Charly Hélaine

Aurélie E Ferré
email: ferre@cyceron.fr

Marine M Leblond
email: leblond.marine.m@gmail.com

Elodie A Pérès

Myriam Bernaudin
email: bernaudin@cyceron.fr

Samuel Valable
email: valable@cyceron.fr

Edwige Petit
email: epetit@cyceron.fr

Charly Helaine
email: helaine@cyceron.fr

Angiopoietin-2 Combined with Radiochemotherapy Impedes Glioblastoma Recurrence by Acting in an Autocrine and Paracrine Manner: A Preclinical Study

Keywords: glioblastoma, angiopoietin-2, vascularization, inflammation, microenvironment, radiotherapy, chemotherapy, radiochemotherapy

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

Introduction

Glioblastoma (GB) is the most common and aggressive primary brain tumor in adults [START_REF] Ostrom | The epidemiology of glioma in adults: A "state of the science[END_REF][START_REF] Louis | World Health Organization Classification of Tumors of the Central Nervous System: A summary[END_REF]. Standard treatment for newly diagnosed patients includes maximal safe resection followed by radiochemotherapy (RCT). RCT consists of fractionated radiotherapy (RT) combined with concomitant Cancers 2020, 12, 3585 2 of 18 and adjuvant cure of chemotherapy (CT) based on temozolomide (TMZ), known as the Stupp's regimen [START_REF] Stupp | European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups, National Cancer Institute of Canada Clinical Trials Group: Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[END_REF]. Despite this intensive multimodal treatment, GB patients constantly develop tumor recurrence, and their median survival rarely exceeds 15 months [START_REF] Stupp | European Organisation for Research and Treatment of Cancer Brain Tumour and Radiation Oncology Groups, National Cancer Institute of Canada Clinical Trials Group: Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial[END_REF]. The limited therapeutic benefit in GB patients can be attributed to inherent tumor cell resistance but also to the tumor microenvironment, such as its vascularization [START_REF] Osuka | Overcoming therapeutic resistance in glioblastoma: The way forward[END_REF].

GB progression is highly dependent on its vascularization, and the intense and aberrant tumor angiogenesis correlates with poor prognosis in patients [START_REF] Jain | Angiogenesis in brain tumours[END_REF]. The major role of the tumor microenvironment in tumor progression and treatment efficacy has led to the development of novel therapeutic options, such as antiangiogenic therapies. Due to its central role in angiogenesis, inhibition of vascular endothelial growth factor (VEGF) with bevacizumab, a humanized monoclonal antibody, was approved for recurrent GB in 2009 and recently as first-line treatment combined with RCT for newly diagnosed GB. However, while initial results with angiogenesis inhibitors were promising, they were subsequently disappointing [START_REF] Gilbert | A Randomized Trial of Bevacizumab for Newly Diagnosed Glioblastoma[END_REF][START_REF] Chinot | Bevacizumab plus Radiotherapy-Temozolomide for Newly Diagnosed Glioblastoma[END_REF]. Indeed, these tumors presented transient response followed by a relapse to anti-VEGF treatment [START_REF] Bergers | Modes of resistance to anti-angiogenic therapy[END_REF]. This is mainly attributable to the overexpression of alternative proangiogenic factors that contribute to an adaptive resistance, as shown for angiopoietin-2 (Ang2) in GB [START_REF] Labussière | Angiopoietin-2 May Be Involved in the Resistance to Bevacizumab in Recurrent Glioblastoma[END_REF][START_REF] Koga | Expression of angiopoietin-2 in human glioma cells and its role for angiogenesis[END_REF][START_REF] Scholz | Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma[END_REF][START_REF] Reiss | The Role of Angiopoietins During Angiogenesis in Gliomas[END_REF][START_REF] Stratmann | Cell Type-Specific Expression of Angiopoietin-1 and Angiopoietin-2 Suggests a Role in Glioblastoma Angiogenesis[END_REF].

Ang2 belongs to the angiopoietin (Ang) family consisting of two major members-Ang1 and Ang2-which act through a common tyrosine kinase receptor Tie2 but with antagonistic activities. Ang2 was originally thought to block the stabilizing effects of Ang1 on the vasculature, thereby facilitating the angiogenic response induced by VEGF or leading to vessel regression in the absence of VEGF [START_REF] Mandriota | Regulation of angiopoietin-2 mRNA levels in bovine microvascular endothelial cells by cytokines and hypoxia[END_REF]. However, conflicting results have been reported in the literature regarding the role of Ang2 in tumor angiogenesis and growth [START_REF] Maisonpierre | Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis[END_REF]. Similar contradictory results have also been reported for GB, suggesting a pro-or antiangiogenic effect of Ang2 in tumors depending on the context [START_REF] Machein | Angiopoietin-1 Promotes Tumor Angiogenesis in a Rat Glioma Model[END_REF][START_REF] Valable | MRI assessment of hemodynamic effects of angiopoietin-2 overexpression in a brain tumor model[END_REF].

The tumor growth-supportive role of VEGF and Ang2 is not restricted to the vascular compartment and is also involved in inflammation [START_REF] Scholz | Angiopoietin-2: A multifaceted cytokine that functions in both angiogenesis and inflammation[END_REF]. During inflammation, endothelial cells are critically involved in regulating vascular permeability and inflammatory cell recruitment [START_REF] Pober | Evolving functions of endothelial cells in inflammation[END_REF]. This close relationship between angiogenesis and inflammation suggests the existence of molecules that trigger both angiogenesis and the recruitment of inflammatory cells. Evidence for the capacity of cytokines to control inflammatory cell recruitment has been previously described, notably for VEGF, which attracts bone marrow-derived circulating cells [START_REF] Zhu | The contribution of tumor-associated macrophages in glioma neo-angiogenesis and implications for anti-angiogenic strategies[END_REF]. In GB, the infiltrating myeloid cells, including tumor-associated macrophages (TAMs), is the major player of the innate immune system and can represent up to 30% of the tumor mass [START_REF] Rossi | Immunohistological study of mononuclear cell infiltrate in malignant gliomas[END_REF]. TAMs have recently been described as potential mediators of resistance to anti-VEGF therapy in these brain tumors as the degree of TAM infiltration is inversely correlated with survival among GB patients [START_REF] Lu-Emerson | Increase in tumor-associated macrophages after antiangiogenic therapy is associated with poor survival among patients with recurrent glioblastoma[END_REF][START_REF] Cho | Increased Antiangiogenic Effect by Blocking CCL2-dependent Macrophages in a Rodent Glioblastoma Model: Correlation Study with Dynamic Susceptibility Contrast Perfusion MRI[END_REF]. Ang2, which is involved in resistance to antiangiogenic therapies in recurrent GB, might also modify the tumor immune microenvironment [START_REF] Cortes-Santiago | Soluble Tie2 overrides the heightened invasion induced by anti-angiogenesis therapies in gliomas[END_REF][START_REF] Park | Normalization of Tumor Vessels by Tie2 Activation and Ang2 Inhibition Enhances Drug Delivery and Produces a Favorable Tumor Microenvironment[END_REF]. This cytokine, derived from endothelial and tumor cells, may promote macrophage and neutrophil infiltration in a paracrine manner depending on either β1-integrin or Tie2, respectively [START_REF] Lemieux | Angiopoietins can directly activate endothelial cells and neutrophils to promote proinflammatory responses[END_REF]. In GB, Ang2 might favor leukocyte infiltration by remodeling the tumor vasculature and the blood-brain barrier integrity [START_REF] Scholz | Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma[END_REF]. In particular, Ang2 has been recognized as a mediator of the homing for a subpopulation of proangiogenic TAMs, identified as Tie2-expressing macrophages (TEMs) in human GB [START_REF] Cortes-Santiago | Soluble Tie2 overrides the heightened invasion induced by anti-angiogenesis therapies in gliomas[END_REF][START_REF] Mazzieri | Targeting the ANG2/TIE2 Axis Inhibits Tumor Growth and Metastasis by Impairing Angiogenesis and Disabling Rebounds of Proangiogenic Myeloid Cells[END_REF].

Based on the key complementary roles of Ang2 and VEGF in tumor angiogenesis, strategies targeting both angiogenesis pathways were initially introduced to overcome anti-VEGF treatment resistance. Accordingly, in a murine GB model, dual inhibition of VEGF and Ang2 decreased vessel density associated with delayed tumor growth and prolongation of animal survival [START_REF] Kloepper | Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival[END_REF][START_REF] Peterson | Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages[END_REF]. Consistent with their inflammatory-modulating function, the survival benefit of this dual therapy was also associated with TAM recruitment as well as the reprogramming of TAMs from the protumor phenotype (M2-like) toward the antitumor (M1-like) phenotype [START_REF] Peterson | Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages[END_REF]. Although this multitarget therapy delays tumor growth, it fails to achieve long-term inhibition of GL261 tumor growth. More importantly, the beneficial effect of this dual therapy may be disturbed when combined with either CT or RT [START_REF] Solecki | Differential Effects of Ang-2/VEGF-A Inhibiting Antibodies in Combination with Radio-or Chemotherapy in Glioma[END_REF]. Vascular changes and slowdown of tumor growth were observed only when the anti-Ang2/VEGF treatment was combined with TMZ. For RT, the best efficacy was obtained with anti-VEGF alone, whereas RT is known to increase Ang2 in the brain [START_REF] Deng | Distinct Expression of Various Angiogenesis Factors in Mice Brain After Whole-Brain Irradiation by X-ray[END_REF]. The discrepancy between these results might reflect the context-dependent effect of Ang2. To date, the role of Ang2 in tumor microenvironment remodeling in response to RCT treatment has not been clearly established. Because Ang2 is upregulated during GB progression, as well as in response to brain RT, we speculated that this pleiotropic cytokine might play a pivotal role in the remodeling of the inflammatory environment in response to RCT. To clarify this point, we generated Ang2-overexpressing GL261 cells and characterized tumor progression, as well as inflammation and vascularization, in response to RCT.

Results

Ang2 Overexpression in Glioblastoma Cells Combined with Radiochemotherapy Results in Improved Animal Survival

To recapitulate the high level of Ang2 observed in GB patients [START_REF] Scholz | Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma[END_REF], we generated a glioma cell line overexpressing Ang2 (GL261-Ang2) and confirmed the efficacy of the transfection at both mRNA and protein levels (Figure S1A,B). GL261-wt and GL261-Ang2 cells exhibited a similar cell cycle distribution (Figure S1C).

We next studied the influence of Ang2 overexpression on tumor growth and its response to RCT following the protocol presented in Figure 1A. Tumor growth rate was monitored using T2w magnetic resonance imaging (MRI). No difference in tumor growth was observed between untreated GL261-Ang2 and GL261-wt groups (p = 0.11) (Figure 1B,C) while the GL261-Ang2 cell line showed a slowdown in growth rate compared to GL261-wt cells in vitro (Figure S1D).

When tumors reached an equivalent volume of about 2 mm 3 (i.e., seven days after cell implantation), animals were treated three times with TMZ (10 mg/kg/day, i.p.) combined with RT (4 Gy/day) for 7, 9, and 11 days. Both groups showed a similar response to RCT with tumor regression 10 days after the last fraction of treatment (Figure 1C). As illustrated in MRI images (Figure 1B (D14 and D18)), the presence of necrosis in the core of tumors attested treatment-induced cell death. Although RCT led to tumor regression (p < 0.05), this one was transient, and tumor recurrence started 14 days after the end of treatment in the GL261-wt group. Interestingly, Ang2 overexpression potentiated the efficacy of RCT (Figure 1B and Figure S2), and this effect was maintained for at least three months post-treatment (Figure 1B,C). According to these results, there was a significant survival improvement in the treated group with the combination of Ang2 overexpression and RCT (p < 0.05). For the four groups of animals, the untreated GL261-Ang2 and GL261-wt had a median survival of 17 and 18 days, respectively (Figure 1D). RCT alone significantly improved the median survival to 35 days. Impressively, for Ang2 overexpression combined with RCT, the median survival was still not reached up to 100 days, and all mice were alive at this time point. At the end of the study (day 100), MRI analysis attested the complete tumor clearance (Figure 1B). We next studied whether the reduction of tumor growth was related to a vascular effect of Ang2 overexpression.

Ang2 Overexpression in Glioblastoma Cells Modulates the Tumor Vascular Change Induced by Radiochemotherapy

We first examined the blood vessel morphology and density in the tumors among the four groups of animals at D14 after cell implantation (i.e., three days after the last fraction of RCT). This time was chosen because it corresponds to an early post-treatment period, allowing the detection of potential early differences in the tumor microenvironment between the groups. Figure 2A depicts the presence of typical vessels in tumors derived from the four groups and in the corresponding healthy contralateral hemisphere. Without treatment, as expected, the quantitative analysis revealed that the vascular area of GL261-wt and GL261-Ang2 tumors was larger than that of the healthy tissue (dashed line) (p < 0.05) (Figure 2B). These vessels were tortuous and displayed an abnormal shape, a feature

Ang2 Overexpression in Glioblastoma Cells Modulates the Tumor Vascular Change Induced by Radiochemotherapy

We first examined the blood vessel morphology and density in the tumors among the four groups of animals at D14 after cell implantation (i.e., three days after the last fraction of RCT). This time was chosen because it corresponds to an early post-treatment period, allowing the detection of potential early differences in the tumor microenvironment between the groups. Figure 2A depicts the presence of typical vessels in tumors derived from the four groups and in the corresponding healthy contralateral hemisphere. Without treatment, as expected, the quantitative analysis revealed that the vascular area of GL261-wt and GL261-Ang2 tumors was larger than that of the healthy tissue (dashed line) (p < 0.05) (Figure 2B). These vessels were tortuous and displayed an abnormal shape, a feature of tumor vessels (Figure 2A). The quantitative analysis confirmed a larger diameter of tumor vessels (p < 0.05) compared to healthy tissue (Figure 2C). When tumors were exposed to RCT, the vascularization was differently affected in the two groups of animals. While the vascularization of the GL261-wt tumors was slightly affected by the treatment, the vascular density decreased significantly for GL261-Ang2 tumors treated with RCT (p < 0.05) (Figure 2A,B). In response to RCT, GL261-wt and GL261-Ang2 tumors displayed a vascular network similar to that of untreated GL261-wt. The vessels of the GL261-wt and GL261-Ang2 groups remained enlarged even after treatment relative to those of the healthy tissue (Figure 2C).

Cancers 2020, 12, x FOR PEER REVIEW 5 of 18 of tumor vessels (Figure 2A). The quantitative analysis confirmed a larger diameter of tumor vessels (p < 0.05) compared to healthy tissue (Figure 2C). When tumors were exposed to RCT, the vascularization was differently affected in the two groups of animals. While the vascularization of the GL261-wt tumors was slightly affected by the treatment, the vascular density decreased significantly for GL261-Ang2 tumors treated with RCT (p < 0.05) (Figure 2A,B). In response to RCT, GL261-wt and GL261-Ang2 tumors displayed a vascular network similar to that of untreated GL261wt. The vessels of the GL261-wt and GL261-Ang2 groups remained enlarged even after treatment relative to those of the healthy tissue (Figure 2C). Collectively, these results suggest that the vascularization induced during the growth of GL261-Ang2 tumors was more sensitive to RCT than the one of GL261-wt tumors. GL261-wt group, # p < 0.05 vs. respective untreated group, $ p < 0.05 vs. healthy tissue, two-way ANOVA followed by Tukey's test.

Ang2 Overexpression in Glioblastoma Cells Combined to Radiochemotherapy Favors Immune Cells Infiltration in Glioblastoma

We then evaluated if Ang2 could affect the recruitment of immune cells into GB. A similar proportion of TAMs (CD68 + ) was identified in the two untreated groups (Figure 3A). The fractionated treatment did not change the CD68 + number in GL261-wt tumors (Figure 3A). In contrast, when GL261-Ang2 tumors were exposed to RCT, the TAM density increased in tumor core at D14 (7.71 ± 0.87% vs. 5.16 ± 0.32% in GL261-wt tumors, p < 0.05) (Figure 3A). Of note, among the CD68 + cells, only few were detected in the tumor cores as Tie2 + (Figure 3B). Fourteen days after cell implantation, no difference was observed in myeloid cell population (Table S1) in the blood and in the spleen of tumor-bearing mice (Figure S3A).

For T lymphocytes (Figure 3C,D), we did not detect any significant differences in the proportion of CD4 + and CD8 + T cells between GL261-Ang2 and GL261-wt tumors. However, a decrease of these immune cells (about 50%) was observed after RCT for both animal groups (Figure 3C,D). With or without RCT treatment, the CD4 + /CD8 + ratio (around 1-1.5/3) was in favor of the CD8 + lymphocytes in the GL261-wt as well as in GL261-Ang2 tumors, whereas this ratio was opposite in the blood and Collectively, these results suggest that the vascularization induced during the growth of GL261-Ang2 tumors was more sensitive to RCT than the one of GL261-wt tumors.

Ang2 Overexpression in Glioblastoma Cells Combined to Radiochemotherapy Favors Immune Cells Infiltration in Glioblastoma

We then evaluated if Ang2 could affect the recruitment of immune cells into GB. A similar proportion of TAMs (CD68 + ) was identified in the two untreated groups (Figure 3A). The fractionated treatment did not change the CD68 + number in GL261-wt tumors (Figure 3A). In contrast, when GL261-Ang2 tumors were exposed to RCT, the TAM density increased in tumor core at D14 (7.71 ± 0.87% vs. 5.16 ± 0.32% in GL261-wt tumors, p < 0.05) (Figure 3A). Of note, among the CD68 + cells, only few were detected in the tumor cores as Tie2 + (Figure 3B). Fourteen days after cell implantation, no difference was observed in myeloid cell population (Table S1) in the blood and in the spleen of tumor-bearing mice (Figure S3A).

For T lymphocytes (Figure 3C,D), we did not detect any significant differences in the proportion of CD4 + and CD8 + T cells between GL261-Ang2 and GL261-wt tumors. However, a decrease of these immune cells (about 50%) was observed after RCT for both animal groups (Figure 3C,D). With or without RCT treatment, the CD4 + /CD8 + ratio (around 1-1.5/3) was in favor of the CD8 + lymphocytes in the GL261-wt as well as in GL261-Ang2 tumors, whereas this ratio was opposite in the blood and in the spleen of tumor-bearing mice (Figure S3B and Table S1). No difference was detected in systemic inflammation irrespective of the tumor group and exposition to treatment (Figure S3A,B), even when the spleen weight of the GL261-Ang2-bearing mice was significantly higher than GL261-wt-bearing mice (p < 0.05) (Figure S3C).

in the spleen of tumor-bearing mice (Figure S3B and Table S1). No difference was detected in systemic inflammation irrespective of the tumor group and exposition to treatment (Figure S3A,B), even when the spleen weight of the GL261-Ang2-bearing mice was significantly higher than GL261-wt-bearing mice (p < 0.05) (Figure S3C).

These data suggest that, in response to RCT, GL261-Ang2 tumors display a more inflammatory environment than GL261-wt tumors. We next carried out an immunohistological study at a later time post-treatment when the tumor was no longer detectable by MRI, i.e., 42 and 100 days after cell implantation (Figure 1B and3E). As illustrated in Figure 3E, microglia/macrophages (CD68 + ) and T cells (CD4 + and CD8 + ) were still detected in the microscopically visible residual core of the tumor (Hoechst). These data suggest that, in response to RCT, GL261-Ang2 tumors display a more inflammatory environment than GL261-wt tumors. We next carried out an immunohistological study at a later time post-treatment when the tumor was no longer detectable by MRI, i.e., 42 and 100 days after cell implantation (Figures 1B and3E). As illustrated in Figure 3E, microglia/macrophages (CD68 + ) and T cells (CD4 + and CD8 + ) were still detected in the microscopically visible residual core of the tumor (Hoechst).

Ang2 Stimulates Migration of Macrophages In Vitro

Our in vivo data suggest that Ang2 might stimulate the recruitment of myeloid cells in the murine glioblastoma model exposed to RCT. To evaluate whether Ang2 could exert a paracrine effect on macrophages, we performed in vitro migration assays with the macrophage murine RAW 264.7 cell line, known to express the two Ang2 receptors, namely, Tie2 and β1-integrin (Figure S4A,B). The migration of RAW 264.7 cells toward Ang2 was first evaluated in response to increasing concentrations of the mouse recombinant protein (rAng2) (Figure 4A). A significant effect of recombinant Ang2 appeared for concentrations greater than 400 ng/mL (p < 0.05). It was partially reversed by the soluble Tie2 receptor (FcTie2, p < 0.05) (Figure 4A), confirming the specificity of the effect of Ang2 on macrophages. We next performed similar experiments using GL261-wt or GL261-Ang2 cells exposed or not to RCT following the protocol illustrated in Figure S4C. Compared to GL261-wt, a significant increase in RAW 264.7 cell migration was detected with GL261-Ang2 cells (Figure 4B). About 1.5 times more cells migrated under these conditions (p < 0.05). However, this effect was similar when GL261-Ang2 cells were pre-exposed to RCT, despite a slight increase in migration of RAW 264.7 cells (Figure 4B). Importantly, like for the experiments with rAng2, the paracrine effect of Ang2 was reversed by the presence of the soluble receptor Tie2 (p < 0.05) (Figure 4).

These in vitro results indicate that Ang2 exerts a chemotactic effect on macrophages.

Cancers 2020, 12, x FOR PEER REVIEW 7 of 18 residual tumor area and correspond to the representative images obtained from the immunohistological study to detect immune cells (CD68 + , CD4 + , and CD8 + ). Scale bar = 100 µm.

Ang2 Stimulates Migration of Macrophages In Vitro

Our in vivo data suggest that Ang2 might stimulate the recruitment of myeloid cells in the murine glioblastoma model exposed to RCT. To evaluate whether Ang2 could exert a paracrine effect on macrophages, we performed in vitro migration assays with the macrophage murine RAW 264.7 cell line, known to express the two Ang2 receptors, namely, Tie2 and β1-integrin (Figure S4A,B). The migration of RAW 264.7 cells toward Ang2 was first evaluated in response to increasing concentrations of the mouse recombinant protein (rAng2) (Figure 4A). A significant effect of recombinant Ang2 appeared for concentrations greater than 400 ng/mL (p < 0.05). It was partially reversed by the soluble Tie2 receptor (FcTie2, p < 0.05) (Figure 4A), confirming the specificity of the effect of Ang2 on macrophages. We next performed similar experiments using GL261-wt or GL261-Ang2 cells exposed or not to RCT following the protocol illustrated in Figure S4C. Compared to GL261-wt, a significant increase in RAW 264.7 cell migration was detected with GL261-Ang2 cells (Figure 4B). About 1.5 times more cells migrated under these conditions (p < 0.05). However, this effect was similar when GL261-Ang2 cells were pre-exposed to RCT, despite a slight increase in migration of RAW 264.7 cells (Figure 4B). Importantly, like for the experiments with rAng2, the paracrine effect of Ang2 was reversed by the presence of the soluble receptor Tie2 (p < 0.05) (Figure 4).

These in vitro results indicate that Ang2 exerts a chemotactic effect on macrophages.

Ang2 Overexpression in Glioblastoma Cells Combined with Radiochemotherapy Promotes Senescence and Mitotic Death of Glioblastoma Cells

We next investigated the nature of the sensitization to RCT induced by Ang2 overexpression in GB cells by focusing on glioma cell death. As depicted in Figure 5A, at D14 after cell implantation Cancers 2020, 12, 3585 8 of 18 (i.e., three days after the last fraction of treatment), RCT induced cell nuclei changes in GL261-Ang2 as well as in GL261-wt tumors compared to the corresponding untreated tumors. These changes included enlarged nuclei with prominent foci of heterochromatin along with cellular enlargement, typical of the irradiation effects [START_REF] Sabin | Cellular Senescence -its role in cancer and the response to ionizing radiation[END_REF]. Morphometric analysis from Hoechst staining showed a two-fold increase in the nuclei size for the treated GL261-wt cells compared to untreated GL261-wt cells. However, this treatment-induced effect was particularly manifest for the GL261-Ang2 cells as the size of nuclei reached three times the one of untreated cells (Figure 5A). According to these characteristic morphological changes, we suspected a senescence phenotype for the treated tumors cells. To validate this hypothesis, GL261-wt and GL261-Ang2 cells were exposed to the identical fractionated RCT protocol that was used for in vivo experiments and were analyzed five days post-treatment. We confirmed that X-rays combined with TMZ caused the senescence of GL261-wt cells. However, this phenomenon was amplified by Ang2 overexpression as the lysosomal beta-galactosidase activity was quantitatively more significant in the GL261-Ang2 group compared to the GL261-wt group (p < 0.05) (Figure 5B).

Cells in senescence may return to the cell cycle, but in most cases, they may undergo cell death via apoptosis or mitotic death. Accordingly, we next addressed the question of whether Ang2 might influence these cellular processes in response to RCT. We performed a flow cytometry study for both cell lines five days post-treatment (Figure 5C). Following RCT, both tumor cells accumulated in a similar proportion in the G2/M phase (about 40% of cells) and in the subG1 phase (about 25% of cells), suggesting the presence of apoptotic cells (Figure 5C). However, at this post-RCT time, we observed a decrease of tumor cells in the G0/G1 phase, which was more marked for GL261-Ang2 (p < 0.05) (Figure 5C). Moreover, we noted an increase in polyploid cells in the two cell lines, but this effect was more pronounced in GL261-Ang2 cells (13.49 ± 1.09%) than in GL261-wt cells (9.28 ± 1.12%, p < 0.05) (Figure 5C).

To strengthen this hypothesis, we also investigated the effects of the combined treatment on DNA double-strand breaks by analyzing γH2AX positive cells. Indeed, persistence of γH2AX foci in time reflects genomic instability, which can lead to irreversible senescence (Figure 5D) [START_REF] Lou | Cellular senescence and DNA repair[END_REF]. One of the consequences of such genomic instability was reflected by the presence of micronuclei. As illustrated in Figure 5D, five days post-RCT, the two cell lines displayed DNA damage (γH2AX + cells) and micronuclei. The cell proportion with micronuclei was higher in GL261-Ang2 cells (64.33 ± 4.16%) than in GL261-wt cells (37.33 ± 6.80%, p < 0.05) (Figure 5D). All together, these results suggest that Ang2 overexpression in GB cells promote mitotic cell death induced by RCT.

Discussion

In GB patients, the upregulation of Ang2 observed during the angiogenesis process and tumor progression is a biomarker of poor prognosis [START_REF] Scholz | Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma[END_REF]. In these brain tumors, the angiogenic factor Ang2 has more recently been recognized as a mediator of homing for a subpopulation of macrophages identified as TEMs [START_REF] Cortes-Santiago | Soluble Tie2 overrides the heightened invasion induced by anti-angiogenesis therapies in gliomas[END_REF][START_REF] Mazzieri | Targeting the ANG2/TIE2 Axis Inhibits Tumor Growth and Metastasis by Impairing Angiogenesis and Disabling Rebounds of Proangiogenic Myeloid Cells[END_REF]. The vascular and inflammatory microenvironment of these tumors may be remodeled by the conventional treatment of GB, which includes RCT [START_REF] Mclaughlin | Inflammatory microenvironment remodelling by tumour cells after radiotherapy[END_REF][START_REF] Larionova | Interaction of tumor-associated macrophages and cancer chemotherapy[END_REF]. The main objective of our study was to evaluate whether Ang2 combined with RCT might modulate the GB microenvironment. To this end, murine GB GL261 cells were genetically modified to overexpress Ang2 and were orthotopically inoculated in mice brain. This syngeneic model allows to investigate, in addition to vascularization, the contribution of the host immune system to tumor progression [START_REF] Mckelvey | Temporal and spatial modulation of the tumor and systemic immune response in the murine Gl261 glioma model[END_REF]. In addition, the tumor biology of GL261 displays the main characteristics as that of human GB and can be used to screen the response of GL261 tumors to different therapies relevant to the treatment of GB patient, such as RCT [START_REF] Newcomb | The Murine GL261 Glioma Experimental Model to Assess Novel Brain Tumor Treatments[END_REF].

The results obtained in this GB preclinical model were in favor of a beneficial effect of Ang2 when combined with fractionated glioma treatment combining TMZ and X-rays. In contrast, without RCT treatment, no difference was detected between tumor progression and survival of animals with GL261-Ang2 or GL261-wt tumors (Figure 1 and Figure S2). Although an autocrine Ang2 effect in tumor cells themselves was observed in vitro, as evidenced by the cell proliferation slowdown, the in vivo effects might also be attributable to its paracrine effect on the vascular and inflammatory compartments (Figures 2 and3). Indeed, we presented evidence that the overexpression of Ang2 by GB cells promoted an increase in tumor vascularization compared to control tumors in the exponential phase of growth (D14) (Figure 2). However, in the GL261-wt and GL261-Ang2 untreated tumors, the vascularization was abnormal and characterized by the presence of enlarged vessels compared to the vascularization of healthy brain tissue. At this time of tumor growth, we could not depict vascular pseudonormalization or regression that we and others had previously observed on the rat 9L gliosarcoma model using a similar in situ Ang2 overexpression approach [START_REF] Machein | Angiopoietin-1 Promotes Tumor Angiogenesis in a Rat Glioma Model[END_REF][START_REF] Valable | MRI assessment of hemodynamic effects of angiopoietin-2 overexpression in a brain tumor model[END_REF]. Ang2 is known to have a complex and contradictory role during tumor angiogenesis. The discrepancy between these effects could be explained by the different levels of Ang2, which could lead to different outcomes [START_REF] Oshima | Different effects of angiopoietin-2 in different vascular beds in the eye: New vessels are most sensitive[END_REF]. However, in this GL261 model and according to Scholz et al. [START_REF] Scholz | Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma[END_REF], the overexpression of Ang2 induced vascular changes but did not modify the overall survival of the mice.

In response to RCT, the vascularization tended to regress in both tumor groups to reach a similar density. This vascular regression was, however, more pronounced in the GL261-Ang2 tumor-bearing mice, and compared to untreated tumors, the decrease in the vascular area following RCT was only significant for the GL261-Ang2 group (Figure 2). In GB patients, the abnormal vascularization of tumors renders this brain tumor less susceptible to standard chemotherapy (TMZ) and radiotherapy (X-ray). Although GL261-Ang2 and GL261-wt tumors displayed disorganized and enlarged vessels, the therapeutic effect of RCT was transient in the GL261-wt tumors, whereas it was drastically prolonged in the GL261-Ang2 tumors. No recurrence was observed three months post-RCT, which corresponds to the last time point studied (Figure 1).

In addition to its function on the vasculature compartment, Ang2 also contributes to myeloid cell infiltration in settings of inflammation and tumors [START_REF] Scholz | Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma[END_REF][START_REF] Coffelt | Angiopoietin-2 Regulates Gene Expression in TIE2-Expressing Monocytes and Augments Their Inherent Proangiogenic Functions[END_REF]. It has been previously shown that the overexpression of Ang2 in endothelial cells, which leads to an immature vascular phenotype, supports the recruitment of inflammatory cells [START_REF] Scholz | Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma[END_REF]. In our model, we showed that the inoculation of GL261-Ang2 cells did not increase the presence of TAMs compared to GL261-wt tumors (Figure 3). However, in response to RCT, the TAM density specifically increased in these GL261-Ang2 tumors. The suspected chemotactic effect of Ang2 was further demonstrated in vitro in migration assays using either the recombinant cytokine or conditioned medium from GL261-Ang2 tumor cells (Figure 4). Our in vitro and in vivo results are in line with those of Cortes-Santiago et al. [START_REF] Cortes-Santiago | Soluble Tie2 overrides the heightened invasion induced by anti-angiogenesis therapies in gliomas[END_REF], although they also suggested that the attractant properties of Ang2 was not restricted to TEMs, as also demonstrated by Murdoch et al. [START_REF] Murdoch | Expression of Tie-2 by Human Monocytes and Their Responses to Angiopoietin-2[END_REF].

The enhanced monocyte infiltration in Ang2-overexpressing tumors could be due to indirect effects via changes in the tumor-associated vasculature induced by Ang2. In the present study, Ang2 overexpression could contribute to vascular regression following RCT, which could lead to hypoxia in GL261-Ang2 tumors [START_REF] Coffelt | Angiopoietin-2 Regulates Gene Expression in TIE2-Expressing Monocytes and Augments Their Inherent Proangiogenic Functions[END_REF]. Hypoxia might favor monocyte extravasation, attraction, and/or retention in the tumor microenvironment in a TIE2-independent manner. Accordingly, in a previous study, we showed that the proportion of CD68 + cells in the tumor increased with the tumor hypoxic status [START_REF] Leblond | Hypoxia induces macrophage polarization and re-education toward an M2 phenotype in U87 and U251 glioblastoma models[END_REF]. Monocyte migration might also be mediated by the hypoxic upregulation of stromal cell-derived factor-1 [START_REF] Mazzieri | Targeting the ANG2/TIE2 Axis Inhibits Tumor Growth and Metastasis by Impairing Angiogenesis and Disabling Rebounds of Proangiogenic Myeloid Cells[END_REF], which is a potent TAM chemoattractant in various tumor models [START_REF] Kozin | Recruitment of myeloid but not endothelial precursor cells facilitates tumor re-growth after local irradiation[END_REF][START_REF] Kioi | Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice[END_REF]. Nevertheless, our in vitro data from migration assay also speak in favor of direct chemoattraction of TAMs by Ang2 (Figure 4).

We confirmed that RCT led to lymphopenia but to a similar extent in both tumor groups, suggesting that Ang2 does not modify T-cell survival in these experimental conditions (Figure S3B). Others have shown that Ang2 may stimulate TEMs to acquire an immunosuppressive function in tumors by acting on Treg activation [START_REF] Coffelt | Angiopoietin 2 Stimulates TIE2-Expressing Monocytes To Suppress T Cell Activation and To Promote Regulatory T Cell Expansion[END_REF]. As for the vascular effect described above, we cannot exclude that the discrepancy between the results of studies on similar GL261 model might be due the different levels of Ang2 [START_REF] Scholz | Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma[END_REF][START_REF] Coffelt | Angiopoietin-2 Regulates Gene Expression in TIE2-Expressing Monocytes and Augments Their Inherent Proangiogenic Functions[END_REF].

Interestingly, the fractionated RCT protocol used in this study in GL261-Ang2 tumors led to tumor clearance (Figure 1 and Figure S2). When we studied the cytotoxic effect of the combined therapy, we found that RCT induced glioblastoma cell senescence and that Ang2 potentiated this effect (Figure 5B). Induced senescence has been widely described for tumor cells treated with various therapeutic agents, including chemotherapies and ionizing radiation. Cells exhibiting senescence can be targeted and cleared by components of the immune system, including macrophages, NK cells, and T-lymphocytes [START_REF] Pawlowska | An Interplay between Senescence, Apoptosis and Autophagy in Glioblastoma Multiforme-Role in Pathogenesis and Therapeutic Perspective[END_REF]. Ang1 and Ang2 have been shown to be involved in senescence in endothelial cells [START_REF] Yang | Angiopoietin-1 Inhibits Mouse Glomerular Endothelial Cell Senescence via Tie2 Receptor-Modulated ERK1/2 Signaling[END_REF]. However, we have shown, for the first time, that Ang2 on glioma cells might exert this effect when the cells are exposed to RCT. A role for the adaptive immune system in the elimination of senescent cells from irradiated melanoma tumors has been reported. [START_REF] Meng | Radiation-inducible Immunotherapy for Cancer: Senescent Tumor Cells as a Cancer Vaccine[END_REF]. We observed a sustained infiltration of inflammatory cells, resulting in tumor regression (Figure 3). In line with an active immune response in treated GL261-Ang2 tumors, the spleen weight of these mice indicates that these treated tumors might induce higher levels of splenocyte proliferation (Figure S3C).

RCT produced long-term survival in mice bearing GL261-Ang2 tumors. Few studies have argued in favor of a beneficial antitumor effect of this cytokine in GB models [START_REF] Machein | Angiopoietin-1 Promotes Tumor Angiogenesis in a Rat Glioma Model[END_REF][START_REF] Valable | MRI assessment of hemodynamic effects of angiopoietin-2 overexpression in a brain tumor model[END_REF]. In contrast, Ang2 has been identified as a resistance factor to anti-VEGF [START_REF] Labussière | Angiopoietin-2 May Be Involved in the Resistance to Bevacizumab in Recurrent Glioblastoma[END_REF][START_REF] Scholz | Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma[END_REF][START_REF] Kloepper | Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival[END_REF] and also shown to mediate the homing of macrophages in human GB [START_REF] Scholz | Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma[END_REF][START_REF] Venneri | Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer[END_REF][START_REF] Gabrusiewicz | Anti-vascular endothelial growth factor therapy-induced glioma invasion is associated with accumulation of Tie2-expressing monocytes[END_REF]. Recently, Ang2/VEGF bispecific antibodies were developed to overcome the anti-VEGF resistance of GB patients and stimulate the host immune as well [START_REF] Kloepper | Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival[END_REF][START_REF] Peterson | Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages[END_REF]. In GB, immune checkpoint therapy might also benefit from Ang2/VEGF blockade. In this way, a tritherapy based on the combined inhibition of PD-1, Ang2, and VEGF might improve survival of tumor-bearing animals. This synergic therapeutic approach normalizes the tumor vessels and promotes an immunostimulatory microenvironment [START_REF] Di Tacchio | Tumor Vessel Normalization, Immunostimulatory Reprogramming, and Improved Survival in Glioblastoma with Combined Inhibition of PD-1, Angiopoietin-2, and VEGF[END_REF]. The divergence of the results between Di Tacchio's study and our study might partly be due to the integration of RCT in our experimental protocol. Recently, it was proposed that conventional therapy (RT or CT) for GB patients could interfere with antiangiogenic strategies. While the combination of anti-Ang2/VEGF with TMZ chemotherapy presented beneficial effects, it was not the case for radiotherapy, which benefited only from anti-VEGF [START_REF] Solecki | Differential Effects of Ang-2/VEGF-A Inhibiting Antibodies in Combination with Radio-or Chemotherapy in Glioma[END_REF]. Currently, no study has evaluated the therapeutic benefit of the combination of anti-Ang2/VEGF and RCT.

The results presented here suggest that the vascular and immune properties of Ang2 probably depend not only on the concentration of this cytokine in the tumor but also on the evolution of the tumor microenvironment (vascularization and inflammatory compartment), which will be remodeled by combined radio-and chemotherapy. According to single or combined treatment (RT plus CT) and the protocols of RT (hypofractionated and normofractionated), the effects on induction or attenuation of antitumor immune response will be divergent [START_REF] Pawlowska | An Interplay between Senescence, Apoptosis and Autophagy in Glioblastoma Multiforme-Role in Pathogenesis and Therapeutic Perspective[END_REF][START_REF] Yang | Angiopoietin-1 Inhibits Mouse Glomerular Endothelial Cell Senescence via Tie2 Receptor-Modulated ERK1/2 Signaling[END_REF].

Altogether, previous studies [START_REF] Scholz | Endothelial cell-derived angiopoietin-2 is a therapeutic target in treatment-naive and bevacizumab-resistant glioblastoma[END_REF][START_REF] Coffelt | Angiopoietin 2 Stimulates TIE2-Expressing Monocytes To Suppress T Cell Activation and To Promote Regulatory T Cell Expansion[END_REF][START_REF] Lee | Sustained Angiopoietin-2 Expression Disrupts Vessel Formation and Inhibits Glioma Growth[END_REF] and our work underline the need to include all modalities of standard treatment to better screen and characterize new therapeutics for GB.

Materials and Methods

Cell Culture

The murine glioblastoma cell line GL261 (NCI-DCTD (Division of Cancer Treatment and Diagnosis), Repository) and murine macrophage cell line RAW 264.7 (ATCC (American Type Culture Collections))

were used in this study. GL261 cells were maintained in RPMI medium (Roswell Park Memorial Institute, Sigma Aldrich, St Quentin Fallavier, France) supplemented with 10% fetal calf serum (Eurobio, Courtaboeuf, France), 2 mM glutamine (Gln, Sigma Aldrich, St Quentin Fallavier, France), and 1 µg/mL penicillin/streptomycin (P/S, Sigma Aldrich, St Quentin Fallavier, France) at 37 • C with 5% CO 2 and 95% air. RAW 264.7 cells were grown in DMEM 4.5 g/L of glucose (Dulbecco's Modified Eagle's Medium, Sigma Aldrich, St Quentin Fallavier, France) supplemented with 10% fetal calf serum, 4 mM Gln, and 1 µg/mL P/S at 37 • C in 5% CO 2 and 95% air.

Establishment of GL261 Cells Overexpressing Ang2

A pCMV6 Kan/Neo expression plasmid was employed for GL261 cells transfection. This construct vector containing murine Ang2 cDNA was transfected into GL261 cells at 80% confluence by lipofection using lipofectamine 2000 (Thermo Fisher Scientific, Illkirch-Graffenstaden, France) according to the manufacturer's instructions. Cells expressing pCMV6-K/N-Ang2 (GL261-Ang2) were selected two days later with geneticin (G-418, Sigma Aldrich, St Quentin Fallavier, France) at 400 µg/mL. GL261-Ang2 cells were grown in RPMI medium and selected with G-418 on a monthly basis. Ang2 mRNA overexpression was routinely checked by qRT-PCR.

Glioblastoma Preclinical Model

Animal investigations were performed under the current European directive (2010/63/EU). Ethical approval was obtained by E.P. and S.V. from the regional committee (CENOMEXA) and the French Ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation with the authorization APAFIS#12496. This tumor model is based on an orthotopic injection of GL261-wt or GL261-Ang2 cells in the right striatum of C57BL/6 mice (male, 20-23 g, Janvier Laboratories, Le Genest-Saint-Isle, France). Anesthesia in mice were induced with 5% of isoflurane in 70% N 2 O and 30% O 2 and then maintained at 2% during surgery. Mice were placed on a stereotactic head holder, and a scalp incision was performed along the median line. A 1 mm diameter burr hole was drilled in the skull, 2 mm laterally from the bregma. Glioblastoma cells (1 × 10 5 cells) in 3 µL of 2 mM Gln-phosphate buffer saline (PBS) were then injected at a depth of 4 mm with a 30G needle and standardized flow of 0.6 µL/min for 5 min. The needle was removed 5 min later to avoid cell reflux. Injections of buprenorphine (Buprecare, 0.05 mg/kg, sc) were administered in perioperative phases.

Tumor volumes were evaluated longitudinally by magnetic resonance imaging (7T MRI, Bruker, CYCERON biomedical imaging platform, Caen, France). Animals were anesthetized with isoflurane as described above and placed inside the magnet for acquisition with T2w sequence. The MRI scan properties were as follows: T2w rapid acquisition with relaxation enhancement (RARE), acceleration factor of 8; TR/TE eff = 5000/60 msec; average = 1; 20 slices de 0.5 mm thick; spatial resolution = 0.07 × 0.07 mm; acquisition time: 2 min. MRI image analyses were completed with ImageJ software (version 1.52k) [START_REF] Schneider | NIH Image to ImageJ: 25 years of Image Analysis[END_REF].

In Vivo Radiochemotherapy

In vivo RCT started seven days after glioblastoma cell implantation when the tumor volume was around 2-5 mm 3 . Mice were treated thrice with chemotherapy (TMZ, T2577, Sigma Aldrich, St Quentin Fallavier, France) and radiotherapy (X-rays). Each fraction of RCT was spaced at 48 h. TMZ (10 mg/kg/day in saline) was administered intraperitoneally. Control animals received the same volume of saline solution (vehicle). Two hours later, ipsilateral hemisphere of mice was irradiated with the X-RAD225 Cx system (Precision X-ray Inc, CYCERON platform, Caen, France) at 4 Gy/day with a dose rate of 3.3 Gy/min. The characteristics of the photon beam delivered by this small animal irradiator were voltage = 225 keV, intensity = 13.3 mA, and energy = 80 keV using 1 mm Cu filter. The schedule of protocol is illustrated in Figure 1A.

Immunohistological Analysis

At the end of the protocol, mice were deeply anesthetized and transcardially perfused with cold heparinized saline solution. Brains were removed and immediately snap-frozen in n-pentane (Sigma Aldrich, St Quentin Fallavier, France) and stored at -80 • C. Thereafter, brains were cut on a cryostat to obtain serial coronal sections of 30 µm. Brains sections were collected on superfrost slides (Thermo Fisher Scientific, Illkirch-Graffenstaden, France) and postfixed 20 min in 4% paraformaldehyde (PFA 4%, Sigma Aldrich, St Quentin Fallavier, France). Slices were blocked 2 h at room temperature with PBS-0.5% triton X100 (Sigma Aldrich, St Quentin Fallavier, France)-0.1% tween (Sigma Aldrich, St Quentin Fallavier, France)-3% bovine serum albumin (BSA, Sigma Aldrich, St Quentin Fallavier, France) and incubated overnight at 4 • C with the following primary antibodies: rat anti-CD31 (1:100; 553370, BD Biosciences, Le Pont-de-Claix, France), rat anti-CD68 (1:800; ab 53444, Abcam, Cambridge, UK), rat anti-CD4 (1:100; 550278, BD Biosciences, Le Pont-de-Claix, France), rabbit anti-CD8 (1:100; ab217344, Abcam, Cambridge, UK), and rabbit-anti-Tie2 (1:200; sc-324, Santa-Cruz, Heidelberg, Germany) in PBS-0.5% triton X100-0.1% tween-1% BSA. Primary antibody was detected with an Alexa-555-conjugated antirat (1:500; A21434, Invitrogen, Carlsbad, CA, USA) or an Alexa-555-conjugated antirabbit (1:500; A21428, Invitrogen, Carlsbad, CA, USA) in PBS-0.5% triton X100-0.1% tween-1% BSA containing Hoechst 33342 (10 µg/mL, Sigma Aldrich, St Quentin Fallavier, France). Slices were then coverslipped with mounting medium Aqua-PolyMount (Tebu-Bio, Le Perray-en-Yvelines, France) and acquired at ×10 magnification with epifluorescence microscope Leica DMi8S (Leitz Leica, Wetzlar, Germany).

Image Analysis and Quantification

All analyses were performed with regions of interest (ROI) of 736 × 736 pixels or 468 × 468 µm, chosen randomly and representative of the whole tumor. Images were then binarized with ImageJ software (version 1.52k) [START_REF] Schneider | NIH Image to ImageJ: 25 years of Image Analysis[END_REF] after applying an automatic threshold. The results obtained were represented as the percentage of immunostaining in the ROI. The blood vessels were analyzed with an ImageJ (version 1.52k) plugging developed in-house [START_REF] Valable | MRI assessment of hemodynamic effects of angiopoietin-2 overexpression in a brain tumor model[END_REF]; this analysis provided the vascular density and the average diameter of vessels.

In Vitro Radiochemotherapy

For in vitro experiments, three fractions of RCT, spaced at 48 h, was applied. Tumor cells were exposed to 100 µM TMZ, which was brought in fresh medium followed by exposition to X-rays at 4 Gy with a dose rate of 2 Gy/min (Precision X-ray Inc, CYCERON platform, Caen, France).

Cell Cycle Analysis

Cell cycle was analyzed by flow cytometry (SFR ICORE, University of Caen platform, France). Propidium iodide (Beckman Coulter SAS, Villepinte, France) staining was realized according to the manufacturer's instruction and detected with Gallios™ flow cytometer (Beckman Coulter SAS, Villepinte, France). All analyses were performed on a minimum of 10,000 events and analyzed with Kaluza ® flow analysis software (Beckman Coulter SAS, Villepinte, France).

SA-β-Galactosidase Assay

Senescence was determined based on alterations in cell morphology (enlargement and flattening) and expression of pH-dependent β-galactosidase [START_REF] Dimri | A biomarker that identifies senescent human cells in culture and in aging skin in vivo[END_REF]. Senescence associated-β-galactosidase staining (SA-β-gal, 9860S, Cell Signaling, Leiden, The Netherlands) was employed according to the manufacturer's instructions five days after the last treatment with RCT.

Migration Assay

For transwell migration assay (5 µm, Sarsted, Nümbrecht, Germany), RAW 264.7 cells were plated in DMEM medium with 0.5% fetal calf serum to the top chambers of transwell, which was then added in a 24-well plate containing GL261 cells previously exposed (24 h before) to RCT. Thirty minutes before seeding RAW 264.7 cells, murine recombinant Ang2 (7186-AN, R&D Systems, Lille, France) was added at increasing doses (0, 200, 400, and 800 ng/mL) in a medium containing 0.5% serum. To evaluate Ang2 contribution, Fc Tie2 at 8 µg/mL (762-T2, R&D Systems, Lille, France) was added 30 min before adding the transwell into the plate. After withdrawing nonmigrating cells on the upper side of the membrane, migrating cells (bottom side of the membrane) were stained with Hoechst 33342 solution (10 µg/mL). Images were acquired on a Leica Aristoplan microscope (Leitz Leica, Wetzlar, Germany) with a 25× objective. Migration was quantified by counting the number of migrated cells in five microscope fields of view.

Immunocytochemistry

Glioblastoma cells were plated in 24-well plates on coverslips. Five days after RCT, cells were fixed with 4% PFA and blocked with PBS-0.1% tween-3% BSA for 1 h at room temperature. Then cells were incubated overnight at 4 • C with the primary antibody phosphohistone H2AX (ser13) (1:200; 2577S, Cell Signaling Technology, Leiden, The Netherlands) in PBS-0.1% Tween-1% BSA. Primary antibody was detected with an Alexa-555-conjugated antirabbit (1:500; A21428, Invitrogen, Carlsbad, CA, USA) in PBS-0.5% triton X100-0.1% tween-1% BSA. Cells were counterstained with Hoechst 33342 (10 µg/mL) for nuclear staining. A micronucleus assay was performed with Hoechst 33342 staining, and a cell with at least one micronucleus was considered positive. All immunocytochemical markers were observed on epifluorescence microscope Leica DMi8S (Leitz Leica, Wetzlar, Germany) with a 25× objective. For each condition, at least three coverslips were analyzed. Images from five representative high-power fields per slide were acquired.

Statistical Analysis

All data are presented as the mean ± standard deviation (SD). Statistical analyses were performed with Statistica ® (Tibco Software Inc, Palo Alto, CA, USA). The tests used and the number of experiments are detailed in each figure legend.

Conclusions

Our results obtained in a murine immunocompetent GB model suggest that an ectopic expression of Ang2 combined with radiochemotherapy impedes tumor recurrence. In these experimental conditions, Ang2 acted in an autocrine manner by increasing GB cell senescence and in a paracrine manner by acting on the innate immune system while modulating the vascular tumor compartment.

Supplementary Materials:

The following are available online at http://www.mdpi.com/2072-6694/12/12/3585/s1, Figure S1: Characterization of Ang2 overexpression in glioblastoma cells, Figure S2: Radiochemotherapy is more effective for glioblastoma derived from GL261-Ang2 cells, Figure S3: Involvement of Ang2 in systemic inflammation after radiochemotherapy in glioblastoma bearing mice, Figure S4: Characterization of Ang2 receptors, Tie2 and β1-integrin, in RAW 264.7 cells, Table S1: List of antibodies used for flow cytometry analyses.

Author Contributions: All authors have made substantial contributions to the conception/design of the work or the acquisition/analysis/interpretation of data and have approved the submitted version as well as agreed to be personally accountable for the authors' own contributions. The individual contributions of authors are as follows: Conceptualization C.H., E.A.P., S.V., and E.P.; methodology C.H., E.A.P., S.V., and E.P.; formal analysis C.H.; investigation C.H., A.E.F., M.M.L., E.A.P., M.B., S.V., and E.P.; writing-original draft preparation C.H., E.A.P., S.V., M.B., and E.P.; writing-review and editing C.H., A.E.F., M.M.L., E.A.P., M.B., S.V., and E.P.; visualization C.H. and E.P.; supervision E.A.P., S.V., and E.P.; project administration M.B., S.V., and E.P.; funding acquisition, M.B., S.V., and E.P. All authors have read and agreed to the published version of the manuscript.

Funding: This study was supported in part by the Région Normandie, the Centre National de la Recherche Scientifique (CNRS), the Université de Caen-Normandie (UNICAEN), the French Ministère de l'Enseignement Supérieur et de la Recherche (MESR), the Advanced Resource Center for Hadrontherapy in Europe (Archade), the Fédération pour la Recherche sur le Cerveau par l'opération Rotary "Espoir en tête" (FRC), and the French National Agency for Research ANR-10-EQPX-1401, HABIONOR European project, cofunded by the Normandy County Council, the French state in the framework of the interregional development contract "Vallée de la Seine" 2015-2020.

Figure 1 .

 1 Figure 1. Combination of radiochemotherapy (RCT) and angiopoietin-2 (Ang2) overexpression in glioblastoma cells improved animal survival and induced complete clearance of brain tumor. (A) Experimental protocol to compare the tumor progression of GL261-wt and GL261-Ang2 glioblastoma models (untreated groups) and in response to RCT (RCT groups). (B) Representative longitudinal images in T2-weighted magnetic resonance imaging (MRI) for the four tumor groups. (C) Longitudinal MRI tumor volume follow-up of GL261-wt untreated (n = 5), GL261-wt + RCT (n = 5), GL261-Ang2 untreated (n = 4), and GL261-Ang2 + RCT (n = 4). Mean ± SD, * p < 0.05 vs. GL261-wt + RCT, $ p < 0.05 vs. respective untreated group, two-way ANOVA followed by Tukey's test, ns = nonsignificant. (D) Comparative Kaplan-Meier survival curves for the four tumors groups: GL261wt untreated (n = 5), GL261-wt + RCT (n = 5), GL261-Ang2 untreated (n = 4) and GL261-Ang2 + RCT (n = 4). * p < 0.05 vs. GL261-wt + RCT, $ p < 0.05 vs. respective untreated group, log-rank test.

Figure 1 .

 1 Figure 1. Combination of radiochemotherapy (RCT) and angiopoietin-2 (Ang2) overexpression in glioblastoma cells improved animal survival and induced complete clearance of brain tumor. (A) Experimental protocol to compare the tumor progression of GL261-wt and GL261-Ang2 glioblastoma models (untreated groups) and in response to RCT (RCT groups). (B) Representative longitudinal images in T2-weighted magnetic resonance imaging (MRI) for the four tumor groups. (C) Longitudinal MRI tumor volume follow-up of GL261-wt untreated (n = 5), GL261-wt + RCT (n = 5), GL261-Ang2 untreated (n = 4), and GL261-Ang2 + RCT (n = 4). Mean ± SD, * p < 0.05 vs. GL261-wt + RCT, $ p < 0.05 vs. respective untreated group, two-way ANOVA followed by Tukey's test, ns = nonsignificant. (D) Comparative Kaplan-Meier survival curves for the four tumors groups: GL261-wt untreated (n = 5), GL261-wt + RCT (n = 5), GL261-Ang2 untreated (n = 4) and GL261-Ang2 + RCT (n = 4). * p < 0.05 vs. GL261-wt + RCT, $ p < 0.05 vs. respective untreated group, log-rank test.

Figure 2 .

 2 Figure 2. The vascularization of GL261-Ang2 tumors was more sensitive to RCT than the vascularization of GL261-wt tumors. (A) Representative images of CD31 immunostaining at D14 in the tumor core of GL261-wt, GL261-Ang2, and in healthy hemisphere. Scale bar = 100 µm. (B) Quantitative analysis of vascular area. (C) Quantitative analysis of vessel diameter. Dashed line corresponds to the mean of healthy tissue. n = 3 animals for each group. Mean ± SD * p < 0.05 vs.GL261-wt group, # p < 0.05 vs. respective untreated group, $ p < 0.05 vs. healthy tissue, two-way ANOVA followed by Tukey's test.

Figure 2 .

 2 Figure 2. The vascularization of GL261-Ang2 tumors was more sensitive to RCT than the vascularization of GL261-wt tumors. (A) Representative images of CD31 immunostaining at D14 in the tumor core of GL261-wt, GL261-Ang2, and in healthy hemisphere. Scale bar = 100 µm. (B) Quantitative analysis of vascular area. (C) Quantitative analysis of vessel diameter. Dashed line corresponds to the mean of healthy tissue. n = 3 animals for each group. Mean ± SD * p < 0.05 vs. GL261-wt group, # p < 0.05 vs. respective untreated group, $ p < 0.05 vs. healthy tissue, two-way ANOVA followed by Tukey's test.

Figure 3 .

 3 Figure 3. Ang2 overexpression in glioblastoma cells combined with radiochemotherapy favored inflammation in glioblastoma. (A) Representative images of CD68 immunostaining at D14 and quantification of CD68 + area. Scale bar = 100 µm. n = 3 animals for each group. Mean ± SD # p < 0.05 vs. the respective untreated group, ANOVA followed by Tukey's test. (B) Representative images of CD68 + /Tie2 + cells in GL261-Ang2 tumor-bearing mice. Scale bar = 100 µm and 50 µm for magnification. (C) Representative images of CD4 immunostaining at D14 and quantification of CD4 + area. Scale bar = 100 µm. n = 3 animals for each group. Mean ± SD # p < 0.05 vs. the respective untreated group, ANOVA followed by Tukey's test. (D) Representative images of CD8 immunostaining at D14 and quantification of CD8 + area. Scale bar = 100 µm. n = 3 animals for each group. Mean ± SD # p < 0.05 vs. the respective untreated group, ANOVA followed by Tukey's test. (E) Representative T2w images of the GL261-Ang2 tumor at D42 and D100 after cell implantation. Dashed lines delimit the

Figure 3 .

 3 Figure 3. Ang2 overexpression in glioblastoma cells combined with radiochemotherapy favored inflammation in glioblastoma. (A) Representative images of CD68 immunostaining at D14 and quantification of CD68 + area. Scale bar = 100 µm. n = 3 animals for each group. Mean ± SD # p < 0.05 vs. the respective untreated group, ANOVA followed by Tukey's test. (B) Representative images of CD68 + /Tie2 + cells in GL261-Ang2 tumor-bearing mice. Scale bar = 100 µm and 50 µm for magnification. (C) Representative images of CD4 immunostaining at D14 and quantification of CD4 + area. Scale bar = 100 µm. n = 3 animals for each group. Mean ± SD # p < 0.05 vs. the respective untreated group, ANOVA followed by Tukey's test. (D) Representative images of CD8 immunostaining at D14 and quantification of CD8 + area. Scale bar = 100 µm. n = 3 animals for each group. (E) Representative T2w images of the GL261-Ang2 tumor at D42 and D100 after cell implantation. Dashed lines delimit the residual tumor area and correspond to the representative images obtained from the immunohistological study to detect immune cells (CD68 + , CD4 + , and CD8 + ). Scale bar = 100 µm.

Figure 4 .

 4 Figure 4. Ang2 induced macrophage migration in vitro. (A) Representative images and quantification of migrated RAW 264.7 cells in response to recombinant Ang2. Scale bar = 50 µm; mean ± SD, * p < 0.05 vs. 0 ng/mL Ang2, # p < 0.05 vs. Fc Tie2 condition, two-way ANOVA followed by Tukey's test. (B) Representative images and quantification of migrating RAW 264.7 cells in response to conditioned medium from tumor cells. Scale bar = 50 µm; Mean ± SD, n = 4; * p < 0.05 vs. GL261-wt group, # p < 0.05 vs. Fc Tie2 condition, two-way ANOVA followed by Tukey's test.

Figure 4 .

 4 Figure 4. Ang2 induced macrophage migration in vitro. (A) Representative images and quantification of migrated RAW 264.7 cells in response to recombinant Ang2. Scale bar = 50 µm; mean ± SD, * p < 0.05 vs. 0 ng/mL Ang2, # p < 0.05 vs. Fc Tie2 condition, two-way ANOVA followed by Tukey's test. (B) Representative images and quantification of migrating RAW 264.7 cells in response to conditioned medium from tumor cells. Scale bar = 50 µm; Mean ± SD, n = 4; * p < 0.05 vs. GL261-wt group, # p < 0.05 vs. Fc Tie2 condition, two-way ANOVA followed by Tukey's test.

Cancers 2020 , 18 Figure 5 Figure 5 .

 20201855 Figure 5. Cont.

Figure 5 .

 5 Figure 5. Ang2 overexpression in glioblastoma cells combined with radiochemotherapy modified cell death. (A) Representative photographs of nuclei (Hoechst) in the tumor core at D14 after cell implantation. Quantification of nuclei area for the four groups, n = 1 image/condition and the nuclei size performed on 250 nuclei/image. Scale bar = 50 µm. (B) Representative photographs of senescent cells detected by β-galactosidase staining performed five days following the last dose of RCT on GL261-wt and GL261-Ang2 cells. The positive cells for senescence assay showed a blue coloration. Scale bar = 50 µm. Quantification of senescence in tumor cells five days after RCT. The proportion of β-galactosidase positive cells (arrows) was expressed relative to total cell number counted by phase contrast microscopy. Mean ± SD, n = 3; * p < 0.001 vs. GL261-wt RCT group, Student's t-test. (C) Cell cycle profile of tumor cells assessed five days after RCT. Quantification of the cell distribution in SubG1 and G0/G1 phases as well as polyploid cells five days after RCT. Mean ± SD, n = 3; * p < 0.05 vs. GL261-wt RCT group, Student's t-test. (D) Representative photographs of DNA double-strand breaks and genomic instability linked to mitotic death and identified by the presence of micronuclei in GL261-wt and GL261-Ang2 cells five days after RCT. DNA double-strand breaks and cell micronuclei were identified with γH2AX and Hoechst staining, respectively. Scale bar = 50 µm. Quantification of mitotic death evaluated by micronuclei assay in GL261-wt and GL261-Ang2 cells five days after RCT. The proportion of positive cells with at least one micronucleus (arrows) was obtained relative to total cell number counted by Hoechst staining. Mean ± SD, n = 3; * p < 0.05 vs. GL261-wt RCT group, Student's t-test.

Acknowledgments:

The authors wish to thank the SFR ICORE (Intéraction Cellules-Organismes-Environnement) platform localized in the University of Caen (France) and Maryline Guillamin for access to the materials used to perform the flow cytometry analyses. We are grateful to Emilie Pacary (Neurocentre Magendie, INSERM U1215, Bordeaux, France) for technical assistance in plasmid construction.

Conflicts of Interest:

No potential conflicts of interest were disclosed.

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Alexis Graux

Sophie Cazottes
email: sophie.cazottes@insa-lyon.fr

Damien Fabrègue

Michel Perez

Frédéric Danoix

An alternative method for the measurement of precipitate volume fractions in microalloyed steels by the means of atom probe tomography

Keywords:

Knowledge of the volume fraction of precipitates is crucial for estimating the impact of precipitation on microstructure evolution or mechanical properties. However, its experimental determination is often a difficult task. In this work, atom probe tomography was applied to an industrial TieNb microalloyed steel, to follow the evolution of austenite solute composition in titanium and niobium as a function of temperature in the austenitic domain. These composition measurements were used to calculate the volume fraction of (Ti x , Nb 1-x )C carbides in austenite, using mass balance. This type of measurement is made possible by the considerable evolutions experienced by tomographic atom probes over the past 20 years in terms of volume analyzed and mass resolution. Atom probe tomography is nowadays not only able to help determining volume fractions below 0.1%, but also provides unique information related to solubility limits as low as a few tens of ppm, most useful for developing phase diagrams, or assessing existing ones.

Introduction

In microalloyed steels, elements such as Ti and Nb form precipitates that control grain growth [START_REF] Gladman | Grain-Size Control in Steels[END_REF] and cause precipitation strengthening [START_REF] Gladman | Precipitation hardening in metals[END_REF]. Precipitate volume fraction and precipitate radii have a key impact on the resulting mechanical properties. Grain growth and precipitate hardening are indeed both controlled by the precipitate size distribution. The precipitate size distribution is usually described by its mean radius 〈r〉 and volume fraction f v , the determination of which is therefore critical to estimate the effect of precipitation on microstructure evolution and mechanical properties.

Transmission electron microscopy (TEM) techniques directly allow the determination of the distribution of precipitate sizes. The determination of f v by TEM is however quite imprecise. From thin foil observations, it is difficult to measure precisely the local thickness of the foil [START_REF] Allen | Foil thickness measurements from convergent-beam diffraction patterns[END_REF], which may not be constant over the observed field of view, leading to large scatter in the observed volume of matrix. Moreover, for low volume fractions, the number of observed precipitates in a single field of view drops to values that often hinders any statistical analysis. This procedure then often turns out to be inaccurate. The estimation of volume fraction using extraction replicas [START_REF] Ashby | On the determination of the number, size, spacing, and volume fraction of spherical second-phase particles from extraction replicas[END_REF] also imposes to estimate the etched volume of matrix, which depends on the studied material, etching time, and many other (uncontrolled) experimental factors. Other techniques are thus needed to estimate the volume fraction of precipitates.

The most common method is to resort to selective matrix dissolution techniques coupled with inductively coupled plasma (ICP) spectroscopy [START_REF] Maugis | Development of methods for the characterisation and modelling of precipitation in steels[END_REF][START_REF] Reyes | Vanadium carbide dissolution during austenitisation of a model microalloyed fecv steel[END_REF][START_REF] Lu | Matrix dissolution techniques applied to extract and quantify precipitates from a microalloyed steel[END_REF]. These techniques aim at isolating the precipitates present in the steel by chemical or electrolytic dissolution of the matrix. Both the precipitates isolated by filtration, and the resulting solution (dissolved matrix) are then analyzed to determine the respective proportions of elements present in precipitates and in solid solution. Selective matrix dissolution has the advantage of providing a global composition of large samples but requires a strong experience in sample preparation, etching solutions, and precipitate filtering.

Small Angle X-rays Scattering (SAXS) and Small Angle Neutron Scattering (SANS) also allow to measure precipitate volume fractions [START_REF] Geuser | Precipitate characterisation in metallic systems by small-angle x-ray or neutron scattering[END_REF][START_REF] Deschamps | Quantitative characterization of precipitate microstructures in metallic alloys using small-angle scattering[END_REF]. With these techniques, the detection limit depends on the type of precipitates. In some cases, volume fraction of approximately 0.1% can be difficult to quantity. It is also hard to distinguish different types of precipitates, having different compositions, if they present similar sizes. Last, these techniques necessitate the access to large-scale facilities.

Alternatively, atom probe field ion microscopy (APFIM) or atom probe tomography (APT) can be applied for indirect precipitate volume fraction determination through the measurement of matrix composition. These techniques have indeed already been applied to high speed steels [START_REF] Rong | The role of alloy composition in the precipitation behaviour of high speed steels[END_REF] or Al-Zn-Mg alloy [START_REF] Dumont | Characterisation of the composition and volume fraction of η and η precipitates in an al-zn-mg alloy by a combination of atom probe, small-angle x-ray scattering and transmission electron microscopy[END_REF] to measure the matrix composition and derive the volume fraction of precipitates. In both studies, volume fractions of precipitates were rather large (of the order of 1 to 10%), leading to low relative uncertainties. In 1994, Palmière et al. [START_REF] Palmiere | Compositional and microstructural changes which attend reheating and grain coarsening in steels containing niobium[END_REF] applied APFIM, the 1D ancestor of current APT, to study the evolution of Nb solute content in austenite for various heat treatments in Nb-microalloyed steels. Although original and promising, this methodology was then limited by the volume sizes (and thus the number of atoms analyzed), the mass resolution of the instrument, and its one-dimensional nature. At last, the instrument they used only provided 1D concentration profiles, rather than 3D reconstruction. As a consequence, they developed a procedure to discriminate between matrix and precipitates. Their procedure can be questioned, but is no longer necessary nowadays, with true 3D atomic reconstructions.

If the atoms are randomly distributed, the standard deviation associated with the measurement of the composition of the analyzed volume by APT is given by [START_REF] Danoix | Standard deviations of composition measurements in atom probe analyses-part ii: 3D atom probe[END_REF]:

= - - σ C C Q N (1 )(1 ) (1)
with C the measured composition, N the total number of atoms collected, and Q the detection efficiency (Q ≈ 0.5). At the time of Palmière et al. study, 10 to 15 samples were needed to collect a total of 100,000 ions. Thus, the standard deviation associated with each experiment was very large, since it is related to the total number of collected ions (Eq. ( 1)): 22 ppm for a measured composition of 100 ppm and a total of 100,000 analyzed ions. During the last two decades, APT has considerably evolved in terms of analyzed volume size (total number of analyzed ions per sample) as well as in mass resolution [START_REF] Miller | The future of atom probe tomography[END_REF][START_REF] Moody | Advances in atom probe tomography instrumentation: implications for materials research[END_REF]. This technique is nowadays capable of analyzing tens of millions of ions in a single sample. For such number of analyzed ions, the standard deviation associated with measurement becomes negligible. These evolutions open new perspectives for using APT as a quantitative tool for solid solution analysis.

In this article, APT was applied to determine the volume fraction of (Ti x ,Nb 1-x )C carbides of the order of 0.1% in an industrial TieNb microalloyed steel. The characterization of the precipitation state within this steel by electron microscopy techniques is presented in [START_REF] Graux | Precipitation and grain growth modelling in ti-nb microalloyed steels[END_REF]. The Nb and Ti concentration in austenite were measured after several heat treatments. Based on these measurements, the volume fraction of (Ti x ,Nb 1-x )C carbides was derived. Experimental data were then compared with Thermo-Calc [START_REF] Andersson | Thermo-Calc & DICTRA, computational tools for materials science[END_REF] equilibrium calculations.

Material and methods

The bulk alloy composition was measured by inductively coupled plasma mass spectrometry (ICP-MS) and is given in Table 1.

The microstructure of the as-received state is mainly composed of granular bainite and is homogeneous (Fig. 1), no noticeable texture was observed. Several heat treatments were performed in the austenite stability domain between 1050 ∘ C and 1280 ∘ C, using parallelepiped samples of approximately 10 × 10 × 1 mm 3 machined from the asreceived steel plate. Heat treatments are listed in Table 2. In order to avoid oxidation and decarburization, samples were placed in primary vacuum sealed quartz tube during heat treatments. The samples were directly inserted in horizontal radiative heating furnaces heated up to the target temperature, followed by water quenching. Martensitic microstructures were obtained after quenching.

The precipitation state of the initial condition and the heat treated samples were investigated by means of electron microscopy. Special emphasis has been placed on the (Ti x ,Nb 1-x )C carbides, responsible for austenite grain growth control. Carbon extraction replicas were prepared and analyzed using a JEOL 2100 TEM operating at 200 kV and a ZEISS Supra 55VP FEG-SEM. To analyze larger precipitates that are not collected on carbon replicas, bulk samples were also investigated in SEM, after polishing down to 1 μm and nital etching. Both TEM and SEM were equipped with Oxford EDX analyzers with silicon drift detector (SDD). EDX measurements were employed to determine the relative contents of the metallic species such as Ti, Nb, and S. SEM-EDX measurements were conducted at 30 kV. Additionally, the crystallographic nature of precipitates was studied using selected area electron diffraction (SAED) on a JEOL 2010F TEM operating at 200 kV. The crystallography data used for indexing the SAED patterns are extracted from [START_REF] Gražulis | Crystallography open databasean open-access collection of crystal structures[END_REF]. Atom probe tomography (APT) was applied to measure the austenite composition, with particular emphasis on Ti and Nb. The measurements were therefore performed on martensitic microstructures obtained by quenching after the heat treatments listed in Table 2. Due to the diffusion-less nature of its transformation mechanism, the composition of martensite is that of the parent austenite.

The specimen tips were prepared using a classical electro-polishing method. First, the tips were thinned using a solution of 75% acetic acid and 25% perchloric acid. Then, a final polishing was completed using a mixture of 2% perchloric acid in 98% 2 butoxyethanol at 15 V until separation of the rod into two pieces with a needle tip radius smaller than 50 nm. The experiments were carried out on a LEAP® 4000HR device from CAMECA under the following conditions: 20% pulse fraction, 50 K temperature and average detection rate of 0.3%. The

Table 1

ICP-MS composition (at.%) of the studied steel and mean APT composition (at.%) of all heat-treated samples. Ti, Nb, and C are not indicated in APT because they are involved in precipitates and vary depending on the heat treatment. N and S were not quantified by APT. reconstruction procedure was conducted using IVAS 3.6 software package. In order to get enough precision in the quantification of microalloying additions, several tens of millions of atoms were collected per heat treated condition.

µm

In the experimental mass spectra generated, a convolution is observed between the peak of the main isotope of Ti ( 48 Ti 2+ ) and that of ( 12 C-12 C) + carbon molecular ions, around 24 Da. This was already observed by Thuvander et al. [START_REF] Thuvander | Quantitative atom probe analysis of carbides[END_REF]. Therefore, the 48 Ti 2+ peak, representing approximately 75% of the total Ti amount was not used for quantification of the Ti. In the same way, 49 Ti 2+ and 50 Ti 2+ peaks were not used due to convolutions with ( 12 C -12 C -12 C -13 C) 2+ , ( 12 C -13 C) + , and 50 Cr 2+ peaks. Thus, the measurement of Ti in solid solution relied on the peaks of the two first isotopes ( 46 Ti 2+ and 47 Ti 2+ ), representing 15% of the total Ti amount. The presented values were corrected according to natural isotopic ratios in order to estimate the actual Ti content.

For several millions of atoms, the standard deviation associated with the measurement of a composition by APT becomes negligible (Eq. ( 1)). Another source of error on this composition measurement can arise from APT very local nature, thus sensitive to possible heterogeneities in composition. For each heat-treated state, several samples were analyzed. The variation on composition measurement is referred to as the standard error of the mean ( - σ x ) and is defined as the standard deviation divided by the number of observations n:

= ∑ - σ C C n ( ) x i i m 2 (2)
where C m is the average composition measured, C i the different individual compositions measured.

Results

Precipitation

In the as-received condition of the alloy, 4 different types of precipitates were identified, either on bulk samples or carbon replicas (Fig. 2): (Ti,Nb)N (a), complex precipitates composed of (Ti,Nb)N and MnS generally nucleated on an aluminum oxide inclusion (b), Ti 4 C 2 S 2 (c), and (Ti x ,Nb 1-x )C (d). Graux et al. [START_REF] Graux | Precipitation and grain growth modelling in ti-nb microalloyed steels[END_REF] provides a more detailed characterization of the precipitation state of this alloy by electron microscopy techniques, EDX, and SAED.

Starting from this initial precipitated state, several reversion heat treatments were performed (Table 2). The precipitation state after each heat treatment was systematically investigated. The same four types of precipitates were also observed after heat treatments at 1050 ∘ C, 1120 ∘ C, and 1200 ∘ C. At 1280 ∘ C, only (Ti,Nb)N and MnS were observed on carbon replicas. A summary of the different precipitates found after each heat treatment is given in Table 3.

Due to their small size (mean radius of 58 nm), (Ti x ,Nb 1-x )C exert a pinning pressure on austenite grain boundaries and are of particular interest for the control of austenite grain growth during heat treatments [START_REF] Graux | Precipitation and grain growth modelling in ti-nb microalloyed steels[END_REF]. Depending on the heat treatment temperature, various amounts of (Ti x ,Nb 1-x )C are dissolved and released in solid solution. The other types of precipitates are often larger than 1 μm, which makes them ineffective for grain boundary pinning. The EDX analysis of (Ti x ,Nb 1-x )C gave a Ti/Nb ratio that was constant for each heat treatment, indicating that the (Ti x ,Nb 1-x )C composition remains stable over the temperature range studied. The Ti/Nb ratio is consistent with (Ti 0.80 Nb 0.20 )C. Apart from very limited Mo amount (< 1 at.%), no other metallic element were found in those precipitates. The large (Ti,Nb)N population only contains slights amounts of Nb. According to EDX analysis and assuming that they are pure nitrides, their Ti/Nb ratio is consistent with (Ti 0.95 , Nb 0.05 )N. This population of precipitates is very stable, formed from the liquid state during casting. It is therefore reasonable to assume that (Ti 0.95 , Nb 0.05 )N do not significantly evolve for the considered heat treatments.

Austenite composition

APT was used to measure the austenite composition for each of the above-cited heat treatments. The total number of analyzed ions for each condition can be retrieved in Table 3. The amount of Ti and Nb in austenite increases with increasing heat treatment temperature, indicating the dissolution of (Ti x ,Nb 1-x )C precipitates. The heat treatments performed are long enough to reach equilibrium regarding the volume fraction of (Ti x ,Nb 1-x )C formed. Thus the austenite composition measurements give the solubility limits of (Ti x ,Nb 1-x )C at each temperature. The analyzed volumes are up to 2 μm long, so that several martensite laths formed during quenching are analyzed. Fig. 3 displays the reconstructed positions of C, Nb, and Ti within a specimen heat treated at 1200 ∘ C, focusing on a martensite lath boundary. As expected for martensitic steels, a segregation of C is observed at lath boundaries. The distribution of Nb and Ti is more homogeneous throughout the sample. No precipitate was intercepted during analysis, therefore the whole tip composition measurement is representative of the matrix composition.

(Ti x ,Nb 1-x )C composition

A composition close to (Ti 0.80 ,Nb 0.20 )C was determined by EDX. This chemical content is inconsistent with the respective evolutions of Ti and Nb in solid solution measured by APT (see Table 3): since a difference in Nb in solid solution of about 200 ppm is observed between 1280 ∘ C and 1050 ∘ C, the difference of Ti content in solid solution between those two temperatures should be approximately 800 ppm (four times higher). A difference of approximately 400 ppm is found instead.

Therefore, an estimation of the composition of the (Ti x ,Nb 1-x )C population is proposed in this section, where x is the unknown to determine. The total amount of Ti and Nb available for the formation of (Ti x ,Nb 1-x )C, respectively X Ti 0 and X Nb 0 , are also estimated.

Since Nb evolution is only related to the (Ti x ,Nb 1-x )C, the total amount of Nb available for the formation of (Ti x ,Nb 1-x )C is directly by the APT measurement at 1280 ∘ C:

= = ∘ X X (1280 C) 201 ppm APT SS Nb 0 Nb, (3)
The Ti evolution from 1200 ∘ C to 1280 ∘ C is affected by the dissolution of both (Ti x ,Nb 1-x )C and Ti 4 C 2 S 2 (Fig. 4). Thus, the amount of Ti involved in both population has to be determined. Below 1200 ∘ C, it is however reasonable to assume that the evolution of Ti and Nb in solid solution is mainly related to the dissolution of (Ti x ,Nb 1-x )C. The amount of Ti 4 C 2 S 2 at 1200 ∘ C and below is considered constant in the following calculations. Thus, the APT measurement at 1200 ∘ C is taken as the reference composition for the derivation of x and X Ti 0 . At 1050 and 1120 ∘ C, the differences in Nb and Ti concentrations relative to the reference, ΔNb and ΔTi, are calculated (Fig. 5). Assuming that the evolution of the amounts of Ti and Nb in solid solution are only related to the (Ti x ,Nb 1-x )C, the value of x can be estimated from the ratio R = ΔTi/ΔNb:

= + x R R 1 (4)
Although R=1.791 and R=1.301 are found at 1050 ∘ C and 1120 ∘ C respectively, R is considered to be constant over the temperature range studied as the composition of the (Ti x ,Nb 1-x )C precipitates was strictly identical from EDX measurement all over the considered temperature range. Therefore, the R value was chosen to be equal to the mean value, 〈R〉 = 1.546. Thus, the precipitate composition coherent with the respective evolutions of Ti and Nb is x = 0.61 ± 0.04.

The total amount of Ti available for the formation of (Ti x ,Nb 1-x )C is estimated using the mean R ratio, 〈R〉:

= + ⋅ - = ∘ ∘ X X R X X (1200 C) ( ( 1200

Estimation of (Ti x ,Nb 1-x )C volume fraction

The evolution of Nb solute content was used as the reference for the calculation of volume fraction. The volume fraction f v was estimated from a mass balance, assuming that Nb atoms lie either in the solid solution or in the precipitates:

= - - ⋅ f X X X X v v v SS P S S P at γ at Nb 0 Nb Nb Nb (6)
where X Nb P is the atomic fraction of Nb within (Ti x Nb 1-x )C, X Nb SS is the atomic fraction of Nb in solid solution in austenite, v γ at is the average atomic volume of austenite, and v P at is the one of (Ti x ,Nb 1-x )C. Here, [START_REF]Thermo-calc Software TCFE8 Steels/Fe-alloys Database[END_REF] and v P at was calculated by a law of mixture between that of pure TiC and NbC:

v γ at = 1.227 × 10 -29 m 3
= ⋅ + -⋅ v xv x v ( 1
)
P at TiC at NbC at [START_REF] Lu | Matrix dissolution techniques applied to extract and quantify precipitates from a microalloyed steel[END_REF] with v TiC at = 1.100 × 10 -29 m 3 , v NbC at = 1.181 × 10 -29 m 3 [START_REF]Thermo-calc Software TCFE8 Steels/Fe-alloys Database[END_REF] and

x = 0.61.
Neglecting the uncertainty on α v , the uncertainty on the volume fraction, Δf v , is given from the partial derivatives of f v :

= | ∂ ∂ |⋅ + | ∂ ∂ |⋅ + | ∂ ∂ |⋅ Δf f X ΔX f X ΔX f X ΔX v v v SS SS v P P Nb 0 Nb 0 Nb Nb Nb Nb ( 8
)
with:

∂ ∂ = ⋅ - f X α X X 1 ( ) v v P S S Nb 0 Nb Nb (9) ∂ ∂ = + ⋅ - f X X X α X X ( )
∂ ∂ = - ⋅ - f X X X α X X ( ) v P SS v P S S Nb Nb Nb 0 Nb Nb 2 (11)
The relative uncertainties on X Nb SS are given in Table 3. The uncertainty on ΔX Nb 0 is equal to the one of X Nb SS at 1280 ∘ C. Following the precipitate chemistry, X Nb P is equal to x/2. Thus, the uncertainty on X Nb P is estimated to 0.02 (0.04/2) according to the estimation of x presented in Section 3.3. The calculated volume fraction after isothermal heat treatments can be retrieved in Table 4. As expected, the precipitated volume fraction increases as the heat treatment temperature decreases.

Discussion

In this work, the calculation of the volume fraction of (Ti x ,Nb 1-x )C relies on a mass balance between the total amount of microalloying elements available for (Ti x ,Nb 1-x )C precipitation (X Ti 0 and X Nb 0

) and the actual amount of Ti and Nb in solid solution at a given temperature.

The measurement of solid solution Ti content was strongly hindered by the peak convolutions, so that only two isotopes representing 15% of the total Ti were used for the measurement. Since carbon will always be present in solid solution in microalloyed steels, this is a major disadvantage of this technique for measuring Ti solid solution contents. For this reason, the calculation of f v was based on Nb, of which measurement suffers less limitations and uncertainties. Note that in a more general view, a limitation of the method is the ability to accurately determine the solute content of the matrix, even for very low content. This can be only performed if there are no peaks overlap in the mass spectrum, or if those peaks overlaps can be corrected.

Each individual composition measurement is affected by uncertainties linked to the analyzed volume and to the analysis of the mass spectrum. Due to the mass balance used, the calculation of f v is not only affected by the uncertainties related to the matrix composition, X Nb SS , but also by those related to X Nb 0 . Therefore, the uncertainty in f v is greater than that on the composition measurements. A poor estimate 0 Nb 0 Fig. 4. Distribution of Nb and Ti for the investigated heat treated states, assuming that solute elements in (Ti,Nb)N remain trapped. Between 1280 and 1200 ∘ C, Nb evolution is only related to the formation of (Ti x ,Nb 1-x )C whereas Ti is affected by the formation of (Ti x ,Nb 1-x )C and Ti 4 C 2 S 2 . of X Nb 0 can lead to completely erroneous estimates of f v . Thus, as for any volume fraction determination technique based on mass balance, the proper determination of X Ti 0 and X Nb 0 is the most critical point for an accurate measurement. In this study, X Ti 0 and X Nb 0 are not equal to the total bulk alloy composition of the steel: a fraction of these elements are involved in other precipitates or may be segregated on defects such as grain boundaries. At 1280 ∘ C, (Ti x ,Nb 1-x )C and Ti 4 C 2 S 2 are completely dissolved. Therefore, Ti and Nb can only be located in (Ti,Nb)N. Performing a simple mass balance, assuming that all N reacts with Ti to form pure (Ti 0.95 Nb 0.05 )N (according to EDX) leads to 754 ppm of Ti and 218 ppm of Nb remaining in solid solution (Table 5). The solid solution content measured by APT are significantly lower: 531 ppm of Ti and 201 ppm of Nb. A combination of two factors may explain this discrepancy. It is first possible that the (Ti,Nb)N nitrides are actually (Ti,Nb)(C,N) carbonitrides, which would increase the amount of Ti and Nb trapped in this population. Indeed, as seen in Fig. 2 a), it seems that slight amounts of carbon are present in these precipitates. It is also possible that a proportion of the total amount of Ti Nb is trapped in austenite grain boundaries and not located within the austenite grains, where the APT samples are probably extracted. In order to avoid these difficulties, X Ti 0 and X Nb 0 are directly evaluated from APT measurements. Therefore, there is no need to locate all possible places where solute atoms may lie (segregated on defects and/or part of other precipitates). The direct measurement of the amount of solute atoms available for precipitation is much more precise than performing a mass balance from the bulk alloy composition.

A comparison of the atom probe measurements with equilibrium calculations is performed in Fig. 6. The measurements performed in this study are comparable with equilibrium calculations since recently published modelling results showed that (Ti x ,Nb 1-x )C reaches equilibrium volume fraction for the considered heat treatment times [START_REF] Graux | Precipitation and grain growth modelling in ti-nb microalloyed steels[END_REF]. Equilibrium calculations were performed using TCFE8 database [START_REF]Thermo-calc Software TCFE8 Steels/Fe-alloys Database[END_REF] on Thermo-Calc [START_REF] Andersson | Thermo-Calc & DICTRA, computational tools for materials science[END_REF] software to predict the stability domain of the (Ti x ,Nb 1-x )C. Calculations were run starting from the X Ti 0 and X Nb 0 determined with APT in Section 3.3. Sulfur and nitrogen were not considered in these calculations. The composition of other alloying elements are extracted from the ICP-MS bulk composition given in Table 1. Nb solid solution measurements by APT, on which volume fraction calculations are based, are in very good agreement with Thermo-Calc predictions.

On the other hand, the agreement regarding Ti is more questionable. Thermo-Calc predicted that the (Ti x ,Nb 1-x )C precipitates contain a greater proportion of titanium than what was estimated based on APT results: x = 0.68 for Thermo-Calc, against x = 0.61 ± 0.04 based on solid solution measurements. The current method of estimating the measurement uncertainties, based on the standard error obtained on several experiment values, might not accurately reflect the intrinsic error done when measuring very low solid solution content. Thus, the measurement of the Ti content, which is made from two minor Ti peaks in the mass spectrum, may actually be affected by a greater uncertainty than that calculated.

Although the analyzed volumes by APT have largely increased these last two decades (typically around 60 × 60 × 1000 nm 3 for this study), APT measurements remain very local. By way of comparison, a few grams of material are analyzed with selective dissolution techniques, which represents a volume 10 13 to 10 14 times larger than those analyzed by APT. APT is thus much more sensitive to heterogeneities in matrix composition, due for example to macro segregations. Nevertheless, in the present case, the standard error observed on several samples of the same state remained limited, of the order of 10% of the measured composition. Moreover, the measurements are comparable with bulk ICP-MS for substitutional alloying elements, as observed in Table 1. The measurements therefore seem to remain fairly precise, with a limited uncertainty.

The results of this study show that APT is nowadays a reliable technique for measuring solute levels as low as a few tens of ppm, which is particularly useful for estimating the volume fraction of precipitates in microalloyed steels. The method used here is relevant and gives consistent results, comparable with equilibrium calculations. It is worth highlighting the complexity of the studied industrial material, which contains several types of precipitates and many alloying elements. For such complex precipitation states, the determination of (Ti x ,Nb 1-x )C volume fraction would have been rather complicated by TEM or SANS. Moreover, this type of analysis can be transposed to other steels with different compositions, as long as a special attention is paid to possible peak convolutions on the mass spectrum. The good resolution and stability of the technique also makes it a prime candidate for conducting more fundamental studies of solubility product determination. Note that such matrix composition measurements could be used to estimate a solubility product of (Ti x ,Nb 1-x )C.

Conclusions

Atom probe analysis was applied to a TieNb microalloyed steel, to follow the evolution of austenite solute composition in Ti and Nb as a function of temperature. The technique is sufficiently precise to measure very low solute contents of the order of a few tens of ppm, which is particularly suitable for microalloyed steels.

Knowing the matrix composition, and the precipitate composition, it is possible to estimate the volume fraction of precipitates by applying a simple mass balance calculation. The most critical issue is the correct estimation of the total amount of solute elements available for the formation of the considered precipitates, here (Ti x ,Nb 1-x )C. This determination classically relies on the knowledge of the bulk alloy composition and the amount of atoms not involved in solid solution (usually involved in other precipitates). Direct atom probe tomography measurements of the amount of solute atoms available for (Ti x ,Nb 1-x )C precipitation avoid the difficult estimation of the amount of atoms possibly involved in many different places (primary precipitates, segregated on defects…).

Since the measurement of precipitate volume fraction in microalloyed steels is often tedious, it appears that atom probe is particularly suited for the measurement of the very low matrix compositions and precipitate volume fractions found in microalloyed steels. The technique also seems relevant to be used for the estimation of solubility products.

Declaration of competing interest

None.

Fig. 1 .

 1 Fig. 1. Optical micrograph of the as-received steel microstructure, etched with Nital. An homogeneous granular bainite microstructure is observed, presenting no particular texture.

Fig. 2 .

 2 Fig. 2. SEM/TEM images of the four types of precipitates observed in the as-received condition of the alloy: (Ti,Nb)N (a), complex assemblies of (Ti,Nb)N and MnS nucleated on an aluminum oxide inclusion (b), Ti 4 C 2 S 2 (c), and (Ti x ,Nb 1-x )C (d). The (Ti x ,Nb 1-x )C (d) are the one of particular interest for austenite grain growth control [16].

Fig. 3 .

 3 Fig. 3. Reconstructed atomic positions of a) carbon, b) niobium and c) titanium of an APT sample heat treated at 1200 ∘ C.

Fig. 5 .

 5 Fig. 5. Determination of x based on the respective evolution of Ti and Nb in solid solution below 1200 ∘ C. The total amount of Ti and Nb available for (Ti x ,Nb 1-x )C is deduced from the analysis.

Table 4

 4 Calculated volume fraction of (Ti x ,Nb 1-x )C from APT for each heat treatment performed.

Table 5 Fig. 6 .

 56 Fig. 6. Comparison of (a) composition of austenite and (b) (Ti x ,Nb 1-x )C volume fraction of precipitates determined by APT and by equilibrium calculations on Thermo-Calc.

Table 2

 2 Heat treatments performed.

	Temperature ( ∘ C)	1050	1120	1200	1280
	Time (min)	240	120	60	60

Table 3

 3 Precipitates observed, total number of ions analyzed by APT, and austenite Ti/ Nb solute content for each heat treatment performed. The standard error is calculated using Eq. (2).

	Heat treatment	Temperature ( ∘ C)	1050	1120	1200	1280
		Time (min)	240	120	60	60
		(Ti x ,Nb 1-x )C	✓	✓	✓	✗
	Precipitates observed	(Ti,Nb)N	✓	✓	✓	✓
	(✓ = yes/✗ = no)	Ti 4 C 2 S 2	✓	✓	✓	✗
		MnS	✓	✓	✓	✓
		Analyzed ions (⋅10 6 )	17.9	60.2	100.4	47.5
		Nb (at ppm)	4	61	155	201
	Austenite composition	Std error of the mean	1	6	11	22
		Ti (at ppm)	141	290	412	531
		Std error of the mean	15	7	33	67

Acknowledgments

The authors gratefully acknowledge the funding received from the European Commission, Research Fund for Coal and Steel, under grant agreement 709803 (NANOFORM). Thanks are due to the CLYM (www. clym.fr) for access to the 2010F microscope, and to Nicholas Blanchard for access and help on the JEOL 2100 microscope. This work was partly carried out owing to experimental GENESIS platform. GENESIS is supported by the Région Haute-Normandie, the Métropole Rouen Normandie, the CNRS via LABEX EMC, and the French National Research Agency as a part of the program "Investissements d'avenir" with the reference ANR-11-EQPX-0020.

Data availability

The raw/processed data will be made available on request.