Bérengère Piron

Salomé Paillas

Boudousq Vincent

André Pèlegrin

Caroline Bascoul- Mollevi

Nicolas Chouin

Isabelle Navarro-Teulon

Jean-Pierre Pouget
email: jean-pierre.pouget@inserm.fr

DNA damage-centered signaling pathways are effectively activated during low dose-rate Auger radioimmunotherapy

Keywords: Radioimmunotherapy, Auger electrons, signaling pathways, DNA damage, 125

Introduction: Low dose-rate radioimmunotherapy (RIT) using 125 I-labelled monoclonal antibodies ( 125 I-mAbs) is associated with unexpected high cytotoxicity per Gy. Methods: We investigated whether this hypersensitivity was due to lack of detection of DNA damage by the targeted cells. DNA damage were measured with the alkaline comet assay, gamma-H2AX foci and the micronucleus test in p53 -/-and p53 +/+ HCT116 cells exposed to increasing activities of internalizing anti-HER1 125 I-mAbs or non-internalizing anti-CEA 125 I-mAbs. The expression of proteins involved in radiation response and progression of cells through the cycle were determined. Results: Cell hypersensitivity to low absorbed doses of anti-CEA 125 I-mAbs was not due to defect in DNA damage detection, since ATM (ataxia telangiectasia mutated gene), gamma-H2AX, p53 and p21 were activated in RIT-treated HCT116 cells and G2/M cell cycle arrest was observed. Moreover, the alkaline comet assay showed that DNA breaks accumulated when cells were placed at 4°C during exposure but were repaired under standard RIT conditions (37°C), suggesting that lesions detected under alkaline conditions (mostly DNA single strand breaks and alkali-labile sites) are efficiently repaired in treated cells. The level of gamma-H2AX protein corroborated by the level of foci measured in nuclei of treated cells was shown to accumulate with time thereby suggesting the continuous presence of DNA double strand breaks. This was accompanied by the formation of micronuclei. Conclusion: Hypersensitivity to non-internalizing 125 I-mAbs is not due to lack of detection of DNA damage after low absorbed dose-rates. However, DNA double strand breaks accumulate in cells exposed both to internalizing and non-internalizing 125 I-mAbs and lead to micronuclei formation. These results suggest impairment in DNA double strand breaks repair after low absorbed doses of 125 I-mAbs.

INTRODUCTION

Auger electrons are produced in cascades during electronic shell rearrangement consecutive to electronic capture and/or conversion processes in unstable atoms [START_REF] Pomplun | A Monte Carlo simulation of Auger cascades[END_REF]. They have a very low energy (from few eV to few keV) and are considered as high LET particles (from 4 to 26 keV/µm) when their energy is less than 1 keV. Consequently, their path length in biological matter is very short, between about 2 nm and 500 nm for most of them (for reviews see [START_REF] Buchegger | Auger radiation targeted into DNA: a therapy perspective[END_REF][START_REF] Howell | Auger processes in the 21st century[END_REF][START_REF] Kassis | Radiotargeting agents for cancer therapy[END_REF][START_REF] Cornelissen | Targeting the nucleus: an overview of Auger-electron radionuclide therapy[END_REF][START_REF] Pomplun | Auger electron spectra--the basic data for understanding the Auger effect[END_REF][START_REF] Yasui | Molecular and Cellular Effects of Auger Emitters: 2008-2011[END_REF][START_REF] Feinendegen | The Auger Effect in biology and medicine. Looking back[END_REF]).

Due to their physical characteristics, efforts have been done to bring Auger electrons in the cell nucleus to obtain the highest cytotoxicity (for review [START_REF] Cornelissen | Targeting the nucleus: an overview of Auger-electron radionuclide therapy[END_REF]).

However, although clinical radioimmunotherapy (RIT) studies using Auger electrons labeled to monoclonal antibodies (mAbs) have not given clear-cut results [START_REF] Li | A Phase II study of antiepidermal growth factor receptor radioimmunotherapy in the treatment of glioblastoma multiforme[END_REF][START_REF] Quang | Radioimmunotherapy as a novel treatment regimen: 125I-labeled monoclonal antibody 425 in the treatment of high-grade brain gliomas[END_REF][START_REF] Welt | Quantitative analysis of antibody localization in human metastatic colon cancer: a phase I study of monoclonal antibody A33[END_REF][START_REF] Welt | Phase I/II study of iodine 125-labeled monoclonal antibody A33 in patients with advanced colon cancer[END_REF], we and others have shown that, in mice, Auger electrons could efficiently delay growth of small solid tumors [START_REF] Santoro | Noninternalizing monoclonal antibodies are suitable candidates for 125I radioimmunotherapy of smallvolume peritoneal carcinomatosis[END_REF][START_REF] Boudousq | Brief intraperitoneal radioimmunotherapy of small peritoneal carcinomatosis using high activities of noninternalizing 125I-labeled monoclonal antibodies[END_REF][START_REF] Behr | Therapeutic advantages of Auger electron-over beta-emitting radiometals or radioiodine when conjugated to internalizing antibodies[END_REF][START_REF] Behr | Therapeutic efficacy and dose-limiting toxicity of Auger-electron vs. beta emitters in radioimmunotherapy with internalizing antibodies: evaluation of 125I-vs. 131I-labeled CO17-1A in a human colorectal cancer model[END_REF][START_REF] Michel | A comparison of 4 radionuclides conjugated to antibodies for single-cell kill[END_REF][START_REF] Govindan | Radionuclides linked to a CD74 antibody as therapeutic agents for B-cell lymphoma: comparison of Auger electron emitters with beta-particle emitters[END_REF]. This efficacy remains to be elucidated because the final localization of 125 I-mAbs (and the subsequent energy deposit [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane[END_REF]) upon binding to their receptors is the cell membrane (noninternalizing mAbs) or the cytoplasm (internalizing mAbs) after a receptor-mediated internalizing process. Therefore, for both types of mAbs, most of the energy is released in extra-nuclear compartments. Moreover, we previously reported in several cell lines that noninternalizing 125 I-mAbs are more toxic than internalizing 125 I-mAbs [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane[END_REF][START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF]. This higher cytotoxicity is p53-independent (differently from internalizing 125 I-mAbs) and does not involve cell membrane-mediated apoptotic mechanisms [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane[END_REF].

In conventional external beam radiation therapy (CEBRT), the nucleus is the key target of ionizing irradiation. However, the comparison between CEBRT and RIT is not straightforward. Indeed, CEBRT delivers high radiation doses (between 40 and 80 Gy) at high dose-rate (1-2 Gy.min -1 ) that are fractionated in daily 2 Gy doses and the biological response to CEBRT is correlated with both dose and dose-rate [START_REF] Goodhead | Initial events in the cellular effects of ionizing radiations: clustered damage in DNA[END_REF][START_REF] Goodhead | Track structure analysis of ultrasoft X-rays compared to high-and low-LET radiations[END_REF]. The cell response to radiation is mostly triggered by DNA double strand breaks (DSBs) the occurrence of which is strictly Submission in the frame of WIPR 2013 -4 proportional to the mean nucleus absorbed dose [START_REF] Goodhead | Initial events in the cellular effects of ionizing radiations: clustered damage in DNA[END_REF]. DSBs initiate different signaling pathways that involve ATM, a DNA damage sensor molecule. In turn, ATM activates proteins (particularly, H2AX and p53) that participate in cell cycle arrest, apoptosis and DNA repair (for reviews see [START_REF] Levine | The first 30 years of p53: growing ever more complex[END_REF][START_REF] Prise | New insights on cell death from radiation exposure[END_REF][START_REF] O'driscoll | The role of double-strand break repair -insights from human genetics[END_REF][START_REF] Meek | Tumour suppression by p53: a role for the DNA damage response?[END_REF]). Conversely, RIT is characterized by protracted exposure (hours to days), resulting in low total doses (10-30 Gy compared to 40 -80 Gy in EBRT) delivered at low dose-rates (<1Gy.h -1 ), and is accompanied by a strong heterogeneity in subcellular energy deposits. These physical features provide cancer cells with an opportunity for DNA repair during RIT. However, several studies in mice have shown that RIT cytotoxicity per unit of absorbed dose is higher than that of a single fraction of radiation delivered at higher dose-rate in CEBRT [START_REF] Knox | Overview of animal studies comparing radioimmunotherapy with dose equivalent external beam irradiation[END_REF][START_REF] Williams | Quantitative comparison of radiolabeled antibody therapy and external beam radiotherapy in the treatment of human glioma xenografts[END_REF]. Hypotheses have been proposed for explaining such increased efficacy per Gy (for review [START_REF] Sgouros | MIRD continuing education: Bystander and low dose-rate effects: are these relevant to radionuclide therapy?[END_REF]).

Here, we investigated whether the higher RIT cytotoxicity per unit of absorbed dose was due to lack of detection of DNA damage induced by low dose-rate RIT in p53 -/-and p53 +/+ HCT116 cells targeted with 125 I-mAbs.

MATERIALS AND METHODS

Cell lines

HCT116 (p53 +/+ ) human colorectal cancer cells were from ATCC. The p53 -/-HCT116 cell line was a gift from Professor Bert Vogelstein (Johns Hopkins University). HCT116 cells express HER1 and basal level of CEA receptors. They were grown in RPMI supplemented with 10% heat-inactivated FBS, 100 µg/mL L-glutamine and antibiotics (0.1 unit/mL penicillin and 100 µg/mL streptomycin). A-431 and SK-OV-3 cells were previously described in [START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF].

Antibodies and radiolabelling

The internalizing m225 mAb [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane[END_REF][START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF] was used for targeting the Epidermal Growth Factor Receptor (EGFR/HER1) expressed in HCT116 and A-431 cells. The internalizing Trastuzumab (Herceptin ® , Genentech Incorporated, San Francisco CA, USA) binds to Epidermal Receptor type 2 (HER2) expressed in SK-OV-3 cells [START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF] and the non-internalizing murine IgG1k mAb 35A7 targets CEA in all the cell lines [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane[END_REF][START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF]. The non-targeting IgG1 mAb PX was used as control [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane[END_REF][START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF]. MAbs were radiolabelled with 125 I using the conventional IODO-GEN method (1,3,4,6-tetrachloro-3α, 6α-diphenylglycoluryl) described in [START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF].

Cellular dosimetry

The mean nucleus absorbed dose was previously calculated for HCT116 cells exposed to increasing activities (0-4MBq/ml) of 125 I-mAbs [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane[END_REF]. Briefly, the MIRD cellular approach [START_REF] Goddu | MIRD Cellular S Values: Selfabsorbed Dose per Unit Cumulated Activity for Selected Radionuclides and Monoenergetic Electron and Alpha Particle Emitters Incorporated into Different Cell Compartments[END_REF] that requires the determination of the total cumulative number of decays (  ˜ A rs ) occurring in cells and the S-values, was used. Then, for each test activity, radioactivity uptake per cell (Bq/cell) was determined as described in [START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF], and used to estimate  ˜ A rs . For all targeting models,  ˜ A rs was then multiplied by the corresponding S-value to obtain the mean nucleus absorbed dose. For S-value calculations, three sources of irradiation were considered: selfirradiation from radiolabelled vectors that were internalized in the cytoplasm (anti-HER1 125 I-mAbs) or bound to the cell membrane (anti-CEA 125 I-mAbs); culture medium irradiation from unbound radiolabelled vectors; and cross-fire irradiation. Cell size was measured by fluorescence microscopy after propidium iodide staining and the cell radius (distributed normally within the HCT116 cell population) was 5.6±1.0 µm when the nucleo-cytoplasmic ratio was about 0.65. The cellular radioactivity was assumed to be uniformly distributed within cytoplasm and at cell surface for internalizing and non-internalizing 125 I-mAbs, respectively.

Alkaline single-cell gel electrophoresis

The alkaline single-cell gel electrophoresis assay (comet assay) was used to measure DNA damage in 3×10 5 HCT116 cells grown in 6-well plates and exposed to 0 and 4 MBq/mL of 125 I-mAbs. The comet assay was carried out either i) under standard RIT conditions (i.e., cells at 37°C for the entire RIT duration) at different time points (15 min, 1h, 2h, 3h, 6h, 12h, 24h and 48h), or ii) following pre-incubation of cells in cold medium at 4°C for 1h and RIT at 4°C for 4h. Samples were collected at 15 min, 30 min, 1h, 2h, 3h and 4h after RIT onset. For both conditions (37°C and 4°C), the methodology described in [START_REF] Laurent | Modulation of DNA damage by pentoxifylline and alphatocopherol in skin fibroblasts exposed to Gamma rays[END_REF] was used. The tail moment (TM) was the parameter retained for analysis. The mean TM, expressed in arbitrary units (a.u.), was measured in 100 randomly chosen cells per slide for each time point and was calculated with the Comet Imager 2.0 software (Metasystems, Hamburg, Germany). Three slides for each condition and each time point were analyzed per experiment and experiments were repeated three times.

Micronucleus assay

3×10 4 HCT116 cells were seeded in 24-well plates. HCT116 cells exposed to activities between 0 and 4MBq/mL of 125 I-mAbs for 2 days. Twenty four hours before selected time points (day 1, day 2 and 3 post onset of RIT), cytochalasin B was added to the culture medium at a final concentration of 5 µg/mL in order to block cytokinesis and was maintained for 24 hours. Cells were then harvested and centrifuged. Supernatant was carefully discarded and the pellet was treated with KCl 125 mM under constant shaking for hypotonic chock. Cells were then fixed three times in acetic acid:ethanol (1:6), dropped onto slides under humidified atmosphere and air dried. Before analysis, slides were stained with propidium iodide (500 µg/mL). Experiments were repeated three times in triplicate.

Protein extraction and western blotting

5×10 5 HCT116 and 4×10 5 A-431 and SK-OV-3 cells were grown in 6-well plates and exposed to 0 and 4MBq/mL of 125 I-mAbs for 2 days. Proteins were extracted as described in [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane[END_REF].

Membranes were pre-incubated with 5% milk in PBS/0.1% Tween 20 and then with anti-p53

(1:10,000), -p21 (1:500) (Santa-Cruz Biotechnology, CA, USA), -gamma-H2AX (1:1000) (Merck Millipore, Guyancourt, France) or p-ATM (1:1000) (Cell Signaling Technology, Danvers, MA, USA) primary antibodies, followed by horseradish peroxidase-conjugated antirabbit secondary antibodies (1:10,000; Sigma-Aldrich, Saint-Louis, MO, USA). GAPDH level was used to evaluate protein loading. Levels of protein expression were quantified using the G-Box system (Syngene; Cambridge UK).

Gamma-H2AX immunofluorescent measurement

For double strand breaks formation, 2× 10 4 cells were grown on coverslip and were exposed for 2 days to 0 and 4 MBq/mL of 125 I-mAbs. Cells were washed twice with PBS and fixed in 3.7% formaldehyde in PBS for 30 min. They were then permeabilized at room temperature for 30 min using PBS/Triton (0.5%). Cells were next washed twice with PBS, saturated with PBS/BSA (1mg/ml) for 1hour before incubation overnight with anti-gamma-H2AX (1:200 PBS/BSA; Merck Millipore). Next, coverslips were incubated for 1h in the dark with FITClabeled goat anti-mouse Ig (Sigma) in PBS-BSA and next washed three times with PBS-BSA and once with PBS before analysis.

Cell cycle

20×10 4 HCT116 cells were grown in 6-well plates one day before RIT. Cells were harvested at day 0, 1, 2, 3, 4 and 7 of RIT by trypsinization and washed twice with PBS. They were then fixed in 70% ethanol at -20°C for 3 hours and stained with cell cycle kit reagent from Merck Millipore (Merck Millipore, Guyancourt, France) in the dark for 30 min at room temperature before analysis using an Muse ® flow cytometer (Merck Millipore, Guyancourt ,France). The percentage of cells in G0/G1, S and G2/M phases was then calculated (mean of three experiments in triplicate).

Statistical analysis

The linear mixed regression model (LMRM) [START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF] was used for statistical analysis of the data.

RESULTS

DNA damage formation and repair occur simultaneously during RIT

We investigated the occurrence of DNA single (SSBs) and double (DSBs) strand breaks and alkali-labile sites (ALSs) in p53 +/+ and p53 -/-HCT116 cells upon exposure to 4 MBq/mL of anti-CEA (35A7), anti-HER1 (m225) or non-targeting (PX) 125 I-mAbs for 2 days. The mean nucleus doses were calculated [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane[END_REF] using the MIRD cellular formalism. Under standard RIT conditions (at 37°C), occurrence of DNA damage (as indicated by the TM parameter) was not significantly different in HCT116 cells treated with anti-HER1 or anti-CEA 125 I-mAbs in comparison to untreated cells (p=0.85 and p=0.72, respectively) (Fig. 1). This finding suggests that radiation-induced DNA damage formation was compensated by DNA repair during RIT. In order to confirm this hypothesis, we investigated the level of DNA breaks in cells incubated with 125 I-mAbs at 4°C for 4h (instead of 48h at 37°C as used under standard RIT). Temperature of 4°C is known to block enzymatic systems involved in DNA breaks repair. The 4h exposure time was determined as the longest time of exposure at 4°C producing no cytotoxicity. We observed under these conditions a progressive increase in DNA damage in p53 +/+ and p53 -/-HCT116 cells exposed to anti-CEA and anti-HER1 125 I-mAbs in comparison to those treated with non-targeting 125 I-mAbs (Fig. 2A andB). These data confirmed that DNA repair occurred during standard RIT while DNA breaks accumulated at 4°C. We next calculated, the corresponding absorbed doses delivered to the nucleus by 4 MBq/mL 125 I-mAbs. We thus considered the cumulative number of decays (  ˜ A rs ) calculated over 4h (and not 48h like under standard RIT). We also considered that Submission in the frame of WIPR 2013 -9 internalization of mAbs was blocked at 4°C such that S-values for cell surface localization were used for internalizing mAbs. Absorbed doses were then shown to be lower than under standard RIT (anti-HER1 125 I-mAbs: from 43 Gy at 37°C to 1.92 Gy at 4°C; anti-CEA 125 I-mAbs: from 1.20 Gy to 0.1 Gy; non-targeting 125 I-mAbs from 0.27 Gy to 0.015).

Corresponding highest TM values were 12.8 (anti-HER1 125 I-mAbs), 6.0 (anti-CEA 125 I-mAbs) and 2.8 (non-targeting 125 I-mAbs) a.u. in p53 +/+ HCT116 cells and 10.4 (anti-HER1 125 I-mAbs), 5.9 (anti-CEA 125 I-mAbs) and 4.5 (non-targeting 125 I-mAbs) a.u. in p53 -/-cells. In cells exposed to anti-HER1 125 I-mAbs, TM increased linearly as a function of the absorbed dose in both p53 +/+ and p53 -/-HCT116 cells (p<0.001). Following exposure to anti-CEA 125 I-mAbs, TM increase was linear in p53 +/+ HCT116 cells (p<0.007), but not in p53 -/-cells (p=0.103), where it exhibited a larger standard error (Fig. 2A andB). However, comparison of the efficacy of anti-CEA and anti-HER1 125 I-mAbs showed that, overall, they produced quantitatively similar effects in both p53 -/-(p=0.251) and p53 +/+ (p=0.172) HCT116 cells, because anti-CEA 125 I-mAbs (with lower mean nucleus absorbed doses) were more efficient per Gy than anti-HER1 125 I-mAbs (with higher mean absorbed doses) in producing DNA damage. Moreover, the effect of each antibody was similar in the two cell lines (p=0.681 for anti-CEA 125 I-mAbs and p=0.372 for anti-HER1 125 I-mAbs).

DNA damage-mediated signaling pathways are efficiently activated in HCT116 cells

Under standard RIT conditions, the comet assay results suggested activation of DNA repair mechanisms. We thus assessed whether such low levels of RIT-induced DNA damage could activate ATM, H2AX and p53 (one of the key proteins of the radiation response) (Fig. 3).

Western blot analysis revealed that phosphorylated ATM (p-ATM) and p53 level increased in p53 +/+ HCT116 cells exposed to RIT at 37°C and were associated with p21 up-regulation (Fig. 3A). The extent of their induction was not correlated with the nucleus absorbed dose because it was only slightly higher upon exposure to anti-HER1 125 I-mAbs than to anti-CEA Submission in the frame of WIPR 2013 -10 125 I-mAbs, although much different doses were delivered to the nucleus (43 Gy versus 1.2 Gy). Detection of gamma-H2AX indicated that DNA DSBs were continuously produced under standard RIT conditions (Fig. 3A), while no increase could be observed using the comet assay which detects all kind of breaks (DSBs + SSBs + ALSs). Similar results were obtained in p53 -/-HCT116 cells for p-ATM, gamma-H2AX, and p21 (Fig. 3B).

Activation of p53 was also observed in A-431 (p53 +/+ ), but not in SK-OV-3 (p53 -/-) cells exposed to 125 I-mAbs (Supplementary Fig. 1). These two cell lines are more sensitive to noninternalizing anti-CEA than to internalizing anti-HER1/HER2 125 I-mAbs [START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF]. In HCT116 and A-431 cells, p53 up-regulation is followed by induction of apoptosis at day 2 post-RIT [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane[END_REF].

These results suggest that the DNA damage induced by mean nucleus absorbed doses as low as 1.2 Gy can be efficiently detected in cells exposed to RIT.

The p21 protein up-regulation measured in HCT 116 cells was accompanied by cell cycle arrest in G2/M phase in both p53 +/+ and p53 -/-cells upon exposure to anti-CEA and anti-HER1 125 I-mAbs (Fig. 6). In both cell lines, the % of cells in G2/M was shown to increase significantly at day 1 post onset of RIT up to day 7.

Double strand breaks detection

Double strand breaks (DSBs) as revealed by gamma-H2AX foci detection were produced in p53 -/-and p53 +/+ HCT 116 cells exposed for 2 days to 4MBq/mL of either internalizing anti-HER1 or non-internalizing anti-CEA 125 I-mAbs (Fig. 4). DSBs were detected as early as 6h post incubation with radiolabeled mAbs and their yield was continuously increasing in a similar way for both types of mAbs, independently of the mean nucleus absorbed dose. Moreover, they were still detected 24h after the radioactivity was removed, namely 72 h post onset of RIT. These data suggest that DSBs are continuously formed under RIT using 125 I-mAbs and that they are not accurately repaired and accumulate with time.

Submission in the frame of WIPR 2013 -11

Yield of micronuclei as a function of the mean nucleus absorbed dose

Micronuclei per binucleated cells (MN/BN) were produced in both p53 +/+ and p53 -/-HCT116 cells during RIT using 125 I-mAbs, suggesting error-prone DNA repair. In p53 +/+ HCT116 cells, their formation at 24h post onset of RIT using either internalizing anti-HER1 or noninternalizing anti-CEA 125 I-mAbs was increasing with the test activity. For non-internalizing 125 I-mAbs, yield of MN at 4MBq/mL was higher at 24h (0.34±0.06 MN/BN versus 0.14±0.06 MN/BN at 0MBq/mL) than at 48h (0.20±0.11 MN/BN) and it decreased to background level at 72h (0.12±0.08). Similar observation could be done with internalizing 125 I-mAbs (0.53±0.02 MN/BN at 24h, 0.22±0.04 MN/BN at 48h, 0.14±0.12 MN/BN at 72h). It must be noted that yield of MN/BN in cells treated with non-specific 125 I-PX mAbs remained at the background level (between 0.08±0.03 and 0.14±0.06MN/BN).

In p53 -/-HCT116 cells, relationship between MN yield and test activity was less pronounced (Fig. 5). This can be partly due to a higher background level in non-treated cells (around between 0.15 and 0.27MN/BN) and to large uncertainties associated to MN measurement.

Similarly, no clear trend of a decrease between 24h and 48h in MN yield was observed.

DISCUSSION

We showed previously in HCT116 cells targeted by 4MBq/mL of 125 I-mAbs (Fig. 7 and [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane[END_REF]) that a mean nucleus absorbed dose of 1.2 Gy of non-internalizing anti-CEA 125 I-mAbs is as effective as 43 Gy of internalizing anti-HER1 125 I-mAbs. As the cell response to ionizing radiation is generally, in CEBRT, triggered by nuclear DNA damage, we thus investigated the relationship between the energy deposited in the nucleus (i.e., the mean nucleus absorbed dose) and several biological endpoints during RIT with 125 I-mAbs. Non-internalizing anti-CEA 125 I-mAbs and, to a lower extent, internalizing anti-HER1 125 I-mAbs, were very efficient in killing cells [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane[END_REF] and also in producing DNA damage despite lower dose-rates (about 0.025 Submission in the frame of WIPR 2013 -12 Gy.h -1 for anti-CEA 125 I-mAbs and 0.9 Gy.h -1 for anti-HER1 125 I-mAbs) than in CEBRT (about 2 Gy.min -1 ). The high efficiency of 125 I-mAbs, particularly of anti-CEA 125 I-mAbs per unit-dose, is reminiscent of low-dose hyper-radiosensitivity and of the inverse dose-rate effect. These two phenomena, which were reported in CEBRT (for review [START_REF] Marples | Low-dose hyper-radiosensitivity: past, present, and future[END_REF]) after low dose (below 0.5 Gy) or low dose-rate (0.02-1.00 Gy.h -1 ) exposure, lead to increased effectiveness of radiation per unit-dose beyond what could be predicted by the conventional linear and linear quadratic radio-biologic models ( [START_REF] Joiner | Renal damage in the mouse: the response to very small doses per fraction[END_REF] and for review [START_REF] Marples | Low-dose hyper-radiosensitivity: past, present, and future[END_REF]). It has been proposed that they are the result of decreased sensing of DNA damage by signaling proteins and of the consequent reduced activation of the early DNA damage response through ATM and p53 or the G2 checkpoint ( [START_REF] Xu | Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation[END_REF] and for review [START_REF] Sgouros | MIRD continuing education: Bystander and low dose-rate effects: are these relevant to radionuclide therapy?[END_REF]). Our findings do not support this hypothesis. Indeed, ATM, H2AX and p21 were efficiently activated in p53 -/-and p53 +/+ HCT116 cells and p53 expression was also increased (in p53 +/+ HCT116 cells and A-431 cells) during RIT with 125 I-mAbs. Moreover, most of the DNA breaks were immediately repaired under standard RIT conditions (comet assay data, Fig. 1 and2). However, the significant increase in expression and accumulation of gamma-H2AX, due to phosphorylation of H2AX at DSB sites [START_REF] Lobrich | gammaH2AX foci analysis for monitoring DNA double-strand break repair: strengths, limitations and optimization[END_REF] in cells treated with anti-CEA or anti-HER1 125 I-mAbs indicates that DSBs were continuously formed and not repaired. Consequently, mis-or un-repaired DSBs might have led to micronuclei formation (Fig. 4) and to mitotic death of damaged cells.

These data indicate that acute cell death associated with low doses and low dose-rate of 125 I-mAbs (particularly of non-internalizing anti-CEA 125 I-mAbs) was not due to defective detection of DNA damage by the cells. Impaired repair of double strand breaks would be involved in low dose rate efficacy of RIT using 125 I-mAbs in a non-dependent dose-effect relationship.

Another intriguing observation is the lack of relationship between the mean nucleus absorbed doses (in Gy) and these biological endpoints when comparing the efficacy/Gy of anti-CEA Submission in the frame of WIPR 2013 -13 and anti-HER1 125 I-mAbs. In CEBRT, the biological effects and, consequently, the survival of irradiated cells are correlated with the mean nucleus absorbed dose [START_REF] Puck | Action of x-rays on mammalian cells. II. Survival curves of cells from normal human tissues[END_REF], according to a linear or linear quadratic relationship depending on whether low-LET or high-LET radiations are used. Here, no linear dose-effect relationship was observed when the effects per Gy of absorbed dose of anti-CEA and anti-HER1 125 I-mAbs are considered. Indeed, the biological response per Gy of non-internalizing anti-CEA 125 I-mAbs was much higher than that of internalizing anti-HER1 125 I-mAbs. Hence, quantitatively similar effects were produced by 125 I-mAbs with much different nucleus absorbed doses. This is true for clonogenic survival [START_REF] Knox | Overview of animal studies comparing radioimmunotherapy with dose equivalent external beam irradiation[END_REF], frequency of DNA breaks and micronuclei formation, induction of DNA damage response and effects on cell cycle progression. Based on the localization of the energy deposits due to decay of anti-CEA 125 I-mAbs at the cell membrane [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane[END_REF] and the lack of correlation between absorbed dose and biological effects we hypothesize that cell membranemediated 125 I-induced bystander effect, as described in CEBRT ( [START_REF] Azzam | Oxidative metabolism, gap junctions and the ionizing radiation-induced bystander effect[END_REF][START_REF] Sowa Resat | Radiation-induced genomic instability: a role for secreted soluble factors in communicating the radiation response to non-irradiated cells[END_REF][START_REF] Mamlouk | Bystander Effect in Tumor Cells Produced By Iodine-125 Labeled Human Lymphocytes[END_REF] and for reviews [START_REF] Sgouros | MIRD continuing education: Bystander and low dose-rate effects: are these relevant to radionuclide therapy?[END_REF][START_REF] Prise | Radiation-induced bystander signalling in cancer therapy[END_REF]), could play a significant role in the higher efficacy of non-internalizing 125 I-mAbs [START_REF] Boyd | Radiation-induced biologic bystander effect elicited in vitro by targeted radiopharmaceuticals labeled with alpha-, beta-, and auger electron-emitting radionuclides[END_REF][START_REF] Chouin | Evidence of extranuclear cell sensitivity to alpha-particle radiation using a microdosimetric model. II. Application of the microdosimetric model to experimental results[END_REF][START_REF] Nagasawa | Involvement of membrane signaling in the bystander effect in irradiated cells[END_REF] and should be further investigated.

We would like to highlight that the hypersensitivity of HCT116 cells to non-internalizing 125 I-mAbs is not dependent on our dosimetric approach based on MIRD formalism since we showed that it was still observed when survival was expressed as a function of cumulated uptake of radioactivity (Figure 7A). Moreover, cytoplasmic and cell surface localizations of m225 and 35A7, respectively, were confirmed in several cell lines using cell fractionation assays and immunofluorescence approaches (unpublished data). However, it must be kept in mind that Bousis et al. showed that for the case where the radiopharmaceutical are either internalized into the cytoplasm or remained bound onto the cell surface (non-internalized), the dose to the cell nucleus determined using Monte Carlo code was found to differ significantly Submission in the frame of WIPR 2013 -14 from the MIRD values. Then, use of MIRD formalism with short range Auger electrons emitters must be done carefully [START_REF] Bousis | Monte Carlo single-cell dosimetry of Auger-electron emitting radionuclides[END_REF].

In conclusion, this study shows that DNA damage produced by low dose of 125 I-mAbs is efficiently detected by targeted cells even after very low absorbed dose, but is associated to accumulation of DNA double strand breaks. G2/M cell cycle arrest does not prevent damaged cells still proceed through cell cycle and to undergo mitotic death. The lack of dose-effect relationship between mean nucleus absorbed dose and several biological endpoints and the property of non-internalizing 125 I-mAbs to deliver localized doses at the cell membrane suggest that cell membrane-mediated bystander effects might be involved in 125 I-mAbs cytotoxicity.

ACKNOWLEDGEMENT

  This work was supported by the Institut National du Cancer, grant R09080FF / RPT09005FFA and by Action Nu1.1 of Plan Cancer 2009-2013 (ASC 13038FSA).

FIGURE LEGENDS FIG. 1 .FIG. 3 .FIG. 4 .FIG. 5 .FIG. 6 .FIG. 7 .Supplementary FIG. 1 .

 LEGENDS1345671 FIGURE LEGENDSFIG. 1. DNA damage in p53 +/+ (panel A) and p53 -/-(panel B) HCT116 cells after RIT at


Yoann Bruneau

Guyve Khalili

Pierre Pillet

Daniel Comparat

Guided and focused slow atomic beam from a 2 dimensional magneto optical trap

Keywords: 67, 85, -d ultracold gases -07, 77, Gx source of atomic beams

We report experimental results which demonstrate how a single laser can be used to both accelerate and guide caesium atoms initially trapped in a two dimensional magneto-optical trap (2D-MOT). The atomic beam size and velocity can be modified using both the laser power and detuning as parameters. In particular, using a 750mW laser detuned 10GHz from resonance, we demonstrate that it is possible to focus an atomic beam to a size of 0.4 mm as far as 60 cm from the output of the 2D-MOT with a flux on the order of 3 × 10 9 atoms/s.

Introduction

Atomic beams have emerged as the preferred method of transport thanks to their high flux and stability. The main difficulty faced with these sources is the control over beam parameters, such as the divergence, longitudinal velocity and flux, which often having no or only minimal tuning.

The manipulation of atomic beams started with the pioneering studies made by A. Ashkin et al. [1], who manipulated the divergence of a sodium atomic beam produced from an oven using dipole trapping. The dipole force can alter the beam divergence and ultimately can be used to focus the atomic beam. However the longitudinal and transverse velocities of the atoms are fixed by the temperature of the oven with no possibility of control. For the case of caesium, we mention Ref. [2], where the authors demonstrated that it is possible to create a bright beam by compressing an effusive Cs beam. However, the experimental apparatus affects only the slowest atoms without controlling their axial velocities. Moreover, the atomic beam still diverges in free space. It is possible to control the longitudinal velocity by using a Zeeman deccelerator [3,[START_REF] Metcalf | Laser Cooling and Trapping[END_REF], however this produces a beam with a non-negligible divergence, and only a small percentage of the atoms are slow down.

In order to have better control of the beam, cold atoms can be used as the source and thus the atomic beam obtained in this case has a narrow velocity spread. Atomic beams from cold atoms can be obtained by the pyramidal MOT [START_REF] Arlt | [END_REF]6], the conical mirror funnel [7] and the low velocity intense atomic source (LVIS) [8][9][10]. Using the now standard two-dimensional MOT [11][12][13][14][15][16][17][START_REF] Catani | Inguscio[END_REF][19][20][21][22][23][24][25][26], high flux, up to 10 11 atoms/s can be obtained. However, the cold atomic beam expands radially once the atoms have left the two-dimensional cooling region, since there is no more transversal cooling. This kind of system is of major interest for atom interferometry, atom optics and precision atomic spectroscopy or even quantum optics applications, such as EIT, entangled photon pair generation, optical quantum memory, and quantum information processing [START_REF] Zhang | A dark-line two-dimensional magnetooptical trap of 85Rb atoms with high optical depth[END_REF]. One typical application is the loading of a 3D magnetooptical trap (MOT) in a ultra-high vacuum environment which allows for the reduction of interactions and collisions with the environment and thus increases the lifetime of the trap. In our group we are interested by another application: the transport of a collimated beam of ceasium atoms far from the MOT magnetic fields in order to be ionized to create a beam of unprecedented brightness and mono-energetic ion or electron beam [28,11].

As demonstrated here, a 2D-MOT with the addition of dipole focusing facilitates atomic transport whilst reducing the beam divergence and providing a control over the beam size. This work is the continuation of the work performed in Ref. [29], where atoms were pushed and guided from a 3D-MOT to load another 3D-MOT. In the present case, by using a similar focusing and guiding technique, but now with a 2D-MOT, we reach a higher flux. The technique allows to transport atoms over large distances while maintaining the high density of the beam. We demonstrate that the atomic beam follows the laser beam shape along its propagation, and especially that focusing of the beam is possible.

We will first describe our experimental setup. Then, we will describe the experiment performed with the dipole pushing laser to guide the atoms.

Experimental results

The 2D-MOT apparatus is available from SYRTE (Observatoire de Paris) and a detailed description can be found in Ref. [START_REF] Cheinet | Conception et Réalisation d ′ un Gravimètre à Atomes Froids[END_REF].

Fig. 1 shows a schematic of the experiment. A cooling laser red-detuned 10 MHz (≃ -2Γ ) from the 6S 1/2 (F = 4) → 6P 3/2 (F ′ = 5) resonance is separated into 3 beams which are retro-reflected through a glass cell, creating 3 regions of cooling aligned along the atomic beam propagation axes. Each region measures 2 cm along the atomic beam axis and 1 cm in the other dimensions, and the regions are separated by 1.25 cm. A repumping laser, locked on the 6S 1/2 (F = 3) → 6P 3/2 (F ′ = 4) transition is sent along one dimension. The cell is surrounded by four magnetic coils which create a quadrupole field gradient of 0.2 T/m. A differential pumping section separates the 2D-MOT chamber from the detection chamber with a 1.5 mm diameter aperture. A Ti:Sa pushing laser is applied along the beam axis with a maximum power of 750 mW at 852 nm. The specifications, such as power, detuning and waist of this laser influence the atomic beam in terms of flux and divergence, and are the subject of this paper. Detection and characterization of the atomic beam are achieved via fluorescence measurements in two areas, respectively 10 cm and 60 cm far from the output of the 2D-MOT, where a probe laser beam is send orthogonally to the atomic beam. This configuration avoids shifts due to Doppler effect. Once the atoms have left the cooling region, there is no repumping laser and the atoms decay to the F=3 hyperfine state and the effect of the pushing laser is negligible. However this implies that we need to add some repumping beam in probe beams in order to improve detection efficiency by having atoms cycling on the 6S 1/2 (F = 4) → 6P 3/2 (F ′ = 5) transition.

By scanning spatially the transverse probe with power below the saturation intensity, and collecting the fluorescence light by lenses and a calibrated photodiode, we can measure the shape and the density of the atomic beam. Indeed, by taking into account the waist of the transverse probe (of 1.3 mm in the first area and 0.75 mm in the second one) it is straightforward to extract the size of the atomic beam, which is found to have a nearly Gaussian spatial distribution. To extract the atomic beam size, we remove the waist size of the probe laser from the raw signal which is a convolution between the Gaussian probe laser and the atomic beam. In this article we always define the "size" of the atomic beam as the (4σ) 1/e 2 diameter of the beam. Except for very large laser beam size (more than few mm) where the depth of field of our optical system reduces the amount of collected light, the atomic beam size is given typically within 15% accuracy. Obtaining quantitative results for the atomic beam density with fluorescence technique can be challenging due to absorption, diffusion or optical pumping effects. Consequently the absolute values for the peak atomic density n 0 and the atomic flux F cannot be considered to be given accurately (the values are probably correct only within 50%). Nevertheless all values are affected by the same systematic factors, such as collection light efficiency, thus relatives values for the integrated fluxes can be considered to be accuratly given within 15%. The flux F is calculated from F = 2πσ 2 n 0 v, where v is the longitudinal mean speed of the atoms. It is extracted from the velocity distribution of the beam obtained using the Doppler effect by scanning the frequency of a longitudinal probe, and collecting the fluorescence as performed previously when a transverse probe was used. For accurate measurement we found it was important to use a weak longitudinal probe in order to not scatter too may photons which can lead to a modification of the velocity of the atoms during their travel.

Initially, we wanted to verify the specifications for Cs atoms comparing to those providing by SYRTE for Rb. With a power of 3 mW for the standard pushing laser collimated with a waist of 1.5 mm, we measured a flux of 1.5 × 10 10 atoms/s along with a longitudinal velocity of 25 m/s. The atomic size is 7.6 mm, in the second area (60 cm from 2D-MOT). We deduce a beam divergence of 2 θ = 11.2 mrad which is in reasonable agreement with a transverse velocity given by the Doppler limit of 0.1 m/s for T = 125 µK. These measurements agree with the specifications provided by the manufacturer, but now for Cs atoms. Further study of the atomic beam velocity versus the power, detuning or even waist of the pushing beam can be performed [17][START_REF] Catani | Inguscio[END_REF][19][20][21][23][24][25], but, one conclusion of these studies is that it does not seem possible to concentrate the flux of an atomic beam with a simple pushing laser due to the large beam divergence.

In an attempt to ameliorate the beam divergence produced with a simple pushing laser, we used a high-power off-resonant laser to both push and trap the beam as demonstrated in Ref. [29] in the case of a 3D-MOT.

In a first experiment, we choose the dipole pushing laser with a waist of 1.2 mm and a Rayleigh length of 5 m, implying that the laser is collimated.

In addition to the pushing effect due to the radiation pressure the laser produced a dipole trap allows guid- ing of the atoms, provided that the trap depth is large enough. However, this makes measurement of the atomic beam parameters more involved because atoms inside the dipole trap are shifted out of resonance by the AC stark shift. The shift depends on the depth of the dipolar potential, which is dependent on the power and detuning of the dipole laser and on the distribution of the atoms in the trap [32]. On the contrary non-trapped atoms are obviously not shifted and, for a transverse probe, have all the resonance frequency of the transition F = 4 → F ′ = 5. Consequently, in order to separate the trapped atoms, from non trapped atoms we need to scan the probe detuning for each position of the probe. Fig. 2 (left panel) shows such raw signals obtained for 5 different vertical positions of the probe. They are taken in the first area of detection for a Ti:Sa power of 750 mW and a detuning of -10 GHz. We clearly see the 2 peaks: one representing the atoms which are not trapped and not sensitive to the dipole pushing laser, and the second peak being blueshifted from the first signal due to the AC Stark shift. This interpretation is confirmed by the fact that for smaller detuning, the intensity of the second peak decreases but its width increases, which is expected because the dipole trap is deeper and closer to resonance the atoms are more heated and so expand in the trap.

In order to have a more quantitative measurement, for each signal, that is, for each position of the probe, we extract the trapped and non trapped part. First by fitting a Lorentzian with fixed linewidth (natural linewidth plus the laser linewidth) to the non-trapped atoms peak, we can estimate the proportion of non-trapped atoms (Fig. 2, square points). Second, we integrate the entire signal (both the trapped and non-trapped atoms) which now is proportional to the total number of atoms present in the area. Using these data, the atomic beam flux and size, still measured in the first area, are shown in Fig. 3. We see that it is possible to push nearly 3 × 10 9 atoms/s. With the dipole pushing laser, the flux is conserved from the first area to the second one, but is slightly lower than the flux obtained when using the standard pushing laser. This can be explained by the fact that the dipole pushing laser modifies the cooling process in the MOT due to the AC Stark shift. We now switch to studies performed using the second area of detection, 60 cm far from the 2D-MOT. This allows us to study in more detail the atomic beam divergence. The raw signals are simpler because of the presence of a single peak. Indeed, the beam is not "polluted" by non trapped atoms since only trapped atoms reach this second zone. We measured the size of the atomic beam in the second area as a function of the laser detuning and the results are shown in Fig. 4. As one would expect, the size of the atomic beam is lower than the previous results obtained with the standard pushing laser. It does not exceed 2.4 mm and is contained in the area of the pushing laser. As expected, the size decreases with the detuning when the depth of the trap increases. Under the same conditions, we measure with the longitudinal probe the velocity of the atoms. Fig. 5 shows the mean velocity obtained as a function of different detuning values. We see that the velocity increases when the detuning is closer to the resonance, which is consistent with our expectations, as atoms absorb more photons and are pushed more efficiently when the frequency is closer to the resonance.

Inside the 2D-MOT, the atoms can be heated as they are accelerated by the pushing laser. However, the trapping lasers temper this effect, allowing atoms to become trapped inside the pushing laser. Outside of the MOT, there is no repumping laser and the atoms are rapidly pumped towards the fundamental F = 3 state. Thus the detuning is decreased by 9.2 GHz compared to the detuning in the 2D-MOT area which considerably reduces the effect of the pushing force and in turn the heating, whereas the dipole force is less affected. This allows atoms to be guided far from the output of the 2D-MOT without to much heating. Indeed, for an atom in the F = 3 state, the scattering rate is 6400 photons.s -1 at a detuning of -10 GHz. This implies that the temperature of the beam increases by 230 µK during the 60 cm of travel. However, for large detunings, the depth of the trap is small (175 µK at a detuning of -10 GHz for 750 mW) and gravity acts to decrease the trap depth. As shown in Fig. 4, the atomic beam size is equal to the size (that is twice the waist) of the pushing laser, implying that the atoms are at the edge of the trap, leading to a loss of atoms from the trap. In Fig. 6, we see this effect, as the shape of the signal is elongated in the direction of gravity. To improve this, one requires a deeper trap, for which there are 2 solutions in this configuration of the pushing laser: increasing the trapping laser power or reducing the detuning. But both solutions would lead to heating. Another solution is to focus the pushing laser after the MOT, which has the additional benefit of beam focusing as well as beam guiding.

To investigate the effect of dipole focusing, we set the focal point of the dipole pushing laser after the 2D-MOT between the 2 detection areas. The position of the waist is now 30 cm before the second detection area. At this position, the waist of the pushing laser is 240 µm and the effect due to the gravity is now negligible. Along the propagation, the waist w(z) of the pushing laser changes and the condition to keep the atoms in the dipole potential is the adiabatic condition (v(z)/ω 2 )(dω/dz) ≪ 1 where ω is the angular frequency of the oscillations of the atoms in the dipole trap. In the harmonic approximation, ω = 2k B T /(w(z) 2 m) where m is the mass of the Cs atoms, k B the Boltzmann constant. The condition becomes: -v(z) (dw(z)/dz) m/(2k B T ) ≪ 1. With a de- tuning of -10 GHz and a power of 750 mW, the mean velocity of the atoms is approximately 12 m/s (from Fig. 5) allowing a maximum modification of the waist size dw(z)/dz of 0.11 mm/cm at T= 125 µK. Our optical setup results in a waist modification of 0.0057 mm/cm, meaning the adiabatic condition is respected. Indeed, we found that the atomic flux of trapped atoms is conserved from the first to the second zone of detection. Fig. 7 shows the experimental points obtained in the second area of detection by scanning the beam with the transverse probe. The flux of 10 9 is confirmed by integrating the signal of Fig. 7. We found a size of the atomic beam of 0.4 mm, which corresponds to the waist of the pushing laser. These result proves that not only have we managed to guide the atomic beam, but in addition focus the beam.

Conclusion

This article shows a simple and robust experiment which is able to modify the divergence of an atomic beam created by a 2D-MOT by changing the detuning, the power and the size of the pushing laser. We note that Ref. [33] presents very similar results, but using a blue detuned Laguerre-Gaussian "doughnut" pushing laser beam. They also achieve a guiding of 100%, but this experiment with Rb atoms hasn't yet demonstrated beam focusing.

In our case, with a high power and far red detuned pushing gaussian laser beam, we can push, trap and even focus the atoms in a small area. This allows the transport atoms to a precise position. We were able to transport more than 10 9 atoms/s over 60 cm with a controllable velocity. This flux was ultimately limited by the available laser power for cooling. With increased power, a higher flux should be achieved. Furthermore, to improve the cooling effect, we can probably adjust the frequency of the cooling lasers to compensate for the AC stark shift [23]. Another solution could be to use 2 cooling lasers with 2 different frequencies : one frequency locked on the transition of the atoms when they are outside the dipole pushing laser and the other frequency locked on the transition shifted by the Stark effect.

Fig. 1 .

 1 Fig. 1. Schematic of the experiment; on the left, the 2D-MOT with the 3 clouds of cold atoms; on the right, the 2 area of detection; the atomic beam properties are extracted from fluorescence measurements, that are performed 10 cm and 60 cm far from the 2D-MOT by the use of two transverse laser beam probes and a longitudinal probe.

Fig. 2 .

 2 Fig. 2. Spatial cuts of the atomic beam for different positions of the transverse probe obtained 10 cm from the output of the MOT. A power of 750 mW and a detuning of -10 GHz was set for the dipole pushing laser. Left, raw signals obtained by scanning the detuning of the probe ; there are 2 peaks for each signals, the resonant signal on the left part and the blue shifted signal on the right part. Right, experimental points obtained by integrated the fluorescent signal for different positions of the probe ; square points present the integration of the resonant signal of the raw signal and stand for the non-trapped atoms (the dashed curve is a fit of these points) ; round points present the full integrated raw signals and stand for the entire beam of trapped and non-trapped atoms (the plain curve is a gaussian fit of the points).

Fig. 2 (

 2 right panel) shows both results, the trapped part and the fully integrated part as a function of probe position. The figure clearly indicates that a 8 mm large and dilute cloud of non trapped atoms surround the trapped atomic beam. A great part of the atoms are trapped and the trapped atomic beam is concentrated in a small area in the center of the pushing laser.

Fig. 3 .

 3 Fig. 3. Evolution of the flux and the size (the accuracy is 0.1 mm) for the entire atomic beam and for the dipole trap for different detuning of the pushing laser at a constant power of 750 mW; results obtained 10 cm far from the output of the 2D-MOT.

Fig. 4 .

 4 Fig. 4. Evolution of the size of the atomic beam, measured 60 cm far from the 2D-MOT, for different detuning of the pushing laser at a constant power of 750 mW.

Fig. 5 .

 5 Fig. 5. Evolution of the speed of the atoms 60 cm from the output of the 2D-MOT versus the laser detuning for a power of 750 mW.

Fig. 6 .

 6 Fig. 6. Shape of the atomic beam, 60 cm from the output of the 2D-MOT, for -10 GHz and 750 mW; inset : potential depth as function of r (vertical direction).

Fig. 7 .

 7 Fig.7. Size of the atomic beam in the second area of detection in the second experiment ; points are experimental datas and the curve is a fit of experimental points ; the signal is the convolution of the waist probe 1.3 mmand the atomic beam shape ; thus, the true atomic beam size is 0.4 mm.

Acknowledgements : The research leading to these results has received funding from the European Research Council under the grant agreement n. 277762 COLDNANO. We thank Andrew McCulloch for the proofreading of the article.