Spiros Kotopoulis

Anthony Delalande

Mihaela Popa

Veronika Mamaeva

Odd Georg Dimcevski

Helge Gilja

Michiel Postema

Tore Bjørn

Emmet Gjertsen

Mc Cormack

Helge Odd

Gilja

Bjørn Tore Gjertsen

PhD Emmet Mccormack
email: emmet.mc.cormack@med.uib.no

Sonoporation-enhanced chemotherapy significantly reduces primary tumour burden in an orthotopic pancreatic cancer xenograft

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

Experimental design: An orthotopic xenograft mouse model of luciferase expressing MIA PaCa-2 cells was developed exhibiting disease development similar to human pancreatic adenocarcinoma. Subsequently, two groups of mice were treated with gemcitabine alone, gemcitabine combined with sonoporation, and saline treated mice were used as a control group.

A custom made focused ultrasound transducer using clinically safe acoustic conditions in combination with SonoVue ® ultrasound contrast agent was used to induce sonoporation in the localised region of the primary tumour only. Whole body disease development was measured using bioluminescence imaging and primary tumour development was measured using 3D ultrasound.

Results: Following just two treatments combining sonoporation and gemcitabine, primary tumour volumes were significantly lower than control groups. Additional therapy dramatically

Introduction

Pancreatic adenocarcinoma (PA) morbidity and mortality remains almost unchanged over the past two decades. Available statistical predictive data estimate 45,220 new cases and 38,460 deaths due to PA in 2013 [START_REF]Cancer Facts and Figures[END_REF]. This type of cancer can rarely be treated radically, and the overall survival rate is less than 4% [START_REF] Greenlee | Cancer statistics, 2000[END_REF]. When surgical intervention is possible survival rates can increase to median of 36 months and surpass 5 years [START_REF] Chen | Long-term survival after pancreatic cancer treatment[END_REF]. However, adjuvant chemotherapy improves the prognosis of the patients even after palliative surgery, and successful newer neoadjuvant chemotherapeutical modalities have recently been reported [START_REF] Hartlapp | Complete Pathological Remission of Locally Advanced, Unresectable Pancreatic Cancer (LAPC) after Intensified Neoadjuvant Chemotherapy[END_REF]. Patients with unresectable tumours undergo chemotherapy and chemoradiation but should be spared the morbidities of surgical resection [START_REF] Tempero | Pancreatic Adenocarcinoma, version 2.2012: featured updates to the NCCN Guidelines[END_REF]. Most of the patients with advanced PA have about a year to live after diagnosis [START_REF] Neoptolemos | A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer[END_REF][START_REF] Neoptolemos | Adjuvant chemoradiotherapy and chemotherapy in resectable pancreatic cancer: a randomised controlled trial[END_REF] and even increase in survival for a few months matters in the pancreatic cancer field. Gemcitabine, a nucleoside analogue, is considered the most effective chemotherapeutic treatment of pancreatic cancer in terms of both response rate and median overall survival [START_REF] El-Kamar | Metastatic pancreatic cancer: emerging strategies in chemotherapy and palliative care[END_REF][START_REF] Marechal | Levels of gemcitabine transport and metabolism proteins predict survival times of patients treated with gemcitabine for pancreatic adenocarcinoma[END_REF]. The benefit it brings is relatively modest, as it does not improve the prognosis much, with a median overall survival of less than 6 months and has rather pronounced systemic side effects [START_REF] Camp-Sorell | Chemotherapy Toxicities and Management[END_REF]. Gemcitabine is approved by the FDA as a single agent, as the first-line treatment for patients with locally advanced or metastatic adenocarcinoma of the pancreas.

Ultrasound is most widely known for its clinical use in diagnostic imaging and modern diagnostic ultrasonography employs contrast agents to improve the signal-to-noise ratio of blood [11][START_REF] Simpson | Techniques for perfusion imaging with microbubble contrast agents[END_REF]. These ultrasound contrast agents typically consist of perfluorocarbon-filled lipid-shelled microbubbles in the size range of 2-10μm (Table 1) [START_REF] Stride | Microbubble ultrasound contrast agents: a review[END_REF] When excited with ultrasound, these microbubbles scatter the incoming ultrasound and volumetrically oscillate generating their own ultrasonic pulse. This results in various complex and controllable actions that can be taken advantage of for therapeutic purpose [START_REF] Postema | Medical Bubbles[END_REF], e.g. sonoporation-enhanced therapy [START_REF] Qiu | Microbubble-induced sonoporation involved in ultrasound-mediated DNA transfection in vitro at low acoustic pressures[END_REF][START_REF] Suzuki | Progress in the development of ultrasound-mediated gene delivery systems utilizing nano-and microbubbles[END_REF][START_REF] Nejad | Optical observations of cell sonoporation with low intensity ultrasound[END_REF][START_REF] Kooiman | Sonoporation of endothelial cells by vibrating targeted microbubbles[END_REF][START_REF] Wu | Sonoporation, anti-cancer drug and antibody delivery using ultrasound[END_REF][START_REF] Delalande | Sonoporation at a low mechanical index[END_REF].

Sonoporation is the transient formation of nanometre-sized pores in cell membranes by the use of ultrasound or ultrasound and microbubbles allowing for intracellular uptake of drugs or genes [START_REF] Delalande | Sonoporation at a low mechanical index[END_REF].

Previous research has elaborated six common mechanistic effects of sonoporation mediated drug and gene delivery [START_REF] Delalande | Sonoporation at a low mechanical index[END_REF][START_REF] Van Wamel | Controlled drug delivery with ultrasound and gas microbubbles[END_REF][START_REF] Emmer | Optical investigation of ultrasound induced encapsulated microbubble oscillations: threshold and hysteresis effects[END_REF][START_REF] Van Wamel | Vibrating microbubbles poking individual cells: drug transfer into cells via sonoporation[END_REF][START_REF] Postema | Sonoporation: Why microbubbles create pores[END_REF][START_REF] Delalande | Ultrasound and microbubble-assisted gene delivery in Achilles tendons: long lasting gene expression and restoration of fibromodulin KO phenotype[END_REF][START_REF] Delalande | Sonoporation: Mechanistic insights and ongoing challenges for gene transfer[END_REF] including push, pull, shear, jetting, inertial cavitation and translation. While "push", "pull", "shearing" and "translation" occur at all acoustic amplitudes resulting in perturbation of the cell membrane, "jetting" [START_REF] Gerold | Bifurcation of ensemble oscillations and acoustic emissions from early stage cavitation clouds in focused ultrasound[END_REF] and "inertial cavitation" [START_REF] Gerold | Lasernucleated acoustic cavitation in focused ultrasound[END_REF] result in partial destruction of the cell membrane which only occur at high acoustic amplitudes. High acoustic amplitudes employed in these studies surpass current ultrasound safety guidelines with the introduction of a cavitation nucleation agent, i.e., microbubbles [START_REF] Piscaglia | The EFSUMB Guidelines and Recommendations on the Clinical Practice of Contrast Enhanced Ultrasound (CEUS): update 2011 on non-hepatic applications[END_REF][START_REF] Ng | International guidelines and regulations for the safe use of diagnostic ultrasound in medicine[END_REF][START_REF]Guidelines for the safe use of diagnostic ultrasound equipment[END_REF].

Furthermore, a large majority of these studies only evaluate the effect of sonoporation in vitro [START_REF] Nejad | Optical observations of cell sonoporation with low intensity ultrasound[END_REF][START_REF] Fan | Intracellular delivery and calcium transients generated in sonoporation facilitated by microbubbles[END_REF][START_REF] Tlaxca | Analysis of in vitro Transfection by Sonoporation Using Cationic and Neutral Microbubbles[END_REF][START_REF] Qiu | The correlation between acoustic cavitation and sonoporation involved in ultrasound-mediated DNA transfection with polyethylenimine (PEI) in vitro[END_REF], in subcutaneous models [START_REF] Czarnota | Tumor radiation response enhancement by acoustical stimulation of the vasculature[END_REF] or for the efficacy of gene delivery [START_REF] Suzuki | Progress in the development of ultrasound-mediated gene delivery systems utilizing nano-and microbubbles[END_REF][START_REF] Fan | Intracellular delivery and calcium transients generated in sonoporation facilitated by microbubbles[END_REF][START_REF] Tlaxca | Analysis of in vitro Transfection by Sonoporation Using Cationic and Neutral Microbubbles[END_REF][START_REF] Lukianova-Hleb | Selective gene transfection of individual cells in vitro with plasmonic nanobubbles[END_REF].

Ultimately in a sonoporation therapeutic approach the goal must be to employ acoustically safe amplitudes but still impact translation of microbubbles and therapeutic across the cell membrane. Thus, "translation" sonoporation offers the preferred clinical modality resulting in microbubbles being forced inside a cell leaving a small pore that re-seals itself, occurring at low, clinically safe acoustic amplitudes.

Preclinical drug development in xenograft models of PA has historically been reliant upon subcutaneous inoculation of PA cells in the flanks of immunodeficient mice. However, subcutaneous models are not sufficiently representative of the clinical paradigm of PA, particularly with respect to metastasis development and drug response [START_REF] Hoffman | Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: A bridge to the clinic[END_REF][START_REF] Bibby | Orthotopic models of cancer for preclinical drug evaluation: advantages and disadvantages[END_REF]. Using an orthotopic model provides many advantages such as the attempts to target local invasion occur at a much more clinically relevant site and blood flow and vascularisation of the tumours closer mimic the human model. Taking into account the importance of an orthotopic model, in this study we aimed to investigate the potential of sonoporation for enhanced localised drug-delivery of a clinical chemotherapeutic (gemcitabine) to the primary tumour of PA in an orthotopic model.

Here we describe the development of an orthotopic PA mouse model exhibiting very similar characteristics to its human counterpart. A custom ultrasound transducer was used to induce sonoporation using clinically approved microbubbles (SonoVue ® ) at an extremely localised area using identical acoustic settings shown to induce sonoporation in our previous work [START_REF] Delalande | Sonoporation at a low mechanical index[END_REF][START_REF] Delalande | Ultrasound and microbubble-assisted gene delivery in Achilles tendons: long lasting gene expression and restoration of fibromodulin KO phenotype[END_REF]. To follow the disease progression, tumour volume was measured weekly using 3D ultrasound and optical imaging was used to observe whole body disease development. Two treatments with combined gemcitabine and sonoporation resulted in statistically significant lower primary tumour volume. This statistical difference was persistent for the further duration of the study. Furthermore bioluminescence imaging indicated that there was a delay in the onset of metastasis in responding mice.

Materials and Methods

Ultrasonic treatment conditions

A custom-made single-element ultrasound transducer consisting of a 25-mm spherically focused element with a geometric focus of 44 mm (Precision Acoustics Ltd., Dorchester, UK) was used as the treatment probe. To ensure correct acoustic alignment with the pancreas depth and location a custom adaptor was designed and built based on the transducer dimensions and beam profile. The adaptor was filled with distilled water and an 80-μm thick nitrile membrane was used at the contact surface to ensure maximum beam propagation. Figure 1a shows the design of the transducer and adaptor.

In order to drive the ultrasound transducer a 40% duty cycle sine wave was generated by an AFG3102, function generator (Tektronix, Inc., Beaverton, OR, USA) and amplified by a 2100L amplifier (Electronics & Innovation Ltd., Rochester, NY, USA). The acoustic field was calibrated with the acoustic adaptor in place using an automated 3D scanning chamber with a 50μm resolution and a calibrated HGL-200 hydrophone (Onda, Sunnyvale, CA) in degassed water.

Based on our previous in-vitro findings [START_REF] Delalande | Sonoporation at a low mechanical index[END_REF][START_REF] Delalande | Ultrasound and microbubble-assisted gene delivery in Achilles tendons: long lasting gene expression and restoration of fibromodulin KO phenotype[END_REF] the acoustic settings shown in Table 2 were used to treat the pancreatic tumour.

Ultrasound contrast agent / microbubbles

A clinically employed ultrasound contrast agent (SonoVue ® , Bracco Imaging S.p.A, Milano, Italy) was prepared according the manufacturers instructions. This results in phospholipid shelled microbubbles containing an SF 6 gas core with a mean diameter of 2.5µm at concentrations between 1x10 8 -5x10 8 microbubbles per mL [START_REF] Greis | Technology overview: SonoVue (Bracco, Milan)[END_REF]. The microbubbles were prepared immediately prior to treatment of the first mouse and the vial was gently agitated prior to each treatment to ensure a homogenous concentration.

Cell lines and cell culture

The human pancreatic adenocarcinoma MIA PaCa-2 cell line were kindly provided by Dr.

Anders Molven (The University of Bergen, Norway). The cells were retrovirally transfected using Phoenix cells (LGC Standards AB, Boras, Sweden) and a luciferase encoding plasmid.

High luciferase expressing cells were selected by puromycin (2.5 μg/ml). Cells were cultured in DMEM (Sigma-Aldrich Co. LLC. St. Louis, MO) complemented by 10% FBS, 2.5% FHS, 100 μg/ml penicillin-streptomycin and 2 mM L-glutamine (Sigma-Aldrich).

Animals

All experiments were approved by The Norwegian Animal Research Authority and conducted according to The European Convention for the Protection of Vertebrates Used for Scientific Purposes. NOD-scid IL2rγ null (NSG) mice (Gades Institute, University of Bergen.

Originally a generous gift of Prof. Leonard D. Shultz, Jackson Laboratories, Bar Harbour, Maine, USA). During surgery, depilation and imaging, mice were anaesthetized with 2% isofluorane (Isoba® vet, Intervet, Kenilworth, NJ).

Orthotopic xenograft model

Animals were fully anesthetized with 250 mg/kg tribromoethanol diluted in 2 methyl-2 butanol, 12.5 mg/ml (Sigma-Aldrich), and placed on a heating pad in dorsal recumbency. Hair was removed by shaving and the abdomen washed with iso-betadine and 70% alcohol. A small incision (0.5 cm) in the abdomen was made below the last rib on the left side, parallel to linea alba. The pancreas was exteriorized and the cells (1×10 6 MIA PaCa-2 luc ) suspended in 20-µL phosphate buffered saline was injected using a 30-gauge needle. After placing the pancreas back in the original position, the muscles and the skin were sutured with Ethilon II 5-0 polyamide sutures (Ethicon, Inc., Somerville, NJ). Prior to return to their holding cages, animals were placed under heat lamps for approximately 30 min, and monitored for any postoperative complications.

All animals were euthanized following institutional guidelines.

Optical imaging

The mice were anaesthetised and injected intraperitoneally (IP) with 150 mg/kg Dluciferin (Biosynth AG, Staad, Switzerland) 10 minutes prior to ventral and dorsal imaging using an In-Vivo FX molecular imaging system (Carestream Health, Inc., Rochester, NY). Total bioluminescence values were measured using manual ROIs in the Carestream MI software (Standard edition, v.5.0.6.20, Carestream Health, Inc.). Two types of ROIs were used, one whole abdomen and one locally in the primary tumour area.

High-resolution ultrasonic imaging

The mice were briefly re-anaesthetised and placed on the mouse handling table set at 40°C in dorsal recumbrency. The abdominal hair on the mice was removed using depilatory cream. A Vevo2100 ultrasound scanner combined with a MS250 (13-24 MHz) probe and 1D stage (VisualSonics Inc., Ontario, Canada) was used to capture 3D images of the primary tumour.

The enhanced abdominal imaging mode in B-mode and 3D mode was used. Doppler imaging was also used to aid primary tumour identification. Respiration gating was used to capture the 3D images. The tumours were measured manually using parallel segmentation in the Vevo2100 software (v1.3.0, VisualSonics Inc.).

Treatment protocol

Three weeks following xenotransplantation, mice were randomly separated into 3 groups; control (n = 3), gemcitabine alone (125 mg/kg, Q7D, n = 3) and combined treatment group, employing SonoVue ® (50 μL), and gemcitabine (125 mg/kg, Q7D, n = 4). Therapy was initiated on week 3 and continued weekly for a total of 8 cycles. Mice were euthanized when they started to demonstrate progressive weight loss below 10% from initial body weight, jaundice and lethargy.

Control mice received 200-μL saline (IP) post-imaging procedures. The gemcitabine group were treated with gemcitabine (125 mg/mg, i.p.) once weekly whilst combination treated animals received gemcitabine (125 mg/mg, i.p.) and Sonovue® ultrasound contrast agent (50-μL, i.v.) 30 minute later. Immediately after Sonovue® injection the mouse was placed on a heated table at 37°C and ultrasound was applied for a total of 10 min (4 min total cumulated ultrasound time). Figure 1b shows how the mice and therapeutic transducer were positioned for the treatment.

Histopathology

Organ samples collected following euthanasia were placed in 4% paraformaldehyde for paraffin-embedding. Sections were stained with hematoxylin and eosin (H&E) and results were analysed by standard light microscopy (Nikon Eclipse Ti, Nikon Instruments Europe BV, Amsterdam, Netherlands) by an experienced pathologist.

Statistics

Three different observers measured the primary tumour volume where two were blind to the groups. Results were expressed as the mean values ± SEM. Comparisons between groups were made using a two-tailed unpaired Student t-test. Survival data was analysed using a Logrank (Mantel-Cox) test. Differences where p < 0.05 were considered statistically significant.

Statistics were analysed using GraphPad PRISM® v5.0a (GraphPad Software Inc., La Jolla, CA) software.

Results

Transducer validation and characterisation

The therapeutic transducer was fabricated in order to induce sonoporation in a very localised region generating a very precise and sharp focal (treatment) zone. The 3D field scan validated the beam profile indicating a focus of Ø4.0 mm and 22-mm long (Fig. 1c). A small focus was chosen to ensure that only the primary pancreatic tumour was treated, as only this region would receive the ideal dose of ultrasound. The non-linearity ratio was 1.00 ensuring there were no shockwaves generated in the acoustic field prohibiting any other adverse effects seen by high-intensity ultrasound treatments and preventing microbubble destruction. Furthermore the adaptor reduced the side lobe intensity allowing for a Gaussian sound intensity profile [START_REF] Lu | Biomedical ultrasound beam forming[END_REF].

By creating such a controllable sound field, with such a precise focus (treatment zone), we can clearly dictate the location and size of the treatment area, without treating the organs behind or in front of the pancreatic tumour.

Pancreatic cancer xerograph model development

Following inoculation of 1×10 6 MIA PaCa-2 luc cells in the head of pancreas the mouse developed PA similar to that seen in the human counterpart. Three weeks after inoculation the primary tumour could be visualised by bioluminescence and high-resolution ultrasound (Fig. 2a and4b respectively). Metastasis was evident 5-6 weeks after inoculation, as demonstrated by the increase of bioluminescence in the whole abdomen when compared to the primary tumour (Fig. 2b). After 8 weeks, metastases have disseminated throughout the whole abdomen and in particular the liver, and in 10 weeks it has engulfed the whole abdomen. The disease developed in a similar fashion to that seen in human patients with an exponential growth of the primary tumour with dense vascular encapsulation. Histology verified that this was a very aggressive form of PA characterised by the nuclear polymorphism, atypical mitosis and invasive growth pattern (Fig 2c).

Sonoporation of gemcitabine with microbubbles is safe in a preclinical model of pancreatic adenocarcinoma

In order to evaluate the safety and efficacy of combined sonoporation and gemcitabine therapy, mice (n = 10) were inoculated and tumour and metastatic development monitored by 3D ultrasound and bioluminescent imaging. Primary tumour development was evident at 3 weeks by bioluminescence and 3D ultrasonic imaging, when mice were randomized into 3 groups. Mice weights were recorded every two days to monitor any adverse effects of the treatment. Following treatment a slight weight loss was recorded in all groups, with subsequent weight recovery following treatment. No significant differences in weight were seen between all three groups (Supplementary Figure 1), and no additional ill effects were noted, suggesting sonoporation combined with chemotherapy was safe in this preclinical setting.

Combined Gemcitabine plus sonoporation significantly inhibits primary pancreatic adenocarcinoma tumour growth

While it was anticipated that therapy might have an impact upon the metastatic pattern of this very aggressive disease the ultimate goal remained to evaluate the effect of therapy within the sonoporation field. Thus, we evaluated the primary pancreatic tumours response to therapy by tumoural bioluminescence imaging (Fig. 3) and 3D ultrasonic imaging (Fig. 4). Quantification of bioluminescence output from the primary tumour revealed reduced photonic flux in combination and gemcitabine treated mice than controls (Fig. 3a, p = 0.0003 and 0.0019 respectively), however scattering of biophotonic light from adjacent metastasis inevitably contaminated the primary tumour photonic quantification. However, it was encouraging to see lower bioluminescence at the primary site compared with controls.

In order to directly quantify primary tumour effects employing 3D ultrasound, we exploited Doppler imaging, which revealed dense vascularisation encapsulating the primary tumour (Fig. 4a-1 and 4a-2). Under high-frequency ultrasound, the primary tumour was seen as an anechoic region (Fig. 4a-3) indicating a very homogenous cellular structure; this was also validated from post-study histology samples (Fig. 6). This was used to generate 3D volumes of primary tumours (Fig. 4a andb). Comparing tumour volumes from week 5 (Fig. 4c) onwards a statistically significant difference between the combined treatment group and the gemcitabine group versus control was seen. There was no statistical difference in primary tumour volume when comparing the gemcitabine group to the control group, however statistically significant differences were observed between the combined treatment group versus both control (p < 0.05 -0.001) and gemcitabine only groups (p < 0.05 -0.001).

Combined treatment prolongs survival in an orthotopic xenograft model of pancreatic adenocarcinoma

Although whole-body bioluminescent imaging revealed a slower onset of metastatic development in combined treated animals (from weeks 6-8, Fig 3c) with lower bioluminescence observed at weeks 9 and 10, all animals eventually succumbed to metastatic disease. While disseminated metastasis of the liver and abdomen were noted in control and to a lesser extent gemcitabine treated animals from weeks 7 to 10, combination treated mice demonstrated restricted metastatic phenotype mostly restricted to the liver (Fig. 3c). Histopathology indicated a very aggressive form of PA with nuclear polymorphism, atypical mitosis and an invasive growth pattern. However, the combination treated group showed a less invasive growth pattern seen at the border between normal pancreatic and tumour tissue (Fig. 5). Furthermore, the combination and gemcitabine treated group demonstrated an increase in survival compared to the gemcitabine alone group with median survivals of 88 and 84 days respectively (p = 0.0570, Fig. 6) whilst the median survival of the control group was 80 days.

Discussion

Sonoporation in combination with gemcitabine was employed to treat orthotopic PA xenografts resulting in a significant reduction of tumour growth, inhibited metastatic development and increased survival when compared to controls. These results indicate potential to enhance localised drug delivery of chemotherapeutics, in consequence reducing the systemic toxic side effects and increasing local drug delivery. In this study we have used very low acoustic conditions that comply with current clinical safety regulations with the inclusion of a contrast agent [START_REF] Piscaglia | The EFSUMB Guidelines and Recommendations on the Clinical Practice of Contrast Enhanced Ultrasound (CEUS): update 2011 on non-hepatic applications[END_REF][START_REF] Ng | International guidelines and regulations for the safe use of diagnostic ultrasound in medicine[END_REF][START_REF]Guidelines for the safe use of diagnostic ultrasound equipment[END_REF] allowing for easy translation from pre-clinical to clinic studies. Clinical increase of local neoadjuvant chemotherapeutic exposure by sonoporation should reduce the growth rate of the primary tumour and allow for the possibility of surgical resection and increase survival [START_REF] Chen | Long-term survival after pancreatic cancer treatment[END_REF].

While gemcitabine therapy is the current standard of care in the therapy of pancreatic cancer, its clinical effect is modest, only extending survival by one month [START_REF] Burris | Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial[END_REF]. Similarly, in our preclinical study, gemcitabine improved survival over controls modestly; with both cohorts exhibiting disseminated metastatic disease. Addition of sonoporation to gemcitabine therapy of the primary tumour not only further increased survival times, but also limited the dissemination of the disease. As such this may add a clinical benefit in limiting primary tumour growth making resection and curable treatment, possible.

Treatment could only be instigated once the tumour could be accurately located and correlated to a physical location on the mouse, only possible 3 weeks following implantation. It was important to be able to unequivocally locate the tumour in 3-dimensions to accurately position the therapeutic probe. As a result, it is not inconceivable that within 3 weeks micrometastasis may have already seeded, undetectable with either imaging modality, prior to initiation of therapy.

Further development of these pre-clinical studies should include near-infrared optical imaging agents [START_REF] Mccormack | Multiplexed mAbs: a new strategy in preclinical time-domain imaging of acute myeloid leukemia[END_REF][START_REF] Mccormack | Nitroreductase, a near-infrared reporter platform for in vivo time-domain optical imaging of metastatic cancer[END_REF] to improve primary tumour delineation. In addition, employing Multispectral Optoacoustic Tomography (MSOT) or Photoacoustic imaging (PA) would permit 3D tumour localisation at an earlier stage. Despite only using ten mice we required an extensive skilled workforce to perform all the imaging in minimal time whilst still allowing the animals to recover. This resulted in a low power to statically analysis. However, the enabling technologies and imaging applications developed in this project should further serve development of this concept in treating patient derived xenografts surgical biopsies as they become available.

Although individual mice will be treated at different times, similar to clinical trial, imaging of the dense vasculature encapsulating the primary tumour (Fig. 4a) combined with optical imaging will aid standardisation of therapy initiation. Implanting and treating mice in this format will also reduce stress and enable greater numbers of animals to be treated.

The encapsulating vascularisation, though maintaining tumour nutrition seems beneficial for the treatment with sonoporation as the microbubbles have better access to the tumour mass, and clinical contrast enhanced ultrasonography demonstrates that microbubbles are not adversely affected by high intratumoral pressures [START_REF] Ozawa | Contrast-enhanced sonography of small pancreatic mass lesions[END_REF]. Developments that may further increase the efficacy of sonoporation would be to introduce a drug into these microbubbles creating anti-bubbles [START_REF] Poortinga | Micron-sized antibubbles with tunable stability[END_REF] and then inducing sonoporation. In this case the toxic chemotherapeutic would only be delivered to the cancer cells. This would ensure maximum efficacy and reduce the side effects of the systemic chemotherapeutics.

As sonoporation has been shown to improve the drug delivery to localized areas, other drug treatments in combination with sonoporation should be explored. Several new drugs are being investigated in-vitro and in pre-clinical trials that have indicated high effectiveness in treating pancreatic cancer cells [START_REF] Zhang | Combination of salinomycin and gemcitabine eliminates pancreatic cancer cells[END_REF][START_REF] Michaelis | Valproic acid as anti-cancer drug[END_REF][START_REF] Kunnumakkara | Curcumin potentiates antitumor activity of gemcitabine in an orthotopic model of pancreatic cancer through suppression of proliferation, angiogenesis, and inhibition of nuclear factor-kappaB-regulated gene products[END_REF]; evaluating the efficiency these drugs in combination with the benefits of sonoporation needs to be further studied.

Following these results we have commenced a Phase I study to evaluate the toxicity and/or efficacy of gemcitabine combined with inducing sonoporation in the clinic on pancreatic cancer tumours (2011/1601/REK).

Conclusion

In this study we have developed a custom ultrasound transducer to induce sonoporation in a very localised region required for preclinical studies and a bioluminescent model of pancreatic adenocarcinoma. We demonstrated that by inducing sonoporation at the location of an orthotopic pancreatic xenograft primary tumour in combination with the chemotherapeutic gemcitabine we are able to significantly inhibit the tumour growth compared with control or gemcitabine treated mice, even in cohorts with low sample number. Our results indicate that it is possible to enhance the efficacy of current chemotherapeutics with the simple addition of low-cost ultrasound contrast agents and a sound source. To further validate the clinical relevance of this study, repeat studies of primary patient derived xenografts, possibly with the addition of near-infrared contrast reagents and MSOT imaging should be considered. On the basis of these preclinical results the combination of gemcitabine and sonoporation is currently undergoing Phase I clinical trial at our centre. Table 1: List of the most common ultrasound contrast agents [START_REF] Postema | Fundamentals of medical ultrasonics[END_REF]. Bold names denote contrast 385 agents clinically approved for human use.

Tables

Contrast agent

Mean diameter (µm) Gas

AI

Figure 1 :Figure 2 :Figure 3 :

 123 Figures

Figure 4 :

 4 Figure 4: Primary pancreatic tumour volume measured by 3D ultrasound of MIA PaCa-2 luc

Figure 5 :

 5 Figure 5: Survival data of MIA PaCa-2 luc model treated with gemcitabine; gemcitabine

Figure 6 :

 6 Figure 6: Histology images showing the invasive border between normal and tumour tissue in

Table 2 :

 2 Acoustic settings used to enhance drug delivery.

Michelsen Research AS. We would like to thank Lars Helgeland (Section for Pathology, The Gade Institute, University of Bergen) for his advice on the histology.

Acknowledgements

This work was supported by the Norwegian Cancer Society (grant number 421828, 732200); the Western Health Board of Norway (grant numbers 911182, 911789) and the Bergen Research Foundation. The study was supported by MedViz (http://medviz.uib.no/), an interdisciplinary

Conflict of Interest 455

The authors have no conflict of interest.


Pauline Lejault

Jérémie Mitteaux

Francesco Rota Sperti

David Monchaud
email: david.monchaud@cnrs.fr

How to untie G-quadruplex knots and why?

For more than two decades now, the prime objective of the chemical biology community studying G-quadruplexes (G4s) has been to use chemicals to interact with and stabilize G4s (i.e., G4-ligands) in cells to obtain mechanistic interpretations. This strategy has been undoubtedly successful, as demonstrated by the spectacular recent advances. However, these insights have also led to a fundamental rethinking of G4-targeting strategies: in light of the prevalence of G4s in the human genome, transcriptome and ncRNAome (collectively referred to as the G4ome), and particularly their involvement in human diseases (cancers and neuropathologies), should we continue to search for and develop G4-stabilizing ligands or would it not be wise to invest efforts in designing and exploiting molecular tools able to unfold G4s? Perhaps provocative, this question invites us to take a new and fresh look at the wealth of data accumulated on the functional relevance of G4s. In this review, we first focus on how, when and where G4s fold in cells; then, we describe the enzymatic systems the cells have evolved to counteract G4 folding (i.e., G4-helicases) and how they have been used as biomolecular tools to manipulate G4s in cells; finally, we present the strategies currently being implemented by chemical biologists on G4-ligands and G4-helicase to devise new molecular G4-unwinding agents.

1.Introduction

The central dogma of biology, heralded by Francis Crick in 1957, [START_REF] Crick | On protein synthesis[END_REF][START_REF] Crick | Central dogma of molecular biology[END_REF][START_REF] Cobb | 60 years ago, Francis Crick changed the logic of biology[END_REF] has been instrumental in the emergence of modern biology. In science, a dogma is "usually based on some recognized authority (here, one of the discoverers of the DNA double helix structure) [START_REF] Watson | Molecular structure of nucleic acids[END_REF][START_REF] Wilkins | Molecular structure of deoxypentose nucleic acids[END_REF][START_REF] Franklin | Molecular configuration in sodium thymonucleate[END_REF] whose pronouncements are unconditionally accepted". [START_REF] Öpik | About dogma in science, and other recollections of an astronomer[END_REF] A dogma is therefore useful as it provides a basis of accepted knowledge that does not need to be re-demonstrated in each new study.

Somewhat paradoxically, a dogma also fuels studies aiming at challenging its ideas, through permanent verification or reconsideration. [START_REF] Kuhn | The structure of scientific revolutions[END_REF] A dogma is therefore bound to evolve, most often at a pace dictated by the technical advances in the field.

Crick's dogma describes the flow of genetic information from DNA to proteins. In his view, DNA is a barcode waiting to be scanned to meet cellular needs. This point has recently been challenged: while the role of DNA as the repository of genetic information is unquestionable, researchers have uncovered that DNA is not structurally placid and that it can fold into a variety of structures that deviate from the Watson-Crick duplex. The exact roles of this structural plasticity are only now unfolding.

The first-in-class example of alternative DNA structures is undoubtedly the G-quadruplex (G4), a four-stranded structure that folds from guanine (G)-rich sequences. [START_REF] Sen | Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis[END_REF] This structure is highly thermodynamically stable and folds spontaneously when a G-rich DNA strand escapes its duplex structure, i.e., when the double helix melts to allow for DNA transactions (replication, transcription). Initially suspected to form at telomeres, [START_REF] Schaffitzel | In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei[END_REF] bioinformatics [START_REF] Todd | Highly prevalent putative quadruplex sequence motifs in human DNA[END_REF][START_REF] Huppert | Prevalence of quadruplexes in the human genome[END_REF][START_REF] Bedrat | Re-evaluation of G-quadruplex propensity with G4Hunter[END_REF] and then sequencing techniques [START_REF] Chambers | High-throughput sequencing of DNA G-quadruplex structures in the human genome[END_REF][START_REF] Hänsel-Hertsch | G-quadruplex structures mark human regulatory chromatin[END_REF][START_REF] Hänsel-Hertsch | Landscape of G-quadruplex DNA structural regions in breast cancer[END_REF][START_REF] Kwok | G-quadruplexes: prediction, characterization, and biological application[END_REF] have progressively raised G4s to the status of key genetic levers, highly prevalent in the human genome, governing gene expression and genomic stability. [START_REF] Spiegel | The structure and function of DNA G-quadruplexes[END_REF][START_REF] Varshney | The regulation and functions of DNA and RNA G-quadruplexes[END_REF][START_REF] Raguseo | Chemical-biology approaches to probe DNA and RNA G-quadruplex structures in the genome[END_REF][START_REF] Rhodes | G-quadruplexes and their regulatory roles in biology[END_REF] After three decades of research, the time is now ripe to look at the wealth of knowledge accumulated with hindsight: are G4s the promising genetic switches able to defeat Crick's dogma? Or are they one-off, ad hoc events that threaten the genetic integrity of human cells? As discussed hereafter, there is no easy way to address these questions and every answer calls for some nuance. Nature has evolved an efficient enzymatic arsenal (helicases) to regulate G4 formation in vivo, as if to comply with Crick's dogma. Scientists are also now conceiving molecular tools to exploit G4 unwinding systems to uncover the roles of G4s in genetic diseases, in an approach that fits the general concept of chemical biology defined by Linus Pauling in 1954 (see section 4a). (22) In the following sections, we will attempt to portray the G4 landscape in its true colors: what is a G4? G4-DNA versus G4-RNA? Where, when and how do G4s fold within cells? At what cost and for which mechanistic uses? We go on to describe the cell's innate strategy to regulate G4 formation using helicases. Next, we detail the approaches currently investigated by chemical biologists to harness nature's own techniques to untie G4 knots and exploit them in oncology, ageing and age-related diseases. One thing is sure: the visionary ideas of Francis Crick, expressed more than 60 years ago, continue to spark debates among scientists and provide a fertile ground on which ever more innovative science is flourishing.

2.

A nucleic acid structure with a strong identity 2a. G-quadruplex-forming sequences are pervasive in the human genome and transcriptome. G4 structure [START_REF] Burge | Quadruplex DNA: sequence, topology and structure[END_REF] hinges on the self-association of four guanines to form a supramolecular macrocycle referred to as the G-quartet (figure 1A). [START_REF] Stefan | Applications of guanine quartets in nanotechnology and chemical biology[END_REF] The stability of the G-quartet originates in the 8 hydrogen (H) bonds on both and Hoogsteen(25) faces, (4 H-bonds per G, whereas canonical GC association involves only 3 Watson-Crick interactions per base) reinforced by either p- [START_REF] Gilli | Evidence for resonanceassisted hydrogen bonding from crystal-structure correlations on the enol form of the. beta.-diketone fragment[END_REF] or s-bonds, [START_REF] Fonseca Guerra | Telomere structure and stability: covalency in hydrogen bonds, not resonance assistance, causes cooperativity in guanine quartets[END_REF] or both. In physiological conditions, the presence of cations such as K + or Na + is instrumental within the G-quartet, being coordinated to the four inwardly pointing carbonyl groups of the Gs, thereby decreasing the high electronic density at the heart of the macrocycle and overcoming the enthalpic penalty of G4 formation. [START_REF] Zaccaria | RNA versus DNA G-Quadruplex: The Origin of Increased Stability[END_REF] A nucleic acid sequence, both DNA and RNA, is termed a putative G-quadruplex forming sequence (PQFS) if it comprises four or more runs of consecutive Gs (at least two, but more often three). [START_REF] Todd | Highly prevalent putative quadruplex sequence motifs in human DNA[END_REF][START_REF] Huppert | Prevalence of quadruplexes in the human genome[END_REF][START_REF] Bedrat | Re-evaluation of G-quadruplex propensity with G4Hunter[END_REF][START_REF] Kwok | G-quadruplexes: prediction, characterization, and biological application[END_REF] Upon strand folding, consecutive Gs will form consecutive G-quartets that will stack on each other, further stabilizing the resulting edifice through p-stacking interactions and cation chelation between G-quartet layers (figure 1A).

Bioinformatics studies identified >300,000 DNA PQFS in the human genome, corresponding to the sequence G≥3NxG≥3NxG≥3NxG≥3 (where G is guanine, and N any intervening nucleobase, and x ranges from 1 to 7). [START_REF] Todd | Highly prevalent putative quadruplex sequence motifs in human DNA[END_REF] Later, the definition of PQFS was extended to higher x values (i.e. longer loops with x ≤ 15,(29) then ≤ 21) [START_REF] Chambers | High-throughput sequencing of DNA G-quadruplex structures in the human genome[END_REF] and, coupled with sequencing-based techniques, [START_REF] Kwok | G-quadruplexes: prediction, characterization, and biological application[END_REF][START_REF] Rodriguez | Unravelling the genomic targets of small molecules using high-throughput sequencing[END_REF] the number of PQFS was raised to > 500,000 sequences corresponding to G≥3NxG≥3NyG≥3NxG≥3 (x ≤ 7, y ≤ 21, in K + -rich conditions). The prevalence of G4-DNA in our genome is thus firmly established; that of RNA PQFS has also been scrutinized, bearing in mind that structures and functions are more intimately correlated in RNA than in DNA. [START_REF] Wan | Understanding the transcriptome through RNA structure[END_REF][START_REF] Mortimer | Insights into RNA structure and function from genome-wide studies[END_REF][START_REF] Ganser | The roles of structural dynamics in the cellular functions of RNAs[END_REF] G4-RNA are pervasive as well, with > 3,000 PQFS corresponding to G≥3NxG≥3NyG≥3NxG≥3 (x ≤ 12, y ≤ 21, in K + -rich conditions) in a pool of purified polyadenylated (polyA) mRNAs, [START_REF] Kwok | rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome[END_REF] and > 6,000 PQFS from rough cell extracts (including coding and non-coding RNAs).( 35) Example of the thermal denaturation of (D) a G4 (G4-DNA, black line; G4-RNA, red line) labeled with a fluorescein (FAM, green circle) at its 5'-end and a tetramethylrhodamine (red circle) at its 3'-end, monitored by the modification of the FAM fluorescence (FRET-melting assay); and (E) an unlabeled G4-RNA, monitored by the modification of its circular dichroism (CD) signal (CD-melting assay).

2b. The formation of G-quadruplexes is intimately linked to genetic transactions. The strong bias in the genomic/transcriptomic distribution of PQFS towards key functional regions lends credence to widespread G4 regulatory roles (telomeres, replication origins and gene promoters for G4-DNA; 3'-and 5'-untranslated regions (UTR) for G4-RNA). [START_REF] Varshney | The regulation and functions of DNA and RNA G-quadruplexes[END_REF][START_REF] Rhodes | G-quadruplexes and their regulatory roles in biology[END_REF][START_REF] Maizels | G4-associated human diseases[END_REF] For instance, the notable enrichment of DNA PQFS in gene promoters (with > 8,000 PQFS localised < 1kb upstream of gene transcription start sites (TSS))( 37) is suggestive of a G4-mediated control of gene expression at the transcriptional level. Similarly, the strong enrichment of RNA PQFS in UTRs (with > 2,000 and > 500 PQFS in 3'-and 5'-UTR, respectively, in polyA mRNA) [START_REF] Kwok | rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome[END_REF] is suggestive of a G4-mediated control of gene expression at the translational level. However, whether G4s contribute to or perturb cellular functions faces systematic analysis. For instance, G4-DNA can, on one hand, act as a roadblock to polymerases (i.e. inhibiting gene expression) and on the other hand help recruit transcription factors (i.e. favoring gene expression). [START_REF] Kim | The interplay between G-quadruplex and transcription[END_REF] Similarly, G4-RNA in 5'-UTRs can, on one hand, act as a physical obstacle to ribosome entry (cap-dependent translation; i.e. inhibiting gene expression) and, on the other hand, facilitate ribosomes' entry (cap-independent translation; i.e. favoring gene expression). [START_REF] Bugaut | 5 '-UTR RNA G-quadruplexes: translation regulation and targeting[END_REF][START_REF] Kharel | Properties and biological impact of RNA G-quadruplexes: from order to turmoil and back[END_REF] To address these conundrums further research is required.

The formation of G4s in cells, whose prevalence has been demonstrated through a wealth of optical methods, (41,[START_REF] Umar | G-Quadruplex-Based Fluorescent Turn-On Ligands and Aptamers: From Development to Applications[END_REF] is thus intimately linked to genetic transactions (figure 1B) that are replication and transcription for G4-DNA, and translation for G4-RNA. ChIP-seq investigations performed with the BG4 antibody confirmed an enrichment of G4s at transcriptionally active sites, linked to nucleosome depletion and open chromatin state. [START_REF] Chambers | High-throughput sequencing of DNA G-quadruplex structures in the human genome[END_REF] Recently, a G4P-ChIP technique, performed with a dimeric, truncated DHX36 helicase (see section 3c), confirmed both the prevalence of QFS in living human cells (> 120,000 G4P peaks, 60% of which in promoter regions) and the transcription-dependence of G4 formation. [START_REF] Zheng | Detection of genomic G-quadruplexes in living cells using a small artificial protein[END_REF] Similarly, while the formation of G4-RNA seems to be globally repressed in vivo, [START_REF] Guo | RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria[END_REF] functional ncRNAs involved in gene expression (among many other biological functions) [START_REF] Cech | The noncoding RNA revolution-trashing old rules to forge new ones[END_REF] exert their cellular effects via the transient formation of G4-RNA. [START_REF] Yang | Transcriptome-wide identification of transient RNA Gquadruplexes in human cells[END_REF] G4 folding in vivo could thus represent another layer of non-genetic, non-epigenetic but structural regulation of gene expression.

2c. G-quadruplexes are structurally defined and thermodynamically stable. The threedimensional structure of G4s is dictated by the sequence they fold from. The G-runs provide the structural basis of the G4 core, allowing G-quartets to stack on top of each other. The number and nature of the intervening nucleobases N (vide supra) offer structural diversity, forming loops of different length (≤ 21), orientation (lateral, diagonal or edgewise loops, figure 1C) and nature (single-stranded or forming bulges). G4-DNA has high structural plasticity (adopting conformations most often referred to as parallel, antiparallel and hybrid-type G4 structure, or types I-III) [START_REF] Karsisiotis | Topological Characterization of Nucleic Acid G-Quadruplexes by UV Absorption and Circular Dichroism[END_REF], while G4-RNA is mostly restricted to a parallel structure, owing to the influence of the additional 2'-OH of the RNA backbone believed to be involved in an additional extended H-bond network. This network might also explain why G4-RNA are generally more stable than their G4-DNA counterparts (vide infra).

Subtle changes in either the nucleic acid sequences (i.e. the nature of Nx) or the experimental conditions in which the PQFS are studied (i.e. the cationic nature of the buffer, the stabilizing Na + or K + versus the non-stabilizing Li + ) have profound effects on the way G4s fold upon themselves and on the stability of the resulting edifices. A wide array of biophysical and spectroscopic methods have been developed to understand the thermodynamic and kinetic parameters that govern G4 folding. [START_REF] Jaumot | Experimental Methods for Studying the Interactions between G-Quadruplex Structures and Ligands[END_REF][START_REF] Murat | Methods for investigating G-quadruplex DNA/ligand interactions[END_REF] Experiments based on an increase of temperature (so-called melting) have been particularly popular: [START_REF] Mergny | Following G-quartet formation by UVspectroscopy[END_REF][START_REF] Rachwal | Quadruplex melting[END_REF][START_REF] Risitano | The stability of intramolecular DNA quadruplexes with extended loops forming inter-and intra-loop duplexes[END_REF] the temperaturepromoted transition from the folded (low temperature) to the unfolded conformation (high temperature) of a PQFS can easily be monitored by fluorescence (e.g. FRET, for fluorescence resonance energy transfer, figure 1D), [START_REF] Rachwal | Quadruplex melting[END_REF][START_REF] De Cian | Fluorescence-based melting assays for studying quadruplex ligands[END_REF] absorbance (UV-vis) [START_REF] Mergny | Following G-quartet formation by UVspectroscopy[END_REF] or circular dichroism (CD, figure 1E) [START_REF] Masiero | A non-empirical chromophoric interpretation of CD spectra of DNA Gquadruplex structures[END_REF][START_REF] Kypr | Circular dichroism and conformational polymorphism of DNA[END_REF] spectroscopy and has been used to conveniently characterize G4 stability via the measurement of the mid-transition temperature (or melting temperature, Tm) at which 50% of the G4 is unfolded. For instance, melting experiments have been instrumental to demonstrate that both the length and nature of nucleobases constituting the G4 loops (Nx) are critical for G4 stability. As an example, the A-to-T substitution in the 3 loops of the G4-DNA d[ 5 ' (G3N3)3G3 3 ' ] triggers a dramatic drop in G4 stability as shown by a decrease of the Tm from 74 (N = T) to 50 °C (N = A) assessed by FRET-melting, in a 1mM K + -containing buffer. [START_REF] Rachwala | Sequence effects of single base loops in intramolecular quadruplex DNA[END_REF] Similarly, increasing the length of the central loop of the G4-DNA d[ 5 ' G3TG3TxG3TG3 3 ' ] destabilizes the resulting G4 as demonstrated by a decrease of the Tm from 84 (x = 2) to 62 °C (x = 15) assessed by UV-melting in a 100 mM K + -containing buffer. [START_REF] Guedin | How long is too long? Effects of loop size on G-quadruplex stability[END_REF] The difference of stability between G4-DNA and G4-RNA was also conveniently demonstrated by both CDmelting, [START_REF] Joachimi | A comparison of DNA and RNA quadruplex structures and stabilities[END_REF] with a Tm = 65 °C for d[ 5 ' G3(T2AG3)3 3 ' ] (the human telomeric sequence) versus 73 °C for r[ 5 ' G3(U2AG3)3 3 ' ] (the telomeric transcript) in 100 mM K + and by FRET-melting (figure 1D), with a Tm of 51 °C for d[ 5 ' G3(T2AG3)

3 3 ' ] (in 10 mM K + ) versus 63 °C for r[ 5 ' G3(U2AG3)3 3 ' ] (in 1 mM K + ).
The stability of G4s determined in vitro corresponds to their ability to trigger genetic instability in vivo. For instance, a study in S. Cerevisiae has demonstrated that shortening the central loop (underlined) of the CEB25 G4 d[ 5 ' A2G3TG3TGTA2GTGTG3TG3T 3 ' ] from 9-to 1-nt loop d[ 5 ' A2G3TG3TG3TG3T 3 ' ] increases the stability of the resulting G4s (with Tm = 55 and 74 °C, respectively), which correlates with a spectacular increase of the genomic instability, from 0.5 to 67% of minisatellites rearrangements in helicase-deficient colonies. [START_REF] Piazza | Short loop length and high thermal stability determine genomic instability induced by G-quadruplex-forming minisatellites[END_REF] These results indicate the extent to which G4s threaten the genetic stability and the importance of the G4-helicases in safeguarding this stability (further discussed below). Initially postulated to be 'melting proteins' by Sen & Gilbert in 1988,(9) the pivotal roles of helicases in managing the threat of G4s was uncovered only nine years later, with the description of the SV40 large T-ag helicase. [START_REF] Baran | The SV40 large T-antigen helicase can unwind four stranded DNA structures linked by Gquartets[END_REF] 3. Enzymatic unwinding of DNA and RNA [START_REF] Abdel-Monem | Enzymic Unwinding of DNA: 1. Purification and Characterization of a DNA-Dependent ATPase from Escherichia coli[END_REF][START_REF] Abdel-Monem | Enzymic Unwinding of DNA: 2. Chain Separation by an ATP-Dependent DNA Unwinding Enzyme[END_REF] 3a. Helicases & genetic diseases. Helicases were discovered in 1976, initially referred to as 'DNA unwinding proteins' [START_REF] Abdel-Monem | Enzymic Unwinding of DNA: 1. Purification and Characterization of a DNA-Dependent ATPase from Escherichia coli[END_REF][START_REF] Abdel-Monem | Enzymic Unwinding of DNA: 2. Chain Separation by an ATP-Dependent DNA Unwinding Enzyme[END_REF][START_REF] Mackay | Selective inhibition of the dnase activity of the recBC enzyme by the DNA binding protein from Escherichia coli[END_REF] and the term helicase was officially coined in 1979. [START_REF] Kuhn | Evidence for two mechanisms for DNA unwinding catalyzed by DNA helicases[END_REF] Helicases are enzymes with the eponymous ability to unwind DNA helices. A total of 95 different helicases, 31 for DNA and 64 for RNA, have now been reported. [START_REF] Brosh | History of DNA Helicases[END_REF] The two most studied families are the RecQ family, including Bloom (BLM), Werner (WRN), and RecQ-like helicase 1, 4 and 5 (RECQL1, RECQL4 and RECQL5, respectively); and the iron-sulfur (Fe-S) family, including xeroderma pigmentosum group D (XPD), Fanconi anaemia complementation group J (FANCJ, also known as BRCA1 interacting protein C-terminal Helicase 1 (BRIP1)), regulator of telomere length helicase 1 (RTEL1) and DEAD/H-Box helicase 11 (DDX11). [START_REF] Estep | RecQ and Fe-S helicases have unique roles in DNA metabolism dictated by their unwinding directionality, substrate specificity, and protein interactions[END_REF] Accumulating evidence points to the critical role of helicases in genome maintenance, [START_REF] Brosh | DNA helicases involved in DNA repair and their roles in cancer[END_REF] being involved in DNA damage response and repair. [START_REF] Brosh | History of DNA Helicases[END_REF] They exert their safeguarding activity via the resolution of higher-order DNA structures that are transiently formed either during DNA repair (e.g. the Holliday junction [START_REF] Holliday | A mechanism for gene conversion in fungi[END_REF][START_REF] Liu | Happy Hollidays: 40th anniversary of the Holliday junction[END_REF] intermediate of homologous recombination) or during genetic transactions (e.g. the 5' flap intermediates during replication, [START_REF] Brosh | Processing of DNA replication and repair intermediates by the concerted action of RecQ helicases and Rad2 structure-specific nucleases[END_REF] or the Rloops during transcription).( 71) Helicases also participate in genome maintenance, resolving higher-order structures that fold from hard-to-replicate sequences, [START_REF] Técher | The impact of replication stress on replication dynamics and DNA damage in vertebrate cells[END_REF][START_REF] Bochman | DNA secondary structures: stability and function of G-quadruplex structures[END_REF] chiefly G4s (vide infra), [START_REF] Mendoza | Gquadruplexes and helicases[END_REF][START_REF] Sauer | G-quadruplex unwinding helicases and their function in vivo[END_REF][START_REF] Lansdorp | Helicases FANCJ, RTEL1 and BLM Act on Guanine Quadruplex DNA in Vivo[END_REF] that can act as physical barriers to polymerase advancement along their DNA substrates.

Most of the information collected about helicases stems from the diseases originating in their dysregulation. For instance, overexpression of RECQL1 [START_REF] Futami | RecQL1 DNA repair helicase: A potential tumor marker and therapeutic target against hepatocellular carcinoma[END_REF] and RECQL5( 78) is a signature of several cancers [START_REF] Su | Human RecQL4 helicase plays critical roles in prostate carcinogenesis[END_REF] and FANCJ is frequently mutated in breast cancers. [START_REF] Cantor | Hereditary breast cancer and the BRCA1associated FANCJ/BACH1/BRIP1[END_REF] Mutations in helicase genes are also responsible for severe inheritable genetic disorders:

Cockayne syndrome (dwarfism, mental retardation, skin and skeletal abnormalities) is due to the mutation of XPD; [START_REF] Lehmann | The xeroderma pigmentosum group D (XPD) gene: one gene, two functions, three diseases[END_REF][START_REF] Broughton | MOLECULAR AND CELLULAR ANALYSIS OF THE DNA-REPAIR DEFECT IN A PATIENT IN XERODERMA-PIGMENTOSUM COMPLEMENTATION GROUP-D WHO HAS THE CLINICAL-FEATURES OF XERODERMA-PIGMENTOSUM AND COCKAYNE-SYNDROME[END_REF] Bloom syndrome (growth retardation and immunodeficiency) to that of BLM; [START_REF] Ellis | The Bloom's syndrome gene product is homologous to RecQ helicases[END_REF] Werner syndrome (or 'adult progeria', early ageing) to that of WRN; [START_REF] Yu | Positional cloning of the Werner's syndrome gene[END_REF] Rothmund-Thomson syndrome (or 'poikiloderma congenitale', abnormalities in skin and skeleton, premature ageing) to that of RecQL4; [START_REF] Kitao | Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome[END_REF] Fanconi anemia (developmental abnormalities, bone marrow failure) to that of FANCJ; [START_REF] Levran | The BRCA1-interacting helicase BRIP1 is deficient in Fanconi anemia[END_REF][START_REF] Levitus | The DNA helicase BRIP1 is defective in Fanconi anemia complementation group[END_REF][START_REF] Litman | BACH1 is critical for homologous recombination and appears to be the Fanconi anemia gene product FANCJ[END_REF] etc.

Collectively, these examples show that the lack of helicase activity triggers the accumulation of unresolved forked DNA that is responsible for severe genetic disorders and cancer predisposition. They also confirm that structure-prone DNA sequences can jeopardize overall genetic stability. A way to tackle this threat is to use helicases as molecular tools to unfold these higher-order structures. As further detailed hereafter, this has been particularly studied with the subfamily of helicases known as the G4-helicases. [START_REF] Mendoza | Gquadruplexes and helicases[END_REF][START_REF] Sauer | G-quadruplex unwinding helicases and their function in vivo[END_REF][START_REF] Lansdorp | Helicases FANCJ, RTEL1 and BLM Act on Guanine Quadruplex DNA in Vivo[END_REF] Table 1. Main mammalian G4-helicases. 3b. An enzymatic machinery to unfold G-quadruplexes. Beyond RecQ and Fe-S helicases, a novel overlapping family is now being classified, the G4-helicases. [START_REF] Brosh | History of DNA Helicases[END_REF][START_REF] Mendoza | Gquadruplexes and helicases[END_REF][START_REF] Sauer | G-quadruplex unwinding helicases and their function in vivo[END_REF][START_REF] Lansdorp | Helicases FANCJ, RTEL1 and BLM Act on Guanine Quadruplex DNA in Vivo[END_REF]107) These helicases unwind G4s in vitro and evidence is now accumulating to support a similar activity in vivo. The most studied mammalian G4-helicases are undoubtedly BLM, WRN, FANCJ and RTEL1, along with Pif1 (Petite integration frequency 1)(108,109) and DHX36 (DEAH (Asp-Glu-Ala-His)-box Helicase 36)(110) (table 1). is supposed to trigger the G4 unfolding in an ATP-independent manner, a mechanical rotation that is dynamically counteracted by spontaneous G4 refolding. This partial opening is disruptive enough to allow for the hybridization of the complementary strand via Watson-Crick base pairing. G4-RNA unwinding is thought to occur via a different mechanism in which ATP hydrolysis is required. [START_REF] Kim | The interplay between G-quadruplex and transcription[END_REF]190) The depletion of DHX36 results in the impairment of a wide range of mRNA, which accumulate in stress granules. (191) The activity of DHX36 was also shown to be impeded by G4 ligands (including the PDS and PhenDC3).( 192) G4-helicases are thus key players for controlling G4 dynamics in cells and their catalytic efficiency can be modulated by a series of DNA-binding protein partners including the replication protein A (RPA), POT1, the proliferating cell nuclear antigen (PCNA) and the DNA repair protein REV1 (Table 1). RPA is a ssDNA binding protein (ssBP) described to greatly enhance the DNA unwinding properties of both Fe-S and RecQ helicases, although RPA was also shown to unfold telomeric G4 in vitro alone. (193,194) This heterotrimeric protein complex, which comprises 3 subunits commonly referred to as RPA70, RPA32 and RPA14 (70, fused ScPif1 with hemagglutinin (HA) to perform a coIP (using anti-HA coated beads) with Pif1-HA and the transcriptional activator and G4 binder, Sub1. Again, it was hypothesized that Sub1 could recruit G4-resolving helicases at G4 sites during transcription, thereby suppressing G4associated genome instability and offering protection against toxicity triggered by a G4 ligand, in this case the porphyrin TMPyP4 (further discussed below, see section 4b).( 226 helicases have proven to be highly efficient and versatile molecular tools to investigate helicase dynamics and function, as well as G4 landscapes in cells. More recently, a series of G4 probes (G4P) were designed on the basis of the dimerization of the G4-binding domain of DHX36 (a 23-amino acid segment referred to as RHAU23) conjugated to either His/Flag tags (for purification purposes) or NLS sequences (for controlling the subcellular localization of the G4P).( 43) As discussed in section 2b, G4Ps enabled the detection of > 120,000 QFS in living cells (70% of which being associated with genes, 60% within 2kb around the TSS) via the G4P-ChIP method, that is, substantially more QFS detected than by G4-ChIP performed with BG4, ascribed to its smaller size (6.7 kDa versus >30 kDa for BG4) and the technical difference in protocols implemented (G4P is incubated live while BG4 is added after cell fixation). 22) Since then, the notion has progressively evolved towards a more general concept in which chemicals are used as "reversible modulators" of biological systems "to gain a deep understanding of cell circuitry and disease biology".( 229)

The latter definition perfectly suits G4s and G4-ligands. Cell-permeable G4-specific small molecules have been used for decades to interact with G4s in cells in the hope of providing mechanistic interpretations. This approach has enabled significant progress beginning in the late 1990s.(230-232) However, most of the reported G4 ligands were intended to be used as drugs,(233-237) rather than chemical biology probes, [START_REF] Raguseo | Chemical-biology approaches to probe DNA and RNA G-quadruplex structures in the genome[END_REF]41,[START_REF] Umar | G-Quadruplex-Based Fluorescent Turn-On Ligands and Aptamers: From Development to Applications[END_REF] bypassing the stage of molecular mechanistic study in order to directly develop novel therapeutics, often in cancer.

From a chemical design point of view, the conformational polymorphism of G4-DNA (see section 2c) might explain why its targeting is challenging. This difficulty is further compounded by the fact that G4 conformation can commonly be ligand-induced. Targeting G4-RNA could be considered simpler for its relative topological simplicity (only forming parallel structures), however this topology lacks well-defined binding sites. Hundreds of G4-ligands have been designed and studied over the past decades,(233-237) with some successful examples (e.g. BRACO19,(238-240) PhenDC3,(241-244) PDS (214,(244)(245)(246) and CX-5461),(244,247) which mostly interact through p-stacking atop the external accessible G-quartet (Figure 4). Notable advances have been made in terms of selectivity for the G4 structure over dsDNA, but this field still faces tremendous challenges: next-generation G4-ligands will have to meet expectations in terms of G4-DNA vs G4-RNA specificity,(248-251) and of their ability to discriminate different G4 topologies. This is particularly important given that an overwhelming majority G4 ligands have been designed against a particular subclass of G4s (e.g. G4s in promoters or telomeres); however, given the very high number of possible PQFS targets in our genome and transcriptome (see section 2a), the chance of success is limited. Yet, the anticancer properties of G4 ligands are undoubtedly established. The mechanisms by which they exert this activity remain to be deciphered. The link between a global antiproliferative effect observed on ligand treatment and the cellular processes involving the G4s for which an in vitro affinity has been determined (e.g. G4-forming sequences from promoters or telomeres in solution) is often hastily established. As G4s are intimately linked to genetic transactions (see section 2b), G4 ligands can stabilize physically barriers to polymerases in general, triggering DNA damage (Figure 4).(252,253) It may be more elegant to consider G4s as genetic switches with specific spatiotemporal control over key cellular processes, compared to considering G4s as genome-wide triggers for creating DNA damage, however this latter, simpler rationale offers a mechanistic model that accounts for most of the cell-based studies collected so far. Therefore, in light of the propensity of G4-stabilising ligands to trigger genetic instability, and the association with severe genetic dysregulations (see section 2c), it is surprising that, to date, most if not all efforts by chemical biologists have been focused on G4 stabilization rather than destabilization. This may stem from the difficulty in designing small molecules able to mimic G4 helicases.

4b. How to destabilize G-quadruplexes and why?

The quest for molecules that could unwind G4s and rescue helicase deficiency is a yet emerging but highly promising topic.( 254)

This research is technically impeded by the lack of standard assays and protocols to reliably assess G4 destabilization, in sharp contrast to the stabilization of G4s (see section 2c).

To date, only a handful of compounds have been described with such a property (figure 4), some of them being a subject of debate (vide infra). However, there is no consensus on the cellular relevance of these compounds as their unwinding capabilities were assessed in vitro with assays tailored for a single study only. Beyond established techniques of nucleic acid denaturation applied to G4s, including heating (see above) and urea,(255) chemicals such as metallic ions (e.g., copper)(256) and complexes(257,258) as well as natural compounds (e.g., polyamines such as spermine) have been used in large excess as a way to study the dynamics of G4 folding and unfolding. The systematic analysis of the G4-unwinding agents really started with TMPyP4 (figure 4B), a ligand that has been thoroughly used for stabilizing G4s, (259,260) owing to its large aromatic structure that overlaps efficiently the external G-quartet of a G4 while its positively charged pyridinium arms interact with the negatively charged G4 grooves.(261) For instance, TMPyP4 was used in the seminal study on the genetic expression regulation of the c-myc oncogene via stabilization of the G4 found in its promoter region.( 262) unfolding presumably through groove binding (kinetic event) followed by the intercalation of the ligand within the G4 structure, in which it acts as a lever that opens the G4 backbone.

Several derivatives were later developed,(290) and studied for their G4-stabilising properties, along with their antiproliferative activity against T. brucei parasite and HeLa cancer cells. NMR titrations confirmed the ability of bispyridinium stiff-stilbenes to disrupt the external G-quartet upon binding (1:2 G4:ligand ratio, in K + -conditions), which was also reported with a bispyridinium dithienylethene (DTE) derivative (at 1:3.6 G4:ligand ratio in both K + and Na + ). DTE can be reversibly photoswitched (lex = 450/635 nm) between a 'closed' and an 'open' conformation, the former behaving as a classical G4 external stacker, while the latter disrupts the external G-quartet through partial intercalation.(291)

Summary & outlook

Over twenty years of research have enabled us to attain our current molecular understanding of the roles that G4s play in human cells and diseases. The functional relevance of G4s has progressed at a pace dictated by the advances in chemical biology methodology.

As the field currently stands, scientists are able to foster G4 cellular effects using G4-stabilizing compounds (such as through inflicting severe damage on the cancer cell genome, thus inducing lethal genetic aberrations in anticancer strategies), as well as alleviate G4-mediated cellular processes using G4-resolving molecular tools, one of the key challenges on the way to providing those eagerly awaited therapeutics to fight neurological disorders and age-related diseases. The genetic diseases listed in Table 1 are currently suffering from severe drug attrition: most of the treatments applied so far are either protective (e.g., protection from sunlight, against aggressive infections for Bloom's syndrome patients)(292) or prospective (e.g., the targeting of p38 MAPK signaling pathway in Werner Syndrome-derived cells),( 293) but none of them tackle the molecular origins of the disease. We do not propose to predict which direction should be followed within G4 research. However, we summarize here the knowledge and tools now available to enable further investigations into G4-unwinding. We described the two types of G4-unwinders that are currently being investigated: the G4helicases on one hand, which are increasingly used as biomolecular tools mostly for optical imaging but also to reverse detrimental G4-mediated cellular effects; and small molecule G4destabilizers on the other hand, with a particular focus on the current issues that must be addressed to make small molecules reliable candidates for manipulating G4s in cells. To date, these are mostly technical issues (e.g. the lack of standard assays to assess G4-destabilization in vitro)(294) but the recent results described here leave no doubts as to the forthcoming development of such promising molecular tools, which will find application ranging from fundamental research tools to potential new genetic disease therapy.

Significance

Small molecule-mediated G-quadruplex (G4)-stabilization has been the privileged chemical biology approach to gain insights into G4 cellular biology. An alternative approach is now emerging, relying on the use of molecular tools able to unfold G4s, which shed new light on G4 regulatory roles. In this review, we describe in detail the different strategies implemented to unwind G4s (using either unfolding proteins, known as G4-helicases, or G4-disrupting smallmolecules), with all their strengths and weaknesses, and what could be the therapeutic consequences of manipulating G4 in cells to treat genetic diseases.

Figure 1 .

 1 Figure 1. Schematic representation of (A) the transient formation of a G-quadruplex-DNA (G4-DNA) when a putative quadruplex forming sequence (PQFS) is freed from the duplex structure (inserts show detailed chemical structures of guanine (G) and G-quartet); (B) the G4-mediated stalling of the RNA polymerase during transcription; and (C) the possible G4 topologies: parallel, antiparallel and hybrid G4. [Created with BioRender]Example of the thermal denaturation of (D) a G4 (G4-DNA, black line; G4-RNA, red line) labeled with a fluorescein (FAM, green circle) at its 5'-end and a tetramethylrhodamine (red circle) at its 3'-end, monitored by the modification of the FAM fluorescence (FRET-melting assay); and (E) an unlabeled G4-RNA, monitored by the modification of its circular dichroism (CD) signal (CD-melting assay).

Figure 2 .

 2 Figure 2. X-ray structures of both BLM (A, PDB ID: 4CGZ)(111) and WRN (B, PDB ID: 4O3M)(112) in complex with a 3'-overhang of a dsDNA, of Pif1 with both 3'-and 5'-overhang of a dsDNA (or forked DNA) (C, PDB ID: 6L3G),(113) and of DHX36 in complex with a 3'-overhang of a G4 (D, PDB ID: 5VHE).(114) The DNA sequences are indicated; nucleotides in orange belong to the overhang. [Created with Chimera] Zakian's and Ferré-d'Amaré's models for G4-helicase-mediated G4-unfolding by Pif1 (E)(115) and DHX36 (F),(114) respectively, based on single-molecule FRET experiments and the X-ray crystallography structure seen in panel D. [Created with BioRender]

32 and 14 Figure 3 .

 143 Figure 3. Optical imaging performed with GFP-labeled Pif1 (A-C) and FANCJ (D) in yeast (S. Pombe strain, A) and human cancer cells (U2OS (B,C) and HeLa cells (D)) showed the subcellular localization of Pif1 (A),(213) the presence of G4s in human cells through the colocalization with both G4-specific small-molecule (PDS-a) and protein (hPif1a) (B),(214) and demonstrated the recruitment of Pif1 (C)(216) and FANCJ (D)(220) to DNA-damage foci.

)

  Pif1 has received a great deal of attention, however other helicases have been genetically engineered and the resulting constructs used to further study their cellular activities. For example, Brosh et al. expressed wild type and mutated FANC-J fused with GFP in chicken DT40 cells for coIP investigations, thus identifying BRCA1 as a FANC-J interaction partner, and visualizing the recruitment of FANC-J to DNA damage sites upon exposure to the DNA cross-linking agent mitomycin C or the G4 ligand TMS.(227) This approach was further developed later, in both DT40 and HeLa cells, expanding the panel of FANC-J mutations studied (defective in dimerization, processivity and helicase activity), testing other DNA damaging agents (TMS, DNA crosslinking agent cisplatin, DNA polymerase inhibitor aphidicholin and DSB-inducer bleomycin), and identifying other interaction partners (e.g. DNA repair protein MRE11 that recruits FANC-J to DSB sites). The investigators used thorough colocalization investigations (with DNA damage marker gH2AX, HR marker RAD51 and interstrand cross-link repair marker XPB) (figure3d).(220,228) Overall, genetically engineered

4 .

 4 Chemical biology with G-quadruplexes 4a. How to stabilize G-quadruplexes and why? The notion of chemical biology dates back to a correspondence in 1975 between Linus Pauling and George W. Beadle, although they officially published the term in 1954.(

Figure 4 .

 4 Figure 4. Schematic representation (A) of the G4-mediated stalling of the RNA polymerase during transcription along with the rescue pathway by the action of G4-helicases (left), a possible rescue pathway by the action of G4-destabilizing compounds (right), and, conversely, reinforcement of the crisis situation by G4-ligands (center). [Created with BioRender] Chemical structures (B) of representative G4-ligands (upper row, i.e. BRACO-19,(238) PhenDC3,(241) PDS(245) and CX-5461)(247) and of G4-detsabilizing compounds (lower row, i.e. TMPyP4,(263) anthrathiophenedione,(264) TAP1(265) and stiff-stilbene).(266) Structures (C) of G4-ligand/G4 complexes obtained with BRACO19 in interaction with a bimolecular human telomeric (HTelo) G4 (PDB ID: 3CE5),(267) PhenDC3 in interaction with Pu24T (from c-myc promoter, PDB ID: 2MGN),(268) and TMPyP4 in interaction with both Pu24I (from c-myc promoter, PDB ID: 2A5R)(261) and the bimolecular HTelo (PDB ID: 2HRI),(269) from either X-ray (HTelo) or NMR investigations (c-myc). [Created with Chimera.] (D) Kaluzhny's and Galan's molecular dynamics models for small molecule-mediated G4-unfolding performed with the telomeric G4 (PDB ID: 143D)(270) and either the anthrathiophenedione(264) or stiff-stilbene.(266) [Created with BioRender]

Acknowledgment

The authors thank the Centre National de la Recherche Scientifique (CNRS), the Agence Nationale de la Recherche (ANR-17-CE17-0010-01), the Université de Bourgogne, Conseil Régional de Bourgogne and the European Union (PO FEDER-FSE Bourgogne 2014/2020 programs, LS 206712) and the INSERM Plan Cancer 2014-2019 (19CP117-00) for financial support. Jo Zell is warmly acknowledged for her help in editing the manuscript.

Author contributions

P.L., J.M., F.R.S. and D.M. wrote the manuscript.

Declaration of interests.

The authors declare no competing interests.

Highlights

• G-quadruplexes (G4s) are pervasive in human genome and transcriptome

• Enzymes referred to as G4-helicases are known to unfold G4s in cells • G4-helicases can be used as molecular tools to manipulate G4s in cells • G4-stabilizing small molecules (G4-ligands) are currently studied as anticancer agents

• G4-disrupting molecules are new molecular tools with high therapeutic potential Graphical abstract