Keywords:

Acknowledgement

As I reach the culmination of my academic journey with this thesis, I would like to express my profound gratitude to the following individuals and entities who have played a pivotal role in my PhD journey.

First and foremost, I would like to praise and thank GOD, the almighty, who has granted me countless blessings, strength, perseverance, knowledge, and opportunities so that I have been finally able to accomplish my thesis.

I sincerely thank Pr. Georges Herbein, my supervisor, for providing me with the opportunity to pursue the PhD under his supervision. I'm beyond grateful for the exceptional support, guidance, presence, and knowledge you've shared with me during my thesis research. Your mentorship has been an invaluable and transformative part of my academic experience. Your supportive input and constructive feedbacks propelled me forward, broaden my knowledge, and challenged me to strive highest standards. Not to forget your presence, both in terms of physical presence during meetings and your willingness to be available when needed, has made a world of difference.

My gratitude knows no bounds for Pr. Mona Diab-Assaf. I appreciate her recommendation to Pr. Georges Herbein, which led to my acceptance as a PhD candidate in his laboratory. Throughout this journey, she offered continuous positive reinforcement and support. I am extremely thankful to the members of my PhD jury including Pr. Olivier Adotevi, Pr. Carine Van Lint, and Pr. Christian Schwartz. I am well aware of the time you invested in reading my manuscript, providing feedback, and offering your expertise which significantly enhanced the quality of this research.

I am indebted to Apex Biosolutions company and the Hariri Foundation for Sustainable Human

Development for their financial assistance that contributed to the successful completion of my PhD.

I extend my heartfelt thanks to my lab colleagues (Sébastien, Zeina, Sandy, Jihan, and Fidaa) for being an essential part of my academic journey. A special thanks to Sébastien who facilitated my integration into the lab environment and acquainted me with various laboratory methods. Thank you for your willingness to share your scientific knowledge and experience with me. I also deeply acknowledge the support of Jihan and Fidaa. I'm thankful for their friendship and encouragement. To all my lab colleagues, may your paths ahead be filled with success, growth, and endless opportunities. Here's to each of you and the bright futures you're destined to create. Here's to your ambitions! To my guardians, "Baba Yehya" and "Mama Ghada", thank you for being the pillar of my PhD journey. You have provided me with strength during life's triumphs and tribulations; you amplified the joy during my successes. Knowing that I can count on you, no matter what, has been a priceless gift. I am forever grateful for your irreplaceable presence in my life. To my gems, my brother "Zakaria" and my sister "Wafaa", your love, support, empathy, listening ear, and words of encouragement have been a source of solace and strength. My love for all of you goes beyond what words can convey. I truly cherish our bond and I love you all! My deepest gratitude goes for my big family (aunts, uncles, and cousins) for their unwavering love and support. Their presence and encouragement have been the cornerstone of my success.

Finally, I would like to extend my sincere gratitude to my close friends who reside in distant corners of the world 'Lebanon, France, Dubai, Canada, Germany, and America'. Although miles apart, your care packages, video calls, love, moral support, and encouragement have been an invaluable source of strength throughout my academic journey. Your friendship has transcended borders and time zones. Your friendship is a treasure that I will forever hold close to my heart.

Abstract

On a worldwide scale, breast cancer, glioblastoma, and ovarian cancer present a significant health obstacle, being responsible for cancer-related fatalities. Cancer etiological factors are assorted into genetic or environmental risk factors of which viruses are estimated to contribute to 20% of all cancer cases. Human cytomegalovirus (HCMV) is a herpesvirus that infects a substantial portion of the global population, ranging from 40% to 95%. The variability in the HCMV genome may contribute to its oncomodulatory potential, as it can promote the initiation and dissemination of cancerous cells. Recently, increasing attention has been directed towards the oncogenic role of HCMV, particularly high-risk HCMV strains that can directly induce transformation in primary cells. Polyploid giant cancer cells (PGCCs), have been definitively shown to exhibit cancer stem cells (CSCs) characteristics. These PGCCs give rise to descendant cells through asymmetric division, the latter express markers associated with epithelial-mesenchymal transition (EMT), which in turn promotes invasion and migration. Research indicated that PGCCs generation can be triggered by anti-cancer therapies. PGCCs presence has been particularly linked to cases with poor prognosis, contributing to tumor relapse and resistance to therapy. Previous investigations by our group have highlighted the presence of PGCCs containing HCMV in cancer biopsies, establishing a noteworthy correlation between the presence of PGCCs and HCMV.

HCMV has been implicated in oncogenesis, particularly in breast cancers, glioblastoma (GBM), and epithelial ovarian cancer. Herein, we investigated the oncogenic potential of HCMV in various cancer contexts and explored the molecular mechanisms and transforming capabilities of different HCMV strains isolated from these distinct cancer types and their impact on patient outcomes. To start with breast cancer, we assessed HCMV-B544 and B693 strains, isolated from EZH2HighMycHigh triple-negative breast cancer (TNBC) biopsies. These strains transformed human mammary epithelial cells (HMECs) resulting in CMV-Transformed-HMECs (CTH cells) and PGCCs displaying stemness phenotype. Notably, HCMV persists in long-term cultures, indicating sustained viral replication with alternance of lytic and latent states. In glioblastoma, HCMV was explored as a reprogramming vector, dedifferentiating mature human astrocytes into CMV-Elicited Glioblastoma Cells (CEGBCs) with glioblastoma-like traits. HCMV influences the transformation, invasion, and spheroid formation processes, correlating with increased EZH2 and Myc expression in GBM biopsies. Eleven clinical HCMV strains isolated from GBM tissues further validated their role in glioblastoma oncogenesis. These CEGBCs exhibit sensitivity to therapeutic interventions, including an EZH2 inhibitor, ganciclovir, and temozolomide triple therapy, offering potential avenues for glioblastoma treatment. In the context of high-grade serous ovarian carcinoma (HGSOC), HCMV-DB and BL high-risk strains transformed ovarian epithelial cells (OECs) into "CMV-transformed Ovarian cells" (CTO). HCMV infection was associated with EZH2 upregulation and the presence of PGCCs with cancer stem cell-like properties expressing EMT markers. From HGSOC biopsies, we isolated three clinical HCMV strains. The high-risk strains transformed OECs resulting in CTO cells that displayed proliferative capacities and revealed an increase in EZH2 levels, along with the generation of PGCCs. Notably, the observed features were curtailed upon the inhibition of EZH2.

All in all, this study emphasizes the diverse oncogenic potential of HCMV in breast cancer, glioblastoma, and ovarian cancer, shedding light on molecular mechanisms, transformation processes, and targeted therapies that may improve the overall survival of cancer patients.

Keywords : Human Cytomegalovirus, High-risk strains, oncogenesis, PGCCs, Myc, EZH2, CTH cells, CEGBCs, CTO cells, breast cancer, glioblastoma, high-grade serous ovarian carcinoma.

Résumé

Le cancer du sein, le glioblastome et le cancer de l'ovaire représentent un problème majeur de santé publique. Les facteurs étiologiques du cancer se répartissent en facteurs de risque génétiques ou environnementaux, parmi ces derniers les virus contribueraient à environ 20% de tous les cas de cancer. Le cytomégalovirus humain (HCMV) est un virus herpès qui infecte une part importante de la population mondiale, avec une prévalence de 40% à 95%. La variabilité génomique du HCMV pourrait contribuer à son potentiel oncomodulateur, car il semble accélérer les mécanismes d'oncogenèse. Les cellules cancéreuses géantes polyploïdes (PGCC) ont des caractéristiques semblables à celles des cellules souches cancéreuses (CSC) et donnent naissance à des cellules « filles» lesquelles expriment des marqueurs associés à la transition épithélio-mésenchymateuse (TEM). La présence de PGCC est particulièrement observée dans les cancers de mauvais pronostic.

De précédentes études de notre groupe ont mis en évidence la présence de PGCC infectées par le HCMV dans les biopsies de cancer du sein, évoquant ainsi un rôle oncogène direct du HCMV, notamment de certaines souches virales « à haut risque ». Le HCMV a été suggéré comme participant à l'oncogenèse, en particulier dans les cancers du sein, le glioblastome et le cancer épithélial de l'ovaire. Notre travail de thèse a consisté à examiner le potentiel oncogène du HCMV dans ces cancers. Nous avons évalué les souches virales HCMV-B544 et B693, isolées de biopsies de cancer du sein triple négatif EZH2 High Myc High . Ces souches virales transforment les cellules épithéliales mammaires humaines (HMEC), donnant naissance à « des cellules HMEC transformées par le CMV » (cellules CTH) d'une grande hétérogénéité cellulaire comprenant notamment des PGCC présentant des traits de cellules souches. Une persistance du HCMV est notée dans les cultures à long terme, indiquant une réplication virale continue avec alternance de phase lytique et de latence. Dans le glioblastome, le HCMV a été étudié en tant que vecteur de reprogrammation, dédifférenciant les astrocytes humains matures en « cellules glioblastomateuses induites par le CMV » (cellules CEGBC) présentant des caractéristiques similaires aux cellules de malades. Le HCMV favorise la transformation cellulaire, l'invasion et la de formation de sphéroïdes, corrélé avec une augmentation de l'expression d'EZH2 et de Myc observée également dans les biopsies de glioblastome provenant de malades. Onze souches cliniques de HCMV ont été isolées à partir de biopsies de glioblastome de malades qui reproduisent la transformation d'astrocytes normaux en cellules glioblastomateuses suite à l'infection virale. Ces cellules CEGBC sont sensibles aux interventions thérapeutiques, notamment à un inhibiteur d'EZH2, au ganciclovir et à une triple thérapie incluant temozolomide-inhibiteur d'EZH2-ganciclovir, offrant ainsi des perspectives nouvelles pour le traitement de ce cancer de très mauvais pronostic. Concernant le cancer séreux épithélial de l'ovaire de haut grade (HGSOC), les souches « à haut risque » HCMV-DB et de BL transforment les cellules épithéliales ovariennes (OEC) en "cellules ovariennes transformées par le CMV" (cellules CTO). Dans ces cellules CTO une augmentation de l'expression d'EZH2 ainsi que la présence de PGCC avec des traits de cellules souches cancéreuses avec TEM ont été observées. À partir de biopsies de HGSOC, nous avons réussi à isoler trois souches cliniques de HCMV qui en infectant les cellules OEC donnent naissance à des cellules CTO hautement prolifératives surexprimant EZH2 et générant des PGCCs ; leur transformation étant réduite par les inhibiteurs d'EZH2 in vitro. En résumé, notre travail met en évidence le potentiel oncogène du HCMV dans trois cancers graves, en dévoilant certains des mécanismes moléculaires mis en oeuvre lors de la transformation cellulaire et ouvre la voie à des thérapies ciblées qui pourraient améliorer la survie des patients.

Mots clés: Cytomégalovirus humain, souches virales à haut risque, oncogenèse, PGCCs, Myc, EZH2, cellules CTH, CEGBCs, cellules CTO, cancer du sein, glioblastome, cancer séreux épithélial de l'ovaire de haut grade Table 1:

List of Figures

List of Tables

gL:

Glycoprotein L gO:

Glycoprotein O GRIM-19:

Gene associated with retinoid/interferon-induced mortality-19 GSCs:

Glioblastoma stem cells HAs:

Human astrocytes HBV:

Hepatitis B virus HBx:

Hepatitis B virus X protein HCC:

Hepatocellular carcinoma HCMV:

Human Cytomegalovirus HCV:

Hepatitis C virus HDACs:

Histone deacetylases HGSIL:

High-grade squamous intraepithelial lesions HGSOC:

High-grade serous ovarian cancer HHV-5:

Human Herpesvirus 5 HIF:

Hypoxia-inducible factor HLTF:

Helicase like transcription factor HMECs:

Human Mammary Epithelial Cells HMTs:

Histone methyltransferases HPV:

Human papillomavirus HR:

High-risk HSCT:

Hematopoietic stem cell transplantation HSV:

Herpes simplex virus hTERT:

Human telomerase reverse transcriptase HTLV-1:

Human T-cell lymphotropic virus-1

ICAM-1:

Intercellular adhesion molecule 1 ICIs:

Immune checkpoint inhibitors ICs:

Intermediate cells IE:

Immediate-early IRS/TRS:

Internal repeat short/terminal repeat short ITAM:

Immunoreceptor tyrosine-based activation motif JCPyV:

John Cunningham Polyomavirus KICS:

KSHV-associated inflammatory syndrome KS:

Kaposi sarcoma KSHV:

Kaposi's sarcoma-associated herpesvirus L:

Late LAcmvIL-10:

Latency-associated viral homolog of IL-10 LAMP1:

Lysosome-associated membrane protein 1 LANA:

Latency-associated nuclear antigen LCs:

Large tetraploid cells LDs:

Lipid droplets LHBs:

HBV large surface protein LMP1:

Latent membrane protein 1 lncRNA HOTAIR: LncRNA HOX antisense intergenic RNA lncRNAs:

Long non-coding RNAs

LR:

Low-risk LTAg:

Large T antigen LUNA:

Latent unique nuclear antigen MAPK:

Mitogen-activated protein kinase MCC:

Merkel cell carcinoma MCD: Multicentric Castleman's disease MCP-1:

Monocyte chemoattractant protein-1 MCPyV:

Merkle Polyomavirus

MES:

Mesenchymal MGMT:

O (6)-methylguanine DNA methyltransferase MHC:

Major histocompatibility complex MIEP:

Major immediate early promoter miR34:

MicroRNA MOI:

Multiplicity of infection NCAM:

Neural cell adhesion molecule ND10:

Nuclear domain 10 NFκB:

Nuclear factor kappa B NIEPs:

Non-infectious enveloped particles NOS:

Nitric oxide synthase NPC:

Nasopharyngeal carcinoma NPC:

Neural progenitor cell OC:

Epithelial ovarian cancer OECs:

Ouman ovarian epithelial cells PBMCs:

Peripheral blood mononuclear cells PCR:

Polymerase chain reaction PD-1:

Programmed cell death protein 1 PD-L1:

Programmed death ligand 1 PEL:

Primary effusion lymphoma PGCCs:

Polyploid giant cancer cells

PI:

Post-infection PI3k:

Phosphatidylinositol 3-kinase Plin4:

Perilipin 4 Plk1:

Polo-like kinase 1 PML:

Promyelocytic leukemia protein PN: Proneural PRC2:

Polycomb repressive complex 2 PTX: Paclitaxel qPCR:

Real-time quantitative PCR RANTES:

Regulated on activation normal T expressed and secreted Rb:

Retinoblastoma protein RNA CLIP:

RNA Cross-linking Immunoprecipitation

RT-PCR:

Reverse transcriptase PCR RT-qPCR:

Quantitative Reverse Transcription PCR SCs:

Small cells

SOT:

Solid organ transplantation sTAg:

Small T antigen STAT-3:

Signal transducer and activator of transcription 3 SV40:

Simian virus 40 TAMs:

Tumor-associated macrophages TGF-β:

Transforming growth factor-β TME:

Tumor microenvironment TMZ:

Temozolomide TNBC:

Triple-negative breast cancer TNFR1:

Tumor necrosis factor receptor 1 TRAIL:

TNF-related apoptosis-inducing ligand TRL/IRL:

Terminal repeat long/internal repeat long TUSC3:

Tumor suppressor candidate 3 UL:

Unique long UNITE:

Universal intracellular targeted expression US:

Unique short VEGF:

Vascular endothelial growth factor vFLIP :

Viral FLICE-inhibitory protein VGCV:

Valganciclovir vIRF-3:

Viral interferon regulatory factor 3 vMIA:

Viral mitochondria-localized inhibitor of apoptosis VZV:

Varicella-zoster virus WHO:

World Health Organization γH2AX:

Gamma histone variant H2AX Lymphocryptovirus, is associated with infectious mononucleosis and various cancers, and Kaposi's sarcoma-associated herpesvirus (KSHV) or (HHV8), belongs to genera Rhadinovirus [START_REF] Grinde | Herpesviruses: latency and reactivationviral strategies and host response[END_REF][START_REF] Roizmann | The familyHerpesviridae: an update[END_REF]. Both viruses are classified as oncogenic pathogens that infect lymphoblastoid cells such as T or B lymphocytes and establish latency. However, it's worth noting that a lytic infection has also been observed in epithelioid and fibroblastic cells [START_REF] Roizmann | The familyHerpesviridae: an update[END_REF].

One of the numerous breakthroughs and advancements documented during the 20th century involved the identification and characterization of HCMV, which stands as one of the most prevalent viruses and opportunistic pathogens encountered worldwide. Within this discovery, several specific dates stand out [START_REF] Ho | The history of cytomegalovirus and its diseases[END_REF][START_REF] Boeckh | Cytomegalovirus: pathogen, paradigm, and puzzle[END_REF]. In 1881, the German pathologist Ribbert documented the presence of cells containing inclusions in the kidney of a stillborn infant affected by syphilis and in the parotid gland of an infant. These same "protozoan-like" cells were also observed by Jesionek and Kiolemenoglou in 1904 in the lungs, kidneys, and liver of an Significantly, the mid-1950s saw a breakthrough with the advent of human cell culture techniques, enabling the isolation of a virus that was later identified as cytomegalovirus [6]. This achievement was the result of independent efforts by three different laboratories: Smith in 1956, Rowe and colleagues in 1956, and Weller and colleagues in 1957. It was subsequently named 'cytomegalovirus' (CMV) [7,8]. This marked a significant milestone in advancing our comprehension of various aspects of the virus, including but not limited to epidemiology, structure, life cycle, replication, latency, pathogenesis, and treatment modalities. These advancements were greatly facilitated by the development of animal models for studying CMV's pathogenesis [START_REF] Crawford | Humanized mouse models of human cytomegalovirus infection[END_REF].

HCMV Genome

HCMV stands out as having the largest genome among human herpesviruses, spanning a length of 235 kilobases (kb) [START_REF] Dolan | Genetic content of wild-type human cytomegalovirus[END_REF]. This genome comprises a linear double-stranded DNA (dsDNA) helix, with more than 751 translated open reading frames (ORFs) [11,[START_REF] Ye | Functional Profile of Human Cytomegalovirus Genes and Their Associated Diseases: A Review[END_REF]. Furthermore, HCMV produces polyadenylated non-coding RNAs, including four notable long non-coding RNAs (lncRNAs), notably RNA2.7, RNA1.2, RNA4.9, and RNA5.0 [START_REF] Gatherer | Highresolution human cytomegalovirus transcriptome[END_REF]. Additionally, it generates nonpolyadenylated RNAs, such as microRNAs, which play pivotal roles in various processes like regulating host cell metabolism, immune evasion, and the maintenance of latency [START_REF] Van Damme | Functional annotation of human cytomegalovirus gene products: an update[END_REF]. The genome of HCMV is highly complex and encodes a wide range of proteins involved in various aspects of the virus's life cycle, including replication, immune evasion, and pathogenesis. The Genome organizations of various human herpesviruses including varicella zoster virus (VZV), human simplex virus (HSV), and Epstein-Barr virus (EBV). The labels within each segment of the genome illustrate the following characteristics: terminal repeat long (TRL), unique long (UL), unique short (US), internal repeat long (IRL), internal repeat short (IRS), terminal repeat short (TRS), and internal repeat (IR). Adapted from Crough et al [START_REF] Crough | Immunobiology of Human Cytomegalovirus: from Bench to Bedside[END_REF].

HCMV displays a remarkably broad tropism, encompassing a diverse range of cell types including epithelial cells, fibroblasts, endothelial cells, monocytes and macrophages, dendritic cells, smooth muscle cells, neural cells, and placental cells [START_REF] Sinzger | Cytomegalovirus Cell Tropism[END_REF]. This trait can significantly impact the pathogenesis of acute HCMV infections in vivo. For instance, infecting epithelial cells facilitates transmission between hosts, while infecting endothelial and hematopoietic cells can support systemic spread within the host. Additionally, infecting ubiquitous cell types, such as fibroblastic and smooth muscle cells, provides a conducive environment for efficient viral proliferation [START_REF] Sinzger | Cytomegalovirus Cell Tropism[END_REF][START_REF] Sinzger | Human Cytomegalovirus Cell Tropism and Pathogenesis[END_REF]. It's worth emphasizing that HCMV tropism varies considerably among different HCMV strains and is primarily influenced by variations within the UL128-131 gene locus. This region has been associated with viral persistence, reactivation, and the ability to infect different cell types.

The resulting genetic differences account for inter-strain variations in viral entry. Notably, the UL128, UL130, and UL131 proteins form complexes with the virion envelope glycoprotein gH/gL, specifically the pentamer gH/gL/UL128-131. This complex is responsible for mediating entry into epithelial and endothelial cells [START_REF] Loughney | Soluble Human Cytomegalovirus gH/gL/pUL128-131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes[END_REF][START_REF] Ryckman | Characterization of the Human Cytomegalovirus gH/gL/UL128-131 Complex That Mediates Entry into Epithelial and Endothelial Cells[END_REF]. Furthermore, laboratory-adapted strains, such as the widely used reference strain AD169 and other well-known strains like Towne or Davis, have lost substantial DNA fragments within their ULb' region due to extensive passaging in human fibroblast cell cultures. Consequently, these laboratory strains replicate efficiently in fibroblasts but exhibit limited replication in other cell types like endothelial or epithelial cells. This is in contrast to clinical isolates, which replicate less efficiently in cultured cells but demonstrate nearly equivalent replication efficiency in endothelial cells [START_REF] Wang | Human Cytomegalovirus UL131 Open Reading Frame Is Required for Epithelial Cell Tropism[END_REF][START_REF] Murphy | Coding potential of laboratory and clinical strains of human cytomegalovirus[END_REF][START_REF] Scrivano | HCMV Spread and Cell Tropism are Determined by Distinct Virus Populations[END_REF]. Understanding the role of the UL128-131 gene locus and its impact on viral entry and tropism is essential for unraveling the complex interactions between HCMV and its host. This knowledge has implications for vaccine development and antiviral strategies aimed at targeting specific strains or cell types.

Virion Structure

The HCMV virion structure consists of several components (Figure 2) that are orchestrated to ensure successful attachment, entry, and establishment of infection in target cells [START_REF] Liu | Structure of human cytomegalovirus virion reveals host tRNA binding to capsid-associated tegument protein pp150[END_REF]. This description has been adapted and altered based on Tomtishen's work [START_REF] Scrivano | HCMV Spread and Cell Tropism are Determined by Distinct Virus Populations[END_REF].

The Envelope

The HCMV virion is enveloped by a lipid bilayer derived from the host cell membrane during the process of virus assembly and budding. The lipid bilayer is approximately 10 nm thick and contains a minimum of 19 integral membrane proteins that play a crucial role in binding to host cells, facilitating viral entry, and, in some instances, evading the host immune response by sequestering human chemokines [START_REF] Ryckman | Characterization of the Human Cytomegalovirus gH/gL/UL128-131 Complex That Mediates Entry into Epithelial and Endothelial Cells[END_REF][START_REF] Weekes | Quantitative Temporal Viromics: An Approach to Investigate Host-Pathogen Interaction[END_REF]. Among these proteins, key players in virus entry, cell-to-cell transmission, and virion maturation include viral glycoproteins like gpUL55 (gB), gpUL73 (gN), gpUL74 (gO), gpUL75 (gH), gpUL100 (gM), gpUL115 (gL), and the pentameric complex composed of gL, gH, and UL128-131 [START_REF] Ryckman | Characterization of the Human Cytomegalovirus gH/gL/UL128-131 Complex That Mediates Entry into Epithelial and Endothelial Cells[END_REF].

The Capsid

Measuring about 100 nm in diameter, the icosahedral HCMV capsid, also known as the nucleocapsid, serves as the innermost core layer of the virion particle. It's a proteinaceous shell that encloses the viral genome. Its primary roles are to safeguard and deliver the genetic material to the host cell's nucleus. This capsid is constructed from a minimum of five distinct proteins, which are meticulously organized into 162 capsomeres. These five proteins encompass the major capsid protein (UL86), the minor capsid protein (UL85), the smallest capsid protein (UL48-49), the assembly protein comprising fragments of UL80, and the minor capsid binding protein (UL46) [START_REF] Yu | Atomic structure of the human cytomegalovirus capsid with its securing tegument layer of pp150[END_REF][START_REF] Wood | Human cytomegalovirus capsid assembly protein precursor (pUL80.5) interacts with itself and with the major capsid protein (pUL86) through two different domains[END_REF].

The Tegument

The tegument layer in the HCMV virion is around 50 nm thick and occupies the space between the lipid envelope and the protein capsid [START_REF] Chen | Three-Dimensional Visualization of Tegument/Capsid Interactions in the Intact Human Cytomegalovirus[END_REF]. This layer is characterized by its high protein content, accounting for approximately half of all viral proteins present in an infectious virion.

Many of these proteins are phosphorylated. These proteins play crucial roles in various aspects of the viral life cycle, including replication, gene expression regulation, viral particle assembly, immune system evasion, and other essential functions. Beyond viral proteins, the tegument layer also contains approximately 70 cellular proteins, as well as viral and cellular RNAs [START_REF] Terhune | RNAs Are Packaged into Human Cytomegalovirus Virions in Proportion to Their Intracellular Concentration[END_REF][START_REF] Varnum | Identification of Proteins in Human Cytomegalovirus (HCMV) Particles: the HCMV Proteome[END_REF].

HCMV Life Cycle

The HCMV life cycle is a complex process involving various stages from viral attachment and entry into host cells to replication, assembly, and release of new virions [START_REF] Beltran | The life cycle and pathogenesis of human cytomegalovirus infection: lessons from proteomics[END_REF].

Attachment and Viral Entry

During the viral entry process, HCMV achieves its tropism by employing cell receptor-mediated endocytosis, which is facilitated by virally encoded glycoproteins binding to specific cellular receptors. This entry mechanism can be delineated into three key stages. Initially, the virus adheres to the host cell's surface by binding to heparan sulfate glycosaminoglycans via the virion glycoprotein B (gB) in conjunction with the glycoprotein M/glycoprotein N complex [START_REF] Compton | Initiation of Human Cytomegalovirus Infection Requires Initial Interaction with Cell Surface Heparan Sulfate[END_REF].

Subsequently, there is an interaction with entry receptors located on the host cell membrane, including integrins and epidermal growth factor receptors (EGFRs), both of which bind to gB [START_REF] Wang | Integrin αvβ3 is a coreceptor for human cytomegalovirus[END_REF][START_REF] Chan | Activation of EGFR on monocytes is required for human cytomegalovirus entry and mediates cellular motility[END_REF]. The third step involves either the internalization of the viral particle or the fusion of the viral envelope with the cell membrane, a process also mediated by gB. In addition to gB, this step may involve the trimeric complex comprised of glycoprotein H, glycoprotein L, and glycoprotein O (gH/gL/gO) in fibroblasts, likely due to interactions with platelet-derived growth factor-α (PDGF-alpha). In epithelial and endothelial cells, it may alternatively entail the pentameric complex of glycoproteins gH/gL, in conjunction with UL128-131 [START_REF] Mosher | Overview of how HCMV manipulation of host cell intracellular trafficking networks can promote productive infection[END_REF][START_REF] Ibig-Rehm | High-content screening to distinguish between attachment and post-attachment steps of human cytomegalovirus entry into fibroblasts and epithelial cells[END_REF].

Lytic Cycle

Viral tegument proteins that are bound to the capsid engage with the host microtubule machinery, leading to the transportation of viral capsids to the nuclear envelope and subsequently into the nucleus, where the viral DNA is released [START_REF] Ogawa-Goto | Microtubule Network Facilitates Nuclear Targeting of Human Cytomegalovirus Capsid[END_REF]. Within this context, two crucial players in nucleocapsid translocation, uncoating, and the release of the viral genome are the large tegument protein (product of UL48) and the large tegument protein binding protein (product of UL47) [START_REF] Bechtel | Human Cytomegalovirus UL47 Tegument Protein Functions after Entry and before Immediate-Early Gene Expression[END_REF][START_REF] Ogawa-Goto | An Endoplasmic Reticulum Protein, p180, Is Highly Expressed in Human Cytomegalovirus-Permissive Cells and Interacts with the Tegument Protein Encoded by UL48[END_REF]. In the nucleus, the processes of viral transcription, genome replication, and encapsidation occur. Lytic replication is a regulated sequence of events, with immediate early genes (IE) being expressed first, followed by early (E) and late genes (L). The IE gene products function as transcription factors and expression regulators for the E and L genes. The E proteins primarily interact with host proteins while also impacting the transcriptional and replication machinery for viral proteins. The L proteins serve primarily as structural components of the virions, and they also encompass proteins crucial for the assembly and release of infectious particles [START_REF] Chambers | DNA Microarrays of the Complex Human Cytomegalovirus Genome: Profiling Kinetic Class with Drug Sensitivity of Viral Gene Expression[END_REF][START_REF] Sanchez | Viable Human Cytomegalovirus Recombinant Virus with an Internal Deletion of the IE2 86 Gene Affects Late Stages of Viral Replication[END_REF][START_REF] Perng | The Human Cytomegalovirus Gene UL79 Is Required for the Accumulation of Late Viral Transcripts[END_REF]. In this intricate process, UL54 (the viral DNA polymerase) and UL70 (DNA primase) play pivotal roles in regulating the replication machinery and ensuring an efficient production of new virus progeny.

Additionally, various viral-encoded proteins contribute to the regulation of cell signaling pathways and cellular metabolism, as well as the inhibition of initial steps in the immune response to support viral replication and immune evasion [START_REF] Loregian | Residues of Human Cytomegalovirus DNA Polymerase Catalytic Subunit UL54 That Are Necessary and Sufficient for Interaction with the Accessory Protein UL44[END_REF][START_REF] Shen | Human Cytomegalovirus Primase UL70 Specifically Interacts with Cellular Factor Snapin[END_REF][START_REF] Yu | Viral effects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection[END_REF][START_REF] Yurochko | Human Cytomegalovirus Modulation of Signal Transduction[END_REF][START_REF] Li | Human Cytomegalovirus Tegument Protein pUL83 Inhibits IFI16-Mediated DNA Sensing for Immune Evasion[END_REF]. For instance, the viral US3 protein sequesters the major histocompatibility complex (MHC) class I within the endoplasmic reticulum (ER), while the US2 and US11 proteins induce the destruction of class I heavy chains, thereby impeding viral clearance by cytotoxic T lymphocytes (CTL) [START_REF] Noriega | Human cytomegalovirus US3 modulates destruction of MHC class I molecules[END_REF].

The nucleocapsid, containing DNA, assembles within the nucleus and exits into the cytoplasm by inducing changes in nuclear lamina components. This process is facilitated by two highly conserved viral proteins, specifically pUL50 and pUL53 [START_REF] Camozzi | Remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pUL50 and pUL53[END_REF]. The assembly and transportation of virions involve the integration of various cellular trafficking pathways such as the hijacking of the endoplasmic reticulum (ER), Golgi apparatus, and endosomal machinery, forming the cytoplasmic viral assembly complex. It is within this complex that capsids acquire their tegument layer and viral envelope from intracellular vesicles [START_REF] Moorman | A Targeted Spatial-Temporal Proteomics Approach Implicates Multiple Cellular Trafficking Pathways in Human Cytomegalovirus Virion Maturation[END_REF]. Subsequently, infectious particles are released either by fusing with the plasma membrane or by causing cell lysis, releasing them into the extracellular space (Figure 3) [START_REF] Homman-Loudiyi | Envelopment of Human Cytomegalovirus Occurs by Budding into Golgi-Derived Vacuole Compartments Positive for gB, Rab 3, Trans-Golgi Network 46, and Mannosidase II[END_REF]. Beside mature virions, two types of defective particles are also released: dense bodies (DBs) and non-infectious enveloped particles (NIEPs) [START_REF] Roby | Characterization of phosphoproteins and protein kinase activity of virions, noninfectious enveloped particles, and dense bodies of human cytomegalovirus[END_REF]. DBs are larger and denser than mature virions, ranging in size from approximately 250 to 600 nm in diameter; they contain most of the structural proteins but lack a viral genome, which is normally packed into the protective capsid. NIEPs are similar in size to virions but lack a viral genome [START_REF] Pepperl | Dense Bodies of Human Cytomegalovirus Induce both Humoral and Cellular Immune Responses in the Absence of Viral Gene Expression[END_REF]. The entire lytic replication cycle for HCMV takes approximately 72 hours before new mature virions are prepared to infect further cells, either through release from the infected cells or via cell-to-cell transmission mechanisms [START_REF] Landolfo | The human cytomegalovirus[END_REF]. The life cycle of HCMV involves a series of steps. Initially, HCMV gains entry into the host cell through the interaction of host receptors with specific viral glycoproteins. Following this, capsid and tegument proteins are liberated into the host cytosol. The capsid then releases the viral genome into the nucleus, initiating the expression of immediate early (IE) genes. These IE proteins, in turn, activate the expression of early (E) genes. Early proteins boost [START_REF] Huang | Efficient Human Cytomegalovirus Reactivation Is Maturation Dependent in the Langerhans Dendritic Cell Lineage and Can Be Studied using a CD14 + Experimental Latency Model[END_REF]. Reactivation of latent HCMV can have significant clinical consequences, ranging from severe inflammatory conditions in immunocompromised patients to complications like allograft rejection and graft-versus-host disease in transplant recipients [START_REF] Walton | Reactivation of Multiple Viruses in Patients with Sepsis[END_REF][START_REF] Nett | Association of Cytomegalovirus Disease and Acute Rejection with Graft Loss in Kidney Transplantation[END_REF].

HCMV

Attachment and penetration

Virus

HCMV Prevalence and Spread

HCMV is widely recognized as a ubiquitous virus, with seroprevalence rates ranging from 40% to 90% in the adult population [START_REF] El Baba | Immune Landscape of CMV Infection in Cancer Patients: From "Canonical" Diseases Toward Virus-Elicited Oncomodulation[END_REF]. HCMV infection is considered endemic and doesn't exhibit seasonal variations [START_REF] Staras | Seroprevalence of Cytomegalovirus Infection in the United States, 1988-1994[END_REF]. The prevalence of HCMV infection varies depending on geographic and socioeconomic factors. In developing regions, more than 90% of preschool children are found to have acquired HCMV during early childhood. In contrast, in industrialized countries, less than 20% of children are seropositive [START_REF] Stagno | Comparative Serial Virologic and Serologic Studies of Symptomatic and Subclinical Congenitally and Natally Acquired Cytomegalovirus Infections[END_REF][START_REF] Revello | Molecular epidemiology of primary human cytomegalovirus infection in pregnant women and their families[END_REF]. As individuals age, the percentage of seropositive individuals continues to rise, reaching levels between 40% and 70% [START_REF] Schottstedt | Human Cytomegalovirus (HCMV) -Revised[END_REF].

The transmission of HCMV is influenced by a multitude of potential sources of exposure [START_REF] Forbes | Acquisition of cytomegalovirus infection: an update[END_REF]. The primary route for acquiring HCMV is the direct contact with body fluids from an infected individual. These fluids encompass saliva, oropharyngeal secretions, urine, cervical and vaginal secretions, semen, breast milk, allografts, and blood products from donors who are seropositive [START_REF] Schottstedt | Human Cytomegalovirus (HCMV) -Revised[END_REF]. Another means of contracting the virus is through perinatal or vertical transmission, where HCMV is transmitted through the placenta, a condition known as congenital infection. It's important to mention that infected infants typically excrete substantial amounts of the virus for months to years following infection, as do older children and adults following a primary HCMV infection [START_REF] Pass | Mother-to-Child Transmission of Cytomegalovirus and Prevention of Congenital Infection[END_REF].

Clinical Aspects of HCMV and HCMV-Related Diseases

HCMV infection is mostly asymptomatic yet it can result in a mild illness in immunocompetent individuals. Primary HCMV infection can occasionally lead to a mild syndrome resembling mononucleosis [START_REF] Lancini | Cytomegalovirus disease in immunocompetent adults[END_REF]. On the contrary, severe CMV disease occurs in individuals with immature, suppressed, or compromised immune systems, such as neonates, transplant recipients receiving immunosuppressive drugs, and patients with AIDS. This severe form of the disease can have lifethreatening consequences or cause significant long-term complications [START_REF] Britt | Manifestations of Human Cytomegalovirus Infection: Proposed Mechanisms of Acute and Chronic Disease[END_REF]. These complications encompass a wide range of potential clinical manifestations due to the virus's ability to spread through the bloodstream and infect various tissues. This includes gastrointestinal issues, respiratory problems, hematological abnormalities, cardiovascular complications, neurological symptoms, and urological manifestations. Additionally, severe HCMV infection can indirectly lead to atherosclerosis acceleration, graft rejection, and increased susceptibility to opportunistic infections [START_REF] Crough | Immunobiology of Human Cytomegalovirus: from Bench to Bedside[END_REF]. In cases of congenital HCMV infection, approximately 90% of infected infants

show no symptoms at birth, although about 5% of them may develop complications later in life [START_REF] Chiopris | Congenital Cytomegalovirus Infection: Update on Diagnosis and Treatment[END_REF]. Common initial symptoms include jaundice, petechiae, and hepatosplenomegaly [START_REF] Dietrich | Congenital Cytomegalovirus Infection[END_REF]. Major long-term complications of congenital HCMV infection encompass prematurity, restricted intrauterine growth, hypotonia, poor feeding, cerebral ventriculomegaly, microcephaly, intracranial periventricular calcifications, sensorineural hearing loss, and, in 10 to 20% of affected infants, chorioretinitis and mental retardation [START_REF] Xia | Congenital Human Cytomegalovirus Infection Inducing Sensorineural Hearing Loss[END_REF][START_REF] Syggelou | Congenital cytomegalovirus infection[END_REF].

HCMV Detection and Diagnosis

Numerous diagnostic approaches are accessible for detecting HCMV infection, primarily encompassing serological testing, virus culture, identification of viral antigens, and CMV DNA detection through polymerase chain reaction (PCR) [START_REF] Grangeot-Keros | Diagnosis and prognostic markers of HCMV infection[END_REF].

Serology

This method relies on assessing the levels of IgM and IgG antibodies in the serum or plasma of patients. The enzyme-linked immunosorbent assay (ELISA) is the most commonly employed technique. The presence or absence of CMV IgG serves as a marker to identify whether the individual has previously experienced a CMV infection. Meanwhile, the detection of IgM antibodies has been employed as an indicator of a recent, acute infection, or viral reactivation [START_REF] Xia | Congenital Human Cytomegalovirus Infection Inducing Sensorineural Hearing Loss[END_REF][START_REF] De Paschale | Positive predictive value of anti-HCMV IgM as an index of primary infection[END_REF]. Furthermore, by determining the avidity of IgG antibodies, it will be possible to differentiate between an early primary infection (low avidity) and a latter infection (high avidity)

during pregnancy [START_REF] Macé | A serological testing algorithm for the diagnosis of primary CMV infection in pregnant women: DIAGNOSIS OF CMV PRIMARY INFECTION IN PREGNANT WOMEN[END_REF].

Virus Culture

The conventional method for detecting HCMV is through traditional cell culture. In this approach, clinical samples are introduced to human fibroblast cells (MRC5 cells), and their development is observed over a period ranging from 2 to 21 days. The presence of cytopathic effects (CPE), characterized by clusters of flattened and swollen cells, is then directly correlated with the virus's titer. However, the limitation of this method is the extended time required, with results often taking 2 to 3 weeks. To expedite this process, an alternative approach involves the detection of viral antigens using monoclonal antibodies directed against CMV immediate-early viral antigens via indirect immunofluorescence after a 16-hour incubation period [START_REF] Ross | Overview of the Diagnosis of Cytomegalovirus Infection[END_REF][START_REF] Drew | Diagnosis of Cytomegalovirus Infection[END_REF].

Identification of Viral Antigens

This assay relies on the detection of the viral pp65 antigen within blood specimens, specifically within leukocytes, as this antigen is expressed in these cells during the early stages of the HCMV replication cycle [START_REF] Revello | Detection of human cytomegalovirus immediate early antigen in leukocytes as a marker of viremia in immunocompromised patients[END_REF]. An immunofluorescence assay that identifies pp65 in a sample of peripheral blood leukocytes not only allows for the quantification of positive leukocyte nuclei but also closely correlates with viremia and the severity of clinical disease in immunosuppressed populations [START_REF] Niubò | Association of quantitative cytomegalovirus antigenemia with symptomatic infection in solid organ transplant patients[END_REF].

However, a significant limitation of this technique is its lack of automation, requiring skilled individuals for accurate test execution and result interpretation. Furthermore, results should be assessed within a 6-hour window, as delays in processing can significantly reduce the assay's sensitivity, potentially leading to false-negative results, especially in patients with neutropenia [START_REF] Schäfer | Minimal effect of delayed sample processing on results of quantitative PCR for cytomegalovirus DNA in leukocytes compared to results of an antigenemia assay[END_REF][START_REF] Boeckh | PLASMA POLYMERASE CHAIN REACTION FOR CYTOMEGALOVIRUS DNA AFTER ALLOGENEIC MARROW TRANSPLANTATION: Comparison with Polymerase Chain Reaction Using Peripheral Blood Leukocytes, pp65 Antigenemia, and Viral Culture1[END_REF].

PCR Amplification

PCR is a widely accessible, rapid, sensitive and specific method for detecting HCMV in various types of samples, including blood, leukocytes, plasma, tissue biopsy specimens, and fluids for instance urine, bronchoalveolar lavage, or cerebrospinal fluid [START_REF] Evans | Qualitative and semiquantitative polymerase chain reaction testing for cytomegalovirus DNA in serum allows prediction of CMV related disease in liver transplant recipients[END_REF]. PCR for HCMV DNA can be conducted in either a qualitative or quantitative manner (Real-Time PCR), enabling continuous monitoring of immunocompromised individuals to identify those at risk for HCMV-related diseases, implement preemptive therapy, and assess treatment response [START_REF] Seehofer | Prospective Evaluation of the Clinical Utility of Different Methods for the Detection of Human Cytomegalovirus Disease after Liver Transplantation[END_REF]. Additionally, the detection of viral mRNA transcripts in peripheral blood leukocytes can be accomplished through reverse transcriptase PCR (RT-PCR) during active HCMV infection. However, it's worth noting that this method is less sensitive than the pp65 antigen test and PCR in diagnosing HCMV-related diseases [START_REF] Meyer-Konig | Human Cytomegalovirus Immediate Early And Late Transcripts In Peripheral Blood Leukocytes: Diagnostic Value In Renal Transplant Recipients[END_REF].

Therapeutic Approaches for HCMV Infection

Effective management of HCMV infection is particularly important for immunocompromised individuals, such as transplant recipients and people with HIV/AIDS, as well as newborns infected with HCMV [START_REF] Ahmed | Antiviral Treatment of Cytomegalovirus Infection[END_REF]. Below are the approved therapies for HCMV, their mechanism of action, and their associated benefits and potential adverse effects in addition to the ongoing research and emerging therapies that hold promise for improving the therapeutic strategies against HCMV infection, offering better patient outcomes [START_REF] Acosta | Advances in the Development of Therapeutics for Cytomegalovirus Infections[END_REF]. recombination with disruptive mutations identified in clinical isolates, even exhibiting rapid evolution within the host [START_REF] Griffiths | Pathogenesis of human cytomegalovirus in the immunocompromised host[END_REF]. In summary, the development of an effective HCMV vaccine has been hindered by these complex challenges, despite the pressing need for such a vaccine.

Antibody Therapy

The FDA has granted approval for passive immunization using HCMV immunoglobulin as a prophylactic measure. This is typically administered alongside GCV, especially in high-risk recipients of lung and cardiothoracic transplants. It is also considered in cases of GCV resistance or poor tolerance. However, when it comes to congenital HCMV, passive immunization with HCMV immunoglobulin has not shown significant reductions in congenital HCMV disease [START_REF] Jückstock | Passive Immunization against Congenital Cytomegalovirus Infection: Current State of Knowledge[END_REF].

Therefore, it is not recommended for use in pregnant women with primary HCMV infection.

Adoptive Cell Therapy (ACT)

T cells isolated from healthy donors who have previously been exposed to HCMV can be expanded in vitro and transferred to immunocompromised patients. These "donor-derived" T cells can help control HCMV reactivation. ACT is now being used as a treatment approach for HCMV reactivation in patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT) and solid organ transplantation (SOT). Research has demonstrated that autologous HCMV-specific T cells can directly eliminate primary glioblastoma (GBM) cells. Some researchers have even devised an innovative adoptive immunotherapy approach targeting CMV antigens for patients with recurrent GBM. Experimental results from this approach have shown it to be both safe and linked to extended progression-free survival in 4 out of 10 patients [START_REF] Long | Insight for Immunotherapy of HCMV Infection[END_REF].

Checkpoint Blockade Therapy

Research has shown that the use of immune checkpoint inhibitors (ICIs) can potentially revive immune function and elicit an immune response to CMV antigens even when in a latent state. One promising approach involves a vector called pS-CIFT-aPD-1, which is designed to express the gene for an antibody targeting the programmed cell death protein 1 (anti-PD-1) [START_REF] Long | Insight for Immunotherapy of HCMV Infection[END_REF].

Novel US28-targeting Strategies

In search of innovative treatments against HCMV infection, a recent approach has emerged focusing on the role of the surface protein US28, which plays a crucial part during both lytic and latent infections. The methods for therapeutically targeting US28 can be broadly categorized into three distinct categories: small molecules, single-domain antibodies (referred to as nanobodies), and fusion toxin proteins (FTPs) [START_REF] Long | Insight for Immunotherapy of HCMV Infection[END_REF][START_REF] Berg | Therapeutic targeting of HCMV-encoded chemokine receptor US28: Progress and challenges[END_REF].

Chapter 2

HCMV-Induced Polyploid Giant Cancer Cells and Their Significance in Tumorigenesis

Morphological Characteristics of PGCCs

Polyploid giant cancer cells (PGCCs) are characterized as either gigantic mononuclear cells with multiple genome copies or multinuclear cells [START_REF] Zhou | Polyploid giant cancer cells and cancer progression[END_REF]. According to the definition by Zhang et al., a PGCC is delineated as a cell three times larger than a typical diploid cancer cell. However, the size of a PGCC can vary significantly depending on the DNA content and the number of nuclei within the cells, sometimes reaching dimensions that are 10 to 20 times larger than a regular diploid cell [START_REF] Zhang | Generation of cancer stem-like cells through the formation of polyploid giant cancer cells[END_REF]. PGCCs have a slow cell cycle and divide through asymmetric cell division patterns, which include budding and bursting. Budding is characterized by the release of numerous small daughter cells resembling the growth and division mechanisms seen in simpler organisms like yeasts.

Additionally, dormant PGCCs are associated with the induction of quiescence and increased storage capacity through the presence of vacuoles and accumulation of lipid droplets, increased metabolic capacity, and elevated energy production [START_REF] Pienta | Cancer recurrence and lethality are enabled by enhanced survival and reversible cell cycle arrest of polyaneuploid cells[END_REF][START_REF] Liu | The life cycle of polyploid giant cancer cells and dormancy in cancer: Opportunities for novel therapeutic interventions[END_REF][START_REF] Liu | Polyploid giant cancer cells: An emerging new field of cancer biology[END_REF].

PGCCs Stimuli and Mechanisms of Generation

PGCCs frequently emerge as a result of various stressors, such as exposure to chemotherapy, ionizing radiation, conditions of hypoxia, viruses, and other stimuli that lead to DNA double-strand breaks (Figure 4). CoCl2 serves as a mimic for hypoxia-induced cellular responses in vitro and activates signaling pathways associated with hypoxia. CoCl2 plays a role in stabilizing hypoxiainducible factor (HIF)-1α by inhibiting proline hydroxylase. Additionally, chemotherapy drugs like capecitabine, oxaliplatin, and irinotecan, as well as radiation therapy, can induce PGCCs formation. Tumor tissues rich in PGCCs are associated with higher risks of recurrence, metastasis, chemoresistance, and poor prognosis [START_REF] Saini | Polyploid giant cancer cell characterization: New frontiers in predicting response to chemotherapy in breast cancer[END_REF][START_REF] Was | Polyploidy formation in cancer cells: How a Trojan horse is born[END_REF].

The generation of PGCCs involves a complex interplay of cellular processes and signaling pathways. The giant cell cycle, which leads to the formation of PGCCs, differs from the regular cell cycle in certain aspects. While the exact mechanisms can vary between different types of cancer, here are some general insights into how PGCCs are generated and the characteristics of the giant cell cycle (Figure 5). PGCCs formation can occur through various mechanisms, including endoreplication, mitotic slippage, cytokinesis failure, cell fusion, or cell cannibalism with the former being the predominant mechanism. While studies have documented the occurrence of PGCCs resulting from cell fusion in Hodgkin's lymphoma and glioblastoma cell lines, it's worth noting that, in MDA-MB-231 and ovarian cancer cells, cell fusion contributes to only a modest fraction, ranging from 10% to 20%, of all PGCCs [START_REF] Saini | Polyploid giant cancer cell characterization: New frontiers in predicting response to chemotherapy in breast cancer[END_REF][START_REF] Chen | Polyploid Giant Cancer Cells (PGCCs): The Evil Roots of Cancer[END_REF]. PGCCs formed through endoreplication and cell fusion have developed the capacity to undergo division using mechanisms similar to those seen in single-celled organisms, such as budding and viral-like amitotic division, which leads to the production of tumor-initiating cells. Adapted from Chen et al. [START_REF] Was | Polyploidy formation in cancer cells: How a Trojan horse is born[END_REF].

Characteristics of PGCCs and Potential Biomarkers

PGCCs exhibit a unique combination of markers, encompassing both normal and cancer stem cell (CSC) indicators [START_REF] Zhang | Generation of cancer stem-like cells through the formation of polyploid giant cancer cells[END_REF][START_REF] Saini | Polyploid giant cancer cell characterization: New frontiers in predicting response to chemotherapy in breast cancer[END_REF]. These markers include OCT4, NANOG, SOX2, CD44, and CD133.

Notably, the daughter cells originating from PGCCs also display characteristics similar to stem cells and continue to express CSC markers, specifically the surface glycoproteins CD44 and CD133. PGCCs maintain a highly dedifferentiated state, reminiscent of embryonic cells, and possess more pluripotency compared to typical CSCs. Furthermore, these PGCC progeny share similarities with blastomeres observed during embryonic development, possessing the capability to differentiate into various cell types representing the three germ layers: ectoderm, mesoderm, and endoderm, as demonstrated in vitro. Their expression profile includes spatiotemporal markers associated with embryonic development and self-renewal, such as NANOG, OCT3, OCT4, aldehyde dehydrogenase-1A (ALDH1A), and SOX-2. These daughter cells exhibit the potential to differentiate into diverse benign cell types, such as adipocytes, chondrocytes, erythrocytes, and osteocytes, or give rise to carcinomas with differing grades [START_REF] Saini | Polyploid giant cancer cell characterization: New frontiers in predicting response to chemotherapy in breast cancer[END_REF]. In a study by Zhang and colleagues, PGCCs progeny derived from the MCF-7 cell line were observed to differentiate into benign stromal cells, including myoepithelial, endothelial, and erythroid cells. Considering the role of the tumor stroma in modulating drug bioavailability and enzymatic degradation, PGCCs can exert a significant influence on promoting resistance to various anticancer therapies [START_REF] Zhang | Generation of cancer stem-like cells through the formation of polyploid giant cancer cells[END_REF]. Further, PGCCs and their progeny often undergo epithelial-mesenchymal transition (EMT). EMT involves significant cytoskeletal transformations that lead to alterations in cell adhesion and morphology, playing a critical role in the metastatic dissemination of cancer cells. PGCCs, along with their progeny, have been observed to exhibit distinct expression patterns, characterized by elevated levels of mesenchymal markers like vimentin, fibronectin, and N-cadherin, accompanied by reduced levels of epithelial markers such as cytokeratin and E-cadherin. This pattern mirrors the connection often seen in cancer cells, where EMT and the acquisition of stem cell-like features are closely intertwined. Likewise, in PGCC progeny cells, there is a notable association between the expression of EMT markers and stemness markers. This association is exemplified by the induction of EMT in human mammary epithelial cells triggered by transforming growth factor-β (TGF-β), which also facilitates the generation of cells exhibiting stem cell-like properties, such as CD44 expression and self-renewal capacity [START_REF] Saini | Polyploid giant cancer cell characterization: New frontiers in predicting response to chemotherapy in breast cancer[END_REF][START_REF] Wang | Epithelial-mesenchymal Transition and Cancer Stem Cells: At the Crossroads of Differentiation and Dedifferentiation: EMT in Differentiation and Dedifferentiation[END_REF]. The giant cell cycle closely mimics the division of blastomeres. Upon initiation by either intrinsic genetic factors or external stresses, a somatic cell enters a phase of self-renewal endoreplication and initiates dedifferentiation or reprogramming. Subsequently, the reprogrammed cell enters a termination phase to start differentiation. During this phase, giant cells employ multiple primitive cell division mechanisms to generate diploid daughter cells. These include (1) horizontal genetic transfer, where DNA migrates horizontally into adjacent cells through cytoplasmic extensions followed by budding, (2) the formation of elongated cells with two giant nuclei, as well as (3) splitting in the middle of the giant cell or (4) direct budding from both (5) mononucleated and ( 6) multinucleated giant cells. In the stability phase, differentiated cells gradually organize themselves out of chaos, reaching a specific developmental level. Dominant clones emerge from this chaos, resulting in the formation of a visible tumor which can behave as benign, malignant, resistant, metastasis or death. Cells that immediately bud off from the giant cells exhibit a high level of stemness, symbolized by a red triangle, and progressively attain stability during the process of differentiation, represented by a blue triangle. Adapted from Liu et al [START_REF] Wang | Epithelial-mesenchymal Transition and Cancer Stem Cells: At the Crossroads of Differentiation and Dedifferentiation: EMT in Differentiation and Dedifferentiation[END_REF].

Potential Involvement of PGCCs in Cancer Progression

Accumulating evidence underscores the significance of genome duplication, or polyploidization, as a fundamental genetic trait in cancer [START_REF] Zhou | Polyploid giant cancer cells and cancer progression[END_REF][START_REF] Liu | Polyploid giant cancer cells: An emerging new field of cancer biology[END_REF][START_REF] Chen | Polyploid Giant Cancer Cells (PGCCs): The Evil Roots of Cancer[END_REF]. Recently, polyploidy was found to be present in approximately 37% of human tumors [START_REF] Matsumoto | Proliferative polyploid cells give rise to tumors via ploidy reduction[END_REF][START_REF] Herbein | Polyploid Giant Cancer Cells, a Hallmark of Oncoviruses and a New Therapeutic Challenge[END_REF]. Polyploidization is recognized for its role in promoting chromosomal instability, primarily through the frequent missegregation of chromosomes during cell proliferation, and by creating conditions conducive to aneuploidy [START_REF] Matsumoto | Proliferative polyploid cells give rise to tumors via ploidy reduction[END_REF].

This, in turn, fosters cancer evolution by introducing genetic diversity. PGCCs have been described in a broad spectrum of high-grade and chemoresistant tumors, as well as in various cancer cell lines [START_REF] Zhou | Polyploid giant cancer cells and cancer progression[END_REF][START_REF] Saini | Polyploid giant cancer cell characterization: New frontiers in predicting response to chemotherapy in breast cancer[END_REF]. These encompass breast and colorectal cancer, glioma, lung, prostate, ovarian, cervical, lymphoma, nasopharyngeal, pancreatic carcinoma, neuroblastoma, renal, thyroid, bowel sarcoma, and melanoma [START_REF] Zhou | Polyploid giant cancer cells and cancer progression[END_REF][START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF]]. It's worth noting that the presence of PGCCs has been preferentially reported in poor prognosis cancers participating in tumor relapse and therapy resistance. PGCCs play a role in contributing to the heterogeneity of tumors (Figure 6) and have been identified in well-established cancer cell lines, including MDA-MB231, MCF-7, U87MG, SKOV-3, HeLa, A549, PC-3, SW620, and HCT-116 cell lines [START_REF] Saini | Polyploid giant cancer cell characterization: New frontiers in predicting response to chemotherapy in breast cancer[END_REF]. Thus, discovering distinctive traits that symbolize polyploid tumors has the potential to enhance the clinical management and classification of tumors based on their ploidy status.

PGCCs and Oncoviruses

Since its inception, the field of viral oncogenesis has yielded groundbreaking insights into the origins of human cancer, illuminating the intricate molecular mechanisms and multifaceted interactions between the host and oncogenic viruses. Approximately 15 to 20% of human cancers worldwide can be attributed to infections caused by oncoviruses. These oncoviruses include human papillomaviruses (HPV), Epstein-Barr virus (EBV), hepatitis B and C viruses (HBV, HCV, respectively), human T-cell lymphotropic virus-1 (HTLV-1), Kaposi's sarcoma herpesvirus (KSHV), and Merkle Polyomavirus (MCPyV) (Table 1) [START_REF] Mui | Viral Oncology: Molecular Biology and Pathogenesis[END_REF]. Although significant advancements have expanded our understanding of how these pathogens designate cellular machinery to establish infection and initiate common pathways leading to tumorigenesis, as well as to replicate and maintain persistence, the development of targeted therapies and definitive curative clinical treatments remains a formidable challenge. This challenge primarily stems from the limitations of equivalent animal models and the mysterious nature of certain mechanistic aspects involved in cancer induction by these agents, given their diverse characteristics.

Nonetheless, polyploidy emerges as a pivotal common trait shared among oncoviruses, where distinct viral proteins can act as catalysts for the induction of polyploidy. Polyploidy is increasingly recognized as an initial trigger for cellular transformation within this context [START_REF] Mesri | Human Viral Oncogenesis: A Cancer Hallmarks Analysis[END_REF][START_REF] Hausen | The search for infectious causes of human cancers: Where and why[END_REF][START_REF] Yasunaga | Viral transformation and aneuploidy[END_REF].

HPV

HPV, a small double-stranded circular DNA virus belonging to the Papillomaviridae family, encompasses more than 200 identified types categorized by tissue tropism (skin or mucosa) and malignant transformation potential (high risk and low risk). Low-risk (LR) HPVs cause benign warts, while high-risk (HR) HPVs are associated with high-grade cervical lesions and cancers, particularly HPV-16 and -18 [START_REF] Wang | Prediction of High-Risk Types of Human Papillomaviruses Using Statistical Model of Protein "Sequence Space[END_REF][START_REF] Burd | Human Papillomavirus and Cervical Cancer[END_REF]. The oncogenic potential of HR-HPVs is primarily attributed to the HPV-E6 and -E7 proteins [START_REF] Yeo-Teh | High-Risk Human Papillomaviral Oncogenes E6 and E7 Target Key Cellular Pathways to Achieve Oncogenesis[END_REF]. Polyploidy is commonly observed in the context of HPV infection, particularly in cervical carcinogenesis. Tetraploidy is an early event, and tetrasomy is detected in cervical squamous intraepithelial lesions caused by HR-HPVs [START_REF] Giannoudis | Basal keratinocyte tetrasomy in low-grade squamous intra-epithelial lesions of the cervix is restricted to high and intermediate risk HPV infection but is not type-specific[END_REF][START_REF] Olaharski | Tetraploidy and chromosomal instability are early events during cervical carcinogenesis[END_REF].

Additionally, polyploidy is linked to high-grade squamous intraepithelial lesions (HGSIL) [START_REF] Lorenzato | DNA image cytometry and human papillomavirus (HPV) detection help to select smears at high risk of high-grade cervical lesions[END_REF][START_REF] Méhes | Chromosomal aberrations accumulate in polyploid cells of high-grade squamous intraepithelial lesions (HSIL)[END_REF]. The expression of E6 and E7 oncoproteins is associated with a higher proportion of cells with >4N DNA content [START_REF] Patel | Human Papillomavirus Type 16 E6 and E7 Cause Polyploidy in Human Keratinocytes and Up-Regulation of G2-M-Phase Proteins[END_REF]. The mechanisms behind HPV-induced polyploidy are diverse.

E6 and E7 can induce abnormal centrosome duplication and erroneous mitotic spindle pole formation, decoupling centrosome duplication from cell cycle division and causing genomic instability [START_REF] Duensing | The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle[END_REF]. Furthermore, E6 disrupts the mitotic checkpoint by interfering with p53-mediated functions, while E7 overcomes the mitotic checkpoint through a p53-independent mechanism, possibly by modulating Rb function [START_REF] Tomaić | Functional Roles of E6 and E7 Oncoproteins in HPV-Induced Malignancies at Diverse Anatomical Sites[END_REF]. Further, E6 and E7 downregulate nuclear p21 localization and upregulate cyclin-dependent kinase 1 (CDK1) upon microtubule disruption, contributing to postmitotic checkpoint abrogation-induced polyploidy [START_REF] Zhang | Role of Cdk1 in the p53-Independent Abrogation of the Postmitotic Checkpoint by Human Papillomavirus E6[END_REF]. This effect is also observed with E7 through its role in Rb downregulation [START_REF] Heilman | Abrogation of the Postmitotic Checkpoint Contributes to Polyploidization in Human Papillomavirus E7-Expressing Cells[END_REF]. Simultaneous expression of E6 and E7 induces polyploidy by disrupting the spindle checkpoint and upregulating G2-M proteins.

Endoreplication, characterized by multiple S phases without mitosis, is associated with HPV-16

E5 and E6 oncoproteins, leading to enlarged nuclei and increased cellular DNA content [START_REF] Araldi | Mutagenic Potential of Bos taurus Papillomavirus Type 1 E6 Recombinant Protein: First Description[END_REF][START_REF] Hu | Expression of HPV16 E5 produces enlarged nuclei and polyploidy through endoreplication[END_REF].

Multinucleated cells with enlarged nuclei result from two consecutive S phases without cytokinesis upon HPV-18 E7 transduction [START_REF] Chien | Alternative Fates of Keratinocytes Transduced by Human Papillomavirus Type 18 E7 during Squamous Differentiation[END_REF]. Re-replication is induced by HPV-16 E7 in response to DNA damage, mediated by the DNA replication initiation factor [START_REF] Fan | Human Papillomavirus E7 Induces Rereplication in Response to DNA Damage[END_REF][START_REF] Dorn | Analysis of re-replication from deregulated origin licensing by DNA fiber spreading[END_REF]. E6 and E7 enhance HIF protein accumulation and activity in human cervical cancers [START_REF] Tang | Overexpression of Human Papillomavirus Type 16 Oncoproteins Enhances Hypoxia-Inducible Factor 1α Protein Accumulation and Vascular Endothelial Growth Factor Expression in Human Cervical Carcinoma Cells[END_REF]. In summary, HPV oncoproteins, particularly E6 and E7, employ various mechanisms to induce polyploidy, contributing to cervical carcinogenesis. These mechanisms encompass centrosome abnormalities, mitotic checkpoint abrogation, endoreplication, multinucleation, and re-replication, with downstream effects on genomic stability and cellular transformation. Additionally, HPV infection is associated with increased HIF activity in cervical cancers.

EBV

EBV, a member of the Herpesviridae family within the Gammaherpesvirinae subfamily, is a linear, double-stranded DNA virus [START_REF] Zanella | A reliable Epstein-Barr Virus classification based on phylogenomic and population analyses[END_REF]. It is the first isolated tumor virus and primarily infects B lymphocytes [START_REF] Farrell | Epstein-Barr Virus and Cancer[END_REF]. EBV is associated with a variety of malignancies, including lymphomas such as Hodgkin's lymphoma, diffuse large B-cell lymphoma, and Burkitt's lymphoma, as well as carcinomas like gastric and nasopharyngeal carcinoma (NPC) [START_REF] Farrell | Epstein-Barr Virus and Cancer[END_REF]. Several viral latent antigens, including Epstein-Barr virus nuclear antigen 1 (EBNA1), EBNA2, EBNA3, and the latent membrane protein 1 (LMP1), play essential roles in B-cell transformation [START_REF] Saha | Epstein-Barr Virus-Associated B-cell Lymphomas: Pathogenesis and Clinical Outcomes[END_REF]. In tissue biopsies of NPC, polynuclear giant cancer cells are observed, often surrounded by small nucleus-containing bodies indicative of budding cells [START_REF] Jiang | A Fraction of CD133+ CNE2 Cells Is Made of Giant Cancer Cells with Morphological Evidence of Asymmetric Mitosis[END_REF]. Multinucleated giant cells are also formed following EBV replication in epithelial NPC hybrid cells [START_REF] Sato | Cytopathic effects induced by Epstein-Barr virus replication in epithelial nasopharyngeal carcinoma hybrid cells[END_REF]. EBV-infected nasal mucosal neoplasms display cells with increased volume and pronounced multinucleation, some containing over 12 nuclei [START_REF] Gelardi | Epsteinbarr virus induced cellular changes in nasal mucosa[END_REF]. Chromosomal integration of the viral genome into primary human B cells has been linked

to polyploidy [START_REF] Humme | The EBV nuclear antigen 1 (EBNA1) enhances B cell immortalization several thousandfold[END_REF]. Infection of B cells with an EBV strain isolated from nasopharyngeal carcinoma predisposes them to polyploidy, with cells exhibiting multiple micronuclei or a single large polyploid nucleus [START_REF] Shumilov | Epstein-Barr virus particles induce centrosome amplification and chromosomal instability[END_REF]. Multinucleation and micronucleus formation have been detected in human laryngeal carcinoma Hep-2 and osteosarcoma U-2 OS cells following EBNA2 expression [START_REF] Pan | Epstein-Barr virus nuclear antigen 2 disrupts mitotic checkpoint and causes chromosomal instability[END_REF]. Stable expression of LMP1 in Burkitt's lymphoma cell lines is associated with multinuclearity, indicating a close correlation between polyploidy and EBV infection or the expression of EBV-latent oncoproteins [START_REF] Parker | Epstein-Barr virus EBNA3C can disrupt multiple cell cycle checkpoints and induce nuclear division divorced from cytokinesis[END_REF]. Additionally, EBV latent genes compromise the mitotic spindle assembly checkpoint, preventing metaphase arrest [START_REF] Leao | Epstein-Barr Virus-Induced Resistance to Drugs That Activate the Mitotic Spindle Assembly Checkpoint in Burkitt's Lymphoma Cells[END_REF]. Cell cycle progression without cytokinesis, G1 checkpoint disruption, and spindle assembly checkpoint disruption can lead to polyploidy upon EBNA3C or EBNA2 expression [START_REF] Pan | Epstein-Barr virus nuclear antigen 2 disrupts mitotic checkpoint and causes chromosomal instability[END_REF]. Expression of EBNA2 or LMP1 triggers cell re-entry into S phase. EBNA-1, EBNA-3, and EBNA-5 expression enhances the synthesis, transcription, and stability of HIF-1α, respectively [START_REF] Wakisaka | Epstein-Barr Virus Latent Membrane Protein 1 Induces Synthesis of Hypoxia-Inducible Factor 1α[END_REF][START_REF] Darekar | Epstein-Barr Virus Immortalization of Human B-Cells Leads to Stabilization of Hypoxia-Induced Factor 1 Alpha, Congruent with the Warburg Effect[END_REF]. In terms of telomere dysfunction, LMP1 expression is associated with an increase in telomeric aggregates, a decrease in total telomere number, an increase in multinucleated cells, and an increase in nuclear volume.

In brief, EBV infection and the expression of EBV-latent oncoproteins are closely linked to polyploidy, which can occur through various mechanisms, including disruption of cell cycle checkpoints, centrosome abnormalities, and telomere dysfunction [START_REF] Lajoie | LMP1 mediates multinuclearity through downregulation of shelterin proteins and formation of telomeric aggregates[END_REF][START_REF] Knecht | LMP1 and Dynamic Progressive Telomere Dysfunction: A Major Culprit in EBV-Associated Hodgkin's Lymphoma[END_REF]. These interactions contribute to the oncogenic potential of EBV in various malignancies.

KSHV

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a double-stranded DNA gamma-herpesvirus capable of infecting various cell types, including monocytes, dendritic cells, keratinocytes, B lymphocytes, oral epithelial cells, and endothelial cells [START_REF] Wen | Kaposi sarcoma-associated herpesvirus (KSHV): Molecular biology and oncogenesis[END_REF]. It exists in both lytic and latent forms and is responsible for several malignancies, including Kaposi sarcoma (KS), primary effusion lymphoma, multicentric Castleman disease, and plasmablastic lymphoma [START_REF] Goncalves | Kaposi sarcoma herpesvirusassociated cancers and related diseases[END_REF]. KSHV encodes several viral oncogenes, such as latencyassociated nuclear antigen (LANA), K cyclin, and viral FLICE-inhibitory protein (vFLIP), which play roles in inactivating tumor suppressor pathways, promoting cell cycle progression and DNA replication, as well as activating nuclear factor kappa B (NFκB) to support cell survival [START_REF] Radkov | The latent nuclear antigen of Kaposi sarcoma-associated herpesvirus targets the retinoblastoma-E2F pathway and with the oncogene Hras transforms primary rat cells[END_REF][START_REF] Zhu | γ-Herpesvirus-encoded miRNAs and their roles in viral biology and pathogenesis[END_REF].

In terms of polyploidy, KSHV infection induces a multinucleation state characterized by enlarged and irregularly shaped nuclei in various cell types [START_REF] Pan | Kaposi's Sarcoma-Associated Herpesvirus Induction of Chromosome Instability in Primary Human Endothelial Cells[END_REF]. Hypoxia, a condition of reduced oxygen availability, is linked to KSHV-induced polyploidy.

KSHV viral interferon regulatory factor 3 (vIRF-3) interacts with and enhances the activity of HIF-1α, leading to increased angiogenesis through vascular endothelial growth factor (VEGF) activation [START_REF] Shin | Kaposi's Sarcoma-Associated Herpesvirus Viral IFN Regulatory Factor 3 Stabilizes Hypoxia-Inducible Factor-1α to Induce Vascular Endothelial Growth Factor Expression[END_REF]. Regarding senescence, viral v-cyclin protein induces senescence during latent infection by disrupting the cell cycle and activating the DNA damage response. However, viral FLICE inhibitory protein (v-FLIP) can bypass senescence, allowing the growth and division of latently infected cell populations, which may include a subpopulation of polyploid cells [START_REF] Leidal | Subversion of Autophagy by Kaposi's Sarcoma-Associated Herpesvirus Impairs Oncogene-Induced Senescence[END_REF]. In summary, KSHV is associated with polyploidy through various mechanisms, including centrosome abnormalities, spindle checkpoint disruption, cell cycle deregulation, and interactions with cellular proteins involved in mitosis and senescence. These mechanisms contribute to the complex interplay between KSHV infection and the development of malignancies. This results in the clonal expansion of T cells harboring the HTLV-1 provirus, eventually leading to cellular immortalization and malignant transformation. HTLV-1 infection can lead to the formation of large lymphoma cells, especially in the presence of Tax [START_REF] Panfil | Human T-cell leukemia virus-associated malignancy[END_REF][START_REF] Mohanty | Mechanisms of Oncogenesis by HTLV-1 Tax[END_REF]. Specifically, HTLV-1-induced multinucleated giant cells, characterized by giant lobulated nuclei or enlarged nuclei, have been observed in mammalian cells with elevated Tax expression [START_REF] Grassmann | Molecular mechanisms of cellular transformation by HTLV-1 Tax[END_REF][START_REF] Ducu | The HTLV-1 Tax oncoprotein represses Ku80 gene expression[END_REF][START_REF] Liang | Human T-Lymphotropic Virus Type 1 Oncoprotein Tax Promotes S-Phase Entry but Blocks Mitosis[END_REF][START_REF] Spencer | Human T Cell Leukemia Virus Type 1 Oncoprotein Tax Targets the Human Mitotic Checkpoint Protein MAD1[END_REF]. HeLa cells expressing Tax also displayed larger cell sizes and DNA content greater than that of cells lacking Tax expression [START_REF] Yang | Complex Cell Cycle Abnormalities Caused by Human T-Lymphotropic Virus Type 1 Tax[END_REF]. HTLV-1-infected CD4+ cell clones exhibited enlarged, wellseparated nuclei in binucleated and multinucleated cells, and this morphological feature correlated with Tax expression levels [START_REF] Sibon | HTLV-1 propels untransformed CD4+ lymphocytes into the cell cycle while protecting CD8+ cells from death[END_REF]. Thus, HTLV-1 plays a significant role in inducing polyploidy, leading to the formation of multinucleated cells and contributing to the pathogenesis of ATL. This occurs through various mechanisms involving disruption of the mitotic checkpoint, alteration of cell cycle regulation, and enhanced HIF-1α expression.

HBV

Hepatitis B virus (HBV), as the prototype member of the Hepadnaviridae family, is an enveloped virus with partly double-stranded DNA. It shares some characteristics with retroviruses, involving the use of reverse transcriptase during its replication cycle [START_REF] Caballero | Hepatitis B virus: The challenge of an ancient virus with multiple faces and a remarkable replication strategy[END_REF]. HBV is primarily a hepatotropic virus, with hepatocytes being its major target and the confirmed site for HBV replication. Infection and replication of HBV in the liver can lead to various complications, ranging from acute hepatitis to severe conditions like liver failure and hepatocellular carcinoma (HCC) [START_REF] Liang | Hepatitis B: The virus and disease[END_REF]. In terms of oncogenic proteins, the hepatitis B virus X protein (HBx or pX) plays a significant role in promoting the progression of the cell cycle, inhibits the expression of tumor suppressor genes, including p53, and affects the transcription of methyltransferases [START_REF] Kew | Hepatitis B virus x protein in the pathogenesis of hepatitis B virus-induced hepatocellular carcinoma[END_REF]. It has been shown that HBx hinders the suppression of E2F1 activity, which is a key promoter of cell cycle progression, through the deactivation of the Rb gene promoter and consequently the tumor suppressor Rb.

Furthermore, HBx augments CDK2 activity, resulting in a disruption of the balance between E2F1

and Rb [START_REF] Sivasudhan | Hepatitis B Viral Protein HBx and the Molecular Mechanisms Modulating the Hallmarks of Hepatocellular Carcinoma: A Comprehensive Review[END_REF]. These functions suggest its potential involvement in the pathogenesis of HCC and cellular transformation [START_REF] Kew | Hepatitis B virus x protein in the pathogenesis of hepatitis B virus-induced hepatocellular carcinoma[END_REF]. Another oncogenic factor is the HBV large surface protein (LHBs), which exhibits properties that may contribute to hepatocarcinogenesis [START_REF] Musa | Hepatitis B virus large surface protein is priming for hepatocellular carcinoma development via induction of cytokinesis failure[END_REF]. Polyploidy, mainly endoreduplication and hyperploidy, have been exclusively identified in peripheral blood cells of individuals with chronic HBV infection when compared to HBV-negative individuals. Nuclear ploidy is positively correlated with poor prognosis and HBV infection [START_REF] Donne | Polyploidy in liver development, homeostasis and disease[END_REF][START_REF] Bou-Nader | Polyploidy spectrum: a new marker in HCC classification[END_REF]. Notably, DNA polyploidy is significantly increased in the liver tissues of HCC patients with a history of HBV infection [START_REF] Gramantieri | Alteration of DNA ploidy and cell nuclearity in human hepatocellular carcinoma associated with HBV infection[END_REF]. This observation is consistent with cellular models where HBV-positive hepatoma cell lines exhibit higher levels of hyperploidy than HBV-negative cells [START_REF] Li | Intrahepatic hepatitis B virus large surface antigen induces hepatocyte hyperploidy via failure of cytokinesis[END_REF]. Mechanistically, the expression of HBV pX can lead to the depletion of p53, suggesting an antagonistic relationship between p53 and pX-induced polyploidy [START_REF] Rakotomalala | Hepatitis B Virus X Protein Increases the Cdt1-to-Geminin Ratio Inducing DNA Re-replication and Polyploidy[END_REF]. Moreover, both LHBs and pX protein contribute to the override of the G2/M checkpoint. This results in the attenuation of the DNA damage checkpoint and inhibition of p53-mediated apoptosis. Furthermore, cells expressing HBV LHBs experience cytokinesis failure compared to controls [START_REF] Li | Intrahepatic hepatitis B virus large surface antigen induces hepatocyte hyperploidy via failure of cytokinesis[END_REF]. In HBV pX-induced polyploid cells, DNA re-replication occurs alongside aberrant mitotic spindle formation [START_REF] Studach | Polo-like Kinase 1 Activated by the Hepatitis B Virus X Protein Attenuates Both the DNA Damage Checkpoint and DNA Repair Resulting in Partial Polyploidy[END_REF]. This DNA rereplication is associated with the upregulation of Cdt1 and Cdc6, which are essential for prereplicative complex assembly [START_REF] Rakotomalala | Hepatitis B Virus X Protein Increases the Cdt1-to-Geminin Ratio Inducing DNA Re-replication and Polyploidy[END_REF]. Lastly, HBx interferes with the degradation of HIF-1α protein and enhances its synthesis [START_REF] Yoo | The carboxy-terminus of the hepatitis B virus X protein is necessary and sufficient for the activation of hypoxia-inducible factor-1α[END_REF][START_REF] Yoo | Hepatitis B Virus X Protein Enhances Transcriptional Activity of Hypoxia-inducible Factor-1α through Activation of Mitogen-activated Protein Kinase Pathway[END_REF]. Hence, HBV, especially through the actions of HBx and LHBs, is strongly linked to the induction of polyploidy, which can contribute to hepatocellular carcinoma development. This occurs through mechanisms involving the disruption of cell cycle checkpoints, cytokinesis failure, DNA re-replication, and interference with HIF-1α regulation.

HCV

Hepatitis C virus (HCV), a member of the Flaviviridae family, is a small, hepatotropic virus with a single-stranded RNA genome that is enveloped and replicates in the cytoplasm. Various cell types, including dendritic cells, epithelial cells, lymph nodes, and others, can support HCV replication [START_REF] Zhu | Hepatitis C virus infection is a risk factor for non-Hodgkin lymphoma: A MOOSE-compliant meta-analysis[END_REF]. Similar to HBV, HCV infection can lead to cirrhosis, severe liver disease, and ultimately HCC [START_REF] Moosavy | Epidemiology, transmission, diagnosis, and outcome of Hepatitis C virus infection[END_REF]. There is also a strong potential association between HCV infection and non-Hodgkin's B-cell lymphoma [START_REF] Zhu | Hepatitis C virus infection is a risk factor for non-Hodgkin lymphoma: A MOOSE-compliant meta-analysis[END_REF]. The development of HCV-mediated HCC is driven by long-standing hepatic inflammation, oxidative stress, and the potential for DNA damage.

Additionally, it involves the alteration of normal cellular signaling pathways and the stimulation of host cell growth, potentially mediated by specific HCV proteins, including the core proteins NS3, NS5A, and NS5B [START_REF] Banerjee | Oncogenic Potential of Hepatitis C Virus Proteins[END_REF]. Polyploidy has been observed in the context of HCV infection. In comparison to peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals, PBMCs from HCV-infected patients have been found to exhibit chromosomal polyploidy [START_REF] Machida | Hepatitis C Virus Causes Uncoupling of Mitotic Checkpoint and Chromosomal Polyploidy through the Rb Pathway[END_REF].

Furthermore, primary human and mouse hepatocytes infected with the HCV strain showed a higher frequency of polyploidy compared to non-infected cells. The expression of the viral core protein was found to induce extensive polyploidy in various cell lines, including the human liver cell line HepG2 and the embryonic kidney cell line HEK293. Moreover, it led to a two-fold increase in polyploidy in primary splenocytes, hepatocytes, and embryo fibroblasts isolated from transgenic mice [START_REF] Machida | Hepatitis C Virus Causes Uncoupling of Mitotic Checkpoint and Chromosomal Polyploidy through the Rb Pathway[END_REF]. The HCV core protein appears to trigger polyploidy by upregulating the transcription of HIF-1α mRNA [START_REF] Abe | Hepatitis C virus core protein upregulates the expression of vascular endothelial growth factor via the nuclear factor-κB/hypoxia-inducible factor-1α axis under hypoxic conditions[END_REF].

MCPyV

Merkel cell polyomavirus (MCPyV) belongs to a family of small, non-enveloped, icosahedral viruses with double-stranded DNA. MCPyV stands out as an oncogenic virus known to cause a rare and aggressive skin cancer called Merkel cell carcinoma (MCC) [START_REF] White | The Rapidly Expanding Family of Human Polyomaviruses: Recent Developments in Understanding Their Life Cycle and Role in Human Pathology[END_REF][START_REF] Prado | Human polyomaviruses and cancer: an overview[END_REF]. The key players in MCPyV-induced transformation and tumorigenesis are the transforming large T antigen (LTAg) and small T antigen (sTAg), which interact with important cellular factors disrupting the role of tumor suppressors like Rb and p53, contributing to the development of cancer [START_REF] Verhaegen | Merkel Cell Polyomavirus Small T Antigen Is Oncogenic in Transgenic Mice[END_REF][START_REF] Dalianis | Human polyomaviruses in disease and cancer[END_REF]. The expression of MCPyV sTAg has been shown to lead to the formation of multinucleated cells and an increase in the population of cells with more than four sets of chromosomes (>4N) in human diploid fibroblastic cells known as WI38 [START_REF] Kwun | Merkel cell polyomavirus small T antigen induces genome instability by E3 ubiquitin ligase targeting[END_REF]. Moreover, when sTAg was induced in C57BL/6 mice, it resulted in the development of poorly differentiated neoplasia. These tumors exhibited pleomorphic nuclei with variations in size and shape, along with multiple nucleoli [START_REF] Shuda | Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model[END_REF]. This phenomenon is similar to the induction of polyploidy observed in the context of John Cunningham Polyomavirus (JCPyV) and BK Polyomavirus (BKPyV) infections, as well as infection with simian virus 40 (SV40), which is a closely related oncogenic primate virus [START_REF] Herbein | Polyploid Giant Cancer Cells, a Hallmark of Oncoviruses and a New Therapeutic Challenge[END_REF]. In general, oncoviruses employ various mechanisms that can be considered as early events leading to polyploidy, tumor advancement, and diversity within tumors. Interestingly, HCMV shares many of these mechanisms involved in inducing polyploidy [START_REF] Herbein | Polyploid Giant Cancer Cells, a Hallmark of Oncoviruses and a New Therapeutic Challenge[END_REF][START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF][START_REF] Hertel | Global Analysis of Host Cell Gene Expression Late during Cytomegalovirus Infection Reveals Extensive Dysregulation of Cell Cycle Gene Expression and Induction of Pseudomitosis Independent of US28 Function[END_REF]. For example, HCMV infection leads to the generation of excess centrosomes and the formation of abnormal mitotic spindles [START_REF] Hertel | Global Analysis of Host Cell Gene Expression Late during Cytomegalovirus Infection Reveals Extensive Dysregulation of Cell Cycle Gene Expression and Induction of Pseudomitosis Independent of US28 Function[END_REF]. Moreover, HCMV proteins interact with the p53 protein, both in vitro and in vivo, resulting in the functional inactivation of p53. Additionally, HCMV affects the Rb protein through the HCMV UL97 protein, leading to Rb inactivation by phosphorylation, and it binds to pp71, which is part of the Rb protein family, ultimately causing their degradation [START_REF] Kwon | Inhibition of p53 transcriptional activity by human cytomegalovirus UL44: Inhibition of p53 by HCMV UL44[END_REF][START_REF] Iwahori | Human cytomegalovirus-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates and inactivates the retinoblastoma protein-related p107 and p130 proteins[END_REF].

Furthermore, HCMV has been shown to activate Myc at both the transcriptional and translational levels and induce the expression of HIF-1α [START_REF] Boldogh | Transcriptional activation of cellular oncogenes fos, jun, and myc by human cytomegalovirus[END_REF][START_REF] Kumar | The Human Cytomegalovirus Strain DB Activates Oncogenic Pathways in Mammary Epithelial Cells[END_REF][START_REF] Mcfarlane | Interaction of the human cytomegalovirus particle with the host cell induces hypoxia-inducible factor 1 alpha[END_REF]. HCMV infection also gives rise to multinucleated giant cells through cell fusion and upregulates the expression of G2/M transition regulators, including polo-like kinase 1 (Plk1) [START_REF] Hertel | Global Analysis of Host Cell Gene Expression Late during Cytomegalovirus Infection Reveals Extensive Dysregulation of Cell Cycle Gene Expression and Induction of Pseudomitosis Independent of US28 Function[END_REF]. These findings suggest a potential connection between HCMV infection, the induction of polyploidy, and the development of tumors. It's worth noting that there may be a possible link between oncogenic viruses, the presence of a polyploid phenotype, and resistance to therapy, metastasis, and disease recurrence, as discussed in previous reviews [START_REF] Herbein | Polyploid Giant Cancer Cells, a Hallmark of Oncoviruses and a New Therapeutic Challenge[END_REF]. This connection is strengthened by observations that chromosomal alterations coincide with morphological transformation [START_REF] Lehman | Early chromosome changes in diploid Chinese hamster cells after infection with simian virus 40[END_REF], and the multinucleated cell phenotype is triggered by the expression of viral oncoproteins rather than simple cell fusion, a phenomenon also observed with other non-oncogenic pathogens [START_REF] Liang | Human T-Lymphotropic Virus Type 1 Oncoprotein Tax Promotes S-Phase Entry but Blocks Mitosis[END_REF][START_REF] Lehman | Regulation of DNA synthesis in macrophages infected with simian virus 40[END_REF]. Additionally, the emergence of a blastomere-like cycle with "CMV-transformed HMECs" or CTH cells in HMECs long-term cultures infected with HCMV as well as the detection of polyploidy and oncogenic potential in CTH cells have been previously shown, by our laboratory [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF]. Several HCMV clinical strains were used to infect Human Mammary Epithelial Cells (HMECs) cultures which were grown for an extended period of time. Cultures of uninfected HMECs were terminated after fifty days due to cellular senescence, similarly the cultures that became senescent when infected with HCMV-SD, HCMV-FS, HCMV-KM, HCMV-RM, or HCMV-PJ strains. While all clinical isolates could be cultured for about two months in HMECs, only HCMV-BL strain, along with the previously described HCMV-DB, exhibited sustained growth. These cells were termed 'CMVtransformed HMECs' or CTH cells. In CTH cultures, several atypical cells with distinct morphologies were observed including giant cells with a blastomere-like appearance, numerous small round daughter cells, a combination of epithelial and mesenchymal cells, filopodia, and patterns indicating asymmetric cell division [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF]. All of these patterns may represent slow self-renewing cells undergoing various stages of the previously described giant cell cycle. This cycle consists of a series of synchronized events characterized by the emergence and dominance of enlarged giant cells, which then produce multiple small daughter cells. Furthermore, using confocal microscopy, common morphological features of PGCCs, characterized by their large size and giant nuclei, were detected, along with a broad range of cell sizes. Nehme et al. detected polyploidy in cultures infected with HCMV-BL as shown by flow cytometric analysis with propidium iodide staining. A significant percentage of cells with polyploidy, including polyploid giant cells (>4N), as well as other subpopulations like large tetraploid cells (LCs), intermediate cells (ICs), and diploid small cells (SCs) were observed. To assess the transforming potential of immortalized CTH cells, they performed a soft agar colony formation assay, which showed an increase in colony formation in CTH-BL and CTH-DB cells. This was associated with a significant increase in cell proliferation, as evidenced by a seven-fold rise in Ki67 antigen expression, increasing from 6.9% in control cells to 51.5% and 53.9% in CTH-BL and CTH-DB cells, respectively. Among these, PGCCs exhibited the highest level of Ki67 antigen expression [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF]. Consequently, there is a growing need for novel treatment approaches that not only disrupt the replicative machinery of viral infections and inhibit the immortalizing and transforming capabilities of oncoviruses but also target PGCCs as a therapeutic strategy.

Chapter 3 2). This concept aligns with the idea of oncomodulation, where disturbances in intracellular signaling pathways, transcription factors, and tumor suppressor proteins in the tumor environment create opportunities for HCMV to exert its oncomodulatory effects, potentially enhancing malignancy [START_REF] Herbein | The Human Cytomegalovirus, from Oncomodulation to Oncogenesis[END_REF][START_REF] Herbein | High-Risk Oncogenic Human Cytomegalovirus[END_REF]. The variability in detecting HCMV, ranging from no detection to a high percentage of positivity, can be attributed to the specific methods employed for its detection, including IHC, PCR, histopathology, serology, antigenemia, etc.. [START_REF] Razonable | Clinical Diagnostic Testing for Human Cytomegalovirus Infections[END_REF]. Hanahan and Weinberg updated their framework to include new additional hallmarks, and the list has continued to evolve to reflect our growing knowledge of cancer (Figure 7). Originally and in 2000, Douglas Hanahan and Robert Weinberg outlined six key hallmarks of cancer including sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, as well as activating invasion and metastasis [START_REF] Hanahan | The Hallmarks of Cancer[END_REF].

HCMV-Mediated Oncogenesis

Afterwards, in 2011, they expanded on the initial hallmarks and introduced two additional ones that involves reprogramming of energy metabolism and evading immune destruction [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF]. In 2022, Douglas Hanahan further incorporated two hallmarks, tumor-promoting inflammation and genome instability and mutation [START_REF] Hanahan | Hallmarks of Cancer: New Dimensions[END_REF] as depicted in the figure below. HCMV fulfills all the criteria associated with cancer hallmarks, as elaborated in the subsequent discussion below and in Table 3.

Sustaining Proliferative Signaling

One of the primary characteristics of cancer cells is their capacity for continuous and uncontrolled proliferation, leading to unlimited growth [START_REF] Feitelson | Sustained proliferation in cancer: Mechanisms and novel therapeutic targets[END_REF]. This phenomenon is primarily achieved through several mechanisms including independent production of growth factors, stimulation of neighboring tumor-associated cells to produce various growth factors that support cancer cells via paracrine signals, and the constant activation of downstream signaling pathways [START_REF] Gutschner | The hallmarks of cancer: A long non-coding RNA point of view[END_REF]. Notably, certain HCMV proteins, including IE1, IE2, pUL44, and pUL84, have been shown to play a role in regulating the tumor suppressor protein p53 by interacting with it and influencing p53-mediated transcription, thereby exerting control over the cell cycle at various checkpoints [START_REF] Castillo | Human Cytomegalovirus IE1-72 Activates Ataxia Telangiectasia Mutated Kinase and a p53/p21-Mediated Growth Arrest Response[END_REF][START_REF] Castillo | Role of Human Cytomegalovirus Immediate-Early Proteins in Cell Growth Control[END_REF][START_REF] Speir | Potential Role of Human Cytomegalovirus and p53 Interaction in Coronary Restenosis[END_REF].

HCMV IE1-72 hindered the ability of p53 to bind to its specific DNA sequences, whereas IE2-86 and/or UL84 augmented p53 binding, resulting in an altered DNA-protein complex [START_REF] Hwang | Human Cytomegalovirus IE1-72 Protein Interacts with p53 and Inhibits p53-Dependent Transactivation by a Mechanism Different from That of IE2-86 Protein[END_REF].

Additionally, the induction of the transcription factor NF-kB by HCMV IE proteins has been found to activate cell survival pathways in both normal and tumor cells. Further, HCMV significantly elevates the activity of mitogen-activated protein kinase (MAPK) p38 and ERK1/2 through various mechanisms [START_REF] Johnson | Activation of the Mitogen-Activated Protein Kinase p38 by Human Cytomegalovirus Infection through Two Distinct Pathways: a Novel Mechanism for Activation of p38[END_REF][START_REF] Boyle | Engagement of the Cellular Receptor for Glycoprotein B of Human Cytomegalovirus Activates the Interferon-Responsive Pathway[END_REF]. The expression of HCMV-US28 has been shown to enhance the secretion of biologically active VEGF and activate multiple cellular kinases, thereby promoting glioma growth and invasion [START_REF] Soroceanu | Human Cytomegalovirus US28 Found in Glioblastoma Promotes an Invasive and Angiogenic Phenotype[END_REF]. Moreover, it induces the production of IL-6 through NF-kB activation, resulting in the potent activation of the signal transducer and activator of transcription 3 (STAT-3) [START_REF] Slinger | HCMV-Encoded Chemokine Receptor US28 Mediates Proliferative Signaling Through the IL-6-STAT3 Axis[END_REF].

Evading Tumor Suppressors

By inactivating p53 function and Rb, DNA oncoviruses overpass the G1/S check point and force the cell to enter into the S phase, resulting in unregulated cell division and tumor formation.

Numerous studies have documented HCMV interference with the cell cycle, showing p53 and Rb being targeted. HCMV-IE1, and IE2 allow the evasion of tumor suppressors p53 and Rb [START_REF] Herbein | The Human Cytomegalovirus, from Oncomodulation to Oncogenesis[END_REF].

HCMV-IE1 and IE2 proteins interact with p53 in vitro and in vivo, transcriptionally inactivating the latter [START_REF] Hwang | Human Cytomegalovirus IE1-72 Protein Interacts with p53 and Inhibits p53-Dependent Transactivation by a Mechanism Different from That of IE2-86 Protein[END_REF][START_REF] Tsai | Human Cytomegalovirus Immediate-Early Protein IE2 Tethers a Transcriptional Repression Domain to p53[END_REF]. The aforementioned HCMV-IE proteins have the potential to deactivate the Rb protein family, which consequently stimulates the entry of cells into the S phase of the cell cycle [START_REF] Hume | Regulation of the retinoblastoma proteins by the human herpesviruses[END_REF]. Additionally, the IE1 protein drives cell cycle progression toward the G1/S transition point by increasing the expression of E2F-responsive genes. Among these genes are cellular regulators of the cell cycle, as well as essential enzymes required for DNA precursor synthesis and the initiation of cellular DNA replication, such as cyclin E and cdk-2 [START_REF] Song | Effect of the human cytomegalovirus IE86 protein on expression of E2F-responsive genes: A DNA microarray analysis[END_REF]. HCMV pUL97 phosphorylates and renders inactive proteins belonging to the Rb family, facilitating the promotion of the cell cycle. Additionally, the pUL82 protein works to reduce the levels of Rb family proteins.

One such protein is encoded by the viral UL82 gene and is termed pp71; HCMV pp71 protein targets the hypo-phosphorylated forms of the Rb proteins for proteasome-dependent degradation through a ubiquitin-independent pathway [START_REF] Kalejta | Proteasome-dependent, ubiquitin-independent degradation of the Rb family of tumor suppressors by the human cytomegalovirus pp71 protein[END_REF]. In HCMV-infected HMECs, there is a decrease in the detection of the Rb protein and an enhanced expression of the UL82 transcript [START_REF] Moussawi | The transcriptome of human mammary epithelial cells infected with the HCMV-DB strain displays oncogenic traits[END_REF]. Further, a study showed that the proliferation of IE1-expressing glioblastoma cells depends on p53 and Rb inhibition and PI3K/AKT activation [START_REF] Dziurzynski | Consensus on the role of human cytomegalovirus in glioblastoma[END_REF].

Activating Invasion and Metastasis

The capacity for invasion and formation of distant metastases are recognized as critical characteristics of tumor cells, regulated by a complex network of interactions and regulatory mechanisms. This enables them to intravasate into blood and lymphatic vessels while evading immune surveillance, displaying anchorage-independent growth. Subsequently, cancer cells extravasate from these vessels into their target tissues, where they establish micrometastases [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF].

The epithelial-mesenchymal transition (EMT) process plays a significant role in this cascade of invasion and metastasis [START_REF] Gutschner | The hallmarks of cancer: A long non-coding RNA point of view[END_REF]. Studies have shown that persistent HCMV infection of neuroblastoma cells leads to increased motility and adhesion of human endothelial cells [START_REF] Cinatl | Molecular mechanisms of the modulatory effects of HCMV infection in tumor cell biology[END_REF]. It's worth noting that the transmigration of tumor cells is facilitated by the focal disruption of endothelial cell integrity, mediated by the activation of β1α5 integrin on the surface of infected tumor cells. This phenomenon is accompanied by HCMV-induced downregulation of neural cell adhesion molecule (NCAM; CD56) receptors, which promotes transendothelial penetration [START_REF] Blaheta | Human Cytomegalovirus Infection of Tumor Cells Downregulates NCAM (CD56): A Novel Mechanism for Virus-Induced Tumor Invasiveness[END_REF].

HCMV infection augments glioma invasiveness by enhancing extracellular matrix-dependent migration and invasion, specifically through the phosphorylation of FAK at Tyrosine397, which is observed exclusively in human malignant glioma cells [START_REF] Cobbs | Human cytomegalovirus induces cellular tyrosine kinase signaling and promotes glioma cell invasiveness[END_REF]. In neuroblastoma cell lines persistently infected with HCMV, gene microarray analysis has revealed an upregulation of genes associated with cancer cell invasion [START_REF] Hoever | Impact of persistent cytomegalovirus infection on human neuroblastoma cell gene expression[END_REF]. Furthermore, vIL-10 and US28 will enhance cancer cell invasion and metastasis [START_REF] Herbein | Tumors and Cytomegalovirus: An Intimate Interplay[END_REF].

Enabling Replicative Immortality

It is widely acknowledged that cancer cells possess the capability for endless replication. This immortality is achieved by their capacity to overcome two significant barriers to proliferation: senescence and crisis, with the latter eventually resulting in cell death [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF]. Additionally, tumor cells find ways to evade the loss of telomeres, which protect the ends of chromosomes, thereby preventing telomere erosion and the entry into crisis, enabling them to sustain unlimited proliferation [START_REF] Gutschner | The hallmarks of cancer: A long non-coding RNA point of view[END_REF]. HCMV has been demonstrated to trigger the continuous expression of human telomerase reverse transcriptase (hTERT) and the activation of telomerase. In this context, CMV-IE and hTERT proteins were observed to co-localize in human primary glioblastoma multiforme cells, and their levels correlated with each other. This effect is facilitated by IE1's ability to stimulate telomerase activity and promote telomere lengthening through a specific interaction with the hTERT promoter [START_REF] Straat | Activation of Telomerase by Human Cytomegalovirus[END_REF]. Previously, in our laboratory, the infection of HMECs with HCMV-DB resulted in the overexpression of hTERT mRNA and increased telomerase activity [START_REF] Kumar | The Human Cytomegalovirus Strain DB Activates Oncogenic Pathways in Mammary Epithelial Cells[END_REF].

Inducing Angiogenesis

During the progression of tumors, an "angiogenic switch" is triggered to ensure the continuous development of new blood vessels that serve not only to sustain tumor growth by providing essential nutrients and oxygen but also facilitate the removal of metabolic waste products and enable the tumor to enter the process of hematogenous metastasis [START_REF] Gutschner | The hallmarks of cancer: A long non-coding RNA point of view[END_REF]. This process primarily involves the induction of pro-angiogenic factors or the suppression of antiangiogenic signals [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF].

HCMV typically promotes angiogenesis through both direct and indirect mechanisms. Infections occurring near blood vessels may stimulate angiogenesis by releasing angiogenic factors or by increasing local inflammation [START_REF] Caposio | The role of cytomegalovirus in angiogenesis[END_REF]. When fibroblasts and endothelial cells are infected, they promote the synthesis and release of various angiogenic factors that facilitate multiple stages of angiogenesis [START_REF] Dumortier | Human Cytomegalovirus Secretome Contains Factors That Induce Angiogenesis and Wound Healing[END_REF]. US28-induced angiogenic activity, characterized by the production of VEGF, is initiated through an increase in cyclooxygenase-2 (COX-2) expression mediated by US28, which activates the NF-κB pathway [START_REF] Maussang | The Human Cytomegalovirus-Encoded Chemokine Receptor US28 Promotes Angiogenesis and Tumor Formation via Cyclooxygenase-2[END_REF]. Furthermore, HCMV infection of endothelial cells induces an angiogenic response through viral binding to the epidermal growth factor receptor, as well as β1 and β3 integrins, leading to the activation of the phosphatidylinositol 3-kinase (PI3k) and mitogen-activated protein kinase signaling pathways [START_REF] Bentz | Human CMV infection of endothelial cells induces an angiogenic response through viral binding to EGF receptor and β 1 and β 3 integrins[END_REF]. HCMV also infects lymphatic endothelial cells and induces the release of a set of molecules that promote angiogenesis and lymphangiogenesis. These molecules include IL-6, granulocyte-macrophage colony-stimulating factor, and IL-8 [START_REF] Fiorentini | Human cytomegalovirus productively infects lymphatic endothelial cells and induces a secretome that promotes angiogenesis and lymphangiogenesis through interleukin-6 and granulocytemacrophage colony-stimulating factor[END_REF][START_REF] Botto | IL-6 in human cytomegalovirus secretome promotes angiogenesis and survival of endothelial cells through the stimulation of survivin[END_REF]. In addition, HCMV induces the production of proinflammatory and angiogenic cytokines in retinal pericytes, contributing to angiogenesis in conditions such as retinopathy and congenital ocular disease [START_REF] Wilkerson | Retinal pericytes and cytomegalovirus infectivity: implications for HCMV-induced retinopathy and congenital ocular disease[END_REF].

Resisting Cell Death

In addition to sustaining continuous proliferation and manipulating cellular growth processes, cancer cells possess the ability to avoid pathways leading to cell death. While three primary pathways apoptosis, necrosis, and autophagy can potentially induce cell death, their activation needs to be regulated by tumor cells. Apoptosis, in particular, has been extensively studied in the context of malignant transformation, where the initiation of apoptosis is finely tuned by balancing pro-apoptotic and anti-apoptotic factors [START_REF] Hanahan | Hallmarks of Cancer: New Dimensions[END_REF][START_REF] Gutschner | The hallmarks of cancer: A long non-coding RNA point of view[END_REF]. Numerous HCMV proteins can modulate apoptosis through diverse mechanisms, ultimately resulting in the inhibition of both intrinsic and extrinsic apoptotic signaling pathways [START_REF] Brune | Inhibition of programmed cell death by cytomegaloviruses[END_REF]. For instance, pUL38, UL37, and US21 have been demonstrated to exert anti-apoptotic effects [START_REF] El Baba | Immune Landscape of CMV Infection in Cancer Patients: From "Canonical" Diseases Toward Virus-Elicited Oncomodulation[END_REF][START_REF] Terhune | Human Cytomegalovirus UL38 Protein Blocks Apoptosis[END_REF][START_REF] Luganini | Human cytomegalovirus US21 protein is a viroporin that modulates calcium homeostasis and protects cells against apoptosis[END_REF]. Notably, HCMV encodes a cell deathsuppressing protein known as viral mitochondria-localized inhibitor of apoptosis (vMIA), which binds to Bcl2 associated X (BAX) and impedes BAX-mediated mitochondrial membrane permeabilization by sequestering BAX within the mitochondria [START_REF] Arnoult | Cytomegalovirus cell death suppressor vMIA blocks Bax-but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria[END_REF]. Another HCMV protein, pUL36, interacts with pro-caspase-8 thereby inhibiting apoptosis initiated through death receptors [START_REF] Chaudhry | UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene[END_REF]. Moreover, HCMV adopts strategies to evade extrinsic pro-apoptotic pathways by downregulating the cell surface expression of death receptors, including tumor necrosis factor receptor 1 (TNFR1), TNF-related apoptosis-inducing ligand (TRAIL) receptor 1, TRAIL receptor 2, and Fas [START_REF] Seirafian | Human cytomegalovirus suppresses Fas expression and function[END_REF]. Additionally, the long non-coding RNA lncRNA2.7 directly interacts with the gene associated with retinoid/interferon-induced mortality-19 (GRIM-19), leading to the stabilization of mitochondrial respiratory chain complex I and the continuous production of ATP [START_REF] Reeves | Complex I Binding by a Virally Encoded RNA Regulates Mitochondria-Induced Cell Death[END_REF]. Furthermore, HCMV employs alternative mechanisms to block apoptosis, such as the expression of the anti-apoptotic Bcl-2 protein or the inhibition of p53-mediated apoptosis by HCMV-IE2 [START_REF] Collins-Mcmillen | Human Cytomegalovirus Promotes Survival of Infected Monocytes via a Distinct Temporal Regulation of Cellular Bcl-2 Family Proteins[END_REF][START_REF] Tanaka | Effects of Human Cytomegalovirus Immediate-Early Proteins on p53-mediated Apoptosis in Coronary Artery Smooth Muscle Cells[END_REF].

While the role of autophagy in cancer regulation is a subject of contention, an increasing body of research predominantly underscores its pro-survival function in promoting cancer progression and metastasis [START_REF] Das | Pro-survival autophagy: An emerging candidate of tumor progression through maintaining hallmarks of cancer[END_REF]. Autophagy has the capacity to limit viral infections, but persistent viruses have developed diverse strategies to evade or hinder multiple stages of the autophagic process [START_REF] Chen | The role of autophagy in viral infections[END_REF].

For instance, HCMV employs mechanisms to counteract autophagy, one of which involves TRS1 in addition to IRS1 that hinder autophagy by binding to Beclin 1 [START_REF] Chen | The role of autophagy in viral infections[END_REF][START_REF] Mouna | Analysis of the role of autophagy inhibition by two complementary human cytomegalovirus BECN1/Beclin 1binding proteins[END_REF]. Autophagy also regulates the properties of CSCs by contributing to the maintenance of stemness, induction of recurrence, as well as tumor resistance [START_REF] Yun | The Roles of Autophagy in Cancer[END_REF]. On the other hand, in early tumorgenesis, it acts as a tumor suppressor and survival pathway by preventing the accumulation of damaged proteins and organelles [START_REF] Li | Autophagy and autophagy-related proteins in cancer[END_REF]. Hence, autophagy plays dual roles in tumor promotion and suppression [START_REF] Yun | The Roles of Autophagy in Cancer[END_REF][START_REF] Li | Autophagy and autophagy-related proteins in cancer[END_REF].

Evading Immune Suppression

HCMV has developed various immune evasion tactics to manipulate the host immune system, enhancing its infection and spread within the host (Figure 8) [START_REF] El Baba | Immune Landscape of CMV Infection in Cancer Patients: From "Canonical" Diseases Toward Virus-Elicited Oncomodulation[END_REF] Compared to healthy tissues, higher LD content have been reported in cancer cells and cancerous tissues such as colorectal cancer, breast and prostate cancers, hepatocellular carcinoma, renal cell carcinoma and glioblastoma. Thereby, during the aggressive process of cancer cells, LDs can function as energy source to support distant dissemination of cells. Notably, not only LD density but also their motility was correlated with cancer aggressiveness [START_REF] Li | Lipid droplet and its implication in cancer progression[END_REF]. Further, HCMV infection disrupts cellular energy metabolism and induces significant alterations in metabolic processes including increased glucose uptake, elevated glycolysis, diversion of glucose carbon, and the induction of the Warburg effect [START_REF] Yu | Viral effects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection[END_REF]. HCMV-IE1 protein appears to modulate the mRNA levels, eliminating glucose transporter 1 (GLUT1) mRNA and increasing GLUT4 mRNA. The elevated GLUT4 in infected cells is driven onto the cell surface, verifying that glucose uptake increases regardless of Akt function [START_REF] Yu | Human Cytomegalovirus Activates Glucose Transporter 4 Expression To Increase Glucose Uptake during Infection[END_REF]. Further, HCMV-pUL38 is capable of initiating glycolytic activation and prompting the breakdown of specific amino acids [START_REF] Rodríguez-Sánchez | The Human Cytomegalovirus UL38 protein drives mTOR-independent metabolic flux reprogramming by inhibiting TSC2[END_REF]; HCMV-pUL13 targets mitochondrial cristae architecture to increase cellular respiration during infection [START_REF] Betsinger | The human cytomegalovirus protein pUL13 targets mitochondrial cristae architecture to increase cellular respiration during infection[END_REF].

3.2.9 Enabling Characteristics: Genomic Instability, Mutation, and Tumor-promoting Inflammation

Throughout the multistep progression of tumors, cancer cells often exhibit an increased rate of mutations, wherein a favorable mutant genotype provides a selective advantage to certain subclones of cells. This advantage supports their proliferation and eventual dominance within a local tissue environment [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF][START_REF] Hanahan | Hallmarks of Cancer: New Dimensions[END_REF]. Several mechanisms contribute to this phenomenon, including increased sensitivity to mutagenic agents, compromised surveillance systems responsible for monitoring genomic integrity (such as p53), as well as increased defects in detecting DNA damage and activating the repair mechanisms responsible for DNA damage repair and deactivating mutagenic substances. On the other hand, the inflammatory response associated with tumor growth has been linked to enhanced tumorigenesis and progression. This is attributed to the release of various chemicals, notably mutagenic reactive oxygen species, as well as factors promoting growth, survival, and angiogenesis. These factors collectively facilitate proliferative signaling, inhibit cell death, and enable processes like angiogenesis, invasion, and metastasis [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF].

In the context of HCMV infection, it has been shown that HCMV-UL76 induces DNA damage as well as the accumulation of chromosome abnormalities, including micronuclei, misaligned chromosomes, and issues like lagging and bridging [START_REF] Siew | Human cytomegalovirus UL76 induces chromosome aberrations[END_REF]. Further, viral replication activates the checkpoint response to DNA double-strand breaks, and this replication alters the localization of checkpoint proteins to the cytoplasm, thus inhibiting the signaling pathway [START_REF] Gaspar | Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins[END_REF]. Furthermore, lytic HCMV infection disrupts ataxia telangiectasia mutated protein (ATM)-related kinases and triggers DNA damage responses [START_REF] Luo | Human Cytomegalovirus Disrupts both Ataxia Telangiectasia Mutated Protein (ATM)-and ATM-Rad3-Related Kinase-Mediated DNA Damage Responses during Lytic Infection[END_REF]. Specifically, IE1 has been shown to activate ATM kinase [START_REF] Castillo | Human Cytomegalovirus IE1-72 Activates Ataxia Telangiectasia Mutated Kinase and a p53/p21-Mediated Growth Arrest Response[END_REF], while E2F1 contributes to the accumulation of the gamma histone variant H2AX (γH2AX)

during HCMV infection or IE protein expression [START_REF] Pickering | An E2F1-Mediated DNA Damage Response Contributes to the Replication of Human Cytomegalovirus[END_REF]. On the other hand, pUS28 exacerbates tumor-related inflammation by inducing the production of various proinflammatory mediators, including IL-6, regulated on activation normal T expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and fractalkine [START_REF] Maussang | Human cytomegalovirus-encoded chemokine receptor US28 promotes tumorigenesis[END_REF][START_REF] Casarosa | Constitutive Signaling of the Human Cytomegalovirus-encoded Chemokine Receptor US28[END_REF]. HCMV augments the recruitment of leukocytes and prompts the production of proinflammatory substances like IL-8 and IL-6, along with the expression of the leukocyte intercellular adhesion molecule 1 (ICAM-1) receptor [START_REF] Craigen | Human cytomegalovirus infection up-regulates interleukin-8 gene expression and stimulates neutrophil transendothelial migration[END_REF][START_REF] Visseren | Interleukin-6 production by endothelial cells after infection with influenza virus and cytomegalovirus[END_REF][START_REF] Moss | Intercellular adhesion molecule-1 expression in endothelial cells is activated by cytomegalovirus immediate early proteins[END_REF]. Furthermore, CMV infection generates intracellular reactive oxygen intermediates in smooth muscle cells and human diploid fibroblasts [START_REF] Speir | Role of Reactive Oxygen Intermediates in Cytomegalovirus Gene Expression and in the Response of Human Smooth Muscle Cells to Viral Infection[END_REF][START_REF] Mao | Suppressive effects of sirtinol on human cytomegalovirus (hCMV) infection and hCMV-induced activation of molecular mechanisms of senescence and production of reactive oxygen species[END_REF].

Tumor Promoting Microenvironment

As cancer evolves, it can resist immune clearance by prompting tolerance in the presence of tumor- Major signaling pathways stimulated by HCMV that modulate the immune landscape. HCMV-infected cells produce elevated levels of interleukin-6 (IL-6) that activates signal transduction via IL-6 receptor (IL6R)-STAT3 axis. US28, an active chemokine receptor, also plays a major role in activating STAT3 in cancer cells. The combination of STAT3 activation and the impact of HCMV on cancer cell apoptosis, invasion, migration, adhesion, angiogenesis, and immunogenicity significantly exacerbates malignancy. In contrast to p53 and Rb, the upregulation of Akt, Myc, PD-L1, and CCL2 strongly exerts immunosuppressive and oncomodulatory effects. HCMV-induced alterations in the TME may contribute to oncomodulation [START_REF] El Baba | Immune Landscape of CMV Infection in Cancer Patients: From "Canonical" Diseases Toward Virus-Elicited Oncomodulation[END_REF].

Inhibits expression of MCH I US2

Intracellular retention of NKG2D UL16

Induces expression of TGF-β1 IE2

Genome Instability and Mutation

Inhibits DNA damage repair HCMV infection

Increases mutation frequency pp65 and pp71

Induces chromosome aberrations in cell lines IE1 and UL76

Tumor Promoting Inflammation

Induces production of RANTES, fraktalkine, MCP-1 HCMV infection NF-κB activation & IL-6 production US28

HCMV and Its Association with Breast Cancer

Globally, breast cancer poses a formidable health challenge as it is the most prevalent malignancy accounting for a high number of cancer deaths in women [START_REF] Sung | Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[END_REF]. Breast cancer consists of a group of heterogeneous diseases driven by a multifactorial etiology involving genetic predisposition, hormones, and environmental factors; viruses are considered indisputable causal factors for nearly 20% of all human malignancies [START_REF] Hausen | Cancers in Humans: A Lifelong Search for Contributions of Infectious Agents, Autobiographic Notes[END_REF]. Triple-negative breast cancer (TNBC) comprises 15% of breast cancers globally [START_REF] Guarneri | Relapsed Triple-Negative Breast Cancer: Challenges and Treatment Strategies[END_REF]. Despite its susceptibility to standard chemotherapy, TNBC is highly invasive, has a high relapse tendency, and is associated with a poor overall prognosis [START_REF] Yin | Triple-negative breast cancer molecular subtyping and treatment progress[END_REF].

Recently, more significant advances include characterizing the molecular features of TNBC which will maximize the efficacy of certain chemotherapeutic agents and aid in actively exploring novel therapeutic targets [START_REF] Yao | Triple-negative breast cancer: is there a treatment on the horizon?[END_REF]. The majority of breast cancers originate from cells lining the milkforming ducts within the mammary gland, known as carcinomas [START_REF] Twite | Sequestration of human cytomegalovirus by human renal and mammary epithelial cells[END_REF]. Following childbirth,

HCMV can be detected in breast milk, potentially spreading to nearby mammary epithelial cells [START_REF] Herbein | The Oncogenic Potential of Human Cytomegalovirus and Breast Cancer[END_REF]. Moreover, HCMV has the ability to infect macrophages and induce an atypical M1/M2 phenotype that closely resembles the phenotype of tumor-associated macrophages. This alteration is linked to the release of cytokines that play a role in the initiation or promotion of cancer, particularly in cases of breast cancer with a poor prognosis [START_REF] Herbein | The Oncogenic Potential of Human Cytomegalovirus and Breast Cancer[END_REF]. Furthermore, HCMV antigens and DNA have been identified in tissue biopsies of individuals with breast cancer [START_REF] Rahbar | Low Expression of Estrogen Receptor-α and Progesterone Receptor in Human Breast Cancer Tissues Is Associated With High-Grade Human Cytomegalovirus Protein Expression[END_REF], and there have been reports of elevated serum HCMV IgG antibody levels occurring before the onset of breast cancer in certain women [START_REF] Cox | Breast cancer, cytomegalovirus and Epstein-Barr virus: a nested case-control study[END_REF]. Further, previous studies demonstrated the ability of HCMV-DB and HCMV-BL strains in transforming primary HMECs into CMV-Transformed HMECs (CTH) cells in vitro [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Kumar | The Human Cytomegalovirus Strain DB Activates Oncogenic Pathways in Mammary Epithelial Cells[END_REF]. CTH cells, which are slow self-renewing cells, undergo diverse stages of the giant cell cycle. They were shown to be heterogeneous, generate PGCCs, exhibit dedifferentiation, and display stemness and EMT/MET features [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF]. HCMV oncomodulation, molecular mechanisms, and ability to support PGCCs generation might underscore its contribution to oncogenesis, especially breast cancers. The heterogeneity of strains can be linked to distinct properties influencing the virus-transforming potential, cancer types induced, and patient's clinical outcomes.

3.4

The Potential Link Between HCMV and Glioblastoma GBM, a subtype of adult diffuse glioma, is a primary central nervous system (CNS) tumor presumed to arise from neuroglial stem cells or their progenitors in the subventricular zone [START_REF] Lee | Human glioblastoma arises from subventricular zone cells with low-level driver mutations[END_REF][START_REF] Louis | The 2021 WHO Classification of Tumors of the Central Nervous System: a summary[END_REF]. There has been a recent paradigm shift, with increasing reliance on molecular information for diagnostic classification and prognostication within gliomas, as seen in the most recent World Health Organization (WHO) classification of CNS tumors [START_REF] Louis | The 2021 WHO Classification of Tumors of the Central Nervous System: a summary[END_REF]. Despite the molecular evolution of GBM, it continues to be an incurable disease with poor survival. HCMV infects neural stem/progenitor cells, and human astrocyte [START_REF] Belzile | Human Cytomegalovirus Infection of Human Embryonic Stem Cell-Derived Primitive Neural Stem Cells Is Restricted at Several Steps but Leads to the Persistence of Viral DNA[END_REF][START_REF] Odeberg | Human Cytomegalovirus Inhibits Neuronal Differentiation and Induces Apoptosis in Human Neural Precursor Cells[END_REF][START_REF] Kossmann | Cytomegalovirus Production by Infected Astrocytes Correlates with Transforming Growth Factor-β Release[END_REF][START_REF] Luo | Human Cytomegalovirus Infection Causes Premature and Abnormal Differentiation of Human Neural Progenitor Cells[END_REF]. Although HCMV DNA and antigens, especially IE1, have been detected in GBM tissue [START_REF] Dziurzynski | Consensus on the role of human cytomegalovirus in glioblastoma[END_REF][START_REF] Mitchell | Sensitive detection of human cytomegalovirus in tumors and peripheral blood of patients diagnosed with glioblastoma[END_REF], there is no conclusive evidence about HCMV oncogenicity in GBM, and the mechanisms by which the virus might contribute/induce oncogenesis remain elusive. The expression of HCMV proteins and oligonucleotides in high percentage of low-and high-grade malignant gliomas was first reported by Cobbs et al. in 2002 [213]. Although these data do not establish a causal role for HCMV in glioma pathogenesis, existing data indicates that HCMV could facilitate glioma progression. A study showed that HCMV-IE1 protein was detected in 100% of glioblastomas and 82% of low-grade gliomas upon using immunohistochemistry. They further reported the detection of HCMVspecific oligonucleotides in the same areas of IE1 expression within the tumor. Their finding, as initially described by Cobbs et al. in 2002, is that HCMV IE1 and virus-specific oligonucleotides weren't detected in necrotic areas or outside the tumor margin [START_REF] Dziurzynski | Consensus on the role of human cytomegalovirus in glioblastoma[END_REF]. Numerous reports have provided evidence of HCMV's presence in GBM, although typically confined to a small subset of cells [START_REF] Santos | Impact of human cytomegalovirus on glioblastoma cell viability and chemotherapy treatment[END_REF]. Studies have also indicated that viral proteins can dysregulate cellular processes and intensify the malignancy of tumors [START_REF] El Baba | Immune Landscape of CMV Infection in Cancer Patients: From "Canonical" Diseases Toward Virus-Elicited Oncomodulation[END_REF][START_REF] Herbein | The Human Cytomegalovirus, from Oncomodulation to Oncogenesis[END_REF][START_REF] Dziurzynski | Consensus on the role of human cytomegalovirus in glioblastoma[END_REF][START_REF] Santos | Impact of human cytomegalovirus on glioblastoma cell viability and chemotherapy treatment[END_REF]. Additionally, it has been observed that HCMV IgG seropositivity is linked to reduced overall survival rates among GBM patients [START_REF] Foster | Human cytomegalovirus seropositivity is associated with decreased survival in glioblastoma patients[END_REF].

These discoveries hold substantial implications for GBM screening and treatment, as anti-HCMV IgG testing may offer valuable prognostic insights. A study revealed a significant association between HCMV seropositivity and diminished overall survival in GBM patients, especially among those with unmethylated MGMT. This observation suggests that prior HCMV infection may have a noteworthy impact on the outcomes of GBM patients [START_REF] Foster | Human cytomegalovirus seropositivity is associated with decreased survival in glioblastoma patients[END_REF]. Consequently, anti-HCMV antibodies may prove to be a valuable prognostic tool in the management of GBM patients.

Moreover, GBM cells exhibit HCMV proteins, presenting a unique opportunity for targeted therapy. A study utilized the universal intracellular targeted expression (UNITE) platform to develop a multi-antigen DNA vaccine encoding HCMV proteins pp65, gB, and IE1. The UNITE platform incorporates lysosomal targeting technology, involving the fusion of lysosome-associated membrane protein 1 (LAMP1) with target antigens. This study demonstrates compelling evidence of enhanced antigen presentation through both MHC-I and -II, resulting in a robust antigenspecific CD4 and CD8 T-cell response, along with a potent humoral response [START_REF] Adhikari | Development and Characterization of an HCMV Multi-Antigen Therapeutic Vaccine for Glioblastoma Using the UNITE Platform[END_REF]. Thus, this research underscores that vaccination with HCMV antigens elicits robust cellular and humoral immune responses, triggering significant anti-tumor activity and leading to improved survival in a mouse model of GBM. Lastly and most recently, it's worth mentioning that Moderna's RNA vaccine for HCMV infection (CMVictory mRNA-1647) is currently undergoing Phase 3 clinical trials (NCT05085366) [START_REF] Modernatx | A Study to Evaluate the Efficacy, Safety, and Immunogenicity of mRNA-1647 Cytomegalovirus (CMV) Vaccine in Healthy Participants 16 to 40 Years of Age[END_REF].

The Interplay Between HCMV and Ovarian cancer

Epithelial ovarian cancer (OC), the most common and life-threatening cancer amongst gynecologic malignancies, has a 5-year age-standardized survival rate of 30-40%. Nearly 75% of all OC cases are diagnosed at late stages [START_REF] Cox | Potential Impact of Human Cytomegalovirus Infection on Immunity to Ovarian Tumours and Cancer Progression[END_REF]. Despite the advances in treatment modalities, the overall survival remains low for stage III and stage IV accounting for 40% and 20%, respectively [START_REF] Delga | 30 Years of Experience in the Management of Stage III and IV Epithelial Ovarian Cancer: Impact of Surgical Strategies on Survival[END_REF][START_REF] Steinberga | Quality Indicators and Survival Outcome in Stage IIIB-IVB Epithelial Ovarian Cancer Treated at a Single Institution[END_REF]. Currently, infection-triggered chronic inflammation is recognized as a crucial mechanism in the progression and spread of OC. In alignment with this observation, recent studies have identified elevated expressions of HCMV proteins in OC tissue samples that have been associated with poor survival outcomes [START_REF] Paradowska | Detection and genotyping of CMV and HPV in tumors and fallopian tubes from epithelial ovarian cancer patients[END_REF]. One study revealed that HCMV-IE protein expression was present in 82% of OC cases and 36% of benign tumors, while pp65 was detected in 97% of EOC cases and 63% of benign tumors. Notably, pronounced HCMV-IE expression was correlated with a shorter median overall survival [START_REF] Yin | Detection of human cytomegalovirus in patients with epithelial ovarian cancer and its impacts on survival[END_REF]. Additionally, the presence of HCMV DNA was found in 70% of cancerous ovarian tissues, significantly exceeding the detection rate in benign tumor cases [START_REF] Paradowska | Detection and genotyping of CMV and HPV in tumors and fallopian tubes from epithelial ovarian cancer patients[END_REF]. These findings suggest that HCMV infection might potentially facilitate the progression of cancer. Within the ovarian cancer TME, active HCMV infection may directly contribute to OC progression. Once HCMV infects host cells, it initiates mechanisms to counteract key antiviral immune responses necessary for infection control. These mechanisms involve the secretion of immunosuppressive cytokines and the impairment of cell-mediated immune responses, which are also crucial for restraining tumor growth [START_REF] Cox | Potential Impact of Human Cytomegalovirus Infection on Immunity to Ovarian Tumours and Cancer Progression[END_REF]. Previously existing data showed the detection of HCMV-gB by PCR in approximately 50% of OC tissues, with 80% of these cases being late-stage invasive tumors, suggesting that HCMV infection within the TME could potentially promote cancer progression or metastasis [START_REF] Cox | Potential Impact of Human Cytomegalovirus Infection on Immunity to Ovarian Tumours and Cancer Progression[END_REF]. Another recent study by Radestad et al. explored the prevalence of HCMV in ovarian cancer and its association with clinical outcomes. Their research indicated higher levels of HCMV-IgG, HCMV-IE proteins, and pp65 proteins in OC patients with malignant or benign ovarian epithelial tumors compared to control subjects [START_REF] Rådestad | Impact of Human Cytomegalovirus Infection and its Immune Response on Survival of Patients with Ovarian Cancer[END_REF]. This observation underscores the need to investigate whether administering anti-HCMV treatment to OC patients experiencing active HCMV reactivation in their TME should be considered in future therapeutic approaches. In conclusion, HCMV proteins and nucleic acids are frequently detected at varying levels in high-grade serous ovarian carcinoma. The shorter median overall survival of patients with HCMV-IE and pp65 in their tumors underscores the imperative to further explore the role of HCMV in ovarian cancer patients. The cumulative body of evidence underscores the pivotal roles played by EZH2 and Myc in the initiation of cancer and maintenance of stem cell properties [START_REF] Wang | EZH2 depletion potentiates MYC degradation inhibiting neuroblastoma and small cell carcinoma tumor formation[END_REF]. Being the enzymatic subunit of polycomb repressive complex 2 (PRC2), EZH2 is a histone-lysine N-methyltransferase responsible of transcriptional silencing [START_REF] Cao | Role of Histone H3 Lysine 27 Methylation in Polycomb-Group Silencing[END_REF]. EZH2 dysregulation was associated with the development, progression, and therapeutic resistance of many tumors [START_REF] Veneti | Polycomb Repressor Complex 2 in Genomic Instability and Cancer[END_REF][START_REF] Kim | Targeting EZH2 in cancer[END_REF]. Additionally, it was shown to expand the stem cell pool and the tumor-initiating cells in glioma, breast, ovarian, and prostate cancer, hence enhancing accelerated initiation, metastasis, invasion and growth [START_REF] Gonzalez | EZH2 expands breast stem cells through activation of NOTCH1 signaling[END_REF][START_REF] Chang | EZH2 Promotes Expansion of Breast Tumor Initiating Cells through Activation of RAF1-β-Catenin Signaling[END_REF][START_REF] Wu | The histone methyltransferase EZH2 promotes mammary stem and luminal progenitor cell expansion, metastasis and inhibits estrogen receptor-positive cellular differentiation in a model of basal breast cancer[END_REF]. In breast cancer pathogenesis, EZH2 overexpression assumes a critical role as it suppresses the expression of early growth response 1 (EGR1), thus inhibiting EGR1-mediated tumorsuppressive signals [START_REF] Guan | EZH2 overexpression dampens tumor-suppressive signals via an EGR1 silencer to drive breast tumorigenesis[END_REF]. Moreover, EZH2 mediates the stability of ribosomal DNA by silencing PHACTR2-AS1, thereby fostering genomic instability and promoting growth and metastasis in breast cancer [START_REF] Chu | The EZH2-PHACTR2-AS1-Ribosome Axis induces Genomic Instability and Promotes Growth and Metastasis in Breast Cancer[END_REF]. Further, EZH2 was demonstrated to be overexpressed in GBM tissues harboring HCMV [START_REF] Ahani | Overexpression of enhancer of zeste human homolog 2 (EZH2) gene in human cytomegalovirus positive glioblastoma multiforme tissues[END_REF]. EZH2 was also shown to be recruited to the major immediate early promoter (MIEP) in CD14+ monocytes where HCMV establishes latent infection in vivo [START_REF] Rossetto | Cis and Trans Acting Factors Involved in Human Cytomegalovirus Experimental and Natural Latent Infection of CD14 (+) Monocytes and CD34 (+) Cells[END_REF].

Furthermore, several studies showed that EZH2 overexpression is associated with HGSOC [START_REF] Leitner | Clinical impact of EZH2 and its antagonist SMARCA4 in ovarian cancer[END_REF][START_REF] Jones | Histone Methyltransferase EZH2: A Therapeutic Target for Ovarian Cancer[END_REF]. A study reported that EZH2 degradation profoundly blocked ovarian tumor cell proliferation and tumorigenesis in vitro and in vivo [START_REF] Chen | EZH2 mediated metabolic rewiring promotes tumor growth independently of histone methyltransferase activity in ovarian cancer[END_REF]. Another study revealed that invading PGCCs possess strong EZH2 expression [START_REF] Zhang | Number of Polyploid Giant Cancer Cells and Expression of EZH2 Are Associated with VM Formation and Tumor Grade in Human Ovarian Tumor[END_REF]. Since EZH2 enhances cell proliferation, inhibits apoptosis, promotes angiogenesis, metastasis and therapy resistance in several tumors, inhibiting EZH2 suggests an effectual therapeutic strategy [START_REF] Coughlan | Exploiting epigenetic dependencies in ovarian cancer therapy[END_REF][START_REF] Reid | Morphologic and molecular correlates of EZH2 as a predictor of platinum resistance in high-grade ovarian serous carcinoma[END_REF].

EZH2 was identified as a downstream target of Myc oncogene, the latter coordinately regulating EZH2 through transcriptional and post-transcriptional mechanisms during tumor initiation and disease progression [START_REF] Sander | MYC stimulates EZH2 expression by repression of its negative regulator miR-26a[END_REF]. Myc has been shown to stimulate EZH2 expression through direct binding and activation of its promoter, as well as by suppressing its negative regulator, miR-26a, or by directly repressing miR-137 [START_REF] Sander | MYC stimulates EZH2 expression by repression of its negative regulator miR-26a[END_REF][START_REF] Nie | Dissecting transcriptional amplification by MYC[END_REF]. The Myc-miR-137-EZH2 pathway has been linked to cisplatin resistance in ovarian cancer [START_REF] Sun | miR-137 mediates the functional link between c-Myc and EZH2 that regulates cisplatin resistance in ovarian cancer[END_REF]. Additionally, bromodomain-4 protein (BRD4) was shown to positively regulate EZH2 transcription through Myc upregulation [START_REF] Wu | BRD4 Regulates EZH2 Transcription through Upregulation of C-MYC and Represents a Novel Therapeutic Target in Bladder Cancer[END_REF]. Interestingly, Myc activation has been widely reported in the progression of breast cancer, especially in triplenegative breast cancer and in tumors that exhibit drug resistance, much like aggressive medulloblastoma tumors where elevated Myc levels are associated with increased EZH2 expression [START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF][START_REF] Fallah | MYC-Driven Pathways in Breast Cancer Subtypes[END_REF]. By uncoupling DNA replication from mitosis, Myc overexpression can induce DNA replication, thus opening the door toward polyploidy [START_REF] Li | c-Myc Overexpression Uncouples DNA Replication from Mitosis[END_REF]. This intricate relationship between Myc, polyploidy, and cancer has been previously demonstrated [START_REF] Anatskaya | Polyploidy and Myc Proto-Oncogenes Promote Stress Adaptation via Epigenetic Plasticity and Gene Regulatory Network Rewiring[END_REF], with Myc correlating with nuclear pleomorphism in both primary and metastatic renal cell carcinomas.

Moreover, Myc has been found overexpressed in GBM; its expression correlates with glioma grade where 60-80% of GBM reveal elevated Myc levels [START_REF] Annibali | Myc inhibition is effective against glioma and reveals a role for Myc in proficient mitosis[END_REF]. In glioma cells, EZH2 knockdown depleted Myc expression [START_REF] Yu | The EZH2 inhibitor GSK343 suppresses cancer stem-like phenotypes and reverses mesenchymal transition in glioma cells[END_REF]. Further, Myc direct transcriptional regulation by EZH2 may establish a new mechanism underlying glioma cancer stem cell maintenance [START_REF] Suvà | EZH2 Is Essential for Glioblastoma Cancer Stem Cell Maintenance[END_REF]. Similarly, enhanced Myc expression have been observed in 20-50% of ovarian carcinoma [START_REF] Vivas-Mejía | c-MYC and Epithelial Ovarian Cancer[END_REF][START_REF] Bast | The biology of ovarian cancer: new opportunities for translation[END_REF].

Viral Variants and Their Oncogenic Potential

Viral variants acquire diverse capacities to manipulate host cell machinery, making them more or less efficient at promoting cellular transformation and cancer development. Some variants may be more efficient at evading the immune response, more prone to causing persistent infections, or more effective at disrupting cellular processes that regulate cell growth and division [START_REF] Latourrette | Determinants of Virus Variation, Evolution, and Host Adaptation[END_REF][START_REF] Dayaram | Effect of Transforming Viruses on Molecular Mechanisms Associated With Cancer[END_REF][START_REF] Bayliss | Masters of manipulation: Viral modulation of the immunological synapse[END_REF].

Oncoviruses could serve to emphasize the significance of HCMV in processes related to cellular transformation and oncogenesis. To start with, HPV strains were classified into high-and low-risk strains after being isolated from different lesions. These discoveries were shown to radically alter the tumor diagnosis, prognosis, and prevention approaches [START_REF] Reddout | High risk HPV types 18 and 16 are potent modulators of oral squamous cell carcinoma phenotypes in vitro[END_REF][START_REF] Pešut | Human Papillomaviruses-Associated Cancers: An Update of Current Knowledge[END_REF]. Low-risk HPV strains were generally not strongly linked to cancer development, for instance HPV-6 and HPV-11. On the other hand, certain high-risk HPV strains were strongly associated with the development of various cancers through persistent infections that may cause various cellular changes. The most notable high-risk HPV strains include HPV-16 and HPV-18, HPV-31, HPV-33, and HPV-45 [START_REF] Yousefi | An Update on Human Papilloma Virus Vaccines: History, Types, Protection, and Efficacy[END_REF][START_REF] Egawa | The low-risk papillomaviruses[END_REF].

Researchers have identified specific genetic sequences within high-risk HPV types that are associated with increased cellular transformation. The HPV E7 carboxyl terminus contains two copies of a CXXC motif that are separated by a 29-amino-acid spacer. This domain may function as a dimerization domain [START_REF] Münger | Mechanisms of Human Papillomavirus-Induced Oncogenesis[END_REF]. Like adenovirus E1A and SV40 T antigen, the HPV E7 proteins interact with pRB and the related "pocket proteins" p107 and p130 through a conserved LXCXE sequence within the conserved region 2 sequences thereby impairing their ability to control cellcycle dependent transcription [START_REF] Münger | Mechanisms of Human Papillomavirus-Induced Oncogenesis[END_REF][START_REF] Münger | Biological activities and molecular targets of the human papillomavirus E7 oncoprotein[END_REF]. These sequences often involve the viral E6 and E7 genes, which produce proteins that interfere with cellular regulatory mechanisms and contribute to the transformation of normal cells into cancerous cells. Variations in these genes can affect the severity of their oncogenic effects. Additionally, KSHV isolates were identified in patients of different geographical regions, indicating the importance of these newly isolated strains in developing better diagnostic procedures and novel treatment approaches in the context of KSHV-associated malignancies as well as enriching potential vaccine studies [START_REF] Bhutani | Kaposi Sarcoma-Associated Herpesvirus-Associated Malignancies: Epidemiology, Pathogenesis, and Advances in Treatment[END_REF][START_REF] Pérez | Diversity of human herpesvirus 8 genotypes in patients with AIDS and non-AIDS associated Kaposi's sarcoma, Castleman's disease and primary effusion lymphoma in Argentina[END_REF][START_REF] Moorad | Whole-genome sequencing of Kaposi sarcoma-associated herpesvirus (KSHV/HHV8) reveals evidence for two African lineages[END_REF]. The cytoplasmic tail of KSHV-K1 protein contains two SH2 binding motifs that together constitute an immunoreceptor tyrosine-based activation motif (ITAM) [START_REF] Wang | Immortalization of Primary Endothelial Cells by the K1 Protein of Kaposi's Sarcoma-Associated Herpesvirus[END_REF]. ITAM motif consisting of six conserved amino acid residues spaced precisely over 26-amino acid sequence,(D/E)X7(D/E)X2YX2LX7YX2L/I [START_REF] Lee | Identification of an Immunoreceptor Tyrosine-Based Activation Motif of K1 Transforming Protein of Kaposi's Sarcoma-Associated Herpesvirus[END_REF], is able to constitutively induce angiogenic and inflammatory responses via AKT and NFkB [START_REF] Cavallin | Molecular and Cellular Mechanisms of KSHV Oncogenesis of Kaposi's Sarcoma Associated with HIV/AIDS[END_REF].

Further, EBV was initially categorized into two primary sub-types, namely type 1 and type 2, based on the genetic sequences of two EBV-encoded genes, EBNA2 and EBNA3. Type 1 is widespread globally, whereas type 2 is primarily found in sub-Saharan Africa. Currently, more than 71 distinct EBV strains have been documented. These EBV variants exhibit varying replication characteristics, and individuals can potentially be infected with multiple strains. It has been observed that certain EBV strains possess a higher oncogenic potential compared to others. For instance, the EBV strain M81, derived from nasopharyngeal carcinoma (NPC), exhibits an elevated rate of replication in B cells and a remarkably strong tendency to infect epithelial cells [START_REF] Chakravorty | EBV-associated diseases: Current therapeutics and emerging technologies[END_REF][START_REF] Tsai | Spontaneous Lytic Replication and Epitheliotropism Define an Epstein-Barr Virus Strain Found in Carcinomas[END_REF]. Ongoing research efforts are increasingly delving into the diversity of EBV latent and lytic genes among the various EBV strains, aiming to discover high-risk EBV strains. This attempt holds the potential to identify individuals at a higher risk of infection and pave the way for the development of effective EBV vaccines and anti-EBV T-cell therapies [START_REF] Chakravorty | EBV-associated diseases: Current therapeutics and emerging technologies[END_REF].

Nonetheless, it is noteworthy that the oncogenic potency of HCMV clinical strains varies between low and high-risk strains. HCMV-DB and HCMV-BL have been classified as high-risk strains in which they possessed their oncogenic potentials in acutely infected HMECs in vitro showing sustained transformation [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF][START_REF] Kumar | The Human Cytomegalovirus Strain DB Activates Oncogenic Pathways in Mammary Epithelial Cells[END_REF][START_REF] Ahmad | Distinct Oncogenic Transcriptomes in Human Mammary Epithelial Cells Infected With Cytomegalovirus[END_REF]. Upon infection with HCMV high-risk strains, there was an increase in the activation of Myc and EZH2 as well as the induction of polyploidy. This activation of the mentioned axis was significantly more pronounced in CTH cells. Moreover, it has been previously shown that the high-risk HCMV-BL strain robustly induces Myc expression while suppressing p53 levels [START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF]. This can enhance the replicative potential of stem cells and the reprogramming of progenitor cells in breast cancer, as Myc is identified as a transcriptional target of p53 in mammary stem cells and is activated upon p53 loss. Furthermore, HCMV-DB increases pRb repression [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF], with the pRB-E2F pathway known to regulate the expression of EZH2 [START_REF] Bracken | EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer[END_REF]. Previously, in experiments involving the infection of HMECs with KM, FS, and TB40/E strains, no significant activation of molecular oncogenic pathways was observed upon acute infection, nor were CTH cells detected in cultures [START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF][START_REF] Ahmad | Distinct Oncogenic Transcriptomes in Human Mammary Epithelial Cells Infected With Cytomegalovirus[END_REF]. This was especially notable when compared to the high-risk HCMV-DB and BL strains. Consequently, it is reasonable to classify HCMV-KM, FS, and TB40/E strains as low-risk strains. This conclusion is consistent with the absence of colony formation in soft agar assays following seeding HMECs infected with HCMV-KM, FS, and TB40/E strains. This underscores the hypothesis that the HCMV high-risk strains, namely DB and BL differentially induce Myc upregulation and consequently stimulating EZH2 overexpression and polyploidy induction, thus pointing to the presence of the Myc/EZH2/PGCCs axis as a key factor underlying these observed results [START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF]. With regard to immune responses, Myc suppresses immune surveillance by modulating the expression of the innate immune regulator (CD47, also known as integrin-associated protein) and the adaptive immune checkpoint namely, programmed death ligand 1 (PD-L1, also known as CD274 and B7-H1) [START_REF] Li | The effects of MYC on tumor immunity and immunotherapy[END_REF]. Further, Myc suppresses thrombospondin-1 gene [START_REF] Chan | Thrombospondin-1 mimetics are promising novel therapeutics for MYC-associated medulloblastoma[END_REF], type 1 IFN [START_REF] Zhou | Silencing of Thrombospondin-1 Is Critical for Myc-Induced Metastatic Phenotypes in Medulloblastoma[END_REF], interleukin-2, and perforin [START_REF] Li | The effects of MYC on tumor immunity and immunotherapy[END_REF]. Hence, Myc initiates and maintains tumorigenesis through the modulation of immune regulatory molecules [START_REF] Casey | MYC regulates the antitumor immune response through CD47 and PD-L1[END_REF]. These high-risk strains were also characterized by • Characterize two HCMV strains, B544 and B693, isolated from TNBC biopsies and assess their transformation potential in vitro.

• Evaluate the resulting cellular phenotype and examine the genetic and molecular features induced by these strains.

• Examine the sensitivity of CTH cells to combination therapy using paclitaxel (PTX) and ganciclovir (GCV).

Objective 2:

With regards to GBM, since HCMV-DB and BL clinical strains previously transformed HMECs into CTH cells and as HCMV has been detected in malignant gliomas, we aimed to discover its transforming potential in human astrocytes (HAs) by setting the following objectives:

• Screening HCMV-DB and BL clinical strains for their transforming capacity in HAs and investigating the molecular and cellular characteristics of CMV-induced glioblastoma cells (CEGBCs) that emerge during long-term cultures.

• Examining the impact of temozolomide (TMZ), Ganciclovir (GCV), and the EZH2 inhibitor (GSK 343) on this glioblastoma model.

• Exploring the potential link between the triad of HCMV, CEGBCs and EZH2, as well as the potential interrelation with Myc in the context of glioblastoma carcinogenesis.

• Investigating the morphological and phenotypic characteristics of CEGBCs and assess the potential implication of Myc and EZH2 in both CEGBCs and GBM biopsies.

• Isolating eleven clinical HCMV strains from GBM biopsies, analyzing their oncogenic, stemness, and invasiveness properties when cultured on HAs, as well as evaluating the potential effectiveness of combination therapy in curtailing these effects.

Objective 3:

In the context of ovarian cancer and since HCMV-DB and BL clinical strains previously transformed HMECs and HAs into CTH and CEGBCs cells, respectively, we decided to investigate the potential association between HCMV infection and epithelial ovarian cancer, particularly in the case of high-grade serous ovarian cancer (HGSOC) through setting the objectives below:

• Assessing the transforming capacities of the two high-risk clinical strains namely, HCMV-DB and HCMV-BL, as well as exploring the molecular and cellular characteristics that emerged during long-term cultures of "CMV-transformed Ovarian cells" (CTO cells).

• Isolating clinical HCMV strains from HGSOC biopsies displaying high EZH2 expression and investigating their oncogenic and stemness properties when cultured on OECs.

• Evaluating the efficacy of EZH2 inhibitors in curtailing CTO's oncogenic and stemness features.

surface epithelial cells (HOSE cells or OECs, as mentioned in our study) were purchased from Innoprot (Derio, Spain), and cultivated in ovarian epithelial cell medium (serum-free) supplemented with ovarian epithelial cell growth supplement (OEpiCGS) and penicillin/streptomycin solution (P60132, Innoprot). To note that, HOSE cells or OECs were isolated from healthy human ovaries, as mentioned in the technical data sheet (P10982, Innoprot).

CTH and CTO cells emerging following chronic HCMV infection were cultured in the same condition as HMECs and OECs, respectively. With regards to CEGBCs isolation and growth, upon the appearance of large cellular clusters/structures in HAs cultures that were infected with HCMV-DB and BL isolates, clusters were gently detached. CEGBCs were initially cultivated in HAs medium complemented with low levels of fetal bovine serum (2%) as per the manufacturer's recommendations. At day 150 post-infection, since CEGBCs resemble stem cells compared to uninfected HAs, serum was excluded and this is to fit with the optimal conditions of serum-free stemness growth as requested for glioblastoma cell cultures [START_REF] Hong | Glioblastoma cell line-derived spheres in serumcontaining medium versus serum-free medium: A comparison of cancer stem cell properties[END_REF]. Serum was omitted at day 5 post-infection of HAs with the HCMV strains that were isolated from GBM biopsies. CTH, CEGBCs, and CTO cultures used in this study were maintained for at least 9 months in culture.

All cells were cultured under standard conditions (37°C, 5% CO2, 95% humidity). Cultures were verified as mycoplasma-free as determined by monthly screenings (VenorGem classic mycoplasma detection, Minerva Biolabs).

HCMV Isolates Growth and Detection

Clinical HCMV strains, namely HCMV-DB (GenBank KT959235), BL (GenBank MW980585), KM, and FS were isolated from patients that were hospitalized at Besançon University Hospital (France) as described previously [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Kumar | The Human Cytomegalovirus Strain DB Activates Oncogenic Pathways in Mammary Epithelial Cells[END_REF]. Data corresponding to TNBC biopsies and other HCMV strains were reported previously [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF]. Cell-free virus stocks and infections were performed as previously detailed [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF]; to prepare these stocks, HCMV strains were propagated in MRC5 cells for only few passages to avoid losing the ULb' region. It is worth mentioning that careful screening of our viral stocks was conducted to rule out the presence of other oncoviruses.

Infections of cells (HMECs, HAs, MRC5, and OECs), quantification of viral replication, and HCMV detection were performed as previously described [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF]. Briefly, the aforementioned cells (1x10 6 ) infection with the clinical isolates was performed at a multiplicity of infection (MOI) of 1. Cells were incubated at 37 °C for two hours, after which the inoculum was dis-carded, and the cell monolayer was washed three times using 1X PBS and afterwards covered with fresh medium. HAs (3x10 6 ) cells infection with HSV was performed at a MOI of 1.

For HCMV quantification, cell-free infectious supernatant was collected, DNA was isolated (EZNA Blood DNA Kit, D3392-02, Omega BIO-TEK, Norcross, GA) and real-time quantitative PCR (qPCR) was performed using a Stratagene Mx3005P thermocycler (Agilent Technologies, Santa Clara, CA) and IE1 primers. Where specified, UL69 as well as HCMV lncRNA4.9 were detected by the qPCR assay. qPCR was performed using a KAPA SYBR FAST Master Mix (KAPA BIOSYSTEMS, KK4601, Potters Bar, UK); reactions were activated at 95 °C for 10 min, followed by 50 cycles (15 seconds at 95 °C and 1 min at 60 °C). Results were collected and analyzed using MxPro qPCR software.

Microscopy

Olympus optical microscope (Olympus Corporation, Tokyo, Japan) and OPTIKA digital camera (Optica Microscopy, Ponteranica, Italy) were used to monitor the short and long-term cultures of infected HMECs, MRC5, HAs, and OECs including CTH, CEGBCs, and CTO cells.

Confocal microscopy imaging for all the aforementioned cells was performed as described previously [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF]. Briefly, cells were washed with 1X PBS, fixed and permeabilized (BD Cytofix/Cytoperm, 554722) and stained with primary antibodies and their corresponding secondary antibodies listed in Table 5. For visualization of the nucleus and the cytoplasm, DAPI (4′, 6′-diamidino-2phenylindole) and phalloidin staining respectively were performed according to the manufacturer's protocol. Post-staining, the slides were assessed using a 63× oil immersion objective lens with a Carl-Zeiss confocal microscope (Jena, Germany); images were analyzed using ZenBlue Software (Carl-Zeiss Microscopy GmbH).

Soft Agar Colony Formation Assay

Colony formation in soft agar seeded with uninfected HMECs, B544, B693, DB, and BL-infected HMECs, untreated and PTX/GCV-treated CTH cells, uninfected HAs, CEGBCs, uninfected OECs, and CTO cells was carried out as mentioned previously [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF]. MCF7 and MDA-MB231

were used as positive controls. Briefly, cells were assayed using Cell Biolabs Cytosolic Cell Transformation Assay kit (Colorimetric assay, CB135; Cell Biolabs Inc., San Diego, CA) as per the manufacturer's protocol. The provided MTT solution and a microplate reader (A570 nm) were used for detection and quantification of the formation of colonies in soft agar (Cayman Chemical, Ann Arbor, MI). Olympus optical microscope (Olympus Corporation, Tokyo, Japan) and OPTIKA digital camera (Optica Microscopy, Ponteranica, Italy) were used to observe the formed colonies.

Tumorsphere and Spheroid formation Assays

Tumorsphere formation by uninfected HMECs and HMECs infected with HCMV-B544 and HCMV-B693 was assayed using StemXVivo serum-free tumorsphere media (R&D Systems) supplemented with heparin (2 U/ml) (Sigma) and hydrocortisone (0.5 microg/ml) (Sigma)

following the manufacturer's protocol and as described previously [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF].

Spheroids of CEGBCs were prepared as described previously [START_REF] Guyon | A 3D Spheroid Model for Glioblastoma[END_REF][START_REF] Daubon | The invasive proteome of glioblastoma revealed by laser-capture microdissection[END_REF]. Single cells (1x10 4 ) isolated by accutase were seeded in a serum-free astrocytes medium containing methylcellulose.

Where indicated, drugs were mixed in serum-free astrocytes medium with 0.4% methylcellulose.

Treatment was renewed daily. Spheroid size and surface area were determined using the ImageJ software, taking the mean length of the major and minor axis of the spheroid at a given time point compared to the initial measurements at time zero. Spheroids of OECs were prepared as described previously [START_REF] Bilandzic | Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time[END_REF]. Single cells (1x10 4 ) isolated by accutase were seeded in a serum-free OECs medium containing methylcellulose.

Flow Cytometry Analysis

Cells (1×10 5 ) were collected from uninfected HMECs, HAs, OECs, as well as CTH cells, HCMVinfected HAs, CEGBCs, HCMV-infected OECs, and CTO cells including treated cells (where indicated). Cells were fixed, permeabilized, and stained as previously reported [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF]. Briefly, cells were fixed and permeabilized using 100 μl of BD Biosciences Cytofix/Cytoperm solution for 20 minutes at 4°C and washed with BD Biosciences 1X BD perm/wash solution. One half of the cells were stained using the respective primary antibody and the remaining cells were mock stained to identify secondary antibody background staining. Cells were incubated at 4°C for 1 hour in the dark, washed twice with BD Biosciences 1X BD perm/wash solution and incubated at 4°C for 1 hour in the dark with the corresponding secondary antibody. Cells were then washed twice and subjected to cytofluorometric analysis. For cell cycle analysis, uninfected OECs or CTO cells were washed in 1X PBS, fixed in 70% ethanol, and resuspended in 50 µg/ml propidium iodide (P3566, life technologies, Eugene, USA) with 0.1 mg/ml RNase (R4642, Sigma-Aldrich, Saint-Louis, MO, USA), then incubated at 37°C for 30 min as described previously. Cytofluorometric analysis was achieved using a BD LSRFortessa X-20 (BD Biosciences) flow cytometer. FACSDiva software (BD Biosciences) was used for data collection and analysis.

Western Blotting

Expression of IE1, pp65, Myc, EZH2, Akt, and pAkt in uninfected HAs and HCMV-infected HAs as well as IE1, pp65, Myc, EZH2, Sox2, Nanog, E-cadherin, and vimentin expression in uninfected OECs, HCMV-infected OECs and CTO cells were assessed as described previously [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF].

Densitometry using ImageJ 1.40 software (National Institutes of Health, Bethesda, MA, USA) was applied to quantify protein levels. β-actin was used as a loading control.

RT 2 Profiler PCR Array

The RT 2 profiler PCR array was performed as detailed previously [START_REF] Ahmad | Distinct Oncogenic Transcriptomes in Human Mammary Epithelial Cells Infected With Cytomegalovirus[END_REF]. Total RNA was extracted from uninfected HAs, and CEGBCs-DB and BL using an RNA extraction kit (EZNATotal kit I,Omega BIO-TEK). Afterwards, cDNA was synthesized using the SuperScript IV First-Strand Synthesis kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. RT 2 Profiler PCR Arrays for human oncogenes & tumor suppressor genes (PAHS502ZR) (from Qiagen,Germantown,MD,USA) were performed using Mx3005P real-time PCR system as per manufacturer's instructions. Fold regulation was analyzed using the delta-delta Ct method [START_REF] Ahmad | Distinct Oncogenic Transcriptomes in Human Mammary Epithelial Cells Infected With Cytomegalovirus[END_REF][START_REF] Schmittgen | Analyzing real-time PCR data by the comparative CT method[END_REF].

6.10 RNA Cross-linking Immunoprecipitation (RNA CLIP) assay RNA CLIP assay was performed on CEGBCs and uninfected HAs as previously reported [START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF][START_REF] Rossetto | Cis and Trans Acting Factors Involved in Human Cytomegalovirus Experimental and Natural Latent Infection of CD14 (+) Monocytes and CD34 (+) Cells[END_REF]. qPCR analysis of EZH2 immunoprecipitated samples (IP EZH2) and negative control (IP IgG) were normalized with respect to each input and expressed as (2(-ΔCt)) x100 (% Input) as previously reported [START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF].

Assessment of telomerase activity

Uninfected OECs and CTO cells were collected, washed with PBS, and resuspended in RIPA lysis buffer and 4% protease inhibitor on ice for 30 minutes. Samples were then centrifuged at 16,000 g for 30 minutes at 4°C and the protein concentration was determined using Bradford assay (DC Protein Assay kit, Bio-Rad Laboratories, Hercules, CA). Telomerase activity was assessed as described previously and according to the manufacturer's instructions.

6.12

Quantitative Reverse Transcription PCR (RT-qPCR)

In CTH cells, the detection of absolute mRNA levels of epidermal growth factor receptor (EGFR), cyclin dependent kinase inhibitor 2A (CNDK2A), cyclin D1 (CCND1), SOX2, Oct4, and Nanog was assessed by RT-qPCR. Furthermore, RT-qPCR was also used to detect the transcripts of IE1, UL69, Myc, EZH2, OLIG2, CD133, SOX2, CD44, EGFR, MET, HCMV lncRNA4.9, and cellular lncRNA HOTAIR in CEGBCs as well as the IE1, UL69, EZH2, Myc, Sox2, Nanog, and Oct4 transcripts in CTO cells. RT-qPCR was performed as detailed previously [START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF][START_REF] Ahmad | Distinct Oncogenic Transcriptomes in Human Mammary Epithelial Cells Infected With Cytomegalovirus[END_REF]. In brief, RNA was extracted from the biopsies or the aforementioned cells using the EZNA® Total RNA Kit I (Omega BIO-TEK, Norcross, GA, USA). Reverse transcription was carried out using the SuperScript IV First-Strand Synthesis kit (Invitrogen, Carlsbad, CA, USA). The expression of markers was assessed by performing real-time qPCR using a KAPA SYBR FAST Master mix (KAPA BIOSYSTEMS, KK4601) and specific primers according to the manufacturer's protocol.

The fold change expression was calculated by adopting the delta-delta Ct method [START_REF] Ahmad | Distinct Oncogenic Transcriptomes in Human Mammary Epithelial Cells Infected With Cytomegalovirus[END_REF].

Invasion Assay

Collagen invasion assay: Collagen I (Corning, New York, NY) of 1 mg/ml concentration was prepared in 1X PBS with 7.2mM NaOH and 0.1% HCl was added. Where indicated, treatments, were mixed directly into the collagen gels. Prepared spheroids were incubated on ice for 30 min and then separately selected, washed in PBS, and subsequently included in the collagen solution.

After 1 hour at 37°C in a cell incubator, serum-free astrocytes medium including the diverse treatments was added. Secondly, 3D astrocyte scaffold invasion assay: scaffolds were formed by HAs in hyperconfluent culture. Afterwards, CEGBCs-DB and BL spheroids were cultured on top of HAs scaffolds. Spheroids' core area and invasion area were measured using ImageJ software; protrusions were measured using OPTIKA Vision Pro software and manually quantified.

Treatments

Where indicated, CTH cells were treated with ganciclovir (GCV) and paclitaxel (PTX). CEGBCs-DB and BL were under GCV, temozolomide (TMZ), and EZH2 inhibitor (GSK343). Treatments was renewed every day. Further, CTO-DB and BL were treated with the two EZH2 inhibitors (GSK343 and EPZ6438). Treatment was renewed every 2 days. CTH, CEGBCs, and CTO cells were also treated with 12-O-45 tetradecanoylphorbol-13-acetate (TPA); treatment was renewed daily. The dosages corresponding to each drug were provided in Table 6.

6.15

Tumor Biopsies and HCMV isolation -Tumor breast biopsies (luminal tumor biopsies n=10 and basal tumor biopsies n=9) and adjacent healthy breast biopsies (n=8). The treatment, clinical outcome and pathological data for these two TNBC patients were described previously [START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF].

-GBM biopsies [n=37; O (6)-methylguanine DNA methyltransferase (MGMT) promoter methylated GBM biopsies n=17, and MGMT promoter unmethylated GBM biopsies n=20] as well as healthy brain biopsies (n=4). Clinical and biological data of the GBM patients were provided in Table 7. -Ovarian biopsies (n=25; HGSOC biopsies n=18, and adjacent non-tumoral biopsies n=7) were provided by the Regional tumor bank (BB0033-00024 Tumorothèque Régionale de Franche-Comté). Clinical data and treatments of the OC patients were provided in Table 8.

Genes and Transcripts Expression As Well As Polyploidy Detection Within Tumor Biopsies

RNA was extracted from TNBC biopsies (B544 and B693) followed by reverse transcription to measure EGFR, CNDK2A, CCND1, SOX2, Oct4, and Nanog mRNA expression by RT-qPCR.

Genomic DNA was isolated from GBM as well as OC biopsies, and HCMV presence was identified by qPCR using specific primers against IE1 and UL69 genes. Further, RNA was extracted from GBM and OC biopsies and reverse transcription was performed as reported previously. For GBM biopsies, expression of EZH2, Myc, Akt, and GAPDH was assessed by realtime qPCR using specific primers according to the manufacturer's protocol. For OC biopsies, the expression of EZH2, Myc, Akt, and β-2-Microglobulin was assessed by real-time qPCR using specific primers according to the manufacturer's protocol. Fold change expression in TNBC, GBM and OC biopsies versus healthy breast and brain biopsies as well as adjacent non-tumoral OC biopsies, respectively was calculated using the delta-delta Ct method.

In TNBC biopsies, hematoxylin and eosin slides were used to detect PGCCs presence based on Zhang et al. description, with PGCCs being defined as a cancer cell with a nucleus at least three times larger than that of a diploid cancer cell [START_REF] Zhang | Generation of cancer stem-like cells through the formation of polyploid giant cancer cells[END_REF]. PGCCs presence in HGSOC biopsies was confirmed by hematoxylin and eosin staining; briefly, PGCCs were counted in five hot spots of each tumor sample (magnification x400, field diameter of 250 µm and 100 µm, Zeiss Axiostar).

HCMV Isolation From Tumor Biopsies

HCMV strains, B544 and B693, were isolated from TNBC patient biopsies by mechanical tissue disruption and filtration of the frozen biopsy through a 0.45 µm filter and initially grown on MRC5 cells. The supernatant from MRC5 culture was filtrated through a 0.45 µm filter and used to infect

HMECs. The purity of our HCMV cultures was confirmed by ruling out the presence of other viruses (Epstein-Barr virus, human papillomavirus, Kaposi sarcoma herpesvirus and adenovirus).

Eleven HCMV-GBM strains were isolated from MGMT promoter methylated (n=4) and promoter unmethylated (n=7) GBM biopsies by mechanical tissue disruption and filtration of the frozen biopsy through a 0.45 µm filter and initially grown on MRC5 cells. The supernatant from MRC5 culture was used to infect fresh human astrocytes. The purity of our HCMV cultures was confirmed by ruling out the presence of other viruses (Epstein-Barr virus and human papillomavirus). Three nor Epstein-Barr virus (EBV) was detected in the OC biopsies.

Statistical Analyses

All quantitative results are reported as mean ± SD of the independent experiments. Statistical To assess the cellular environment achieved by HCMV, B544 and B693 were isolated from TNBC biopsies and grown in MRC5 cells, revealing a viral growth at days 3 and 5 post-infection (PI) (Figure 10A). At day 1 post-HMECs infection, HCMV-IE1 and pp65 were detected with confocal microscopy (Figure 10B). HCMV-B544 and B693 promoted the transformation of the infected HMECs toward CTH cells as previously reported [START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF]. Uninfected HMECs were used as controls which underwent cellular senescence in long-term cultures. At day 110 PI, we detected a wide variety of spheroids and giant cells distributed between round dense cells, flat, and elongated spindle-like cells. Afterwards, lipid droplet-packed cells, multinucleated giant cells, cell budding, and filopodium protrusions were observed in CTH cultures (Figure 10C,D). Hence, this population displayed mesenchymal and fibroblastic-like structures in addition to epithelial and small cells. The above-mentioned detailed cell morphology is close to that of CTH cells which were previously detected in HMEC cultures acutely infected with high-risk BL and DB strains [START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF]. Thus, the CTH-B544 and CTH-B693 heterogeneous cell population represent transformed and self-renewing cells that are engaged in distinct phases of the giant cell cycle [START_REF] Liu | The life cycle of polyploid giant cancer cells and dormancy in cancer: Opportunities for novel therapeutic interventions[END_REF][START_REF] Liu | Polyploid giant cancer cells: An emerging new field of cancer biology[END_REF].

CTH Cells Promote Embryonic Stemness and Develop an Epithelial/Mesenchymal Hybrid State

When cultured in serum-free tumorsphere medium, CTH-B544 and B693 cells gave rise to mammospheres at day 14 and expressed stemness markers versus uninfected HMECs (Figure 13A). CTH cells revealed a rise in CD44 and CD24 expression when compared to uninfected HMECs (Figure 13B), in line with CTH-DB and BL data. Activated expression of the embryonic stem cell markers, SSEA-4 and Nanog, was recognized in CTH-B544, CTH-B693, CTH-DB, and CTH-BL cells by performing flow cytometry (Figure 14A) and confocal microscopy imaging (Figure 14B,C). Moreover, CTH-B544 and B693 cells gained embryonic stem-like properties by highly expressing Oct4 and SOX2 as demonstrated by confocal microscopy imaging (Figure 14D,E). Similar to CTH-DB and BL, the newly discovered CTH cells showed an elevated expression of the stemness marker CD49f or Integrin alpha-6, and a limited EpCAM expression (Figure 15). CTH-B544 and B693 as well as CTH-DB and BL cells were positive for both vimentin and E-cadherin staining (Figure 15) thereby signifying their ability to dynamically oscillate between the epithelial-hybrid-mesenchymal spectrum as reported previously in tumors with poor prognosis [START_REF] Liao | Hybrid Epithelial/Mesenchymal State in Cancer Metastasis: Clinical Significance and Regulatory Mechanisms[END_REF][START_REF] Grosse-Wilde | Stemness of the hybrid Epithelial/Mesenchymal State in Breast Cancer and Its Association with Poor Survival[END_REF]. As a result, CTH-B544 and B693 cells exhibited the following two phenotypes: stemness and hybrid epithelial/mesenchymal phenotypes.

7.1.5

A Specific Molecular Landscape Unveiled in the Tumor Microenvironment of TNBC Harboring High-Risk HCMV To evaluate the variation in strains' aggressiveness, we compared the transcriptomic profile corresponding to the two high-risk biopsies. The absolute mRNA level of epidermal growth factor receptor (EGFR), cyclin dependent kinase inhibitor 2A (CNDK2A), cyclin D1 (CCND1), SOX2, Oct4, and Nanog was assessed by RT-qPCR. EGFR, a proto-oncogene that enhances cell proliferation and survival, the cell cycle regulator CNDK2A, the proliferation marker CCND1, and the three embryonic markers were overexpressed in biopsy 693 compared to biopsy 544 (pvalue ≤ 0.05 for all markers) (Figure 17A). Our results indicated that biopsy 693, from which we isolated the HCMV-B693 strain, is associated with higher tumor aggressiveness and poor prognosis.

Restricting Soft Agar Colony Formation, Controlling PGCCs Count and Proliferation by Paclitaxel and Ganciclovir Therapy

To assess the colony formation in soft agar, CTH cells were treated by PTX/GCV combination therapy to target the oncogenic cellular environment as well as HCMV. Breast cancer cell lines, MDAMB231 and MCF7 were used as positive controls. PTX/GCV treatment of CTH-B544 cells resulted in the disappearance of colonies; however, colony formation was restricted in treated CTH-B693 cells versus untreated cells (Figure 17B). Post therapy, the PGCCs count was reduced by 25% in CTH-B544 while it remained constant in CTH-B693 as measured by FACS (Figure 17C). Further, the proliferation of PGCCs was assessed by Ki67 Ag measurement using flow cytometric analysis; Ki67 Ag expression was reduced in PTX/GCV-treated CTH-B544, but not in CTH-B693 (Figure 17D). Our outcomes revealed that CTH-B544 cells are more responsive to PTX/GCV therapy compared to CTH-B693 cells implying that the latter exhibits aggressive behavior.

EZH2-Myc Driven Glioblastoma Elicited by Cytomegalovirus Infection of Human Astrocytes

HCMV Clinical Isolates Permissively Infect HAs Inducing Increased Myc and EZH2 Expression

The cellular environment induced by HCMV infection was assessed by studying the tropism of DB and BL high-risk HCMV strains (Figure 18) as well as KM and FS low-risk HCMV strains (Figure 19) to HAs. HCMV-DB and BL strains replicated in HAs with a burst of viral growth (6 logs for DB and 3 logs for BL) followed by occasional blips (Figure 18A and Figure 20). Acute infection was then confirmed through immediate early gene (IE1), pp65, and the late HCMV antigens detection (Figure 18B,C and Figure 19). In addition, IE1 and early/late gene (UL69) transcripts were detected in HAs infected with HCMV-DB and BL compared to controls (Figure 18D). At day 3 post-infection, Myc was overexpressed in HAs-DB and BL compared to controls (p-value = 0.09), mostly in HAs-DB. Elevated EZH2 expression was detected in HAs-DB and BL compared to uninfected HAs (p-value = 0.02) (Figure 18E). Myc and EZH2 transcripts were detected in HAs-DB and BL compared to controls (Figure 18F). Lower apoptosis levels were recognized with the two strains (Figure 18G) in line with Akt and pAkt-Ser473 upregulation as confirmed by western blot and FACS, particularly with HCMV-DB (Figure 18H). In contrast to the high-risk DB and BL strains, the low-risk FS and KM strains did not elicit any of the abovementioned behavior (Figure 19). Taken together, a Myc High EZH2 High molecular profile was observed with both high-risk strains, preferentially with HCMV-DB.

7.2.2

Emergence of a Glioblastoma-Like Phenotype With CEGBCs in HAs Chronically Infected With High-Risk HCMV Strains.

In contrast to the low-risk HCMV strains that didn't allow long-term replication in HA cultures and were senescent (Figure 19), HAs infected with HCMV-DB and BL were maintained in culture for an extended period of time (Figure 20). Around day 80-90 post-infection, dense cellular aggregates appeared in HCMV-BL and DB cultures with invasive-like cells irradiating from the main cellular structures resembling the formerly described "go or growth" phenotype of glioblastoma cells [START_REF] Engwer | WWU Münster, Institute for Computational und Applied Mathematics and Cluster of Excellence EXC 1003[END_REF] (Figure 21A,B). Cells with a glioblastoma-like phenotype were termed "CMV-Elicited GlioBlastoma Cells" or CEGBCs similar to the previously reported "CMV-Transformed Human mammary epithelial cells" or CTH cells [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Kumar | The Human Cytomegalovirus Strain DB Activates Oncogenic Pathways in Mammary Epithelial Cells[END_REF].

We next assessed the protein expression of EZH2 and Myc in CEGBCs in which increased expression levels were observed compared to controls (Figure 21C). CEGBCs characterization was achieved by assessing oncogenes, tumor suppressor genes and cell cycle genes. Oncogenes and cell cycle genes were mainly upregulated in CEGBCs-DB; however, tumor suppressor genes were down-regulated mostly in CEGBCs-BL (Figure 21D,E). CEGBCs-DB and BL were seeded on a soft agar to evaluate their tumorigenic potential and colony formation was detected; uninfected HAs and HAs infected with herpes simplex virus (HSV) showed no colony formation (Figure 21F). Primary GBM experiences the subtype switch during relapse, shifting from the proneural (PN) subtype to the mesenchymal (MES) one namely the proneural-mesenchymal transition (PMT), thus acquiring a therapy-resistant phenotype [START_REF] Fedele | Proneural-Mesenchymal Transition: Phenotypic Plasticity to Acquire Multitherapy Resistance in Glioblastoma[END_REF]. With regards to PMT markers, vimentin was elevated mostly in CEGBCs-DB, and to a lesser extent in CEGBCs-BL (Figure 21G). CD44, a widely accepted marker for cancer stem cells and a mesenchymal marker regulating both stemness and epithelial-mesenchymal plasticity, was shown to be predominantly upregulated in CEGBCs-DB (Figure 21H). EMT genes were mostly upregulated in CEGBCs-DB compared to CEGBCs-BL (Figure 21I). CEGBCs-DB were shown to be close to the transcriptome profile of mesenchymal glioblastoma (mGB) whereas CEGBCs-BL expressed more PN traits (mesenchymal markers: p-value (CEGBCs-DB:CEGBCs-BL) = 0.002; proneural markers: pvalue (CEGBCs-DB:CEGBCs-BL) = 0.06) (Figure 21J). Taking into account the proteomic and transcriptome data, CEGBCs-DB mostly displayed a mesenchymal phenotype compared to and Figure 23A,B). Hence, the identified stemness features in CEGBCs indicated their relevance to glioblastoma stem cells (GSCs). Assessing the spheroid formation potential of CEGBCs, spheroids were generated 24-48 hours post-seeding; no spheroid formation was detected in HAs infected with HSV (Figure 22B). Nestin and IE1 were concomitantly expressed in CEGBCs-DB and BL spheroids (Figure 22C).

7.2.3

CEGBCs Productively Infected With High-Risk HCMV Display Invasiveness CEGBCs from spheroids readily invaded astrocyte scaffolds, by aligning along and intercalating between astrocytes and penetrating all scaffold layers as measured by nestin detection. After 7 days, nestin was present in the spheroids' core and invasive part; cells were IE1 and nestinpositive. HCMV-IE1 was predominantly located in the spheroid core and present in the individual cells detaching from the core (Figure 22D). Uninfected HAs expressed GFAP in the absence of nestin. PGCCs and neural progenitor cell (NPC)-like cells, positive for nestin and IE1, were also present (Figure 22E,F); supernatants were positive for HCMV-IE1 indicating ongoing viral replication. Further, using a 3D collagen-invasion assay, invasiveness was noticed for CEGBCs-DB and to a lesser extent for CEGBCs-BL as measured by the invasion area (Figure 22G,H); the protrusions' number and length were also recorded (Figure 24). Within the CEGBCs-DB cultures, the majority of invading cells adopted a neural progenitor-like phenotype with a round small cell body and a long leading process characterized by high cell motility (Figure 25, left panel).

Cellular heterogeneity occurred among the invasive cells with random morphology in which low motility cells co-existed with highly motile cells (Figure 25). Three mechanisms of invasiveness were detected in CEGBCs-DB and BL cultures as recently reported [START_REF] Venkataramani | Glioblastoma hijacks neuronal mechanisms for brain invasion[END_REF] (Figure 26A,B, andC).

Filopodia and lamellipodia were also observed (Figure 26D). Microscopic images showing the high and low motility cells that are invading from the CEGBCs-DB spheroid; magnification x100, scale bar 100µm. Graphs showing the variation in the migration distance of the selected cells using ImageJ.

7.2.4

Detection of lncRNA4.9/EZH2 and HOTAIR/EZH2 Complexes in CEGBCs Cultures HCMV latency in CEGBCs cultures was established by IE1 expression that was observed at day 1 post-TPA treatment (Figure 27A) parallel to the detection of HCMV gene (lncRNA4.9) (Figure 27B). In agreement with the presence of the lncRNA4.9 gene in EZH2-expressing CEGBCs, we observed the interaction of HCMV lncRNA4.9 and cellular lncRNA HOX antisense intergenic RNA (HOTAIR) transcripts with EZH2 using RNA CLIP assay (Figure 27C,D). Cellular lncRNA HOTAIR transcript, reported as a poor prognostic factor in cancers [START_REF] Xin | LncRNA HOTAIR: A Potential Prognostic Factor and Therapeutic Target in Human Cancers[END_REF] was detected particularly in the EZH2 immunoprecipitated samples corresponding to CEGBCs-DB compared to CEGBCs-BL (Figure 27D).

Upregulation of EZH2 and Myc in HCMV-positive GBM Tissues

To further decipher the role of HCMV and EZH2-Myc pathway in vivo, we analyzed 37 GBM biopsies (MGMT promoter methylated n = 17 and MGMT promoter unmethylated n = 20) for the presence of HCMV as well as EZH2 and Myc expression. Tumor biopsies displayed an enhanced EZH2 and Myc expression in both MGMT promoter methylated and unmethylated tissues, particularly in MGMT promoter unmethylated ones (Figure 28B). HCMV was detected in all GBM samples (100%) (Table 7). In all GBM biopsies, there was a statistically significant strong correlation between Myc and EZH2 expression (Figure 28C). A significant strong correlation was found between HCMV presence (IE1 gene) and Myc/EZH2 expression in unmethylated GBM biopsies (r = 0.690, p-value = 0.001; r = 0.589, p-value = 0.006; respectively) (Figure 28D). In unmethylated GBM biopsies, HCMV presence (UL69 gene) strongly correlated with Myc/EZH2 expression (r = 0.507, p-value = 0.02 and r = 0.544, p-value = 0.01, respectively) (Figure 28E).

On the other hand, a weak to moderate correlation was detected between HCMV presence and Myc/EZH2 expression in methylated GBM biopsies (Figure 28D,E). Hence, we reported the detection of HCMV in GBM tumor biopsies displaying enhanced EZH2, Myc, and Akt expression (Figure 28 and Figure 29). 9). Following HAs infection with the eleven HCMV-GBM strains, we detected cell clusters with irradiating low and high motility cells displaying a neural progenitor-like phenotype (Figure 30B) parallel to the sustained viral replication confirmed by FACS (Figure 30C) and IE1 gene detection by qPCR (Figure 30D). Viral transcripts (IE1 and UL69) were detected in HAs infected with methylated and unmethylated HCMV-GBM strains compared to uninfected HAs (Figure 30E). Upregulated Myc and EZH2 proteins and transcripts were detected in HAs infected with methylated and unmethylated HCMV-GBM strains, unlike uninfected HAs (Figure 30F,G,H and Table 9). All HCMV-GBM isolates transformed HAs as measured by soft agar colony formation assay (p-value (UI HAs: HCMV-GBM) = 0.02; p-value (UI HAs:

HCMV-GBM-M) = 0.04, p-value (UI HAs: HCMV-GBM-UM) = 0 . 03) (Figure 30I). (A) Microscopic images of the spheroids generated from the isolated GBM HCMV strains; magnification ×100, scale bar 20 μm. Histograms representing the number of spheroids generated in all HCMV GBM strains as well as methylated and unmethylated HCMV-GBM strains; UI HAs were used as a control. Confocal microscopic images of SOX2 (B) and concomitant Nestin/IE1 (C) staining in spheroids generated from the isolated HCMV-GBM strains. Nuclei were counterstained with DAPI; magnification ×63, scale bar 10 μm. (D) Microscopic images showing the invasion potential of CEBGCs through protrusions and cell migration (red arrows); magnification ×200, scale bar 20 μm. (E) lncRNA 4.9 gene detection in the supernatants of HAs infected with the isolated HCMV-GBM strains using qPCR; UI HAs were used as a control. (F) Interaction of lncRNA4.9 and HOTAIR transcripts with EZH2 in CEGBCs-GBM using RNA cross-linking immunoprecipitation (CLIP) assay.

EZH2 inhibitor, TMZ, and GCV tritherapy Curtails CEGBCs Growth

Although TMZ is known as the first-choice chemotherapeutic agent in glioblastoma, TMZ resistance often becomes a limiting factor in effective glioblastoma treatment [START_REF] Singh | Mechanisms of temozolomide resistance in glioblastoma -a comprehensive review[END_REF][START_REF] Virrey | Gliomaassociated endothelial cells are chemoresistant to temozolomide[END_REF]. Herein, we evaluated EZH2 inhibitor GSK343, GCV and TMZ efficacy as single therapies, as well as bior tri-combination therapy on CEGBCs-DB, BL, and GBM spheroids (Figure 32). 33A and Figure 34A). The peak level of HCMV productive infection was 6 and 4 logs in OECs-DB and BL, respectively (Figure 33A). HCMV-IE1 and pp65 proteins were detected in OECs-DB and BL (Figure 33B). We noticed the existence of large-sized cells having large nuclei that were detected only in OECs chronically infected with the high-risk HCMV-DB and BL strains compared to uninfected OECs (Figure 36A) and OECs chronically infected with HCMV-KM and FS strains (Figure 34B). Further, around three months post-infection, cellular survival was noted only in the chronically infected OECs-DB and BL compared to OECs-KM and FS (Figure 35). The emerging cells were termed "CMV-Transformed Ovarian epithelial cells" or CTO similar to the transformed cells that were previously reported by our group, namely, "CMV-Transformed Human mammary epithelial cells" or CTH cells and "CMV-Elicited GlioBlastoma Cells" or CEGBCs [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Kumar | The Human Cytomegalovirus Strain DB Activates Oncogenic Pathways in Mammary Epithelial Cells[END_REF][START_REF] El Baba | EZH2-Myc driven glioblastoma elicited by cytomegalovirus infection of human astrocytes[END_REF]. After applying a morphology-based classification, cellular heterogeneity was detected in CTO-DB and BL cultures including giant cells, blastomeres, blastocytes, multinucleated, mesenchymal, budding, and lipid droplets-rich cells as well as cells exhibiting filopodia and cytoplasmic vacuolization (Figure 36B). Some of the aforementioned morphologies were further confirmed by confocal microscopy showing mainly multinucleated and mesenchymal cells, asymmetric division, budding, and cells with giant nuclei (Figure 36C). A high percentage of tetraploidization and polyploidy cells was detected in CTO-DB and BL compared to UI OECs (Figure 36D). CTO-small cells (SCs of 2 N) (Figure 36D). As a positive control, cobalt chloride (CoCl2) was used to induce PGCCs formation in OECs cultures (Figure 36D).

Since the blockade of tumor suppressors and decreased telomerase activity have been correlated with tetraploidization in several human cancers [START_REF] Davoli | Telomere-Driven Tetraploidization Occurs in Human Cells Undergoing Crisis and Promotes Transformation of Mouse Cells[END_REF], p53 and Rb expression as well as telomerase activity were assessed in uninfected OECs, CTO-DB, and CTO-BL (Figure 36E,F . 03) (Figure 36E). Notably, telomerase activity was relatively low or undetectable in CTO-DB and BL compared to uninfected OECs (p-value = 0 . 03) (Figure 36F).

Hence, p53 and Rb downregulation along with decreased telomerase activity are sufficient to drive polyploidization in CTO-DB and BL, as previously reported [START_REF] Davoli | Telomere-Driven Tetraploidization Occurs in Human Cells Undergoing Crisis and Promotes Transformation of Mouse Cells[END_REF]. . 03) (Figure 40C, and Figure 41B). Besides PGCCs appearance in CTO-DB and BL cultures, it's worth mentioning the appearance of spontaneous spheroids in CTO-BL cultures (Figure 40D). Further, in the presence of methylcellulose, spheroids were generated in CTO-DB and BL cultures (Figure 40E). Finally, CD44, a marker of stemness and invasiveness, was upregulated in CTO-DB and BL cultures compared to uninfected OECs (Figure 42). EMT fuels cancer progression, tumor cell invasion, and therapy resistance [START_REF] Dudás | Epithelial to Mesenchymal Transition: A Mechanism that Fuels Cancer Radio/Chemoresistance[END_REF]. PGCCs gain strong invasiveness and migration ability after they undergo EMT [START_REF] Zhao | High Migration and Invasion Ability of PGCCs and Their Daughter Cells Associated With the Nuclear Localization of S100A10 Modified by SUMOylation[END_REF]. 8) for the presence of HCMV, PGCCs count, as well as EZH2, Myc, and Akt expression (Figure 50, Figure 51, and Figure 52). PGCCs with giant or multiple nuclei were detected in the HGSOC biopsies (Figure 50A). HCMV was detected in 72% of HGSOC biopsies (Figure 50B). Elevated PGCCs count was mainly detected in HCMV-positive HGSOC biopsies (Figure 50C). HCMV-positive tumor biopsies displayed mostly an enhanced EZH2 and Akt with a limited Myc expression (Figure 50D, Figure 51, and Figure 52A). A significant positive correlation was found between HCMV presence and EZH2 as well as Akt expression (r = -0.598, p-value = 0 . 009 and r = -0.466, p-value = 0 . 05, respectively, based on Ct values) (Figure 50E, and Figure 52B). A significant positive correlation was detected between Akt expression and EZH2 as well as Myc expression (r = 0.420, p-value = 0 . 015 and r = 0.620, p-value = 0 . 006, respectively) (Figure 52B).

Among the eighteen HCMV-positive HGSOC biopsies, three biopsies with the highest EZH2 expression were considered for HCMV isolation. The three HCMV-OC strains were isolated by tissue disruption and filtration, and were subsequently grown in MRC5 cells showing a peak of viral load of 3 logs around day 7 post-infection (Figure 50F). Following OECs infection with the three HCMV-OC strains, HCMV presence was confirmed by the detection of IE1 gene in addition to the morphological changes that appeared in the OECs infected cultures, for instance, giant and multinucleated cells showing cell budding as well as mesenchymal cells (Figure 50F,G). Post-EZH2 inhibition, using GSK343, no HCMV replication was detected in addition to the absence of morphological heterogeneity in the infected cultures (Figure 50G,H). Upregulation of Ki67Ag, EZH2, and Myc expression was observed in CTO-HCMV-OC compared to uninfected OECs and GSK-treated CTO-HCMV-OC cells (Ki67Ag p-value (CTO:GSK343-CTO) = 0 . 01; EZH2 pvalue (CTO:GSK343-CTO) = 0 . 002; Myc p-value (CTO:GSK343-CTO) = 0 . 002). GSK343 reduced Ki67Ag, EZH2, and Myc expression by 56%, 79%, and 63%, respectively (Figure 50I). EZH2 inhibition reduced PGCCs percentage in infected cultures compared to untreated CTO cells (p-value = 0 . 002) (Figure 50J). Chapter 8

Discussion

Understanding the potential connections between viral infections and oncogenesis has been an ongoing area of research. In the context of breast cancer and to the best of our knowledge, our study demonstrated the oncogenic transformation and stemness potential of HCMV-B544 and B693 strains that were isolated from TNBC biopsies and indicated a differential treatment response depending on the HCMV strain present in the tumor. CTH-B544 and CTH-B693 cells are heterogeneous cellular populations that give rise to PGCCs, and display dedifferentiating phenotypes with stemness features as well as hybrid epithelial/mesenchymal phenotypes, resembling the morphological features found in aggressive cancers.

Myc activation has been widely described in breast cancer progression and can be used as a predictive marker for cancer staging, therapy resistance, and prognosis [START_REF] Fallah | MYC-Driven Pathways in Breast Cancer Subtypes[END_REF]. It is noteworthy that both B544 and B693 HCMV strains were isolated from EZH2 High Myc High -expressing TNBC [START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF], further indicating a potential link between the presence of these HCMV strains and cancer progression. The coupling of c-Myc overexpression with Akt pathway activation observed in CTH cells is in line with previous findings [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF]. Moreover, colony formation was detected in CTH-B544 and CTH-B693 cells associated with a high expression level of Ki67 Ag and c-Myc overexpression, thus revealing cellular transformation and their oncogenic potential which is consistent with the previously reported CTH data [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF]. Ki67, a prognostic biomarker in invasive breast cancer, is not only required for cell proliferation in tumors but is also strongly linked to tumor initiation, growth, and metastasis [START_REF] Davey | Ki-67 as a Prognostic Biomarker in Invasive Breast Cancer[END_REF]. PGCCs formation was promoted in hypoxic environments. It is worth noting that hypoxia inducible factor 1 alpha (HIF-1α) expression is induced by HCMV infection [START_REF] Mcfarlane | Interaction of the human cytomegalovirus particle with the host cell induces hypoxia-inducible factor 1 alpha[END_REF]. In PGCCs, the evaluation of metabolic reprogramming revealed the presence of PLIN4, a perilipin covering the lipid droplets especially in chemoresistant tumors [START_REF] Liu | Polyploid giant cancer cells: An emerging new field of cancer biology[END_REF]; the Warburg effect [380] and the involvement of the glycolytic pathway were also found to be induced in cancer environments and upon HCMV infection [START_REF] Liu | The life cycle of polyploid giant cancer cells and dormancy in cancer: Opportunities for novel therapeutic interventions[END_REF]. PGCCbearing CTH cells acquired embryonic-like stemness and an epithelial-mesenchymal hybrid phenotype. Studies have shown that the acquisition of the EMT and stemness properties lead to an increase in the invasiveness and the metastatic potential of cancer cells within tumors [START_REF] Liao | Hybrid Epithelial/Mesenchymal State in Cancer Metastasis: Clinical Significance and Regulatory Mechanisms[END_REF]. The embryonic stem cells transcriptional network is based on the presence of master pluripotency regulators, Oct4, SSEA-4, SOX2, and Nanog [START_REF] Hadjimichael | Common stemness regulators of embryonic and cancer stem cells[END_REF]. Besides mammosphere generation, CTH-B544 and CTH-B693 gained a stemness phenotype with increased expression of Oct4, SOX2, and Nanog promoting tumor progression; CTH cells were positively stained for SSEA4 which is associated with EMT and drug resistance [START_REF] Hadjimichael | Common stemness regulators of embryonic and cancer stem cells[END_REF]. SOX2, Nanog, and Oct4 expression was associated with poor differentiation, advanced cancer stages, and the worst outcomes in breast cancer patients [START_REF] Sridharan | Novel and Alternative Targets Against Breast Cancer Stemness to Combat Chemoresistance[END_REF]. Studies have implicated CD44 in breast cancer cell adhesion, proliferation, motility and migration, angiogenesis, and metastasis. Limited CD24 expression in breast cancer cells was shown to augment their growth and metastatic potential through a chemokine receptor response [START_REF] Hadjimichael | Common stemness regulators of embryonic and cancer stem cells[END_REF]. The CD44 high /CD24 low phenotype was recognized in CTH-B544 and B693 cells indicating a tumor-initiating phenotype similar to that of the highly tumorigenic breast cancer cells [START_REF] Velasco-Velázquez | The Role of Breast Cancer Stem Cells in Metastasis and Therapeutic Implications[END_REF]. The existence of an intermediate state between epithelial and mesenchymal phenotypes is considered a hybrid E/M state which is associated with elevated cellular plasticity, migration, stem-cell-like properties, metastatic potential, and therapy resistance [START_REF] Liao | Hybrid Epithelial/Mesenchymal State in Cancer Metastasis: Clinical Significance and Regulatory Mechanisms[END_REF]. In CTH cells, the coexistence of vimentin and E-cadherin resembled a partial EMT hence ensuring their plasticity while preserving the same tumor-propagating potential [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF]. Since in breast tumors CD49f was considered a marker for distant metastasis and recurrence, CD49f+/CD44 high /CD24 low CTH cells represented an aggressive phenotype which is associated with an increased risk for disease recurrence with poor clinical outcomes [START_REF] Ye | CD49f Can Act as a Biomarker for Local or Distant Recurrence in Breast Cancer[END_REF].

On the transcriptomic level, biopsy 693 overexpressed EGFR, CNDK2A, CCND1, SOX2, Oct4, and Nanog. It is known that EGFR promotes TNBC progression through JAK/STAT3 signaling [START_REF] Song | EGFR Promotes the Development of Triple Negative Breast Cancer Through JAK/STAT3 Signaling[END_REF], CNDK2A drives TNBC tumorigenesis [START_REF] Nie | CDK2-mediated site-specific phosphorylation of EZH2 drives and maintains triple-negative breast cancer[END_REF], CCND1 has a prognostic significance in TNBC [START_REF] Velasco-Velázquez | Examining the role of cyclin D1 in breast cancer[END_REF], and the three embryonic markers correlate with stemness, metastasis, tumor relapse, and poor clinical outcomes of TNBC [START_REF] Fultang | Regulation of cancer stem cells in triple negative breast cancer[END_REF]; therefore, we proposed that biopsy 693 maintains a tumor signature that is associated with an aggressive behavior predicting poor clinical outcome.

Studies reveal that the initiation of KSHV and EBV lytic cycles supported malignancies driven by the aforementioned oncogenic viruses [START_REF] Jary | Kaposi's Sarcoma-Associated Herpesvirus, the Etiological Agent of All Epidemiological Forms of Kaposi's Sarcoma[END_REF][START_REF] Münz | Latency and lytic replication in Epstein-Barr virus-associated oncogenesis[END_REF]. HCMV persistence was established in CTH cells by detecting IE1 and pp65 throughout long-term cultures [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF][START_REF] Kumar | The Human Cytomegalovirus Strain DB Activates Oncogenic Pathways in Mammary Epithelial Cells[END_REF]. High-risk strains express immediate-early (IE), early (E), and late (L) viral antigens including IE1 in agreement with a viral lytic cycle following the acute infection of permissive cells such as MCR5 cells. High-risk strains are detected in chronically infected cells, for instance CTH cells in our study, which is in line with the HCMV latency observed in Hodgkin's disease and Non-Hodgkin's lymphoma revealing the latent viral UL138 protein expression [START_REF] Mehravaran | Association of Human Cytomegalovirus with Hodgkin's Disease and Non-Hodgkin's lymphomas[END_REF]. Nonetheless, a dynamic state of latency is recommended by novel transcriptomic studies. Since HCMV develops a complex relationship with the host, to define lytic and latency phases, several studies used the ratio of replicative and latency genes as a phase indicator [START_REF] Forte | Cytomegalovirus Latency and Reactivation: An Intricate Interplay With the Host Immune Response[END_REF][START_REF] Collins-Mcmillen | Control of Immediate Early Gene Expression for Human Cytomegalovirus Reactivation[END_REF]. To further highlight the role of the HCMV-IE1 gene, a study showed the potential of HCMV in regulating stemness in glioblastoma cells by specifically increasing SOX2 and Nestin, thus upregulating stemness and proliferation markers [START_REF] Soroceanu | Cytomegalovirus Immediate-Early Proteins Promote Stemness Properties in Glioblastoma[END_REF].

A study revealed the potential of combining GCV with certain chemotherapeutic agents to suppress EBV-positive NPC tumor growth [START_REF] Feng W Hai | Chemotherapy Induces Lytic EBV Replication and Confers Ganciclovir Susceptibility to EBV-positive Epithelial Cell Tumors[END_REF]. In HPV-infected cervical cancer cells, cidofovir and cisplatin inhibited cellular proliferation, reduced E6 protein expression, and restored the activity of p53 [START_REF] Yang | Inhibition of antiviral drug cidofovir on proliferation of human papillomavirus-infected cervical cancer cells[END_REF]. A third study showed the effectiveness of anti-herpetic drugs, GCV and cidofovir, as single therapies or in combination with chemotherapy in treating KSHV-associated primary effusion lymphoma (PEL) [START_REF] Coen | KSHV Targeted Therapy: An Update on Inhibitors of Viral Lytic Replication[END_REF]. Based on our results, the heterogeneity of HCMV strains including their distinct behavioral aspects had a major impact on CTH cells' response post-therapy.

CTH-B544 cells were therapy sensitive whereas CTH-B693 cells displayed an aggressive behavior with lower sensitivity to PTX/GCV combination therapy. Generally, isolating distinct HCMV strains from tumors that possess potential prognostic biomarkers and behave differently depending on their own heterogeneity and various cell types may improve the diagnostic process and treatment options, provide effective follow-up strategies, and may be essentially pertinent in breast cancer pathophysiology and other adenocarcinomas, particularly of poor prognosis.

While our primary aim in this study was to investigate the potential link between breast cancer and the high-risk HCMV strains, we must not overlook the importance of the secondary goal of our research which was to examine the impact of these high-risk HCMV strains on the progression and clinical outcomes of GBM patients. Within the context of GBM, we presented the first experimental evidence for HCMV as a reprogramming vector, straight through the dedifferentiation of mature human astrocytes, and generation of CMV-Elicited Glioblastoma Cells (CEGBCs) possessing glioblastoma-like traits. HCMV counterparts the progression of the perceived cellular and molecular mechanisms succeeding the transformation and invasion processes with CEGBCs involved in spheroid formation and invasiveness. Our study was conducted to assess the potential transforming capacities of HCMV-DB and BL following the HAs infection which were previously classified as high-risk transforming strains [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF][START_REF] Herbein | Tumors and Cytomegalovirus: An Intimate Interplay[END_REF][START_REF] El Baba | Oncogenic and Stemness Signatures of the High-Risk HCMV Strains in Breast Cancer Progression[END_REF].

HAs infection with the high-risk HCMV-DB and BL strains resulted in a pro-oncogenic cellular environment and sustained growth of CEGBCs with soft agar colonies formation, unlike HAs infected with the low-risk HCMV-KM and FS strains that showed no transforming potentials and resulted in cell death in the long term cultures. CEGBCs displayed a "go and growth" phenotype in 2D monolayer cultures, dedifferentiated and displayed stemness as well as PMT features, and finally resulted in spheroid formation and invasion in 3D cultures. PGCCs appearance as well as cellular heterogeneity were previously allied to cultures of mammary epithelial cells infected with the high-risk HCMV strains [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Liu | The dualistic origin of human tumors[END_REF]. Similar to HMECs transformed with the high-risk HCMV strains, around day 80 post-infection, we observed the appearance of dense cell aggregates, followed by the emergence of a wide array of morphologically distinct cells in HCMV-DB and BL cultures. We named these cells CMV-elicited glioblastoma cells (CEGBCs) with reference to the CMV-transformed HMECs (CTH) cells. PGCCs, NPC-like, neuron-like and mesenchymallike cells were detected as well as filopodia, lamellipodia, and asymmetric cell division patterns.

The described patterns could be representative of self-renewing cells undergoing diverse stages of the previously described giant cell cycle [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Liu | The dualistic origin of human tumors[END_REF], although blastomere-like structures weren't so far detected as reported previously in CTH cells [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF]. A replication-competent virus susceptible to reactivation from latency upon TPA treatment has been detected in CEGBC cultures. Activation of the Myc/EZH2 axis was observed in acute and sustained chronic infection with both high-risk HCMV strains. In agreement with EZH2 activation by HCMV, we observed a direct interaction between EZH2 and HCMV lncRNA4.9 transcript, likewise between EZH2 and cellular lncRNA HOTAIR transcript, a poor prognosis oncogenic factor for glioma patients. In line with EZH2 and HCMV involvement in our glioblastoma model, combination triple therapy (GSK343/GCV/TMZ) curtailed the growth of CEGBCs-derived spheroids. In vivo, all GBM tumor biopsies were found to harbor HCMV with enhanced EZH2 and Myc expression, possessing a strong positive correlation between EZH2 and Myc expression as well as a strong correlation between EZH2/Myc and HCMV presence. Eleven HCMV-GBM strains were isolated from GBM tumors which acutely transformed HAs toward CEGBCs with increased EZH2/Myc expression that undergo dedifferentiation towards glioblastoma stem cells with spheroid formation and invasiveness capacities that could be curtailed by GSK343/GCV/TMZ triple therapy.

Among the mechanisms studied to transform HAs and promote disease progression in addition to poor prognosis in GBM, is the coupling of Myc and EZH2 overexpression as well as the depletion of retinoblastoma protein (Rb) which was observed in our study [START_REF] Suvà | EZH2 Is Essential for Glioblastoma Cancer Stem Cell Maintenance[END_REF][START_REF] Furnari | Malignant astrocytic glioma: genetics, biology, and paths to treatment[END_REF]. Although limited Myc upregulation and Rb downregulation were observed, none of the two low-risk HCMV-KM and FS strains transformed HAs as measured by soft agar colony formation in addition to the cell death observed in prolonged cultures. On the other hand, the high-risk clinical isolates HCMV-DB and BL can drive HAs towards oncogenic transformation in vitro. Our findings conform to the "astrocyte dedifferentiation theory" corresponding to glioblastoma origin [START_REF] Stiles | Glioma Stem Cells: A Midterm Exam[END_REF][START_REF] Beiriger | The Subventricular Zone in Glioblastoma: Genesis, Maintenance, and Modeling[END_REF][START_REF] Sher | Ezh2 Expression in Astrocytes Induces Their Dedifferentiation Toward Neural Stem Cells[END_REF]. In contrast to uninfected HAs, the distinct transcriptome profile including oncogenes, tumor suppressor genes and cell cycle genes facilitated the characterization of CEGBCs that possess a glioblastoma-like phenotype [START_REF] Sasmita | Biomarkers and therapeutic advances in glioblastoma multiforme[END_REF][START_REF] Senhaji | Molecular and Circulating Biomarkers in Patients with Glioblastoma[END_REF]. Stemness acquisition, commonly described in metastasis and poorly differentiated tumors [START_REF] Guerra-Rebollo | Targeting of replicating CD133 and OCT4/SOX2 expressing glioma stem cells selects a cell population that reinitiates tumors upon release of therapeutic pressure[END_REF][START_REF] Ben-Porath | An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors[END_REF][START_REF] Lou | Stage-specific embryonic antigen-4 as a potential therapeutic target in glioblastoma multiforme and other cancers[END_REF], is in accordance with previous findings where GB-generated spheroids are composed of glioma stem cells (GSCs). The concomitant presence of the stemness marker nestin and HCMV-IE1 was detected in the spheroid structures generated from CEGBCs, as reported for nestin in the cell lines derived from GBM [START_REF] Veselska | Nestin expression in the cell lines derived from glioblastoma multiforme[END_REF]. Highly motile Nestin/IE1-positive cells were spotted leaving the core which are similar to the neural-progenitor-like tumor cells detected in glioblastoma, especially the ones adopting the Lévy-like movement patterns [START_REF] Lee | Human glioblastoma arises from subventricular zone cells with low-level driver mutations[END_REF][START_REF] Veselska | Nestin expression in the cell lines derived from glioblastoma multiforme[END_REF].

The concomitant presence of viral proteins and nestin within transformed cells has been reported

for the two herpes oncoviruses EBV and KSHV [START_REF] Kondo | Epstein-Barr Virus Latent Membrane Protein 1 Induces Cancer Stem/Progenitor-Like Cells in Nasopharyngeal Epithelial Cell Lines[END_REF][START_REF] Li | Evidence for Kaposi Sarcoma Originating from Mesenchymal Stem Cell through KSHV-induced Mesenchymal-to-Endothelial Transition[END_REF]. In agreement with enhanced CD44 and CD133 expression in CEGBCs, their expression in glioblastoma stem cells correlates with cell proliferation, intra-tumor heterogeneity, invasion and poor prognosis in CD44-expressing glioma [START_REF] Guerra-Rebollo | Targeting of replicating CD133 and OCT4/SOX2 expressing glioma stem cells selects a cell population that reinitiates tumors upon release of therapeutic pressure[END_REF][START_REF] Xiao | CD44-Mediated Poor Prognosis in Glioma Is Associated With M2-Polarization of Tumor-Associated Macrophages and Immunosuppression[END_REF]. The presence of vimentin + /CD44 + cells in CEGBC cultures as well as the detection of stem cells expressing SOX2 and nestin confirms the PMT plasticity. In agreement with the proneural-mesenchymal plasticity described upon oncogenic stress activation highlighting astrocyte plasticity/reactivity during tumorigenesis [START_REF] Fedele | Proneural-Mesenchymal Transition: Phenotypic Plasticity to Acquire Multitherapy Resistance in Glioblastoma[END_REF][START_REF] Yabo | Cancer cell heterogeneity and plasticity: A paradigm shift in glioblastoma[END_REF][START_REF] Li | Rapid Reprogramming of Primary Human Astrocytes into Potent Tumor-Initiating Cells with Defined Genetic Factors[END_REF], a high invasive potential was observed in CEGBCs-DB compared to BL with increased mesenchymal traits indicating a more aggressive behavior that might drive therapeutic resistance. Accumulated evidence highlighted Myc and EZH2 as key players in both oncogenesis and stemness. Myc stimulates EZH2 expression by activating the EZH2 promoter [START_REF] Nie | Dissecting transcriptional amplification by MYC[END_REF], repressing miR-26a [START_REF] Sander | MYC stimulates EZH2 expression by repression of its negative regulator miR-26a[END_REF], or directly suppressing miR-137. Bromodomain-4 protein (BRD4) positively regulates EZH2 transcription through Myc upregulation [START_REF] Wu | BRD4 Regulates EZH2 Transcription through Upregulation of C-MYC and Represents a Novel Therapeutic Target in Bladder Cancer[END_REF]. Myc activation was reported in glioblastoma progression, particularly in poor prognosis and therapy resistant-tumors [START_REF] Nie | Dissecting transcriptional amplification by MYC[END_REF][START_REF] Chen | MYC-mediated upregulation of PNO1 promotes glioma tumorigenesis by activating THBS1/FAK/Akt signaling[END_REF].

EZH2 mediates proliferation, migration, and invasion in GBM. High-risk HCMV clinical strains DB and BL differentially induce Myc upregulation, and consequently stimulate EZH2 overexpression as well as CEGBCs induction, pointing toward the presence of Myc/EZH2/CEGBCs axis underlying the described results. Though, the interrelationship between HCMV and EZH2 is further complexed by the detection of HCMV lncRNA4.9 gene in CEGBCs which is in line with Rossetto et al. report [START_REF] Rossetto | Cis and Trans Acting Factors Involved in Human Cytomegalovirus Experimental and Natural Latent Infection of CD14 (+) Monocytes and CD34 (+) Cells[END_REF]. Consistent with our data, the cellular lncRNA HOTAIR was described to interact with EZH2 in glioblastoma, thus linked to tumor dissemination, PMT, and drug resistance [START_REF] Chen | MYC-mediated upregulation of PNO1 promotes glioma tumorigenesis by activating THBS1/FAK/Akt signaling[END_REF][START_REF] Yadav | LncRNAs associated with glioblastoma: From transcriptional noise to novel regulators with a promising role in therapeutics[END_REF]. The noticeable detection of high lncRNA HOTAIR in the EZH2 IP samples corresponding to CEGBCs-DB explicates the aggressiveness of this particular high-risk HCMV strain, predicting poor prognosis. Indeed, EZH2-mediated stemness could underlie the appearance and maintenance of CEGBCs expressing a high degree of embryonic stemness, as EZH2 expression in astrocytes induced their dedifferentiation toward stem-like cells expressing nestin, SOX2, and CD133 [START_REF] Sher | Ezh2 Expression in Astrocytes Induces Their Dedifferentiation Toward Neural Stem Cells[END_REF]. Further, we reported the detection of HCMV in GBM tumor biopsies displaying enhanced EZH2, Myc, and Akt expression. HCMV-induced Myc and EZH2 expression along with the embryonic stem-like phenotype in the IE1-expressing CEGBCs could establish a significant model in the context of GBM. Since EZH2 and Myc have been implicated in tumor initiation and proven to impact glioblastoma appearance and development with the two high-risk HCMV DB and BL strains isolated from biological fluids (cervical swab and urine respectively), we evaluated EZH2/Myc expression and recovered HCMV strains directly from GBM biopsies thereby assessing their oncogenic potential. Eleven HCMV strains were isolated from GBM tumors (with unmethylated and methylated MGMT promoters). After HAs infection, CEGBCs were generated with morphological features matching the previously described CEGBCs-DB and BL and led to the appearance of spheroids with invasiveness potential.

HCMV-IE1 protein detection parallel to stemness markers and the upregulated Myc and EZH2 expression parallel to the detection of lncRNA4.9 gene, lncRNA4.9 and HOTAIR transcripts in cultures infected with the eleven HCMV-GBM strains recapitulates the previously observed molecular phenotype induced by HCMV-DB and BL strains. The expression of Myc was predominantly elevated in IE1-positive HAs. Altogether, HCMV strains are present in GBM tumors retaining tumor-promoting abilities, therefore considered as oncogenic strains.

Highlighting the critical role of EZH2 and HCMV in our glioblastoma model, the impact of EZH2 inhibitor (GSK343) and anti-HCMV drug ganciclovir (GCV) alone and in combination with TMZ was assessed. TMZ possessed a very limited effect on the growth of CEGBCs spheroids derived from HCMV-DB, HCMV-BL and the eleven HCMV-GBM strains. In agreement with our results, valganciclovir possessed a positive effect on glioblastoma tumors with an unmethylated or methylated MGMT promoter gene [START_REF] Pantalone | Valganciclovir as Add-on to Second-Line Therapy in Patients with Recurrent Glioblastoma[END_REF], potentially through its antiproliferative effect [START_REF] Ding | Antiviral drug ganciclovir is a potent inhibitor of microglial proliferation and neuroinflammation[END_REF][START_REF] Zhang | Antiviral Drug Ganciclovir Is a Potent Inhibitor of the Proliferation of Müller Glia-Derived Progenitors During Zebrafish Retinal Regeneration[END_REF].

Although GSK343 single therapy had a limited effect on the growth of CEGBCs spheroids, its combination with TMZ enhanced the restriction of the CEGBCs spheroids growth derived from DB and BL, and to a lesser extent HCMV-GBM strains. EZH2 may modulate TMZ resistance where blocking EZH2 reverses TMZ chemosensitivity in GBM patients; an increased number of apoptotic cells were detected by knocking down EZH2 [START_REF] Fan | Inhibition of EZH2 reverses chemotherapeutic drug TMZ chemosensitivity in glioblastoma[END_REF]. Although encouraging responses were detected post-dual therapy (GSK343/TMZ) in CEGBCs-DB and BL, and to a lesser extent from the eleven GBM HCMV strains, the triple therapy (TMZ/GSK343/GCV) was the most effective in CEGBCs derived from DB, BL, and the eleven GBM HCMV strains. Hence, triple therapy provides the foundation for a combinational therapeutic strategy to improve overall patient survival, reduce viral resistance, and lower drug toxicity.

While exploring the potential association between HCMV, BC, and GBM, we also recognized the significance of understanding how diverse HCMV viral strains may influence OC progression.

Finally, we presented the experimental evidence for HCMV as a reprogramming vector that elicited human ovarian epithelial cells (OECs) transformation leading to the generation of "CMV-transformed Ovarian cells" (CTO). Herein, we assessed the potential transforming capacities of the high-risk HCMV-DB and BL strains, following the OECs infection. The OECs infection with the high-risk HCMV-DB and BL strains resulted in a pro-oncogenic cellular environment and sustained growth of CMV-transformed OECs with soft agar colonies formation. The CTO cells dedifferentiated, displayed stemness as well as EMT-MET hybrid phenotype, and finally resulted in PGGCs generation and spheroid formation. HCMV presence accompanied by polyploidy, EZH2 upregulation, and malignant phenotype potentially confirm the transformation process. In vivo, 72% of HGSOC biopsies were found to harbor HCMV with elevated PGCCs count as well as enhanced EZH2 expression, revealing a strong correlation between HCMV, PGCCs, and EZH2 expression. Three HCMV-OC strains were isolated from EZH2 high OC tumors that transformed OECs toward CTO possessing increased EZH2, Ki67Ag, and Myc expression parallel to polyploidy induction. The expression of the aforementioned markers and polyploidy were curtailed by EZH2 inhibitors therapy.

PGCCs play a fundamental role in tumor progression and in regulating tumor heterogeneity [START_REF] Zhang | Generation of cancer stem-like cells through the formation of polyploid giant cancer cells[END_REF][START_REF] Richards | Polyploid giant cancer cells and ovarian cancer: new insights into mitotic regulators and polyploidy[END_REF]. Accumulating evidence reveals the presence of PGCCs in OC, particularly the HGSOC, where PGCCs act as stem-like, self-renewing cells that are considered prognostic factors for OC [START_REF] Zhang | Generation of cancer stem-like cells through the formation of polyploid giant cancer cells[END_REF][START_REF] Bowers | Autophagy modulating therapeutics inhibit ovarian cancer colony generation by polyploid giant cancer cells (PGCCs)[END_REF]. In our study, CTO cells generated PGCCs, and were heterogeneous showing distinct morphological features including budding, filopodia, lipid droplets-filled cells, blastomere-like cells, and multinucleated cells that were previously detected in OC especially HGSOC [START_REF] Richards | Polyploid giant cancer cells and ovarian cancer: new insights into mitotic regulators and polyploidy[END_REF][START_REF] Niu | Dedifferentiation into blastomere-like cancer stem cells via formation of polyploid giant cancer cells[END_REF][START_REF] Iwahashi | Lipid Droplet Accumulation Independently Predicts Poor Clinical Prognosis in High-Grade Serous Ovarian Carcinoma[END_REF][START_REF] Yoshihara | Filopodia play an important role in the trans-mesothelial migration of ovarian cancer cells[END_REF][START_REF] Zhang | iTRAQ-Based Proteomic Analysis of Polyploid Giant Cancer Cells and Budding Progeny Cells Reveals Several Distinct Pathways for Ovarian Cancer Development[END_REF][START_REF] Lv | Polyploid giant cancer cells with budding and the expression of cyclin E, S-phase kinase-associated protein 2, stathmin associated with the grading and metastasis in serous ovarian tumor[END_REF][START_REF] Davidson | Epithelial-Mesenchymal Transition in Ovarian Carcinoma[END_REF] and reported upon HCMV infection of human mammary epithelial cells [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF][START_REF] Nehme | Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus[END_REF][START_REF] El Baba | Oncogenic and Stemness Signatures of the High-Risk HCMV Strains in Breast Cancer Progression[END_REF]. Our findings indicated that polyploidy harboring HCMV might induce the acquisition of a malignant phenotype via the giant cell cycling [START_REF] Liu | Polyploid giant cancer cells: An emerging new field of cancer biology[END_REF]. p53 and Rb inactivation have been correlated with tetraploidization in human tumors [START_REF] Castedo | Tetraploid cancer cell precursors in ovarian carcinoma[END_REF]. Indeed, p53 was shown to be mutated in more than 50% of human tumors, especially HGSOC; most of the p53 mutations acquired an oncogenic function.

It's worth mentioning that tumor-promoting viruses reduce p53 activity [START_REF] Aloni-Grinstein | p53 and the Viral Connection: Back into the Future ‡[END_REF]. Studies have shown that the incidence of tetraploidy occurs in p53-deficient cells possessing prolonged DNA damage due to persistent telomere dysfunction [START_REF] Davoli | Persistent Telomere Damage Induces Bypass of Mitosis and Tetraploidy[END_REF]. p53 and Rb expression was decreased in CTO-DB and BL that showed high percentages of tetraploidization and decreased telomerase activity, in line with the telomere-driven tetraploidization induced by critically short telomeres with the potential to promote tumorigenesis in early cancerous lesions [START_REF] Davoli | Telomere-Driven Tetraploidization Occurs in Human Cells Undergoing Crisis and Promotes Transformation of Mouse Cells[END_REF]. Several HCMV products have been involved in the cellular transformation including IE1, IE2, pp65, US28, cmvIL-10, UL76, UL44, and UL84, etc. Such expression of HCMV gene products could impair the pathways of p53 and Rb [START_REF] El Baba | Immune Landscape of CMV Infection in Cancer Patients: From "Canonical" Diseases Toward Virus-Elicited Oncomodulation[END_REF][START_REF] Herbein | The Human Cytomegalovirus, from Oncomodulation to Oncogenesis[END_REF]. HCMV-IE1, IE2, and UL97 allow the evasion of p53 and Rb [START_REF] Herbein | The Human Cytomegalovirus, from Oncomodulation to Oncogenesis[END_REF]. Hence, the impaired p53 and Rb pathways in CTO cells was due to the transforming potential of HCMV contrasting the Ras, human telomerase reverse transcriptase (hTERT), and SV40-mediated transformation of human ovarian cells [START_REF] Liu | A Genetically Defined Model for Human Ovarian Cancer[END_REF].

Cells possessing sphere-forming potentials were shown to reside in the malignant ascites of OC patients [START_REF] Velletri | Single cell-derived spheroids capture the self-renewing subpopulations of metastatic ovarian cancer[END_REF][START_REF] Liao | Ovarian Cancer Spheroid Cells with Stem Cell-Like Properties Contribute to Tumor Generation, Metastasis and Chemotherapy Resistance through Hypoxia-Resistant Metabolism[END_REF][START_REF] Shepherd | Principles of dormancy evident in high-grade serous ovarian cancer[END_REF]. In OC, the aforementioned cells are strong contributors to tumor progression, metastasis, chemotherapy resistance, and disease relapse [START_REF] Liao | Ovarian Cancer Spheroid Cells with Stem Cell-Like Properties Contribute to Tumor Generation, Metastasis and Chemotherapy Resistance through Hypoxia-Resistant Metabolism[END_REF]. The spontaneous spheroid generation along with the high expression of Nanog and Oct4 in CTO-DB and BL is in line with the previously described OC cell phenotype [START_REF] Pease | Spontaneous spheroid budding from monolayers: a potential contribution to ovarian cancer dissemination[END_REF]. Given their role in maintaining pluripotency and long-term self-renewal, the embryonic transcription factors Oct4 and Nanog are recognized as part of the stem cell signature that strongly correlates with spheroid formation, tumor initiation, and chemoresistance in ovarian cancer cells [START_REF] Pease | Spontaneous spheroid budding from monolayers: a potential contribution to ovarian cancer dissemination[END_REF][START_REF] Robinson | Characterization of SOX2, OCT4 and NANOG in Ovarian Cancer Tumor-Initiating Cells[END_REF]. CD44 was highly expressed in CTO-DB and BL cells; it has been previously shown to drive the progression of several tumors [START_REF] Martincuks | CD44 in Ovarian Cancer Progression and Therapy Resistance-A Critical Role for STAT3[END_REF][START_REF] Gao | Up-regulation of CD44 in the development of metastasis, recurrence and drug resistance of ovarian cancer[END_REF], maintain stem cell quiescence, and promote EMT in OC [START_REF] Zhou | CD44 Expression Predicts Prognosis of Ovarian Cancer Patients Through Promoting Epithelial-Mesenchymal Transition (EMT) by Regulating Snail, ZEB1, and Caveolin-1[END_REF]. Further, CD44 has been linked to the sphere-forming, self-renewing and tumor-initiating potential of OC cells [START_REF] Lupia | Ovarian cancer stem cells: still an elusive entity?[END_REF]. The coexistence of vimentin and E-cadherin in CTO-DB and BL highlights the high cellular plasticity.

EMT plasticity with EMT and MET alternately taking place was revealed during HGSOC progression where cells co-express epithelial and mesenchymal determinants [START_REF] Davidson | Epithelial-Mesenchymal Transition in Ovarian Carcinoma[END_REF][START_REF] Loret | The Role of Epithelial-to-Mesenchymal Plasticity in Ovarian Cancer Progression and Therapy Resistance[END_REF][START_REF] Ricci | Ovarian carcinoma tumor-initiating cells have a mesenchymal phenotype[END_REF].

Besides cellular plasticity, the hybrid E/M state promotes stem-like properties as well as metastatic and tumorigenic potential [START_REF] Liao | Hybrid Epithelial/Mesenchymal State in Cancer Metastasis: Clinical Significance and Regulatory Mechanisms[END_REF][START_REF] Tedja | Generation of Stable Epithelial-Mesenchymal Hybrid Cancer Cells with Tumorigenic Potential[END_REF].

Upon infecting OECs with HCMV-DB and BL strains, IE1 and pp65 proteins were detected which is in line with the recent studies that have identified high expressions of HCMV-IE or pp65 in ovarian tumor samples that were associated with poor survival outcomes [START_REF] Cox | Potential Impact of Human Cytomegalovirus Infection on Immunity to Ovarian Tumours and Cancer Progression[END_REF][START_REF] Carlson | Human cytomegalovirus in high grade serous ovarian cancer possible implications for patients survival[END_REF], suggesting that HCMV infection could potentially promote cancer progression. All in all, HCMV-induced EZH2 expression along with the embryonic stem-like phenotype and cellular plasticity in the IE1expressing OECs/CTO cells could establish a significant model in the context of OC. Several studies shed light on the existence of herpesviruses DNA and proteins in ovarian tissues that may hold potential in the process of OC tumorigenesis [START_REF] Cox | Potential Impact of Human Cytomegalovirus Infection on Immunity to Ovarian Tumours and Cancer Progression[END_REF][START_REF] Carlson | Human cytomegalovirus in high grade serous ovarian cancer possible implications for patients survival[END_REF][START_REF] Pathak | Factors in Oncogenesis: Viral Infections in Ovarian Cancer[END_REF]. Herein, we reported the detection of HCMV in HGSOC biopsies displaying elevated PGCCs count as well as enhanced EZH2 expression. It is worth mentioning that, neither HPV nor EBV was detected in the eighteen tested HGOSC biopsies.

Three HCMV strains with a strong transforming potential, so called high-risk strains, were isolated from EZH2 High HGOSC biopsies. After OECs infection, CTO cells were generated with morphological features matching the previously described CTO-DB and BL. Additionally, we evaluated EZH2 expression in the high-risk HCMV strains that were recovered directly from HGSOC biopsies thereby assessing their oncogenic and stemness potential. Unlike GSK-treated CTO, the expression of EZH2, Myc, and Ki67Ag was predominantly elevated in untreated CTO-HCMV-OC cells parallel to the PGCCs appearance. In summary, HCMV clinical strains reside in HGOSC biopsies retaining cancer-promoting potentials.

Targeting HCMV might have a vital role in treating OC [START_REF] Carlson | Human cytomegalovirus in high grade serous ovarian cancer possible implications for patients survival[END_REF]. Post-anti-CMV therapy, a remarkably high extended survival was observed in both newly diagnosed and recurrent glioblastoma patients [START_REF] Stragliotto | Valganciclovir as Add-on to Standard Therapy in Glioblastoma Patients[END_REF]. Current vaccine candidates have focused on several HCMV antigens/epitopes such as gB, gH, pentamer complex, pp65 and IE1 [START_REF] Nelson | A new era in cytomegalovirus vaccinology: considerations for rational design of next-generation vaccines to prevent congenital cytomegalovirus infection[END_REF][START_REF] Yu | Human cytomegalovirus in cancer: the mechanism of HCMV-induced carcinogenesis and its therapeutic potential[END_REF]. Recently, CMVspecific immunotherapy including cytotoxic T lymphocyte (CTL) or dendritic cell (DC)-based vaccines has contributed to minimal successful outcomes in glioblastoma treatment [8,[START_REF] Yu | Human cytomegalovirus in cancer: the mechanism of HCMV-induced carcinogenesis and its therapeutic potential[END_REF]. In addition, using EZH2 inhibitors to specifically target ovarian tumors could ultimately improve patients outcomes [START_REF] Jones | Histone Methyltransferase EZH2: A Therapeutic Target for Ovarian Cancer[END_REF]. Two inhibitors triggering EZH2 degradation (DZNep and YM281) exhibited potent efficacy in OC cell lines and xenografts [START_REF] Chen | EZH2 mediated metabolic rewiring promotes tumor growth independently of histone methyltransferase activity in ovarian cancer[END_REF]. GSK343 significantly induced apoptosis and inhibited the invasion of ovarian epithelial cells in 3D cultures which more closely mimics the tumor microenvironment in vivo [START_REF] Amatangelo | Threedimensional culture sensitizes epithelial ovarian cancer cells to EZH2 methyltransferase inhibition[END_REF]. In patient-derived glioma stem cells, GSK343 was shown to suppress the stemness traits [START_REF] Yu | The EZH2 inhibitor GSK343 suppresses cancer stem-like phenotypes and reverses mesenchymal transition in glioma cells[END_REF]. Interestingly, in the present study, EZH2 inhibitors totally blocked HCMV replication. It's worth noting that the HCMV major immediate early promoter (MIEP) transcriptional repressor, growth factor independence1 (GFI1), is controlled by the EZH2-NDY1/KDM2B-JARID2 axis. Therefore, EZH2 inhibitors might result in an enhanced GFI1 expression which could block viral replication [START_REF] Sourvinos | They include pathogens like SARS-CoV, MERS-CoV, and the highly infectious SARS-CoV-2, responsible for the COVID-19 pandemic. Severe outcomes of COVID-19 are linked to older age and compromised immune systems, often involving a cytokine storm marked by excessive inflammation. The molecular processes governing coronavirus infection encompass interactions between the virus and host, affecting viral entry, replication, escape, and immune control. Exploring coronavirus infections and COVID-19 from an epigenetic perspective may lead to altered gene expression, potentially limiting infection. Clinical trials of epigenetic therapies, approved epigenetic agents, and combining antivirals with epigenetic drugs are viewed as effective strategies for controlling viral replication and excessive inflammation[END_REF]. Hence, combinational therapies including EZH2 inhibitors may prove to be a promising milestone in developing therapeutic strategies for ovarian cancer treatment.

Overall, the existence of diverse strains expressing potential biomarkers and possessing various replication potentials in different tissues and/or cell types explain the distinct oncogenicity. So far, sixteen high-risk oncogenic clinical HCMV strains were isolated directly from tumor biopsies (breast cancer, N=2; glioblastoma, N=11; HGSOCs, N=3). These strains shared the same transforming potential. PGCCs were detected in the aforementioned cell cultures that were shown to highly express Myc and EZH2 pointing toward a potential link between HCMV, PGCCs, EZH2, and Myc. CTO and CTH cells dedifferentiated and revealed stemness as well as EMT traits. On the other hand, CEGBCs dedifferentiated and displayed stemness, PMT as well as invasiveness features. The phenotypic changes were similar in HCMV-infected ovarian and mammary epithelial cells. However, the difference was mainly the detection of high percentages of tetraploid cells in the CTO cultures compared to the >4N population detected in CTH cells. In both models, a high degree of cellular plasticity was detected knowing that ovarian and mammary epithelial cells are isolated from human glandular organs. Thus, discovering distinct viral strains will adjust the different adopted approaches to tumor diagnosis, prognosis, prevention, advanced treatment, as well as therapy monitoring and will enrich potential vaccine studies. In the forthcoming studies, it would be essential to sequence the isolated high-risk HCMV strains aiming to define the specific viral gene(s) that are responsible for driving the transformation process. Further, it is of high importance to conduct in vivo experimental studies since they can serve as a crucial bridge for extrapolating our findings to the patient context as well as translating the exact relevance of such models in the pathogenesis of breast cancer, GBM, and ovarian cancer. This could play a pivotal role in indicating and confirming HCMV as an oncogenic virus.

Chapter 9 9. Conclusion

As a conclusion, our findings elucidate the concept of how HCMV is linked to breast cancer, glioblastoma, and ovarian cancer as depicted in the figures below (Figure 53, Figure 54, and Figure 55). The data that were provided proved that HCMV is not solely associated with oncomodulation; it is also recognized for its involvement in oncogenesis. The oncogenic and stemness signatures of the high-risk HCMV strains accentuate the oncogenic potential of HCMV in breast cancer, glioblastoma, and ovarian cancer progression and support the tumorigenic properties of EZH2 and Myc which might be highly pertinent in the pathophysiology of the aforementioned tumors. With respect to breast and ovarian cancer, HCMV triggered EZH2 and Myc expression, generated PGCCs, displayed dedifferentiating phenotypes with stemness features as well as hybrid EMT/MET phenotypes parallel to the existence of giant cell cycling. Further, HCMV induced a CEGBCs phenotype with tumor heterogeneity, proneural to mesenchymal plasticity, and embryonic-like stemness leading to spheroid formation and invasiveness in vitro and in GBM biopsies.

In accordance with the previous findings discussed, and considering that HCMV has been detected primarily within tumors correlating positively to poor prognosis, along with the potential influence of HCMV gene products on the regulation of tumorigenic pathways and processes associated with key cancer characteristics, and taking into account HCMV's wide tissue tropism, we deduce that HCMV harbors distinctive mechanisms that contribute to cancer progression. In response to HCMV infection (1), the 2N human mammary epithelial cells (HMECs) enter the initiation phase of the giant cell cycle through endoreplication [START_REF] Sehrawat | Herpesviruses: Harmonious Pathogens but Relevant Cofactors in Other Diseases?[END_REF]. Polyploid (>4N) cells in the dedifferentiation stage display a luminallike phenotype with high to intermediate expression of epithelial and stemness markers. Cells directly bud from multinucleated or mononucleated giant cells (3) or alternatively continue endoreplication (4). The termination/differentiation step characterized by intermediate 2-4N cells profile gradually achieve stability into diploid 2N small cells (SCs) [START_REF] Boeckh | Cytomegalovirus: pathogen, paradigm, and puzzle[END_REF], where the latter are triple-negative with lower expression of epithelial and stemness markers. A small population of SCs could revert and show a mesenchymal-to-epithelial transition (6). A gradual decrease in HCMV presence is seen from left to right. Adapted from Nehme et al [START_REF] Nehme | Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus[END_REF]. Future studies, with more detailed analyses of target genes within the CMV-transformed cells namely, CTH, CEGBCs, and CTO cells and their corresponding response to inhibitors may establish new avenues to understand the complex pathogenesis of breast cancer, glioblastoma, and ovarian cancer. Additionally, large-scale experiments are highly encouraged to further validate our findings. Meanwhile, the presented data provides new insights into the oncogenic role of HCMV in cancer progression, thereby uncovering novel targeted therapeutic approaches and innovative clinical interventions that will improve cancer patient outcomes and their overall survival rates. Lately, human cytomegalovirus (HCMV) has been progressively implicated in carcinogenesis alongside its oncomodulatory impact. CMV-Transformed Human mammary epithelial cells (CTH) phenotype might be defined by giant cell cycling, whereby the generation of polyploid giant cancer cells (PGCCs) could expedite the acquisition of malignant phenotypes. Herein, the main study objectives were to assess the transformation potential in vitro and evaluate the obtained cellular phenotype, the genetic and molecular features, and the activation of cellular stemness programs of HCMV strains, B544 and B693, which were previously isolated from triple-negative breast cancer (TNBC) biopsies. The strains' sensitivity to paclitaxel and ganciclovir combination therapy was evaluated. A unique molecular landscape was unveiled in the tumor microenvironment of TNBC harboring high-risk HCMV. Overall, the explicit oncogenic and stemness signatures highlight HCMV potential in breast cancer progression thus paving the way for targeted therapies and clinical interventions which prolong the overall survival of breast cancer patients.

Publication N°10

El Baba R, Haidar Ahmad S, Monnien F, Mansar R, Bibeau F, Herbein G. Polyploidy, EZH2 upregulation, and transformation in cytomegalovirus-infected human ovarian epithelial cells. Oncogene 2023. https://doi.org/10.1038/s41388-023-02813-4.

The present study investigates the connection between human cytomegalovirus (HCMV) infection and epithelial ovarian cancer (OC), particularly high-grade serous ovarian carcinoma (HGSOC). Herein, we identified the presence of polyploid giant cancer cells (PGCCs) with stem cell-like characteristics in HGSOC. Additionally, we highlighted the role of the oncogene EZH2, which correlates with OC tumor grade and proliferation. Our study presents evidence of HCMV's involvement in transforming human ovarian epithelial cells, leading to the creation of "CMVtransformed Ovarian cells" (CTO). High-risk clinical HCMV strains, including the three strains isolated from HGSOC biopsies, induced distinct cellular and molecular changes, including EZH2 upregulation and increased cell proliferation. These findings support the notion of an HCMVinduced model for epithelial ovarian cancer and suggest the tumorigenic properties of EZH2. This research may have implications for targeted therapeutics in the treatment of ovarian tumors.

Figure 1 :

 1 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 2 :

 2 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 3 :

 3 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 4 :

 4 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 5 :

 5 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 6 :

 6 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 7 :

 7 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 8 :

 8 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 9 :

 9 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 10 :

 10 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 11 :

 11 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 12 :

 12 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 13 :

 13 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 14 :

 14 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 15 :

 15 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 16 :

 16 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 17 :

 17 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 18 :

 18 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 19 :

 19 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 20 :

 20 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 21 :

 21 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 22 :

 22 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 23 :

 23 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 24 :

 24 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 25 :

 25 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 26 :

 26 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 27 :

 27 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 29 :

 29 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 30 :

 30 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 31 :

 31 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 32 :

 32 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 33 :

 33 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 34 :

 34 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 35 :

 35 Figure 1: Schematic representations comparing genome organizations. .............................................Figure 2: HCMV Virion. ..........................................................................................................................Figure 3: HCMV Life cycle. .....................................................................................................................Figure 4: Scheme illustrating PGCCs formation mechanisms. ............................................................Figure 5: A scheme illustrating the giant cell cycle. ...............................................................................Figure 6: Generation of PGCCS in various cancer types. .....................................................................Figure 7: The Hallmarks of Cancer. .......................................................................................................Figure 8: Modulation of the host immune system. .................................................................................Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment. ....................Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains. .........Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells. ..........Figure 12: CTH proliferation capacities and AKT activation. .............................................................Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells. ...........Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells. ......................Figure 15: Penotypic analysis of CTH cells. ...........................................................................................Figure 16: Sustained viral replication in CTH cells. ..............................................................................Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo. ...................................................................Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates. ..............................................................................Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains. ...................Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures. .......................Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs. ...............Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration. ..........Figure 23: Stemness potential of CEGBCs-DB and BL. .....................................................................Figure 24: The invasion capacities of CEGBCs-DB and BL. ..............................................................Figure 25: Cellular migration of CEBGCs. ..........................................................................................Figure 26: The three major invasion mechanisms of CEGBCs-DB and BL. ....................................Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures................................................................................................................. Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies. ........Figure 29: Akt expression in glioblastoma biopsies. ............................................................................Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies. .............................................Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts. .............................................................................................Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth. ...............Figure 33: Replication of high-risk HCMV strains in OECs cultures. ..............................................Figure 34: Replication of low risk HCMV strains in OECs cultures. ................................................Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs. .....................................................................................................................

Figure 36 :

 36 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 37 :Figure 38 :

 3738 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 39 :

 39 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 40 :Figure 41 :

 4041 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 42 :

 42 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 43 :

 43 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 44 :

 44 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 45 :

 45 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 46 :

 46 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 47 :

 47 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 48 :

 48 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 49 :

 49 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 50 :

 50 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 51 :

 51 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 52 :

 52 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 53 :

 53 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 54 :

 54 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

Figure 55 :

 55 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures. ....................................................................................................................Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs. ................................................................................................................................. Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells. ...Figure 39: Expression of SUZ12 in CTO-DB and BL cells. ................................................................Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential..................................................................................................................... Figure 41: Expression of Oct4 in CTO-DB and BL cells. ....................................................................Figure 42: Expression of CD44 in CTO-DB and BL cells. ..................................................................Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits. ........................................Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures. .............................................................................................................................................Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs. .....................................................................................................Figure 46: Sustained HCMV replication in chronically HCMV-infected OECs. .............................Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS. ........................Figure 48: CTO cultures post-TPA treatment. ....................................................................................Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS. ..........................................................................................................Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies. ....................................................................................................................................................Figure 51: Myc Expression in ovarian cancer biopsies. ......................................................................Figure 52: Akt Expression in ovarian cancer biopsies. .......................................................................Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle. .........................................................................................................................................................Figure 54: Glioblastoma generation scheme. .......................................................................................Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs. .

  Oncoviruses. ............................................................................................................................... Table 2: HCMV Gene Products. .............................................................................................................. Table 3: HCMV enabling the characteristics of cancer. ...................................................................... Table 4: Primers Used. ............................................................................................................................ Table 5: Antibodies Used. ......................................................................................................................... Table 6: Treatments Used. ....................................................................................................................... Table 7: Clinical and biological data of the GBM patients. .................................................................. Table 8: Clinical data and treatments of the OC patients. ................................................................... Table 9: Characteristics of HCMV-GBM strains. ...............................................................................

1 . 1

 11 Herpesviridae Family and HCMV DiscoveryHerpesviridae is a family of viruses that includes a large and diverse group of viruses known as herpesviruses. These viruses are characterized by their ability to establish latent infections by remaining dormant within the host's body and reactivate later under certain conditions.Herpesviruses can infect a wide range of species[START_REF] Grinde | Herpesviruses: latency and reactivationviral strategies and host response[END_REF][START_REF] Sehrawat | Herpesviruses: Harmonious Pathogens but Relevant Cofactors in Other Diseases?[END_REF]. The family Herpesviridae is divided into three subfamilies: Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae. Each subfamily has distinct characteristics in terms of virus-host interactions, replication strategies, and clinical manifestations. To start with Alphaherpesvirinae, these viruses are known for their rapid replication cycle and ability to establish lytic and latent infections, they are often associated with lesions and sores on the skin and mucous membranes. Examples of alphaherpesviruses include Herpes simplex virus 1 (HSV-1), Herpes simplex virus 2 (HSV-2), and Varicella-zoster virus (VZV), which causes chickenpox and shingles. Second, the Betaherpesvirinae comprising betaherpesviruses that have a slower replication cycle compared to alphaherpesviruses; they are often associated with persistent infections and can cause long-term health issues. Human Cytomegalovirus (HCMV), also known as Human Herpesvirus 5 (HHV-5), belongs to this subfamily, within the genera Cytomegalovirus. On the other hand, we have HHV6A, HHV6B, and HHV7, which belongs to the genera Roseolovirus. Third, the Gammaherpesvirinae including Gammaherpesviruses that are associated with certain types of cancers, particularly in immunocompromised individuals. Epstein-Barr virus (EBV) or (HHV4), belonging to genera

  organization of the HCMV genome adheres to the characteristic architecture of herpesviruses classified under class E[15]. HCMV genome features two inverted domains: the unique long (UL) and unique short (US) domains, each flanked by a pair of inverted repeats, one at the terminal end (terminal repeat long/internal repeat long (TRL/IRL)) and the other at the junction with the other unique domain (internal repeat short/terminal repeat short (IRS/TRS)). This results in a genome organization denoted as TRL-UL-IRL-IRS-US-TRS. Consequently, HCMV gene annotations are based on their respective positions within these genome segments, specifically UL, US, IRL, IRS, TRL, and TRS (Figure1)[START_REF] Martí-Carreras | Human cytomegalovirus genomics and transcriptomics through the lens of next-generation sequencing: revision and future challenges[END_REF].

Figure 1 :

 1 Figure 1: Schematic representations comparing genome organizations.

Figure 2 :

 2 Figure 2: HCMV Virion.Double-stranded DNA genome is enclosed within an icosahedral symmetry capsid which is further enveloped by a layer called tegument. Mature virions are enveloped; viral glycoproteins protrusions are found on the envelope surface. This description has been adapted and altered based on Tomtishen's work[START_REF] Scrivano | HCMV Spread and Cell Tropism are Determined by Distinct Virus Populations[END_REF].

Figure 3 :

 3 Figure 3: HCMV Life cycle.

Figure 4 :

 4 Figure 4: Scheme illustrating PGCCs formation mechanisms.

Figure 5 :

 5 Figure 5: A scheme illustrating the giant cell cycle.

Figure 6 :

 6 Figure 6: Generation of PGCCS in various cancer types.

  Constitutive expression of LANA in different cell lines results in a dramatic increase in the multinucleated phenotype, characterized by cells with two or more polarized nuclei. K cyclin expression leads to cells with enlarged nuclei and multiple large multi-lobular nuclei, highlighting a strong association between KSHV and polyploidy. Several mechanisms contribute to KSHV-induced polyploidy. KSHV can induce abnormal centrosome duplication and multipolar or monopolar spindle formation, mediated by LANA and K cyclin proteins. Aurora kinase B cleavage by serine protease-N terminus in KSHVlatently infected tumor cells promotes the transition from metaphase to telophase, enhancing mitotic progress and tumorigenesis. LANA induces S-phase entry and multinucleation by protecting cells from cell cycle arrest; it also interacts with the spindle checkpoint protein Bub1, leading to Bub1 ubiquitination and degradation, favoring multinucleation. K cyclin expression results in DNA replication and cell nuclei division without simultaneous cell division [169,170].

2. 5 . 4

 54 HTLV-1 Human T-cell lymphotropic virus type 1 (HTLV-1), the first human retrovirus discovered, belongs to the Retroviridae family. While it can infect various human cell types in vitro, HTLV-1 primarily undergoes productive replication within CD4+ T helper cells. HTLV-1 is the causative agent of adult T-cell leukemia/lymphoma (ATL), a highly aggressive non-Hodgkin's peripheral T-cell malignancy. The viral oncoprotein Tax plays a central role in the development of ATL. Tax activates genes that prevent cell death and disrupts the normal cell cycle, leading to DNA damage.

Figure 7 :

 7 Figure 7: The Hallmarks of Cancer.

  . A significant strategy involves hindering MHC class I-restricted antigen presentation. During the immediate early phase of HCMV infection, cytotoxic T-lymphocytes (CTLs) counteract antigenic peptides produced by the IE1 transcription factor. However, the matrix protein pp65, which possesses kinase activity, phosphorylates IE1, specifically blocking the presentation of IE-derived antigenic peptides. This allows HCMV to evade immune recognition of early viral proteins. Additionally, pp65 is introduced directly into cells during viral fusion, enabling HCMV to escape immunological surveillance until other immune evasion-related proteins are released. HCMV-specific viral proteins and genes associated with host interferon responses further inhibit NK cell detection and activation, as well as hinder the recognition of CD4+ and CD8+ T-cells by preventing MHC Class I and II antigen processing and presentation. Furthermore, HCMV-infected cells produce a viral IL-10 homolog, which suppresses CD4+ and CD8+ T-cell responses [70].

Figure 8 :

 8 Figure 8: Modulation of the host immune system.

  associated inflammatory cells. Consequently, a tumor microenvironment (TME) is generated and controlled by tumor-induced molecular and cellular interactions in which immune cells not only fail to exert anti-tumor effector functions, but also promote tumor development[START_REF] Tiwari | Tumor microenvironment: barrier or opportunity towards effective cancer therapy[END_REF]. HCMV expresses several gene products that modulate the host immune response and promote modifications in non-coding RNA and regulatory proteins. These changes are linked to several complications, such as immunosenescence and malignant phenotypes leading to immunosuppressive TME and oncomodulation (Figure9)[START_REF] El Baba | Immune Landscape of CMV Infection in Cancer Patients: From "Canonical" Diseases Toward Virus-Elicited Oncomodulation[END_REF]. NF-κB and PI3K activity play a role in facilitating the HCMV-induced transformation of monocytes into macrophages with a dual phenotype, exhibiting characteristics of both inflammatory macrophages (M1) and immunosuppressive macrophages (M2). In this context, M1 macrophages are responsible for releasing inflammatory factors such as TNF-alpha, IL-6, and nitric oxide synthase 2, while M2 macrophages exhibit an immunosuppressive profile similar to tumor-associated macrophages (TAMs), characterized by the secretion of immunosuppressive cytokines like TGF-β and IL-10[280]. It's worth noting that a significant portion of TAMs in breast cancer cells display activated M2 features, accompanied by the secretion of elevated levels of immunosuppressive cytokines, including IL-10, TGF-β, and, to a lesser extent, pro-inflammatory cytokines[START_REF] Tang | Tumor-associated macrophages as potential diagnostic and prognostic biomarkers in breast cancer[END_REF]. Notably, research from our laboratory has demonstrated that the highly macrophage-tropic HCMV-DB strain can induce the activation of an M2-like state[START_REF] Khan | Bcl-3-Regulated Transcription from Major Immediate-Early Promoter of Human Cytomegalovirus in Monocyte-Derived Macrophages[END_REF]. This underscores the potential contribution of HCMV infection to the development of neoplastic transformation in breast epithelial cells, partially through the recruitment and polarization of macrophages[START_REF] Pasquereau | Cytomegalovirus, Macrophages and Breast Cancer[END_REF]. The oncomodulatory potential of HCMV catalyzes an oncogenic process by producing viral proteins, helping tumor cells to evade the immune system, and preventing and/or delaying cell death. The lack of HCMV specific cellular immune responses in these immune-privileged tumor sites would definitely enhance HCMV replication. On the other hand, cancer cells on their own can escape immune responses by diverse mechanisms. Thus, the combination of intrinsic cellular with viral immune escape machineries in cancer cells may offer an environment which enhances HCMV replication and boost cancer cells to evade from immune surveillance showing the bidirectional relationship between tumor cells and HCMV[START_REF] El Baba | Immune Landscape of CMV Infection in Cancer Patients: From "Canonical" Diseases Toward Virus-Elicited Oncomodulation[END_REF].

Figure 9 :

 9 Figure 9: HCMV Oncomodulation and Its Significance in Tumor Microenvironment.

4 . 1

 41 Decoding The Molecular Pathways in Cancer Development

PI3K/ 1 :

 1 Akt pathway activation. PI3K/Akt activation induced inflammation and immunosuppression through nitric oxide synthase (NOS) overexpression; thus, resulting in tumor initiation via the activated Notch pathway leading to tumor progression. On the other hand, suppression or mutation of p53 has been shown to decrease MHC-I presentation, increase STAT3 phosphorylation, upregulate PD-L1 via microRNA (miR34), elevate pro-inflammatory chemokine and cytokine production, and indirectly upregulate the expression of chemokine receptors (CXCR4 and CXCR5). Loss of Rb leads to the increase in CCL2 and IL6 secretion and this is because of the elevated fatty acid oxidation (FAO) activity and enhanced mitochondrial superoxide (MS) production. Indeed, those molecular alterations have been linked to immune suppression in the TME [70]. In the context of breast cancer, previous studies that were conducted by our research team demonstrated the ability of HCMV-DB and HCMV-BL strains in transforming primary HMECs into CMV-Transformed HMECs (CTH) cells in vitro. CTH cells, which are slow self-renewing cells, undergo diverse stages of the giant cell cycle. They were shown to be heterogeneous, generate PGCCs, exhibit dedifferentiation, and display stemness and EMT/MET features. Herein, we aimed to:

  were provided by the Regional tumor bank (BB0033-00024 Tumorothèque Régionale de Franche-Comté). It's worth noting that a written informed consent for participation was obtained from all patients. The studies were authorized by the local ethics committees of Besançon University Hospital (Besançon, France) and the French Research Ministry (AC-2015-2496, CNIL n°1173545, NF-S-138 96900 n°F2015).

7 . 1

 71 analyses were done using Wilcoxon and Mann-Whitney tests; a p-value≤0.05 was considered to be statistically significant [*: ≤0.05; **: ≤0.01; ***: ≤0.001]. Correlation analyses were done using Spearman, Pearson, and Kendall's Tau correlation tests. Microsoft Excel was used to construct the plots and histogram data. Oncogenic and Stemness Signatures of the High-Risk HCMV Strains in Breast Cancer Progression 7.1.1 Growth of Two HCMV Clinical Strains Isolated from TNBC in HMECs and the Emergence of Morphologically Distinct Cells

7. 1 . 2

 12 Transformation Capacity of CTH Cells and the Induction of an Oncogenic EnvironmentTo evaluate the transformation of HCMV-B544 and B693 immortalized infected HMECs, cells were seeded in soft agar. Colony formation was detected at day 14 post-seeding in CTH-B544 and CTH-B693 cells (p-value ≤ 0.05) in contrast to uninfected HMECs which showed no changes (Figure11A). The resulting anchorage-independent growth in CTH cells is a crucial phase in the acquisition of malignancies. With regard to oncogenes, the expression of c-Myc was assessed by performing confocal microscopy imaging where large and elongated CTH cells showed remarkable c-Myc staining compared to uninfected HMECs (Figure11B). Using flow cytometry, a slightly higher expression of the proliferation marker (Ki67Ag) was detected in CTH-B693 compared to CTH-B544 cells with a limited expression in the uninfected HMECs. Only CTH-B544 and B693 cells were positively stained with Ki67 Ag as detected by confocal microscopy imaging (Figure12A). On the other hand, a slight increase in the expression of phosphorylated Akt (pAkt-ser473) along with a limited to nonexistent increase in Akt expression levels was shown in all CTH cells compared to uninfected HMECs (Figure12B). Overall, the acquisition of an immortal phenotype in CTH-B544 and B693 cells reflects their transformation potential.

Figure 10 :

 10 Figure 10: Replication of B544 and B693 strains in MRC5 cultures, and the appearance of morphologically distinct cells following the infection of HMECs with these high-risk strains.

Figure 11 :

 11 Figure 11: Colony formation in soft agar and Myc expression in CTH-B544 and B693 cells.

(

  A) Colony formation in soft agar seeded with uninfected HMECs, CTH-B544 and CTH-B693 cells. At day 15 postseeding, quantification of colonies was performed; Histogram represents the mean data ± SD of three independent experiments. * p-value ≤ 0.05, (B) Confocal microscopic images of Myc and DAPI staining in CTH cells. Magnification ×63, scale bar 10 μm.

Figure 13 :

 13 Figure 13: Tumorspheres formation and the expression of stemness markers in CTH cells.

(

  A) Tumorspheres were observed under an inverted light microscope in CTH-B544 and CTH-B693 cells. Magnification ×100 and ×200, scale bare 100 µm. Uninfected HMECs were used as a negative control. (B) FACS staining of CD44 and CD24 was performed in CTH cells. Results are representative of three independent experiments.

Figure 14 :

 14 Figure 14: Expression of embryonic stem cell markers in CTH-B544 and B693 cells.

(

  A) Detection of SSEA4 and Nanog in CTH cells by FACS. Results are representative of three independent experiments. Confocal microscopy imaging demonstrating the expression of (B) SSEA-4, (C) Nanog, (D) Oct4, and (E) SOX2. Nuclei were counterstained with DAPI; magnification ×63, scale bar 10 μm.

Figure 15 :

 15 Figure 15: Penotypic analysis of CTH cells.

Figure 16 :

 16 Figure 16: Sustained viral replication in CTH cells.

Figure 17 :

 17 Figure 17: Distinct responses of CTH cells to paclitaxel/ganciclovir (PTX/GCV) treatment in vitro recapitulates distinct TNBC molecular signatures in vivo.

(

  A) EGFR, CNDK2A, CCND1, SOX2, Oct4, and Nanog mRNA expression was measured by RT-qPCR in TNBC biopsies B544 and B693. Histograms represent the mean ± SD of three independent experiments. * p ≤ 0.05, determined by Mann-Whitney U test. (B) Soft agar seeded with CTH-B544 and CTH-B693 cells treated with PTX (20 nM)/GCV (20 μM) combination therapy. Untreated cells were used as negative controls; MCF7 and MDA-MB231 cells were used as positive controls. Magnification ×200, scale bar 100 μm. (C) Propidium iodide (PI) staining for detection of PGCCs in untreated and treated CTH cells by FACS analysis. (D) Ki67 Ag expression in PGGCs of untreated and treated CTH cells. Histograms represent the mean data ± SD of three independent experiments.

Figure 18 :

 18 Figure 18: Replication of two high-risk oncogenic HCMV strains in HAs cultures, the activation of oncogenic pathways, and reduced apoptosis rates.

Figure 19 :

 19 Figure 19: Long-term cultures of HAs infected with the two low-risk HCMV strains.

(

  A) Time-course of the viral titer in the supernatant of infected HAs as measured by IE1-qPCR along with the cellular analysis of HAs-KM and FS based on their size (FSC) and granularity (SSC). (B) Confocal microscopic images of HCMV-IE1, pp65, and late antigen staining in HAs infected with HCMV-KM and FS (day 1 post-infection). Nuclei were counterstained with DAPI; magnification ×63, scale bar 10 μm. (C) Myc, Akt, pAkt-Ser473, and Rb protein expression as measured by FACS in uninfected HAs and HAs infected with HCMV-KM and FS. Data are represented as mean ± SD of two independent experiments.

Figure 20 :

 20 Figure 20: Long-term replication of two high-risk HCMV strains in HAs cultures.

Figure 21 :

 21 Figure 21: Chronic infection of HAs with HCMV clinical isolates, the appearance of CEGBCs as well as colony formation in soft agar, and the phenotypic characterization of CEGBCs.

Figure 22 :

 22 Figure 22: Spheroid-forming potentials of CEGBCs as well as invasiveness and migration.

Figure 23 :

 23 Figure 23: Stemness potential of CEGBCs-DB and BL.

Figure 24 :

 24 Figure 24: The invasion capacities of CEGBCs-DB and BL.

Figure 25 :

 25 Figure 25: Cellular migration of CEBGCs.

Figure 27 :

 27 Figure 27: Detection of replicative HCMV and identification of the lncRNA4.9 and HOTAIR/EZH2 complex in CEGBCs cultures.

Figure 28 :

 28 Figure 28: HCMV detection as well as EZH2, and Myc expression in glioblastoma biopsies.

Figure 29 :

 29 Figure 29: Akt expression in glioblastoma biopsies.

Figure 30 :

 30 Figure 30: Isolation of oncogenic HCMV strains from GBM biopsies.

Figure 31 :

 31 Figure 31: Spheroid forming and invasion potentials of the HCMV-GBM strains and the detection of lncRNA 4.9 and HOTAIR transcripts.

  TMZ reduced spheroids' size by 23% only in CEGBCs-BL cultures, unlike CEGBCs-DB which displayed more mesenchymal traits (p-value (CEGBCs-DB:CEGBCs-BL) = 0.03). GCV reduced the spheroids' size by 21% and 24% in CEGBCs-DB and BL, respectively (p-value (CEGBCs-DB:CEGBCs-BL) = 0.35). On the other hand, GCV/TMZ combination therapy lead to a 27% size reduction of CEGBCs-BL spheroids, meanwhile having a very limited effect in CEGBCs-DB in which the spheroids' size was reduced by 9% (p-value (CEGBCs-DB:CEGBCs-BL) < 0.01) (Figure32A). Spheroids of CEGBCs-DB and BL were treated by GSK343, GSK343/GCV, GSK343/TMZ, and GSK343/GCV/TMZ. CEGBCs-DB were resistant to mostly all therapies except the triple therapy (p-value (CEGBCs-DB:CEGBCs-BL) = 0.06) unlike CEGBCs-BL that were mainly responsive to GSK343/TMZ (p-value (CEGBCs-DB:CEGBCs- BL) < 0.001) and triple treatment (p-value (CEGBCs-DB:CEGBCs-BL) = 0.06) (Figure32A). Under triple therapy (GSK343/GCV/TMZ), spheroids' size was reduced by around 90% in CEGBCs-DB and BL at day 10 post-treatment (p-value (triple therapy: TMZ) = 0.02) (Figure32B). Spheroids' size was reduced by around 60% with all the eleven HCMV-GBM strains at day 10 post-triple treatment (GSK343/GCV/TMZ) (p-value (triple therapy: TMZ) < 0.001) (Figure32C), similar to that reported for DB and BL strains.

Figure 32 :

 32 Figure 32: The effect of diverse single and combination therapies on CEGBCs' growth.

Figure 33 :

 33 Figure 33: Replication of high-risk HCMV strains in OECs cultures.

(

  A)Time-course of the viral titer in the supernatant of OECs infected with HCMV-DB and BL as measured by IE1-qPCR. (B) Immunoblotting data of IE1 in uninfected OECs lysates and OECs infected with HCMV-DB and BL (day 5 post-infection). β-actin was used as loading control. IE1 expression by FACS in acutely infected OECs-DB and BL; UI OECs were used as a control.

Figure 35 :

 35 Figure 35: Flow cytometric analysis based on FSC and SSC of uninfected OECs as well as chronically infected OECs.Only chronically-infected OECs-DB and BL, namely CTO-DB and CTO-BL cells, and not the chronically-infected OECs-KM and FS were alive at day 86 post-infection (black arrows).

  ). Downregulation of p53 and Rb proteins was noticed in CTO-DB and BL compared to controls (pvalue (UI OECs:CTO-HCMV) = 0 . 03), unlike pRb which was upregulated in CTO-DB and BL (p-value (UI OECs:CTO-HCMV) = 0

Figure 36 :

 36 Figure 36: Chronic infection of OECs with the high-risk HCMV clinical isolates and polyploidy detection in OECs cultures.

(

  A) Microscopic images including confocal images of DAPI and phalloidine staining in OECs infected with HCMV-DB and BL; uninfected OECs were used as a negative control. Left panel: magnification ×100 and ×200, scale bar 100 and 50 μm; Right panel: magnification ×63, scale bar 10 μm. Red arrows showing the PGCCs detected in OECs cultures. (B) The appearance of distinct cellular morphologies of the giant cell cycle including (a) filopodia, (b, c, d) blastomeres and blastocytes, (e) lipid droplets-filled cells, (f) multinucleated, (g, h) budding, (i) mesenchymal cells as well as (j, k, l) few atypical morphologies; magnification ×100, scale bar 100 μm. Uninfected OECs were used as a control. (C) Confocal microscopic images of DAPI and phalloidine staining in CTO-DB and BL. Uninfected OECs were used as a negative control; magnification ×63, scale bar 10 μm. (D) Propidium iodide (PI) staining for polyploidy detection in HCMV-transformed OECs. Cobalt chloride (CoCl2)-treated OECs (450 μM) were used as a positive control. Microscopic images of uninfected OECs as well as the PGCCs generated in CTO-DB and BL cultures and post-CoCl2 treatment; magnification ×100, scale bar 100 μm. (E) p53, Rb, and p-Rb expression in uninfected OECs and CTO-DB and BL by FACS. (F) Histograms representing the relative telomerase activity in uninfected OECs as well as CTO-DB and BL. Data are represented as mean ± SD of two independent experiments. *p-value ≤ 0 . 05. 7.3.2 CTO cells Display Dedifferentiation, Stemness, and EMT Characteristics CTO-DB and BL were seeded in soft agar to assess their oncogenic transforming potential. Colony formation was detected in the cultures seeded with CTO-DB and BL, compared to uninfected OECs (Figure 37A). On the proteomic level, EZH2 and Myc upregulation was detected in CTO-DB and BL compared to uninfected OECs (p-value (UI OECs:CTO-HCMV) = 0 . 03) (Figure 37B,C, and D); on the contrary, a limited EZH2 and Myc upregulation was observed in OECs-KM and FS (pvalue (UI OECs:OECs-HCMV) = 0 . 02) (Figure 34C). A significant positive correlation between EZH2and Myc protein expression was detected in CTO cells (r = 0.983, p-value < 0 . 001) (Figure38).Further, EZH2 and Myc transcripts were upregulated in CTO-DB and BL compared to uninfected controls (p-value (UI OECs:CTO-HCMV) = 0 . 03) (Figure37E). Altogether, a remarkable EZH2 upregulation parallel to the limited increase in Myc expression was noticed in CTO infected with the high-risk HCMV-DB and BL strains. EZH2 and Myc were expressed mainly in the PGCCs subpopulation of CTO-DB and BL (Figure37G). A limited increase in SUZ12 expression was observed in CTO-DB and BL compared to uninfected OECs (p-value (UI OECs:CTO-HCMV) = 0 . 03) (Figure39A,B). Upon assessing the proliferative potential of OECs chronically infected with HCMV high-risk strains, Ki67Ag was highly prominent in CTO-DB and BL compared to uninfected OECs (p-value (UI OECs:CTO-HCMV) = 0 . 03) (Figure37F). High Ki67Ag expression was detected in the large cells (Figure37G).

Figure 37 :

 37 Figure 37: Colony formation in soft agar and the phenotypic characterization of HCMVtransformed OECs.

Figure 38 :

 38 Figure 38: The assessment of the correlation between EZH2 and Myc expression in CTO cells.

Figure 39 :

 39 Figure 39: Expression of SUZ12 in CTO-DB and BL cells.

Figure 40 :

 40 Figure 40: HCMV-transformed OECs display an embryonic stemness phenotype and possess spheroid-forming potential.

Figure 42 :

 42 Figure 42: Expression of CD44 in CTO-DB and BL cells.FACS staining of CD44 in CTO-DB and BL whole and subpopulations; uninfected OECs were used as a control.

  Vimentin was strongly upregulated in DB and BL-infected OECs (p-value (UI OECs:CTO-HCMV) = 0 . 03), whereas a slight decrease in E-cadherin was noticed compared to uninfected OECs (p-value (UI OECs:CTO- HCMV) = 0 . 06) (Figure43A,B, and C). Vimentin and E-cadherin were expressed mainly in the PGCCs and ICs subpopulations of CTO-DB and BL (Figure43D). Altogether, the co-existence of vimentin and E-cadherin was detected in CTO-DB and BL, indicating a mesenchymal/epithelial hybrid state that was also accompanied with occasionally existing small cells possessing an elevated E-cadherin expression (Figure43and Figure44). The co-existence of mesenchymal and epithelial phenotypes confirms the cellular plasticity of CTO-DB and BL. As a control, EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 protein expression in the subpopulations of uninfected OECs was provided in Figure45.

Figure 43 :

 43 Figure 43: HCMV infection of OECs enhances EMT/MET hybrid traits.

Figure 44 :

 44 Figure 44: Detection of high expression of E-cadherin in several small cells present in CTO-DB and BL cultures.

Figure 45 :

 45 Figure 45: FACS staining of EZH2, Myc, Ki67Ag, Vimentin, E-cadherin, and CD44 in the subpopulations of uninfected OECs.

Figure 47 :

 47 Figure 47: Expression of IE1 in CTO-DB and CTO-BL subpopulations by FACS.

Figure 48 :

 48 Figure 48: CTO cultures post-TPA treatment.

Figure 49 :

 49 Figure 49: EZH2 expression in untreated CTO-DB/BL and CTO-DB/BL treated with 0.1 µM of GSK343 and EPZ6438 by FACS.

Figure 50 :

 50 Figure 50: HCMV detection, PGCCs presence as well as EZH2 expression in ovarian cancer biopsies.

Figure 51 :

 51 Figure 51: Myc Expression in ovarian cancer biopsies.

Figure 52 :

 52 Figure 52: Akt Expression in ovarian cancer biopsies.

Figure 53 :

 53 Figure 53: Potential model depicting the course of events leading to the initiation of the giant cell cycle.

Figure 55 :

 55 Figure 55: A schematic representing the giant cell cycling following HCMV infection of OECs.

  Pasquereau S, Haidar Ahmad S, Diab-Assaf M, Herbein G. Oncogenic and Stemness Signatures of the High-Risk HCMV Strains in Breast Cancer Progression. Cancers 2022;14:4271. https://doi.org/10.3390/cancers14174271.

  8-month-old fetus with syphilis and by Löwenstein in 1907. These cells, characterized by their large eccentrically placed nuclei, appear to represent the earliest descriptions of typical cytomegalic cells, which were later termed 'cytomegalia' in 1907 [6]. In 1921, Goodpasture and Talbert first suggested a definitive connection between viral diseases and cells exhibiting characteristic intranuclear inclusion bodies. This concept was further elucidated by Wyatt et al., who later in 1932 referred to it as "generalized

cytomegalic inclusion disease (CID)." This notion was in alignment with the observations and hypotheses put by other researchers, such as Von Glahn, Pappenheimer, Minder, and others.

Relea se Release viral

  genome replication and the expression of late (L) genes. The L gene expression leads to capsid assembly and the expression of tegument and glycoproteins. The genome loaded capsid exits the nucleus through nuclear egress and associates with the tegument proteins. Subsequently, the capsid acquires a viral envelope by budding into intracellular vesicles. Finally, these enveloped viral particles are released into the extracellular space, completing the HCMV life cycle. Figure has been adapted from Crough et al[START_REF] Crough | Immunobiology of Human Cytomegalovirus: from Bench to Bedside[END_REF].

	1.4.3 Latent Cycle
	Latency in the context of HCMV infection refers to a dormant phase where the viral genome is
	retained without active virus replication or the release of infectious viral particles, occasionally
	interrupted by reactivation events [56]. Multiple latency reservoirs have been identified in the
	blood and bone marrow including mainly CD34+ hematopoietic progenitor cells and CD14+
	monocytes. HCMV reactivation could be triggered by monocytes harboring latent viral genomes
	differentiating into macrophages or dendritic cells (DCs) [57,58]. During latency, only a limited
	set of genes are expressed, including genes from the UL133-UL138 locus, UL144, the latent
	unique nuclear antigen (LUNA), the US28 viral G-coupled receptor and chemokine receptor
	homologue, the latency-associated viral homolog of IL-10 (LAcmvIL-10) encoded by UL111A, a
	shorter variant of the UL123-encoded IE1 protein (IE1×4), a transcript associated with latency
	originating from a distant promoter within the MIE region, and long non-coding RNAs of 2.7 kb
	and 4.9 kb [59]. The establishment of HCMV latency is a complex process that involves epigenetic
	silencing of the viral genome through chromatin remodeling around the viral major immediate
	early promoter (MIEP) and the presence of latency-associated repressors. Histone
	methyltransferases (HMTs) and histone deacetylases (HDACs) play roles in maintaining latency
	[60-62]. Cellular defenses and controls, such as nuclear domain 10 (ND10) structures or
	promyelocytic leukemia protein (PML) nuclear bodies, also contribute to suppressing viral
	replication by modifying chromatin around the viral MIEP [63-65]. On the other hand, reactivation
	involves the transition to lytic replication [66]. Various factors can trigger this switch, including
	cellular differentiation, stress conditions, and certain drugs like histone deacetylase inhibitors; the
	latter have been shown to alleviate the repressive marks on chromatin, resulting in IE gene
	expression

Table 1 :

 1 Oncoviruses.

	Oncogenic	Genome	Human Cancers	Mode of infection	Oncoproteins
	Viruses
	EBV	dsDNA	Burkitt's lymphoma, Hodgkin's	Saliva, milk, blood, or-	EBNA1,
			lymphoma, posttransplantation	gan transplantation	EBNA2,
			lymphoma, nasopharyngeal carci-		EBNA3A,
			noma		EBNA3B,
					EBNA3C, LMP1
	HBV	dsDNA	Hepatocarcinoma	Transplacentally, milk,	HBx
				sexually, parenteraly,
				percutaneously
	HCV	ssRNA	Hepatocarcinoma	Injection equipment,	Core protein
				blood transfusion, sex-
				ually
	HPV	dsDNA	Cervical cancer, penis cancer, anal	Sexually	E6, E7
			cancer, head and neck carcinoma
	HTLV-1	ssRNA	Adult T-cell leukemia (ATL)	Transplacentally, milk,	Tax, HBZ
				sexually, blood
	KSHV	dsDNA	Kaposi's sarcoma, primary effu-	Sexually, via drug in-	LANA, RTA,
			sion lymphoma (PEL), multicentric	jection, blood transfu-	vFLIP, vIL-6,
			Castleman's disease (MCD),	sion, solid organ trans-	vGPCR
			KSHV-associated inflammatory	plantation
			syndrome (KICS)

Table 2 : HCMV Gene Products. HCMV Gene Products Mechanism of Action Oncogenic Characteristic US2, US3, US6, US10, US11

 2

		▪ MHC class I expression impairment, reducing	➢ Preventing host cell MHC
		HCMV antigen presentation to CD8+ cells	class I antigen expression
		and evasion of CD8+ T cell immunity,	that is required for CD8+
		superinfection	cytotoxic tumor killing
		▪ US2 down regulates MHC class II and
		reduces HCMV antigen presentation to CD4+
		cells
	US18 and US20	▪ Interfere with B7-H6 surface expression	➢ HCMV-immune	evasion
		involving endosomal degradation, evades NK	might indirectly affect tumor
		cells' immune recognition	environment
	US28 (viral GPCR)	▪ Promotes chemotaxis	➢ Cellular proliferation, tumor growth, enhanced
			angiogenesis	and	cell
			survival

Table 3 : HCMV enabling the characteristics of cancer.

 3

	Cancer Hallmarks	HCMV Mechanism of Action	HCMV Proteins
		Induces cell cycle progression to S phase	IE2
	Sustaining Proliferative Signaling	Induces expression of E2F genes	pp71
		Phosphorylates Rb	UL97
		Activates EGFR	HCMV infection
		Dysregulates Cyclin E expression	IE1
	Evading Growth Suppressors	Inhibits p53 degradation
		Decreases levels of p21 Induces expression of p53	mtrII
		Binds to p53
	Activating Invasion and Metastasis	Activation of RhoA dependent motility of U373 cells as well as smooth muscle cells	US28
	Enabling Replicative Immortality	Activation of telomerase	IE1
	Inducing Angiogenesis	Induction of VEGF expression Induction of IL-8	US28 IE1
		Inhibits apoptosis	IE1
	Resisting Cell Death
		Activates PI3-K/Akt pathway	IE2, vMIA, vICA
	Deregulating Cellular Energetics	Increases flux through glycolytic pathway, acetyl CoA, flux of carbon, nucleotide biosynthesis	HCMV infection
	Avoiding Immune Destruction	Production of homologs to immunosuppressive cyto-kines	HCMV IL-10

Table 7 : Clinical and biological data of the GBM patients.

 7

	GBM	Age	Sex	CMV	EZH2 Fold gene	Myc Fold gene	Akt Fold gene
	Pa-	(years)	(F/M)	detection	Expression	Expression	Expression
	tients			(IE1/UL69	(Low/Intermedi-	(Low/Intermedi-	(Low/Intermedi-
	N°			Expres-	ate/ High)	ate/ High)	ate/ High)
				sion)
			MGMT PROMOTER METHYLATED GBM BIOPSIES (n=17)
	1		M	+/+	*	*	**
	2		M	+/+	**	**	***
	3		F	+/+	**	**	*
	4		F	+/+	***	**	**
	5		M	+ /+	*	*	*
	6		F	+/+	**	**	*
	7		M	+/+	**	**	*
	8		F	+/+	*	*	*
	9		F	+/+	**	*	*
	10		M	+/+	***	**	*
	11		F	+/+	*	*	*
	12		M	+/+	*	*	*
	13		M	+/+	***	**	**
	14		M	+/+	*	*	*
	15		F	+/+	*	*	**
	16		F	+/+	*	*	**
	17		M	+/+	*	*	*
	Mean	69.29	[8F/9M]	[+/+]	[9*/5**/3***] 53-	[10*/7**/0***]	[11*/5**/1***]
			47-53%		29-18%	59-41-0%	65-29-6%
			MGMT PROMOTER UNMETHYLATED GBM BIOPSIES (n=20)
	18		M	+/+	***	**	*
	19		M	+/+	*	*	*
	20		M	+/+	***	*	*
	21		M	+/+	***	***	**
	22		F	+/+	*	*	*
	23		M	+/+	**	**	*
	24		M	+/+	**	**	*
	25		M	+/+	**	**	***
	26		M	+/+	*	*	*
	27		F	+/+	*	*	*
	28		M	+/+	*	*	*
	29		F	+/+	**	*	**
	30		F	+/+	**	*	**
	31		M	+/+	***	**	***
	32		M	+/+	**	*	*
	33		F	+/+	**	**	*
	34		F	+/+	*	*	*
	35		M	+/+	**	*	**
	36		F	+/+	*	*	*

Table 8 : Clinical data and treatments of the OC patients.

 8

	Biopsy	Age Tumor	Metastasis	Chemotherapy
	#
	1	67	HGSOC	+	carboplatin -gemcitabine -bevacizumab
	2	64	HGSOC	+	carboplatin -paclitaxel
	3	50	HGSOC	+	bevacizumab-carboplatin -paclitaxel -pegylated liposomal doxorubi-
					cin-HIPEC
	4	82	HGSOC	+	carboplatin -gemcitabine -bevacizumab -paclitaxel
	5	63	HGSOC	+	carboplatin -paclitaxel -pegylated liposomal doxorubicin
	6	48	HGSOC	+	bevacizumab -carboplatin -paclitaxel -pegylated liposomal doxo-
					rubicin
	7	57	HGSOC	+	topotecan
	8	79	HGSOC	-	carboplatin -paclitaxel
	9	70	HGSOC	+	carboplatin -gemcitabine -bevacizumab
	10	57	HGSOC	-	carboplatin -cyclophosphamide
	11	70	HGSOC	+	carboplatin -paclitaxel -pegylated liposomal doxorubicin -gemcita-
					bine -topotecan
	12	54	HGSOC	+	carboplatin -paclitaxel -bevacizumab
	13	56	HGSOC	+	carboplatin -paclitaxel
	14	88	HGSOC	+	carboplatin -paclitaxel
	15	60	HGSOC	+	carboplatin -paclitaxel
	16	64	HGSOC	+	carboplatin -paclitaxel
	17	57	HGSOC	+	docetaxel -carboplatin -paclitaxel
	18	44	HGSOC	+	carboplatin -paclitaxel

Table 9 : Characteristics of HCMV-GBM strains.

 9

	HCMV iso-lated from GBM	Myc	Human Astrocytes Infected with HCMV-GBM EZH2 SOX2 Soft Agar Spheroids For-	Invasion
	N° (HCMVGBM) Biopsy	FACS sion by Expres-	FACS sion by Expres-	Staining cal by Confo-Expression	Colonies	mation
		MGMT PROMOTER METHYLATED GBM BIOPSIES (n=4)
	10	+	+	+	+	+	+/-
	11	+	+	+	+	+	+
	12	+	+	+	+	+	+
	13	+	+	+	+	+	+
	Positive	[4/4]	[4/4]	[4/4]	[4/4]	[4/4]	[3/4]
		MGMT PROMOTER UNMETHYLATED GBM BIOPSIES (n=7)
	18	+	+	+	+	+	+
	20	+	+	+	+	+	+
	21	+	+	+	+	+	+
	23	+	+	+	+	+	+
	25	+	+	+	+	+	+
	31	+	+	+	+	+	+
	33	+	+	+	+	+	+
	Positive	[7/7]	[7/7]	[7/7]	[7/7]	[7/7]	[7/7]
			TOTAL ISOLATED GBM BIOPSIES (n=11)
	Positive	[11/11]	[11/11]	[11/11]	[11/11]	[11/11]	[10/11]

CD24

BD-555428/BD Biosciences (Franklin Lakes, USA) To determine the sustained HCMV presence, HCMV-IE1 antigen was detected using flow cytometric analysis. IE1 was strongly expressed in CTH-B544 and CTH-B693; uninfected HMECs showed no staining for IE1 (Figure 16A). Using qPCR, we detected HCMV (IE1 DNA)

in the supernatant of CTH-B544 and CTH-B693 cultures (Figure 16B). Further, CTH cells were positively stained for HCMV-pp65 antigen versus uninfected HMECs (Figure 16C). CTH cells were treated with TPA to assess latency relevance (Figure 16D,E). Post TPA treatment, the proliferation of CTH-B544 and CTH-B693 cells was promoted (Figure 16D). IE1 detection was elevated at day 1 and day 2 post-treatment in CTH-B544 and CTH-B693, respectively (p-value= 0.33), and subsequently decreased (Figure 16E). The scheme illustrates the four phases of the CEGBCs cycling including initiation, self-renewal, proliferation, and dedifferentiation, termination/differentiation, and stability. Large red arrows showed the large cells and PGCCs; Small red and black arrows showed the intermediate/small heterogeneous cells. In the termination/differentiation phase, NPC-like (1, 2), neuron-like [START_REF] Roizmann | The familyHerpesviridae: an update[END_REF][START_REF] Ho | The history of cytomegalovirus and its diseases[END_REF], and mesenchymal-like (5, 6) cells were detected.