domain: spi chim phys

text:
M.Sc Diringer Mc

Ph.D Tran Vl

Ph.D F Lux

M.D Ghoroghchian Pp
email: paiman_ghoroghchian@dfci.harvard.edu

Ph.D Alexandre Detappe
email: a.detappe@icans.eu

M.D. Donald Kufe
email: donald_kufe@dfci.harvard.edu

M.D P Peter Ghoroghchian

Anti-MUC1-C Antibody-conjugated Nanoparticles Potentiate the Efficacy of Fractionated Radiation Therapy Short-running title (46 characters): Anti-MUC1-C/NP radiotherapy enhancer

Purpose: Heavy-metal chelators and inorganic nanoparticles (NPs) have been examined as potential radioenhancers to increase the efficacy of external beam radiation therapy of various cancers. Most of these agents have, unfortunately, displayed relatively poor pharmacokinetic properties, which limit the percentages of injected dose (%ID/g) that localize to tumors and which shorten the window for effective radiation enhancement due to rapid tumor washout.

Materials and Methods:

To address these challenges, we sought to conjugate gadoliniumbased ultra-small (<5 nm) NPs to an antibody directed against the oncogenic MUC1-C subunit that is overexpressed on the surfaces of many different human cancer types. Note that the binding of the anti-MUC1-C antibody 3D1 to MUC1-C on the surface of a cancer cell is associated with its internalization and, thereby, to effective intracellular delivery of the antibody-associated payload, promoting its effective tumor retention. As such, we examined whether systemically administered anti-MUC1-C antibody-conjugated, gadolinium-based NPs (anti-MUC1-C/NPs) could accumulate within cell-line xenograft models of MUC1-Cexpressing (H460) lung and (E0771) breast cancers to improve the efficacy of radiation therapy (XRT).

Results:

The %ID/g of anti-MUC1-C/NPs that accumulated within tumors was found to be similar to that of their unconjugated counterparts (6.6 ± 1.4 vs. 5.9 ± 1.7 %ID/g, respectively).

Importantly, the anti-MUC1-C/NPs demonstrated prolonged retention in in vivo tumor microenvironments; and, as a result, the radiation boost was maintained over the course of fractionated therapy (3 x 5.2 Gy). We found that by administering anti-MUC1-C/NPs with XRT, it was possible to significantly augment tumor growth inhibition and to prolong the animals' overall survival (46.2 ± 3.1 days) when compared to the administration of control NPs with XRT (31.1 ± 2.4 days) or with XRT alone (27.3 ± 1.6 days; p < 0.01, log-rank).

Conclusion:

These findings suggest that anti-MUC1-C/NPs could be employed to enhance the effectiveness of radiation therapy and potentially to improve clinical outcomes.

Introduction

Gold nanoparticles (NPs) were the first NP-based radioenhancers to be tested in small animals for tumor therapy [START_REF] Hainfeld | The use of gold nanoparticles to enhance radiotherapy in mice[END_REF][START_REF] Schuemann | Roadmap to Clinical Use of Gold Nanoparticles for Radiation Sensitization[END_REF] . Their ability to augment the efficacy of external bean radiation was found to be mediated via the photoelectric effect and by Auger electron showers that arise due to the interactions between gold atoms and low energy photons produced by the external beam [START_REF] Butterworth | Physical basis and biological mechanisms of gold nanoparticle radiosensitization[END_REF][START_REF] Berbeco | Low Z target switching to increase tumor endothelial cell dose enhancement during gold nanoparticle-aided radiation therapy[END_REF][START_REF] Detappe | The effect of flattening filter free delivery on endothelial dose enhancement with gold nanoparticles[END_REF] . Based on these early findings, various inorganic NPs have been subsequently developed to similarly boost the efficacy of radiation therapy [START_REF] Liu | Metal-based NanoEnhancers for Future Radiotherapy: Radiosensitizing and Synergistic Effects on Tumor Cells[END_REF] , including ones comprised of bismuth, hafnium, [START_REF] Chen | Hafnium-doped hydroxyapatite nanoparticles with ionizing radiation for lung cancer treatment[END_REF][START_REF] Bonvalot | NBTXR3, a first-in-class radioenhancer hafnium oxide nanoparticle, plus radiotherapy versus radiotherapy alone in patients with locally advanced soft-tissue sarcoma (Act.In.Sarc): a multicentre, phase 2-3, randomised, controlled trial[END_REF][START_REF] Bonvalot | First-in-Human Study Testing a New Radioenhancer Using Nanoparticles (NBTXR3) Activated by Radiation Therapy in Patients with Locally Advanced Soft Tissue Sarcomas[END_REF] and gadolinium, [START_REF] Lux | AGuIX((R)) from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine[END_REF][START_REF] Detappe | Key clinical beam parameters for nanoparticle-mediated radiation dose amplification[END_REF][12][START_REF] Du | Engineered gadolinium-doped carbon dots for magnetic resonance imaging-guided radiotherapy of tumors[END_REF] amongst others [START_REF] Hainfeld | Iodine nanoparticles enhance radiotherapy of intracerebral human glioma in mice and increase efficacy of chemotherapy[END_REF][START_REF] Minafra | Radiosensitizing effect of curcumin-loaded lipid nanoparticles in breast cancer cells[END_REF] . Simulation studies conducted by McMahon and colleagues demonstrated that localized dose escalation is directly linked to the amount of internalized NP per cell and not to the atomic number of the metal comprising the NP [START_REF] Mcmahon | Biological consequences of nanoscale energy deposition near irradiated heavy atom nanoparticles[END_REF] . Since this seminal report, various approaches have been adopted to improve the internalization of radioenhancers in preclinical tumor models, including through functionalization of NPs with antibodies [START_REF] Chattopadhyay | Role of antibody-mediated tumor targeting and route of administration in nanoparticle tumor accumulation in vivo[END_REF][START_REF] Retif | Nanoparticles for Radiation Therapy Enhancement: the Key Parameters[END_REF] to aide in tumor targeting. [START_REF] Retif | Nanoparticles for Radiation Therapy Enhancement: the Key Parameters[END_REF][START_REF] Kunjachan | Nanoparticle Mediated Tumor Vascular Disruption: A Novel Strategy in Radiation Therapy[END_REF] Efforts have also focused upon optimization of the timing of radiation via imaging of the same NP construct by computed tomography (CT) [START_REF] Deng | Tumor targeted, stealthy and degradable bismuth nanoparticles for enhanced X-ray radiation therapy of breast cancer[END_REF][START_REF] Detappe | Ultrasmall Silica-Based Bismuth Gadolinium Nanoparticles for Dual Magnetic Resonance-Computed Tomography Image Guided Radiation Therapy[END_REF] or by magnetic resonance imaging (MRI). [START_REF] Detappe | Key clinical beam parameters for nanoparticle-mediated radiation dose amplification[END_REF]12 The aforementioned preclinical studies have provided the rationale for clinical trials based on 2 separate NP compositions and via 2 distinct routes of administration. Hafnium oxide-based NPs (NBTXR3) injected intratumorally in a hydrogel have been imaged effectively by CT, demonstrating persistence inside the tumor bed post-implantation as well as limited diffusion outside of the injection site [START_REF] Bonvalot | NBTXR3, a first-in-class radioenhancer hafnium oxide nanoparticle, plus radiotherapy versus radiotherapy alone in patients with locally advanced soft-tissue sarcoma (Act.In.Sarc): a multicentre, phase 2-3, randomised, controlled trial[END_REF][START_REF] Bonvalot | First-in-Human Study Testing a New Radioenhancer Using Nanoparticles (NBTXR3) Activated by Radiation Therapy in Patients with Locally Advanced Soft Tissue Sarcomas[END_REF] . In parallel, gadolinium-containing NPs (AGuIX) that were administered intravenously (IV) have been successfully tracked by MRI, enabling radiation therapy only after tumor localization [START_REF] Lux | AGuIX((R)) from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine[END_REF][START_REF] Verry | Treatment of multiple brain metastases using gadolinium nanoparticles and radiotherapy: NANO-RAD, a phase I study protocol[END_REF] . Both studies have demonstrated promising results and support the generalized capabilities of inorganic NPs to serve as radioenhancers in clinical applications. While IV injection of imaging agents enables accessibility to a multitude of cancers, NPs administered via the IV route have been shown to rapidly wash out from tumors if not internalized by tumor cells as was observed in the NANO-RAD trial (NCT02820454). In that study, the gadolinium-based NPs were injected once weekly over the course of radiation therapy. After a single administration, the NPs initially localized within tumor environments but were then rapidly washed out, which was a phenomenon that could be attributed to their accumulation in perivascular spaces and their inability to specifically bind or to be taken up by tumor cells. The observed tumor wash out likely underlies their inability to enhance radiation over the full course of therapy and highlights the necessity for repeated NP administration to maintain early radiation boost effects.

Given these findings, we hypothesized that NPs that are engineered to persist within the tumor environment could more effectively enhance the dose of fractionated radiation treatment and could obviate the need for repeated radioenhancer administration, which could decrease potential morbidities and/or treatment-related costs. In the present study, we conjugated multiple NPs to a single tumor-specific monoclonal antibody (mAb) to increase the dose of radioenhancer that is delivered to tumor cells. As the target for these antibodyconjugated NPs, we selected mucin 1 (MUC1) based on its high expression levels across a variety of solid and hematologic malignancies [START_REF] Nath | MUC1: a multifaceted oncoprotein with a key role in cancer progression[END_REF] . MUC1 consists of an extracellular subunit (MUC1-N) and a transmembrane subunit (MUC1-C). Effective antibody-based targeting of MUC1-N has proven elusive due to its continuous shedding from the tumor cell surface [START_REF] Kufe | Mucins in cancer: function, prognosis and therapy[END_REF][START_REF] Kufe | Functional targeting of the MUC1 oncogene in human cancers[END_REF] .

In contrast, the transmembrane MUC1-C subunit, which is not shed, functions as a potent oncoprotein, driving an aggressive tumor phenotype [START_REF] Kufe | MUC1-C oncoprotein as a target in breast cancer: activation of signaling pathways and therapeutic approaches[END_REF] . We have recently demonstrated targeting of the MUC1-C extracellular domain with the mAb 3D1 27 . 3D1 binds to a conserved alpha3 helix that is not expressed in other proteins and selectivity reacts with malignant cells across a variety of cancer types. In order to compare the radioenhancement properties of 3D1-conjugated NPs (i.e., anti-MUC1-C/NPs) to their unconjugated counterparts, we utilized the same type of nanoparticles that were employed in the NANO-RAD trial 28 (Fig. S1); both compositions were administered in combination with either a single high dose of external beam or with fractionated radiation therapy; and, treatment effects were compared in various models of lung and triple-negative breast cancer (Fig. 1A).

Results and Discussion

Synthesis of gadolinium-containing ultra-small nanoparticles. NPs were synthesized by following a previously established protocol 28 that incorporates the silane precursors tetraethyl orthosilicate (TEOS) and aminopropyl triethoxysilane (APTES) with the macrocyclic chelator DOTAGA anhydride (1,4,7,10-tetraazacyclododecane-1-glutaric anhydre-4,7,10-triacetic acid) in a one-pot synthesis; the resultant APTES-DOTAGA conjugates are then complexed with Gd 3+ , using a solution of GdCl3. Thereafter, they are isolated and mixed with other organic silanes to generate the NPs (Fig. 1B, Fig. S1). The advantages of NPs produced by this method, versus those constructed by a bottoms-up approach that is typically employed to synthesize other gadolinium-based NPs for clinical applications, are the facility afforded by swapping and comparing the activities of various metallic atoms that can be complexed with different chelators in order to control physical properties through reproducible experimental conditions. With an average hydrodynamic diameter of 6.3 ± 1.7 nm (as determined via dynamic light scatting; DLS) and a favorable stability profile (as examined over 3 days in solution; Fig. S2), these ultra-small gadolinium-based NPs were found to be comparable to those examined previously in the NANO-RAD trial, vide supra, and were advanced for further anti-MUC1-C mAb conjugation.

Conjugation of nanoparticles to anti-MUC1-C antibodies.

Modification of the free amino groups of 3D1 with transcyclooctene (TCO) enabled coupling to tetrazine (Tz) groups present on the surfaces of NPs 29 via established click chemistry protocols (Fig. 1C). TCO binding to Tz was validated by MALDI-MS (Fig. S3) and was optimized by using model reactions that examined various ratios of PEG4-TCO to Tz-modified antibody. Bulk UV-VIS measurements were utilized to follow modification of NPs, wherein the change in ABS of Tz at 517 nm was correlated to TCO-Tz coupling over time (Fig. S4). These experiments demonstrated that 33.6 TCO per anti-MUC1-C 3D1 mAb provided for optimal conjugation. Lower amounts of TCO did not result in sufficiently high levels of NP conjugation, yielding no detectable levels of fluorescently-labeled NPs after incubation with MUC1-C+ E0771 (murine triple negative breast cancer [START_REF] Domcke | Evaluating cell lines as tumour models by comparison of genomic profiles[END_REF] ) cells in culture; similarly, higher amounts of TCO per mAb did not result in better binding (Fig. S3). The final composition of the resultant anti-MUC1-C/NPs was determined by ICP-MS after removal of excess unreacted reagent, using a tangential filtration device equipped with a 15 nm size cutoff and by following previously published protocols 31 . These experiments determined that 4.1 ± 1.8 NPs bound to a single 3D1 mAb, which was consistent with a previously study 31 .

To examine the capabilities of the anti-MUC1-C/NPs to bind tumor cells, we labeled the NPs with Cy5.5 prior to mAb coupling and confirmed their specific uptake by MUC1-C+ human E0771 breast cancer cells via fluorescence microscopy (Fig. 1D). The presence of the Cy5.5 dye also enabled confirmation of cellular labeling with anti-MUC1-C/NPs via flow cytometry, which was conducted after NP incubation with (i) MUC1-C+ E0771 cells (Fig. 1E) and (ii) MUC1-C+ human H460 lung cancer cells [START_REF] Shames | The evolving genomic classification of lung cancer[END_REF] before and after silencing of MUC1-C expression, using a lentiviral vector encoding a MUC1-C shRNA [START_REF] Kharbanda | MUC1-C confers EMT and KRAS independence in mutant KRAS lung cancer cells[END_REF] (Fig. S5). For both cell lines, >95% cellular-labeling with Cy5.5 was observed only after incubation with Cy5.5-conjugated anti-MUC1-C/NPs as compared to various controls. The specificity of MUC1-C binding by the Cy5.5-conjugated anti-MUC1-C/NPs was further confirmed in a blocking experiment wherein pre-incubation of the same cells with free and unlabeled anti-MUC1-C mAb (i.e, 3D1) was conducted prior to addition of the labeled NPs, yielding <15% cellular labeling with the fluorophore and confirming the binding specificity of anti-MUC1-C/NPs.

In vitro studies of radiation enhancement. The radioenhancement properties of the anti-MUC1-C/NPs were examined using the E0771 and H460 cancer cell lines prior to advancing to animal studies. Clonogenic assays were performed after irradiating cells with 0 to 10 Gy doses, which were delivered in a single fraction by the small animal radiation research platform (SARRP). Augmentation of radiation-induced inhibition of in vitro cellular expansion was observed for cells treated with either unconjugated NPs or anti-MUC1-C/NPs as compared to those treated with radiation alone (p = 0.019, and p = 0.014, Mann Whitney test, respectively). The sensitive enhancement ratio (SER) values, which are defined as the radiation dose needed to reach a survival fraction of 50% in the radiation-containing treatment groups without and with the addition of radioenhancers [START_REF] Detappe | AGuIX nanoparticles as a promising platform for image-guided radiation therapy[END_REF] , were similar for anti-MUC1-C/NPs and their unconjugated (free NP) counterparts, equaling 1.69 (vs. 1.32) and 1.86 (vs. 2.00) in E0771 and in H460 cells, respectively. These SER values are also similar to those obtained with AGuIX NPs as reported with various cell lines [START_REF] Sancey | The use of theranostic gadolinium-based nanoprobes to improve radiotherapy efficacy[END_REF] , which confirmed a macroscopic radiation boost increase of approximately 25%. Note that there were no differences observed between the passive (NP) and the actively targeted (anti-MUC1-C/NP) groups (p = 0.42, Mann Whitney test) (Fig. 2A). The local dose enhancement was further confirmed by a DNA-damage assay based on staining for γH2AX foci at 15 min after exposure to a 2 Gy dose of radiation (Fig. 2B). Quantification of the number of γH2AX foci (Fig. 2C), which can be directly correlated to the numbers of double-stranded DNA breaks within the genome of cells, confirmed a significant increase observed with both XRT + nanoparticle-containing treatment groups (XRT + NP and XRT + anti-MUC1-C/NP groups) in comparison to the XRT only group (i.e., p = 0.021 and p = 0.027, Mann-Whitney test, respectively).

In vivo applications.

A preliminary evaluation of the in vivo pharmacology of anti-MUC1-C/NPs was conducted in healthy Balb/c mice. Animals were injected intravenously with nanoparticles (350 mg/kg of Gd 3+ in 0.2 mL PBS) alone or after conjugation to anti-MUC1-C mAb (i.e., anti-MUC1-C/NPs). The circulatory persistence of the anti-MUC1-C/NPs was found to be enhanced when compared to that of unconjugated NPs, demonstrating a half-life of 28.6 vs. 16.8 min, respectively (Fig. S6). At 72 h post-administration of either formulation, only traces amounts of Gd 3+ were detected in the liver, lungs, or other major organs of treated mice as quantitively determined via inductively-coupled plasma mass spectrometry (ICP-MS) (Fig. S7). These findings are consistent with those of previous studies with the base nanoparticle formulation (NPs) upon conjugation to an mAb against the B cell maturation antigen (BCMA) and when examined in preclinical models of multiple myeloma 31 .

In the current study, histologic examination of H&E stained tissues sections was performed by a veterinary pathologist upon necroscopy at 20 days after treatment administration and demonstrated no overt signs of acute tissue toxicities (Fig. S8), which was similarly consistent with the previous study 31 .

Based on these encouraging findings, we conducted further experiments to evaluate the potential utility of our NP constructs to serve as radioenhancers for cancer therapy. We employed two distinct murine models: (i) a subcutaneous xenograft model of lung cancer that was generated via implantation of human H460 lung cancer cells in the flanks of Balb/c nude mice; and, (ii) an orthotopic and syngeneic model of triple negative breast cancer that was generated by implantation of E0771 cells in the mammary fat pads of B6(Cg)-Tyrc-2J/J mice.

In order to determine the optimal window for radiation enhancement, which is defined as the time period whereby the maximal treatment effect is observed for the first irradiation fraction while avoiding concomitant toxicities to healthy surrounding tissues, the biodistribution patterns of fluorescently-labeled NPs and anti-MUC1-C/NPs were examined via in vivo optical imaging, using the orthotopic triple-negative breast cancer model. Imaging was conducted at various time points after IV administration of each formulation (Fig. 3A); at time points >1 h post-injection, negligible signals were observed in the heart, lungs, and healthy breast tissues of the treated mice. This time period after administration further allowed for substantial uptake of the nanoparticles within the tumor bed (6.6 ± 1.4%ID/g at 1 h, 4.3 ± 1.1%ID/g at 24 h, 3.8 ± 0.9%ID/g at 48 h), which was sufficient to generate a significant radiation boost. At 24 h post injection, residual NPs that were neither internalized nor surface-associated through MUC1-C-binding to tumor cells were observed to clear from the tumor microenvironment (Fig. 3B). Note that at this dose and (IV) mode of administration, no treatment group (i.e., unconjugated NPs, anti-IgG/NPs, nor anti-MUC1-C/NPs) whether alone or in combination with XRT was found to promote overt healthy-tissue toxicities as determined by serial body-weight measurements in the orthotopic (E0771) breast tumor model (Fig. 3C).

We hypothesized that the prolonged tumor retention characteristics exhibited by anti-MUC1-C/NPs could potentially obviate the practice of employing multiple injections to provide radiation enhancement, which has previously been deemed necessary when utilizing untargeted NPs to impart treatment effects [START_REF] Lux | AGuIX((R)) from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine[END_REF][START_REF] Bort | EPR-mediated tumor targeting using ultrasmall-hybrid nanoparticles: From animal to human with theranostic AGuIX nanoparticles[END_REF] . To test this possibility, we first employed a single fraction of high-dose stereotaxic radiation (10 Gy), which was administered in concert with different nanoparticle and control formulations. Irradiation was performed with a 220 kVp X-ray beam (SARRP platform), whereby 2 orthogonal beams were used to uniformly treat the entire tumor of each animal (Fig. S9) 12 . Using the subcutaneous (H460) lung cancer model (n=5 mice/group), we compared the radiation enhancement properties of anti-MUC1-C/NPs to those of the untargeted nanoparticle formulations (i.e., unconjugated NPs and anti-IgG/NPs) that were administered at the same total dose (350 mg/kg of Gd 3+ ) (Fig. 4A).

Based on the preliminary pharmacology experiments, vide supra, all nanoparticle and control (PBS) groups were administered via a single IV (tail-vein) injection at 1 h prior to irradiation in order to provide sufficient time for their accumulation within tumor environments. After animal irradiation, tumor sizes and body weights were monitored twice per week. For each subject, the study was terminated once its tumor reached size > 3 cm in any axis or when it experienced a >20% loss in body weight. Compared to mice in the control group (i.e., PBS + XRT alone) that lived for 26.4 ± 3.2 days, animals that were similarly administered radiation in conjugation with any nanoparticle formulation demonstrated significant improvements in tumor growth delay (p = 0.033, Mann Whitney test) as well as a prolongation in survival (to 29.3 ± 2.1 days in the unconjugated NP, 30.1 ± 1.6 days in the anti-IgG/NP, and 30.3 ± 1.6 days in the anti-MUC1-C/NP groups; p<0.001, Log-Rank test). Note that when administered in combination with a single fraction of high-dose radiation, there were no differences in survival imparted between any NP-containing treatment groups (p>0.05, Log-Rank test).

To investigate the potential for anti-MUC1-C/NPs to impart preferential enhancements in combination with fractionated radiation treatment (3x5.2Gy), a second therapeutic study was conducted whereby radiation was delivered at 1, 24, and 48 h after anti-MUC1-C/NPs, untargeted NPs (i.e., unconjugated NPs or anti-IgG/NPs), or after control (PBS) administration and by using the same dosimetry approach. Note that the total radiation dose in this study was equivalent to the single 10 Gy that was administered in the first study, vide supra, as based on the linear quadratic model equation. We hypothesized that with fractionated -as opposed to single high-dose -administration, the radiation boost to the tumor bed could be maximized for subjects in the anti-MUC1-C/NP group due to their enhanced persistence that was imparted by specific surface binding and/or internalization by tumor cells. By again employing the subcutaneous (H460) lung tumor model, we observed significant enhancements in tumor growth delay (p= 0.024, Mann-Whitney test) and in overall survival (p<0.001, Log-Rank test) for animals in the anti-MUC1-C/NP group (median survival of 46.2 ± 3.1 days) when compared to mice treated with either untargeted NPs (median survival of 31.1 ± 2.4 and 29.1 ± 2.2 days with unconjugated NPs and with anti-IgG/NPs, respectively) or with control (PBS + XRT) administration (median survival of 27.3 ± 1.6 days;

Fig. 4B).

Given the potential confounder of enhanced tumor vascularity, and thereby nanoparticle delivery, that may be exhibited by subcutaneous xenotransplanted tumors in murine model systems [START_REF] Hofmann | NIR-emissive PEG-b-TCL micelles for breast tumor imaging and minimally invasive pharmacokinetic analysis[END_REF] , we sought to further employ the orthotopic and syngeneic (E0771) breast tumor model to validate the putative radioenhancement properties imparted by combining anti-MUC1-C/NPs with fractionated XRT and in comparison to untargeted NPs and (PBS) control. E0771 cells that are implanted in the mammary fat pad have previously been shown to recapitulate the same vascular, metastatic and treatment resistance patterns of human triple-negative breast cancer [START_REF] Maeda | MUC1-C Induces PD-L1 and Immune Evasion in Triple-Negative Breast Cancer[END_REF] . Irradiation, which was conducted by using 2 orthogonal beams and which focused upon the mammary fat pad of the animal, was performed at 1, 24, and 48 h after (IV) administration of each treatment group. The same endpoints were adopted as with the previous therapeutic study (in the subcutaneous model, vide supra); and, the results were consistent between the two model systems, demonstrating significant improvements in overall survival for mice that received fractionated radiation in combination with any NPcontaining treatment group and when compared to those that received radiation or the same NP treatments alone (Fig. 4C). The greatest effects where seen with animals that received fractionated radiation therapy in conjunction with anti-MUC1-C/NPs, exhibiting a >15-day improvement in overall survival when compared to all other groups. Upon reaching the study endpoint, the tumor from each mouse was extracted and its histologic features were examined after H&E staining (Fig. 4D). Large fractions of necrosis (28.8 ± 5.4%) were observed in the tumors of animals that were treated with both fractionated radiation and anti-MUC1-C/NPs, confirming the observed radiation boost when compared with the animals that were treated with XRT+NP (7.6 ± 3.2 % necrosis, p<0.01, Mann Whitney test) or with XRT alone (3.5 ± 1.9% necrosis, p<0.001, Mann Whitney test)

Conclusions

Upon systemic administration of untargeted NPs, a small fraction accrues in the perivascular spaces of tumors, wherein constructs that are comprised of heavy metals may be used as radioenhancers to augment treatment effects. Over time, the majority of these extravasated NPs are cleared from tumor environments via the lymphatics, by reentry into the systemic circulation and/or via non-specific internalization by select tumor and stromal cells. Here, we demonstrate that ultra-small Gd 3+ -containing NPs that are conjugated to an anti-MUC1-C antibody exhibit prolonged retention in both subcutaneous and orthotopically-implanted tumors in murine model systems, thereby improving radioenhancement properties when combined with fractionated radiation therapy. Such augmentation in properties suggest that these anti-MUC1-C/NPs may afford for less frequent and/or smaller total dose administrations to achieve comparable effectiveness to that observed with their untargeted NP counterparts. If validated in future translational studies, our exemplified method may be utilized with other antibody-targeted NPs, rendering a potentially powerful approach for radiation enhancement that is generalizable and mediated entirely by antibody specificity.

The applications of such constructs may further translate into lower costs, reduced radiation doses, improved safety profiles, and/or improved clinical outcomes for patients.

Material and Methods

Anti-MUC1-C monoclonal antibody: Dry aliquots of the anti-MUC1-C mAb 3D1 were freshly prepared by dissolution in PBS (5 mL) and thereafter stored at 4 o C prior to use.

Synthesis of NP formulations:

To a freshly prepared solution of APTES-DOTAGA (11.7 mM, pH 4) was added GdCl3 in 3 equal aliquots to a final molar ratio of APTES-DOTAGA:Gd of 1:0.9; the solution pH was thereafter monitored and maintained. Upon 48 h of incubation at 80 °C, the solution was adjusted to pH 9; TEOS (2 eq) and APTES (1 eq) were added; and, the volume of water was adjusted to obtain final concentrations of APTES-DOTAGA(Gd 3+ ), APTES-DOTAGA, TEOS and APTES of 9, 1, 20, and 10 mM, respectively. This mixture was subsequently stirred for an additional 18 h at 25 °C, adjusted to pH 4.5, and further incubated for 18 h at 80 °C to generate the NP suspensions. The solution pH was then adjusted to 2; and, NPs were concentrated and purified by tangential ultrafiltration, using the Vivaspin ® device (MWCO = 3 kDa; SigmaAldrich). The NP suspension was further filtered through a 0.2 mm membrane (SigmaAldrich) and freeze-dried for longer-term storage. NP compositions were characterized by DLS and by HPLC analyses. Prior to biological application, they were resuspended with Gd 3+ (10 mM). To generate fluorophore-labelled NP constructs, small volume additions (0.1 mL) of a stock solution of Cy5.5-NHS ester (10 mM in DMSO) were added to NPs slowly and under agitation to a final molar ratio of 1:500 Cy5.5 to Gd 3+ in solution. For antibody coupling, NPs were further surface modified upon small volume additions (1 mL) of a stock solution of Tz-PEG4-NHS ester (10 mM in DMSO) and to a final molar ratio of 1:10 Tz to Gd 3+ . All modified NP suspensions were then stirred for 5 h at RT and thereafter purified by tangential ultracentrifugation, using the Vivaspin ® device (MWCO = 3 kDa). The number of Tz groups in Tz-PEG4-NP suspensions were determined by UV-vis spectroscopy. The complexes were then purified by using a tangential filtration device equipped with a 50 kDa molecular weight cutoff membrane (Millipore) as previously described 31 . The final concentrations of the anti-MUC1-C/NPs were determined by ICP-MS, using an Agilent 7900 instrument (Agilent Technologies, Inc.). Clonogenic assay: Various NP-based treatment groups (0.4 mg/mL of Gd 3+ ) were incubated with E0771 and H460 cells for 45 min prior to cellular irradiation with a dose of 2, 4, 6, 8, or 10 Gy, which was administered with a 220 kVp X-ray beam (SARRP, Xstrahl). Following previously reported protocols, [START_REF] He | DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells[END_REF][START_REF] Drane | TIRR regulates 53BP1 by masking its histone methyl-lysine binding function[END_REF] the treated cells were subsequently incubated for 6 h, thereafter counted, re-seeded in 10 cm dishes (at 300 cells per plate), and allowed to grow for an additional 10 d before staining with a dye solution comprised of 1% crystal violet in 10% ethanol; foci were then counted manually.

Quantification of DNA damage: Various NP-based treatment groups (0.4 mg/mL of Gd 3+ ) were incubated with E0771 for 30 min prior to irradiation with 2 Gy. The treated cells were then fixed with 4% paraformaldehyde in PBS for 30 min at RT. Fixed cells were blocked with 1% BSA, 10% FBS, and 0.3% tritonX-100 in PBS prior to staining overnight at 4 o C with anti-γH2AX antibody (Millipore). Following previously reported protocols, [START_REF] He | DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells[END_REF][START_REF] Drane | TIRR regulates 53BP1 by masking its histone methyl-lysine binding function[END_REF] Therapeutic studies: 5-week old female Balb/c nude mice were implanted with H460 cells (1 million cells under the skin) and B6(Cg)-Tyrc-2J/J mice were implanted with E0771 cells (1 million cells via mammary fat pad injection) to establish the subcutaneous (lung) and orthotopic (triple negative breast) tumor models, respectively. Animals were included in the study when their tumors reached >7 mm in largest axis and were administered one of the following treatments (n = 5 mice/group): PBS (control), unmodified NPs, or anti-MUC1-C/NPs (350 mg/kg of Gd 3+ ; 5 mg/kg of anti-MUC1-C) alone or in further combination with external beam radiation (vide infra); untargeted (i.e., anti-IgG) NPs administered at the same dose and in combination with XRT served as an additional control. Mice were administered a single dose of each NP group by IV tail-vein injection at 30 min prior to irradiation with either a single dose of 10 Gy or with 3 equal fractions of 5.2 Gy each, which were delivered with a 220 kVp X-ray beam collimated to the tumor size and by using the SARRP platform 12 . Tumor sizes were thereafter monitored daily by using calipers; and, animals were removed from

  a secondary antimouse AlexaFluor-594 conjugated IgG (Abcam) was used for staining and micrographs were obtained by using an upright Carl Zeiss microscope with an HXP 120C light source and a 63x/1.4 oil plan-apochromat objective. Semiquantitative analyses were conducted to compare the numbers of foci per cell expressing γH2Ax. The signal intensities of individual foci were quantified by using the CellProfiler cell imaging software (version 3.1.8). Animal experiments: All animals experiments were performed in accordance with the rules and regulations set-forth by the IACUC of the hospital and under an approved protocol (#03-029). Toxicity studies: Healthy female Balb/c nude mice were injected (IV) with a single dose of either PBS, NP, NP-IgG, anti-MUC1-C mAb (3D1), or anti-MUC1-C/NPs. Their body weights were monitored daily, starting on the day of injection. Blood was collected by submandibular puncture to determine the half-life of the different NP formulations. After 20 days, the major organs of the animals were collected and stained with H&E prior to histological analyses by a board-certified veterinary pathologist.. Biodistribution study: 5-week old female B6(Cg)-Tyrc-2J/J mice were implanted with E0771 cells (1 million via mammary fat pad injection). They were included in study when their tumors reached >7 mm in largest axis and were administered one of the following treatments (n = 5 mice/group per timepoint): PBS (control), unmodified NPs, or anti-MUC1-C/NPs (350 mg/kg of Gd 3+ ; 5 mg/kg of anti-MUC1-C mAb). Cy5.5-bound NPs were tracked by imaging after IV administration, using an IVIS Spectrum-bioluminescence and fluorescence imaging system (Perkins Elmer) for qualitative quantification. ICP-MS measurements were performed to confirm the presence of NPs in the major organs at different timepoints after IV administration, following previously established protocols 31 .

FiguresFigure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. Gadolinium-containing nanoparticles enhance the effects of radiation therapy on cultured tumor cell lines. A) Clonogenic assay on H460 (lung) and E0771 (triple-negative breast) cancer cells treated with different doses of radiation alone (XRT) or in combination with either untargeted or anti-MUC1-C/NPs that were incubated with the cells for 3 h and at equivalent doses (0.5 mg/mL). B) Fluorescence imaging of γH2AX foci in micrographs of E0771 cells (dotted) that were incubated for 3 h with various treatment groups before (-XRT) and after radiation (+XRT) administration (2Gy). C) Quantification of the numbers of γH2AX foci (observed amongst 250 counted cells) for E0771 (top) and H460 cells (bottom) that were treated with various control or NP formulations either alone or in combination with radiation (2 Gy).

Figure 3 .

 3 Figure 3. Pharmacology, biodistribution and gross toxicity experiments with anti-MUC1-C/NPs. A) In vivo optical imaging and B) quantitative biodistribution (via ICP-MS for Gd content in excised tissues) at various timepoints after the systemic administration of Cy5.5-labeled untargeted (i.e., NP and anti-IgG) NPs and antibody-conjugated (anti-MUC1-C) NPs in the orthotopic breast tumor model (i.e., E0771 cells implanted in the mammary fat pads of C57BL/6 mice); untargeted NPs demonstrated rapid tumor washout while the antibody-conjugated constructs were found to persist within tumor environments for > 48 h. C) Body weight measurements of breast tumor-bearing mice at various time points after a single administration of various treatment groups alone (control) or after either single highdose (XRT (10 Gy)) or fractionated radiation therapy (XRT (3x5.2 Gy)).

Figure 4 .

 4 Figure 4. Potential therapeutic utility of combining anti-MUC1-C/NPs with radiation. A) Tumor growth and Kaplan Meier survival curves for orthotopic breast-tumor bearing mice (i.e., B6(Cg)-Tyrc-2J/J mice with E0771 cells implanted in their mammary fat pads) that were administered various NP or control formulations alone or in combination with a single high dose of external beam radiation (10 Gy). Tumor growth and survival were followed in B) the subcutaneous (H460) lung tumor-bearing model and C) the orthotopic breast-tumor model as a function of administration of the same NP and control groups alone or in combination with fractionated radiation therapy (3x5.2 Gy). C) Representative images of H&E-stained tumor micrographs after the last day of RT (20 days after tumor implantation), confirming increased necrosis in a tumor from an animal that was administered anti-MUC1-C/NPs followed by fractionated radiation (3x5.2 Gy).

Funding statement: A.D. acknowledges support from the Philippe Foundation. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under grant numbers CA97098, CA166480, CA229716 and CA233084 awarded to D.W.K. P.P.G. acknowledges support from the Charles W. and Jennifer C.

Johnson Clinical Investigator Fund and from the Kathryn Fox Samway Foundation.

All data generated and analyzed during this study are included in this published article (and its supplementary information files). Raw data can be requested to by joining the corresponding authors.

study when their tumor reached >3 cm in the largest axis, when they exhibited >15% loss in body weight or when they were moribund.


Périne Millot

Carine San

Evangeline Bennana

Baptiste Porte

Nicolas Vignal

Jacques Hugon

Claire Paquet

Benoit Hosten

François Mouton-Liger
email: francois.mouton-liger@inserm.fr

François Mouton- Liger

STAT3 inhibition protects against neuroinflammation and BACE1 upregulation induced by systemic inflammation

Keywords: STAT3, Neuroinflammation, microglial activation, LPS, Stattic, BACE1 ABBREVIATIONS

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

INTRODUCTION

Interleukin 6 (IL-6)/Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway is involved in various physiological processes, including immune reaction, cell growth and hematopoiesis. JAK/STAT3 pathway is activated by cytokines such as IL-6 and intercellular mediators [START_REF] Mori | IL-1beta and TNFalpha-initiated IL-6-STAT3 pathway is critical in mediating inflammatory cytokines and RANKL expression in inflammatory arthritis[END_REF]. In non-stimulated cells, STAT3, like other STATs proteins, remains in an inactive cytoplasmic conformation. Then, once turned on, STAT3 translocates into the nucleus where it behaves as a transcription factor. This transcriptional activation of STAT3 is induced by phosphorylation on a critical tyrosine residue (Tyr705) and dimerization of the protein [START_REF] Villarino | Mechanisms of Jak/STAT signaling in immunity and disease[END_REF].

Abnormal activation of STAT3 signaling was also associated with neurodegenerative disorders such as Huntington disease or Alzheimer Disease (AD) [START_REF] Ben Haim | The JAK/STAT3 pathway is a common inducer of astrocyte reactivity in Alzheimer's and Huntington's diseases[END_REF]. The pathology of AD is characterized by the presence of extracellular amyloid-β (Aβ) aggregates, forming Aβ plaques, due to an increase of the expression of β-APP cleaving enzyme-1 (BACE1), the enzyme responsible for the first cleavage of the amyloid precursor protein (APP) [START_REF] Hugon | PKR involvement in Alzheimer's disease[END_REF][START_REF] Mouton-Liger | Oxidative stress increases BACE1 protein levels through activation of the PKR-eIF2alpha pathway[END_REF].

Other features include neurofibrillary tangles, neuroinflammation and neuronal loss [START_REF] Duyckaerts | Classification and basic pathology of Alzheimer disease[END_REF]. In AD patients, neuropathological analyses revealed an elevation of STAT3 Tyr705 phosphorylation in the hippocampus [START_REF] Ben Haim | The JAK/STAT3 pathway is a common inducer of astrocyte reactivity in Alzheimer's and Huntington's diseases[END_REF][START_REF] Wan | Tyk2/STAT3 signaling mediates beta-amyloid-induced neuronal cell death: implications in Alzheimer's disease[END_REF] and phosphor-STAT3 is strongly increased in hippocampus of APP/PS1 transgenic mice [START_REF] Wan | Tyk2/STAT3 signaling mediates beta-amyloid-induced neuronal cell death: implications in Alzheimer's disease[END_REF]. In this model, STAT3 activation may especially act as an inducer of astrocyte reactivity [START_REF] Ben Haim | The JAK/STAT3 pathway is a common inducer of astrocyte reactivity in Alzheimer's and Huntington's diseases[END_REF] and its selective down-regulation in astrocytes improved reduces memory decline [START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF][START_REF] Reichenbach | Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model[END_REF]. Neuroinflammation is marked by increasing proinflammatory cytokines production and microglial cells overactivation, in particular around Aβ plaques [START_REF] Heneka | Neuroinflammation in Alzheimer's disease[END_REF]. STAT3 can interfere with BACE1 levels and neuroinflammation in AD.

The translation of BACE1 is controlled by a pro-apoptotic kinase involved in several pathological hallmarks of AD: the Protein kinase R (PKR) [START_REF] Hugon | PKR involvement in Alzheimer's disease[END_REF] inducing the phosphorylation of eukaryotic initiation factor 2 alpha (eIF-2α) [START_REF] Mouton-Liger | Oxidative stress increases BACE1 protein levels through activation of the PKR-eIF2alpha pathway[END_REF]. BACE1 is also regulated at the transcriptional level, notably by STAT3 activation [START_REF] Carret-Rebillat | Neuroinflammation and Abeta accumulation linked to systemic inflammation are decreased by genetic PKR down-regulation[END_REF][START_REF] Liu | Palmitate induces transcriptional regulation of BACE1 and presenilin by STAT3 in neurons mediated by astrocytes[END_REF][START_REF] Wen | Transcriptional regulation of beta-secretase by p25/cdk5 leads to enhanced amyloidogenic processing[END_REF]. STAT3 phosphorylation plays a role in cytokines secretion, involved in AD neuroinflammation in AD, such as IL-1β or tumor necrosis factor (TNF-α) [START_REF] Samavati | STAT3 tyrosine phosphorylation is critical for interleukin 1 beta and interleukin-6 production in response to lipopolysaccharide and live bacteria[END_REF].

We have previously shown that systemic inflammation induced by intraperitoneal (i.p.) lipopolysaccharide (LPS) administration can trigger neuroinflammation and increase brain Aβ production in wild type (WT) mice [START_REF] Carret-Rebillat | Neuroinflammation and Abeta accumulation linked to systemic inflammation are decreased by genetic PKR down-regulation[END_REF]. In the same study, the genetic invalidation of PKR, led to the reduction of neuroinflammation and Aβ accumulation associated with a reduction of STAT3 activation [START_REF] Carret-Rebillat | Neuroinflammation and Abeta accumulation linked to systemic inflammation are decreased by genetic PKR down-regulation[END_REF]. Inhibition of STAT3 is an emerging field of research for cancer therapy [START_REF] Igelmann | STAT3 and STAT5 Activation in Solid Cancers[END_REF][START_REF] Akiyama | The antitumor activity of the STAT3 inhibitor STX-0119 occurs via promotion of tumor-infiltrating lymphocyte accumulation in temozolomide-resistant glioblastoma cell line[END_REF][START_REF] Wei | Targeting colon cancer with the novel STAT3 inhibitor bruceantinol[END_REF][START_REF] Bai | A Potent and Selective Small-Molecule Degrader of STAT3 Achieves Complete Tumor Regression In Vivo[END_REF]. Several inhibitors have been developed and are currently tested in clinical trials for solid and hematopoietic tumors [START_REF] Ogura | Phase I study of OPB-51602, an oral inhibitor of signal transducer and activator of transcription 3, in patients with relapsed/refractory hematological malignancies[END_REF][START_REF] Yoo | Phase I Dose-Finding Study of OPB-111077, a Novel STAT3 Inhibitor, in Patients with Advanced Hepatocellular Carcinoma, Cancer research and treatment[END_REF]. For this study, we selected Stattic, a small molecule that selectively inhibits activation, dimerization, and nuclear translocation of STAT3 [START_REF] Schust | Stattic: a small-molecule inhibitor of STAT3 activation and dimerization[END_REF][START_REF] Zhang | Impairment of Hepcidin Upregulation by Lipopolysaccharide in the Interleukin-6 Knockout Mouse Brain[END_REF] and used the LPS model to assess the consequences of a chemical STAT3 inhibition on neuroinflammation and hippocampal BACE1 levels.

MATERIAL & METHODS

Animals and treatments

All experimental procedures were approved by the Ethical Committee on Animal Experiments of Paris Nord (CEEA n°121). Eight-week-old C57BL/6 male mice were obtained from Janvier Labs (Le Genest-Saint-Isle, France). Mice were housed in cages with alternative periods of light and dark (12h/12h) with water and food ad libitum. Efforts were made to minimize the number of mice used in accordance with European guidelines for care of laboratory animal's 2010/63/UE by using the same brain for immunohistochemistry and protein quantification. Every day, i.p. injection of 1 mg/kg of LPS from Salmonella enterica serotype typhimurium (Sigma Aldrich, L'Isle d'Abeau Chesnes, France) or equivalent volume of saline was performed for 3 days with a syringe. In addition, mice were injected i.p with 20 mg/kg Stattic (solubilized in 2% DSMO+30% polyethylene glycol) or vehicle (containing 2% DMSO+30% polyethylene glycol) at the same time and following the same procedure. The concentration of Stattic used in this study was determined based on several publications using this dosage in in vivo models [START_REF] Das | Rapamycin protects against myocardial ischemia-reperfusion injury through JAK2-STAT3 signaling pathway[END_REF][START_REF] Han | Inhibition of STAT3 signaling targets both tumor-initiating and differentiated cell populations in prostate cancer[END_REF]. Four groups of mice were explored: saline controls, Stattic, LPS+Saline and LPS+Stattic. The number of mice used for each group is detailed in the figure legend corresponding to each experiment. 24 hours after the last injection, mice were anesthetized with lethal dose of ketamine/xylazine. Brains were extracted and sliced on the middle of the sagittal plan. One hemisphere was immerged in paraformaldehyde (PFA 4%) for immunohistochemistry. The second part was dissected.

Hippocampus were separated on ice and frozen in liquid nitrogen for biochemical analyses.

Immunohistochemistry

Hemispheres were successively placed in 0.4% PFA (5 days), 30% sucrose (24h), frozen in isopentane and then sectioned using a cryostat. Sagittal sections (30 µm) were chosen according to the Mouse Brain Atlas at 0.84 to 1.32 lateral positions. After PBS rinsing, brain sections were blocked with 5% BSA (bovine serum albumin, Sigma Aldrich) and 0.4% Triton (Janssen Chemical, Beerse, Belgium) for 1 hour. The brain sections were then incubated with the primary antibody for IBA-1 (1:1000, Wako, Richmond, VA), MAC-1/CD11b (1:200, Bio-Rad/AbD Serotec) or NeuN (Merck-Millipore, Darmstadt, Germany) overnight at 4°C. The sections were washed and incubated for 2 hours with secondary antibodies goat anti-rabbit or goat anti-rat AlexaFluor 488 (ThermoFisher Scientific, Waltham, MA). Sections were mounted on blades with mounting medium containing DAPI (Vectashield, Vector Laboratory, Burlingame, CA) and imaged using Dmi8 microscope confocal (Leica, Wetzlar, Germany) and LASX (Leica).

Microglial morphological analyses were performed by combining several ImageJ plugins [START_REF] Young | Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ[END_REF] including AnalyzeSkeleton (2D/3D) [START_REF] Arganda-Carreras | 3D reconstruction of histological sections: Application to mammary gland tissue[END_REF] and FracLac [START_REF] Karperien | Quantitating the subtleties of microglial morphology with fractal analysis[END_REF]. NeuN Immunoreactive cells were counted in each hippocampal sub-regions by an experimenter blind to group identity. Anatomical regions were determined as per Franklin and Paxinos (2007). CA1 and CA2 were combined (CA1/2) due to the relatively amorphous boundary separating these two regions.

Immunobloting and ELISA

Proteins were extracted from hippocampus by sonication with Bioruptor (Diagenode, Denville, NJ) until homogenization in RIPA lysis buffer containing phosphatase and protease inhibitors. Proteins quantification was determined by a colorimetric method (BCA, Thermo scientific) and absorbance was measured using Victor technology (PerkinElmer, Wellesley, MA). After denaturation, 50 µg of protein lysate were separated on a gradient gel 4-20% polyacrylamide Bis-Tris (Bio-Rad). Blots were transferred on nitrocellulose membranes (BioRad, Hercules, CA). Membranes were blocked 1h in 5% milk (Fisher BioReagent, Bio-Rad) and then incubated with primary antibody overnight at 4°C. After TBST rinses, membranes were incubated with a secondary antibody (HRP Goat Anti-Rabbit IgG, Jackson ImmunoResearch, Cambridgeshire, UK). Chemiluminescence signals were detected by placing blots in Clarity Western ECL (Bio-Rad) and using an imaging system (Chemi-doc XRS, Bio-Rad). Results were normalized with GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and analyzed on Multigauge software (Fuji-Film, Tokyo, Japan).

Quantification of cytokines, TNF-, IL-1β, IL-6, IFN-γ (Interferon γ), IL-10 and IL-17-, was performed using Luminex multiplexing system (Bio-Rad) according to the manufacturer's protocols.

Statistical analyses

STAT3 inhibition decreases LPS-induced microglial activation in the hippocampus.

To assess the impact of Stattic on neuroinflammation, we performed an immunohistochemistry using two markers of microglial cells/ IBA-1 (ionized calcium-binding adapter molecule 1) (Figure 2A-D) and MAC-1 (CD11b) (Supplementary Figure 1A). We observe a strong increase (+139%) of IBA-1 expression in LPS-treated mice hippocampus compared to untreated mice (Figure 2A, 2C and 2F). Quantification of MAC-1 staining reveal a +284% increased expression (Supplementary Figure 1B). The enhanced intensity of these two markers is associated with morphological changes typical of activated microglia, such as enlarged amoeboid cell bodies with shorter ramified projections (see right boxes under Figure 2A-D). By combining several ImageJ plugins [START_REF] Young | Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ[END_REF], we have precisely quantified microglial ramification length. In the LPS condition, we found a 49% reduction of cell process length (Figure 2G). Stattic injections in LPS treated mice reduce both IBA-1 and MAC-1 levels (respectively -39% and -44%) (Figure 2C-D, F and Supplementary Figure 1B) and microglial morphological switch from ramified to amoeboid state (+64% for cell ramification length) compared to LPS only treated mice (Figure 2G). No statistical difference is observed between the saline group and the saline+Stattic group for the two microglial markers (Figure 2A-B, 2F and Supplementary Figure 1B). To assess the potential impact of STAT3 inhibition on neuronal death, we perform an immunohistochemistry with the neuronal marker, NeuN (Supplementary Figure 2A-D

). Quantification of NeuN immunoreactive cells in the different

Millot et al. STAT3 inhibition protects against neuroinflammation and BACE1 upregulation regions of the hippocampus, Cornu Ammonis (CA)1/2, CA3 and Dentate gyrus (DG), did not reveal any difference between the 4 groups of our study (Supplementary Figure 2E)

Stattic reduces cytokines production triggered by LPS administration.

To determine the impact of Stattic on cytokines production, we measured using Luminex six mediators known for their role in brain inflammation: TNF-, IL-1β, IL-6, IFN-γ, IL-10 and IL-17 . IL-1β, TNF-α and IL-6 are significantly increased in LPS-injected mice hippocampus in comparison to saline treated animals, respectively +104% (Figure 3A), +50% (Figure 3B), and +44% (Figure 3C). These level changes are drastically reduced following Stattic injections (-34% for IL-1β, -39% for TNF-α and -35% for IL-6). IFN-γ production is also significantly increased following LPS production and Stattic injections reverse this augmentation (Figure 3D). The administration of Stattic alone does not affect cytokines production (Figure 3A-D).

LPS or Stattic exposure does not modify the levels of the two other tested cytokines, IL-10 and IL-17 (data not shown).

Stattic reduces hippocampal BACE1 levels in LPS-treated mice

BACE1 protein levels are assessed with immunoblot analyses in hippocampal extracts from saline, saline+Stattic, LPS and LPS+Stattic treated mice (Figure 4A). We observe a 33% increase of BACE1 levels in the hippocampus of LPS-treated mice compared to controls.

Stattic injections in LPS-treated mice reduce BACE1 concentrations to control levels. No significant difference between saline-group and saline-Stattic-treated group is detected (Figure 4B).

DISCUSSION

Our results show that peripheral injections of Stattic, a selective STAT3 inhibitor, protect in vivo against, (i) neuroinflammation by reducing microglial activation and pro-inflammatory cytokines production, and (ii) excess hippocampal BACE1 protein levels induced by LPS.

In this study, we used a mouse model previously developed by our group showing that systemic inflammation produced by LPS administration can induce neuroinflammation and BACE1 upregulation in WT mice, two pathophysiological hallmarks involved in AD [START_REF] Carret-Rebillat | Neuroinflammation and Abeta accumulation linked to systemic inflammation are decreased by genetic PKR down-regulation[END_REF].

Although LPS models are commonly used to study aberrant innate immune activation in AD models [START_REF] Qin | Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration[END_REF][START_REF] Sheng | Lipopolysaccharide-induced-neuroinflammation increases intracellular accumulation of amyloid precursor protein and amyloid beta peptide in APPswe transgenic mice[END_REF], few of them also induce deregulation of Aβ metabolism [START_REF] Sheng | Lipopolysaccharide-induced-neuroinflammation increases intracellular accumulation of amyloid precursor protein and amyloid beta peptide in APPswe transgenic mice[END_REF].

Preliminary works of our group has demonstrated the involvement of PKR, a pro-apoptotic kinase involved in several pathways leading to AD brain lesions [START_REF] Hugon | PKR involvement in Alzheimer's disease[END_REF][START_REF] Ill-Raga | Activation of PKR causes amyloid ss-peptide accumulation via derepression of BACE1 expression[END_REF][START_REF] Mouton-Liger | Oxidative stress increases BACE1 protein levels through activation of the PKR-eIF2α pathway[END_REF][START_REF] Tible | PKR knockout in the 5xFAD model of Alzheimer's disease reveals beneficial effects on spatial memory and brain lesions[END_REF], in the severity of hippocampal neuroinflammation as in the regulation of BACE1 expression [START_REF] Carret-Rebillat | Neuroinflammation and Abeta accumulation linked to systemic inflammation are decreased by genetic PKR down-regulation[END_REF]. Aberrant phosphorylation of STAT3 seems to be the key mechanism linking phosphorylated PKR, neuroinflammation and BACE1 deregulation. The group of Kroemer has proposed that PKR and STAT3 could directly interact by forming a protein complex that may modulate autophagy [START_REF] Niso-Santano | Direct interaction between STAT3 and EIF2AK2 controls fatty acid-induced autophagy[END_REF][START_REF] Shen | Cytoplasmic STAT3 represses autophagy by inhibiting PKR activity[END_REF]. The existence of a similar interaction in microglia and neurons remains to be explored.

STAT3 is already a therapeutic target, particularly in certain cancers, and numerous analyzes on the side effects and toxicity of STAT3 inhibitors have already been carried out [START_REF] Wei | Targeting colon cancer with the novel STAT3 inhibitor bruceantinol[END_REF][START_REF] Pan | Stat3 inhibitor Stattic exhibits potent antitumor activity and induces chemo-and radio-sensitivity in nasopharyngeal carcinoma[END_REF].

Among STAT3 inhibitors, we chose Stattic for two reasons: (i) the demonstration in a previous study that the i.p. injection of Stattic decreased the phosphorylation of STAT3 in the brain of mice suggesting that Stattic crosses the blood-brain barrier; (ii) the existence of numerous pre-clinical models using this molecule [START_REF] Zhang | Impairment of Hepcidin Upregulation by Lipopolysaccharide in the Interleukin-6 Knockout Mouse Brain[END_REF].

Our results showed that STAT3 could mitigate microglia activation state and their proinflammatory cytokines production, IL-1β, IL-6, IFN-γ and TNF-α. If several previous works carried out in mouse models of AD support a role of STAT3 activation in astrocytes reactivity [START_REF] Ben Haim | The JAK/STAT3 pathway is a common inducer of astrocyte reactivity in Alzheimer's and Huntington's diseases[END_REF][START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF][START_REF] Reichenbach | Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model[END_REF], its roles in microglial activation in the AD pathological context have been poorly explored. Nevertheless, inhibition of the JAK-STAT3 pathway in a Parkinson disease rat model abolished neuroinflammation by suppressing microglial activation, macrophage infiltration and production of proinflammatory cytokines. These findings using a different STAT3 inhibitor and our results suggest a potential protective effect of STAT3 inhibition in neurodegeneration [START_REF] Qin | Inhibition of the JAK/STAT Pathway Protects Against alpha-Synuclein-Induced Neuroinflammation and Dopaminergic Neurodegeneration[END_REF].

Finally, we have shown that Stattic could modulate Aβ metabolism by decreasing BACE1 protein level induced by systemic LPS administration (Figure 4C). This result confirms previous observations revealing a role of STAT3 in Aβ-induced neuronal death [START_REF] Wan | Tyk2/STAT3 signaling mediates beta-amyloid-induced neuronal cell death: implications in Alzheimer's disease[END_REF]. Previous works suggest that the regulation of BACE1 by STAT3 may occur at the transcriptional level and it was shown that phosphorylated STAT3 act as a transcription factor binding mRNA of BACE1 [START_REF] Liu | Palmitate induces transcriptional regulation of BACE1 and presenilin by STAT3 in neurons mediated by astrocytes[END_REF]. To deepen this study, other STAT3 inhibitors should be tested in our model.

Moreover, an evaluation of beneficial effects of Stattic on AD hallmarks (amyloid plaques and tau phosphorylation) should be assessed in AD mouse models, such as 3xTg-AD or APP/ PS1 mice [START_REF] Lok | Characterization of the APP/PS1 mouse model of Alzheimer's disease in senescence accelerated background[END_REF][START_REF] Sterniczuk | Characterization of the 3xTg-AD mouse model of Alzheimer's disease: part 2. Behavioral and cognitive changes[END_REF].

Targeting neuroinflammation is a promising field of pharmaceutical drug development in AD [START_REF] Dong | Drug Development for Alzheimer's Disease: Microglia Induced Neuroinflammation as a Target?[END_REF]. Recently, molecules inhibiting inflammatory pathway, such as the protein kinase p38 MAPK (p38 MAPKα), or the NLRP3 inflammasome have shown promising results for reduce neuroinflammation and slow cognitive decline in AD patients [START_REF] Alam | Neflamapimod: Clinical Phase 2b-Ready Oral Small Molecule Inhibitor of p38alpha to Reverse Synaptic Dysfunction in Early Alzheimer's Disease[END_REF][START_REF] Burstein | Development of Azeliragon, an Oral Small Molecule Antagonist of the Receptor for Advanced Glycation Endproducts, for the Potential Slowing of Loss of Cognition in Mild Alzheimer's Disease[END_REF][START_REF] Dempsey | Inhibiting the NLRP3 inflammasome with MCC950 promotes non-phlogistic clearance of amyloid-beta and cognitive function in APP/PS1 mice[END_REF][START_REF] Duffy | The Discovery of VX-745: A Novel and Selective p38alpha Kinase Inhibitor[END_REF]. With its powerful antiinflammatory action [START_REF] Yu | STATs in cancer inflammation and immunity: a leading role for STAT3[END_REF], STAT3 inhibition may be considered as an interesting candidate to normalize brain inflammation in AD context.

In conclusion, this study provides a proof of concept of the beneficial effects of STAT3 inhibition on neuroinflammation and BACE1 metabolism and paves the way for future explorations of Stattic in animal models of AD.

AKNOWLEDGMENTS

The authors thank Fortune Hontonou, Calypso Nepost, Cindie Courtin, Luana Spano, Marion Brault, Bruno Saubaméa and Véronique Cochois for their technical help.

FUNDINGS

RESULTS 3 . 1 .

 31 Statistical analyses were performed with Prism 6 (GraphPad Software). Pooled results are expressed as means ± SEM. Two-way ANOVA was used to analyze the effect of treatments (LPS, Stattic). Unpaired Student's t tests (unless the data were shown not to be normally distributed) or Mann and Whitney tests were used for other comparisons. For all tests, p values <0.05 were considered statistically significant. Millot et al. STAT3 inhibition protects against neuroinflammation and BACE1 upregulation 3. In the hippocampus, Stattic reverses STAT3 phosphorylation induced by systemic injection of LPS We first evaluated the impact of Stattic on STAT3 phosphorylation (Figure 1A). Administration of LPS increases the phosphorylation of STAT3 on Tyrosine 705 in the hippocampus (+187%) of LPS-treated mice compared to controls. Peripheral injection of Stattic in LPS-treated mice lead to a significant decrease of phosphoSTAT3Tyr705/STAT3 ratio (-34.6%) compared with LPS-treated only animals. No significant difference is seen between saline-injected mice and saline + Stattic-treated animals. (Figure 1B)

Figure 1 .

 1 Figure 1. Stattic injections reduce LPS-induced phosphorylation of STAT3 in mice hippocampus. A. Immunoblot of hippocampal protein extracts revealed by different antibodies specific for phospho-STAT3, STAT3 full and GAPDH. B. Corresponding quantification of phospho-STAT3 /STAT3 ratio after normalization on GAPDH level in the four groups of mice non-exposed to LPS and Stattic (Saline, n=9), treated with Stattic (Saline+Stattic, n=7) or LPS only (LPS, n=8) and injected in parallel with LPS and Stattic (LPS+Stattic, n=6). The error bars indicate the ±SEM. AU: Arbitrary unit. ***p<0.001

Figure 2 .

 2 Figure 2. Effect of Stattic on microglial activation in the hippocampus after LPS exposure. A-D. Double-labeling in immunofluorescence of DAPI (blue) and IBA1 (green) in hippocampus sagittal sections of WT mice treated with saline (A), saline+Stattic (B), LPS (C) and LPS+Stattic (D). Left boxes: "MICROGLIAL CELL". Higher magnification of hippocampal microglial cells (boxed indicated by white arrow and enlarged on the respective bottoms). Cellular contours of microglial cells (white line) are obtained by image J analyses. Right boxes: "SKELETAL ANALYSIS". Higher magnification of microglial cells after skeletal and fractal analyses using Image J software. Microglial processes are highlighted in red. E. Schematic sagittal representation of hippocampal sections used for immunohistochemistry.

F.

  Relative IOD (integrated optical density) of IBA1 immunoreactivity in activated microglial cells. G. Microglial processes length determined using Image J software. n=8 for each group.

Figure 3 .

 3 Figure 3. Protective role of Stattic injections on cytokines production in mice hippocampus after LPS treatment. Hippocampus protein extracts analyzed with Luminex technology to determine cytokines levels of respectively IL-1β (A), TNF-α (B), IL-6 (C) and IFN-γ (D) in saline and LPS-treated mice with or without Stattic injection. n is comprised for each group between 5 and 8 animals (IL-1β), 8 and 15 animals (TNF-α, IL-6 and IFN-γ). The error bars indicate the ±SEM (standard error of the mean). AU: Arbitrary unit. *p<0.05, **p<0.01, ***p<0.001

Figure 4 .

 4 Figure 4. Inhibition of STAT3 decreases BACE1 levels and in mice hippocampus after LPS systemic challenge. A. Immunoblot analysis of mature BACE1 protein. B. Corresponding quantification after normalization on GAPDH level in the four groups: untreated mice (Saline, n=9), mice treated with Stattic (Saline+Stattic, n =7) or LPS only (LPS, n=7) and mice injected in parallel with LPS and Stattic (LPS+Stattic, n=7). AU: Arbitrary unit. The error bars indicate the ±SEM. **p<0.01. C. Schematic representation of Stattic protective effects on neuroinflammation and BACE1 levels. Peripheral inflammation and Stattic communicates with the brain through the blood-brain barrier to induce microglial activation and proinflammatory mediator's production involving STAT3 activation. Cytokines, including TNF-α and IL-6, induce STAT3 nuclear translocation in hippocampal neurons leading to an increase of BACE1 mRNA transcription.

This work was partially supported by University of Paris, Institut national de la santé et de la recherche médicale (INSERM), and Assistante Publique des Hôpitaux de Paris (APHP).

AUTHORS' CONTRIBUTIONS

BIBLIOGRAPHY


P Pin

H Roumanille

Frémont

E Ben Romdhane

A Guédon-Gracia

S Pin

P Roumanille

H Frémont

joints on thermomechanical response and reliability of ball grid array components

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

Introduction

Reliability of electronic equipment for automotive and aircraft applications is a major importance due to multiple environmental stresses (e.g. thermal, mechanical, thermomechanical). With the RoHS directive averting the use of lead (Pb), fatigue characterization of lead-free solder joint has widely interested industries and academics [START_REF] Lee | Solder joint fatigue models: Review and applicability to chip scale packages[END_REF]. Although several researches study thermomechanical behaviour of solder joints on a macroscopic scale, few studies have considered the effect of the initial microstructure of SnAgCu solder alloy on the stress state of solder joints.

The microstructure of SAC solder joints after reflow is characterised by diverse tin grain morphologies with random crystalline orientations. Several studies have shown the complex microstructure observed in the initial state of SAC305 [START_REF] Henderson | The microstructure of Sn in near-eutectic Sn -Ag -Cu alloy solder joints and its role in thermomechanical fatigue[END_REF] [START_REF] Gracia | Statistical study of SAC solder joints in QFN and BGA assemblies[END_REF]. Due to this stochastic as-soldered microstructure and coupled with the highly anisotropic properties of the tin phase, the thermomechanical response o²f SAC solder joints greatly depends on the morphology and crystal orientation of its Sn grains [START_REF] Syed | Accumulated Creep Strain and Energy Density Based Thermal Fatigue Life Prediction Models for SnAgCu Solder Joints[END_REF]. For these reasons, early failures may occur unexpectedly and predicting the first failed joint is quite difficult [START_REF] Lehman | Microstructure and damage evolution in Sn-Ag-Cu solder joints[END_REF] [START_REF] Smetana | Pb-free solder joint reliability in a mildly accelerated test condition[END_REF]. Failures may be more or less randomly located and not always at the position of highest global strain or distance to neutral point [START_REF] Zhou | Crack development in a low-stress PBGA package due to continuous recrystallization leading to formation of orientations with [001] parallel to the interface[END_REF] [START_REF] Bieler | Influence of Sn Grain Size and Orientation on the Thermomechanical Response and Reliability of Pb-free Solder Joints[END_REF]. The main goal is hence to predict the position of the first cracked joints using Finite Elements (FE) model that considers initial solder joint microstructure. Some authors already began to study the effect of anisotropy of coarsegrained joints by considering microstructure in their models [START_REF] Mukherjee | Multiscale modeling of the anisotropic transient creep response of heterogeneous single crystal SnAgCu solder[END_REF]. Lovberg et al. investigated the anisotropic elasticity and position of a solder joint in a package modeled at assembly level and showed the influence of the grain orientations of neighbouring single grained joints on the stress/strain state on the corner joint [START_REF] Lovberg | Simulations of the impact of single-grained leadfree solder joints on the reliability of ball Grid Array components[END_REF] [START_REF] Lovberg | The Stress State of BGA Solder Joints Influenced by the Grain Orientations of Neighboring Joints[END_REF] .

In this study, the influence of location and orientation of each single-grained solder joint in a BGA package is investigated by finite-element simulation. The goal is to provide a numerical tool making it possible to study the effect of initial microstructure on the stress state of each solder joint. In an ageing test campaign done in parallel, similar packages are submitted to different thermal aging and thermal cycling conditions. Microstructural analyses using electron backscatter diffraction (EBSD) as well as polarized light microscopy will be performed for comparison.

This paper presents the first steps of this approach:

1) an analytical parametric study to highlight the effect of initial microstructural characteristics of solder joints;

2) implementation of different combinations of crystalline orientations of solder joints in FE simulations to discuss the influence of orientations and location on the stress distribution of BGA solder joints 3) comparison with some published numerical and experimental data [START_REF] Lovberg | Simulations of the impact of single-grained leadfree solder joints on the reliability of ball Grid Array components[END_REF].

Microstructure of lead-free solder joints

Distributions of microstructure

The microstructure of a solder joint and its mechanical properties are very impacted by conditions and reactions that take place when SnAgCu solder alloy solidify. This microstructure has a truly random distribution with very varied morphologies. Several statistical studies showed that in typical SAC solder joints, the as-reflowed microstructure will consist of few large β-Sn grains and can be described as single-grained, cyclic twinned (also known as beach ball structure), twinned interlaced and mixed; The beach ball and interlaced microstructures are known to be both due to the sixfold twinning of tin, with three dominant grain orientations [START_REF] Arfaei | Failure Mechanism and Microstructural Evolution of Pb-free Solder Alloys in Thermal Cycling Tests : Effect of Solder Composition and Sn Grain Morphology Universal Instruments Corporation[END_REF] The occurrence of these morphologies in BGA components depends on solder volume, alloy composition, surface finish and cooling rate. 16% to 80% of solder joints are single-grained and their orientations tend to be more or less random [START_REF] Lovberg | Simulations of the impact of single-grained leadfree solder joints on the reliability of ball Grid Array components[END_REF]. Zhao et al. showed that there is a relation between the temperature gradient during solidification and the orientation of tin grains, where the c-axis tends to depart from the direction of temperature gradient by 45 to 88 degrees. However, these results are established on only four joints (10 grains in total) [START_REF] Zhao | Formation of highly preferred orientation of β-Sn grains in solidified Cu/SnAgCu/Cu micro interconnects under temperature gradient effect[END_REF].

Failure Mechanism

When a lead-free solder joint is exposed to thermal cycling, recrystallization will happen in the high strain region near the solder pad. The recrystallization is characterized by the production of a large number of randomly orientated subgrains in the higher deformation areas. A network of high angle grain boundaries along the high strain region during thermal cycling is formed after a global recrystallization [START_REF] Arfaei | Effect of Sn Grain Morphology on Failure Mechanism and Reliability of Lead-Free Solder Joints in Thermal Cycling Tests[END_REF]. This network represents a high energy area that provide favourable intergranular crack propagation paths until failure [START_REF] Arfaei | Reliability and failure mechanism of solder joints in thermal cycling tests[END_REF] [START_REF] Terashima | Recrystallization of Sn grains due to thermal strain in Sn-1.2Ag-0.5Cu-0.05Ni solder[END_REF]. Initially, recrystallization is slowed down due to precipitation hardening caused by intermetallic (IMC) particles but strainenhanced coarsening will take place during thermal cycling. Despite recrystallization has been localized at twin boundaries and voids, it is most often favoured in the neck region due to the strain-enhanced precipitate coarsening that occurs fastest there. During thermal cycling, the spacing between inter-particles increases in the high strain area; the material softens which will further accelerate the coarsening of the precipitates. This coarsening will decrease the pinning effect of the secondary precipitates, allowing the formation and rotation of subgrains which will finally lead to high angle grain boundaries.

As the degree of coarsening and recrystallization is impacted by the strain level, the orientation of tin grains will strongly affect the damage evolution; an unfavourable orientation and position of a solder joint can cause early failures (Figure 2). The reliability of solder joints is affected by Sn grain morphology and orientation. In Ref. [START_REF] Henderson | The microstructure of Sn in near-eutectic Sn -Ag -Cu alloy solder joints and its role in thermomechanical fatigue[END_REF], single-grained solder joints with certain orientations were observed to fail unexpectedly early, as compared to multi-grained SnAgCu solder joints.

The presence of an interlaced Sn grain morphology was correlated with a delay in crack initiation and the degree of interlaced tin grain morphology has an impact on the fatigue lifetime of solder joint. Arfaei et al. showed that the variation of lifetimes with changes in surface finish in LGA solder joints was directly related with these changes in Sn grain morphology for six different surface finishes (Figure 3) [START_REF] Arfaei | Failure Mechanism and Microstructural Evolution of Pb-free Solder Alloys in Thermal Cycling Tests : Effect of Solder Composition and Sn Grain Morphology Universal Instruments Corporation[END_REF]. Solder joints with mixed cyclic and interlaced twin structures may be detrimental for the fatigue lifetime of solder joints. As the interlaced region is supposed to be stiffer, because of the larger undercooling resulting in a larger degree of precipitation hardening, it may induce a high stress level in the area that is not interlaced where the interparticle spacing is higher. If this area also has a critical orientation that maximizes the CTE mismatch to the component, very early failures may occur [START_REF] Tegehall | Impact of various combinations of PCB laminate , solder pad finish and solder composition on the fatigue life of solder joints to various types of components[END_REF].

Analytical model

In order to verify the effect of crystal orientation of tin grains on thermomechanical response of each SAC solder joint, a first analytical approach is used.

For the theoretical analysis of a BGA assembly, Timoshenko proposed a theory that studies bending of bi-metal strip submitted to thermal loading [START_REF] Timoshenko | Analysis of bi-metal thermostats[END_REF]. Using this result, a simple analytical model permitted us to quickly calculate the shear stress in solder joints from dimensions and mechanical properties of the various elements of the assembly. To simplify the configuration, bumps are considered as beams with different Young's moduli so as to mimic various crystallographic orientations. Mechanical properties (α, E, ν) of moulding compound and PCB are first-order parameters to evaluate thermal-mechanical loading in solder joints. Dimensions and properties of the different elements of the package are described in Figure 4. A parametric study allowed us to set up multiple Young's Modulus distributions and to study the effect of changing Young's modulus of each bump along the length of the assembly. The different Young's Modulus are randomly generated. Fig. 5a shows an example for two Young's Modulus combinations randomly performed. The evolution of shear stress with position of joints for this two Young's Modulus combinations is presented in Fig. 5b. From these two scenarios, we can see that the most stressed solder joint is not necessarily located at the highest distance to neutral point (scenario 1). We can also see that the most stressed joint is not always the one with the highest Young's Modulus (the 4 th joint in scenario 1) and each combination of Young's modulus gives a different stress state along length of the assembly. From these reasons, we can conclude that the stress state of each joint strongly depends on its orientation. So, to facilitate relevant and accurate lifetime predictions, modelling and simulation should be performed with many grain orientation combinations. This first analytical approach therefore let us suspect that the stresses experienced by a joint depend not only on its crystalline orientation, but also on that of its neighbours. On the other hand, the assumptions used in the models are too simplistic to conclude to quantitative results. So we started to take these parameters into account in finite element models.

Description of the finite element model

To generate a finite element model that allows implementation of different combinations of grain orientations, a model of a quarter Chip Array BGA with anisotropic properties is set up to simulate thermomechanical loads (Figure 6). A temperature drop from the reflow point (230°C) to the ambient (25°C) followed by one thermal cycle (-55/125°C) has been applied on the model. The response of the lead-free solder joints to thermomechanical loading are very impacted by the anisotropy of the tin phase; Tin is one of the most anisotropic metals in the periodic table and is characterized by a body-centered tetragonal crystal structure with 4 additional atoms at four faces, as shown in Figure 7:

In order to change the orientation of the solder joints in the model, the elements of the joint are rotated using Euler angles. This rotation is done according to a first rotation φ around the zaxis, followed by θ around the new x-axis and finally Ψ around the updated z-axis and based on the rotation matrix:

𝑅 = ( 𝑐 3 𝑐 1 -𝑠 3 𝑐 2 𝑠 1 -𝑐 3 𝑠 3 -𝑠 3 𝑐 2 𝑐 1 𝑠 3 𝑠 2 𝑠 3 𝑐 1 + 𝑐 3 𝑐 2 𝑠 1 -𝑠 3 𝑠 1 + 𝑐 3 𝑐 2 𝑐 1 -𝑐 3 𝑠 2 𝑠 2 𝑠 1 𝑠 2 𝑐 1 𝑐 2 ) ( 2
)
Where 𝑐 1 , 𝑐 2 , 𝑐 3 and 𝑠 1 , 𝑠 2 , 𝑠 3 are the cosine and sine of the rotation angles φ, θ and Ψ. All other materials in the model are modelled as linear elastic.

Results and discussion

Several works have pointed out the importance of the c-axis orientation with respect to the board surface on the fatigue life of solder joints to area array packages. Bieler et al. [START_REF] Bieler | Influence of Sn Grain Size and Orientation on the Thermomechanical Response and Reliability of Pb-free Solder Joints[END_REF] observed a clear dependence of the thermomechanical response of SAC solder joints on their Sn grain orientation. They showed that Sn crystal orientations with very high CTE values (c-axis orientation parallel to the substrate) generated a maximal CTE mismatch in shear at the joint interface, and also had maximal tensile stress states in the hot part of the thermal cycle. Thus, these orientations presented the worst cases and hence the best cases are characterized by orientations with low CTE values (caxis orientation perpendicular to the substrates).

For these reasons, we have chosen in this study to model the two extreme orientations with another third orientation with a caxis which makes a 45 degree angle to the board surface.

Influence of crystalline Orientation on the stress state of the corner joint

In order to assess the influence of orientations on the stress state of the corner joint, three different cases of grain orientation are modelled. In all three cases, all joints have the same orientation.

Figure 8 shows the stress state in the corner joint as a function of orientation. As supposed from analytical approach results, the stress state in the solder joints strongly depends on their orientations. Although the highest stressed area was in the neck of the joint for all orientations, we noted a variation of the maximum Von Mises stress between 5% and 15% depending on orientation. Tegehall et al. argued that the orientation has an impact on the behaviour of the corner joint and therefore on its degree of recrystallization [START_REF] Lovberg | Simulations of the impact of single-grained leadfree solder joints on the reliability of ball Grid Array components[END_REF]. As a consequence, the orientation of the solder joint plays a significant role in the time to recrystallization and subsequently in the fatigue life of the solder joints. The objective from this part of the study, is to understand the impact of corner joint orientation on the stress state of neighbouring joints. We studied also three configurations but only by changing the orientation of the corner joint; the neighbouring bumps orientations are defined by a c-axis parallel to the substrate for each simulation. For the corner joint (A1), the three orientations chosen at the beginning of this section were implemented.

Results of two configurations are presented in Figure 9. Balls of the first range of the FE model were cut in half to show the representative stress state of each solder joint. As expected, we can observe that the corner joint with a c-axis orientation parallel to the substrate is more stressed than that with a c-axis orientation perpendicular to the substrate which validates that orientations with very high CTE value are more detrimental to the fatigue life than orientations with low CTE values.

This figure shows also that the stress state of their surrounding balls was impacted by the orientation of the corner joint. This effect is more remarkable on the stress distribution of the joints A2 and A5 and depends on the value of the variation of the orientations from one bump to another.

These observations show that orientation has an important effect on the behaviour of the solder joints and therefore it influences their fatigue life.

Simulations of combinations of different orientations

To get closer to reality and have a more representative model, different cases of joint orientation combinations were modelled. Each case is characterized by a random c-axis orientations distribution; the crystal orientations implemented are also those defined at the beginning of this paragraph.

Simulations of three different combinations of bumps orientations are presented in Figure 10. As shown in this figure, by generating more realistic combinations, the effect of crystalline orientation on the behavior of the joints became clearer. Due to the combined effect of orientation of joint, its position and orientations of neighbouring bumps, the stress distribution of each solder joint is unique and locations of most stressed joints change from one combination to another.

On the other hand, by comparing the stress levels along the simulations carried out in this study, we observed that a model which contains joints with different orientations has a higher stress level (Figure 10) with differences of 37% compared to the stress level observed in the simplest cases (Figure 8) and 25% for configurations where the orientation of the corner joint has been modified (Figure 9). These results explain the dispersion in times of failure and lead to the conclusion that fatigue life of an area array component will be strongly affected by the initial microstructure of solder joints. For that, it is necessary to make lifetime predictions based on a certain combination of joint orientations.

Since the initial microstructure has a significant role in the study of solder joints reliability, the fatigue life of an area array component can be also strongly affected by the morphology of joints. Further studies on the influence of grain morphologies on the stress state of BGA solder joints will be presented in a future publication.

A6 A5 A4 A3 A2 A1 (

Conclusion

The investigations detailed in this study aim at giving numerical tools for designing solder joints reliability taking into account crystalline grain orientation. Simulations confirm that the stress state of each single-grained solder joint is unique and affected by the response of its neighbouring joints which gives a unique thermal fatigue behaviour for each combination of orientations. More realistic simulations are now possible in order to study the lifetime of the solder joints as a function of their initial microstructure. This tool will be used in future work to run an important number of simulations with different sets of joint orientations; A sensitivity analysis based on Monte Carlo method will allow us to define multiple distributions of orientations. The results will be compiled into a response surface in order to define the most critical case. These response surfaces will also be useful for studying the distribution of predicted lifetime as a function of crystalline orientations and positions of solder joints. This work is also supported by a statistical study of crystalline orientations of joints in the initial state; after cycling the influence of the crystal orientation of the tin grains on the failure mode of SAC305 solder joints will be evaluated. This also allows us to better understand the as-reflowed microstructure and its evolution during thermo-mechanical loading in order to improve the interpretations of the modelling results.

  [START_REF] Lehman | Cyclic twin nucleation in tin-based solder alloys[END_REF]. Examples of these microstructures are shown in Figure1.

Figure 1 .

 1 Figure 1. Polarized light observations of BGA solder joints microstructure: (a) beach ball structure, (b) interlaced grain

Figure 2 .

 2 Figure 2. EBSD analysis (IPFy orientation map) of failed solder joint after thermal cycling, -55/125°C 2.3. Impact of microstructure on solder joint fatigue life

Figure 3 .

 3 Figure 3. Plot showing the correlation between the lifetime ofLGA samples and percentage of interlaced joints[START_REF] Arfaei | Failure Mechanism and Microstructural Evolution of Pb-free Solder Alloys in Thermal Cycling Tests : Effect of Solder Composition and Sn Grain Morphology Universal Instruments Corporation[END_REF].

Figure 4 .

 4 Figure 4. Simplified BGA assembly for the analytical model The objective of this first analytic calculation is to assess the stress level during a temperature drop of 100 ° C of each bump for different Young's Modulus distributions, representative of variations related to the dispersion of bibliographic data and the orientation of the -Sn phase.

Figure 5 .

 5 Figure 5. Random Young's modulus values as a function of joint position (a), shear stresses as a function of joint position (b)

Figure 6 .

 6 Figure 6. Model of a quarter of BGA.

  𝒑 , 𝑬 𝒑 , 𝝂 𝒑 ) Composant (𝜶 𝒄 , 𝑬 𝒄 , 𝝂 𝒄 )

Figure 7 .

 7 Figure 7. Unit cell of body centered tetragonal ß-SnAnisotropic properties of β-Sn were implemented in ABAQUS through its elastic coefficient matrix in stiffness form. This matrix is defined by 6 independent constants due to the tin crystal symmetry:

Figure 8 .

 8 Figure 8. Von Mises stress in the corner joint as function of c-axis orientation 5.2. Effect of the orientation of the corner joint on the neighbouring balls

Figure 9 .Figure 10 .

 910 Figure 9. Effect of the c-axis orientation of the corner joint A1 on the neighbouring bumps; (a) A1 with a c-axis orientation parallel to the substrate, (b) A1 with a c-axis orientation perpendicular to the substrate A6 A5 A4 A3 A2 A1

Acknowledgment

This work is in the framework of the IRT Saint Exupery's research project FELINE. We acknowledge the financial and inkind support (background knowledge and services) from the IRT Saint Exupery's industrial and academic members and the financial support of the French National Research Agency.