)x(r2)x(r3). Similar experiments were performed in C57BL/6 mice (WT) receiving either probenecid intraperitoneally (50 mg/Kg BW at day 0, 4 and 8) or saline (controls). All animal procedures were performed in accordance with the European Union Guidelines for the Care and Use of laboratory animals and approved by the local ethical committee and by the French Ministry of Research (Authorization for the document reference 00515.01).

Cell cultures. B16-F10 cells were cultured in DMEM medium (Gibco, France), containing 10% fetal bovine serum (Biowest, France) and supplemented with Hepes 10mM (Gibco) and penicillin/streptomycin 50 U/ml (Gibco). Human umbilical vein endothelial cells (HUVECs) were cultured in Endothelial Cell Basal Medium 2 (ECBM2; PromoCell) and used for experiments between passage 2 and 10.

Calpain activity assay. Intracellular calpain activity was determined in melanoma cells and endothelial cells, as previously described [START_REF] Letavernier | Calpains contribute to vascular repair in rapidly progressive form of glomerulonephritis: potential role of their externalization[END_REF][START_REF] Raimbourg | The calpain/calpastatin system has opposing roles in growth and metastatic dissemination of melanoma[END_REF]. For measuring calpain activity in the extracellular milieu, these cells were cultured in 24-well tissue culture dishes in RPMI 1640 medium (3 × 10 6 cells in 500 μl). After the indicated culture period, cell-conditioned medium was diluted in Krebs-Ringer HEPES (KRH) solution (pH 7.4) containing 4 mM CaCl2, with or without 100 μM calpain inhibitor-1, and incubated for 10 min before the addition of 50 μM calpain substrate N-succinyl-Leu-Leu-Val-Tyr-7amino-4-methylcoumarin (AMC) (Sigma-Aldrich). After a 90-min incubation period, calpain activity was determined as the difference between fluorescence (measured at 360 nm excitation and 430 nm emission) with and without calpain inhibitor 1 [START_REF] Letavernier | Calpains contribute to vascular repair in rapidly progressive form of glomerulonephritis: potential role of their externalization[END_REF][START_REF] Perez | Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression[END_REF][START_REF] Raimbourg | The calpain/calpastatin system has opposing roles in growth and metastatic dissemination of melanoma[END_REF].

SiRNA. To modulate ABCA1 expression, B16-F10 cells were transfected with small interfering RNA targeting ABCA1murine gene, 4 siRNAs for Entrez gene 11303: SI02652692; SI02652685; SI02652678 and SI02652671 (FlexiTube siRNA) or 3'-Alexa Fluor 488 AllStars Negative control siRNA (QIAGEN). B16-F10 cells were seeded in a 24 wells plate at a density of 40 000 cells/ well in DMEM 10% FBS. Twenty-four hours after, cells were transfected with 5nM siRNA through the use of 3 μl HiPerFect transfection reagent (QIAGEN). Two days after transfection, cells were incubated with or without 100µM Probenecid for 4 h. Cells were lysed and RNA was isolated with an EZ-10 Spin Column Kit (Proteogenix, Schiltigheim, France) and reverse-transcribed with a First-Strand cDNA Synthesis Kit (Thermo Scientific,Illkirch, France). cDNA was amplified on a Light-Cycler 480 system (Roche) using SYBR Green (Roche) and specific primers for ABCA1 and Gusb as housekeeping gene.

Quantitative RT-PCR. Total cellular RNA was collected from melanoma tumors using a RNA Miniprep Super Kit (Bio Basic), checked for quality by measuring the ratio of optical densities at 260 and 280 nm, reverse transcribed into cDNA with Superscript II (Life Technologies BRL), and amplified by PCR using a LightCycler 480 (Roche Diagnostic) with SYBR Green (Fast Start DNA Master SYBR Green I; Roche Applied Science, Roche Diagnostic) and specific primers for mouse IL-2, IL-6, FOXP3, and RORγt and housekeeping genes (β-actin and Gusb).

Immunohistochemical analyzes. Melanoma tumors embedded in paraffin were cut into 3 μm sections. Endothelial cells were immunostained with rat anti-mouse panendothelial cell antigen (MECA-32; BD Biosciences) and MAX-PO (rat) Histofine (Nichirei Biosciences).

Two independent investigators (JP and ET) counted the number of capillaries in 10 fields at 200x magnification by using ImageJ grids. The mean number of capillaries/field was taken into consideration for each melanoma tumor.

Melanoma flow cytometry.

Melanoma from 7 CRP/CAST and 7 WT mice were collected at day 16 and dissociated by using a gentleMACS Dissociator (Miltenyi-Biotec). Tissue was then passed through a 30µm sieve. Cells were platted during 30 mn at +4°C with mouse Fc Block (Miltenyi-Biotec). Antibodies were incubated during one hour at +4°C before flow cytometry analysis on a MACSquant analyser (Miltenyi-Biotec). Antibodies used were: mouse anti-CD45 PERCP (130-102-469), mouse anti-F4/80 PE (130-102-422), anti-CD4 APC (17-0042-82) and anti-CD8 FITC (130-102-490) (Miltenyi-Biotec).

Cell proliferation and death. To perform BrdU incorporation assays, B16-F10 cells or endothelial cells (500×10 3 cells/well) were cultured in medium supplemented with probenecid (0-0.5 mM). BrdU was added for 30 min after 4 hours incubation (B16-F10 cells) or for 2 hours after 2 hours incubation (endothelial cells). Thereafter, cells were fixed and immunostained with the anti-BrdU antibody (Cell Proliferation ELISA, BrdU, Roche) before flow cytometry analysis.

To quantify cell death, B16-F10 cells (500×10 3 cells/well) were cultured in medium supplemented without or with probenecid (10-500 µM). After 24h, cell necrosis was quantified using the Cell Death Detection ELISA (Roche).

Endothelial cell migration. For endothelial monolayer repair assay, 7x10 4 HUVEC were cultured to confluence in Culture-Insert (Ibidi). After 24h, the Culture Insert was removed and the cell monolayer including a central cell-free gap of 0.5 mm was covered with fresh medium ±probenecid (Fischer Scientific). Gap surface area was analyzed at 9h by phase contrast microscopy.

In vitro angiogenesis assay. 6 x10 4 endothelial cells in suspension were seeded in 24-wells on 150 µL Matrigel and incubated for 20h with or without calpastatin (20 µg/mL in the presence of 2.0% FCS) or human µ-calpain (4 µg/mL in the presence of 0.5% FCS) with or without probenecid (0.5 mM). The density of formed tubes was analyzed by measuring total capillary length by hpf, as previously described [START_REF] Letavernier | Calpains contribute to vascular repair in rapidly progressive form of glomerulonephritis: potential role of their externalization[END_REF].

Aortic ring outgrowth assay. The thoracic aorta from WT mice was cut into 1mm segments that were placed on Matrigel and covered with culture medium supplemented with or without probenecid (0-0.5 mM) and/or µ-calpain (4 µg/mL every 2 days). After 7 days, 6 mean total vessel outgrowths were measured for each ring at 200x magnification, by using Analysis software.

Figure 1: Inhibition of extracellular calpain activity limits melanoma growth in vivo.

Tumor volume was significantly lower in CRP/CAST mice than in control (WT) mice 15 days after the subcutaneous injection of 1 million B16F10 cells (p=0.037, n=9 animals/group at day 0-9 and n=7 and 8 mice/group at day 15, Figure 1a). Melanoma weight was lower in CRP/CAST than in controls at day 16 (p= 0.02, n=7 and 8 mice/group, Figure 1b).

Figure 2: Extracellular calpain activity does not affect melanoma cell proliferation but promotes CD8+ immune infiltrate and tumoral neo-angiogenesis. In vitro, exposure of B16F10 melanoma cells to calpains at various concentrations or inhibition of extracellular calpain activity by calpastatin did not affect tumoral cell proliferation (n=4 experiments, p=NS, Figure 2a). In vivo, there was no impact of extracellular calpain inhibition in CRP/CAST mice on the expression of the main nuclear transcription factors and cytokines involved in antitumoral immune response (n=7 tumors analysed/group, p=NS, Figure 2b). IL-2: interleukin 2; IL-6: interleukin 6. Immune cell infiltrate was quantified by flow cytometry to assess the percentage of leucocytes (CD45+), macrophages (F4/80), and lymphocytes (CD4+ and CD8+) among tumoral cells (n=7 tumors analysed/group, p= 0.03 for CD8+ cells, Figure 2c-f). Tumoral neoangiogenesis was quantified by MECA32 immunostaining in whole melanoma of animals sacrificed at day 10, CRP/CAST mice had significantly less capillaries/field than WT mice (n=16 and 15 animals/group respectively, p=0.0067, Figure 2 g-i). In vitro, exposure of B16F10 melanoma cells to probenecid decreased extracellular calpain activity in a dose-dependent manner (p=0.016 probenecid 500µM vs control, n=5 experiments, Figure 3a). In parallel, probenecid increased intracellular calpain activity in a dose dependent manner (p=0.016 probenecid 100 and 500µM vs control, n=5 experiments, Figure 3b). Exposure of B16F10 melanoma cells to glyburide decreased extracellular calpain activity in a dose-dependent manner (p=0.03 glyburide 50 and 100µM vs control, n=4 experiments, Figure 3c). In parallel, there was a significant increase in intracellular calpain activity in cells exposed to 100 µM glyburide (p=NS, n=4 experiments, Figure 3d). Transfection of B16F10 with SiRNA targeting ABCA1 decreased significantly extracellular calpain activity (p= 0.009 SiRNA vs control, n=6 experiments, Figure 3e) and increased intracellular calpain activity (p=0.002 SiRNA vs control, n=6 experiments, Figure 3f). Exposure of B16F10 melanoma cells to probenecid at various concentrations did not affect tumoral cell proliferation (p=NS, n=5 experiments, Figure 4a). In vivo, Probenecid administration reduced significantly tumor volume in comparison to control mice 15 days after the subcutaneous injection of 1 million B16F10 cells (p=0.002, n=6 animals/group, Figure 4b). Melanoma weight was lower in mice receiving probenecid than in controls at day 16 (p= 0.008, n=6 mice/group, Figure 4c). Tumoral neoangiogenesis was quantified by MECA32 immunostaining in whole melanoma of animals sacrificed at day 10, probenecid-treated mice had significantly less capillaries/field than control mice (p=0.0079, n=5 animals/group respectively, p=0.01, Figure 4d). Ex vivo, probenecid inhibited the outgrowth of vessels from aortic ring in a dose dependent manner (p=0.003 at 100µM and p=0.001 at 500µM vs control, n=7 experiments, Figure 5a). The exposure of aortic ring to calpains (4µM every 2 days) did not increase significantly the outgrowth of vessels, but the addition of calpains to a medium containing 500µM probenecid inhibited partly the antiangiogenic effect of probenecid) (p=0.05, n=5 experiments, Figure 5bf). In vitro, probenecid inhibited the formation of capillaries by endothelial cells (HUVECs) in matrigel (p=0.0079, n= 5 experiments, Figure 5g). Extracellular calpain inhibition by calpastatin inhibited capillary formation in a similar manner, alone or in addition to probenecid (p=0.0079, n=5 experiments, Figure 5g). In a medium containing low amounts of fetal calf serum, extracellular calpain (4µg/mL) increased capillary formation (p=0.011, n=7 experiments, Figure 5h-j). Probenecid (500µM) decreased capillary formation significantly in similar conditions (p=0.0006, n=7 experiments, Figure 5h,i,k). The addition of 4 µg/mL calpain to medium containing 500 µM probenecid restored capillary formation (p=0.0041, n=7 experiments, Figure 5h,k,l). Scale bar=200 µM. In vitro, exposure of HUVECs to probenecid at various concentrations did not increase cell death (p=NS, n=4 experiments, Figure 6a). Probenecid did not decrease HUVECs proliferation (p=NS, n=5 experiments, Figure 6b). In vivo, intact fibronectin was increased in melanoma in the presence of probenecid, suggesting that probenecid actually limited fibronectin cleavage due to calpain activity (p=0.0079, n=6 experiments, Figure 6c, Figure 6d: representative western-blot). In vitro, probenecid inhibited the migration of endothelial cells in a dose-dependent manner (p=0.05 at 100µM and p=0.037 at 500µM vs control, n= 5 experiments, Figure 6e). Extracellular calpain (4 µg/mL) increased endothelial cell migration (p=0.026, n=6 experiments, Figure 6f-h). The addition of 4 µg/mL calpain to a medium containing 500 µM probenecid restored endothelial cell migration properties (p=0.007, n=6 experiments, Figure 6f,i,j).

Figure 3 :

 3 Figure 3: Probenecid, glyburide and ABCA1 SiRNA inhibit extracellular calpain activity.In vitro, exposure of B16F10 melanoma cells to probenecid decreased extracellular calpain activity in a dose-dependent manner (p=0.016 probenecid 500µM vs control, n=5 experiments, Figure3a). In parallel, probenecid increased intracellular calpain activity in a dose dependent manner (p=0.016 probenecid 100 and 500µM vs control, n=5 experiments, Figure3b). Exposure of B16F10 melanoma cells to glyburide decreased extracellular calpain activity in a dose-dependent manner (p=0.03 glyburide 50 and 100µM vs control, n=4 experiments, Figure3c). In parallel, there was a significant increase in intracellular calpain activity in cells exposed to 100 µM glyburide (p=NS, n=4 experiments, Figure3d). Transfection of B16F10 with SiRNA targeting ABCA1 decreased significantly extracellular calpain activity (p= 0.009 SiRNA vs control, n=6 experiments, Figure3e) and increased intracellular calpain activity (p=0.002 SiRNA vs control, n=6 experiments, Figure3f).

Figure 4 :

 4 Figure 4: Probenecid limits tumor growth and tumoral neoangiogenesis in vivo.Exposure of B16F10 melanoma cells to probenecid at various concentrations did not affect tumoral cell proliferation (p=NS, n=5 experiments, Figure4a). In vivo, Probenecid administration reduced significantly tumor volume in comparison to control mice 15 days after the subcutaneous injection of 1 million B16F10 cells (p=0.002, n=6 animals/group, Figure4b). Melanoma weight was lower in mice receiving probenecid than in controls at day 16 (p= 0.008, n=6 mice/group, Figure4c). Tumoral neoangiogenesis was quantified by MECA32 immunostaining in whole melanoma of animals sacrificed at day 10, probenecid-treated mice had significantly less capillaries/field than control mice (p=0.0079, n=5 animals/group respectively, p=0.01, Figure4d).

Figure 5 :

 5 Figure 5: Probenecid inhibits ex-vivo and in vitro angiogenesis by limiting extracellular calpain activity.Ex vivo, probenecid inhibited the outgrowth of vessels from aortic ring in a dose dependent manner (p=0.003 at 100µM and p=0.001 at 500µM vs control, n=7 experiments, Figure5a). The exposure of aortic ring to calpains (4µM every 2 days) did not increase significantly the

Figure 6 :

 6 Figure6: Probenecid does not affect tumoral cell proliferation or death but limits fibronectin cleavage by extracellular calpains and inhibits endothelial cell migration. In vitro, exposure of HUVECs to probenecid at various concentrations did not increase cell death (p=NS, n=4 experiments, Figure6a). Probenecid did not decrease HUVECs proliferation (p=NS, n=5 experiments, Figure6b). In vivo, intact fibronectin was increased in melanoma in the presence of probenecid, suggesting that probenecid actually limited fibronectin cleavage due to calpain activity (p=0.0079, n=6 experiments, Figure6c, Figure6d: representative western-blot). In vitro, probenecid inhibited the migration of endothelial cells in a dose-dependent manner (p=0.05 at 100µM and p=0.037 at 500µM vs control, n= 5 experiments, Figure6e). Extracellular calpain (4 µg/mL) increased endothelial cell migration (p=0.026, n=6 experiments, Figure6f-h). The addition of 4 µg/mL calpain to a medium containing 500 µM probenecid restored endothelial cell migration properties (p=0.007, n=6 experiments, Figure6f,i,j).

Western blots. Total melanoma tumors were isolated from mice at day 10 and cell lysates were extracted using a RIPA buffer. Equivalent amounts of proteins (5 µg/lane) were separated on an SDS-PAGE gel and transferred to nitrocellulose membrane incubated with primary anti-fibronectin antibody (Santa Cruz 9098). Intact human fibronectin appeared with an apparent molecular weight >250 kD. Intact fibronectin expression was indexed to GAPDH expression (optical density, Image J).