Jacques

Ellie, Maor, Cyril Josefine Tobias

SELENIUM NANOPARTICLES INDUCE GLOBAL HISTONE METHYLATION CHANGES IN OVARIAN CANCER CELLS

Keywords:

for the funding during the three years of my PhD. They allowed me to fulfil this PhD between Swansea and Grenoble in the best conditions sharing my time between the two labs. I would like to thank all technicians, secretary and doctoral school administrators for their helpful support when I was going through the different administrative challenges. I'd like to thank my PhD directors. Laurent that trusted me in the first place, trusted the fact that even after 3 years into a teaching program I would still be able to adapt to the research world. Steve that decided since the beginning to set recurrent meeting that helped me a lot in leading my research to high scientific standards allowing publications. Lewis that was their everyday for 3 years, supporting and helping me at any time, correcting my clumsy English writings with the same patience everytime. I can't be more grateful for all what you did for me. Alexandra for the MITI bursary we obtained that released a lot of pressure on my research expenses for those three years. I'd like to thank the post-doc dream team of the RGBO Jetzi, Seydou, Marcos, Andrea, without you the lab wouldn't be the same. Dorine and Antoine for hosting me in Lyon every time I had to go to the ENS for my experiments. Martina, Francesca, Carla, Roberto, Claudia, Dom for those numerous diners we cooked altogether during the hard winter months in Swansea. Andrew for being an amazing roommate as well as an amazing friend, slowing your

Introduction I. Ovarian cancer 1. The disease

Cancer is characterized by an abnormal and uncontrolled growth of cells 1 . This transformation is due to genomic instability and specific gene mutations that have been linked to cancer cells being able to adapt to, and regulate, their local micro environment 1 . Tumorigenic processes are distinct and have been well characterised, exhibiting hallmarks linking to biological functions that are unique to cancer cells 1 (Figure 1). Cancer cells have a unique ability to sustain proliferative signalling by activation of growth factors expression, evade growth suppressors notably p53 (signalling DNA damages) mutation, activate invasive and metastatic processes, enable replicative immortality, induce angiogenesis to increase oxygen and nutrient availability, and resist cell death by circumventing apoptosis 2 . In addition, cancer cells have been shown to deregulate their energy metabolism and avoid the immune cell surveillance mechanisms 1 . 1 Cancer cells are cells that divide without control and resist to cell death due to genomic instability. The growth of tumours can lead to induced angiogenesis responsible for metastatic dissemination.

Figure 1 Hallmarks of cancer

i. Epidemiology

Ovarian cancer is the most common cancer in women with 295,400 cases in 2018 and the 6 th most lethal with 184,800 mortalities reported worldwide that year 3 . The incidence has been increasing during the last decade with 1.3% of the women in the UK and 3.8% in France being affected by the disease 4 . Ethnicity has been correlated with the prevalence with 1.64 times more black women than other ethnic groups developing ovarian cancer.

Different symptoms of early stage such as abdominal and pelvic pain, irregular menarche, change in bowels habits and increase urinary frequency 5 have been identified however these are not specific to ovarian cancer. More severe symptoms are often indicating that ovarian cancer has developed. Survival rates of late detection drops with only 35% of UK patients surviving within 5 years 6 whereas the early detection lead to an 90% chance of survival 4,7 . Despite the fact that the survival rate have been increasing in the last 20 years due to better detection and development of new chemotherapies (PARP inhibitors for example), amongst the high grade carcinomas ovarian cancer remains the deadliest gynaecological cancer (35% survival rate) compared to an 80% 5-years survival for breast cancer, 70% for endometrial cancer and 60% for cervical cancer 8,9 .

ii. High Grade Serous Ovarian Cancer (HGSOC) Risk Factors

Ovarian cancer most often affects women between the ages of 75 and 80 years 10,11 . Two hypotheses have been suggested to explain the development of High Grade Serous Ovarian Cancer (HGSOC). Firstly ovulation creates a lesion of the ovarian epithelium that needs to be repaired. This highly inflammatory microenvironment may lead to DNA damage, replication errors and malignant transformations 12,13 . Incessant ovulation with an early menarche has been related to an increase prevalence of low grade ovarian cancer 14,15 .

Secondly, the development of Ovarian Cancer has been related to the onset of menopause. During menopause the ovaries are unable to respond to hormonal stimuli stopping the feedback of gonadotropins 16 . The higher level of oestrogen compared to progesterone then results in higher oestrogen exposure by ovarian epithelial cells increasing the risk to develop ovarian cancer 5,17 .

and by 27% for BRCA2 mutation 19 . BRCA1 and 2 are involved in DNA repair, maintenance of genome stability and function as tumour suppressors 20 . The use of progesterone-based oral contraceptives for at least 4 years has been related to a decrease of ovarian cancer risk by 50% in women with a BRCA mutation 21 by blocking ovulation. Genetic mutations are not limited to BRCA1/2 genes inducing instability in genome [START_REF] Pappano | The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia[END_REF] with deficiencies in homologous recombination, impairing the repair of the DNA also reported [START_REF] Vinceti | Selenium for preventing cancer[END_REF] . Several suppressor genes and oncogenes have been associated with ovarian cancer. P53 and mismatch repair (MMR) or double strand break repair system (CHEK, RAD1) mutations have also been related to cancer development 4,11,17 .

Finally, epigenetic modifications have also be related to malignant development and progression of ovarian cancer. Hypermethylation of BRCA1 and 2 promoters has been related to a decrease in the efficacy of DNA repair of spontaneous mutations in ovarian epithelial cells 24 . Hypermethylation of CpG islands have been found to be related with tumour development in comparison with normal tissues [START_REF] Bai | Selenium nanoparticles-loaded chitosan/citrate complex and its protection against oxidative stress in d-galactose-induced aging mice[END_REF] .

Metastasis

Cancer metastasis is the leading cause of cancer death accounting for 90% of ovarian cancer cases [START_REF] Campaner | The methyltransferase Set7/9 (Setd7) is dispensable for the p53mediated DNA damage response in vivo[END_REF] . Ovarian cancer has a unique mode of development, disseminating locally in the peritoneal cavity and rarely beyond [START_REF] Clare | One-Carbon Metabolism: Linking Nutritional Biochemistry to Epigenetic Programming of Long-Term Development[END_REF][START_REF] Bothwell | Se-Adenosyl-l-selenomethionine Cofactor Analogue as a Reporter of Protein Methylation[END_REF] . Peritoneal dissemination occurs by movement of ovarian cancer cells in the peritoneal fluid (also called ascites) [START_REF] Malkowski | Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing[END_REF] . Dissemination happens either when the tumour has grown extensively in the organ and caused rupture of the ovary surface or when tumour arises from the surface of the ovary. This dissemination is accompanied by molecular alterations in cells and notably through a cadherin switch involving overexpression of E-cadherin (Figure 2), and activation of N-cadherin expression which is a mesenchymal marker and vimentin expression [START_REF] Davis | Dietary Folate and Selenium Affect Dimethylhydrazine-Induced Aberrant Crypt Formation, Global DNA Methylation and One-Carbon Metabolism in Rats[END_REF] . Moreover the phenotype of the cells is modified as they undergo an epithelial to mesenchymal transition [START_REF] Campaner | The methyltransferase Set7/9 (Setd7) is dispensable for the p53mediated DNA damage response in vivo[END_REF][START_REF] Davis | Dietary Selenium and Arsenic Affect DNA Methylation In Vitro in Caco-2 Cells and In Vivo in Rat Liver and Colon[END_REF] . Once in the peritoneal cavity, ovarian cancer cells undergo two different paths. Isolated cells undergo anoikis as they lost interaction with extracellular matrix and other cells while multicellular aggregates [START_REF] Barroso | The Link Between Hyperhomocysteinemia and Hypomethylation: Implications for Cardiovascular Disease[END_REF] formed in the peritoneal cavity form spheroids that can seed in multiple distal sites. The invasion of secondary sites is facilitated by the remodelling of the extracellular matrix of the mesothelial lining at these locations by matrix metalloproteases. [START_REF] Davis | Dietary Folate and Selenium Affect Dimethylhydrazine-Induced Aberrant Crypt Formation, Global DNA Methylation and One-Carbon Metabolism in Rats[END_REF] . Dissemination of ovarian cancer cells from the primary tumour to the peritoneal cavity through ascitic fluid is accompanied with cadherin switch allowing the formation of spheroids which can land on the mesothelial lining of the abdominal cavity. This attachment leads to a second set of modifications of the cellular properties in their interactions with other cells and cancer cells acquire the ability to go through the peritoneum.

Figure 2: Modification of the membrane expression profile of the cells in the primary tumour to their dissemination

The colonisation of secondary sites involves the interaction between ovarian cancer cells and mesothelium cells of the peritoneal cavity [START_REF] Bothwell | Se-Adenosyl-l-selenomethionine Cofactor Analogue as a Reporter of Protein Methylation[END_REF] switching the cancer cells from a proliferative to an invasive phenotype that is translated by an increase of integrin expression [START_REF] Decelle | Subcellular Chemical Imaging: New Avenues in Cell Biology[END_REF] .

The adhesion of the spheroids on the surface of the mesothelium causes a decrease of Ecadherin and increase of CD44 34,35 . This docking triggers the expression of fibronectin by the mesothelium increasing the interaction with the integrin of the cancer cells 36 . CD44 and L1CAM are crucial for secondary tumour formation 35 . Blocking CD44 or L1CAM expression has been shown to reduce mesothelial adhesion 37 (Figure 2). Once docked the spheroids initiate infiltration and spread to surrounding tissues.

The dissemination through the peritoneum is a passive mechanism involving the circulation and accumulation of ascitic fluid [START_REF] Clare | One-Carbon Metabolism: Linking Nutritional Biochemistry to Epigenetic Programming of Long-Term Development[END_REF] . In comparison with the surrounding environment of other solid tumours, the malignant ascitic fluid accumulating in the peritoneal cavity during ovarian cancer progression is uniquely constituted forming of highly inflammatory environment due to macrophage activation 38 . The circulation of ascitic fluid transports the spheroids allowing them to spread and attach throughout the peritoneal cavity forming nodules mainly on the omentum but also on the diaphragm, liver or lungs [START_REF] Vinceti | Selenium for preventing cancer[END_REF]39 . The ascitic fluid is constantly changing with the evolution of the pathology and plays a major role in tumour progression, spheroid formation, tumour dissemination. For example, lysophosphatic acid which is present in ascites or ovarian cancer patients promotes motility and invasiveness of cancer cells via induction of expression of metalloproteases that modify the extracellular matrix of the mesothelium [START_REF] Decelle | Subcellular Chemical Imaging: New Avenues in Cell Biology[END_REF] . Moreover the increase of CXCL12 released by epithelial ovarian tumoral cells in the ascitic fluid acts as autocrine and paracrine stimulation inducing increased expression of integrins by ovarian cancer cells leading to increased migratory potential. Finally after cancer cell implantation, synthesis of pro-inflammatory TNF-α by ovarian cancer cells stimulates endothelial cells 40 to secrete interleukins enhancing angiogenesis at metastatic tumour sites. The accumulation of ascitic fluid is not well understood but it is thought that vascular endothelial growth factor (VEGF) is involved 41 . VEGF promotes angiogenesis inducing in vitro the formation of confluent microvascular endothelial cells that invade collagen gels and form capillary structures. Its overexpression has been detected in some cancer patients and allow the creation of a more favourable environment for the new implant.

iii. Different forms of Ovarian Cancer

Ovarian cancer cells can originated from cells of ovarian epithelial surfaces [START_REF] Rosen | Ovarian Cancer: Pathology, Biology, and Disease Models[END_REF] , from the epithelium of distal fallopian tubes [START_REF] Kroeger | Pathogenesis and Heterogeneity of Ovarian Cancer[END_REF] or from peritoneal cavity epithelium [START_REF] Campaner | The methyltransferase Set7/9 (Setd7) is dispensable for the p53mediated DNA damage response in vivo[END_REF] . Epithelial ovarian cancer is the most common tumour type accounting for 90% of the cases [START_REF] Erickson | The Role of the Fallopian Tube in the Origin of Ovarian Cancer[END_REF] . They are separated in 2 categories depending on the pathway of tumorigenesis (Histotypes are detailed in figure 3).

Type I is comprised of low grade serous, endometrioid, mucinous and clear cell carcinomas originating from lesions in the ovary [START_REF] Koshiyama | Recent Concepts of Ovarian Carcinogenesis: Type I and Type II[END_REF] . They are slow growing tumours and characterized by a stable genome and do not carry a p53 mutation, however they have a mutation in KRAS gene, which is involved in the RAS/MAPK pathway controlling cell growth, proliferation or maturation [START_REF] Koshiyama | Recent Concepts of Ovarian Carcinogenesis: Type I and Type II[END_REF] .

Figure 3 52 : Histopathology of ovarian cancer

Ovarian cancer is defined by any any primary malignant tumour initiating from the ovary, the endometrium or from fallopian tubes. More than 85% of ovarian cancers are carcinomas, meaning they are derived from epithelium. Amongst them 70-74% are High Grade Serous Carcinoma, 3-5% are Low-Grade Serous Carcinoma, 10 to 26% are Clear Cell Carcinoma, 2-6% are Mucinous Carcinoma and 7-24% are Endometrioid Carcinoma.

Biomechanical process during cancer progression

Dissemination of cancer cells following Epithelial to Mesenchymal Transition (EMT) is sustained by modification of cell-cell, cell-matrix interactions and cytoskeleton modifications. The cell cytoskeleton is formed of actin filaments, microtubules and intermediate filaments that all influence cell morphology [START_REF] Yamaguchi | Regulation of the Actin Cytoskeleton in Cancer Cell Migration and Invasion[END_REF] . These different structures interact with each other providing mechanical stability of cells. The effect of chemical drugs targeting cytoskeleton been used to reveal the role of each type of fibre in cell elasticity. For example the depolymerization of actin fibres resulted in rounder cells related to softening of the cells [START_REF] Ramos | The Softening of Human Bladder Cancer Cells Happens at an Early Stage of the Malignancy Process[END_REF] .

Invasive properties in HEY/HEYA8 ovarian cells have revealed that modified morphology is linked to cytoskeleton modifications increased the migration capacity of these cells [START_REF] Cross | Nanomechanical Properties of Glucans and Associated Cell-Surface Adhesion of Streptococcus Mutans Probed by Atomic Force Microscopy under in Situ Conditions[END_REF][START_REF] Cross | AFM-Based Analysis of Human Metastatic Cancer Cells[END_REF][START_REF] Guck | Optical Deformability as an Inherent Cell Marker for Testing Malignant Transformation and Metastatic Competence[END_REF] . Such observations are not directly applicable to in vivo mechanisms as in the tumour environment cell-cell contacts and extracellular matrix are strongly affecting cell stiffness [START_REF] Rebelo | Comparison of the Viscoelastic Properties of Cells from Different Kidney Cancer Phenotypes Measured with Atomic Force Microscopy[END_REF][START_REF] Levental | Soft Biological Materials and Their Impact on Cell Function[END_REF][START_REF] Krouskop | Elastic Moduli of Breast and Prostate Tissues under Compression: Ultrasonic Imaging[END_REF][START_REF] Paszek | Tensional Homeostasis and the Malignant Phenotype[END_REF] . Notably during the metastatic process, the cells acquire motility and increased deformability. Those morphological changes influence cell stiffness [START_REF] Cross | Nanomechanical Properties of Glucans and Associated Cell-Surface Adhesion of Streptococcus Mutans Probed by Atomic Force Microscopy under in Situ Conditions[END_REF] .

The adhesion of cells to the extra cellular matrix is a key property that has important functions in cell physiology. Indentation experiments in breast epithelial cancer cells have shown that matrix stiffness dictates intracellular mechanical state of those cells [START_REF] Baker | Cancer Cell Stiffness: Integrated Roles of Three-Dimensional Matrix Stiffness and Transforming Potential[END_REF] . Moreover the comparison between cirrhosis tissue and hepatocellular carcinoma tissue did not show any stiffness differences suggesting the hardness of the liver is increased during carcinogenesis [START_REF] Gang | Measuring Microenvironment Mechanical Stress of Rat Liver during Diethylnitrosamine Induced Hepatocarcinogenesis by Atomic Force Microscope[END_REF] .

Such modifications are studied using instruments, such as the atomic force microscope (AFM) which can measure nanomechanical changes. The measure of a force necessary to indent a cell (Young's modulus) in different cancerous cells such as breast [START_REF] Lekka | Cancer Cell Detection in Tissue Sections Using AFM[END_REF][START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] , prostate [START_REF] Lekka | Cancer Cell Detection in Tissue Sections Using AFM[END_REF][START_REF] Faria | Measurement of Elastic Properties of Prostate Cancer Cells Using AFM[END_REF] , ovaries [START_REF] Xu | Cell Stiffness Is a Biomarker of the Metastatic Potential of Ovarian Cancer Cells[END_REF] , or kidney [START_REF] Rebelo | Comparison of the Viscoelastic Properties of Cells from Different Kidney Cancer Phenotypes Measured with Atomic Force Microscopy[END_REF] cancer has been shown to be less than their normal counterparts. It appears that modification of the Young's modulus can indicate the transition to a cancerous state for individual cells.

Current diagnostics and new developments

Current diagnostics

Different symptoms of early stage ovarian cancer have been identified, however, they are not specific to ovarian cancer, including abdominal and pelvic pain, irregular menarche, change in bowels habits and increased urinary frequency 5 . These benign gastrointestinal and gynaecological problems are often symptoms attributed to stomach or colon diseases.

The familial history of cancers plays an important role in deciphering the cancer risk. Importantly it can be related to the presence of an inherited mutation in the germline such as a BRCA genes mutation [START_REF] Bergfeldt | Risk of Ovarian Cancer in Breast-Cancer Patients with a Family History of Breast or Ovarian Cancer: A Population-Based Cohort Study[END_REF] .

Ovarian cancer detection is currently based on circulating cancer antigen 125 (CA-125) glycoprotein concentrations as is has been shown to be elevated in 50% of cases with early stage ovarian cancer [START_REF] Scholler | CA125 in Ovarian Cancer[END_REF] , but is also increased in pregnancy and endometriosis and other benign clinical conditions 70 , which reduces its specificity. The lack of specificity and sensitivity of current early detection biomarkers severely impacts screening efficacy 71,[START_REF] Vaughan | Rethinking Ovarian Cancer: Recommendations for Improving Outcomes[END_REF] . Population screening is also limited due to the rarity of the disease, and therefore cost implications related to such testing. However CA-125 remains an effective approach for sequentially monitoring the response to chemotherapy from patients and detecting relapse 208 . Transvaginal ultrasonography (TVU) is used in addition with CA-125 to screen symptomatic patients to detect ovarian cancer and rule out the false positives caused by weak or absent CA-125 signal. TVU enables precise imaging of the ovaries and helps to identify simple cysts, complex pelvic masses and solid tumours. However, only a fraction of metastatic tumours reach a sonographically-detectable size which may lead to false negatives in the detection of earlystage ovarian cancers [START_REF] Mathieu | Screening for Ovarian Cancer: Imaging Challenges and Opportunities for Improvement[END_REF] . [START_REF] Balter | Natural Variations of Copper and Sulfur Stable Isotopes in Blood of Hepatocellular Carcinoma Patients[END_REF][START_REF] Albarède | Metal Stable Isotopes in the Human Body: A Tribute of Geochemistry to Medicine[END_REF][START_REF] Chamel | Retrospective Evaluation of Blood Copper Stable Isotopes Ratio 65Cu/63Cu as a Biomarker of Cancer in Dogs[END_REF][START_REF] Gourlan | Copper Isotopes as Possible Neoplasia Biomarkers in Captive Wild Felids[END_REF] . Alkaline earth metals such as Calcium and Magnesium and the transition elements Iron, Copper, and Zinc have been studied due to their functional roles in biology and because their turnover rates in the body are relatively short.

Dietary intake of copper in human needs to be around between 1 and 3mg/day 82,[START_REF] Micronutrients | Copper[END_REF] . A portion is absorbed by the intestinal cells, after being reduced from Cu 2+ to Cu + by the membrane protein STEAP [START_REF] Knutson | Steap Proteins: Implications for Iron and Copper Metabolism[END_REF] , through the CTR1 transporter. In cells, chaperons ATOX1 or COX17 85,86 bind to copper Cu + and deliver copper to different organites where it is used as cofactor of cytochrome c oxidase in mitochondria, or the superoxide dismutase SOD1 that catalyses the scavenging of ROS producing oxygen and hydroperoxide. Copper is transported through the intestinal cells and delivered to the blood through the ATP7A copper transporter 85 . Copper is then transported in blood by the ceruloplasmin to the liver [START_REF] Turnlund | Human Whole-Body Copper Metabolism[END_REF] . The liver is the main site of copper accumulation, controlling concentrations in blood [START_REF] Olivares | Limits of Metabolic Tolerance to Copper and Biological Basis for Present Recommendations and Regulations[END_REF] . Liver synthesizes ceruloplasmin [START_REF] Frieden | Ceruloplasmin: The Copper Transport Protein with Essential Oxidase Activity[END_REF] which can transport up to seven copper [START_REF] Fox | Structure, Oxidant Activity, and Cardiovascular Mechanisms of Human Ceruloplasmin[END_REF] atoms due to a methionine rich domain and cysteine-histidine domains.

Excess copper is excreted in the duodenum or in urine via the kidneys. Demands of copper in organs depend on their metabolic functions such as mitochondrial content and activity. For example in muscles, the high amount of mitochondria increase the demand of copper for cytochrome c function. This transmembrane protein contains 2 copper centres [START_REF] Iwata | Structure at 2.8 Å Resolution of Cytochrome c Oxidase from Paracoccus Denitrificans[END_REF] . Their functions are to transport electrons from the soluble cytochrome c to the oxygen that is reduced into water.

Modifications of Cu concentration and relative abundance of Cu isotopes (fractionation) have been linked to modified metabolic processes (oxidative phosphorylation, hypoxia) or in angiogenesis, and thus to health and disease [START_REF] Albarède | Medical Applications of Isotope Metallomics[END_REF] . In different cancers it has been shown that copper is required for angiogenic processes [START_REF] Nasulewicz | Role of Copper in Tumour Angiogenesis--Clinical Implications[END_REF] , stimulating proliferation and migration of endothelial cells [START_REF] Hordyjewska | The Many "Faces" of Copper in Medicine and Treatment[END_REF] . In the liver tissue of colon tumour bearing mice, gene expression the copper transporters ceruloplasmin, and CTR1 and ATP7B was increased significantly, which can explain elevated copper serum levels [START_REF] Babich | Non-Hepatic Tumors Change the Activity of Genes Encoding Copper Trafficking Proteins in the Liver[END_REF] and suggesting its potential use as a diagnostic marker of cancer. Isotopic ratio between heavy isotope [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu and the light isotope [START_REF] Gang | Measuring Microenvironment Mechanical Stress of Rat Liver during Diethylnitrosamine Induced Hepatocarcinogenesis by Atomic Force Microscope[END_REF] Cu has been measured in different healthy and human materials 96 (Table 2). Cu is more concentrated in erythrocytes compared with serum (Table 2). Moreover Cu isotopic composition is lower in serum (-0.28‰) and higher in erythrocytes (0.46 to 0.67‰). To explain this difference, it has been suggested that enrichment of [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu in red blood cells is due to the strong binding of [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu with nitrogen of histidines and sulphurs of cysteines of the superoxide dismutase 1 or ceruloplasmin inducing enrichment of [START_REF] Gang | Measuring Microenvironment Mechanical Stress of Rat Liver during Diethylnitrosamine Induced Hepatocarcinogenesis by Atomic Force Microscope[END_REF] Cu in the serum [START_REF] Albarède | Isotopic Evidence of Unaccounted for Fe and Cu Erythropoietic Pathways[END_REF][START_REF] Fujii | Density Functional Theory Estimation of Isotope Fractionation of Fe, Ni, Cu, and Zn among Species Relevant to Geochemical and Biological Environments[END_REF] . Although Cu is more concentrated in serum in women relative to men [START_REF] Albarède | Metal Stable Isotopes in the Human Body: A Tribute of Geochemistry to Medicine[END_REF][START_REF] Iyengar | Reevaluation of the Trace Element Content in Reference Man[END_REF] , the slight isotopic enrichment in women blood compared to men (𝛿 [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cumen= -0.24 ± 0.36 ‰ and 𝛿 65 Cuwomen= -0.28 ± 0.40 ‰) shows that sex does not affect copper fractionation in humans [START_REF] Albarède | Isotopic Evidence of Unaccounted for Fe and Cu Erythropoietic Pathways[END_REF] .

The potential of measuring the variability of copper isotopes as a new diagnostic tool for cancer detection has been evaluated in colorectal and breast cancer patient serum. For all patients a decrease of [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu concentration was observed 100 and related with an increase of tumour [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu concentration. By following the evolution of the isotopic composition over time a faster shift of 𝛿 [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu was correlated with a more severe tumour, and with the CA15-3 (MUC1 blood concentration) marker. A decrease by 0.25‰ in serum of breast cancer patients, 0.14‰ 100 in colorectal cancer patients and 0.5‰ in cirrhosis patients 101 has been measured. Moreover, this shift in Cu isotopic composition has also been observed on other mammals such as dogs [START_REF] Chamel | Retrospective Evaluation of Blood Copper Stable Isotopes Ratio 65Cu/63Cu as a Biomarker of Cancer in Dogs[END_REF] and felines [START_REF] Gourlan | Copper Isotopes as Possible Neoplasia Biomarkers in Captive Wild Felids[END_REF] .

So far no direct link has been established between the increase of the copper concentration in the blood and the modifications of the isotopic compositions. However the analysis of tumour and peri-tumoral hepatocarcinoma cells has highlighted an increase of 𝛿 65 Cu by 0.5 to 1‰ in tumour cells 102 . Moreover hypoxic growth of primary tumour cells have revealed the same type of shift of the 𝛿 65 Cu [START_REF] Albarède | Metal Stable Isotopes in the Human Body: A Tribute of Geochemistry to Medicine[END_REF]102 . These results suggest that 𝛿 [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu ratio could be a cancer marker candidate for detection of Ovarian Cancer.

'Classic' Ovarian Cancer treatments

Ovarian cancer treatment depends on disease stage. If the tumours are confined to one ovary (type Ia), the surgical removal of one ovary is performed 103 . If both ovaries have developed tumours (stage Ib), platinum and taxane (cisplatin and paclitaxel) treatment is given. Treatment for stage II and above where cancer has spread beyond the ovaries, involves chemotherapy and surgical reduction of the tumour mass 104,105 . The timescale for medical intervention depends on the size of the tumours. A high tumour load involves chemotherapy treatment before surgery 106 . Intravenous or more recently intraperitoneal delivery of platinum based chemotherapy with paclitaxel infusion can be used 105,106 . In the case of severe side effects of the treatment, the use of liposomal doxorubicin can also be used 107 To gauge treatment efficacy, the monitoring of CA-125 levels and physical examination are performed 108 .

A major obstacle in the use of chemotherapy to treat ovarian cancer is its high recurrence rate (70%) due to the development of resistance to platinum treatment within 18 months which reduces dramatically the survival rate 19 . Resistance to treatment is defined by patients not responding to treatment or relapse within 6 months after first treatment 109 . The recurrence is due to the multiplication of subpopulation of cells that have adapted to the chemotherapy 110,111 .

In most cases this is due to acquired resistance to chemotherapy via overexpression of drug efflux pump 35,112 . Restoration of BRCA genes in subpopulations of tumour cells thus regaining more effective DNA repair capabilities results in enhanced resistance to chemotherapy 20,113 . BRCA gene mutation is therefore both a risk factor for ovarian cancer development due to the lack of repair of DNA breakage, but also a risk factor if it is unregulated in ovarian cancer cells which can overcome chemotherapy.

To overcome the acquired resistance to treatment, Paclitaxel has been developed and is considered as the standard for platinum resistant ovarian cancer. Paclitaxel blocks cell division by binding to beta-tubulin that stabilize microtubules which leads to cell death 104 . However, like platinum treatment, taxane based chemotherapy induces oxidative stress and selects cancer cell populations that resist treatment. The side effects of chemotherapy are a reduced immunity, gastrointestinal disruption, neuropathy and breathing difficulties 114 .

The development of more specific and less harmful treatments is highly needed in order to target specific cancer cell populations allowing better life preservation and quality of life 103

'New' Ovarian Cancer treatments

New therapeutics have been developed that are more effective against ovarian cancer than platinum and taxane based treatments, and can be used as second line treatments. Olaparib and Veliparib, PARP inhibitors, have shown good efficacy in 50% of HGSC patients [START_REF] Vinceti | Selenium for preventing cancer[END_REF] carrying BRCA1 and 2 mutations 5,116 . The inhibition of PARP leads to accumulation of single strands breaks and unrepaired forks in DNA. PARP inhibitors have been proven to selectively kill cells with defects in DNA repair pathway 116 . PARP inhibitors are preferentially used in treatment of recurrent, BRCA-associated ovarian cancer patients but can also be used in maintenance following platinum-based chemotherapy for recurrent epithelial ovarian cancer 117 .

Antibody therapies targeting human epithelial growth factor receptor 2 (HER2) and folate receptor 118,119 have been developed which block signalling that drives cell proliferation. Recent use of Trastuzumab (Herceptin) in ovarian cancer, a therapeutic monoclonal antibody directed against HER2, which is overexpressed in some ovarian cancer patients and related with poor prognosis 120 has raised good hopes. However only 6.7% of advanced ovarian carcinomas overexpresses HER2 121 therefore only a limited number of patients would benefit from this treatment.

Nanomedicines and Ovarian Cancer Definition

Nanomedicines include a range of nanomaterials and nanosize biological entities (e.g. exosomes and antibody drug conjugates) that have been 'engineered' and are applied to cancer treatment. In some cases nanocarriers have been developed to transport drugs in order to overcome the low solubility, low stability or strong side effects of classic chemotherapy 122 . Nanocarriers have specific properties such as their size, high surface/volume ratio, physical or chemical specificities, while loading drugs into nanocarriers improves the pharmacokinetic and dynamic profiles of drugs enhancing therapeutic index by increasing accumulation to tumour sites 123 . Other nanomedicines use intrinsic properties of nanomaterials to elicit an anti-cancer effect. For example, gold nanoparticles have been used for local thermal ablation of tumour due to the ability of such particles to be manipulated by magnetic fields of radio frequency to generate heat locally 124 .

Passive targeting

Nanocarriers/nanoparticles can be passively targeted to tumour sites via the so called EPR (Enhanced Permeability and Retention) effect to deliver pharmacologically active compounds.

The EPR effect involves the accumulation of drugs in the tumour sites due to the movement of particles from the circulatory system through 'leaky' intratumoral blood vessels 125 with fenestrated endothelium allowing the passive transport of the nanomedicines through gaps in the vessels. The main limitations of passive targeting are heterogeneity 126 of the tumours impacting delivery of the drugs, interstitial pressure, extracellular matrix secreted by tumours and accumulation of nanocarriers to other organs. The escape from the opsonization by the immune system is also important to prevent the clearance of the drug before it has any effect on tumours.

Active targeting

Actively targeted nanomedicines are directed to tumours via high affinity ligand attached to their surfaces, selectively binding to a receptor of the targeted cells. Targeting moieties can include sugars, proteins, antibodies, and oligonucleotides. The targeting molecule needs to be stable in the blood in order to deliver specifically the nanocarrier to a tumour. The particle is still reliant on the passive EPR effect to reach the tumour, but then targets the tumour once escaped from the neovascular system 126 . Once at the tumour site, cancer cells can internalise the nanocarriers allowing the accumulation of drugs.

Diversity of nanocarriers in cancer care

Various type of nanomedicine have been developed and tested in clinical trials including drug conjugates, lipid based nanocarriers, polymer-based nanocarriers or inorganic nanoparticles.

Drug conjugates are defined as binding of the drug of interest with antibodies, peptides or polymers. Polymer HPMA (hydroxypropyl methacrylamide) drug conjugates are passive targeting nanocarriers based on the EPR effect that have been used due to the high biocompatibility of HPMA. Conjugated with oxaliplatin such nanomedicines have reached phase 2 clinical trials for recurrent ovarian cancer patients with equal or superior efficacy than oxaliplatin alone and demonstrating excellent tolerability with low accumulation in liver 127 .

Active targeting such as antibodies drug conjugates (ADCs) have been developed against specific receptors overexpressed on cancer cell membrane such as HER2 (Human Epidermal growth factor Receptor 2) in breast cancer and in some ovarian cancers. Herceptin has been in clinical use and proved its efficacy as adjuvant in conjugation with emtansine inhibiting microtubule polymerisation increasing Herceptin efficacy.

Lipid based nanocarriers such as liposomes or micelles are able to transport greater amounts of drug in comparison with ADCs and use the EPR effect to accumulate tumour tissue. Paclitaxel loading lipid nanoparticles have been used in ovarian cancer and proved equivalent to Paclitaxel infusions in phase 2 trials, and effectively reduced side effects 128 . Despite the ability of liposomes nanocarriers to concentrate drugs, they do not actively target tumours. However functionalisation of liposomes with anti HER2, anti EGFR, anti VEGFR2 antibodies has been developed, and overcame the multidrug resistance in xenograph mouse models and in breast and gastric cancer patients [129][130][131] Polymer based nanocarriers include protein or peptide nanocarriers, polymers such as PEGylation or sugars. Albumin (BSA) based nanocarriers are mainly used due to its high bioavailability and stability in blood. Albumin coated particles/conjugates allow increased solubility of the chemotherapeutic drugs such as paclitaxel. A major drawback is the immunogenicity induced by this kind of coating leading to opsonization. Glycan nanocarriers such as chitosan based nanoparticles have been used for loading gemcitabine resulting in increased uptake by intestinal cells compared to free oral gemcitabine improving the stability of the drug in preclinical trials 132 . Chitosan coated nanocarriers are uptaken by endocytosis after binding to the phospholipids of the membrane. With a pKa of 6.5 for its primary amine groups, chitosan is highly soluble at acidic pH allowing the swelling of the chitosan nanostructure leading to the leak of drug of interest through the nanoparticle to the cells 133 .

Inorganic nanoparticles are made of different materials and used for variety of applications including theragnostic. For example MRI studies have been using superparamagnetic iron oxide nanoparticles to image tumours 134 . Moreover iron oxide nanoparticles are used for their magnetic properties to induce thermal ablation 135 (magnetic hyperthermia). Gold nanoparticles are also extensively used in the synthesis of nanocarriers. Pegylated gold nanoparticles binding TNF-α to deliver necrosis factor to solid tumours have been tested in phase one clinical trial but hasn't reached the next stage yet 136 .

Development of nanocarriers for ovarian cancer treatment

Nanocarriers such as those described above have been developed in both in vitro and in vivo ovarian cancer models (summarised in table 3), with several progressing to clinical trials (summarised in table 4).

II. Epigenetics 1. Definition

Epigenetics refers to reversible 137 chemical modifications of chromatin organization and structure 138 that result in alterations in gene expression profiles, cellular function, and which are in some cases heritable. These chromatin related epigenetic mechanisms allow cells to modify gene expression in response to chemical and environmental stimuli for example. Distinct mechanisms of epigenetic alterations include DNA methylation, histone acetylation, methylation or phosphorylation, and microRNA (miRNA) expression and regulation 138 . These mechanisms (except miRNA) are affected by enzyme families that transfer or remove small chemical moieties that function as activating and repressing 'marks' from histones or DNA, that are then recognised by regulatory proteins that control transcription.

Histone structure and modifications

Epigenetic changes can result in aberrant oncogene activation or inactivation of tumour suppressor genes, allowing cancer progression 137 . DNA is packaged in the nucleus, wound around nucleosomes that are composed of segments of 147 base pairs of DNA wrapped around a histone core, comprising of 2 copies of histones 2A, 2B, 3 and 4 [START_REF] Guck | Optical Deformability as an Inherent Cell Marker for Testing Malignant Transformation and Metastatic Competence[END_REF][START_REF] Rebelo | Comparison of the Viscoelastic Properties of Cells from Different Kidney Cancer Phenotypes Measured with Atomic Force Microscopy[END_REF] . These 14kDa basic proteins are positively charged due to high numbers of lysine and arginine amino acids, enabling them to bind to the negatively charged DNA. Sequences of nucleosomes are then organized in a relatively open and uncompacted form, termed euchromatin associated with open, active chromatin regions or more densely packed, closed chromatin, termed heterochromatin which is associated with silent or inactive gene expression. Chromatin compaction is controlled by combinations of post translational modification of histones tails 141 . The main histone modifications include methylation, acetylation, phosphorylation or ubiquitination 142 to lysines and arginines, present in the histone tails (Figure 4). The different modifications are controlled by enzyme families such as methyltransferases or demethylases, acetylases or deacetylases 139,141,143 .

Histone methylation

Histone H3 consists of a main globular domain and a long N-terminal tail that contains 6 sites of modifications for methylation that have different consequences on chromatin compaction and genetic expression. The methylation sites are constituted of lysines (K) or arginines (R) and their methylation is catalysed by a range of different histone methyltransferases (HMT) (Figure 5). Methylation of lysines and arginines is mediated by the transfer of a methyl from SAM by HMTs (Figure 6). The product of this reaction is a methylated lysine/arginine and Sadenosylhomocystein (SAH) which is cleared into homocysteine and recycled into methionine through remethylation by Betaine Homocysteine MethylTransferase (BHMT) (Figure 6).

Transcription activation mark (H3K4)

The methylation of H3K4 has been shown to result in gene activation 141,142,147 (Figure 7). H3K4me3 is highly enriched near the Transcription Start Sites. 148 , and appears to be an epigenetic signature in tumour-suppressor genes in normal cells 147 . Genetic domains covered by H3K4me3 are broader (>4kb) in genes controlling cell-type specific functions in normal cells, where transcription is increased 147,149 . The shortening of broad H3K4me3 in cancers is associated with repression of tumour suppressors. Patient studies have demonstrated that decreased levels of H3K4me3 are associated with poor prognostic factors in lung and kidney cancers 150,151 . H3K4 methylation is mediated by SET1 complex which is composed of methyltransferase and seven subunits 152 . The PR/SET domain gene family (PRDM) encodes for 19 zinc-finger domain containing proteins involved in gene expression regulation modifying chromatin structure through methyltransferase activity or recruitment of chromatin remodelling complex 153,154 . While PRDMs have been mainly identified as tumour suppressors, some family members have been associated with mutations, epigenetic silencing or overexpression in multiple cancer types.

PRDMs have two isoforms differing by the presence of a PR domain with the short isoform being oncogenic 153 .

SMYD histone methyltransferases have also been implicated in cancer development with increased expression of SMYD3 in ovarian cancer inducing high H3K4me3 155 . SMYD3 is known to interact with RNA Pol II and H3K4me3, and functions as a selective transcriptional amplifier for oncogenes. A meta-analysis of 1474 patients from 10 clinical studies accross different cancers indicated that lower levels of H3K4me2 were characteristic of shorter overall survival, whereas patients with lower level of H3K4me3 expression had a longer overall survival 149 and raises the importance of investigating patterns of H3K4 methylation on prognosis of patients with malignant tumours.

Transcription repression mark (H3K27)

H3K27me is regulated by EZH2 a catalytic component of polycomb repressive complex 2 (PRC2). Through H3K27me2 and H3K27me3, EZH2 represses gene expression (Figure 7), triggering the differentiation or maintenance of stem cell self-renewal capacity [156][157][158] . The expression of EZH2 increases in prostate cancer, esophageal squamous cell carcinoma, and breast cancer 159160 161 . EZH2 knockdown often leads to reduced invasive potential of cancer cells 1,2 . H3K27 methylation has also been related to increased DNA breaks, since EZH2 is found in PRC2 complex that is recruited to damage sites through PARP activity (poly ADPribose polymerase) where it increased H3K27 methylation [163][164][165] . The induction of apoptosis by staurosporine lead to PARP cleavage and increased H3K27me in osteosarcoma cells 166 . EZH2 accumulates in promoters of actively transcribed genes inducing repression upon DNA damage by recruiting remodelling factors, which may facilitate repair of DNA lesions and organize response to DNA damage 167 .

Heterochromatin mark (H3K9)

The H3K9me2 or 3 marks are catalysed by the G9a (EHMT2) methyltransferases 168 . The position of these marks are important in their role in chromatin expression. H3K9me2 and 3 have been associated with gene silencing and heterochromatin 169 (Figure 7) when they accumulate in the 5'UTR regions of genes. In contrast increase of H3K9me3 within the body of a gene has been linked to active gene expression. H3K9me2 is rarely found within silenced genes 170 . H3K9me2 has been described as a repressive mark and has been located within LaminB1 bound regions (nuclear periphery, nuclear lamina, associated with inactive genes). LaminB1 regions are areas of low gene expression indicating that Lamin B1 rich regions represents a repressive chromatin environment. H3K4me3 and RNAPol II are also absent from LaminB1 regions which strengthens the idea of H3K9me2 being a repressive mark that separates active and inactive genes 171 .

G9a is highly expressed in different cancers such as hepatocellular carcinoma 172 , colorectal cancer 173 and breast cancer 174 . The knockdown of G9a induced apoptosis and growth inhibition with increased cell population in sub-G1 phase 143,175 . Moreover, G9a downregulation induced centrosome disruption and chromosomal instability leading to cell senescence in prostate cancer cells 176 .

Figure 7 177 : Histones methylation regulating the euchromatin and heterochromatin forms.

Among the different histones marks described in the literature, we focus here on three important ones. H3K4me3 has been described as translational activation mark and H3K27me3 and H3K9me3 have been described as repressive marks. The methylation is orchestrated by different regulators which are specific of each mark. Addition of methylation is permited by histone methyltransferase (HMT) while methyl removal is mediated by histone demethylase (HDM). H3K9 is methylated by G9a, SUV39H1/2 or SETDB1 and lead to chromatin condensation, e.g. hetetochromatin. The JMJD family of HDMs remove methyl from H3K9 leading to decondensation of the chromatin. In euchromatin, the marks H3K4 and H3K27 are respectively modified by SET1 complex (SMYD or PRDM families) and EZH1/2 Polycom complex. These marks are present on the histones and the balance between both leads to activation or repression of the transcription.

Current knowledge of the effect of epigenetics in ovarian cancer

Recent studies of ovarian cancer cell populations identified sub-populations that are stem-cell like nature 178 . This is supported by the presence of the methylation bivalent chromatin mark H3K4/H3K27 at the transcription start site of silenced genes 179 . This mark is required for silencing of developmental genes which keeps cells in a stem cell nature conducive to the formation of tumours.

The balance between the different epigenetic marks has been linked to the aggressiveness of the ovarian cancer 179 . In high grade serous ovarian tumours H3K4me3 and H3K27me3 marks at transcription start sites are specific for malignancy progression 179 . The overexpression of EZH2 lead to H3K27me3 mediated gene silencing driving tumorigenesis in subpopulation of cells.

Following treatment with chemotherapy, subsequent development resistance to treatment by ovarian cancer cells is prevalent 4,[START_REF] Vaughan | Rethinking Ovarian Cancer: Recommendations for Improving Outcomes[END_REF] , with specific populations of cancer cells thought to retain pluripotent embryonic stem cell-like features, with high H3K4me3 and H3K27me3 repressing transcription factor networks and subsequent patterns of gene expression 180 . In platinumresistant PEO4 cell lines (derived from malignant effusion from the peritoneal ascites), the presence of the bivalent mark (H3K4me3 and H3K27me3) or repressive H3K27me3 mark were generally expressed at a lower level than in PEO1 platinum-sensitive cell lines.

In ovarian cancer spheroids, SMYD3 expression was elevated 155 and associated with increased H3K4 methylation. A knockdown of SMYD3 decreased spheroid invasion and adhesion associated with a downregulation of integrin family members. Patients with high SMYD3 expression have been related with ovarian cancer cells proliferation 181 .

In OVCAR-3 cells, H3K27me3 relates to the development of resistance to cisplatin and tumour progression 179 . However in human studies, the expression of H3K27me3 was lower in ovarian cancer tissues than in normal tissues 182 . This has also been observed in clinical studies comparing different ovarian cancer (cystadenomas, borderline tumour or carcinomas) where between 30 to 50% of cases displayed a decrease in H3K27me3 mark expression 183 . The use of inhibitors of PRC2 like metformin caused reduced methylation of H3K27, reducing cancer cells proliferation and migration, and triggering apoptosis 184 . In SKOV-3 cells, when epithelial to mesenchymal transition was triggered by TGF-β, EZH2 expression was reduced leading to reduced H3K27me3 162 . Moreover, the inhibition of EZH2 triggered EMT-like changes in SKOV-3 cells. EZH2 is thought to be required for the maintenance of epithelial phenotype in ovarian cancer cells 162 . During metastatic process, cells undertake the EMT. However in ovarian cancer, after dissemination from primary tumour site, cells adopt epithelial phenotype to adhere to the mesothelium. EZH2 is thought to facilitate this process during tumour metastasis, as it is overexpressed, tilting the balance EMT/MET in favour of MET 162 .

G9a overexpression has been identified as a marker of aggressiveness and can promote the peritoneal metastasis 185 . Knockdown of G9a expression suppressed prometastatic cellular activities including adhesion, migration and invasion in cell lines. It significantly attenuated the development of ascites and tumour nodules in a peritoneal dissemination model 185 . The expression levels of G9a were higher in metastasis in comparison with primary tumours. The expression of G9a is correlated with late stage of ovarian cancer, predicting a shorter survival in patients expressing high levels of G9a 185 .

III. Selenium in cancer treatment 1. Dietary window of selenium and metabolism

Dietary window

The amount of Selenium (Se) in food and thus in the diet is highly dependent on the amount of Se in soil and water which is regionally dependent 186 . The optimal dose range of Se intake in human nutrition is narrow and has been shown to be between 100 and 200 µg/day (Figure 8) 187,188 . In contrast, consumption of over 1500 µg/day of Se can induce single and double strand DNA breaks 189 that progressively worsen with increasing dose leading to selenosis 190191 characterised in acute phase by necrosis and haemorrhage resulting from capillary damage and in chronic poisoning by degenerative and fibrotic changes of the liver and skin 192 .

To prevent adverse effects due to excessive intake of selenium, the USA Institute of Medicine set a tolerable upper intake level of selenium at 400µg/day 193 . The consumption of selenium between 200 and 400µg/day has been shown to be protective against liver necrosis by increasing glutathione peroxidase and thioredoxin reductase levels 194 . The role of selenium in ROS protection lead to the study of the effect of supranutritional doses of selenium (>200µg) in cancer. While doses of selenium between 200 and 400µg/day 186,195 of selenium were shown as protective, doses above 400µg/day were shown to have a strong inhibitory effect on cancer cells growth and impaired cancer development in vitro and in vivo. Various organic (selenomethionine, methylseleninic acid) and inorganic (selenite, selenate) selenium forms have been tested over the years allowing to describe different biological activities highlighting the need of better characterisation of toxicology of each selenium species 193,[196][197][198] . Those studies raise the challenge of the definition of the dietary window of selenium.

Cell Metabolism

Assimilation of selenium in human nutrition occurs through different mechanisms. As elemental selenium 0 is insoluble, it is not likely to cross the cell membrane 200 . To transit through the gut wall, selenium can enter the cells as selenite (SeO3 2-) using sulphate transporters 200 . Moreover, phosphate transporters can also be involved in selenium transport. Selenite uptake kinetics have been correlated with phosphate uptake kinetics 201 . While high affinity phosphate transporters are not affected by the presence of selenite in vitro, low affinity phosphate transporters poorly discriminate selenite from phosphate, enabling selenite to enter cells when phosphate concentration increases 201 . Once inside the body, selenate and selenite can be directly transported to other cells through blood transport bound to proteins 202 and/or transformed into selenocysteine in intestinal cells 203 . Selenoaminoacids (selenomethionine and selenocysteine) from food are transported through the bloodstream to the liver (Figure 9) where they are incorporated into selenoprotein P (SELENOP) that is then transported to cells requiring Se through blood. SELENOP is a secreted glycoprotein that bind to glycosaminoglycans using heparin of endothelial cell membrane. SELENOP have antioxidant properties and the numerous selenocysteines it transports is a source of selenium for the cells absorbing it by endocytosis. In the cells, selenium spontaneously reacts with other compounds producing organic selenium metabolites such as selenides (R2Se), diselenide (R2Se2) and selenols (RSeH) 204 (Figure 9). Selenite is metabolized by glutathione (GSH) or glutathione reductase as a seleno-diglutathione (GS-Se-SG) which can be utilised by cells as an antioxidant in the presence of reactive oxygen species 195,205,206 . However, seleno-di-diglutathione has a short lifetime due to its catabolism by glutathione reductase 207 , which results in its conversion back to selenide (H2Se) and GSH. 203,208 Selenium from diet (as selenite or seleno amino acids) enters intestinal epithelial cells and is transported through the blood to the liver where its involved in synthesis of the SePP1 or selenosugars. Those selenium forms are circulating forms and are distributed to organs where they are involved in selenoprotein synthesis. Selenium can be methylated via the methyl donor SAM into dimethylselenium which is volatilized through lungs or into trimethylselenium in kidneys where it is then excreted through the bladder. Selenium as selenite can also circulate in the blood and be absorbed in organs through sulphate or phosphate transporters. Once in the cells, selenium is metabolized through the glutathione system leading to the production of H2Se that can either enter the pathway for synthesis of selenocysteine or be methylated and excreted.

Figure 9 : Dietary and metabolic forms of selenium in cells

The proximity between sulphur (S) and selenium (Se) allows for substitutions in organic molecules to occur, especially in proteins such as selenomethionine, which is randomly included in proteins or selenocysteine, the 21 st amino acid 209 . This seleno amino acid is inserted into proteins in the same way as serine; each selenoprotein is synthetized by a selenoprotein mRNA that contains a UGA codon and a unique SElenoCysteine Insertion Structure (SECIS). The Selenocysteine-Serine tRNA is used for the integration of the selenocysteine into the amino acid sequence that forms selenoproteins 204,205 . This system produces a group of around 25 identified proteins in the human proteome 210 that are mainly involved in antioxidant and antiinflammatory activities 211 .

Modifications of the level of selenium in the cells has been shown to influence selenoprotein activity and production 1,2 . Thioredoxine reductase for example, which contain selenocysteines, is present in the cytosol (TrxR1, thioredoxin glutathione reductase TGR-TrxR3) and the mitochondria (TrxR2). These proteins are involved in the reduction of oxidized thioredoxins, can catalyse NADPH, control ascorbate levels and regulate metabolism 212 . Glutathione peroxidases (GPX) are found in the cytosol and mitochondria (GPX1), extracellular matrix (GPX3), and embryonic cells (GPX4) 212,213 . GPXs have been shown to protect cells against oxidative damage by reducing lipo hydroperoxides and hydrogen peroxide 212 . GPX1 is known to be highly sensitive to selenium content and oxidative conditions in cells. Selenoprotein P meanwhile, is a selenocysteine rich secreted glycoprotein is endocytosed 214 through ApoER2 receptor by cells that subsequently break down the protein and use selenium as SeCys, therefore regulating Se distribution in the body 212 . Importantly, selenoproteins in combination with vitamin C, E beta-carotenes, has been proven to enhance the control of free radicals, protecting cellular functions 215,216 .

Selenium is also involved in the biosynthesis of diverse molecular components that are required for important cellular functions, from deoxyribonucleoside triphosphates (dNTPs) for DNA, to the reduction of oxidized proteins and/or membranes, to roles in diverse regulation mechanisms such as redox, apoptosis, immunomodulation, thyroid hormones and the formation of methyl donor compound 217 (S/Se adenosylmethionine). Selenium, is therefore an important trace element which is fundamental to human health.

Low dose of selenium triggers ROS scavenging

As a naturally occurring element with both nutritional and toxicological properties, selenium deficiency has been linked to cancer development. Indeed, a meta-analysis of the epidemiological literature shows that selenium deficiency is linked with higher cancer development risk 218 . Cancer risk is 2 to 6 times lower when blood serum selenium concentrations are between 100 and 400 ng/mL; corresponding to a consumption of 55 to 200 µg/day of selenite 219 . However a stratified analysis of SELECT results based on genotype examining the nutritional prevention of prostate cancer, with a group of men taking 200µg of selenium per day over a period of 7 years, showed a no effect of Selenium and Vitamin addition for cancer prevention compared to the treated group 220 . Moreover, analysis of randomized controlled trials has failed to show any beneficial effect of Se supplements in reducing cancer risk in humans 193 .

Major impacts of different selenium forms on human health are currently not well understood. This is mainly due to the lack of knowledge in requirements of the different selenium forms for therapeutic application. In vitro and murine model studies have focused on the treatment of various cancer cell models with selenite (SeO3 2-, the most toxic form of free selenium), MethylSelininic Acid (MSA) or SeMet. It appears that at levels below 5µM most forms of selenium tend to have a protective effect against DNA damages and ROS production 221,222,223- 225 . However above 10µM studies have shown a strong inhibitory and cytotoxic effect of different forms selenium leading to cell cycle arrest and ROS production leading to mitochondria or DNA damages triggering cell-type dependent cell death 225,226,[START_REF] Erickson | The Role of the Fallopian Tube in the Origin of Ovarian Cancer[END_REF][START_REF] Koshiyama | Recent Concepts of Ovarian Carcinogenesis: Type I and Type II[END_REF][START_REF] Karst | Ovarian Cancer Pathogenesis: A Model in Evolution[END_REF][START_REF] Labidi-Galy | High Grade Serous Ovarian Carcinomas Originate in the Fallopian Tube[END_REF][START_REF] Kohn | Whence High-Grade Serous Ovarian Cancer[END_REF][START_REF] Bavle | Mitosis at a Glance[END_REF][START_REF] Malpica | Grading Ovarian Serous Carcinoma Using a Two-Tier System[END_REF] . Several mechanisms have been suggested to explain the effect of selenium in cancer therapy depending on the concentration of treatment applied to the cell culture and murine models (summarised in Table 2).

High doses of selenium are cytotoxic i. Effect of selenium on ROS production

Reactive oxygen species (ROS) are free radicals formed within cells during oxidation processes that are characterized by one or more unpaired electrons 238 . The major ROS found in cell systems are superoxide (O2°-), hydroxyl radical (HO°) and nitric oxide (NO°). These transitory but highly reactive species are implicated in cellular reactions as second messengers at physiological concentrations 238 . When ROS concentrations increase in cells, a range of cellular damage can be induced such as lipid oxidation, DNA and protein damage 239 . The control of ROS concentration is mediated by antioxidant molecules such as vitamins (A, E, C), peptides (Glutathione GSH) and proteins (GPx, TRx). In cancer, high metabolic activity, cellular signalling and mitochondrial dysfunction elevate ROS levels 238 . Supplementation with selenium in human studies have failed to prove beneficial chemopreventive effects 193 . However in cells, low doses of selenium have been proven to decrease the amount of ROS 225 , with higher doses inducing levels of ROS 223,225 . As a result, two parameters for ROS production need to be studied; the time of exposure with selenium and the concentration of the selenium species used (Figure 8).

In vitro, glutathione spontaneously reacts with selenite (SeO3 2-) to form various selenocompounds: selenodiglutathione (GS-Se-SG), glutathioselenol (GS-Se-), hydrogen selenide (HSe -) and elemental selenium (Se 0 ) 240 through reduction by thiols and NADPHdependent reductases (Figure 10). This leads to an oxidized inactive thioredoxin system by oxidation of structural cysteine 241 and depletion of NADPH 242 . Hydrogen selenide is known to quickly and spontaneously react with dioxygen to form elemental selenium Se 0 , water, and ROS (Figure 10). It can also react with sulphur forming intramolecular disulphide bonds that have been related with inactivation of voltage sensors in the mitochondrial permeability transition pore 243 . Moreover H2Se promotes inhibition of heme containing enzymes belonging to respiratory chain 244,245 . This reaction leads to leakage of electrons which react with oxygen forming superoxide ions (ROS). This formation of ROS is then amplified by mitochondria 246 .

Figure 10 : Effect of selenium on ROS production 247

Selenate is transformed into selenite in cells under redox conditions. Selenite interacts with the glutathione system and is processed into selenide. The reaction of selenite with oxygen liberates ROS and aggregates of Se°. Selenide can also be used in selenocystein production and can be eliminated through a methylation process.

ii. Effect of selenium on cell death

Apoptosis

Apoptosis is a regulated suicide mechanism that is involved in development and defence of multicellular organisms and can occur via two (intrinsic and extrinsic) pathways 248 . The extrinsic pathway is initiated by attachment of a ligand to the death receptor on cell membrane activating caspase 8 inside the cells. The intrinsic pathway is initiated within the cell through caspase 9 and caspase 3 due to DNA damage or internal stress, such as mitochondria membrane leakage 249 . BCL-2 family proteins regulate this pathway, controlling the release of the mitochondrial cytochrome c which activates the caspases (Figure 11).

Autophagy

Autophagy, related to cell survival, is a cellular process characterized by the formation of autophagosomes including cytoplasmic contents, such as proteins and organelles, in response to starvation or oxidative stress 238,261,262 . The formation of autophagosomes is initiated by the formation of protein complex ULK, PI3K (which contains Beclin-1) and ATG5-ATG12. The fusion of autophagosomes with lysosomes induces degradation of the autophagosome content 262 (Figure 11). Selenite treated cancer cells have been shown to display increased levels of autophagy 230,263,264 . In colon cancer cells, 10 µM selenite induced ROS-related DNA damages leading to p53 (protein signalling DNA damages) mediated apoptosis in HCT116. However, in p53 Knock Out HCT116, autophagy was triggered through selenite treatment 250 .

In both cell lines, when autophagy was inhibited, the apoptotic response to treatment was increased suggesting that the autophagic response is actually a defence mechanism against selenite treatment 263 . This is also supported by the fact that cell death decreased following autophagy activation in these cells 264 . This crosstalk was mediated via BCL-2 family proteins that are bound to Beclin-1 under normal conditions, which is cleaved by apoptotic caspases 262 . This prosurvival autophagy activation against selenite treatment phenotype has not been shown for all cancer cells. In glioma cells 7 µmol/L of selenite triggered autophagy that lead to increased cell death 225 through ROS overproduction, suggesting that the type of cell death is dependent on the cancer cell line.

Effect of selenium on epigenetic mechanisms

Cell and murine models have both revealed that selenium triggers DNA methylation modifications through dose dependent DNA methyltransferases (DNMT) activation/inhibition 207,217 . Se deficiency have been reported to inhibit liver expression of enzymes involved in the one-carbon metabolism 207,217 . Low doses of selenium increase DNA methylation while higher doses decrease levels of methylation of promoters 217 through modification of the activity of DNMTs.

The effect of selenite and methylseleninic acid on H3K9 modifications has been measured in prostate cancer cell lines. In LNCap prostate cancer cells, treatment with 1.5µM of selenite for 7 days reduced the activity of histone deacetylases either by binding to the catalytic site of the enzyme or by modifying cysteine residues in HDAC proteins leading to increased acetylated H3K9. Investigators have also measured a decreased amount of H3K9me in this cell line. Overall this induced the activation of gene expression. Decreased methylation of histone might be due to a decrease of the DNMT1 following selenium treatment as inhibition of DNMT by 5aza-dC has been shown to result in decrease of H3K9 methylation 265 . This was concomitant with decrease of the DNA methylation on promoter of tumour suppressor genes. In glioblastoma spheroids, LN229 cells were treated with 2.5µM selenite resulting in a 30% reduction in of H3K9 methylation in comparison to control, whereas treatment of U87 (O( 6)methyguanine-DNA-methyltransferase (MGMT) negative cells) with 10µM selenite treatment increased the methylation of H3K9 233 , and methylseleninic acid (MSA) inhibited DNMT1 expression and decreased methylation of H3K9 266 .

Selenium Nanoparticles

The toxicity of selenium is critically dependent on its redox state and concentration making it difficult to use in pharmacology. Selenium in solution has been extensively studied in different cancer cell types overall showing induction of redox functions at low doses 212,267 and apoptosis at higher doses 268 depending on the form of selenium and the type of cells used.

Preclinical and clinical studies have suggested a protective effect of selenium against prostate cancer 220 however higher (>90µg/day) didn't prove any chemotherapeutic effect. Higher doses of aqueous selenium are toxic and lead to selenosis 190,269 . To overcome the limitations of high dose systemic toxicity the use of selenium in the form of nanoparticles (SeNPs) has been initiated, with initial promising results including preferential uptake by cancer cells in comparison with normal cells 257,270 . Similarly to the aqueous selenium studies, SeNPs demonstrate an antioxidant activity in cancer cell lines 208 at low doses and demonstrating cytotoxic effect at higher doses. Moreover, SeNPs reduced toxicity of selenium up to four times in mouse models 271 in comparison with selenium in solution and the toxic effect on livers have been significantly reduced 272 . A major drawback of bare SeNPs appears to be the poor cellular intake, which has been overcome by conjugation to stabilizing and targeting ligands on the exterior surface of the nanoparticles [271][272][273][274][275] .

Coated SeNPs

In order to increase the reactivity, bioavailability and stability of SeNPs but also to control their size, different coatings can be added during the process of synthesis. The addition of proteins (albumin), oligosaccharides (sucrose) and polysaccharides (chitosan) at different concentrations or at different times of preparation of the SeNPs influences size, morphology and stability 276 of the NPs in liquid dispersion 277 .

In early 2000's SeNPs were synthesized in presence of different concentrations of albumin (BSA). It appeared that the higher the concentration of BSA, the smaller the nanoparticles 274,278 . The albumin bind the selenium through interaction by the cysteines as Cys-S-Se 274,279 . It allows the stability of the nanoparticles which would aggregate as micro particles in absence of proteins.

BSA-SeNPs have been proven to be 7-fold less toxic than selenite in mice with respectively 113 and 14mg Se/kg body weight to reach toxic doses. However in hepatic cancer cells (HepG2) no differences have been measured in growth inhibition between selenite and SeNP treatment after 72h of 25µM of Se 279 while GPx and TRx were upregulated. BSA allow stabilization of the nanoparticles and doesn't elicit toxic biological responses as well as being largely available. However non specific binding of albumin to other proteins inhibit their functions disturbing cellular processes. While paving the way for selenium nanoparticles study, BSA-SeNPs effect on cancer cells have been barely studied. The use of chitosan for SeNP coating results in the nanoparticles having the ability to bind cell membrane phospholipids via exposed NH3 + groups 280 . Compared to bare SeNPs, chitosan coated SeNPs are better internalized by cancer cells through endocytosis 281,282 . In mouse model, chitosan coated SeNPs were 10 fold less toxic than aqueous selenite with a 50% mouse mortality of 24 mg selenite/g of body mass and 250 mg/g of bodymass chistosan-SeNPs/g 276 .

Cancer cell specific properties can also be used to enable the targeting of SeNPs. The overexpression of folate receptor (vitamin B9) in different cancer tumours (kidney, liver, skin, lung) 164,165 has been used in order to specifically target cancer cells. In 4TI breast cancer cells, folic acid (C19H19N7O6) modified SeNPs increased cell mortality by 68% compared to bare SeNPs 285 . Moreover, folic acid targeting of coated SeNPs induced apoptosis [285][286][287] in liver HepG2, osteosarcoma MG-63 and kidney HK-2 cells. In mouse models SeNP-folate decreased the tumour growth rate 285 .

Interestingly chitosan and folate combined coatings resulted in targeting folate receptor and negatively charged membrane 286 . After internalization, when pH increase above 6.5, the amino groups of chitosan become positively charged and chitosan precipitate which induce an increased intracellular drug accumulation 288 .

SeNPs combined with chemotherapy

Classic chemotherapy increases the oxidative stress in cancer cells but also in other normal cells. The preclinical and clinical studies using inorganic selenium supplements increased the antioxidant capacities of the cells 193,197 . Recent studies conjugated SeNPs with chemotherapeutics such as paclitaxel (PTX) or cisplatin, have any combined (additive or synergistic) cytotoxic effect 254,289,290 against cancer cells. Further development is needed to investigate the low impact of these treatment toward normal cells.

Paclitaxel has been loaded on 20-100 nm SeNPs by adsorption on pluronic F-127 detergent. The NPs demonstrated an anti-proliferative activity against lung (A549), breast (MCF7) and cervical (Hela) cancer cells 289 . Cell cycle analysis demonstrated a G2/M arrest in a dose dependent manner of SeNP-PTX leading to apoptosis due to ROS induced mitochondrial membrane disruption and activation of caspases without deciphering whether SeNPs or PTX had this effect. PTX-SeNPs used on these cancer cell types showed a greater cytotoxic effect at much lower concentrations than treatment with SeNPs or PTX alone and is thought to decrease the side effects of the different drugs 289 .

Cisplatin the most widely used treatment in ovarian cancer causes side effects including nephrotoxicity and genotoxocity mediated via activation of the inflammatory pathway due to high oxidative stress in cells 204 . SeNPs reduced cisplatin toxicity against reproductive system in Wistar rats 290 . SeNP-cisplatin also reduced toxicity in mice against osteoblasts 291 , thyroid gland 292 , intestinal cells 293,294 by limiting nuclear and mitochondrial damages and apoptosis In brain and hepatocytes SeNP-cisplatin reduced the number of pro-apoptotic B-cells 295 .

Building a more comprehensive approach of the effect of selenium nanoparticles in cancer models, we decided to review the literature in order to describe the different type of SeNPs that have been synthesized and used as treatment toward cancer cells or tumour bearing mice since the beginning of the 21 st century. Table 3 sums up the main outcomes of those trials.

IV. Objectives

Hypothesis: Selenium nanoparticles accumulate in ovarian cancer cells and limit the growth of these cells more effectively then free forms of selenium that display inherent in vivo toxicity. Moreover the use of copper isotopes ratio in blood and samples are informative in Ovarian Cancer early detection. Main objectives:

1. Determine the effects of selenium nanoparticles on ovarian cancer cells by investigating nanoparticle accumulation, cell death, cytotoxicity, migration of cancer cells. 2. Characterise the effects of selenium nanoparticles on cell biomechanics to understand if selenium impairs metastatic potential by affecting cytoskeleton. 3. Determine the molecular process triggered by selenium nanoparticles in ovarian cancer cells to understand underlying mechanisms of action 4. Evaluate [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu/ [START_REF] Gang | Measuring Microenvironment Mechanical Stress of Rat Liver during Diethylnitrosamine Induced Hepatocarcinogenesis by Atomic Force Microscope[END_REF] Cu ratios (𝛿 [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu) in serum samples from cancer patients as a potential complementary ovarian cancer biomarker.

Selenium Nanoparticles triggers alterations in ovarian cancer cells biomechanics I. Presentation of the article

Metastasis is the leading cause of cancer death in 90% of ovarian cancer cases [START_REF] Campaner | The methyltransferase Set7/9 (Setd7) is dispensable for the p53mediated DNA damage response in vivo[END_REF] . The ability to limit this process is paramount in the treatment of ovarian cancer, where metastasis seems to be triggered by the accumulation of highly inflammatory peritoneal ascitic fluid, which contains growth factors that can induce epithelial to mesenchymal transition (EMT). In addition to these biochemical factors, the sheer stresses that cells are exposed to as a result of ascitic fluid circulation, may help tumour cell dispersion [START_REF] Clare | One-Carbon Metabolism: Linking Nutritional Biochemistry to Epigenetic Programming of Long-Term Development[END_REF] . EMT is defined by the loss of cellular polarity, modifying their interactions with neighboring cells while gaining migratory and invasive properties. During this process, cells exhibit modified mechanical properties as a result of altered architectural changes of their cytoskeleton 2,[START_REF] Yamaguchi | Regulation of the Actin Cytoskeleton in Cancer Cell Migration and Invasion[END_REF] . Principal cytoskeletal components such as actin microfilaments, intermediate filaments and microtubule polymer networks play important roles in locomotion and cell integrity, influencing cell adherence, interactions with other cells and motility [START_REF] Rebelo | Comparison of the Viscoelastic Properties of Cells from Different Kidney Cancer Phenotypes Measured with Atomic Force Microscopy[END_REF][START_REF] Levental | Soft Biological Materials and Their Impact on Cell Function[END_REF][START_REF] Krouskop | Elastic Moduli of Breast and Prostate Tissues under Compression: Ultrasonic Imaging[END_REF] .

With Ovarian cancer motility and mechanical transformation being a central theme to both local and distal metastasis, selenium nanoparticle effect on ovarian cancer cell lines, SKOV-3 and OVCAR-3 biomechanical properties were assessed in vitro. SKOV-3 are metastatic epithelial cancer cells originally derived from the ascitic fluid. OVCAR-3 are also epithelial cancer cells, however are derived from slow growing adenocarcinoma and are associated with resistant phenotypes. Expression of EMT markers in SKOV-3 and OVCAR-3 cell were assessed using western blotting, alongside cellular morphology, membrane roughness and mechanical properties (elasticity and adhesion), in the presence and absence of selenium nanoparticles coated with BSA and chitosan. BSA is used in nanoparticles synthesis for its ease of use and high bioavailability and stability in blood. However, it can non selectively bind to proteins impairing cellular functions. Chitosan is highly soluble when pH<6.5 thanks to the positive charges of the nitrogen groups and the coating disaggregate allowing the drug release 133 . Free selenite was used as a control to show evidence for the increased efficacy and uptake of selenium nanoparticles.

Dissemination of cancer cells following EMT is sustained by modification of cell-cell, cellmatrix interactions and cytoskeleton modifications. Such modification can be studied using nanomechanical tool such as atomic force microscopy. Atomic Force Microscopy is composed of a mobile cantilever on which a laser detects its deflection through its interaction with the surface studied. While the cantilever is moving, the surface elasticity and roughness impacts the way the cantilever bends. This is detected by the laser receptor which translates the properties of the material studied into force curves using the Hertz model allowing the estimation of Young's Modulus (force necessary to indent into the cells). Literature data demonstrate [START_REF] Yamaguchi | Regulation of the Actin Cytoskeleton in Cancer Cell Migration and Invasion[END_REF][START_REF] Cross | AFM-Based Analysis of Human Metastatic Cancer Cells[END_REF]300 that cellular elasticity is strongly correlated with cell cytoskeleton.

Graphical Abstract: Selenium nanoparticles triggers nanomechanic properties modifications in ovarian cancer cell lines Treatment of high grade serous ovarian cancer cells OVCAR-3 and SKOV-3 with coated selenium nanoparticles highlighted two opposite behavior depending on the aggressiveness of the cell type. Biomechanical modifications in metastatic SKOV-3 and aggressive OVCAR-3 assessed by Atomic Force Microscopy measurement led us to decrease the metastatic potential of SKOV-3 cells and decrease viability of OVCAR-3 cells.

We assessed the EMT markers and the mechanical properties of SKOV-3 and OVCAR-3 using AFM before and after treatment with selenite or coated selenium nanoparticles (see Figure above). SeNP penetration in SKOV-3 and OVCAR-3 ovarian cancer cells was assessed by measuring the levels of selenoprotein transcript GPX1 after treatment. It appeared that selenium can't affect EMT markers in the two different ovarian cancer cell lines. The analysis of nanomechanical parameters revealed opposite phenotype between SKOV-3 and OVCAR-3 after treatment with SeNPs. While SKOV-3 increased stiffness and roughness, OVCAR-3 became more elastic and softer. In addition we acquire we hypothesized the SeNPs are effective for cell proliferation inhibition but in different mech anisms depending on the cell type. We tried to elucidate those mechanisms in the following part constituted by the paper entitled "Selenium nanoparticles induce global histone methylation changes in ovarian cancer cells".

II. Article

This paper is Published in Nanomedicine: Nanotechnology, Biology and Medicine https://www.sciencedirect.com/science/article/pii/S154996342030112X

Selenium nanoparticles trigger alterations in ovarian cancer cell biomechanics.

Benoit Toubhans 1,2 (PhD Student) Salvatore Andrea Gazze 1 (Dr) Caroline Bissardon 3 (Dr), Sylvain Bohic 3,4 (Dr) Alexandra T. Gourlan 2 (Dr) Deyarina Gonzalez 1 (Pr) Laurent Charlet 2 (Pr) R. Steven Conlan 1 (Pr) and Lewis W. Francis 1 (Dr).

Affiliations:

Selenium nanoparticles induce global histone methylation changes in ovarian cancer cells I. Presentation of the article

Selenium has been proven to be useful as a potential cancer preventive agent, especially in populations with demonstrable poor intake. Despite selenium toxicity increases at doses slightly higher than nutritional requirements, clinical trials investigating selenite as an anti-cancer therapy, have revealed its protective effect against liver cancer. Null or adverse effects have been found in breast cancer. These in vivo results contrast with the promising results found in in vitro cell culture models 301 . SeNPs, have led to increased possibilities when compared to aqueous selenium, due primarily to their increased accumulation and specificity against cancer cells 257,270 .

Selenium nanoparticles have been shown to induce cell death mechanisms in hepatocarcinomas and breast cancers 282,290 . Moreover, selenium has been shown to influence the epigenome, regulating cancer development and influencing the expression of selenoproteins. High doses of selenium treatment have been shown to inhibit DNA methyltransferase activity and expression; however, the mechanism remains unclear. At low selenium doses, reduced DNA methylation has been observed, due to disrupted restoration of the S-adenosylmethionine methyl donor, as a result of redirection of homocysteine toward glutathione synthesis. High doses of selenium have also been shown to trigger DNA hypomethylation due to DNMT inhibition and competition of selenium with DNA for the methyl group. At intermediate doses, DNA methylation is increased in a dose dependent manner. Epigenetic mechanisms are abundant, complex and altered in cancer cells. The link between selenium and other relevant post translational modifications such as histone methylation has never been studied and is an important gap in the epigenetic knowledge related to selenium. In this study we investigated the effect of sub lethal dose selenium treatment on histone H3 methylation, particularly on lysines K4, K27 and K9. These marks are gross marks of DNA compaction transcription activation. We utilized 2D and 3D culture in the presence and absence of SeNP (24 to 72 hours) and combinations of epigenetic enzyme inhibitors to reveal potential pathways for histone methylation mediated selenium mechanisms in ovarian cancer cells (see Figure below).

Graphical Abstract: Simplified proposed selenium mechanisms of action for histone methylation induction in ovarian cancer cells Selenium can influence histone methylation through three different pathways. The selenite from the addition of SeNPs or sodium selenite to cancer cells reacts with the glutathione GSH through the Transulfuration pathway pulling the equilibrium of the methylation SAM-SAH cycle towards the homocystein clearance into transulfuration pathway leading to the formation of selenide H2Se. H2Se can then react with oxygen forming ROS damaging cells and aggregates of Se0. The increase of H2Se also increases the synthesis of SeCys through selenocysteine lyase increased expression which could enter in the Methionine cycle. Moreover, selenium increased the activity of the HMTs by increasing their level of expression.

The lysine methyltransferase (KMT) inhibitors used were provided by the Structural Genomic Consortium. We targeted G9a which is the main histone methyltransferase allowing methylation of H3K9 which is a mark of heterochromatin. We also targeted EZH2 regulating the methylation of H3K27, associated with repression of the transcription. Finally we targeted PRDM9 regulating the methylation of H3K4, an active transcription mark. We studied the effect of selenium treatment after inhibition of those marks, showing that selenium, at sublethal doses, increased histone methylation by increasing the activity of histone methyltransferases. Treatment may also drive the clearance of S-adenosylhomocysteine through the transulfuration pathway, avoiding its inhibitory effect on histone methyltransferase, leading to increased methylation of histone lysines. Moreover when HMT were inhibited, selenium is able to increase histone methylation. This increase may be related to the increased expression of other methyltransferases, upregulated after selenium treatment. Interestingly the activated methyltransferases were different between SKOV-3 and OVCAR-3 cell types, as shown using RNA-seq analysis, with increased autophagy in OVCAR-3 and increased apoptosis in SKOV-3. The consequence of these different phenotypes highlight an increased resistance of OVCAR-3 against SeNP treatment.

Abstract

The trace element selenium plays a key role in redox reactions through its incorporation in selenocysteine in antioxidant enzymes. Selenium has also been shown to affect DNA methylation through modulating the expression of DNMT1. Here we identified novel effects of selenium on histone methylation using ovarian cancer cell models treated with inorganic selenium nanoparticles (SeNP). As well as inducing oxidoreductase expression, ROS activity and cancer cell cytotoxicity, selenium caused significant increases in histone methylation. Specifically, selenium triggered an increase in the methylation of histone 3 at lysine's K4, K9 and K27, histone marks involved in both the activation and repression of gene expression, suggesting a fundamental role for selenium in these epigenetic processes. This direct function was confirmed using chemical inhibitors of the histone lysine methyltransferases EZH2 (H3K27) and G9a/EHMT2 (H3K9), both of which blocked the effect of selenium on histone methylation. This novel role for selenium supports a distinct function in histone methylation that is likely to occur though interference in the one-carbon metabolism pathway responsible for providing the methyl donor S-adenosylmethionine in both DNA and histone methylation. These observations provide important insights into the action of selenium, and the effects of SeNPs which, unlike selenite, are well tolerated in vivo. It will be important to consider both the classic antioxidant and novel methylation effects of this key redox element in its development in cancer therapy and other applications.

Introduction

Selenium compounds contribute to the maintenance and integrity of cellular systems by influencing cellular redox states and capacity to detoxify compounds, free radicals and reactive oxygen species (1). Thioredoxin reductases for example, which contain selenocysteine, are present in the cytosol (TrxR1) and mitochondria (TrxR2) and involved in the reduction of oxidized thioredoxins, can catalyse NADPH, control ascorbate levels and regulate metabolism. Selenium is also involved in the biosynthesis of diverse molecular components that are required for important cellular functions including deoxyribonucleoside triphosphates for DNA, the reduction of oxidized proteins, and has roles in diverse regulatory mechanisms such as redox, apoptosis, immunomodulation and the formation of methyl donor compounds. In cancer increased H3K27me3, catalysed by histone lysine methyltransferases (HMTs) including EZH2, has been associated with chemoresistance ( 2) and demethylation linked to more aggressive phenotypes (3). Similarly knockdown of G9a/EHMT2, which catalyzes H3K9me2, is linked to ovarian cancer peritoneal metastasis and decreased invasiveness in ovarian cancer cell models ( 4), as is a second putative HMT SMYD3 ( 5). Here we show that exposure of two pathologically distinct ovarian cancer cell models to selenium results in different redox responses and effects on cell viability, and that selenium delivery via SeNP, that are well tolerated in vivo, is as effective as selenite (6, 7). Interestingly we found that selenium treatment stimulated an increase in histone methylation at the distinct epigenetic marks H3K4me3, H3K27me3 and H3K9me2.

Remarkably this effect was inhibited by specific histone lysine methyltransferase inhibitors targeting EZH2 (H3K27) and G9a/EHMT2 (H3K9) demonstrating that selenium can directly modulate histone methylation, and thus cellular epigenomics. These findings highlight the importance of this micro-nutrient and that its role in redox biology should be evaluated together with its effects on epigenetic processes, particularly when considering potential applications in cancer therapy.

Results

SeNP penetration and response in ovarian cancer cells.

In contrast to selenite, SeNPs are well tolerated in vivo and appear to offer a route to unlocking the potential of Se as a therapeutic agent (6). We compared the effect of SeNPs to selenite using two distinct 3D spheroid ovarian cancer models, OVCAR-3 and SKOV-3, and demonstrated that SeNPs were able to penetrate at least 80 µm into tumor models. 30 nm electron dense particles corresponding to selenium aggregates were observed by TEM in vacuoles and mitochondria in cells treated with SeNP-BSA (Figure 1A, Figure 1B) or SeNP-chitosan (Supplementary Figure 1) and confirmed using FITC-tagged SeNPs (Figure 1C). To determine whether the observed vacuolar structures were autophagosomes, autophagy markers were assessed ( 8) and ATG5 levels found to be up-regulated by SeNP-BSA, but unaffected by selenite or SeNP-chitosan in SKOV-3 (Figure 1C), whereas there was an increase of LC3B with selenite, but not with either SeNP (Figure 1C). In OVCAR-3, ATG5 expression and levels of LC3 maturation increased following all treatments, consistent with these cells being more resistant to selenium, and with constitutively activated autophagy (9). SKOV-3 and OVCAR-3 cell spheroids treated for 24 h with selenite or SeNPs and profiling autophagy markers (C). Control and selenite treated SKOV-3 displayed similar levels of ATG5 (respectively 0.53 and 0.59), whereas SeNP-BSA treated cells showed a significant increase (0.79, p=0.05) of ATG5 levels.

SeNP-chitosan treated SKOV-3 cells showed non-significantly elevated level of ATG5 (0.68). Selenite, but not SeNPs, significantly increased LC3B levels in SKOV-3 cells. OVCAR-3 cells displayed a general increase the expression of ATG5 in the different conditions (selenite 0.65, SeNP-BSA 0.56, SeNPchitosan 0.87) compared to the control (0.46) but was only significant for SeNP-chitosan (p<0.05).

Selenium treatments increased LC3A to LC3B maturation. Data represent mean +/-SD of three biological replicates.

Gene expression analysis by PCR demonstrated an expected increase in thioredoxin reductase 1 (TrxR1) expression following treatment (Figure 2A). Additionally, RNA-seq analysis identified several selenium-related genes that were differentially regulated in response to treatment (Figure 2B&C). Expression of selenoproteins I, S and T and the selenocysteine lyase (SCLY) were increased in both cell types, with increased SCLY expression suggesting the transformation of selenium to selenocysteine SeCys is likely to be occurring (10). Oxidative stress response decreased by 20-50% over the first 3 h following SeNP treatment, but steadily increased thereafter (Figure 2D&E). SKOV-3 (A) and OVCAR-3 (B) cells were treated for 24 h with sublethal doses of selenite or coated selenium nanoparticles. RNA was extracted and sequenced by the sequencing platform of the Ecole Normale Supérieure de Lyon (IGFL). Heatmaps show the log10 of the ratio between the selenium treated and control conditions for selenium related proteins. SKOV-3 (D) and OVCAR-3 (E) cells were grown as a 2D layer at 50 x 10 3 cells per well, incubated with ROS probes, and treated IC50 concentrations of selenite, SeNP-BSA or SeNP-chitosan. ROS red fluorescent assay results showed that selenite, and SeNPs treatments at IC50 decreased the production of ROS in SKOV-3 cells. In OVCAR-3 cells, selenite and SeNP-BSA ROS production peaked above control levels after 6h. The data represents the mean +/-SD of three individual experiments.

Treatment of spheroids resulted in significant reductions in cell viability with selenite (>0.6 µg/mL), SeNP-BSA (>1.25 µg/mL) and SeNP-chitosan (>3 µg/mL) for SKOV-3 (Figure 3A), and OVCAR-3; selenite (5 µg/mL) or SeNP-BSA or SeNP-chitosan (10 µg/mL for both).

Further analysis revealed that SeNP exposure increased caspase-3 cleavage levels in SKOV-3

(2.5 fold, Figure 3C) suggesting increased apoptosis (11), whereas apoptosis was not induced in OVCAR-3 (Figure 3D). Gene ontology analysis supported these observations showing that in SKOV-3 SeNPs triggered intrinsic pathways involved in apoptotic signalling in response to DNA damage and response to oxidative stress (Table 1), whereas in OVCAR-3 exposure caused changes in cell mobility, with the activation of epithelial cell migration, epithelial to mesenchymal transition (EMT) and extracellular matrix organisation (Table 1). Only moderate increase in caspase 3 cleavage was observed for SeNP-BSA treatment (0.09). In OVCAR-3 no changes in caspase 3 (D) were seen for selenite (0.47), SeNP-BSA (0.62) and SeNP-chitosan (0.67) in comparison with control (0.55), and cleaved caspase levels were very low for each condition. The data represents the mean +/-SD of three individual experiments. Selenium has been shown to modulate DNA methylation, mainly through the regulation of DNMT expression; any wider involvement in epigenetic mechanisms involving methylation have yet to be explored (12, 13). We investigated whether selenium had a role in histone methylation and very interestingly observed that SeNPs triggered an increase in the levels of H3K4me3 (Figure 4A), H3K27me3 (Figure 4B) and H3K9me2 (Figure 4C), thus revealing an important role for selenium in this process. Detailed RNAseq analysis was undertaking to understand whether, as for DNA methylation, these effects were due to selenium-induced changes in HMT expression. The H3K4me3 HMT PRDM9 was not expressed in the cell lines used, whereas SETD7 ( 14) and SUV39H2/KMT1B (15, 16) expression was consistently upregulated by selenium. EZH2, which methylates H3K27me3 was also upregulated, whilst the expression levels of the H3K9me2 HMT EHMT2/G9a along with EHMT1/GLP were relatively unaffected (Figure 5 A&B). Changes in the expression level of other potential HMTs (as well as lysine demethylases, KDMs) were observed, although these HMTs require further experimental validation to define their precise roles (Supplementary Figure 2), and DNMT3A and DNMT3B (18) expression was also increased. In OVCAR-3 selenite increased levels by 3 fold (p<0.001) and by 2 fold with SeNPs (p<0.05). The presence of the inhibitor MRK740 did not affect H3K4me3 levels in control or treated samples (p<0.05).

Figure 4B:

Cells were treated for 24 h with the epigenetic probe GSK343, an inhibitor of the H3K27 HMT EZH2 followed by 24 h with selenite or SeNPs. No control probe was available for GSK343.

Selenium treatments significantly (p<0.01) increased H3K27me3 levels by 2 fold in SKOV-3. In OVCAR-3 selenite and SeNP-chitosan increased levels by 4 fold (p<0.001) and by 2 fold with SeNP-BSA treatment (p<0.01). The presence of GSK343 inhibited H3K27me3 methylation in SKOV-3 cells (p>0.05) but not significantly in OVCAR-3 although expression was slightly decreased. Selenium induced histone methylation occurs via histone methyltransferase activity.

Whilst upregulation of SETD7 and EZH2 gene expression by selenium could account for increases in H3K4me3 and H3K27me3 methylation, there were no changes in EHMT2 expression. This suggested that for H3K9me2 at least selenium may also affect other histone methylation processes. To determine if this directly involved the methylation of histones, inhibitors blocking the activity of specific HMTs was investigated. As expected inhibition of PRDM9 using MRK740 (19) (or the inactive analogue MRK740N) did not result in a decrease in H3K4 tri-methylation as PRDM9 was not expressed in either cell line. Inhibition of the H3K27 methylase EZH2 using GSK343 (20) resulted in a significant decrease in H3K27me3 levels in SKOV-3 demonstrating that the effect of selenium occurs via the activity of EZH2 in these cells (Figure 4C), and likely also in OVCAR-3 cells where, whilst the effect of GSK343 was less pronounced, a decrease in H3K27me3 levels was apparent. Finally, two different EHMT2 inhibitors, UNC0642 (21) and A-366 (22), essentially ablated H3K9me2 in SeNP treated SKOV-3 (Figure 4D), and again there was a general decrease in H3K9me2 in OVCAR-3. These data support a mechanism whereby selenium-mediated increases in histone methylation occur through a biochemical process involving HMTs, as despite selenium-induced increases in expression of some HMT genes including EZH2, HMT inhibitors can abrogate this effect.

Discussion

Selenium in the form of selenite has been explored as a mode of cancer therapy but has to date failed due to systemic toxicity (23). Here we have shown that as well as triggering enhanced redox activities, SeNPs induce cytotoxicity in ovarian cancer cell lines, with cell-type specific responses, and could therefore offer significant benefits as these nanoparticles can be delivered at cytotoxic doses in vivo (24, 25). In OVCAR-3 cells autophagy was constitutively activated resulting in the intracellular accumulation of SeNPs offering an explanation as to the greater resistance of these cells to high levels of selenium. In contrast, SKOV-3 cells displayed increased levels of apoptosis. To understand the mechanisms underlying the differential responses to SeNPs, we investigated the possibility of selenium inducing epigenetic effects beyond DNA methylation and identified increased levels of histone H3K4me3, H3K27me3 and H3K9me2. To understand whether this selenium induced histone methylation occurred through HMTs known to methylate each of these histone marks, specific HMT inhibitors were evaluated. The PRDM9 inhibitor MRK740 had no effect on reducing methylation H3K4 as this HMT is not expressed in the cell lines used, H3K4 methylation increases could be attributed to SeNP induced expression of SETD7 (26). Inhibition of EZH2 by GSK343 blocked SeNP induced H3K27 methylation despite associated increases in EZH2 gene expression levels, suggesting that selenium-induced H3K27 methylation may in part be due to HMTs, or mechanisms impacting EZH2 function. Increases in H3K9 methylation were effectively blocked by the EHMT2 inhibitors A-366 and UNC0642, and as EHMT2 mRNA expression was unchanged following SeNP treatment supporting the view that a mechanism distinct from the modulation of G9a/EHMT2 expression is involved in increasing histone methylation.

The inhibition of SeNP mediated methylation by HMT-specific inhibitors led to the notion that this could occur through a ubiquitous process linked to histone methylation. The methionine metabolic pathway generates SAM, the principal substrate for DNA and histone methylation (27). One possibility (Figure 5) that would result in an increased pool of SAM is the upregulation of MAT1, that synthesises SAM from methionine, by SeNP. This was effectively ruled out in the current study as no significant changes in MAT gene expression levels were observed (Figure 5). Uniquely the elements selenium and sulphur share many chemical properties, and as they are so tightly coupled it has been assumed that selenium may follow the same metabolic pathways as sulphur. More speculatively therefore, selenium could be incorporated into Se-adenosylmethionine (SeAM) and function as a SAM analogue to increase the methyl-donor pool. The synthetic SeAM analogue ProSeAM appears to be processed by G9a/EHMT2 in a cellular context suggesting that SeAM could be used as an HMT substrate (28). However, genetic studies in yeast lacking both sam1/sam2 (MAT1/MAT2 orthologs) demonstrated whilst SeAM synthesis can occur it is highly toxic accounting for the toxicity associated with its precursor Se-methionine (SeMet) (29). Whilst SeMet was not present in the cell culture media used here, it can be synthesised from seleno-homocysteine (SeHCys) by betaine-homocysteine S-methyltransferase (BHMT) (10, 27) which is expressed in the cell lines used here (Figure 5). Another explanation is that the product of histone methylation reactions, SAH, which is an HMT inhibitor, is removed from the cellular system due to the presence of high levels of selenium. Indeed selenium supplementation in murine models has been shown to decrease the ratio of SAM/SAH suggesting that this clearance of SAH occurs in vivo (30, 31).

SAH levels could be decreased following SeNP treatment, resulting in increased HMT activity, by diverting HCY into the transulfurication pathway as the introduction of selenite to this pathway leads to the formation of selenoglutathione (GS-Se-SG) and ultimately selenide (H2Se) (32).

Conclusion

We propose that selenium driven increases in histone methylation are likely to occur through distinct processes (Graphical Abstract) including 1) increasing the activity of HMT due to increasing the levels of expression of the genes encoding these enzymes and 2) clearance of SAH, likely due to a 'pull' of homocysteine to H2Se due to selenium activating the transsulfuration pathway.

The discovery that selenium, though the activity of HMTs, can directly modulate histone methylation, a key process in the regulation of global gene expression, highlights the importance of this micro-nutrient. Selenium's pivotal role in redox biology, and its potential applications in cancer and viral therapy, should now also consider its wider role in the mechanism of action pertaining to epigenetic processes.

Materials

Nanoparticles BSA and chitosan coated SeNPs were purchased from NANOCS (New York, USA) with a manufacturer defined diameter of 25 -50 nm for both nanoparticles. SeNP characterisation and IC20 treatment levels used in 2D culture experiments have been reported previously (7). and 5% CO2 and routinely passaged using 0.25% trypsin-0,1%EDTA (v/v).

Spheroids growth, treatment and viability assay

Cell viability was determined using a CellTiterGlo assay (PROMEGA, UK). 5,000 cells/well were plated in 96-well Ultra Low Attachment coated round bottom plates (Corning, UK). After spheroid formation (usually after 24h), 100 µL of fresh medium containing a 2X concentration of Sodium Selenite (Na2SeO3) or selenium nanoparticles (BSA or chitosan) were added. For the viability assay, an increasing dose range (0.01 µg/mL to 20 µg/mL) was applied by dilution in appropriate medium for 24, 48 or 72 h. After the treatment, 100µL of media was removed from wells and 100 µL of CellTiterGlo added. Plates were shaken for 5 min and equilibrated at room temperature for 25 min before luminescence measurements were taken (BMGLabtech Fluostar Omega, UK). IC20 and IC50 doses were determined as the concentration required to reduce the luminescence signal by 20/50%. The IC20/50 values shown are the result of a minimum of five independent experiments performed with 4 technical repeats.

High-pressure freezing and freeze substitution TEM sections were prepared as previously described (33). Briefly spheroids were pelleted and vitrified by high pressure freezing (HPM100, Leica Microsystems) to -90°C for 80 h in acetone with 1%OsO4. The temperature was then raised 1°C/h to 30°C and samples rinsed 4 times in acetone. Samples were infiltrated with agar low viscosity resin (LVR, Agar scientific) in acetone for 3h. After polymerisation for 24 h at 60°, 70 to 400nm sections were obtained using an ultra-microtome (UC7, Leica Microsystems). Sections were collected on formvar-carboncoated 100mesh copper grids and post-stained for 10min with 2% aqueous uranyl acetate, rinsed and incubated for 5 min with lead citrate. Grids were analysed using Tecnai 12 FEIMicroscope (120kV) at different magnification.

ROS assay

SKOV-3 and OVCAR-3 cells were seeded as monolayers at 20,000 cells per well in a dark 96well plates and cultured overnight. Following removal of media, cells were washed once with PBS, then incubated for 1 h with the Cellular Reactive Cellular Reactive Oxygen Species Detection reagent (Red Fluorescence, Abcam186027). A 6x IC50 concentration of selenite or SeNPs was then added and the plate incubated at 37°C for the duration of the assay.

Fluorescence was analysed at different time points from 30 min to 10 h (excitation filter 520nm, emission filter 605nm, BMGLabtech Fluostar Omega, UK).

Protein blotting

Total cellular protein was extracted, and equal amounts of protein separated by SDS-PAGE and transferred to an acrylamide membrane (BioRad, UK). After blocking in 5%BSA TBS-T for 1 h, blots were incubated with primary antibody (Caspase 3: rabbit polyclonal (CellSignal9662, UK), ATG5: rabbit polyclonal (CellSignal 2630, UK), H3K4me3: rabbit polyclonal (Thermo PA517420), H3K27me3: rabbit polyclonal (Thermo PA531817), H3K9me2: rabbit polyclonal were normalized to values obtained for the reference gene (GAPDH) and relative expression expressed as the mean fold induction ± standard deviation. Statistical differences between the treatment groups and the control were determined by analysis of variance (ANOVA) (where p<0.05 was considered significant).

RNA-Sequencing

For each condition a total of 96 independently cultured spheroids were pooled. Extracted RNA from pooled samples underwent quality control assessment using the RNA Tapestation 2200 (Agilent). cDNA libraries were prepared using the SENSE mRNA-Seq Library Prep Kit V2

(Lexogen) prior to RNA-sequencing (RNA-Seq, genomic platform, Ecole Normale Supérieure de Lyon). Raw fastq files were quality-checked using FastQC, a quality-control tool for high throughput sequencing data, prior to alignment to the hg38 indexed transcriptome using Bowtie2 (34). The eXpress software (35) was used to quantify expression from the transcriptome mapping and derive count data and the differential expression tool package DESeq2 (36), implemented within R, was used to correct for multiple hypothesis testing and determine significantly modified transcripts (FDR < 0.1) (Supplementary 1). Raw and processed RNA-Seq data is deposited in the GEO Dataset with accession number GSE149397.

The PANTHER platform was used to perform statistical overrepresentation/enrichment tests (38, 39). All major PANTHER terms were tested for over-representation (GO-Biological Processes or Reactome, e.g. binomial) and gene-set enrichment comparing the lists of genes expressed in different experimental conditions. The results are displayed showing the differential distribution of significantly enriched clusters of genes compared to the overall expression tendency within samples.

Epigenetic probes

Epigenetic probes were supplied by the Structural Genomics Consortium under an Open Science Trust Agreement: https://www.thesgc.org/click-trust. Probes were diluted in DMSO to a final concentration of 20 µM. UNC1999 and GSK343 inhibitors and an inactive control probe UNC2400 were used to evaluate EZH1/2. The inhibitor MRK-740 and inactive control probe MRK-740N were used for PRDM9. UNC0642 and A-366 were used to evaluate G9a/EHMT2.

Cells were treated for 1 to 3 days with the different probes to reach IC90 of the targeted

Cu isotope ratios are meaningful in ovarian cancer diagnosis I. Presentation of the article

Ovarian cancer detection is currently based on intraperitoneal cavity imaging and a blood test to measure the levels of circulating CA-125 concentrations. CA-125, or MUC16, is a glycoprotein secreted by the ovarian epithelium during cancer development, particularly in advanced high-grade serous sub types, with well-known limits to sensitivity and specificity for other cancer sub types. New biomarkers are required to complement CA-125 testing to increase effectiveness. However, it remains an effective approach for following patient response to chemotherapy and detecting relapse.

In the last decade, there has been significant interest in the use of blood-based metal concentration and isotopic variations in terms of their diagnostic application in oncology and other fields. Such measurements, performed using multiple-collector inductively coupled plasma mass spectrometer (MC-ICP-MS), have been growing in interest as a result of their ease of use and rapid measurement. Copper isotopes are present in the form of two ions Cu(I) and Cu(II) in cells and in blood. Transport and uptake of these two Cu ions is the cause of the selective distribution (fractionation) of the copper isotopes [START_REF] Gang | Measuring Microenvironment Mechanical Stress of Rat Liver during Diethylnitrosamine Induced Hepatocarcinogenesis by Atomic Force Microscope[END_REF] Cu(I/II) and [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu(I/II) between the cells and the blood. Copper isotopes are linked to fundamental biological functions such as extracellular matrix remodulation or mitochondrial metabolism. Ratios between those isotopes are determined using MC-ICP-MS and the delta value 𝛿 [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu obtained is a measure of the report of Cu isotope abundances relative to a reference :

This represents the relative deviation of the [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu/ [START_REF] Gang | Measuring Microenvironment Mechanical Stress of Rat Liver during Diethylnitrosamine Induced Hepatocarcinogenesis by Atomic Force Microscope[END_REF] Cu ratio in the measured sample from its value in the reference material NIST SRM 976 in parts per 1000 (‰).

With a typical reproducibility on 𝛿 [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu at the 95 percent confidence level as determined from multiple replicates of serum samples is 0.05‰ in samples containing as low as 30ng of copper, the quantity of material needed for precise measurement is very low (200µL of serum typically) Moreover, copper turnover in the body is around 6 weeks, with a concentration of 1mg/L in blood allowing enough material to measure copper isotopes variations and being sure the differences in 𝛿 [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu are due to recent copper metabolism modifications. Recent studies have focused on the evolution of copper isotopic composition in serum of breast cancer patient 100 (Télouk et al., 2015) (Télouk et al., 2015). For all patients tested, a decrease of 𝛿 65 Cuserum by 0.25 ‰, which was relative to an increase in [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] ‰ have also been measured in colorectal cancer patients 100 , and together indicate that 𝛿 [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu could be a useful biomarker for cancer detection and progression (See Figure below).

Graphical Abstract: Proposed mechanisms of copper fractionation between normal cells and ovarian cancer cell.

Changes in the circulating [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu levels from patients with ovarian cancer may be multifactorial. The tumour environment is hypoxic and results in an increase in tumour cellular lactate metabolism leading to the preferentially chelation of heavy copper by lactate thus retaining this isoform of Cu in the tumour cells. In addition, amino acid sequence composition is selectively transporting light copper isotope [START_REF] Gang | Measuring Microenvironment Mechanical Stress of Rat Liver during Diethylnitrosamine Induced Hepatocarcinogenesis by Atomic Force Microscope[END_REF] Cu. When patients are treated with platinum chemotherapy, resistance to treatment can occur. We proposed the following mechanism: In platinum treated cancer cells the copper transporter ATP7A would selectively export [START_REF] Gang | Measuring Microenvironment Mechanical Stress of Rat Liver during Diethylnitrosamine Induced Hepatocarcinogenesis by Atomic Force Microscope[END_REF] Cu. This would result in [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu being selectively retained in the tumour cells by lactate and increased expression of efflux copper transporter increasing the relative amount of [START_REF] Gang | Measuring Microenvironment Mechanical Stress of Rat Liver during Diethylnitrosamine Induced Hepatocarcinogenesis by Atomic Force Microscope[END_REF] Cu in blood.

Using sample biobank of Swansea University we measured copper isotopes in the serum of 44 ovarian cancer patients and in some biopsies we have been able to obtain from the hospital. We have been able to measure a significant decrease of the 𝛿 65 Cu in the serum of ovarian cancer patients in comparison with controls. This preliminary work brought additional results to current studies proving the efficacy of isotopic measurements to detect cancer. However a larger clinical study will be required to define 𝛿 [START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu thresholds that would be indicative of the presence of the disease.

Conclusion and Perspectives

I. Conclusions

The close relationship between the environment and human health is well known, with geomedical links furthering our understanding of trace element bioavailability, leading to innovative therapeutics in a range of diseases. Selenium, a crucial trace element available to humans through soil, has long been postulated to have anti-cancer properties despite its narrow toxicity window. This project utilises the principles of geochemistry and biochemistry to test and develop the use of selenium, in nanoparticulate form, in the treatment of ovarian cancer.

The multidisciplinary approach utilised here, highlights the potential benefits of SeNP for ovarian cancer treatment, identifying novel histone methylation associated mechanisms of action and optimal nanoparticle coatings for increased cellular uptake and spheroid penetration.

In ovarian cancer research, SeNP have been used only once as a carrier of doxorubicin in vitro 302 and never as a stand alone chemotherapeutic despite their high bioavailability and proved inhibition of tumour cancer cell growth. To determine the role of SeNPs in ovarian cancer we adopted a multiscale approach. We have been able to reproduce results from other cancer research groups proving the anticancer effect of SeNPs in different cancer cell line. We have also been able to measure cell physical properties after selenium treatment and, for the first time, the effect of selenium on histone modifications in cancer cells and overall impact on ovarian cancer.

In this Ph.D. dissertation we evaluated the effect of two types of coated SeNP (chitosan-coated and albumin-coated) on two ovarian cancer cell lines, OVCAR-3 and SKOV-3. These cell lines were selected as they represent metastatic and non metastatic phenotypes of high-grade serous cancer. In addition they have distinctly different cellular phenotypes with regard to growth characteristics and resistant phenotypes, prevalent in heterogenous in vivo context. SKOV-3 cells are epithelial cells derived from the ascitic fluid, they are metastatic cancer cells. OVCAR-3 are epithelial cancer cells from slow growing adenocarcinoma. Both SeNPs are cytotoxic in the two cell lines, however SKOV-3 is more sensitive than OVCAR-3. Moreover, cell monolayers (2D models) were less sensitive to selenium treatments than cell spheroids (3D models). Analysis of the mechanical cell membrane properties by AFM revealed an increased cell surface roughness and cellular stiffness in SKOV-3, while with OVCAR-3 cells, a decreased cellular stiffness is observed, indicative of altered cytoskeletal dynamics, alongside decreased vimentin expression level. SeNPs triggered early production of ROS, and a cell line dependent induction of apoptosis (in SKOV-3) or autophagy (in OVCAR-3), indicative of an enhanced resistance to SeNP in this cell type. SeNPs stimulated a global increase in histone methylation, as shown by elevated levels of H3K4me3, H3K27me3 and H3K9me2, involved in both gene activation and repression. Transcriptome analysis revealed SeNPs had a limited effect on pathways involved in the metabolism of the ubiquitous methyl-group donor Sadenosylmethionine (SAM). Whilst some effects appeared to be due to modulation of HMT activity, a clearance route for the HMT inhibitor S-adenosyl-homocysteine (SAH), possibly due to enhanced activity of the transulfuration pathway, also appears likely. It appears that reduction in cell viability following SeNP exposure occurs through different mechanisms that result in contrasting perturbations in cellular mechanics in serous ovarian cancer subtypes.

As well as the development of new, advanced therapeutics for ovarian cancer, there is additional need for the development of new or additional diagnostic procedures. Currently diagnosed via a blood test for elevated CA-125; Ovarian cancer diagnosis lacks specificity and sensitivity and is limited to the detection of advanced disease. Numerous attempts are being made to source alternative or complimentary diagnostics. Copper metabolism is affected in a large range of disease in human and in mammals. Cancer development have been related to increase of copper concentration in blood and modification of the copper isotope ratios in liver, colon and breast cancer patients. Blood copper ratio indicate enrichment in light copper isotopes. On the other hand, heavy copper isotopes are found preferentially in tumoral cells in comparison with surrounding tissues. We hypothesized the role of intracellular oxidative conditions and cancer adaptations to explain the modifications of copper ratios. The accumulation of lactate would explain the chelation of heavy copper. Moreover copper transporters would also be involved in the process. Following copper isotopes ratio in blood would give good tool to detect cancer and follow its treatment.

II. Perspectives

The potential impact of Selenium on cancer is the topic of intense discussion. Understanding novel SeNP mechanisms of action on cancer cells will enable more effective SeNP-based treatment to be developed against cancer, not least in ovarian cancer, a complex and multifaceted disease with a very poor prognostic outcome. As shown in this study, the inclusion of advanced cell culture models in pre-clinical evaluation, such as the spheroid cultures used here, will provide significant added value in terms of clinical translation. This project could be expanded with further explorative work, enhancing our understanding of Selenium speciation, functional genomics and further establishing links to cell and tissue mechanical properties. Amongst a myriad of future research lines, I would recommend the following;

Mapping the speciation of selenium in tumours

Conducting 2D or 3D selenium elemental (and speciation) mapping in spheroids and tumours would enable the nature of the selenium aggregates observed in the cells to be determined. Extra-and intracellular speciation would to reveal whether assimilatory selenate reduction occurs, in analogy to S, and whether blood SeMet is preferentially taken up by tumours in humans. Such a quantitative understanding of S/Se metabolism and its intracellular localisation would allow known S species, metabolites (sulphate, Cys, Met, SAM, APS and PAPS) and (potential) Se analogues to be targeted using standard commercially available or newly synthesised compounds. The identity of the synthesized organic Se compounds will be determined in situ by XAS technique, and could be compared to ex-situ determination, after extraction by LC-ICP-MS/MS and -if required -high resolution LC-ESI-MS/MS, for identification of species for which no analytical standards are available. Synchrotron-based XAS techniques (HERFD-XAS and µ-XANES) could fingerprint in-situ the presence of S/Se organic or inorganic forms in tissues close to the natural, hydrated state. The recent upgrade (30 times X-ray brilliance increase) at the European Synchrotron Radiation Facility (ESRF) will allow bulk speciation tomography to be performed with 30 nm and 0.3 pm spatial resolution and detection limit, respectively, and in very short times. Larger sections of ovarian tissue can be exposed to [START_REF] Weber | Laparoscopic Surgical Staging of Early Ovarian Cancer[END_REF] Se, and NanoSIMS maps obtained after their metabolism (with a 100 nm, 10 ppm spatial resolution and detection limit). In addition, spatial speciation of S or Se in a thin layer of flat tissue surface or within a cell, both down to 100 nm thickness, can be obtained at LBNL, Berkeley, CA, with prototype NMR imaging system using confocal imaging.

Overall, µ-XRF and µ-XAS around the Se K-edge will be used to distinguish between Se(IV) or Se°NP uptake by spheroids and their distribution within cell compartments. Moreover it would allow to decipher the mechanism of aggregation of selenium inside the mitochondria and vacuoles we observed in the paper entitled "Selenium nanoparticles induce global histone methylation changes in ovarian cancer cells" whether it is native SeNPs or secondary aggregated forms.

Expanding knowledge of selenium nanoparticle effect on the genome

We have been able to prove the increase of histone 3 lysine methylation through selenium nanoparticles treatment in ovarian cancer cells. We also used the RNAseq dataset in order to determine gross modifications of hallmarks through selenium treatment. We tried to combine treatment of selenium with DNMT inhibitor (5-aza) and HDAC inhibitor (SAHA) and haven't been able to prove any additive cytotoxic effect. This first approach needs to be complemented with gene expression screening as drug resistance is dependent from chromatin regulators that cannot be analysed only with genomic analysis but need the help of functional genomic. We preferentially would study the effect of SeNPs on DNA methylation as it has been done only with aqueous selenium.

The genetic screening using RNAi of specific epigenetic complexes such as KRAB, GLP, EED, LSD1 or DNMT3A would lead us to increase the knowledge of the effect of selenium on transcription factors and the most essential amongst them in drug resistance. Epigenetic and genetic mediators are influenced by selenium and lead cells to death. We would use this RNAi genetic screen in order to determine the main cell death actors that are regulated through selenium treatment. For example, the transient KO experiments of autophagy would lead us to determine which factors are leading cells to greater cell death. Moreover we would target genes associated with histone modifications based on stable knockdown cell lines from sensitive and resistant to selenium ovarian cancer cells. RNAi screening would lead to identify regulatory molecules responsible for epigenetic modifications in those cell lines.

Increasing our ability to systematically screen gene expression affected by selenium treatment would lead us to draw a precise pathway of action of selenium within cancer cells and normal cells.

Better models for ovarian cancer study

The environment surrounding a tumour influences the evolution of this tumour in many cancers and especially ovarian cancer. In comparison with the surrounding environment of solid tumours, the malignant ascitic fluid accumulating in the peritoneal cavity during ovarian cancer progression constitutes a unique form of highly inflammatory environment. The ascitic fluid is constantly evolving with the evolution of the pathology and plays a major role in tumour progression, spheroid formation, tumour dissemination. Ascitic fluid is constituted of a high cell density fluid with high concentration of growth factors such as vascular endothelial growth factors (VEGF), transforming growth factors (TGFB, IL6, CXCL2). Dissemination of epithelial ovarian cancer is mediated through the ascitic fluid which is circulating around the ovaries and allow the detachment and the survival of ovarian cancer cells leading to the formation of spheroids, and then tumours, on secondary sites, notably on the peritoneal cavity epithelium (highly receptive to ovarian cancer seeding) which further lead to ovarian cancer metastasis to other organs when the epithelial ovarian cancer undergo epithelial-to-mesenchymal-transition through the peritoneal cavity. However during the dispersion from the primary site to the peritoneum, epithelial ovarian cancer cells must undergo adaptative changes in tumour to progress to the next step and most of the time only a fraction of cancer cells are circulating in the intraperitoneum before landing to the secondary site. This led us to develop a microfluidic model of the circulation of fluid surrounding a spheroid and assess the effect of selenium on the structure of the spheroid.

Improving mechanical properties investigation

Importantly, in vivo, tissue shaping, tissue repair and cancer invasion is done through collective movement of cells. This is possible thanks to the orientation of migration of the cells and made possible thanks to coordination between the cells that require the cytoskeleton. This coordination is regulated by multiscale process of mechanical changes within cells occurring at different timescales.

Following the monolayer mechanical properties analysis we performed, building a nanomechanical model on 3D spheroids would make these mechanical measurement closer to the tumoral reality. We applied a Cell-Tak coating as a thin layer on glass slides. The Cell-Tak is a polyphenolic protein from marine mussels which can bind to the sugar coating of the human cells in order to immobilize them (https://pubmed.ncbi.nlm.nih.gov/17779975/). The stiffness of the slide is greater than the spheroid allowing us to be sure we measure the modification of stiffness of the spheroid without parasite effect of the substrate. It also allows us to place securely the spheroid avoiding its movement while measuring. We have been able to assess the effect of selenite on SKOV-3 spheroids measuring a decrease of the overall stiffness of 5,000 cell spheroids. A decrease of the spheroid stiffness is interpreted as a decrease of the interactions between the cells leading to an erosion of the spheroid structure liberating single cells which are easier to treat than aggregates and are more prone to anoïkis due to lost interactions with other cells. These types of measurements are giving us insights on what would be the effect of intraperitoneal selenium treatment and further development might lead to combined treatment with other intraperitoneal injected drugs to kill cancer cells.

Finally combining AFM and confocal we would be able to draw the relationship between cell structure, cell mechanics and functions of those parameters. It would allow us to link changes in cell stiffness following selenium treatment and the metastatic potential of ovarian cancer cells. We would also be able to draw the relation between changes in nanomechanical properties and cell death or loss of viability.

Improving Ovarian Cancer detection

In order to improve the detection of ovarian cancer development, we would also improve our pilot study on copper isotopes. With only 11 paired serum-biopsy we haven't been able to find any correlation between the 𝛿Cu of serum and 𝛿Cu of the tumoral biopsies. Going further would involve increasing the number of tumoral biopsy samples paired with serum. Moreover we would track copper ratio in blood over time through treatment process and assess if the copper biomarker can detect relapse of cancer. Finally on a biochemistry part it would be interesting to decipher the mechanism underlying this shift of copper isotopes which remains hypothetical.

Figure 4 :

 4 Figure 4: Main post translational modification sites of the histones affecting epigenetics. Nucleosomes are composed of segments of 147 base pairs of DNA wrapped around a histone core, comprising of 2 copies of histones 2A, 2B, 3 and 4 57,58 . These 14kDa basic proteins are positively charged due to high numbers of lysine and arginine amino acids, enabling them to bind to the negatively charged DNA. The post translational modifications of the histone on the amino acids of their N-terminal end, also called tail, induce modifications in gene expression by altering chromatin structure and recruiting transcription factors. These modifications activate or inactivate transcription, induce chromosome packaging or recruit factors for DNA repair.

Figure 5 :

 5 Figure 5 : Enzyme responsible for histone tails methylation 144 Histones H3 and H4 are part of the nucleosomes and their N terminal domain (tails) can be modified through the activity of different methyltransferases using S-adenosylmethionine (SAM) methyl donor.

Figure 6 :

 6 Figure 6 : Histone lysine methylation 144 SAM methyl donor can transfer up to 3 methyl groups onto lysines or arginines in histone by methyltransferase enzymes (HMTs). The methyl group changes the interaction of the histone tails with the DNA and modulating the binding of transcription factor hence the DNA expression.

Figure 8 :

 8 Figure 8 : Dietary range of selenium in human diet199 Selenium is a nutrient with a narrow dietary range with a requirement of 100 to 200 µg of selenium per day. Under this intake selenium deficiency can cause diseases, notably Kashin-Beck disease which is a skeletal disorder due to necrosis of the growth plates of bones and joint cartilage. Above this dietary window, selenium may prevent cancer development up to 400µg per day however becomes toxic above this dose. Selenosis is an intoxication to selenium characterized by nail and hair loss which can lead to death.

Figure 11 :

 11 Figure 11 : Effect of selenium on cell death 250-253 Selenite triggers the production of ROS which leads to DNA, ER and mitochondrial damage triggering apoptosis. The accumulation of ROS can trigger the activation of Beclin-1 which triggers the autophagy in cells.

Figure 1 :

 1 Figure 1: SeNP accumulation in SKOV-3 and OVCAR-3 SeNP penetrate and accumulate in SKOV-3 and OVCAR-3 spheroids. SKOV-3 (A) and OVCAR-3 (B) cells spheroids were treated with BSA-SeNPs at sublethal doses for 24 h and imaged by TEM. Scale bars are displayed on the images (between 0.5 and 5µm). Loose contact between OVCAR-3 cells a high number of autophagosomes were observed. SeNP accumulation was observed in vesicles and mitochondria. SKOV-3 show limited accumulation of SeNPs. All images are representative of a minimum 3 biological repeats. In order to determine nanoparticle penetration, SKOV-3 cells were treated with FITC-tagged-SeNP-BSA (D) for 24 h. Confocal microscope (Ex 495nm / Em 521nm) imaging shows 50µm z-stacks of a 300µm diameter spheroid (scale bare 100µm). Local fluorescence was observed inside the spheroid demonstrating nanoparticle penetration.

Figure 2 :

 2 Figure 2: Effect of selenite and coated selenium nanoparticles on selenium related gene expression and ROS production in OVCAR-3 and SKOV-3 cells. Increases in expression of Trx1 (D) after 24 h of sublethal treatment with selenite or SeNPs demonstrated an expected effect of selenium on cells. Selenium treatments increased significantly (p<0.001) Trx1 RNA levels in SKOV-3 (Relative value of 0.01 control, 0.05 selenite and SeNP-BSA, 0.03 SeNP-chitosan) and OVCAR-3 (Relative value of 0.01 control, 0.06 selenite, 0.07 SeNP-BSA and 0.04 SeNP-chitosan).

Figure 3 :

 3 Figure 3: Ovarian Cancer Cytotoxicity in the presence of SeNP formulations. SKOV-3 (A) and OVCAR-3 (B) were grown as 5 x 10 3 cell spheroids for 24 h then treated with an increasing range of concentration (0 to 20 µg/mL) of selenite, BSA-SeNP or chitosan-SeNPs over 24 h and cellular cytotoxicity monitored. Both cell lines were treated for 24h with an. Cytotoxicity was evaluated by CellTiterGlo endpoint experiment. OVCAR-3 cells were more resistant to selenium treatment than SKOV-3 cells. Mean (+/-SD) relative to control luminescence values are shown from five independent experiments. In SKOV-3 relative Caspase 3 levels (C) were similar between control and selenium treated levels, 0.65 control, 0.68 selenite, 0.70 SeNP-BSA and 0.68 SeNP-chitosan. Selenite and SeNP-chitosan treatments caused significant levels (p<0.05) caspase 3 cleavage with levels of 0.29 and 0.23 detected respectively.

Figure 4 :

 4 Figure 4: Histone methylation markers in ovarian cancer cells treated with selenium and epigenetic probes.SKOV-3 (left) and OVCAR-3 (right) cells were grown as a monolayer then treated for 72 h with the epigenetic probe MRK740 (A), 24h with GSK343 (B) or 72h with A366 or UNC0642 (C) then 24 h with selenium treatments.

Figure 4A :

 4A Figure 4A: Cells were treated for 72 h with the epigenetic probe MRK740, an inhibitor of the H3K4 HMT PRDM9, followed by 24h with selenite or SeNPs. An inactive probe MRK740N was used as a control. Selenium treatment significantly (p<0.001) increased H3K4me3 levels in SKOV-3 by 2.5 fold.

Figure 4C :

 4C Figure 4C:Cells were treated for 24 h with epigenetic probes A-366 or UNC0642, inhibitors of the H3K9 HMT EHMT2/G9a followed by 24 h with selenite or SeNPs. No control probe was available for A-366 or UNC0642. Selenium treatments significantly (p<0.001) increased H3K9me2 levels by 2 fold in SKOV-3. In OVCAR-3 selenite and SeNP-chitosan increased levels by 2 fold (p<0.001) and 1.5 fold with SeNP-BSA treatment (p<0.01). In SKOV-3 UNC0642 and A-366 decreased levels of H3K9me2 to almost undetectable levels after 24 h and blocked any effect of selenium treatments. In OVCAR-3, UNC0642 and A-366 decreased levels of H3K9me2 (p<0.05), and reduced the ability of selenium treatments to increase H3K9me2 levels Figure 4D: Methyl transferase expression patterns in SKOV-3 and OVCAR-3 spheroids treated with sublethal doses selenium nanoparticles.Heatmap of methyltransferase(D left SKOV-3, right OVCAR-3) gene expression after 24h of selenium treatments of SKOV-3 and OVCAR-3 at sublethal doses.

Table 1 :

 1 Summary of the international staging system for ovarian carcinomas.

	Stage1	Limited to ovary or ovaries
	IA	One ovary, surface involvement or rupture
	IB	Both ovaries, surface involvement or rupture
	IC	Malignant ascites
	Stage II	Pelvic extension
	IIA	Involvement of the uterus or the fallopian tubes
	IIB	Involvement of the other pelvic organs (bladder, rectum)
	Stage III	Involvement of the upper abdomen or lymph nodes
	IIIA	Microscopic peritoneal metastases outside pelvis
	IIIB	Macroscopic peritoneal metastases , 2cm diameter
	IIIC	Macroscopic peritoneal metastases >2cm diameter
	Stage IV	Distant organ involvement
	IVA	Pleural effusion with positive cytology
	IVB	Metastases to extra-abdominal sites

Development of a new blood-based biomarker

Stable and radioactive isotopes have been used in earth science in numerous fields (paleoclimate, paleocirculation, chemical evolution of earth, pollution). Recent improvements of Multi-Collector Inductively Coupled Plasma Mass Spectrometry (MC-ICP-MS), thermic ionization (TIMS) and isotopic ratio mass spectrometry (IRMS) allowed new measurements of small isotopic elemental variations. While those techniques were common in archaeology, they have recently been used in human and animal medicine

Table 2 96

 2

	é			ù
	ë ê ê	(	) ref	û ú ú	x10 3

Cu isotope composition in blood and bones of human samples Cu isotope data were obtained by MC-ICP-MS and expressed as 𝛿 65 Cu (‰) notation with a comparison between 65 Cu/ 63 Cu ratio of the samples with the ratio of the Cu standard solution and calculated by the formula : ¶ 65 Cu= 65 Cu/ 63 Cu ( ) sample -65 Cu/ 63 Cu ( ) ref 65 Cu/ 63 Cu

Table 3 :

 3 In vitro and in vivo nanocarrier development in ovarian cancer models (adapted from 126 ).

	Passive	System	Drug	Description	Model
	targeting	Nanoparticles Cisplatin	Poly ethylene glycol (PEG) based polymer SKOV-3
	nanocarriers		Paclitaxel		A2780
					Female
					athymic
					mice
		Polymeric	Cisplatin	Chitosan	A2780
		nanocarriers	Paclitaxel
		Lipid based	Cisplatin	Phosphatidylcholine, and cholesterol PEG	OVCAR-3
		nanocarriers	Paclitaxel		A2780
			Doxorubicin		SKOV-3
		Polymeric	Paclitaxel	Pegylated liposomes, poly-ethylene based,	CAOV-3
		micelle	Doxorubicin	polystyrene based	SKOV-3
		Nanocapsule Cisplatin	DOPC based or alginic core shell	OVCAR-3
			Paclitaxel		Female nude
					mice
		Dendrimer	Cisplatin	Polyamidoamine dendrimers	SKOV-3
			Paclitaxel		A2780,
					female
					athymic
					nude mice
		Hydrogel	Paclitaxel	Hyaluronic acid based hydrogel	SKOV-3,
					Female
					BALB/cmice
		Polymer-drug	Doxorubicin	PolyL-lysine citramide and DOX copymer	SKOV-3,
		conjugate			Female
					BALB/cmice

Active targeting nanocarriers Targeted receptor Carrier system Description Model

	Folate	AuNP	PEG conjugated AuNP	SKOV-3
	receptor	Liposome	Phosphatidylcholine-PEG-cholesterol
		NP	Folicacid-PEG-chitosan	OVK18
			Paclitaxel-glucose	OVCAR-3
	Luteinizing	Nanogel	PEG based + cisplatin	A2780
	hormone	Magnetic NP	Iron-platinum-PEG copolymer
	releasing
	hormone
	receptor
	HER2 rceptor Polymeric NP	Paclitaxel loaded poly lactic acid PEG NP	SKOV-3
		Dendrimers	Polyamidoamine dendrimers	OVCAR-3
	Transferrin	Micelles	Tf PEG-PE micelles	A2780
	receptor
	Integrin	NP	Cyclic pentapeptide containing gemcitabine	SKOV-3
	receptors		hydrochlorine
	CA125	Liposome	Chain of anti CA25 fused to streptavidin	OVCAR-3
	Angiogenesis NP	SilicaNP loaded with candesartan	SKOV-3
	Magnetic NP NP	Carboplatin Fe3O4NP	A2780
	SKOV-3, A2780, OVCAR-3, CAOV-3 cited in the table are commercial ovarian cancer cell
	lines.

Table 4 :

 4 Nanocarriers under clinical trials (from searchtrials.com) When typing Nanoparticles in Ovarian Cancer in clinicaltrials.gov, we summed up the actual drugs in test in clinical trial.

	Name	Chemotherapeutic agent	Description	Number patients of	Clinical phase trial	Goal / Effect
	PIPAC	Paclitaxel	Albumin (nab) shell	Recruiting Phase 1	Single-agent activity and
			covering paclitaxel			a favourable toxicity
			Pressurized			profile
			intraperitoneal aerosol
	Paclitaxel-	Intraperitoneal	Albumin shell	27	Phase 1	Determine the potential
	Albumin	injection of	covering paclitaxel			pharmacokinetic
	Stabilized	Paclitaxel				advantage of the
	NP					nanoparticles
						Determine the favorable
						ratio of nab-paclitaxel
						(Abraxane)
						concentration in the
						peritoneal cavity vs.
						plasma
	Paclitaxel-	Blood infusion of	Albumin shell	51	Phase 2	30% of reduction of
	Albumin	Paclitaxel	covering paclitaxel			tumour size
	Stabilized
	NP
	CriPec	Docetaxel	Polymeric NP loading	27	Phase IIa	Inhibit VEGF, cell cycle
			docetaxel (analogue of		in Pt-	arrest in G2/M, Inhibit
			paclitaxel)		resistant	microtubule
					patients	disassembly.
	IMX10	Curcumin	Curcumin Doxorubicin	70	Phase 1	Coupling the role of
		Doxorubicin	encapsulated NP			curcumin as a signal
						transducer, activator of
						transcription Stat3,
						NFkB to doxorubicin
						which is antineoplasic
	EGEN-	Doxorubicin	IL12 based	16	Phase 1	Stimulate immune
	001		immunotherapeutic			system, stop tumour
			coupled with pegylated			growth
			liposomal doxorubicin			Increased cell death in
						comparison with
						separated drugs
	CRLX101 Bevacizumab	Camptothecin in	63	Phase 2	Would inhibit together
			nanoparticle (structure			distinct step along HIF->
			not detailed)			CAIX->VEF-VEGFR2
						pathway.
						CRLX101 inhibits
						HIF1α hypoxia
						inductible transcription
						factor and HIF1α
						associated resistance to
						VEGFR inhibitors.
	9-ING41		Single agent therapy	350	Phase 2	GSK3b inhibitor

Table 2 : Main effects of different dietary forms of selenium on cancer cell lines and in vivo models.

 2

	Cells		Selenium form	Dose	Effect		Ref
	PC3	Prostate	Methylseleninic	10µM for 12h	Reducing DNA damage	221,222
	cancer		acid
	LNCaP Prostate	Selenite	1µM
	Cancer
	Whole blood	Selenite	30nM
	Mouse		Selenomethionine	10µM 15h	Improving DNA repair	234-236
	embryonic
	fibroblasts
	Human			10µM 24h
	fibroblast
	LNCap Prostate	Selenite	30nM 72h
	cancer
	MCF7 Breast		30nM 72h
	cancer
	Human Blader	Selenite	10µM 12h	Increasing DNA damage	225,226
	Cancer Cell
	Glioma cells		7µM 24h
	Rat pancreatic	Selenite	30nM 1 to 6	Reduction of oxidative stress by	223-225
	islets			days	increase	of	selenoproteins
	Prostate cancer		1.5µM 6h	expression
	LnCaP
	Glioma cells		1µM 24h
	Glioma cells	Selenite	7µM 24h	Induce ROS production	225,226
	Human bladder
	cancer cells		10µM 12h	Mitochondria damages
	RT-112
	Prostate cancer	Selenite	1µM	Alteration of DNA methylation for	213,217
	PC3 LNCaP and			tumour suppressor genes
	Du145 cells
	DU145 prostate	Selenomethionine		Cell cycle arrest in G1	227-229
	cancer
	HT1080				Cell cycle arrest in G2
	Fibrosarcoma	Methylseleninic	5µM	Altering functions of cyclins (C,
	cells		acid		D1 cyclin-dependent kinases (1, 2,
	TM6 mammary			4) and protein kinases AKT
	hyperplastic
	epithelial cell
	SW982		Selenite	10µM	Induce apoptosis	230-233
	Synovial
	sarcoma			2.5µM 5d
	LNCaP Prostate
	cancer
	OVCAR-3		10µM 72h
	Ovarian cancer
	Glioblastoma		5µM 24h
	RT-112 Blader	Selenite	10µM 24h	Necroptosis		226,237
	cancer
	PC3	Prostate
	cancer
	Glioma cells	Selenite	7µM 12h	Autophagy

Table 3 :

 3 2000 to 2020 selenium nanoparticle preparations and effect in cancer cells.

	NP Type	Size	Shell	Model	Outcomes	Ref
				HeLa cercical
	SeO2 in vitamin C	NC	None	cancer cells breast cancer MDA-MB-231	Cell cycle arrest in S phase	296
				cells
				MCF-7 breast	SeNP induce cell death
	Nano Se	40 to 90nm	None	MDA-MB-231 cancer (ERα+)	ERα+ cells. Scavenging expression Bax/Cytc in	297
				(ERα-)	ROS
	Nano Se	5 to 200nm	BSA	In vitro ROS scavenging	Better lower size NP free scavenging efficiency for radical	274,279
	Nano Se	36 to 90nm	BSA	GST/GPx activity measurement in	Size effect of NanoSe as chemopreventive	278
				mice liver/blood
					Lower	toxicity	than
	Nano Se	20-60nm BSA	Mice	increase selenomethionine selenoenzyme with	272
					producton
	Folic modified SeNPs Acid	70nm	Folic Acid	MCF-7 breast cancer cells	Antiproliferative, apoptosis Mitochondria-dependent	287
	Light and heavy Chitosan on synthesized SeNP	50 to 103nm	Chitosan	BABLC-3T3 skin cells cells Caco2 viscera	Inhibit ROS Production Chitosan stabilize NP (GPx increase).	276
	Sialic coated SeNPs Acid	70-170nm	Sialic Acid	cancer cells HeLa Cervical	uptake Apoptosis and increased	298
				MCF-7 breast
				HepG2
	Transferrin conjugated doxorubicin loaded SeNP	130nm	Transferrin Doxorubicin (DOX)	hepatocellular carcinoma umbilical vein HUVEC A375 melanoma	Cytotoxicity ROS overproduction) against apoptosis (p53 activation, cancer cells through	256
				endothelial
				cancer cells
	5FU functionalized surface SeNP	70nm	fluorouracil 5-	melanoma A375 human	subG1 arrest apoptosis Mitochondrial disruption damages (casp9) due do DNA	269,299
				A549 lung
	Paclitaxel charged SeNP	74nm	Paclitaxel	MCF7 breast HeLa cervical HT29 colon	G2/M arrest apoptosis (casp3) Mitochondrial disruption
				cancer cells

Table 1 Sample Over-representation GO biological processes

 1

	Fold
	Enrichment	FDR
	(log value)

  Serial dilutions of cDNA were used to plot a calibration curve, and gene expression quantified by plotting threshold cycle values. Expression levels

	GTCCACTGGCGTCTTCAC, Reverse: CTTGAGGCTGTTGTCATACTTC) and TrxR1
	(TrxR1	Forward:	CTACAGACCATTGCCTTGCT,	Reverse:
	ACCTCCTACCCACAAGATCC).
	(CellSignal 4658, UK), H3 (1B1B2): mouse monoclonal (CellSignal 14269, UK) or GAPDH:
	mouse monoclonal (Santa Cruz sc-47724, UK)) at a concentration of 200 μg/ml overnight, at
	4°C. Blots were washed, then incubated with the appropriate secondary antibodies (goat anti-
	mouse Abcam ab150113 or goat anti-rabbit Abcam ab6721 HRPsecondary, UK) at a
	concentration of 400 μg/ml. Cross-reacting proteins were visualised (ChemiDoc XRS, BioRad,
	UK), and band intensities quantified using ImageLab software normalising expression to
	GAPDH.
	qPCR
	Following RNA extraction and quantification, qPCR was carried out in accordance with the
	manufacturers' recommendations, using the RETROscript® kit two-step method (Invitrogen

Ltd., UK). Following cDNA synthesis from 100 ng of RNA, each sample was analysed by qPCR in triplicate using iQ SYBR Green supermix (BioRad, UK) and gene specific primers (Sigma-Aldrich, UK) to evaluate different gene expression GAPDH (GAPDH Forward:

  Cu concentration in tumours, was observed. Measuring the temporal evolution of isotopic composition in blood demonstrated that a rapid shift in 𝛿[START_REF] Coceano | Investigation into Local Cell Mechanics by Atomic Force Microscopy Mapping and Optical Tweezer Vertical Indentation[END_REF] Cu corresponded to a more advanced tumour, and correlated with CA15-3 (MUC1 blood concentration) levels in breast cancer patients100 . Decreases in 𝛿 65 Cuserum by 0.14 ¶ 65 Cu= 65 Cu/ 63 Cu

	é ë ê ê	(	) sample -65 Cu/ 63 Cu ( ) ref 65 Cu/ 63 Cu ( ) ref	ù û ú ú	x10 3

Acknowledgments

Acknowledgments This project received financial support from Welsh Government ERDF SMART Expertise 2014-

I would like to thank the Université

2020 West Wales and the Valleys Grant CEAT (Cluster for Epigenetics and Advanced Therapeutics, 2017/COL/004); the Medical Research Council UK Confidence in Concept grant (MC_PC_19053) s, and INSERM Grant SEDMAC (PC201607). Benoit Toubhans received a scholarship co-funded by the Université Grenoble Alpes and Swansea University. TEM images: The electron microscope image was supported by the Rhône-Alpes Region, FRM, FEDER, CNRS, CEA, the University Grenoble Alpes, EMBL, GIS-IBISA, the Grenoble Instruct-ERIC Centre (ISBG : UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB) platform (D. Fenel , C. Moriscot, B. Gallet and G. Schoen (PSB, ISBG, UMS 3518)).

II. Article

This paper is Published in Journal of Trace Elements in Medicine and Biology https://www.sciencedirect.com/science/article/pii/S0946672X20301760 from Ovarian Cancer Patients. Cancer Res. 2004, 64 (18), 6476-6481. https://doi.org/10.1158/0008-5472.CAN-04-1529. (25) Wei, S. H.; Balch, C.; Paik, H. H.; Kim, Y.-S.; Baldwin, R. L.; Liyanarachchi, S.; Li, L.; Wang, Z.; Wan, J. C.; Davuluri, R. V.; Karlan, B. Y.; Gifford, G.; Brown, R.; Kim, S.; Huang, T. H.-M.; Nephew, K. P. Prognostic DNA Methylation Biomarkers in Ovarian Cancer. Clin Cancer Res 2006, 12 (9), 2788-2794. https://doi.org/10.1158/1078-0432.CCR-05-1551. (26) Lengyel, E. Ovarian Cancer Development and Metastasis. Am. J. Pathol. 2010, 177 (3), 1053-1064. https://doi.org/10.2353/ajpath.2010.100105. (27) Tan, D. S.; Agarwal, R.; Kaye, S. B. Mechanisms of Transcoelomic Metastasis in Ovarian Cancer. The Lancet Oncology 2006, 7 (11), 925-934. https://doi.org/10.1016/S1470-2045(06)70939-1. (28) Burleson, K. M.; Boente, M. P.; Pambuccian, S. E.; Skubitz, A. P. N. Disaggregation and Invasion of Ovarian Carcinoma Ascites Spheroids. J Transl Med 2006, 4, 6. https://doi.org/10.1186/1479-5876-4-6. (29) Ahmed, N.; Stenvers, K. Getting to Know Ovarian Cancer Ascites: Opportunities for Targeted Therapy-Based Translational Research. Front. Oncol. 2013, 3. https://doi.org/10.3389/fonc.2013.00256. (30) Moffitt, L.; Karimnia, N.; Stephens, A.; Bilandzic, M. Therapeutic Targeting of Collective Invasion in Ovarian Cancer. International Journal of Molecular Sciences 2019, 20 (6), 1466. https://doi.org/10.3390/ijms20061466. (31) Patel, I. S.; Madan, P.; Getsios, S.; Bertrand, M. A.; MacCalman, C. D. Cadherin Switching in Ovarian Cancer Progression. International Journal of Cancer 2003, 106 (2), 172-177. https://doi.org/10.1002/ijc.11086. (32) Elloul, S.; Silins, I.; Tropé, C. G.; Benshushan, A.; Davidson, B.; Reich, R. Expression of E-Cadherin Transcriptional Regulators in Ovarian Carcinoma. Virchows Arch 2006, 449 (5), 520-528. https://doi.org/10.1007/s00428-006-0274-6. (33) Yeung, T.-L.; Leung, C. S.; Yip, K.-P.; Au Yeung, C. L.; Wong, S. T. C.; Mok, S. C. Cellular and Molecular Processes in Ovarian Cancer Metastasis. A Review in the

II. Article

This paper is Under Review in Redox Biology (19/11/2020) Selenium nanoparticles induce global histone methylation changes in ovarian cancer cells.

Benoit Toubhans 1,2 , Marcos Quintela 1 , Deyarina Gonzalez 1 , Alexandra T. Gourlan methyltransferase. Cells were then treated for 48 h with selenite or SeNP at sub-lethal doses, protein was extracted and probed using antibodies targeting specific histone modifications as described above.

Statistical Analysis.

All data presented are from a minimum of three biological repeats, with technical repeats included per sample, as denoted. Data normality was analysed using the Kolmogorov Smirnov test, with normally distributed data analysed with the one-way and two-way analysis of variance (ANOVA) followed by the Mann-Whitney pairwise test for non-parametric data. In all cases in which ANOVA was significant, multiple comparison methods were used. Differences were considered significant for P ≤ 0.05 (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). All data were analysed in MiniTab 14.

Supplementary

Supplementary 1: TEM images of SKOV-3 (A) and OVCAR-3 (B) treated with IC20 of SeNPchitosan Supplementary 2 : Heatmap of methyltransferase and demethylases (A and C SKOV-3, B and D OVCAR-3) gene expression after 24h of selenium treatments at sublethal doses.

Author contribution Data collection, data analysis and manuscript drafting was performed by Benoit Toubhans. Marcos Quintela and Philipp Rathert analysed the RNA sequencing dataset. R Steven Conlan, and Lewis W Francis contributed to study design, critical interpretation of data and drafting of the manuscript. Deyarina Gonzalez, Laurent Charlet and Alexandra Gourlan contributed to the review of the manuscript. The authors read, amended and approved the final manuscript.