Salomé Paillas

Riad Ladjohounlou

Catherine Lozza

Alexandre Pichard

Vincent Boudousq

Marta Jarlier

Samuel Sevestre

Marion Le Blay

Emmanuel Deshayes

Jane Sosabowski

Salome ´paillas

Thierry Charde

Isabelle Navarro-Teulon

Robert J Mairs

Jean-Pierre Pouget

Localized Irradiation of Cell Membrane by Auger Electrons Is Cytotoxic Through Oxidative Stress-Mediated Nontargeted Effects

Keywords:

Introduction

T he highly localized cytotoxicity of Auger elec- trons is particularly attractive for targeted radionuclide therapy of microscopic or metastatic disease because it allows the irradiation of tumor cells while sparing healthy tissues. Auger electrons are produced in atoms decaying by internal conversion or electron capture. These processes create a vacancy in the inner electron shell that is subsequently filled by electrons dropping in from higher energy levels. Thus, the vacancy moves to the outer shells, and the transition is accompanied by cascades of complex emissions of Auger, Coster-Kronig, and super Coster-Kronig electrons (collectively called Auger electrons) and soft X-rays [for review, Kassis (25)]. The majority of Auger electrons have very low energies (between a few eV and 1 keV) and thus very short range in tissue (<1 lm). They release high amounts of energy in a spherical volume of a few cubic nanometers [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125 I-labeled monoclonal antibodies targeting the cell membrane[END_REF][START_REF] Pomplun | A Monte Carlo simulation of Auger cascades[END_REF][START_REF] Sastry | Dosimetry of low energy electrons[END_REF] and behave like medium-to-high linear energy transfer (LET) radiation (4-26 keV/lm). Hence, they produce locally dense ionizations that are very destructive when incorporated into nuclear DNA [START_REF] Bradley | The radiotoxicity of iodine-125 in mammalian cells. I. Effects on the survival curve of radioiodine incorporated into DNA[END_REF][START_REF] Hofer | Radiotoxicity of intranuclear tritium, 125 iodine and 131 iodine[END_REF][START_REF] Kassis | Lethality of Auger electrons from the decay of bromine-77 in the DNA of mammalian cells[END_REF][START_REF] Kassis | Radiotoxicity of an 125 I-labeled DNA intercalator in mammalian cells[END_REF][START_REF] Kassis | Radiotoxicity of 125 I in mammalian cells[END_REF][START_REF] Kassis | Kinetics of uptake, retention, and radiotoxicity of 125 IUdR in mammalian cells: Implications of localized energy deposition by Auger processes[END_REF] or the nucleus [START_REF] Anthony | Indium-111-pentetreotide prolongs survival in gastroenteropancreatic malignancies[END_REF][START_REF] Chen | Antitumor effects and normal tissue toxicity of 111 In-labeled epidermal growth factor administered to athymic mice bearing epidermal growth factor receptorpositive human breast cancer xenografts[END_REF][START_REF] Commerford | Radiotoxicity of intranuclear 125 I atoms not bound to DNA[END_REF][START_REF] Costantini | 111 In-labeled trastuzumab (herceptin) modified with nuclear localization sequences (NLS): an Auger electron-emitting radiotherapeutic agent for HER2/ neu-amplified breast cancer[END_REF][START_REF] Costantini | Antitumor effects and normal-tissue toxicity of 111 In-nuclear localization sequence-trastuzumab in athymic mice bearing HER-positive human breast cancer xenografts[END_REF]. Although they have high LET features, they have been shown to contribute to cytotoxicity or DNA lesion formation indirectly via reactive oxygen species (ROS) [START_REF] Walicka | Indirect mechanisms contribute to biological effects produced by decay of DNA-incorporated iodine-125 in mammalian cells in vitro: clonogenic survival[END_REF][START_REF] Walicka | Indirect mechanisms contribute to biological effects produced by decay of DNA-incorporated iodine-125 in mammalian cells in vitro: double-strand breaks[END_REF].

The radionuclides iodine 125 ( 125 I), iodine 123 ( 123 I), and indium 111 ( 111 In) are the most widely used Auger electron emitters for in vitro and in vivo studies. Clinical trials have evaluated the efficacy, toxicity, or tumor distribution of Auger electron emitters conjugated to (i) thymidine analogs that are incorporated into the DNA of cells in S phase [START_REF] Galanis | Phase I trial of sequential administration of raltitrexed (Tomudex) and 5-iodo-2'-deoxyuridine (IdUrd)[END_REF][START_REF] Macapinlac | Pilot clinical trial of 5-[ 125 I]iodo-2¢deoxyuridine in the treatment of colorectal cancer metastatic to the liver[END_REF][START_REF] Mariani | Tumor targeting in vivo and metabolic fate of 5-[iodine-125]iodo-2¢-deoxyuridine following intratumoral injection in patients with colorectal cancer[END_REF], (ii) octreotide, a somatostatin analog targeting neuroendocrine and other cancers [START_REF] Delpassand | Safety and efficacy of radionuclide therapy with high-activity In-111 pentetreotide in patients with progressive neuroendocrine tumors[END_REF][START_REF] Krenning | The role of radioactive somatostatin and its analogues in the control of tumor growth[END_REF][START_REF] Limouris | Selective hepatic arterial infusion of In-111-DTPA-Phe1-octreotide in neuroendocrine liver metastases[END_REF], and (iii) monoclonal antibodies (mAbs) with specificity for cancer cellular antigens [START_REF] Li | A Phase II study of anti-epidermal growth factor receptor radioimmunotherapy in the treatment of glioblastoma multiforme[END_REF][START_REF] Quang | Radioimmunotherapy as a novel treatment regimen: 125 I-labeled monoclonal antibody 425 in the treatment of high-grade brain gliomas[END_REF][START_REF] Welt | Phase I/II study of iodine 125-labeled monoclonal antibody A33 in patients with advanced colon cancer[END_REF] and human epidermal growth factor receptor [START_REF] Vallis | Phase I trial to evaluate the tumor and normal tissue uptake, radiation dosimetry and safety of (111)In-DTPA-human epidermal growth factor in patients with metastatic EGFR-positive breast cancer[END_REF]. The latter treatment is known as radioimmunotherapy (RIT).

Conventionally, Auger electron emitters are targeted to the nucleus or DNA because it is considered that Auger electrons need to be within the nucleus to achieve maximal cell kill. Therefore, RIT using Auger electron emitters has been regarded as comparatively disadvantageous because the localization of the radionuclide, after receptor binding, is not the nucleus, but the cytoplasm (internalizing mAbs) or the cell membrane (noninternalizing mAbs).

However, we previously showed, using in vitro and in vivo models, substantial antitumor efficacy of noninternalizing monoclonal antibodies labeled with 125 I ( 125 I-mAbs). Furthermore, the cytotoxicity of noninternalizing mAbs was greater than that achieved by internalizing 125 I-mAbs [START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF][START_REF] Santoro | Noninternalizing monoclonal antibodies are suitable candidates for 125 I radioimmunotherapy of small-volume peritoneal carcinomatosis[END_REF] and was not due to inefficient detection of DNA damage related to low absorbed dosage. We proposed that, instead, nontargeted effects could be involved [START_REF] Piron | DNA damage-centered signaling pathways are effectively activated during low dose-rate Auger radioimmunotherapy[END_REF]. This is in agreement with the work by [START_REF] Xue | Bystander effect produced by radiolabeled tumor cells in vivo[END_REF] showing in vivo that nontargeted effects are produced by LS174T cells radiolabeled with the DNA base analog 5-[(125)I]iodo-2¢deoxyuridine ( 125 I-UdR), indicating that Auger electrons can kill cells beyond their path length [START_REF] Xue | Bystander effect produced by radiolabeled tumor cells in vivo[END_REF]. Other reports indicate that they have also been observed during radionuclide therapy using tritiated thymidine (3H3H-dThd) [START_REF] Bishayee | Evidence for pronounced bystander effects caused by nonuniform distributions of radioactivity using a novel three-dimensional tissue culture model[END_REF], meta-[ 211 At]astatobenzylguanidine ( 211 At-MABG), meta[ 123 I]iodobenzylguanidine ( 123 I-MIBG) [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], and 213 Bi-mAbs [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].

Radiation-induced nontargeted effects (also called bystander effects) occur in cells that are not directly traversed by ionizing particles, but are in contact with irradiated cells. They have been mainly observed after low-dose (<0.5 Gy) external beam radiotherapy (EBRT), for both low and high LET irradiation, and are associated with a lack of dose-effect relationships [for reviews, Hamada [START_REF] Hamada | Evidence and significance of non-targeted effects of ionizing radiation[END_REF] and Prise and O'Sullivan [START_REF] Prise | Radiation-induced bystander signalling in cancer therapy[END_REF]]. Bystander effects include cell death, DNA damage, apoptosis [START_REF] Lyng | Initiation of apoptosis in cells exposed to medium from the progeny of irradiated cells: A possible mechanism for bystanderinduced genomic instability?[END_REF], yield of micronuclei and chromosomal aberrations [START_REF] Belyakov | Delayed lethality, apoptosis and micronucleus formation in human fibroblasts irradiated with X-rays or alpha-particles[END_REF][START_REF] Nagasawa | Involvement of membrane signaling in the bystander effect in irradiated cells[END_REF], and malignant transformation [START_REF] Sawant | The bystander effect in radiation oncogenesis: I. Transformation in C3H 10T1/2 cells in vitro can be initiated in the unirradiated neighbors of irradiated cells[END_REF]. The bystander response depends both on the cell type and on radiation LET and involves the release of soluble factors in the extracellular environment together with the transmission of signaling molecules through gap junctions when cells are in contact [START_REF] Lehnert | Extracellular factor(s) following exposure to alpha particles can cause sister chromatid exchanges in normal human cells[END_REF][START_REF] Mothersill | Cell-cell contact during gamma irradiation is not required to induce a bystander effect in normal human keratinocytes: Evidence for release during irradiation of a signal controlling survival into the medium[END_REF]. ROS and reactive nitrogen species (RNS), Ca 2+ ions, ATP, and cytokines have been shown to be involved [START_REF] Azzam | Oxidative metabolism, gap junctions and the ionizing radiation-induced bystander effect[END_REF][START_REF] Lyng | The involvement of calcium and MAP kinase signaling pathways in the production of radiationinduced bystander effects[END_REF].

In this study, we show that oxidative stress-induced nontargeted effects are involved in the cytotoxicity of 125 I-mAbs targeting cell surface receptors. This phenomenon involves lipid raft formation followed by subsequent activation of signaling pathways. Moreover, the potency of the cytotoxic nontargeted effect induced by targeting the nucleus with 125 I-UdR was comparable to that resulting from exposure to 125 I-mAbs against cell surface receptors, suggesting that it was independent of the localization of Auger electron emitters. Finally, our data support the existence of nontargeted cytotoxic effects, both in vitro and in vivo.

Results

I-mAbs induce nontargeted effects

Clonogenic survival of p53 +/+ HCT116 cells targeted by 4 MBq/ml of noninternalizing anti-carcinoembryonic antigen (CEA) 125 I-mAb (resulting in cell surface localization of 125 I) or internalizing anti-Human Epidermal Receptor type 1 (HER1) 125 I-mAb (resulting in cytoplasmic localization of 125 I) (donor cells) was reduced to 58% -2.5% and 57.8% -7%, respectively. These values were significantly different ( p < 0.001) from values measured using the nonspecific 125 I-PX mAb (Fig. 1A, left panel). We also investigated the role of p53 by performing the same experiments in p53 -/-HCT116 cells and showed a similar reduction in clonogenic survival (to 54.6% -1.2% and 56.5% -1.9%, respectively) (Fig. 1B, left panel). Again survival was statistically significantly different from survival measured after nonspecific 125 I-PX mAb exposure ( p < 0.001), where no toxicity was observed (Fig. 1B, left panel).

To test whether nontargeted effects were involved in the toxicity induced by 125 I-mAb, nonirradiated p53 +/+ and p53 -/- HCT116 cells (recipient cells) were incubated with medium in which donor cells had been cultured for 2 h following a 2-day exposure to 4 MBq/ml of anti-CEA or anti-HER1 125 I-mAbs. Clonogenic survival of recipient cells was significantly reduced when compared to that of cells incubated with medium from cells exposed to nonspecific 125 I-PX mAb ( p < 0.01). For example, in p53 +/+ HCT116 recipient cells, survival decreased to 71.2% -7.0% after incubation with medium from p53 +/+ HCT116 donor cells exposed to anti-CEA 125 I-mAb and to 70.6% -8.4% after exposure to medium from cells exposed to anti-HER1 125 I-mAb (Table 1 and Fig. 1A, right panel). Similarly, in p53 -/-HCT116 recipient cells, survival decreased to Innovation Because of their physical properties, Auger electron emitters, such as iodine 125 ( 125 I), are usually targeted to the nucleus to maximize their cytotoxicity. In this study, we show that monoclonal antibodies labeled with 125 I ( 125 I-mAbs) and targeting the cell membrane are cytotoxic through oxidative stress-mediated nontargeted effects. As this nontargeted response is comparable to that observed with 125 IdUrd, bystander effects induced by cell membrane irradiation could compensate for the anticipated inferior efficacy of the absence of nuclear targeting, particularly when vectors do not gain access to every tumor cell. Furthermore, Auger emitter-labeled mAbs bypass the disadvantages of using labeled deoxyribonucleotides. 73.3% -5.6% following incubation with medium from p53 -/- HCT116 donor cells exposed to anti-CEA 125 I-mAb and to 74.5% -5.1% upon switch to medium from cells exposed to anti-HER1 125 I-mAb (Fig. 1B, right panel).

No cell killing was observed after incubation of recipient cells with medium from donor cells treated with the nontargeting 125 I-PX mAb (Fig. 1B, right panel) or with unlabeled mAbs (Supplementary Fig. S1A; Supplementary Data are available online at www.liebertpub.com/ars). This suggests that the effect was specific and required 125 I-mAb binding to the cell surface. Similar results were obtained in A-431 CEA (Fig. 1C) and SK-OV-3 CEA cells (Supplementary Fig. S1B) treated with the same 125 I-mAbs. Gamma-H2AX foci (markers of DNA double strand breaks, DSBs) were detected in both p53 +/+ HCT116 donor (exposed to anti-CEA or anti-HER1 125 I-mAbs) and recipient cells, again indicating the occurrence of nontargeted effects in recipient cells (Fig. 2A,B). It must be noted that cells were proliferating and that a larger number of gamma-H2AX foci are likely to be produced during the S phase. The proportion of donor cells showing foci was assessed to be about 10% in nonirradiated cells, while it increased up to 25% in cells exposed to the nonspecific 125 I-PX mAb and 100% after exposure to the specific anti-HER1 and anti-CEA 125 I-mAbs.

Corresponding values were 15% and 100% in recipient cells (data not shown).

Nontargeted effects do not require cellular internalization of 125 I

Localization of the anti-CEA and anti-HER1 mAbs at the cell membrane and cytoplasm, respectively, was confirmed by cell fractionation followed by Western blotting (Fig. 2C) and by immunofluorescence (Fig. 2D) in p53 +/+ HCT116 and A-431 CEA cells. Coincubation of p53 +/+ HCT116 donor cells with anti-CEA or anti-HER1 125 I-mAbs and sodium azide, a drug preventing antibody internalization after receptor binding, did not modify the nontargeted response in recipient cells (Fig. 2E). These results suggest that internalization of 125 I is not required for radiation-induced nontargeted effects.

The role of lipid rafts in 125 I-mAb cytotoxicity in vitro

Lipid rafts can be ceramide-enriched platforms in the cell membrane that are produced through sphingomyelin hydrolysis to ceramide and phosphorylcholine following acid sphingomyelinase (ASMase) activation by ROS [START_REF] Scheel-Toellner | Reactive oxygen species limit neutrophil life span by activating death receptor signaling[END_REF]. To investigate whether 125 I-mAbs affected the cell membrane organization, the formation of lipid raft domains, as detected by cholera toxin, was monitored in donor p53 +/+ HCT116 cells exposed to 4 MBq/ml of anti-CEA or anti-HER1 125 I-mAbs for 48 h. In these cells, lipid raft formation was increased compared with nonirradiated cells (NT) or with cells incubated with the irrelevant 125 I-PX mAb (Fig. 3A). Flow cytometric analysis showed that the membrane concentration of ceramide (indicative of lipid raft formation) in cells exposed to anti-CEA or anti-HER1 125 I-mAbs was increased compared with nonirradiated cells (NT) and cells treated with unlabeled mAbs (Fig. 3B). Codetection of ceramide and Annexin (apoptosis marker) by immunofluorescence was observed in some of the p53 +/+ HCT116 cells (Fig. 3C). However, we previously showed that apoptosis was not involved in the cytotoxic effects of 125 I-anti-CEA mAb [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125 I-labeled monoclonal antibodies targeting the cell membrane[END_REF].

The formation of ceramide was corroborated also by the significantly higher ASMase activity measured in whole cell lysates of p53 +/+ HCT116 treated with anti-CEA or anti-HER1 125 I-mAbs (three times higher compared to unlabeled mAbs, p < 0.001) (Fig. 3D). We also found that the level of sphingosine-1-phosphate (S1P) resulting from hydrolysis of ceramide was strongly increased in cells exposed to anti-CEA or anti-HER1 125 I-mAbs (Fig. 3E), while no such increase was observed in control cells.

The involvement of lipid raft formation in 125 I-mAb cytotoxicity was confirmed by the finding that lipid raft formation was reduced in p53 +/+ HCT116 donor cells coincubated with anti-CEA or anti-HER1 125 I-mAbs and filipin (a lipid raft disruptive agent) compared with cells exposed only to 125 I-mAbs (Fig. 4A). Moreover, coincubation of donor cells with filipin significantly reduced the formation of phosphorylated histone H2AX (c-H2AX) foci in both donor and recipient cells (Fig. 4B). Finally, 125 I-mAb cytotoxicity in both donor (Fig. 4C) and recipient cells (Fig. 4D) was significantly reduced when donor cells were incubated in the presence of filipin. Indeed, clonogenic survival increased from 58% -2.5% to 84.4% -2.6% and from 57.8% -7% to 81.0% -1.6% in donor cells coincubated with 4 MBq/ml anti-CEA or anti-HER1 125 I-mAbs and filipin, respectively (Table 1). In recipient cells, survival increased from 71.2% -7% to 92.8% -2.3% and from 70.6% -8.4% to 94.2% -2.4%, respectively. Similar protection was afforded by methyl-b-cyclodextrin (MBCD), another lipid raft disruptor (Supplementary Fig. S2). These findings indicate that lipid raft formation is involved in both targeted and nontargeted cytotoxicity of 125 I-mAbs.

Phosphorylated proteins associated with 125 I-RIT

To identify the signaling pathways activated in cells exposed to anti-CEA or anti-HER1 125 I-mAbs, the phosphorylation profiles of 46 kinases were assessed by array-based proteomics (Fig. 5 and Supplementary Fig. S3). Only extracellular signal-related kinase ½ (ERK1/2) and C-AMP Response Element-binding protein (CREB) were strongly activated after treatment with unlabeled anti-CEA mAb (Fig. 5A). Conversely, upon incubation with the unlabeled anti-HER1 mAb, several proteins were induced, including ERK1/2, CREB, p53, and AKT (Fig. 5B).

Thus, only the anti-CEA 125 I-mAb was used to investigate the effect of targeting 125 I to the cell surface. When cells were exposed to anti-CEA 125 I-mAb (Fig. 5A), the ERK1/2, AKT, p38, and c-Jun N-terminal kinase ( JNK) signaling pathways

ZSTK474) and subsequently of downstream AKT signaling pathway, p38 (SB203580), and JNK (SP600125) indicated that, among the identified proteins activated, p38 and JNK were mainly involved in the response of cells to 125 I-mAb exposure (Fig. 6). No effect of EGTA on clonogenic survival was observed (Supplementary Fig. S1C).

ROS are involved in Auger electron-induced nontargeted response

The involvement of ROS in the mediation of the bystander response was examined by culturing recipient cells in medium from p53 +/+ HCT116 donor cells that had been treated To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars with 125 I-mAbs in the presence or absence of the free radical scavengers dimethyl sulfoxide (DMSO) or N-acetylcysteine (NAC) (Fig. 7). We previously checked (using immunofluorescence detection of mAbs in the presence of cells grown on coverslips) that NAC did not prevent anti-HER1 or anti-CEA mAbs from binding to their receptors.

Survival of both donor and recipient cells was significantly increased compared with controls treated with 125 I-mAbs in the absence of free radical scavengers ( p < 0.001 and p < 0.01, respectively). This suggests that oxidative stress is involved in both targeted and nontargeted cytotoxic effects of 125 I-mAbs.

Nontargeted effects of 125 I directed to the cell membrane or the nucleus are equipotent

To compare the effect of targeting 125 I to the cell membrane or the nucleus, p53 +/+ HCT116 donor cells were incubated with 125 I-mAbs. Subsequently, clonogenic survival was assessed in both donor and recipient cells. Results are summarized in Table 1. The uptake of 125 I-UdR and 125 I-mAbs was measured for every test activity for 6 days, and the total number of decays occurring in cells was calculated as described previously [START_REF] Paillas | Apoptosis and p53 are not involved in the anti-tumor efficacy of 125 I-labeled monoclonal antibodies targeting the cell membrane[END_REF]. The results of the clonogenic survival assay showed that, in donor cells, 125 [START_REF] Santoro | Noninternalizing monoclonal antibodies are suitable candidates for 125 I radioimmunotherapy of small-volume peritoneal carcinomatosis[END_REF]. Treatment with MBCD removed the statistical significance of RITinduced tumor growth delay such that no difference was observed between NaCl and RIT+MBCD groups ( p = 0.2481) (Fig. 8A).

Oxidative DNA damage occurs beyond the range of Auger electrons in vivo Digital autoradiographic analysis performed on sections of tumors collected in mice injected with 125 I-anti-CEA mAb FIG. 4. Role of lipid rafts in the nontargeted response. (A) Disruption of lipid rafts was confirmed by immunofluorescence analysis using Alexa-488 (Green)-conjugated cholera toxin (a marker of lipid rafts) in p53 +/+ HCT116 cells coincubated with 125 I-mAbs (noninternalizing anti-CEA or internalizing anti-HER1) and filipin. Nuclei were stained with Hoechst (Blue). (B) Images of gamma-H2AX foci (immunofluorescent detection of Alexa-488 [Green] conjugated anti mouse-anti gamma-H2AX antibody) in p53 +/+ HCT116 donor cells incubated 2 days with 125 I-mAbs in the presence or absence of filipin (top panels) and in the corresponding recipient cells (bottom panels). The number of foci per cell was counted in 100 cells. Clonogenic survival was assessed in p53 +/+ HCT116 donor cells coincubated for 2 days with and without 125 I-mAbs and filipin (C), and in the corresponding recipient cells (D). Results are the mean -SD of four experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 compared to cells treated with 125 I-mAbs. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars showed that radioactivity (and thus the absorbed dose) was mainly localized at the tumor periphery in A-431 CEA tumor cell xenografts (Fig. 9A, inset in the bottom left corner). Conversely, DNA DSBs occurred homogeneously throughout the tumor, as indicated by immunofluorescence detection of p53-binding protein 1 (53BP1), a DNA damage sensor that is recruited to damaged chromatin (Fig. 9A). The average number of foci per cell was 4.4 at the center and 4.3 at the periphery of the tumor. The corresponding regional activities were 2.3 and 34.7 cpm per mm 2 . Our observation of similar levels of DNA DSBs at the center of the tumor, independent of the absorbed dose and beyond the range of Auger electrons, supports the involvement of nontargeted effects also in vivo. 53BP1 expression was also investigated in A-431 CEA tumor cell xenograft sections from mice injected with the irrelevant 125 I-PX mAb (Fig. 9B). The numbers of foci per cell was 0.8 (tumor center) and 1.0 (tumor periphery). These values were lower than those observed following treatment with anti-CEA 125 I-mAb, de-spite the higher uptake of 125 I-PX mAb exemplified by activity per area values of 28 and 18.2 cpm/mm 2 at the tumor center and periphery, respectively. A similar level of 0.7 --0.1 53BP1 foci was found in tumors of NaCl or anti-CEA-treated mice (Fig. 9C).

This ruled out the possibility that the signal detected in tumors treated with anti-CEA 125 I-mAb was caused by nonspecific induction of 53BP1 due to cross-irradiation from the radioactivity present in nonmalignant regions of the animal.

Discussion

In the present study, we show that 125 I translocation to the nucleus is not required for cell killing and that radiationinduced nontargeted effects contribute to 125 I cytotoxicity when labeled with monoclonal antibodies ( 125 I-mAbs) (Table 1). We also identify the signaling pathways involved in these nontargeted effects. In particular, we show using p53 -/- and p53 +/+ HCT116 cells that the latter protein did not play a was used to detect the phosphorylation level of 46 kinases. For each kinase, the Image J software was used to determine pixel number. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars significant role in anti-CEA 125 I-mAb cytotoxicity since no difference was observed between the two cell lines (Fig. 1A, B and Supplementary Fig. S4). We chose the nuclear p53 protein as it is one of the main proteins involved in signaling pathways activated by nuclear damage.

The potency of nontargeted cell kill, assessed using a medium transfer protocol, was independent of 125 I localization (nucleus, cytoplasm, or cell surface) and of the magnitude of targeted cytotoxicity in donor cells (Table 1). In our in vitro experimental RIT model, donor cells were incubated with 125 I-mAbs and were killed both by targeted and nontargeted effects. Therefore, it is possible to determine the relative contribution of targeted and nontargeted cytotoxicity to donor cells by comparing the survival values of donor and recipient cells reported in Table 1. As death of recipient cells (incubated in culture medium in which donor cells had been cultured for 2 h) ranged between 28.8% and 29.4% (Fig. 1B), it may be assumed that a similar (or even higher) magnitude of nontargeted effects also occurred in donor cells that were directly exposed to 125 I-mAbs for 48 h. Accordingly, cell death by targeted effects in donor cells would be equal to or less than 13.2% (42.0% minus 28.8%) and 12.8% (42.2% minus 29.4%) for 125 I localized at the cell surface or cytoplasm, respectively, and equal to or less than 50.7% (77.9% minus 27.2%) for 125 I within the nucleus (Table 1). These data confirm the greater cytotoxicity of 125 I when incorporated into DNA [START_REF] Hofer | Radiotoxicity of intranuclear tritium, 125 iodine and 131 iodine[END_REF][START_REF] Desombre | Therapy of estrogen receptor-positive micrometastases in the peritoneal cavity with Auger electron-emitting estrogens-Theoretical and practical considerations[END_REF][START_REF] Walicka | Indirect mechanisms contribute to biological effects produced by decay of DNA-incorporated iodine-125 in mammalian cells in vitro: double-strand breaks[END_REF] in cells that are directly targeted.

Our results indicate that the cell membrane plays a major role not only in the targeted cytotoxic effects of 125 I-mAbs but also in nontargeted cytotoxic effects (Fig. 1). According to the fluid mosaic model [START_REF] Singer | The fluid mosaic model of the structure of cell membranes[END_REF], the membrane contains sphingolipids (predominantly sphingomyelin), cholesterol, and glycophospholipids. Sphingolipids associate with each other and with cholesterol to form sphingolipid-and FIG. 6. Pharmacological inhibitors of MAPK pathways. p53 +/+ HCT116 donor cells were incubated for 48 h with 125 I-anti-CEA mAb in the presence of (A) SB203580 (p38 inhibitor), (B) SP600125 ( JNK inhibitor), (C) ZSTK474 (PI3K inhibitor and indirectly of its downstream AKT pathway), or (D) U0126 (MEK1/2 inhibitor and indirectly of its downstream ERK1/2 pathway). Results are the mean -SD of four experiments performed in triplicate. *p < 0.05, **p < 0.01, compared to cells treated with 125 I-mAbs. ERK1/ 2, extracellular signal-related kinase ½; JNK, c-Jun Nterminal kinase; MAPK, mitogen-activated protein kinase.

cholesterol-enriched domains called lipid rafts that are stabilized in some cells by caveolin [START_REF] Lajoie | Caveolin-1 regulation of dynamindependent, raft-mediated endocytosis of cholera toxin-B sub-unit occurs independently of caveolae[END_REF]. It has been shown that ASMase activated by hydroxyl radicals [START_REF] Scheel-Toellner | Reactive oxygen species limit neutrophil life span by activating death receptor signaling[END_REF] translocates from the cytoplasm to the outer layer of the cell membrane, where it hydrolyzes sphingomyelin into hydrophobic ceramide and hydrophilic phosphorylcholine [START_REF] Smith | The unexpected role of acid sphingomyelinase in cell death and the pathophysiology of common diseases[END_REF][START_REF] Zhang | Ceramide-enriched membrane domains-Structure and function[END_REF]. Ceramide production is accompanied by the coalescence of lipid raft microdomains into ceramide-enriched large platforms that can participate in the spatial reorganization of membrane receptors and in the activation of multiple signaling pathways. Several mechanisms leading to protein activation through membrane reorganization have been identified in response to EBRT [START_REF] Chalfant | Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid[END_REF][START_REF] Cordes | Human pancreatic tumor cells are sensitized to ionizing radiation by knockdown of caveolin-1[END_REF][START_REF] Patel | Caveolae as organizers of pharmacologically relevant signal transduction molecules[END_REF].

Our results confirm that similar mechanisms might be involved in 125 I-mAb efficacy after binding to the cell surface because we show that the localized energy deposition associated with 125 I decays was accompanied by ASMase activation (Fig. 3D), formation of ceramide-enriched large platforms (Fig. 3A), and subsequent activation of signaling pathways (Fig. 5). ROS scavenging or disruption of lipid rafts, using MBCD or filipin, reduced not only the targeted cytotoxic effect of 125 I-mAbs but also the nontargeted cytotoxic effect (Figs. 4 and7). The use of filipin, a cholesteroldepleting agent, reduced the cytotoxic effect of 4 MBq/ml anti-CEA or anti-HER1 125 I-mAbs by about half in donor cells and by 75%-80% in recipient cells (Table 1), showing the involvement of lipid rafts in the cytotoxic effects of 125 I-mAbs. These results are supported by in vivo data showing that MBCD treatment removes the statistical significance of the therapeutic efficacy of 125 I-anti-CEA mAb (Fig. 8).

A schematic representation of the mechanisms that might be involved in 125 I-mAbs targeted cytotoxicity is proposed in Figure 10. Formation of ceramide-enriched large platforms is induced following ASMase activation mediated by ROS [START_REF] Scheel-Toellner | Reactive oxygen species limit neutrophil life span by activating death receptor signaling[END_REF] originating from water radiolysis (presumably in a dosedependent manner) (Fig. 3D). ROS could also be generated following lipid raft-mediated activation of NAD(P)H oxidase [START_REF] Li | Lipid rafts and redox signaling[END_REF][START_REF] Zhang | Acid sphingomyelinase and its redox amplification in formation of lipid raft redox signaling platforms in endothelial cells[END_REF]. ROS contribute to ''chronic'' oxidative stress and the subsequent DNA damage, including the formation of micronuclei in nontargeted cells [START_REF] Azzam | Oxidative metabolism modulates signal transduction and micronucleus formation in bystander cells from alpha-particle-irradiated normal human fibroblast cultures[END_REF][START_REF] Narayanan | Alpha particles initiate biological production of superoxide anions and hydrogen peroxide in human cells[END_REF]. This is in agreement with our previous finding that the abundance of micronuclei in cells exposed to 125 I-mAbs was not correlated with the mean absorbed dose to the nucleus [START_REF] Piron | DNA damage-centered signaling pathways are effectively activated during low dose-rate Auger radioimmunotherapy[END_REF][START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF]. The involvement of oxidative stress in EBRT-induced nontargeted effects has been extensively reviewed by Havaki et al. [START_REF] Havaki | The role of oxidative DNA damage in radiation induced bystander effect[END_REF]. Irradiated cells produce ROS and RNS through radiolysis and cytokines. ROS and RNS are then directly transmitted to bystander cells through gap junctions or through release in the extracellular medium [START_REF] Havaki | The role of oxidative DNA damage in radiation induced bystander effect[END_REF]. In bystander cells, different signaling pathways participate in the upregulation of genes encoding cytokines, cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS) that generate intracellular ROS and RNS [START_REF] Havaki | The role of oxidative DNA damage in radiation induced bystander effect[END_REF]. However, the involvement of such mechanisms following low dose rate and protracted exposure (as in RIT) needs to be confirmed. The present study focused on the mechanisms involved in irradiated donor cells, and we assume that the main source of oxidative stress was associated with radiation and hydroxyl radical formation. Elucidation of the precise mechanisms involved in recipient cells would require further work.

It is noteworthy that 125 I decays must occur at the surface of targeted cells to initiate the nontargeted response. Indeed, our data show that low LET radiation (including electrons and soft X-rays) emitted by the irrelevant PX 125 I-mAb, which does not interact with cells, did not produce a significant nontargeted response. This suggests that nontargeted effects are induced mainly by high LET Auger electrons (of very low range) generated at the cell surface when 125 I-mAbs bind to CEA or HER1 receptors.

The relative contribution of the various signaling pathways activated by cell membrane reorganization following lipid raft formation is complex and remains to be elucidated. We show that the mitogen-activated protein kinases (MAPKs), ERK1/2, P38, JNK, and AKT, and their downstream proteins, are activated during exposure to 125 I-mAbs. The role of these kinases in our model is still unclear because AKT and ERK1/ 2 promote cell survival, while ceramide, p38, JNK, and its downstream target c-JUN participate in cell death. However, the use of p38 or (to a lesser extent) of JNK inhibitors was accompanied by a stronger effect on clonogenic survival (increase) than the use of PI3K or MEK inhibitors, suggesting the cell death mechanisms predominate (Fig. 6). We also found that S1P, the hydrolysis product of sphingosine that is generated from ceramide by ceramidase (Figs. 3E and 10), was upregulated following exposure to anti-CEA 125 I-mAb. Contrary to ceramide, S1P inhibits apoptosis and stimulates cell proliferation through activation of the AKT and e-NOS pathways [for review, Oskouian and Saba [START_REF] Oskouian | Cancer treatment strategies targeting sphingolipid metabolism[END_REF]]. Paxillin also was activated by incubation with anti-CEA 125 I-mAb (Fig. 5). Paxillin can be phosphorylated by focal adhesion kinase (FAK) or PYK-2, and phosphorylated paxillin serves as a scaffold protein to facilitate the functional integration of focal adhesion proteins. Paxillin is phosphorylated during EBRT as a downstream protein target (together with AKT and ERK1/2) of FAK [START_REF] Hehlgans | Targeting FAK radiosensitizes 3-dimensional grown human HNSCC cells through reduced Akt1 and MEK1/2 signaling[END_REF].

Moreover, our data suggest a possible role of ion channels located in lipid rafts, such as Ca 2+ channels. The role of Ca 2+ in EBRT-associated nontargeted effects has clearly been demonstrated [START_REF] Lyng | The involvement of calcium and MAP kinase signaling pathways in the production of radiationinduced bystander effects[END_REF]. The influx of extracellular Ca 2+ can be regulated by ceramide [START_REF] Church | TNFR1-induced sphingomyelinase activation modulates TCR signaling by impairing storeoperated Ca2+ influx[END_REF], but also by PLC-c that is activated upon exposure to anti-CEA 125 I-mAb (Fig. 5A). During EBRT, PLCc localized at the plasma membrane produces inositol triphosphate (IP3) that consequently induces Ca 2+ release from the endoplasmic reticulum through IP3 receptors [START_REF] Todd | Ionizing radiation stimulates existing signal transduction pathways involving the activation of epidermal growth factor receptor and ERBB-3, and changes of intracellular calcium in A431 human squamous carcinoma cells[END_REF]. Such an increase in Ca 2+ cytoplasmic concentration could also contribute to the phosphorylation of PYK-2 observed in cells exposed to anti-CEA 125 I-mAb (Fig. 5A) [START_REF] Lev | Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions[END_REF]. PYK-2, a member of the FAK family, is involved in cell proliferation and phosphorylates paxillin. However, incubation of donor cells with EGTA, a chelator of extracellular calcium, was not accompanied by modifications in clonogenic survival. In a previous study, Lyng et al. have used EGTA in combination with verapamil and nifedipine to block also voltage-dependent calcium channels [START_REF] Lyng | The involvement of calcium and MAP kinase signaling pathways in the production of radiationinduced bystander effects[END_REF] to investigate the role of Ca 2+ ions. Such an approach merits further investigation in our models also.

Finally, we investigated in A-431 CEA tumor xenografts from mice injected with anti-CEA 125 I-mAb whether the induction of DNA DSBs could be correlated with the distribution of radioactivity. We observed that radioactivity was mainly localized at the periphery of tumors, whereas DNA DSBs were homogenously distributed throughout the tumor (Figs. 8B and9A).

A lower DSB induction was detected after 125 I-PX mAb treatment, despite the higher nonspecific uptake of radioactivity, for some of the tumors, compared to anti-CEA 125 I-mAb. These data indicate that the DSBs observed after treatment with anti-CEA 125 I-mAb were not due to nonspecific irradiation. Indeed, we analyzed 125 I-PX mAb-treated tumors showing high tumor uptake to be able to exclude the possibility that DNA DSBs measured in the center of the tumor were caused by nonspecific irradiation of 125 I (mainly soft X-rays and electrons) from the tumor periphery to the center. We could also exclude any contribution from blood circulating 125 I-mAbs in mouse. As the major contribution to the absorbed dose from 125 I is due to Auger electrons, radioactivity distribution may be considered to be indicative of the distribution of the absorbed dose. The reason for the observed higher uptake in some areas of tumor of 125 I-PX mAb-treated mice compared with those treated with 125 Ianti-CEA mAb (especially the center) is unclear.

It could be that while the nonspecific antibody is able to freely diffuse to the center of the tumor, the CEA binding antibody binds to receptors, and the first of those encountered would be in the periphery. Unless these receptors in the periphery become saturated, substantial penetration of further unbound antibody molecules would be prevented. Notwithstanding this effect, whatever the distribution of radioactivity, we have previously shown that 125 I-PX mAb does not have a therapeutic effect in vivo [START_REF] Santoro | Noninternalizing monoclonal antibodies are suitable candidates for 125 I radioimmunotherapy of small-volume peritoneal carcinomatosis[END_REF]. Accordingly, our results indicate that DNA DSB formation did not correlate with FIG. 9. Radioactivity distribution and 53BP1 induction do not overlap in A-431 CEA tumor cell xenografts treated with 125 Ianti-CEA mAbs. Digital autoradiography (inset on the bottom left side of each panel) and immunohistochemical detection of 53BP1 in tumor sections from mice xenografted intraperitoneally with A-431 CEA cells and treated 24 h before tissue collection with a single intraperitoneal injection of 37 MBq of (A) 125 I-anti-CEA mAb, (B) 125 I-anti-PX mAb, or (C) injected with NaCl. The distribution of 53BP1 foci was determined in 100 cells of relevant areas of tumors. Between 6 and 7 areas of tumors in different mice were selected. To see this illustration in color, the reader is referred to the web version of this article at www .liebertpub.com/ars the absorbed dose and that DNA damage occurred to a similar extent both within and beyond the range of the 125 I Auger electrons. In a similar approach using 212 Pb-mAbs for treating A-431 CEA tumor xenografts, we also observed that distribution of DNA damage and induction of abnormal mitosis do not correlate with tumor absorbed dose, a phenomenon not observed with 212 Pb-PX nonspecific mAb (data not shown). These data support the involvement, in vivo, of nontargeted effects during 125 I-mAb RIT, but this needs to be confirmed in further experiments.

One of the issues raised by our study is related to radiation protection since nonirradiated tissues can show DNA damage and subsequent possible cell death or cell transformation. This may have implications for nuclear medicine where about one atom in two ( 99m Tc, 123 I, 125 I, 201 Tl, 51 Cr, 67 Ga, etc.) emits Auger electrons associated with other particles. It was generally considered that Auger binding DNA, or localized in the cell nucleus, produces high LET radiation. However, there is no guidance on how to calculate the equivalent dose for these radionuclides. It has been proposed by the American Association of Physicists in Medicine that the component of dose from the Auger electrons for radionuclides bound to DNA should be given a preliminary radiation weighting factor of 10 for deterministic effects and 20 for stochastic effects for nuclear localization while ICRP has recommended considering each situation (isotope and localization) separately [START_REF] Humm | Dosimetry of Augerelectron-emitting radionuclides: Report no. 3 of AAPM Nuclear Medicine Task Group[END_REF][START_REF] Icrp | The 2007 Recommendations of the International Commission on Radiological Protection[END_REF]. The dose equivalent calculated with these weighting factors must be modulated by experimentally determined subcellular distributions of the Auger electron emitters. Our study indicates that the nontargeted effect of Auger electron emitters bound to the cell surface has to be considered.

In conclusion, we have shown that Auger electron emitters, such as 125 I, conjugated to mAbs directed against the cancer cell surface can be cytotoxic by a cell oxidative, stress-FIG. 10. Schematic of the mechanisms involved in RIT using 125 I-mAbs. 125 I-mAbs targeting the cell membrane deliver high and localized energy at the cell surface. ASMase is activated by ROS produced by radiation energy deposition. ASMase catalyzes sphingomyelin hydrolysis to ceramide and phosphorylcholine. Ceramide modifies the cell membrane biophysical properties and participates in the coalescence of sphingolipid-and cholesterol-enriched domains (called lipid rafts) into large ceramide-enriched platforms, leading to cell membrane receptor reorganization and activation of the ERK1/2, AKT, p38/JNK signaling pathways, and their downstream targets. Activation of phospholipase C-c and of Ca 2+ -dependent PYK-2 and paxillin is also observed. Lipid raft-dependent radiation-induced nontargeted effects, including cytotoxicity and cH2AX formation, are observed in neighboring cells, whatever the localization of 125 I (cell surface or nucleus) and independently of the decay number (1047 Bq$s for 125 I-mAbs and 441 Bq$s for 125 I-UdR). Nontargeted effects are strongly reduced by filipin or MBCD. 125 I-UdR, 5-[(125)I] iodo-2¢-deoxyuridine; PLC-c, phospholipase C-c; PYK-2, proline-rich tyrosine kinase 2; ROS, reactive oxygen species. To see this illustration in color, the reader is referred to the web version of this article at www .liebertpub.com/ars mediated nontargeted mechanism involving lipid rafts. This nontargeted effect is comparable to that induced by 125 I-UdR concentrated in the nucleus of tumor cells. Compared with 125 I-UdR, lower targeted cytotoxicity is expected when using 125 I-mAbs, but the induction of nontargeted effects could compensate for the inferior efficacy of non-nuclear targeting. Moreover, 125 I-mAbs should also be less toxic toward proliferating normal tissue, thus allowing an increase in the injected activities.

These findings are particularly relevant for targeted therapy in which vectors cannot gain access to every tumor cell. Their extrapolation to targeted alpha radiotherapy, now routinely used in clinical radionuclide therapy, should be evaluated.

Materials and Methods

Cell lines

A-431 CEA and SK-OV-3 CEA cells were obtained by transfecting A-431 vulvar squamous and SKOV-3 ovarian carcinoma cells (from ATCC, Molsheim, France), respectively, using vectors encoding the CEA. p53 +/+ HCT116 colon adenocarcinoma cells were obtained from ATCC. The p53 -/-HCT116 cell line was a kind gift from Professor Bert Vogelstein. HCT116 cells were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum (FCS), 2 mM l-glutamine, and antibiotics (0.1 U/ml penicillin and 100 lg/ml streptomycin) at 37°C in a humidified atmosphere with 5% CO 2 . A-431 CEA and SKOV-3 CEA cells were grown as described [START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF]. All cell culture reagents were from Life Technologies (Thermo Fisher Scientific, Waltham, MA).

Radiolabeled vectors for cell targeting

To target the cytoplasm, 125 I was coupled to the internalizing mouse m225 mAb. 125 I-labeled m225 mAb is internalized within the cytoplasm after binding to its receptor (HER1). For cell surface localization, 125 I was coupled to the noninternalizing mouse IgG1k mAb 35A7. For the CEA Gold 2 epitope (50), 35A7 is specific. The nonspecific PX antibody was used as a control (irrelevant antibody). PX is an IgG1 mAb purified from mouse myeloma P3-X63 cells [START_REF] Kohler | Fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines[END_REF]. The three mAbs were obtained from mouse hybridoma ascites fluids by ammonium sulfate precipitation followed by ion exchange chromatography on DE52 cellulose (Whatman, Balston, United Kingdom).

Antibody radiolabeling was performed according to the IODO-GEN (1,3,4,6-tetrachloro-3a, 6a-diphenylglycoluryl) (Sigma-Aldrich, St. Louis, MO) method [START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF]. 125 I-mAbspecific activity was 370 MBq/mg of protein, and immunoreactivity was 60%-95%. All experiments used 125 I-mAb activities £4 MBq/ml. 125 I was targeted to the nucleus using the thymidine base analogue 125 I-UdR at a specific activity of 81.4 TBq/mmol. 125 I-UdR was purchased from PerkinElmer (Waltham, MA).

RIT, medium transfer, and clonogenic survival of irradiated and nonirradiated cells p53 +/+ or p53 -/-HCT116, SK-OV-3 CEA , and A-431 CEA cells were cultured in 6-cm Petri dishes (100-500 cells/dish) with 3 ml of medium. The following day, cells were incubated with 125 I-mAbs at activities £4 MBq/ml for 48 h (donor cells). For targeting of the nucleus, due to the higher toxicity of 125 I-UdR, between 100 and 1200 p53 +/+ HCT116 cells were seeded in 6-cm Petri dishes containing 3 ml of medium and exposed, the following day, to 125 I-UdR activities of £20 kBq/ml for 48 h. Culture medium was then removed, and cells were washed twice with 4 ml phosphate buffered saline (PBS). For clonogenic assays, performed as described in [START_REF] Pouget | Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons[END_REF], fresh medium (3 ml) was added and donor cells were cultured for 12 days. For investigating the nontargeted response, fresh medium (3 ml) was added to donor cells for 2 h and then transferred to dishes in which cells had been seeded the day before (recipient cells: 100-500 cells/6 cm dish). The transferred medium was checked for radioactivity (values were generally very low, comprising between 0.08% and 0.10% of the activity incubated with donor cells). Donor and recipient cells were then grown for 12 days and colonies stained with crystal violet (2.5 g/l in 45:5 30% [v/v] methanol/paraformaldehyde). Colonies containing 50 or more cells were scored, and the surviving fraction was calculated. The toxicity of unlabeled mAbs was assessed using the same experimental conditions.

Drug treatment

To determine the role of mAb internalization in the bystander response, p53 +/+ HCT116 cells were exposed for 2 days to 125 I-mAbs of activity concentration £4 MBq/ml in the presence of 0.02% (w/v) sodium azide, a drug that blocks mAb transport into the cytoplasm.

The role of cell membrane lipid raft formation in 125 I-mAb-induced bystander effects was investigated by coincubating p53 +/+ HCT116 donor cells with 125 I-mAbs and 4 mM MBCD (Sigma-Aldrich) for 6 h or with 2.5 lg/ml filipin (Sigma-Aldrich) overnight.

The potential role of oxidative stress in the induction of bystander effects was assessed by coincubating p53 +/+ HCT116 donor cells with 125 I-mAbs and 50 lM NAC (Sigma-Aldrich) (for 2 days) or 0.5% DMSO (Sigma-Aldrich) (overnight).

Immunofluorescence analysis of mAb localization, lipid rafts, and gH2AX foci For immunofluorescence studies, p53 +/+ HCT116 cells were plated on 12-mm glass coverslips in culture dishes. They were fixed in 3.7% (v/v) formaldehyde and then permeabilized in 0.1% (v/v) Triton X-100 for 15 min followed by incubation with 1% (v/v) PBS/bovine serum albumin (BSA) for 1 h. To confirm the mAb localization, an Alexa-488-conjugated anti-mouse secondary mAb was used at a dilution of 1:200 (Invitrogen, Saint Aubin, France). F-actin was stained with conjugated phalloidin (1:5000; Sigma-Aldrich), and nuclei with 0.1 lg/ml Hoechst (Sigma-Aldrich). To visualize lipid rafts, p53 +/+ HCT116 cells were incubated with Alexa-488-conjugated cholera toxin (Sigma-Aldrich) at a dilution of 1:1000. cH2AX foci were quantified in p53 +/+ HCT116 cells after exposure to 125 I-mAbs for 2 days according to previously described methodology [START_REF] Piron | DNA damage-centered signaling pathways are effectively activated during low dose-rate Auger radioimmunotherapy[END_REF]. For immunofluorescent colocalization of ceramide and apoptosis marker, cells were incubated with anticeramide 15B4 antibody at dilution 1:10 (Alexis Biochemicals, Heidelberg, Germany) for 1 h at 37°C followed by three PBS washing steps and incubation with PE-conjugated goat antimouse IgM at a dilution of 1:200 ( Jackson Immuno Research, Baltimore, PA) and annexin V conjugated with FITC dilution 1:100 (Beckman Coulter, Marseille, France). Coverslips were then mounted in Mowiol Ò (Sigma-Aldrich), and images recorded using a 63 • or 100 • NA objective and a Leica (Leica Microsystems, Wetzlar, Germany) inverted microscope.

Proteome array, subcellular fractionation, and Western blotting

The Human Phospho-Kinase array (Proteome Profiler Array; R&D Systems, Minneapolis, MN) was used to detect the relative phosphorylation levels of 46 kinases in p53 +/+ HCT116 cellular extracts according to the manufacturer's protocol. To confirm the localization of the targeting mAbs, a subcellular protein fractionation kit (Thermo Scientific) was used according to the manufacturer's protocol. For Western blotting, membrane and cytosolic protein fractions and total lysates (40 lg of each) from p53 +/+ HCT116 cells and A-431 CEA were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Uppsala, Sweden). Membranes were incubated with the 35A7 (anti-CEA), m225 (anti-EGFR), sodium/potassium ATPase (1:1000; Abcam, Cambridge, United Kingdom), and anti-tubulin (1:10,000; Abcam) primary antibodies that were then detected with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit (Sigma-Aldrich) secondary antibodies. p-ERK, p-AKT, and p-p38 were detected by primary anti-p-ERK, anti-p-AKT, and anti-p-p38 antibodies (Cell Signaling Technology, Danvers, MA) followed by incubation with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit (Sigma-Aldrich) secondary antibodies. Proteins were detected by enhanced chemoluminescence (ECL) using the ECL detection system from Amersham Biosciences (Saclay, France).

Ceramide measurement by flow cytometric analysis

Ceramide levels present in the cell membrane of p53 +/+ HCT116 cells were assessed by flow cytometry. Cells were treated with unlabeled mAbs (20 lg/ml) or 125 I-mAbs (4 MBq/ml) at 37°C for 1 h. After three washes, cells were fixed in 3.7% (v/v) paraformaldehyde for 15 min. Then, cells were washed three times in PBS buffer and incubated with the anti-ceramide 15B4 mAb at a dilution of 1:50 (Alexis Biochemicals) at 37°C for 1 h. After three washes in PBS-2% FCS, cells were incubated with PE-conjugated goat antimouse IgM at a dilution of 1:100 ( Jackson ImmunoResearch) in the dark for 1 h. Cells were then washed three times and suspended in PBS for analysis using a Muse flow cytometer (Merck Millipore, Fullerton, CA). The y axis corresponds to the total number of analyzed cells (arbitrary units), while the x axis represents the fluorescence signal intensity (PEconjugated goat mAb anti-ceramide 15B4 mAb). The numbers provided in the red boxes correspond to the proportion of ceramide-positive cells within the total population of cells compared with the nontreated control.

Inhibitors of MAPK pathways and chelation of extracellular Ca 2+

To confirm the involvement of MAPK proteins, including AKT, ERK1/2, p38, and JNK on final survival outcome, HCT116 donor cells were incubated for 48 h with 125 I-anti-CEA mAb in the presence of 1 lM of PI3K inhibitor (ZSTK474), and indirectly of downstream AKT signaling pathway, 1 lM of MEK1/2 inhibitor (U0126), and indirectly of downstream ERK1/2 signaling pathway, 10 lM of SP600125 ( JNK inhibitor), 10 lM of SB203580 (p38 inhibitor), or 5 mM EGTA (to chelate extracellular Ca 2+ ions). Donor cells were preincubated with inhibitors 1 h before addition of 125 I-mAbs. After 48 h, medium was removed and standard medium transfer protocol was followed as previously described. Clonogenic survival of both donor and recipient cells was measured. The efficacy of ZSTK474 and of U0126 in inhibiting AKT and ERK1/2 was confirmed by Western blotting (Supplementary Fig. S3B, right panel).

Measurement of ASMase activity

ASMase activity in whole cell lysates of p53 +/+ HCT116 cells exposed to 125 I-mAbs (0-4 MBq/ml) for 2 days was measured using the Amplex Red Sphingomyelinase Assay Kit (Molecular Probes, Saint Aubin, France). The ASMase activity of experimental samples and positive control was calculated as a percentage of the ASMase activity of the negative control.

Measurement of S1P concentration

The level of S1P was determined in p53 +/+ HCT116 cells exposed to 125 I-mAbs (4 MBq/ml) for 2 days using the Sphingosine 1 Phosphate Assay Kit according to the manufacturer's protocol (Echelon Biosciences, Salt Lake City, UT).

Animal model

Swiss nude mice (7-week-old females) from Charles River were acclimated for 1 week before experimental use. They were housed at 22°C and 55% humidity with a light-dark cycle of 12 h. Food and water were available ad libitum. Body weight was determined weekly, and the mice were clinically examined throughout the study. All animal experiments were performed in compliance with the guidelines of the French government and the INSERM standards for experimental animal studies (agreement B34-172-27).

In vivo experiments

Swiss nude mice (7-week-old females) from Charles River were xenografted intraperitoneally (i.p.) with 1.5 • 10 6 A-431 CEA cells suspended in 0.3 ml PBS. The A-431 CEA cell line was chosen for in vivo 125 I experiments because it expresses high levels of CEA receptors (compared with HCT116 cells), which enables treatment with a tumoricidal dose.

RIT experiments with or without MBCD. For these experiments, 32 mice were divided in four groups (n = 8/each) that were treated as follows: (i) two i.p. injections of NaCl at day 8 and 11 postgraft, (ii) two i.p. injections of anti-CEA mAb (100 lg) at day 8 and 11, (iii) two i.p. injections of 37 MBq of 125 I-anti-CEA mAb at day 8 and 11, (iv) daily i.p. injections of 300 mg/kg MBCD from day 6 to 15 postgraft, and (v) daily i.p. injections of MBCD from day 6 to 15 and two injections of 37 MBq of 125 I-anti-CEA mAb at day 8 and 11 postgraft. At day 30 postgraft, mice were sacrificed, tumors collected, and the total tumor mass was determined.

For this purpose, length, width, and depth of each tumor nodule was measured and used for calculating the nodule volume and a density of 1.05 g/cm 3 was used to determine each nodule mass. The average total tumor mass for each mouse was calculated by summing up the mass value of each nodule.

Digital autoradiography and immunohistochemical detection of 53BP1. Ten days postgraft, eight mice were injected i.p. with 37 MBq of 125 I-PX or of 125 I-anti-CEA mAbs. Four and 24 h later, mice were anesthetized, bled, and dissected. Tumors were collected and frozen after inclusion in optimal cutting temperature embedding matrix. Two consecutive 10 lm-thick frozen sections were analyzed by digital autoradiography (DAR) or by immunohistochemical analysis. DAR acquisition was performed using a Beta Imager 2000 instrument (Biospace, Paris, France) over 24 h and analyzed using M3vision software to determine the radioactivity distribution. Immunofluorescence analysis of 53BP1 expression was performed to assess DNA DSB formation. Frozen sections were rehydrated in PBS for 10 min, fixed in 3.7% (v/v) formaldehyde, and then permeabilized in 0.5% (v/v) Triton X-100 for 15 min followed by incubation with 10% (v/v) PBS/BSA for 1 h. Sections were incubated at 37°C with an anti-53BP1 mAb (1:400) for 1 h followed by incubation with an Alexa-594-conjugated anti-rabbit secondary antibody (1:500; Invitrogen). Nuclei were stained with 0.1 lg/ml Hoechst. The distribution of 53BP1 foci was determined in 100 cells in two relevant areas of representative tumors. All animal experiments were performed in compliance with the guidelines of the French government and the INSERM standards for experimental animal studies (agreement B34-172-27). They were approved by the local ethic committee of ''Institut de Recherche en Cance ´rologie de Montpellier'' (IRCM/INSERM) and by the Ethic Committee of Languedoc Roussillon (CEEA LR France No. 36) for animal experiments under the number 1056.

Statistical analysis

All in vitro data were obtained from four independent experiments in triplicate. Data were described using mean and standard deviation. Analysis of variance was used for multigroup comparisons (significance level set at a = 0.05). Pairwise comparisons were performed using the Student's t test, and to control for multiple testing, the significance threshold was set at 0.013 in these comparisons.

Data were analyzed using Stata software, version 13 (StataCorp, College Station, TX).

FIG. 1 .

 1 FIG. 1. Clonogenic survival. Clonogenic survival was assessed in donor and recipients cells 12 days following either exposure to activities of 125 I-mAbs from 0 to 4 MBq/ml for 2 days (donor cells) or incubation in culture medium in which donor cells had been cultured for 2 h (recipient cells). Donor cells were treated with noninternalizing 125 I-anti-CEA, internalizing 125 I-anti-HER1, or nontargeting 125 I-PX mAbs. Clonogenic survival was measured in (A) p53 +/+ HCT116, (B) p53 -/- HCT116, and (C) A-431 CEA cells. Results are the mean -SD of four experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 compared with 125 I-PX mAbtreated cells. CEA, carcinoembryonic antigen; HER1, Human Epidermal Receptor type 1; 125 I, iodine 125; mAbs, monoclonal antibodies; SD, standard deviation.

HCT116 donor cells exposed to 4

 4 MBq/ml 125 I-anti-CEA, 125 I-anti-HER1, 125 I-PX mAbs, or 20 kBq/ml 125 I-UdR and of the corresponding recipient cells. Cells were incubated with 125 I-vectors for 2 days. Donor cells were exposed to 125 I-mAbs in the absence [Filipin (-)] or presence [Filipin (+)] of 2.5 lg/ml of Filipin, a lipid raft disruptor, for 6 h. Donor cells were in reality both donor and recipient cells. Therefore, the specific contribution of targeted effects to donor cell killing was calculated by subtracting the percentage of cell death in recipient cells from the percentage of cell death in donor cells. Due to the long incubation with 125 I-vectors, the contribution of nontargeted effect in donor cells might be underestimated. Italic numbers show cell death percentages compared with untreated cells. CEA, carcinoembryonic antigen; HER1, Human Epidermal Receptor type 1; 125 I, iodine 125; 125 I-UdR, 5-[(125)I]iodo-2¢-deoxyuridine; mAbs, monoclonal antibodies. 4 PAILLAS ET AL. were induced. Activated downstream AKT proteins included p70S6 kinase (p70S6K), glycogen synthase kinase 3 (GSK3), and endothelial nitric oxide synthase (e-NOS) (61). Signal transducer and activator of transcription (STAT) 1 and 4, 90 kDa ribosomal S6 kinase (RSK), mitogen-and stressactivated protein kinase (MSK), and CREB proteins, which are downstream targets of ERK1/2 signaling, were also activated, as well as c-JUN, which is downstream of p38 and JNK (for review (61)). Phosphorylated proteins involved in Ca 2+ fluxes, such as phospholipase C-c (PLC-c), proline-rich tyrosine kinase 2 (PYK-2), and paxillin, were strongly activated by exposure to anti-CEA 125 I-mAb. Phosphorylation of AKT, p38, JNK, CREB, GSK3, STAT 1 and 4, e-NOS, MSK, PLC-c, PYK-2, and paxillin was abolished, whereas phosphorylation of ERK1/2, c-JUN, p53, RSK, SAT 2, and p70S6K was diminished in cells coincubated with filipin (Fig. 5), confirming that these proteins are downstream ef-fectors of lipid raft formation and, thus, are likely to be involved in 125 I-mAb nontargeted effects. Moreover, the use of different pharmacological inhibitors of MEK (U0126) and subsequently of downstream ERK1/2 signaling pathway, PI3K

FIG. 2 .

 2 FIG. 2. Gamma-H2AX foci detection and optional role of 125 I-mAb internalization for nontargeted cytotoxicity. (A) Detection of gamma-H2AX foci in p53 +/+ HCT116 donor (top panels) and recipient cells (bottom panels) by immunofluorescence assay. (B) The number of foci per cell was counted in 100 cells. Antibody localization was assessed in p53 +/+ HCT116 and A-431 CEA cells by (C) subcellular fractionation and (D) immunofluorescence. F-actin was stained with conjugated phalloidin (Red) to visualize cytoplasm, nuclei with Hoechst (Blue), and mAbs with Alexa-488-conjugated anti-mouse secondary mAbs. (E) Clonogenic survival was assessed in p53 +/+ HCT116 donor cells coincubated with 0.02% sodium azide (a drug blocking antigen internalization) and a range of activities of 125 I-mAbs (noninternalizing anti-CEA, internalizing anti-HER1, or nontargeting PX mAbs). Clonogenic survival of recipient cells was assessed after incubation with conditioned medium from donor cells exposed to 125 I-mAbs in the presence of sodium azide. Results are the mean -SD of three experiments performed in triplicate. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to NT cells (B) or to the 125 I-PX mAb-treated cells (E). Mb, membrane; Cyto, cytoplasm. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

  FIG. 3. Lipid rafts are formed upon exposure to 125 I-mAbs via ASMase-induced ceramide formation. (A) Lipid rafts were detected by immunofluorescence analysis using Alexa-488-conjugated cholera toxin (a marker of lipid raft) in untreated (NT) and treated p53 +/+ HCT116 cells (noninternalizing 125 I-anti-CEA, internalizing 125 I-anti-HER1, nontargeting 125 I-PX mAbs). (B) Ceramide was measured in p53 +/+ HCT116 cells by flow cytometry analysis after treatment with unlabeled mAbs (top panels), or with 125 I-mAbs (bottom panels). Ctrl+, positive control (100 ll of 0.4U/ml Bacillus cereus SMase and 10 ll sphingomyelin). (C) Colocalization of ceramide and Annexin V in apoptotic cells was determined by immunofluorescence, in untreated (NT) and 125 Ianti-CEA-treated p53 +/+ HCT116 cells. (D) ASMase activity was determined in whole p53 +/+ HCT116 cell lysates using the Amplex Red Sphingomyelinase Fluorimetry Kit after incubation with unlabeled or 125 I-mAbs. Ctrl+, positive control (100 ll of 0.4 U/ml B. cereus SMase and 10 ll sphingomyelin); Ctrl-(Amplex Red working solution without sphingomyelin). (E) S1P concentration was determined in whole p53 +/+ HCT116 cells lysates using the Sphingosine-1-Phosphate Assay Kit after incubation with unlabeled or 125 I-mAbs or in nontreated cells (NT). Results are the mean -SD of four experiments performed in triplicate. **p < 0.01 and ***p < 0.001 compared to cells treated with unlabeled mAbs. ASMase, acid sphingomyelinase; S1P, sphingosine-1phosphate. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

  I-UdR (nuclear localization) was more cytotoxic than anti-CEA (cell surface localization) or anti-HER1 (cytoplasm localization) 125 I-mAbs. Conversely, in recipient cells, cell survival was similar and independent of 125 I localization, indicating that 125 I at the cell membrane is as efficient as nuclear 125 I in killing bystander cells. The role of lipid rafts in 125 I-mAb cytotoxicity in vivo Mice bearing intraperitoneal tumor xenografts were treated with NaCl, anti-CEA mAb (100 lg/injection at day 8 and 11), 125 I-anti-CEA mAb (37 MBq/injection at day 8 and 11), or MBCD (300 mg/kg; daily, from day 6 to 15), or with both 125 Ianti-CEA mAb and MBCD. At day 30, tumor mass was significantly reduced in the group treated with 125 I-anti-CEA mAb (RIT) compared with the NaCl group ( p = 0.0227). No difference was observed between the groups treated with MBCD, NaCl ( p = 0.9349), or anti-CEA mAb ( p = 0.9563). The latter result is in agreement with a previously published study indicating that anti-CEA mAbs have no effect on mouse tumor growth

FIG. 5 .

 5 FIG. 5. Signaling pathways activated in 125 I-mAbtreated p53 +/+ HCT116 cells and role of oxidative stress in the nontargeted response. (A) Signaling pathways activated after incubation of p53 +/ + HCT116 donor cells with unlabeled anti-CEA, anti-CEA 125 I-mAbs, or anti-CEA 125 I-mAbs and filipin. (B) Signaling pathways activated after incubation of p53 +/+ HCT116 donor cells with unlabeled anti-HER1, anti-HER1 125 I-mAbs, or anti-HER1 125 I-mAbs and filipin. The Human Phospho-Kinase array (Proteome Profiler Array; R&D Systems, Minneapolis, MN) was used to detect the phosphorylation level of 46 kinases. For each kinase, the Image J software was used to determine pixel number. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 7 .

 7 FIG. 7. Oxidative stress is involved in direct and nontargeted cytotoxicity: Effect of NAC on RIT-induced effects. p53 +/+ HCT116 donor cells were coincubated for 2 days with 50lM NAC or 0.5% DMSO and 125 I-anti-CEA (A, C, respectively) or 125 I-anti-HER1 (B, D, respectively) mAbs. Clonogenic survival of the donor cells and of the corresponding recipient cells was measured. Results are the mean -SD of four experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 compared to cells treated with 125 I-mAbs. DMSO, dimethyl sulfoxide; NAC, N-acetyl-L-cysteine; RIT, radioimmunotherapy.

FIG. 8 .

 8 FIG. 8. In vivo therapeutic efficacy of 125 I-mAbs with or without MBCD. (A) Mice bearing intraperitoneal A-431 CEA tumor xenografts were treated with NaCl, unlabeled anti-CEA mAb (100 lg/injection at day 8 and 11), or with 125 I-anti-CEA mAb (RIT) at day 8 and 11 postgraft, daily with MBCD from day 6 to 15 postgraft (MBCD), or with 125 I-anti-CEA mAb (day 8 and 11 postgraft) and MBCD (daily from day 6 to 15 postgraft) (RIT+MBCD) (n = 8 mice/ group). Tumors were collected at days 30 postgraft, and the total tumor mass was determined. (B) The level of 53BP1 was determined by immunofluorescence in cryocut slices of tumors of mice treated with 37 MBq of 125 I-anti-CEA mAbs, 125 I-PX mAbs (RIT), or with NaCl. The level was determined in 100 cells of a selected area and was correlated with the level of radioactivity (cpm/mm 2 ) of the same area. Between 6 and 7 areas of tumors in different mice were selected. In A, results are the mean-SD of eight values. *p < 0.05 compared to mice treated with NaCl. 53BP1, p53binding protein 1; MBCD, methyl-b-cyclodextrin.

Table 1

 1

	. 125 I Subcellular Localization and Nontargeted Effects	p53 +/+ HCT116 cells survival (%) at 4 MBq/ml	Contribution to HCT116 donor	Donor cells Recipient cells cell killing (%) at 4 MBq/ml	Filipin (-) Filipin (+) Filipin (-) Filipin (+) Targeted cytotoxicity Nontargeted cytotoxicity	92.9 -2.2 (7.1) 98.3 -1.5 (1.7) <5.4 >1.7	58 -2.5 (42) 84.4 -2.6 71.2 -7 (28.8) 92.8 -2.3 (7.2) <13.2 >28.8	57.8 -7 (42.2) 81.0 -1.6 (19) 70.6 -8.4 (29.4) 94.2 -2.4 (5.8) <12.8 >29.4	22.1 -1.3 (77.9) 72.8 -2.8 (27.2) <50.7 >27.2
			Total No. of decays	(Bq$s/cell) at 4 MBq/ml	Nucleus Membrane+cytoplasm	125 I-PX 49.8 -2 350.4 125 I-anti-CEA 45 -1.4 1047 125 I-anti-HER1 91 125 I-UdR -1.7 14267 441 -27.4 14 -21 -76 -317 -1.6

Total number of decays occurring in the nucleus and in the extranuclear subcompartments and clonogenic survival (% relative to untreated cells) of p53 +/+

PAILLAS ET AL.

PAILLAS ET AL.

Acknowledgments

This work was supported by Action Nu1.1 of Plan Cancer 2009-2013 (ASC 13038FSA), by the French National Research Agency under the program ''Investissements d'avenir'' Grant agreement LabEx MAbImprove: ANR-10-LABX-53, Grant INCa-DGOS-Inserm 6045, and the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement No. 608765 and Marie Curie WHRI Academy COFUND programme.

Author Disclosure Statement

No competing financial interests exist.