Dr Véronique M Braud

Professor Marie-Dominique Galibert-Anne

Alain Sarasin

Alexander Steinle

Maria Gonçalves-Maia

Miguel Basante

Yannick Gache

Estelle Cosson

Emie Salavagione

Margot Muller

Françoise Bernerd

Sébastien Schaub

DR Thierry Magnaldo
email: thierry.magnaldo@univ-cotedazur.fr

CLEC2A regulates natural killer cell and fibroblast-mediated control of cutaneous squamous cell carcinoma invasion

Keywords: Xeroderma pigmentosum, squamous cells carcinoma, tumoral microenvironment, immune surveillance, CLEC2A

My sincere thanks to Thierry, my thesis supervisor, for

really could write a dissertation containing only the multitude of the things that you do for us on a daily basis. The most important though, is your availability to take the time to listen to us and our problems. You were a constant and reassuring presence during these past years and I know we will keep in touch for years to come. Thank you.

Margot and Julia "mes copinettes"! The first acknowledgement lines I wrote were actually to thank you two! Not only for our surrealistic discussions that reassured me that I wasn't alone in my thinking but also for your friendship, for your continuous support, and for making my days in the Pasteur tower so much fun! Finally, thank you for running with me our incredible Instagram account @lavieaulabo. I will always cherish the friendship we have created. I lOvE yOu! I also want to thank all the former members of the laboratory especially Sahar, Sophie and Miguel. Thank you for your support and the assistance you gave towards this work.

Valérie and Nicolas, thank you for always taking the time to help me deal with the incredibly complex work of orders and shipments. I appreciate all of your help.

To the people of the second floor of the Pasteur tower, thank you for your genuine concern for my wellbeing and for all of the fun and interesting discussions we had over these past years that helped me growth as a researcher and most importantly as a person.

A particular thanks also goes to Marina Shkreli that generously let me use one of her team's desks for almost a year now, and to all of Paquis team, for the laughter and good moments we shared together.

To my family, the ones with whom I share a bloodline and the ones with whom I chose to share life with. For them, I'm the cousin, the niece, the godmother, the best friend, the chief, the lucky one, the fearless... For me, they are the reason I wake up happy every morning. Having you in my life has made this path a lot easier. Thank you for your love and friendship, for all of your understanding that I am a bit too far away to be able to see you as much as I would like and for coming to visit as much as you can. I feel blessed to have you in my life.

Ivo, thank you for your support, encouragement and understanding of my goals and my aspirations. Your love is a blessing and has been my major source of strength. Without your help, I would not have been able to complete much of what I have done and become who I am today. Thank you for being with me every single day and for never letting me feel lonely in this solitary journey. There are no words to express how much I value and love you.

Aos meus pais, meus exemplos maiores, que são a constante da minha vida, a presença de todas as horas e a certeza de que o Amor é o unico que importa. Obrigada. Résumé de la thèse L'étiologie des cancers cutanées est liée à des mutations génétiques résultant de l'exposition aux rayonnements ultraviolets (UV) émis par le soleil. La propagation des cellules cancéreuses dépend aussi des interactions avec les cellules présentes dans le microenvironnement circulant, notamment des fibroblastes associés au cancer (FAC) et des cellules immunitaires.

Xeroderma pigmentosum (XP) est une maladie génétique qui comprend 7 groupes de complémentation génétique (XP-A à XP-G). Les patients XP présentent une déficience du mécanisme de réparation des lésions de l'ADN provoquées par les UV. Ces patients sont susceptibles au développement précoce de très nombreux cancers cutanées. XP-C est le groupe de complémentation le plus représenté en Europe. Chez ces patients, les carcinomes spino-cellularies (CSC) sont plus fréquents que les carcinomes baso-cellulaires (CBC) (taux Is there anything that skin can't do? -Albert Kligman [START_REF] Chuong | What is the 'true' function of skin?[END_REF] The word skin has roots in both Greek and Latin and ultimately means surface envelope. Greek and latin medicine pointed the skin as being purely an enveloping tissue of the internal organs, therefore underestimating its true importance. Partially because of that, the study of skin and its diseases did not have an official name -dermatology -till the 18 th -19 th centuries [START_REF] Santoro | Skin over the centuries. A short history of dermatology: Physiology, pathology and cosmetics[END_REF]. We know today that besides from "enveloping" the entire body, skin is an organ that plays vital functions in the mammalian body.

Table of contents

List of figures

List of tables

Skin is commonly described as the largest human organ. It has a surface area of 1.2 to 2m 2

and accounts for about 12 to 15% of total body weight of an adult (Millington 2009, Lai-Cheong and[START_REF] Lai-Cheong | Structure and function of skin, hair and nails[END_REF]. Recent studies enlightened that even if this estimate surface area is accurate when talking about heat, water loss or topical drug delivery calculations, the measurement fails by not considering the approximately 5 million of appendages openings.

These appendages openings put the total surface area of the skin between 25 and 30 m 2 (Gallo 2017).

1. Structure of the skin Skin comprises three main layers: the epidermis, the dermis and the hypodermis. The epidermis is a stratified cellular layer separated from a dermis of connective tissue by a dermalepidermal junction. The hypodermis is the deepest layer of the skin and is mainly composed of adipose cells (figure 1) (McGrath and Uitto 2016).

Epidermis

The epidermis is a self-renewing, stratified squamous terminally differentiated epithelium.

Keratinocytes are the major cell type present in the epidermis accounting for 90 to 95% of the cells. Keratinocytes migrate from the epidermal basement membrane to the outermost part of the epidermis during a differentiation process and form four main different layers during the transit: basal layer, spinous layers, granular layers and cornified layers (figure 2). These different layers contribute to maintain of the structure of the skin.

(McGrath and Uitto 2016) [START_REF] Eckhart | Cell death by cornification[END_REF] Figure 1. The skin and its appendages. The basal layer of the epidermis consists on a single layer of small and cuboidal keratinocytes.

These keratinocytes express keratins K5, K14 and K15 and can be divided in three subtypes depending on their mitotic activity: epidermal stem cells, transient amplifying cells and postmitotic keratinocytes [START_REF] Lyle | The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells[END_REF][START_REF] Eckhart | Cell death by cornification[END_REF].

The epidermal stem cells are located in special niches of the basal layer. They are quiescent cells, that divide unfrequently upon request for epidermal renewal or wound healing and, when they do, they give rise to the transient amplifying (TA) cells. These TA proliferating cells divide to generate post-mitotic keratinocytes that start terminal differentiation [START_REF] Blanpain | Epidermal stem cells of the skin[END_REF][START_REF] Koster | Mechanisms Regulating Epithelial Stratification[END_REF][START_REF] Eckhart | Cell death by cornification[END_REF]. Potten et al. and Bickenbach et al. were among the first to identify epidermal stem cells by labeling basal cells with a nucleotide analog. Once labeled, stem cells are able to retain the label for prolonged periods of time in what had been termed as "label retaining cells" (Potten, Kovacs et al. 1974, Bickenbach and[START_REF] Bickenbach | Identification and Localization of Label-Retaining Cells in Hamster Epithelia[END_REF]. More recently, following the identification strategy proposed by Ami Li et al. in 1998, authors were able to estimate the percentage of stem cells in the basal layer. The identification is based on the patterns of expression of two surface markers: the adhesion molecule α6 integrin and the proliferationassociated marker CD71 also known as transferrin receptor. Epidermal cells expressing high levels of α6 integrin and low levels of CD71 are considered as stem cells, and conversely epidermal cells expressing α6 integrin and high levels of CD71 are considered as being TA cells (Li, Simmons et al. 1998) [START_REF] Schneider | Measuring stem cell frequency in epidermis: a quantitative in vivo functional assay for long-term repopulating cells[END_REF][START_REF] Webb | Location and phenotype of human adult keratinocyte stem cells of the skin[END_REF].

By constantly providing post-mitotic keratinocytes that undergo differentiation, the basal layer contributes to assure the mechanical stability and homeostasis of the epidermis.

1.1.b. Spinous layer or stratum spinosum

Post-mitotic keratinocytes move upwards to spinous layers changing in shape and protein expression. Spinous keratinocytes are adjacently attached through desmosomes and stop expressing K5 and K14 and K15. Instead, they express K1 and K10 [START_REF] Fuchs | Epidermal differentiation: the bare essentials[END_REF][START_REF] Lyle | The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells[END_REF]. The cytoplasm of the upper spinous cells contains lamellar bodies (LB) enriched with the precursors of stratum corneum lipids. Enzymes involved in lipogenesis such as lipid hydrolases, proteases and antimicrobial peptides (AMPs) are also present in these LB. Due to keratin synthesis and lipogenesis, cells from the upper spinous layers become larger and ultimately reach another differentiation stage to form granular layers [START_REF] Feingold | Thematic review series: Skin Lipids. The role of epidermal lipids in cutaneous permeability barrier homeostasis[END_REF].

1.1.c. The granular layers or stratum granulosum

The granular keratinocytes are the uppermost nucleated cells of the epidermis. They are characterized by the presence of LB and two types of keratohyalin granules containing mostly profilaggrin, and loricrin [START_REF] Steven | Biosynthetic pathways of filaggrin and loricrin--two major proteins expressed by terminally differentiated epidermal keratinocytes[END_REF]. Profilaggrin is the precursor of filaggrin, known for its role as an aggregator of keratin filaments, as well as in maintaining epidermal hydration (Dale, Resing et al. 1985, Kezic and[START_REF] Kezic | Filaggrin and Skin Barrier Function[END_REF]. Loricrin is the major component of the corneocyte envelope [START_REF] Mehrel | Identification of a major keratinocyte cell envelope protein, loricrin[END_REF]). LB migrate from the cytoplasm to the cell membrane and ultimately release their lipid components into the intercellular space between the stratum granulosum and the stratum corneum. This process, is primarily regulated by calcium signaling (i.e. depend on minimal Ca 2+ concentrations) and promotes the formation of a unique hydrophobic film as initially described by [START_REF] Gray | Lipid compositions of cells isolated from pig, human, and rat epidermis[END_REF][START_REF] Gray | Lipid compositions of cells isolated from pig, human, and rat epidermis[END_REF][START_REF] Feingold | Role of lipids in the formation and maintenance of the cutaneous permeability barrier[END_REF].

1.1.d. Cornified layers or stratum corneum

Cornified layers are the outermost layers of the epidermis. Keratinocytes present in these layers have lost nuclei and cytoplasmic organelles and are called corneocytes. Under the influence of the now active filaggrin, keratin filaments aggregate and align and cells become flattened (Steinert andMarekov 1995, Kezic and[START_REF] Kezic | Filaggrin and Skin Barrier Function[END_REF]. Corneocytes possess a cornified envelope within the plasma membrane that is composed by structural proteinsincluding involucrin and loricrin -cross-linked between each other through transglutaminases.

The intercellular spaces between corneocytes are filled with a lipid lamellae composed by a complex series of lipids, derived from the secretion of the LB. The terminal differentiation process ultimately results on a rigid and hydrophobic structure that provides both mechanical resistance and an active permeable barrier preventing transepidermal water loss and blocking the entry of external compounds (Candi, Schmidt et al. 2005, Feingold and[START_REF] Feingold | Role of lipids in the formation and maintenance of the cutaneous permeability barrier[END_REF].

Corneocytes are attached to each other by a modified type of desmosomes called corneodesmosomes. During the last stages of the differentiation process, corneodesmosomes are degraded by proteases allowing the desquamation of dead corneocytes. The equilibrium between cell loss by desquamation and epidermal renewal is essential for epidermal homeostasis [START_REF] Marks | Studies on isolated corneocytes[END_REF][START_REF] Candi | The cornified envelope: a model of cell death in the skin[END_REF][START_REF] Eckhart | Cell death by cornification[END_REF]).

1.1.e. Other epithelial cells

Langerhans cells

Langerhans cells reside in the basal and suprabasal layers of the epidermis and account for about 2% to 4% of epidermal cells. They are antigen-presenting dendritic cells (DC) that originate from bone marrow and derive from primitive erythro-myeloid progenitors [START_REF] Milne | Langerhans cell origin and regulation[END_REF]. The most important role of Langerhans cells is to detect and transport antigens to skin local lymph nodes therefore contributing to the adaptive immune response of the skin.

They also have a role in maintaining immune tolerance against innocuous and self-antigens of the skin [START_REF] Romani | Langerhans cells -dendritic cells of the epidermis[END_REF][START_REF] Stoitzner | The Langerhans cell controversy: are they immunostimulatory or immunoregulatory cells of the skin immune system?[END_REF][START_REF] Tay | The Skin-Resident Immune Network[END_REF].

Merkel cells

Merkel cells are neuroendocrine cells mainly located in the basal layer of the epidermis and account for about 0,2% to 5% of epidermal cells. The embryonic origin of Merkel cells has been a matter of intense debate on whether they originate from embryonic epidermal or neural crest progenitors. A recent review unify this two hypothesis and clarify that Merkel cells derive from both neural and embryonic epidermis during early intrauterine life and they only undergo further differentiation once in epidermis [START_REF] Abraham | Merkel Cells: A Collective Review of Current Concepts[END_REF]. Merkel cells have mechanosensory properties and are more concentrated in high tactile and sensitive areas.

Their cytoplasm contains neurosecretory granules and they are tightly connected with sensory nerve endings forming the Merkel cell-neurite complex. Throughout this association Merkel cells actively participate in touch reception [START_REF] Boulais | Merkel cells[END_REF].

Melanocytes

Dermal-epidermal junction

The dermal-epidermal junction (DEJ) is at the interface between dermis and epidermis. It promotes epidermal adhesion to the dermis and is essential to the crosstalk and cell migration between the two compartments.

DEJ is composed by a network of proteins, the main ones being type IV collagen and laminins, and is divided in different layers. Lamina lucida, the upper layer of the DEJ, is structurally attached to basal keratinocytes through hemidesmosomes. Lamina densa is below lamina lucida and interacts with the upper dermis through anchoring fibrils. Besides its importance in maintaining the cohesion and integrity of skin, DEJ also plays a role in the protection against mechanical shearing. Both epidermal keratinocytes and dermal fibroblasts are implicated in the synthesis of the components of the DEJ and therefore in its homeostasis (El Ghalbzouri, Jonkman et al. 2005, Has and[START_REF] Has | Epidermal Basement Membrane in Health and Disease[END_REF].

Dermis

The dermis is the major component of the human skin and its thickness varies among different regions of the body. It consists of an extracellular matrix (ECM) of connective tissue that provides strength and flexibility to the skin and has a strong capacity to retain water. Unlike the epidermis, dermis is an abundantly vascularized and sparsely cellular layer with fibroblasts being the most abundant cell type. The blood vessels present in the dermis provide nutrients and oxygen to the skin and also have a role in thermoregulation [START_REF] Plager | 33 -Structure of the Skin and Cutaneous Immunology[END_REF].

There are three classes of ECM components: fiber-forming structural molecules, nonfiberforming structural molecules, and matricellular proteins (figure 3).

Adapted from [START_REF] Tracy | Extracellular Matrix and Dermal Fibroblast Function in the Healing Wound[END_REF] Collagens and elastin are important fiber forming molecules providing structure to the dermis. Fiber-forming molecules are the ones providing structure to the ECM. The major fiber-forming molecule is collagen comprising approximately 80% of the dry weight of the skin. There are nearly 30 different types of collagen identified so far (Ricard-Blum 2011). Type I and type III are the most abundant in the dermis while type IV is the principal type in the DEJ. Whereas collagen fibers provide tensile strength to the skin, elastic fibers are the ones responsible for skin elastic properties. The main component of elastic fibers is elastin [START_REF] Kielty | Elastic fibres[END_REF]. Other fiber-forming proteins that are mediators in cell migration and hemostasis are fibrins, fibronectins and vitronectins [START_REF] Weinstein | Collagen and Elastin of Human Dermis**From the Divisions of Dermatology and Cardiology of the Department of Medicine[END_REF][START_REF] Bosman | Functional structure and composition of the extracellular matrix[END_REF][START_REF] Tracy | Extracellular Matrix and Dermal Fibroblast Function in the Healing Wound[END_REF].

The majority of the dermis interstitial space is filled by the nonfiber-formin moleculesproteoglycans and glycosaminoglycans (GAGs). Their hydrophilic nature assures the hydration of the dermis. Matricellular proteins do not affect the structure of the dermis. They are very little present in a healthy dermis but increases expression during tissue injury contributing to cell-matrix interactions [START_REF] Bosman | Functional structure and composition of the extracellular matrix[END_REF]Stamenkovic 2003, Tracy, Minasian et al. 2014).

ECM began to be considered as more than a structural support when it was shown that some cells needed to be anchored to ECM to grow in a process called "anchorage dependence". We now know that besides its structural support and anchoring capacities, the complex composition of the ECM is essential to the homeostasis of the skin, by regulating certain aspects of cell behavior such as proliferation and migration [START_REF] Stoker | Anchorage and growth regulation in normal and virus-transformed cells[END_REF][START_REF] Bosman | Functional structure and composition of the extracellular matrix[END_REF][START_REF] Tracy | Extracellular Matrix and Dermal Fibroblast Function in the Healing Wound[END_REF]).

1.3.a. Fibroblasts

Mesenchymal dermal fibroblasts, the main cell type of the dermis, are responsible for both the synthesis and degradation of ECM components. Skin fibroblasts represent a heterogeneous population of cells. Vimentin and fibroblasts specific protein 1 (FSP-1) are two of the most known fibroblasts markers in the post-natal skin. However, they cannot be considered as specific markers as they are present in other types of cells such as endothelial and myoepithelial cells (vimentin) and hematopoietic, endothelial cells and vascular smooth muscle cells (FSP-1) [START_REF] Sorrell | Fibroblast heterogeneity: more than skin deep[END_REF][START_REF] Kong | Lack of specificity of fibroblastspecific protein 1 in cardiac remodeling and fibrosis[END_REF][START_REF] Chang | Mesenchymal Stem Cell-Like Properties in Fibroblasts[END_REF][START_REF] Driskell | Understanding fibroblast heterogeneity in the skin[END_REF].

Three different subpopulations of fibroblasts can easily be identified in different locations:

papillary layer, reticular layer and fibroblasts associated with hair follicles. Papillary layer is the upper layer of the dermis. This layer is thinner, has high cellular density and a poorly organized fiber bundles comparing to the reticular layer. Fibroblasts from papillary layer proliferate at higher rate, and interfere with keratinocyte viability and growth when cultured in a skin equivalent model [START_REF] Harper | Human skin fibroblasts derived from papillary and reticular dermis: differences in growth potential in vitro[END_REF]Grove 1979, Janson, Rietveld et al. 2017). Fibroblasts from the reticular layer are mostly involved in wound healing [START_REF] Janson | Different Gene Expression Patterns in Human Papillary and Reticular Fibroblasts[END_REF][START_REF] Janson | Differential effect of extracellular matrix derived from papillary and reticular fibroblasts on epidermal development in vitro[END_REF]. Driskell et al. show that in mouse, fibroblasts from papillary and reticular dermis arise from two different lineages which may be a coherent explanation for how different and specific the functions of these two subpopulations are [START_REF] Driskell | Distinct fibroblast lineages determine dermal architecture in skin development and repair[END_REF].

The characteristics of papillary and reticular fibroblasts are summarized in table 1 The third subpopulation of fibroblasts, associated with hair follicles, plays a role in the homeostasis of the hair follicle [START_REF] Yang | Review of hair follicle dermal cells[END_REF].

Papillary fibroblast Reticular fibroblasts Size (diameter of cells in suspension)

small large Adapted from [START_REF] Stunova | Dermal fibroblasts-A heterogeneous population with regulatory function in wound healing[END_REF] Matrix metalloproteinase (MMP), Hepatocyte growth factor (HGF), Transforming growth factor-β (TGF)

Shape

Table 1 Characteristics of papillary and reticular dermis.

For a long time, fibroblasts were considered as only having a structural role in the homeostasis of the dermis through the synthesis and remodeling of the ECM. Although they are still a very enigmatic heterogeneous population, it is now established that fibroblasts interact with numerous distinct cell typesepidermal, endothelial, dendritic and immunocompetent cellsplaying a central role in a diversity of processes. They are essential in wound healing, in mediating inflammatory and immune responses, in cancer cell progression and could be considered as a barometer for aging [START_REF] Sorrell | Chapter 4 Fibroblasts-A Diverse Population at the Center of It All[END_REF]. The functional diversity of dermal fibroblasts is not totally understood. Besides of the obvious differences observed in fibroblasts arising from different lineages, Xu et al. proved that the different stimuli to which fibroblasts are subjected in different anatomic sites also contribute to the heterogeneity of fibroblast population [START_REF] Xu | Analysis of Mechanical Behavior of Dermal Fibroblasts Obtained From Various Anatomical Sites in Humans[END_REF]. The role of fibroblasts in immune responses and cancer cell progression will be further discussed later in this manuscript.

1.3.b. Other dermal cells

Mast cells

Mast cells derive from myeloid progenitors and are present in connective tissues throughout the body. In skin, they are concentrated in the papillary dermis near blood vessels, cutaneous appendages and nerves. In 1950, Hellstrom and Holmgren determined that there are about 7000 to 10000 mast cells per mm 3 in normal skin. Mast cells play a role in immune responses and are particularly well known for their role in the immediate allergic reaction through the release of histamine. Although they reside in the dermis, mast cells can migrate to the epidermis upon inflammation [START_REF] Hellstrom | Numerical distribution of mast cells in the human skin and heart[END_REF]Holmgren 1950, Harvima and[START_REF] Harvima | Mast cells as regulators of skin inflammation and immunity[END_REF].

Macrophages

Macrophages are phagocytic cells that derive from monocyte differentiation. Besides from their classical phagocytic properties removing dead cells and metabolites from tissues, they also play a role in would healing and angiogenesis. Macrophages are activated after a cutaneous injury and transit between a pro-inflammatory and an anti-inflammatory phenotype. They contribute to both the synthesis of ECM and the re-epithelialization [START_REF] Koh | Inflammation and wound healing: the role of the macrophage[END_REF].

Dermal dendritic cells

Dermal DCs are a heterogeneous population that can be distinguish from other dendritic cells by the expression of CD209, a dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non integrin receptor. Like Langerhans cells in the epidermis, dermal DCs are antigen-presenting cells (APC), playing a role in both the initiation of primary immune responses and in the maintenance of immune tolerance [START_REF] Geijtenbeek | Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses[END_REF][START_REF] Ebner | Expression of C-type lectin receptors by subsets of dendritic cells in human skin[END_REF]).

Lymphocytes

T cells are the main type of lymphocytes present in the dermis. There are about 20 billion T cells in skin, making it the largest reservoir of T cells in body tissues. Some T cells remain resident cells never leaving the dermis, while others have the capacity to re-enter the blood circulation. T cells are able to memorize and recognize specific antigens presented by APCs, hence leading to specific immune responses [START_REF] Clark | Skin-Resident T Cells: The Ups and Downs of On Site Immunity[END_REF]. Natural killer (NK) cells are another type of lymphocytes present in the dermis. NK cells are endowed with cytotoxic properties to kill external pathogens, infected or cancer cells without previous need of antigen presentation [START_REF] Ebert | Homing and function of human skin gammadelta T cells and NK cells: relevance for tumor surveillance[END_REF]). The role of T cells and NK cells in cancer microenvironment will be discussed n chapter III.

Hypodermis

The hypodermis is the deepest layer of the skin and is mainly composed of adipocytes. It provides insulation and cushioning to the internal organs and store energy in the form of lipids.

Within the hypodermis, adipocytes are organized in two distinct tissue subsets: the dermal adipose tissue and the subcutaneous adipose tissue. Studies have shown that murine adipocytes from these two adipose tissues have different developmental origins. Moreover, murine adipocytes from dermal adipose tissue shared the same progenitor with reticular fibroblasts [START_REF] Driskell | Distinct fibroblast lineages determine dermal architecture in skin development and repair[END_REF][START_REF] Driskell | Defining dermal adipose tissue[END_REF]). In addition to the classic metabolic function, adipocytes also play a role in regulation of hair follicle growth, angiogenesis and wound healing [START_REF] Rivera-Gonzalez | Adipocytes in Skin Health and Disease[END_REF]. For a long time, hypodermis was not considered as responsive to UV radiation because UVs can't penetrate the hypodermis. Of note, UVA penetrates deeply into the dermis, while UVB penetrate only up to the superficial dermis [START_REF] Christensen | Ultraviolet Photobiology in Dermatology[END_REF]. However, studies from Ji Ho Chung laboratory show that UV indirectly affect adipocytes through keratinocytes and fibroblasts, promoting a pro-inflammatory state that unbalances the homeostasis of the adipose tissue [START_REF] Kim | Adipochemokines induced by ultraviolet irradiation contribute to impaired fat metabolism in subcutaneous fat cells[END_REF]).

Skin appendages

Skin appendages are structures that are essential for skin to fulfil all its functions properly.

1.5.a. Pilosebaceous units

Pilocebaceous units are composed of hair follicles, sebaceous glands and the arrector pili muscle that, when contracted, is responsible for the "raiser" of the hair. Hair follicles derive from epidermal tissue and project into the dermis. The dermal papilla is located at the base of the hair follicle. It is surrounded by a rich extracellular matrix and acts as a mesodermal signaling center. The bulge region, another component of the hair follicle, is a reservoir of stem cells that when activated is responsible for the production of new hair [START_REF] Alonso | The hair cycle[END_REF]Fuchs 2006, Schneider, Schmidt-Ullrich et al. 2009). Hair growth depends on cycles of growth (anagen), apoptosis-mediated regression (catagen) and relative quiescence (telogen). In each cycle, a new hair is formed, and the old hair eventually shed. For more information about hair cell cycle please refer to Alonso and Fuchs review [START_REF] Alonso | The hair cycle[END_REF].

The superficial part of the hair is mainly composed by keratin filaments that derive from heavily keratinized and pigmented cells. Hair length and structure differs in different parts of the body.

Hair acts as a mechanoreceptor due to its sensitivity to touch and has other specific functions dependent on the location. For example head hair protects the scalp against UV radiation and eyelashes and eyebrows helps keeping foreign particles out of the eyes [START_REF] Arda | Basic histological structure and functions of facial skin[END_REF]). The complete structure of the hair follicle is represented in figure 1.

1.5.b. Glands

There are two types of skin glands: sebaceous glands and sweat glands. Sebaceous glands are typically associated to hair follicles. They secrete a complex lipid mixture called sebum that allows to maintain skin suppleness and protects the upper part of the epidermis from the external environment. Sebum also presents antimicrobial properties. Sweat glands are subdivided in eccrine and apocrine glands. Eccrine glands are functional after birth and apocrine glands became functional during puberty. Both eccrine and apocrine glands have a thermoregulatory function releasing water to the cell surface when the body temperature rises [START_REF] Arda | Basic histological structure and functions of facial skin[END_REF][START_REF] Fuchs | Epithelial Skin Biology: Three Decades of Developmental Biology, a Hundred Questions Answered and a Thousand New Ones to Address[END_REF]).

Function

In a 2002 textbook, a large council of biologists and clinicians answered the question "What is the "true" function of the skin?". The first thoughts come from Albert Kligman, a dermatologist, who allowed himself to reformulate the question to "Is there anything that skin can't do?" [START_REF] Chuong | What is the 'true' function of skin?[END_REF]. In this section a description of skin functions supports his idea of the skin as a complete, unparalleled, multi-tasking organ.

Skin is frequently described as a barrier, protecting the body from external insults. This barrier is at the same time mechanical, antimicrobial, chemical and hydric.

Mechanical barrier:

The viscoelastic properties of the skin reflect both its rich fiber composition and the different cell-to-cell interactions. This complex structure confers to the skin resistance against internal and external forces and, at the same time, is capable of providing physiological responses when needed [START_REF] Tissot | The body's tailored suit: Skin as a mechanical interface[END_REF].

Antimicrobial barrier and immunesurveillance: Skin cells like keratinocytes and mast cells, sweat glands and commensal bacteria are able to produce and secrete AMPs that are part of skin defense mechanisms against pathogens and external insults. These AMPs have the capacity to directly kill pathogens and trigger an immune response via the activation of immune cells. Furthermore it has been shown that skin inflammatory diseases such as atopic dermatitis and psoriasis exhibit a different AMP profile compared to healthy skin [START_REF] Nakatsuji | Antimicrobials from human skin commensal bacteria protect against Staphylococcus aureus and are deficient in atopic dermatitis[END_REF][START_REF] Patra | Unique profile of antimicrobial peptide expression in polymorphic light eruption lesions compared to healthy skin, atopic dermatitis, and psoriasis[END_REF]. As already mentioned in this manuscript, Langerhans and NK cells play essential roles in the immune system surveillance, permanently screening for the presence of external pathogens in the skin. In presence of microbial invasion, they are able to either initiate immune responses (Langerhans) or directly kill the threat (NK).

Chemical and hydric barrier:

The structure of the corneocytes together with the specific combination of skin lipids confer at the same time a barrier against passive water loss and chemical penetration. Once again, Langerhans cells can act as sensitizers, triggering immune responses after a chemical assault [START_REF] Cumberbatch | Epidermal Langerhans cell migration and sensitisation to chemical allergens[END_REF][START_REF] Wickett | Structure and function of the epidermal barrier[END_REF][START_REF] Jia | The mechanism of skin lipids influencing skin status[END_REF]. Finally, skin is also an effective barrier against UV radiation through melanocytes and their secreted melanin, and cutaneous appendages such as hair and nails.

Besides its function as a barrier, skin play other essential roles in the homeostasis of the human body.

First, in thermoregulation. There are two mechanisms responsible for the control of body temperature. One involves the ability of the sympathetic nervous system to control vasoconstriction and vasodilatation of blood vessels, therefore regulating heat lost through the skin. The other one involves the capacity of sweat glands to cool the body via the release of water [START_REF] Charkoudian | Skin Blood Flow in Adult Human Thermoregulation: How It Works, When It Does Not, and Why[END_REF].

Second and third, in providing insulation and cushion to the internal organs and in storing energy. These two functions are fulfilled by the hypodermis and are related to both structural and cellular characteristics of this layer.

Fourth, as an endocrine organ. Skin specialized cells are able to convert androgens in different activated forms and to produce insulin-like growth factors. After UV exposure, keratinocytes are the ones responsible for the synthesis and metabolism of vitamin D which is essential to regulate the absorption of calcium [START_REF] Zouboulis | The skin as an endocrine organ[END_REF].

Fifth, skin is part of the somatosensory system. Through a complex mechanism, involving keratinocytes, Merkel cells, nerve endings and hair, the mechanical information is transformed in the sensation of touch [START_REF] Tissot | The body's tailored suit: Skin as a mechanical interface[END_REF].

Last, but not least, skin has to be considered as a psychosocial determinant, regarding social interactions. Dermatological patients can easily be stigmatized and stigma often lead to anxiety and depression scenarios. It is important to build strategies to help these patients be more confident, to avoid social reclusion [START_REF] Dimitrov | Stigmatization in dermatology with a special focus on psoriatic patients[END_REF].

Skin cancers

Cutaneous cancers are the most frequent cancer worldwide and their prevalence has highly increased over the last thirty years. BCC is the most common cancer and its incidence varies around the world. The highest incidence rate is in Australia (>1000/100000 person per year) and the lowest in Africa regions (<1/100000 person per year) [START_REF] Lomas | A systematic review of worldwide incidence of nonmelanoma skin cancer[END_REF]. BCCs arise either from interfollicular basal keratinocytes of the interfollicular epidermis or from the hair follicle [START_REF] Youssef | Identification of the cell lineage at the origin of basal cell carcinoma[END_REF], Wang, Wang et al. 2011). Studies have shown that activation of the Sonic HedgeHog pathway signaling due to mutations of core genes like PTCH and the G-protein coupled receptor smoothened (SMO) promote oncogenic signaling and drive the growth of BCCs [START_REF] Fan | Induction of basal cell carcinoma features in transgenic human skin expressing Sonic Hedgehog[END_REF][START_REF] Xie | Activating Smoothened mutations in sporadic basal-cell carcinoma[END_REF][START_REF] Aszterbaum | Ultraviolet and ionizing radiation enhance the growth of BCCs and trichoblastomas in patched heterozygous knockout mice[END_REF][START_REF] Hutchin | Sustained Hedgehog signaling is required for basal cell carcinoma proliferation and survival: conditional skin tumorigenesis recapitulates the hair growth cycle[END_REF]. BCCs rarely metastasize (incidence rates varies from 0.0028% to 0.55%) and are often associated with good prognosis (von Domarus and Stevens BCC is characterized by the presence of aggregations of basaloid keratinocytes that typically stay in contact with the epidermis [START_REF] Stanoszek | Histologic Mimics of Basal Cell Carcinoma[END_REF].

SCC represents less than 20% of NMSC. Like BCC, the higher incidence rate of SCC is also in Australia with > 380/100000 person per year. The incidence of both BCC and SCC changes between geographic areas. In North America there is a clear distinction between SCC rates in northern (60/100000 person per year) and southern (290/100000 person per year) regions [START_REF] Lomas | A systematic review of worldwide incidence of nonmelanoma skin cancer[END_REF]). This difference is most probably related to the UV radiation levels of these two areas. UV radiation is one of the main risk factors in the development of skin cancer and will be discussed later in this chapter. A cohort study from Schmults et al. puts the risk of cutaneous SCC metastasis at 3,7 %, which is significantly higher than in BCC and is frequently associated with poor outcomes [START_REF] Schmults | Factors predictive of recurrence and death from cutaneous squamous cell carcinoma: a 10-year, single-institution cohort study[END_REF]. SCC commonly arises in areas with existing actinic keratoses (AK) benign lesions. AK lesions are caused by accumulation of UV exposure and clinically manifest as rough, scaly and erythematous patches. Many studies over the years tried to estimate the rate of progression of AK into SCC.

A systematic review from 2013 ranged progression rates between 0% and 0,075% per lesionyear, while a previous study puts the rates between 0,025% and 20% per lesion-year [START_REF] Quaedvlieg | Actinic keratosis: how to differentiate the good from the bad ones?[END_REF][START_REF] Werner | The natural history of actinic keratosis: a systematic review[END_REF]. In mice, SCC derives from both hair follicle and interfollicular stem cells however, these observations are highly dependent on the genetic background of the studied mouse as well as their genetically engineered modifications [START_REF] Lapouge | Identifying the cellular origin of squamous skin tumors[END_REF][START_REF] White | Defining the origins of Ras/p53-mediated squamous cell carcinoma[END_REF]. Histologically, SCC presents strands of atypical keratinocytes that infiltrate the dermis and produce high amounts of keratin resulting in the formation of keratin pearls that express terminal markers of epidermal differentiation. They range from well differentiated to poorly differentiated cells showing frequent mitoses [START_REF] Yanofsky | Histopathological variants of cutaneous squamous cell carcinoma: a review[END_REF][START_REF] Stratigos | Diagnosis and treatment of invasive squamous cell carcinoma of the skin: European consensus-based interdisciplinary guideline[END_REF].

Melanoma skin cancer

Melanoma is the most threatening skin cancer. The IARC estimations indicated in 2018 that the 5-year prevalence of melanoma worldwide is > 960 000 cases. In 2018, the worldwide incidence was > 280 000 and the number of deaths > 60 000, putting the mortality rates at about 18%. Like with NMSC, Australia is the region with the highest incidence and, UV radiation is the main risk factor [START_REF] Karimkhani | The global burden of melanoma: results from the Global Burden of Disease Study 2015[END_REF] Approximately 50% of melanomas arise from mutations in BRAF, a gene that codes for a protein kinase that activates the MAP kinase/ERK-signaling pathway. V600E mutation represents 90% of BRAF mutations described in melanoma [START_REF] Davies | Mutations of the BRAF gene in human cancer[END_REF].

Independent of BRAF mutations (i) individuals exhibiting a low pigmentation, due to polymorphisms of the melanocortin 1 receptor gene (MC1R), are more prone to develop melanoma and (ii) the presence of mutations in microphthalmia-associated transcription factor (MITF) predisposes to co-occurring melanoma and renal-cell carcinoma [START_REF] Bertolotto | A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma[END_REF][START_REF] Dessinioti | Melanocortin 1 receptor variants: functional role and pigmentary associations[END_REF][START_REF] Leonardi | Cutaneous melanoma: From pathogenesis to therapy (Review)[END_REF].

The melanoma-related mortality is due to the ability of melanoma cells to form distant metastases. To avoid a late diagnosis that could lead to poor prognoses, in 1985, an ABCD acronym nomenclature system -coding for Asymmetry, Border, Color and Diameter -was created to help melanoma self-diagnosis. This system was later improved with the notion of lesion evolution (E) and became the ABCDE criteria (table 2) (Friedman, Rigel et al. 1985, Robinson and[START_REF] Robinson | Skills training to learn discrimination of ABCDE criteria by those at risk of developing melanoma[END_REF]. in their majority in skin exposed to sunlight areas, most probably due to mutagenic UV radiations [START_REF] Savoye | Patterns of Ultraviolet Radiation Exposure and Skin Cancer Risk: the E3N-SunExp Study[END_REF].

The spectrum of solar radiation contains UV, visible light and infrared wavelengths of radiation.

UV spectrum ranges from 100 nm to 400 nm and can be further subdivided in UVA (315-400 nm), UVB (280-315 nm) and UVC (315-400 nm). Since UVC is (normally) completely blocked by the ozone layer therefore barely reaching earth surface, UVA and UVB are the sun light UV subtypes that actually penetrate skin (figure 6). 95% of the UV radiation reaching the Earth surface correspond to UVA and the remaining 5% to UVB [START_REF] Sage | Distribution and repair of photolesions in DNA: genetic consequences and the role of sequence context[END_REF]. UVA longer wavelength penetrates deeper in skin reaching reticular dermis and is mostly implicated in photo aging due to its capacity to deteriorate dermal fibers. It can also promote oxidative DNA damage through the generation of oxidative base modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) [START_REF] Kozmin | UVA radiation is highly mutagenic in cells that are unable to repair 7,8-dihydro-8-oxoguanine in Saccharomyces cerevisiae[END_REF][START_REF] D'orazio | UV radiation and the skin[END_REF]) UVB radiations (UVBs) are able to penetrate epidermis and superficial dermis and has a strong carcinogenic potential through its direct mutagenic effect in DNA strand [START_REF] De Gruijl | Photocarcinogenesis: UVA vs UVB[END_REF][START_REF] D'orazio | UV radiation and the skin[END_REF]. UVBs mainly induce the formation of two specific DNA photoproducts -cyclobutane pyrimidine dimers (CPDs) and 6,4 pyrimidine-pyrimidone (6-4 PP) (figure 7). UVA are also capable of inducing CPDs but not 6-4 PPs, albeit following much higher doses of radiation [START_REF] Douki | Bipyrimidine photoproducts rather than oxidative lesions are the main type of DNA damage involved in the genotoxic effect of solar UVA radiation[END_REF]. [START_REF] Li | Similarities and differences between cyclobutane pyrimidine dimer photolyase and (6-4) photolyase as revealed by resonance Raman spectroscopy: Electron transfer from the FAD cofactor to ultraviolet-damaged DNA[END_REF] The cell machinery is able to either repair UV-induced DNA lesions or to trigger apoptosis if the amount of DNA damage is too high and considered as "saturating" to the repair mechanisms. However even with fully functional UV protecting mechanisms, some lesions could persist leading to mutations [START_REF] Valejo Coelho | The dark side of the light: mechanisms of photocarcinogenesis[END_REF]. The nucleotide excision repair (NER) is the mechanism responsible for the repair of UV-induced CPDs and 6-4PP and will be further discussed in the next chapter. Base excision repair (BER) is the mechanism responsible for the removal of the damaged bases like 8-oxoG.

3.3.a. Pollution

Atmospheric aerosols containing particles, and biological and gaseous contaminants are considered as the main risk factor towards multiple diseases, especially respiratory and cardiovascular related-diseases. Evidences show that some of the contaminants of these aerosol particles also have a negative impact on skin and are associated with the development of skin cancer [START_REF] Kim | Air pollution and skin diseases: Adverse effects of airborne particulate matter on various skin diseases[END_REF]. Examples are the black carbon, cigarette smoke and polycyclic aromatic hydrocarbons (PHA). Occupational exposure to black carbon increases the incidence of melanoma ,and cigarette smoke increases incidence and exacerbation of NMSC through the formation of reactive oxygen species (ROS) that ultimately lead to oxidative DNA damage [START_REF] Puntoni | Occupational exposure to carbon black and risk of cancer[END_REF][START_REF] Coppe | A role for fibroblasts in mediating the effects of tobacco-induced epithelial cell growth and invasion[END_REF]). After bio activation, PHA turn into carcinogenic metabolites and are therefore considered as general carcinogens [START_REF] Siddens | Polycyclic aromatic hydrocarbons as skin carcinogens: comparison of benzo[a]pyrene, dibenzo[def,p]chrysene and three environmental mixtures in the FVB/N mouse[END_REF].

3.3.b. Phototype

Fitzpatrick first proposed a phototype classification (FPC) in 1975 based on the response of different skin colors to the UV radiation, in terms of tanning and tendency to burn. After a modification of the initial Fiztpatrick classification to include people with darker skin colors, individuals falls in six (I to VI) different categories spreading from those who burn easily and never tan and those who never burn and tan profusely [START_REF] Fitzpatrick | Soleil et Peau[END_REF][START_REF] Fitzpatrick | The validity and practicality of sun-reactive skin types I through VI[END_REF].

Melanins are considered as skin photoprotectors due to their respective capacity to absorb UV radiations. Epidemiological studies show a correlation between the FPC and the development of skin cancer. High FPCs, (i.e. darker skin phototypes IV -VI) are less prone to develop malignancies while fairly pigmented (i.e. skin phototypes I-III) are more susceptible to skin cancers). Today, FPC values are used by researchers and clinicians as prognostic criteria to assess the risk of developing skin cancer [START_REF] Sitek | Skin color parameters and Fitzpatrick phototypes in estimating the risk of skin cancer: A casecontrol study in the Polish population[END_REF][START_REF] Fajuyigbe | Melanin distribution in human epidermis affords localized protection against DNA photodamage and concurs with skin cancer incidence difference in extreme phototypes[END_REF][START_REF] Holm-Schou | Skin cancer phototype: A new classification directly related to skin cancer and based on responses from 2869 individuals[END_REF].

3.3.c. Aging

Most cancers have high incidence and are more prevalent upon aging. About 95% of the NMSC cases between 2014 and 2018 were reported in people of at least 50 years (International Agency for Research on Cancer 2019).

The accumulation of DNA damage during life is the "primary suspect" in the correlation between skin cancer and aging. Both cancer and aging are processes that share a genomic instability and it can lead to different outcomes from an uncontrolled cellular growth to senescence or apoptosis [START_REF] Moskalev | The role of DNA damage and repair in aging through the prism of Koch-like criteria[END_REF][START_REF] Aunan | The Biology of Aging and Cancer: A Brief Overview of Shared and Divergent Molecular Hallmarks[END_REF].

Importantly, senescence process plays a paradoxical role in cancer progression acting both as a protection mechanism against uncontrolled cellular proliferation and a promoter of epithelial growth and tumorigenesis. This notion will be further discussed in chapter III [START_REF] Krtolica | Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging[END_REF][START_REF] Coppe | The senescence-associated secretory phenotype: the dark side of tumor suppression[END_REF][START_REF] Ghosh | The Senescence-Associated Secretory Phenotype: Critical Effector in Skin Cancer and Aging[END_REF].

3.3.d. Immunosupression

The immune system has an important role in controlling cancer progression. Over the years, thousands of studies described the dramatically higher rates of NMSC incidence in organ transplant patients during immunosuppressive periods [START_REF] Nair | Long-term immunosuppression and malignancy in thoracic transplantation: Where is the balance?[END_REF][START_REF] Manyam | Inferior outcomes in immunosuppressed patients with high-risk cutaneous squamous cell carcinoma of the head and neck treated with surgery and radiation therapy[END_REF][START_REF] Iannacone | Prevalence of Skin Cancer and Related Skin Tumors in High-Risk Kidney and Liver Transplant Recipients in Queensland, Australia[END_REF][START_REF] Pinho | Non-melanoma skin cancer in Portuguese kidney transplant recipients -incidence and risk factors[END_REF].

One of the first drug used in post-transplant regimens was the anti-metabolite azathioprine (AZ). It impairs DNA synthesis and was used to lower immune response in post-transplant patients to avoid rejection. However, AZ treatment in monotherapy correlates with occurrence of cancers in post-transplanted patients [START_REF] Scharf | Carcinoma of the bladder with azathioprine therapy[END_REF][START_REF] Van Scoik | The pharmacology and metabolism of the thiopurine drugs 6-mercaptopurine and azathioprine[END_REF]. With the discovery of calcineurin as a key signaling enzyme in T-cell activation, new drugs attempting to inhibit this pathway were created. The first one was cyclosporine that, like AZ, is related with higher rates of skin cancer among transplant patients [START_REF] Dantal | Effect of long-term immunosuppression in kidney-graft recipients on cancer incidence: randomised comparison of two cyclosporin regimens[END_REF]. In 2000, the introduction of mTOR (mammalian target of rapamycin) sirolimus inhibitor started a new era in post-transplant therapies. TOR inhibitors prevent cytokine receptors from activating the cell cycle and the use of TOR inhibitors to maintain immunosuppression are associated with a lower risk of post-transplant NMSC comparing to the "first era" treatments [START_REF] Eng | Activity of rapamycin (AY-22,989) against transplanted tumors[END_REF][START_REF] Bernat-Garcia | The role of new immunosuppressive drugs in nonmelanoma skin cancer in renal transplant recipients[END_REF][START_REF] Iannacone | Prevalence of Skin Cancer and Related Skin Tumors in High-Risk Kidney and Liver Transplant Recipients in Queensland, Australia[END_REF][START_REF] Pinho | Non-melanoma skin cancer in Portuguese kidney transplant recipients -incidence and risk factors[END_REF][START_REF] Okut | Nonmelanoma Skin Cancers After Kidney Transplant: Our 15 Years of Experience With Mammalian Target of Rapamycin Inhibitors[END_REF].

The role of the immune system in cancer progression is an important part of this manuscript and will be further discussed in chapter III. [START_REF] Fine | Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification[END_REF][START_REF] Mellerio | Management of cutaneous squamous cell carcinoma in patients with epidermolysis bullosa: best clinical practice guidelines[END_REF].

Familial melanoma (FM) is the main cause of melanoma diagnosis in the youth. More than 50% of autosomal dominant mutations transmitted to the offspring are on genes involved in tumor suppression and telomere maintenance. Germline mutations in cyclin-dependent kinase inhibitor 2A (CDKN2A) were the first to be associated with familial melanoma susceptibility and account for 20% of the cases (Kamb, Shattuck-Eidens et al. 1994, Goldstein and[START_REF] Goldstein | Genetic epidemiology of cutaneous melanoma: a global perspective[END_REF].

Individuals with familial history of melanoma should be followed by a dermatologist at least once a year to a skin exam and informed of the advantages of the self-evaluation [START_REF] Aoude | Nonsense mutations in the shelterin complex genes ACD and TERF2IP in familial melanoma[END_REF][START_REF] Soura | Hereditary melanoma: Update on syndromes and management: Genetics of familial atypical multiple mole melanoma syndrome[END_REF].

Xeroderma pigmentosum (XP) is a rare autosomal genetic disease caused by mutations on genes involved in the repair of DNA lesions after a UV stress. The accumulation of unrepaired lesions in the DNA strand lead to a propensity to develop cutaneous cancers at an early age.

They develop 10,000 more BCC and SCC and 2000 more melanomas than the general population [START_REF] Bradford | Cancer and neurologic degeneration in xeroderma pigmentosum: long term followup characterises the role of DNA repair[END_REF]. In my thesis work, I used XP as a model to study development and progression of cutaneous cancers. The next chapter will be dedicated to this disease.

Treatment of skin cancers

Surgical excision is the standard treatment for both BCC, SCC and melanoma. Small and superficial BCC can still be treated with liquid nitrogen or abrasive therapies. Topical treatments with imiquimod, an immunomodulatory agent that induces the release of proinflammatory cytokines, or photodynamic therapies are also a possibility. For the rare cases of inoperable BCC, mostly due to its location, the first choice is radiation therapy. Some Hedgehog pathway inhibitors are in clinical testing and seemed to be a good option especially with advanced or metastatic BCCs [START_REF] Berking | Basal cell carcinoma-treatments for the commonest skin cancer[END_REF].

For SCC, besides the inoperable ones that are treated mostly with radiotherapy, the surgical excision is always the first choice as it allows to definitely confirm the type and extension of tumor invasion and clear the margins. In patients that cannot undergo surgery, radiation therapy is, like with BCC the first choice. In presence of late stages of SCC presenting aggressive and metastatic features, chemotherapy with combinations of the neoplastic alkylating agent cisplatin and the neoplastic anti-metabolite 5-fluoracil could also be considered [START_REF] Stratigos | Diagnosis and treatment of invasive squamous cell carcinoma of the skin: European consensus-based interdisciplinary guideline[END_REF][START_REF] Strom | Management of BCC and SCC of the Head and Neck[END_REF].

In melanoma, as for the NMSC, radiotherapy is only an option when surgery is not possible.

When metastases are detected or a high metastatic risk is associated, adjuvant therapies like interferon-α, immunotherapy (further discussed in chapter III), target therapy or chemotherapy (with the neoplastic alkylating agent dacarbazine) can be proposed [START_REF] Garbe | Diagnosis and treatment of melanoma. European consensus-based interdisciplinary guideline -Update 2016[END_REF].

Chapter II: Xeroderma pigmentosum

"The prognosis, [of xeroderma pigmentosum] according to our experience is absolutely

unfavorable" -Kaposi 1874 [START_REF] Hebra | On diseases of the skin including exanthemata[END_REF] Xeroderma pigmentosum (XP) is a rare autosomal recessive genetic disease characterized by the patient's higher susceptibility to develop skin cancers in sun exposed areas. Hebra and Kaposi were the first to describe XP in 1874 pointing out "a remarkable abnormality of pigmentation" and a "parchment-like dryness, thinness, and wrinkling of the epidermis" [START_REF] Hebra | On diseases of the skin including exanthemata[END_REF]. A century later, James Cleaver classified XP as a genetic disease reporting defects in the DNA repair process in XP patients' cells after UV radiation [START_REF] Cleaver | Defective repair replication of DNA in xeroderma pigmentosum[END_REF]). Not long after, De Weerd-Kastelein proved that there is a genetic heterogeneity in XP disease and finally seven groups of genetic complementation were identified (XP-A to XP-G) plus one variant (XP-V) [START_REF] Cleaver | Xeroderma pigmentosum: variants with normal DNA repair and normal sensitivity to ultraviolet light[END_REF][START_REF] Weerd-Kastelein | Genetic heterogeneity of xeroderma pigmentosum demonstrated by somatic cell hybridization[END_REF][START_REF] Keijzer | A seventh complementation group in excision-deficient xeroderma pigmentosum[END_REF]). The clinical and genetic features of XP patients provide a model to study both DNA repair and carcinogenic mechanisms [START_REF] Kraemer | Forty years of research on xeroderma pigmentosum at the US National Institutes of Health[END_REF].

Epidemiology and clinical traits

Estimated incidences of XP vary according to geographical location. The most recent epidemiological study in five Western Europe countries estimates the incidence in autochthonous population in 1 per million births but when considering immigrant population this ratio doubles to 2,3 per million births [START_REF] Kleijer | Incidence of DNA repair deficiency disorders in western Europe: Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy[END_REF]. XP is more prevalent in regions with an history of consanguinity. The prevalence of XP disease is estimated in 1 in 80,000 in Morocco and 1 in 10,000 in Tunisia. In both countries, as in all the Mediterranean region, defects in XPC gene are responsible for more than 80% of cases [START_REF] Zghal | Xeroderma pigmentosum. Cutaneous, ocular, and neurologic abnormalities in 49 Tunisian cases][END_REF][START_REF] Soufir | A prevalent mutation with founder effect in xeroderma pigmentosum group C from north Africa[END_REF][START_REF] Doubaj | Carrier frequency of the recurrent mutation c.1643_1644delTG in the XPC gene and birth prevalence of the xeroderma pigmentosum in Morocco[END_REF]. In Japan, where XP prevalence is about 1 in 20,000 the main affected gene is XPA [START_REF] Hirai | Heterozygous individuals bearing a founder mutation in the XPA DNA repair gene comprise nearly 1% of the Japanese population[END_REF].

XP patients present a sensitivity to sunlight and freckling and pigmentation spots in sun exposed areas as soon the age of two years old. Skin is frequently dry, scaling and atrophic and presents early signs of photoaging. Skin pigmentation is heterogeneous intermixing between hypopigmented and hyperpigmented areas (Kraemer, Lee et al. 1987[START_REF] Lehmann | Xeroderma pigmentosum[END_REF]. Ocular tissue exposed to UV radiation is frequently affected in these patients. Conjunctivitis, corneal neovascularization and eye dryness are the most observed abnormalities (Kraemer, Lee et al. 1987[START_REF] Brooks | Ocular Manifestations of Xeroderma Pigmentosum: Long-Term Follow-up Highlights the Role of DNA Repair in Protection from Sun Damage[END_REF]. About one fourth of XP patients also present neurological symptoms including a progressive mental deterioration and hearing loss, deterioration of speech function, ataxia, peripheral neuropathy, abnormal gait, and psychomotor alterations affecting walking [START_REF] Kraemer | Xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome: A complex genotypephenotype relationship[END_REF][START_REF] Bradford | Cancer and neurologic degeneration in xeroderma pigmentosum: long term followup characterises the role of DNA repair[END_REF].

The most common cause of death in XP patients is skin cancer. They have a 10,000 fold risk of developing NMSC and a 2000 fold risk of developing melanoma under the age of 20 compared to the general population [START_REF] Bradford | Cancer and neurologic degeneration in xeroderma pigmentosum: long term followup characterises the role of DNA repair[END_REF]. The most comprehensive XP case-study until now dates from 1987 and puts the median age of the first skin cancer at 8 years old, 50 years earlier than the general population (figure 8) (Kraemer, Lee et al. 1987). (Kraemer, Lee et al. 1987) A more recent follow-up of 106 XP patients paid special attention to data related to the median age of the first NMSC and the median age of the first melanoma in XP patients compared to the general population. In the general population, the average age of the first melanoma is 67 years old and the one of the first NMSC is 55 years old. In XP patients, the median age of the first diagnosis of melanoma is 22 years old whereas the one of the first NMSC stays under 10 years old. These data show that not only XP patients develop skin cancers at early age compared to the general population, but also that there is a reversal regarding the type of the first cancer diagnosed. In general population melanomas tend to arise first and the opposite occurs in XP patients with a "preference" for the development of NMSC. This reversal suggests that there are different mechanisms of carcinogenesis between the two types of cancer [START_REF] Bradford | Cancer and neurologic degeneration in xeroderma pigmentosum: long term followup characterises the role of DNA repair[END_REF].

In a study from 1984, a 10-to 20-fold increase in the frequency of internal malignancies in XP patients was also reported [START_REF] Kraemer | DNA repair protects against cutaneous and internal neoplasia: evidence from xeroderma pigmentosum[END_REF]. Immune abnormalities were also identified in XP patients. Several studies from the late 80s / early 90s report that NK from XP patients present a defect in their lytic activity suggesting an implication of the NK cell dysfunction in the cancer proneness of XP (Norris, Limb et al. 1988, Norris, Limb et al. 1990, Mariani, Facchini et al. 1992). Furthermore, activation with poly inosinic cytidylic acid, showed that compared with peripheral blood lymphocytes from the general population (PBL), XP PBL produced reduced levels of interferon gamma and alpha (IFN-γ, IFN-α) that are both potent inductors of NK cell activation, [START_REF] Gaspari | Impaired interferon production and natural killer cell activation in patients with the skin cancer-prone disorder, xeroderma pigmentosum[END_REF]. XP patients also present reduced levels of CD3 + CD4 + circulating lymphocytes [START_REF] Wysenbeek | Immunologic alterations in xeroderma pigmentosum patients[END_REF], Mariani, Facchini et al. 1992).

Etiology

The "classic" form of XP disease is due to constitutive defects in the mechanism involved in the repair of DNA lesions after UV exposure. James Cleaver was the first researcher identifying the link between XP phenotype and a failure in the DNA repair after UV exposure [START_REF] Cleaver | Defective repair replication of DNA in xeroderma pigmentosum[END_REF]). Using a technique that was later named "unscheduled DNA synthesis" (UDS), he

showed that the majority of XP cells were deficient in UDS after UV irradiation. The principle of UDS is to incorporate labeled nucleotides into the newly DNA patches that are synthetized upon DNA repair. Historically, radionucleotides such as 3 H-thymidine or 3 H-bromodeoxyuridine were used; quantitation of the incorporation was made by autoradiography, or liquid scintillation counting [START_REF] Lehmann | A rapid procedure for measurement of DNA repair in human fibroblasts and for complementation analysis of xeroderma pigmentosum cells[END_REF]. Today, it is possible to measure UDS with a non-radioactive label such as 5-Ethynyl-2´-deoxyuridine (EdU) and it remains a robust criterion to confirm XP diagnosis (see point 3 for more details) [START_REF] Limsirichaikul | A rapid nonradioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU)[END_REF].

XP disease can result from mutation in any one of eight genes (XPA to XPG plus XPV i.e. DNA polymerase eta). XPA to XPG are encoding proteins whose expression is altered in the DNA repair mechanism called nucleotide excision repair (NER). Pol eta encodes for a DNA polymerase from the eta DNA polymerases family, and essentially manages the translesion DNA synthesis.

Nucleotide Excision Repair (NER)

In 1964, Rasmussen and Painter published in Nature the first evidence of a DNA repair mechanism after UV damage in mammalian cells [START_REF] Rasmussen | Evidence for Repair of Ultra-Violet Damaged Deoxyribonucleic Acid in Cultured Mammalian Cells[END_REF]. Three decades later, the major steps of the NER mechanism were reported through in vitro approaches of DNA repair complex reconstruction [START_REF] Aboussekhra | Mammalian DNA nucleotide excision repair reconstituted with purified protein components[END_REF][START_REF] Mu | Reconstitution of human DNA repair excision nuclease in a highly defined system[END_REF].

There are two converging pathways of the NER distinguished in the initial steps of the recognition of the DNA lesion: Global Genome Repair (GGR) and Transcription couplednucleotide excision repair (TC-NER), also called sometimes "preferential repair" (figure 9). (Stary and Sarasin 2002) Figure 9. Nucleotide Excision Repair mechanism.

The GGR pathway is activated when the lesion stands in a non-transcribed region of the genome while TC-NER is involved in repair lesions in DNA regions that are being transcribed into RNA (including non-transcribed DNA strand). During GGR, DNA distortion led by the lesion is first recognized by a XPC-HR23B-centrine2 protein complex helped by a DDB2/XPE-DDB1complex when the degree of helix distortion is lower (i.e. in presence of CPD). TC-NER is initiated when RNA polymerase II is thought to stall when encountering a lesion in the transcribed strand. However, the number of blocking DNA transcription following a minimal erythema dose (MED) equivalent (about 2 CPD /1kb) irradiation is far insufficient to explain the global block of transcription, including in active genes with no detectable DNA lesion [START_REF] Perdiz | Distribution and repair of bipyrimidine photoproducts in solar UV-irradiated mammalian cells. Possible role of Dewar photoproducts in solar mutagenesis[END_REF]. Signaling pathways connecting DNA distortion and/or cellular components to command transcriptional block deserve further investigations. The classical interpretation is based on the fact that the CSA and CSB proteins are recruited to the lesion site due to stalling of RNA polymerase II. After the recognition of the lesion through both GGR and TC-NER sensors, the multiprotein transcription factor TFIIH is recruited to the recognition complex and the NER pathway shared by GGR and TC-NER is turned on [START_REF] Spivak | Nucleotide excision repair in humans[END_REF]. TFIIH is a complex composed of ten subunits -XPB, XPD, p62, p52, p44, p34, p8/TTD-A, CDK7, cyclin H, and MAT1. The ATP-dependent helicases XPB and XPD are responsible for the unwinding of the DNA double strand around the lesion. To this end, the P44 protein of TFIIH phosphorylates the carboxyterminal end of RNA pol II [START_REF] Coin | Phosphorylation of XPB helicase regulates TFIIH nucleotide excision repair activity[END_REF]). The repair complex is then stabilized by the XPA protein allowing subsequent 3' a 5' incisions [START_REF] Riedl | The comings and goings of nucleotide excision repair factors on damaged DNA[END_REF][START_REF] Camenisch | Recognition of helical kinks by xeroderma pigmentosum group A protein triggers DNA excision repair[END_REF]). The DNA damaged strand is incised and excised thanks to the intervention of the endonucleases XPF/ERCC1 5' and XPG 3'. After the dual incisions, DNA polymerases ε/δ/κ synthetize a new strand using as matrix the remaining single strand DNA protected by SSB proteins; the newly synthetized strand is covalently linked by DNA ligase I, IIIα and XRCC1 [START_REF] Moser | Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner[END_REF][START_REF] Marteijn | Understanding nucleotide excision repair and its roles in cancer and ageing[END_REF].

NER is only one of the different pathways that can be triggered after induction of DNA damage.

The other main DNA repair pathways: BER (Base Excision Repair), HR (Homologous Recombination), MMR (Mismatch Repair) and NHEJ (Non-Homologous End-Joining) are summarized in figure 10. 3). For instance, XP-C and XP-E are less prone to develop acute sunburn and XP-A (De Sanctis-Cacchione syndrome) is related to severe neurological symptoms [START_REF] Moriwaki | Xeroderma pigmentosum clinical practice guidelines[END_REF].

Figure 10. DNA repair pathways. [START_REF] Lehmann | Xeroderma Pigmentosum -Facts and Perspectives[END_REF] The variant (XP-V) syndrome is not related to mutations in the NER pathway. NER defect can thus not be considered per se as a criteria of diagnostic. Instead XP-V cells present problems during the DNA replication of UV-damaged strands, a process called translesion synthesis (TS). In absence of DNA damage, DNA polymerases (I) is responsible for DNA replication but when in presence of a damage in the DNA template, there is a switch taking place between DNA pol I and other specialized DNA polymerases that are able to process throughout the DNA damage. DNA polymerase eta (pol eta) participates in the replication of DNA strands that present UV related lesions. The gene that encodes pol eta, POLH is mutated in XP-V patients.

XP-V patients present a typical XP phenotype but they generally display milder symptoms and some delay in the onset age of skin cancer development [START_REF] Cordonnier | Impaired translesion synthesis in xeroderma pigmentosum variant extracts[END_REF][START_REF] Gratchev | Molecular genetics of Xeroderma pigmentosum variant[END_REF].

XP is not the only genetic disease associated with defects in NER pathway. There are two other identified genodermatoses -trichothiodystrophy (TTD), and Cockayne' syndrome (CS)

and several related or overlapping disorders that derive from mutations in NER pathway associated genes (figure 11). One disease may be the product of mutations in different genes, like what happens with XP, but the inverse can also occur, meaning that different mutations in the same gene may lead to different diseases (DiGiovanna and Kraemer 2012).

Table 3. Xeroderma pigmentosum complementation groups: insight in frequency, proneness to skin cancer development, neurological involvement and cellular repair capabilities.

Adapted from (DiGiovanna and Kraemer 2012)

Trichothiodystrophy

Trichothiodystrophy (TTD) was first identified by Pollitt et al in 1968 [START_REF] Pollitt | Sibs with mental and physical retardation and trichorrhexis nodosa with abnormal amino acid composition of the hair[END_REF]).

Clinically, TTD patients present with brittle hair and normally have an outgoing personality.

TTD features range from only hair involvement to massive developmental and neurological abnormalities. Some forms of TTD are related to photosensitivity but patients neither develop skin cancers nor present pigmentary abnormalities like XP patients [START_REF] Brauns | Photosensitive form of trichothiodystrophy associated with a novel mutation in the XPD gene[END_REF]). The disease is related to mutations in XPD, XPB or TTDA genes, that all belong or, contribute, to the multifunctional protein complex TFIIH. TFIIH has roles in both DNA repair and basal transcription [START_REF] Hoeijmakers | TFIIH: a key component in multiple DNA transactions[END_REF][START_REF] Yew | Understanding photodermatoses associated with defective DNA repair: Photosensitive syndromes without associated cancer predisposition[END_REF]. Mutations affecting only the repair function of the complex lead to the XP disease whereas mutations affecting the transcription function lead to TTD [START_REF] Taylor | Xeroderma pigmentosum and trichothiodystrophy are associated with different mutations in the XPD (ERCC2) repair/transcription gene[END_REF]

Cockayne syndrome

Cockayne syndrome (CS) was first described by [START_REF] Cockayne | Dwarfism with retinal atrophy and deafness[END_REF][START_REF] Cockayne | Dwarfism with retinal atrophy and deafness[END_REF]. CS patients present microcephaly, stunted growth, progressive neurologic dysfunction and generally premature aging. Alike TTD patients, they also can present with photosensitivity but neither pigmentation abnormalities nor higher incidence of skin cancer as compared to the general population [START_REF] Nance | Cockayne syndrome: review of 140 cases[END_REF]Berry 1992, Yew, Giordano et al. 2016). The disease results typically from mutations in ERCC8 and ERCC6 genes, encoding for CSA and CSB proteins, respectively. These proteins are part of the lesion recognition complex formed during the TC-NER branch of the NER [START_REF] Stefanini | Genetic analysis of twenty-two patients with Cockayne syndrome[END_REF]. Less frequently, CS can result from mutation in XPF gene [START_REF] Kashiyama | Malfunction of nuclease ERCC1-XPF results in diverse clinical manifestations and causes Cockayne syndrome, xeroderma pigmentosum, and Fanconi anemia[END_REF]. Overlap syndromes of XP/CS and TTD/CS have been described. XP/CS patients show with the classic symptoms of CS plus a severe photosensitivity and an increased risk of skin cancer development due to additional mutations on XPB, XPD, XPG or XPF genes [START_REF] Natale | Xeroderma pigmentosum-Cockayne syndrome complex[END_REF]. Ultraviolet sensitive syndrome (UVSS) and Cerebro-oculo-facio-skeletal (COFS) syndrome are CS-associated disorders [START_REF] Karikkineth | Cockayne syndrome: Clinical features, model systems and pathways[END_REF]. There is a debate whether they can be considered as variant forms of CS presenting different severities or as different diseases.

UVSS patients possess mutations in CSA, CSB, or UVSSA genes. Clinical features are mild comparing to classical CS. UVSS patients present acute sunburn, dryness, freckling, and pigmentation anomalies without neurological abnormalities [START_REF] Itoh | UVs syndrome, a new general category of photosensitive disorder with defective DNA repair, is distinct from xeroderma pigmentosum variant and rodent complementation group I[END_REF][START_REF] Karikkineth | Cockayne syndrome: Clinical features, model systems and pathways[END_REF]. COFS can be caused by mutations in CSB, ERCC1, XPD, or XPG genes. Clinical features of COFS often overlap with severe forms of CS -microcephaly with cerebral atrophy as well as severe mental and growth retardation.

However, microphthalmia and arthrogryposis that are commonly found in COFS are rare in CS patients [START_REF] Laugel | Cerebro-oculo-facio-skeletal syndrome: three additional cases with CSB mutations, new diagnostic criteria and an approach to investigation[END_REF][START_REF] Yew | Understanding photodermatoses associated with defective DNA repair: Photosensitive syndromes without associated cancer predisposition[END_REF].

Diagnosis and treatment

The diagnosis of XP is first based on both clinical symptoms and DNA repair tests. The most frequent DNA repair test is the measurement of the unscheduled DNA synthesis (UDS) after UV irradiation of fibroblasts, already described in point 1. The impairment of UDS synthesis can confirm the XP diagnosis except in the presence of XP-V variant cell. In the latter case, cells should be cultured in presence of caffeine when exposed to UV radiation, and cell viability is checked 2 to 4 days after irradiation. The sensitivity (through survival measurement) of cells to UV in presence of caffeine is a particularity of XP-V cells and together with the normal UDS can confirm the XP-V diagnosis [START_REF] Broughton | Molecular analysis of mutations in DNA polymerase eta in xeroderma pigmentosum-variant patients[END_REF]. In fact, caffeine, through the inhibition of checkpoint kinases ATM and ATR, is able to enhance the inhibition of cell proliferation induced by UV. However this effect is reversed by expression of DNA polymerase η and this is why this is a specific test to confirm an XP-V diagnosis [START_REF] Kauffman | Maintenance immunosuppression with target-of-rapamycin inhibitors is associated with a reduced incidence of de novo malignancies[END_REF].

After the diagnosis, complementary experiments can determine which gene exactly is altered, by either genetic tests (complementation analysis, DNA sequencing) or, when applicable, by quantification of the suspected defected gene product either by reverse transcriptase-PCR or protein quantification.

Complementation analysis by cell fusion from independent patients were one of the earliest methods to pose the diagnosis of XP. Such approach revealed the existence of the different XP complementation groups. Briefly, cells from different XP patients were fused to form heterokaryons and the levels of UDS were measured after UV irradiation (notably UVC).

Recovery of normal UDS levels implied that the two cells belong to two different complementation groups; conversely, absence of UDS improvement meant that cells from the two patients belonged to the same complementation group [START_REF] Kraemer | Genetic heterogeneity in xeroderma pigmentosum: complementation groups and their relationship to DNA repair rates[END_REF][START_REF] Kraemer | Five complementation groups in xeroderma pigmentosum[END_REF][START_REF] Arase | A sixth complementation group in xeroderma pigmentosum[END_REF][START_REF] Keijzer | A seventh complementation group in excision-deficient xeroderma pigmentosum[END_REF]). After the identification of the different groups, and for diagnosis purposes, the cells from the suspected XP patient were fused with cells for which the complementation groups were already known. Because complementation analysis by cell fusion was time-consuming and difficult to perform some alternatives methods were set up. One of the first alternatives to the cell fusion procedure relied in the transfer of vectors (expression plasmid or replication deficient retroviruses expressing the various genes (cDNAs) known to be involved in NER-deficient cells, such as XPD or XPC [START_REF] Carreau | Development of a new easy complementation assay for DNA repair deficient human syndromes using cloned repair genes[END_REF].

Although it is not currently used as a diagnostic method, the measurement of UV suspicious cells survival using clonal analysis remains a good practice to determine if we are in presence of the XP-C complementation group. Keratinocytes from XP-C patients shows changes in the representation of the different colony types compared to WT and XP cells from other complementation groups with a preference for abortive ones (Otto, Riou et al. 1999).

One hundred and forty five years have passed since Kaposi first described the prognosis of the XP syndrome as "absolutely unfavorable" [START_REF] Hebra | On diseases of the skin including exanthemata[END_REF]. Even if many palliative therapeutics have emerged since then, no curative medicine can yet be proposed to these XP patients. Today, treatments are mostly based on cancer prevention or progression. Among those, strict sun avoidance is a crucial preventive measure so long as diagnostic is early enough with age i.e. before 2. Under these circumstances prevention of sun exposure has proven essential to limit progressive alterations of the quality of life of XP patients, at least at the cutaneous level. Regarding cancer progression, the most frequent treatment to avoid dissemination is the surgical resection of skin lesions.

The potential of gene therapy brings new hope to the possibility of a curative treatment but despite the efforts that have been made, as skin is the largest organ of the human body, its full transplantation following genetic correction remains a challenge. De Luca's team was until now the only one, to successfully replace almost the entire epidermis -of a patient suffering from junctional epidermolysis bullosa disease -with autologous transgenic epidermal cultures [START_REF] Hirsch | Regeneration of the entire human epidermis using transgenic stem cells[END_REF]. However, taking into account the complexity of skin as a multiple embryologic origin, it can be suspected, including in the studies of the laboratory, that full recovery of NER after gene transfer cannot alone normalize all the "side" impacts of XP gene mutations [START_REF] Arnaudeau-Begard | Genetic correction of DNA repair-deficient/cancer-prone xeroderma pigmentosum group C keratinocytes[END_REF], Frechet, Warrick et al. 2008[START_REF] Warrick | Preclinical corrective gene transfer in xeroderma pigmentosum human skin stem cells[END_REF].

For further details on XP diagnosis and treatment, I invite you to read our review "Genetic therapy of Xeroderma Pigmentosum: analysis of strategies and translation" enclosed in page 192 of this manuscript (Goncalves-Maia and Magnaldo 2016).

The XP-C genetic group of complementation

The XP-C genetic group of complementation is the most frequent in USA, Europe and North Africa. XP-C patients present severe skin abnormalities and are particularly hypersensitive to ocular damage [START_REF] Fassihi | Deep phenotyping of 89 xeroderma pigmentosum patients reveals unexpected heterogeneity dependent on the precise molecular defect[END_REF]). Together with group XP-E and XP-V groups, the majority of XP-C patients don't have abnormal sunburn reactions on minimal sun exposure probably due to the fact that they retain fully efficient TC-NER mechanism (van Steeg and [START_REF] Van Steeg | Xeroderma pigmentosum and the role of UV-induced DNA damage in skin cancer[END_REF][START_REF] Kleijer | Incidence of DNA repair deficiency disorders in western Europe: Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy[END_REF][START_REF] Sethi | Patients with xeroderma pigmentosum complementation groups C, E and V do not have abnormal sunburn reactions[END_REF][START_REF] Jerbi | Clinical, genealogical and molecular investigation of the xeroderma pigmentosum type C complementation group in Tunisia[END_REF]). In the rare cases where XP-C patients develop neurological symptoms, the cause seems to be unrelated with the XPC gene defect but perhaps on other yet undisclosed impacts of ascending consanguinity [START_REF] Khan | XPC initiation codon mutation in xeroderma pigmentosum patients with and without neurological symptoms[END_REF]). There is one report of XP-C patients showing an impairment of basal cutaneous cell-mediated immunity response after a sensitization test using the contact allergen diphenylcyclopropenone (DPCP) [START_REF] Anttinen | Neurological symptoms and natural course of xeroderma pigmentosum[END_REF]. Furthermore, in early 90-th, some XP-C patients showed defects in NK cell lytic activity in blood samples (Norris, Limb et al. 1990).

The XP-C group is highly predisposed to develop aggressive skin cancers. XP-C patients develop at least 5 times more SCC than BCC whereas the incidence ratio SCC:BCC in the general population under 45 years old is rather 1:4 (Giglia, Dumaz et al. 1998[START_REF] Chahal | Incidence ratio of basal cell carcinoma to squamous cell carcinoma equalizes with age[END_REF]. Internal afflictions such as pyramidal syndrome, thyroid nodules, as well as internal cancers (leukemia, non HPV-cervix cancer, kidney cancers and thyroid adenocarcinoma) were also reported in XP-C patients but the physiopathological basis of these "associated diseases" remains poorly understood [START_REF] Khatri | Xeroderma pigmentosum in Libya[END_REF][START_REF] Hadj-Rabia | Unexpected extradermatological findings in 31 patients with xeroderma pigmentosum type C[END_REF]).

The human XPC gene was (partly) identified in 1992 by [START_REF] Legerski | Expression cloning of a human DNA repair gene involved in xeroderma pigmentosum group C[END_REF]. It is located on chromosome 3p25.1 and has a length of 33 kb with 16 exons and 15 introns (figure 12) [START_REF] Khan | The human XPC DNA repair gene: arrangement, splice site information content and influence of a single nucleotide polymorphism in a splice acceptor site on alternative splicing and function[END_REF]. Of note, Legerski and Peterson published the cDNA sequence, truncated from a part of its NH2-terminal sequence. Thus, the initial XPC protein was supposed to comprise about 100 kDa, instead of the full length 125 kDa actual sequence. The results from Legerski and Peterson were contradicted by [START_REF] Masutani | Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23[END_REF][START_REF] Masutani | Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23[END_REF]. Actually, the misinterpretation of Legerski and

Peterson were due to (3) internal codons of initiation of translation contained in the 5' region of the XPC cDNA [START_REF] Masutani | Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23[END_REF][START_REF] Khan | XPC initiation codon mutation in xeroderma pigmentosum patients with and without neurological symptoms[END_REF].

Adapted from [START_REF] Khan | The human XPC DNA repair gene: arrangement, splice site information content and influence of a single nucleotide polymorphism in a splice acceptor site on alternative splicing and function[END_REF] Structure of human XPC gene. The 16 exons are numbered and indicated by blue bands.

The XPC gene encodes a 940-aa DNA-binding protein (figure 13) known for its role in the "lesion" recognition step of the GGR mechanism [START_REF] Masutani | Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23[END_REF]). The Cterminal region of XPC (residues 492-940) contains the major protein-protein interaction sites -including binding regions to DDB2/XPE, RAD23B, centrin2, p62 and XPB -and the DNA binding domain ; the N-terminal region (residues 1-491) includes the XPA, p62 and 8-Oxoguanine glycosylase 1 (OGG1) binding regions [START_REF] Yokoi | The xeroderma pigmentosum group C protein complex XPC-HR23B plays an important role in the recruitment of transcription factor IIH to damaged DNA[END_REF][START_REF] Bunick | Biochemical and structural domain analysis of xeroderma pigmentosum complementation group C protein[END_REF][START_REF] Puumalainen | Xeroderma pigmentosum group C sensor: unprecedented recognition strategy and tight spatiotemporal regulation[END_REF].

XPC in GGR

XPC was first found to be complexed with the homologous of RAD23B (RAD23) in yeast in 1994 and a few years later, centrin 2 was identified as the third element of the heterotrimeric complex [START_REF] Masutani | Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23[END_REF][START_REF] Araki | Centrosome protein centrin 2/caltractin 1 is part of the xeroderma pigmentosum group C complex that initiates global genome nucleotide excision repair[END_REF][START_REF] Popescu | Xeroderma pigmentosum group C protein possesses a high affinity binding site to human centrin 2 and calmodulin[END_REF]. The XPC subunit of the complex is the one responsible for the DNA-binding and the recognition of the DNA distortion. Indeed, XPC recognizes bulky distortions in DNA, rather than the lesion itself. It shows higher affinity to 6-4PP probably because 6-4PP are associated with more important helix distortions compared to CPD [START_REF] Kim | Contrasting structural impacts induced by cis-syn cyclobutane dimer and (6-4) adduct in DNA duplex decamers: implication in mutagenesis and repair activity[END_REF][START_REF] Mcateer | Solution-state structure of a DNA dodecamer duplex containing a Cis-syn thymine cyclobutane dimer, the major UV photoproduct of DNA[END_REF][START_REF] Sugasawa | A multistep damage recognition mechanism for global genomic nucleotide excision repair[END_REF]. RAD23B is a 26S proteasome-interacting factor that protects XPC from proteasomal degradation [START_REF] Ng | A novel regulation mechanism of DNA repair by damage-induced and RAD23-dependent stabilization of xeroderma pigmentosum group C protein[END_REF]. Centrin 2 is a calcium-binding protein, initially found in centrosomes. It stabilizes the XPC-RAD23B complex and strengthen the DNA-binding activity of XPC [START_REF] Araki | Centrosome protein centrin 2/caltractin 1 is part of the xeroderma pigmentosum group C complex that initiates global genome nucleotide excision repair[END_REF], Nishi, Okuda et al. 2005). In the presence of CPD lesions, the XPC-RAD23B-centrin2 complex is assisted by DDB2/XPE DNA-binding protein, whose role is to facilitate the interaction of XPC with CPD sites [START_REF] Kulaksiz | Xeroderma pigmentosum complementation group E protein (XPE/DDB2): purification of various complexes of XPE and analyses of their damaged DNA binding and putative DNA repair properties[END_REF][START_REF] Fei | Regulation of nucleotide excision repair by UV-DDB: prioritization of damage recognition to internucleosomal DNA[END_REF]. DDB2/XPE together with DDB1 forms the heterodimeric UV-DDB (UV-DNA damage binding protein) complex. After the recognition of the distorted DNA, RAD23B rapidly dissociates from the complex. The remaining heterodimeric XPC-centrin2 complex mediates the recruitment of TFIIH via interaction with p62 and XPB [START_REF] Evans | Mechanism of open complex and dual incision formation by human nucleotide excision repair factors[END_REF][START_REF] Bernardes De | Dissection of the molecular defects caused by pathogenic mutations in the DNA repair factor XPC[END_REF][START_REF] Bergink | Recognition of DNA damage by XPC coincides with disruption of the XPC-RAD23 complex[END_REF]).

The UV-DDB complex is associated with Cul4A/B-RING ubiquitin ligases essential to the ubiquitination of both XPC and DDB2/XPE upon UV radiation [START_REF] Sugasawa | UV-induced ubiquitylation of XPC protein mediated by UV-DDB-ubiquitin ligase complex[END_REF]).

The p67 segregase, an ATP-driven molecular chaperone, then recognizes the ubiquitinated proteins and is responsible for its proteasomal degradation or recycling. While DDB2/XPE is rapidly degraded by proteosomal activity, the ubiquitination of XPC is reversible. Lubin et al.

proposed that the deubiquitinase OTUD4 was involved in the recycling of XPC after ubiquitination [START_REF] Lubin | A human XPC protein interactome--a resource[END_REF]. The removal of both XPC and DDB2/XPE from DNA upon lesion recognition is crucial since a retention of these proteins in the DNA lesion site impairs further steps of NER [START_REF] Sugasawa | UV-induced ubiquitylation of XPC protein mediated by UV-DDB-ubiquitin ligase complex[END_REF][START_REF] Puumalainen | Chromatin retention of DNA damage sensors DDB2 and XPC through loss of p97 segregase causes genotoxicity[END_REF].

4.2. Biological roles of XPC beyond NER

4.2.a. Repair pathways

The role of XPC in DNA repair is not restricted to NER. XPC is also recruited during the BER pathway (presented in figure 10). It has a DNA binding affinity to 8-oxoG and methylformamidopyrimidine lesions that are typical substrates of BER. This affinity is consistent with the publications describing that XPC recognizes helix destabilizations rather than specific lesions [START_REF] Sugasawa | A multistep damage recognition mechanism for global genomic nucleotide excision repair[END_REF][START_REF] Menoni | Nucleotide excision repair-initiating proteins bind to oxidative DNA lesions in vivo[END_REF][START_REF] Shell | Xeroderma pigmentosum complementation group C protein (XPC) serves as a general sensor of damaged DNA[END_REF]) [START_REF] Sugasawa | A multistep damage recognition mechanism for global genomic nucleotide excision repair[END_REF], D'Errico, Parlanti et al. 2006[START_REF] Menoni | Nucleotide excision repair-initiating proteins bind to oxidative DNA lesions in vivo[END_REF][START_REF] Shell | Xeroderma pigmentosum complementation group C protein (XPC) serves as a general sensor of damaged DNA[END_REF]. XPC is also implicated in the stimulation showing that p53 mutations found in XP-C primary internal cancers are compatible with oxidative damage mutagenesis (Giglia, Dumaz et al. 1998, D'Errico, Parlanti et al. 2006).

XPC has also been implicated in nonhomologous end joining (NHEJ) pathway (presented in chapter 1). XPC -/-HeLa cells present reduced NHEJ after sensitization with double single breaks (DSB) inducers. Furthermore the XP14BR fibroblast cell line, derived from a XP-C patient, show defects in the repair of DSB after ionizing radiation [START_REF] Arlett | Clinical and cellular ionizing radiation sensitivity in a patient with xeroderma pigmentosum[END_REF][START_REF] Despras | Longterm XPC silencing reduces DNA double-strand break repair[END_REF]). The evidences of the implication of XPC in other DNA repair mechanisms is compatible with the idea that XPC protein may act as a "sensor" of DNA damage across different DNA transaction pathways.

4.2.b. Transcription and chromatin remodeling

There is evidence of the implication of XPC in gene regulation independently of its role in DNA repair. The XPC-RAD23B-centrin2 complex was described as a co-factor that controls transcriptional activation of OCT4/SOX2. OCT4 and SOX2 are pluripotency factors that together with NANOG are part of the mechanism that controls the embryonic stem cell (ESC)

state. The knockdown of the XPC-RAD23B-centrin2 complex in murine ESC lead to a downregulation of pluripotency genes and up-regulation of differentiation markers [START_REF] Cattoglio | Functional and mechanistic studies of XPC DNA-repair complex as transcriptional coactivator in embryonic stem cells[END_REF]. In agreement with these results, Fong et al. showed that the depletion of XPC compromise the induction of pluripotency in mouse embryonic fibroblasts (MEFs) [START_REF] Fong | A DNA repair complex functions as an Oct4/Sox2 coactivator in embryonic stem cells[END_REF]).

The presence of XPC was also detected in the promoter of RARβ2 (retinoic acid receptor β2) The implication of XPC in chromatin remodeling has been described during the last decade.

in
Ray et al. showed that XPC associates and co-localizes with SNF5 -a component of the ATPdependent chromatin remodeling complex SWI/SNF -at sites of UV DNA damage. In SNF5-/fibroblasts, XPC is able to bind DNA lesion but is unable to recruit ATM (ataxia telangiectasia mutated) to the damage site [START_REF] Ray | Human SNF5/INI1, a component of the human SWI/SNF chromatin remodeling complex, promotes nucleotide excision repair by influencing ATM recruitment and downstream H2AX phosphorylation[END_REF] . ATM together with ATR (ATM and Rad3 related) are two protein kinases that initiate a phosphorylation cascade leading to cell cycle checkpoint arrest during DNA repair process of SS DNA breaks. Both ATM and ATR were found associated with XPC in UV damage sites. It has been proposed that the association between SWI/SNF and XPC facilitates ATM access to the lesion and allows the downstream pathway to occur [START_REF] Ray | NER initiation factors, DDB2 and XPC, regulate UV radiation response by recruiting ATR and ATM kinases to DNA damage sites[END_REF]. Finally, a possible interaction between XPC and a subunit of the chromatin assembly factor 1 (CAF-1) was identified but still needs confirmation.

CAF-1 is implicated in the restoration of intact chromatin following DNA replication and repair (Green andAlmouzni 2003, Zhu, Wani et al. 2009 [START_REF] Li | Characterization of molecular defects in xeroderma pigmentosum group C[END_REF][START_REF] Khan | Xeroderma pigmentosum group C splice mutation associated with autism and hypoglycinemia[END_REF][START_REF] Sheth | Novel mutation in the XPC gene: a case report of a patient with xeroderma pigmentosum[END_REF]. Until a decade ago, studies reporting mutational analysis in XPC were conducted in small cohort of XP-C patients, which made it difficult to access the possibility of revealing the existence of hotspots or founder mutations. Exceptions were made with two studies. Gozukara et al. proposed in 2001 the existence of a founder mutation after reporting that XP-C patients from Turkey and Italy were homozygous for the same XPC stop mutation c.1735C>T. They resorted to microsatellite markers, and discarded the possibility of hotspot mutations, suggesting that the founder mutation came from a common ancestor about 300-500 years ago [START_REF] Gozukara | A stop codon in xeroderma pigmentosum group C families in Turkey and Italy: molecular genetic evidence for a common ancestor[END_REF]). In 2007, Cleaver et al. after mutational analysis of patients of an isolated Guatemala village proposed c.940delC as a founder mutation in that area [START_REF] Cleaver | Xeroderma pigmentosum group C in an isolated region of Guatemala[END_REF].

A major breakthrough for the molecular diagnosis of XP-C in Maghreb came from a large cohort study in 2010 with the discovery that 74% of maghrebian patients shared the same XPC mutation -c.1643_1644delTG [START_REF] Soufir | A prevalent mutation with founder effect in xeroderma pigmentosum group C from north Africa[END_REF]. Haplotype microsatellite analysis confirmed that this is a founder mutation and suggested that the original mutation occurred 1,260 years ago, right in the middle of the Muslim conquests of the Maghreb and Iberia. In light of these data, it is not surprising that the same founder mutation was found in patients from Italy, Middle East and Sudan [START_REF] Chavanne | Mutations in the XPC gene in families with xeroderma pigmentosum and consequences at the cell, protein, and transcript levels[END_REF][START_REF] El-Harith | Diagnosis of Xeroderma pigmentosum C by detection of the founder mutation c.1643_1644delTG (p.Val548Ala fsX25) in a Sudanese Family[END_REF].

The geographical situation of Maghreb together with the history of frequent consanguineous marriages are important factors that contribute to the higher prevalence of XP-C in this region. Although there is a line of work suggesting that it could be a correlation between XP-C patients' phenotype and XPC mutation profile, until now there is not a major cohort study that can validate the results obtained in small groups of XP-C patients. For instance, the proposition that the c.2033+2A>C splice mutation could be associated with hyperglycinemia comes from a case report of only one XP-C patient [START_REF] Khan | Xeroderma pigmentosum group C splice mutation associated with autism and hypoglycinemia[END_REF]. Besides, XP-C patients are often described as a homogeneous group regarding their phenotypic characteristics [START_REF] Fassihi | Deep phenotyping of 89 xeroderma pigmentosum patients reveals unexpected heterogeneity dependent on the precise molecular defect[END_REF] (ratio 5:1); second, the majority of XP-C skin cancers present TP53 mutations. These mutations are located at dipyrimidine sites and predominantly correspond to transitions C to T or tandem CC to TT mutation that both, constitute a signature of UV-induced lesions (Giglia, Dumaz et al. 1998). The "preference" for the CC to TT tandem mutations seems to be specific from XP-C group. In the general population, TP53 mutations are found in at least 50% of skin cancers, and about 60% correspond to CG to TA transitions [START_REF] Giglia-Mari | TP53 mutations in human skin cancers[END_REF].

The P53 protein is implicated in cell-cycle regulation, apoptosis and DNA repair (Lane 1992, Balint and[START_REF] Balint | Activation and activities of the p53 tumour suppressor protein[END_REF]. Interestingly, p53 seems to control the UV inducible expression of XPC through the binding to XPC promoter [START_REF] Adimoolam | p53 and DNA damage-inducible expression of the xeroderma pigmentosum group C gene[END_REF] XP-C fibroblasts also show similarities with senescent cells. They have photo-aged like features such as elongated dendritic shape and high levels of reactive oxygen species (ROS) accumulation (Frechet, Warrick et al. 2008). XPC -/-mice show increase in skin cancer after massive and chronic UV exposure, reproducing in part the human XP-C phenotype [START_REF] Sands | High susceptibility to ultravioletinduced carcinogenesis in mice lacking XPC[END_REF]. Surprisingly, these mice also present high levels of internal cancers, particularly in lung -a tissue that is constantly exposed to oxygen. Mutational spectra of the rare internal cancers found in XP-C patients and Xpc defective mice shows an accumulation of oxidative DNA damage (i.e compatible with 8oxoG mutagenesis) (Giglia, Dumaz et al. 1998[START_REF] Wijnhoven | Age-dependent spontaneous mutagenesis in Xpc mice defective in nucleotide excision repair[END_REF][START_REF] Melis | Slow accumulation of mutations in Xpc-/-mice upon induction of oxidative stress[END_REF][START_REF] Nemzow | XPC: Going where no DNA damage sensor has gone before[END_REF]. The implication of XPC in BER could be directly linked to these phenomena, and the accumulation of oxidative lesions explain both the incidence of internal cancers and the aged phenotype of XP-C cells. Furthermore, young XPC-/-mice present features of premature skin aging including overexpression of p 16INK4a , senescence-associated beta-galactosidase (SA-β-gal) activity and NADPH oxidase 1 (NOX). The activation of NOX triggers a disturbed redox homeostasis and its inhibition is sufficient to rescue premature skin aging in XPC -/-mice [START_REF] Hosseini | Premature skin aging features rescued by inhibition of NADPH oxidase activity in XPC-deficient mice[END_REF] . These results strongly strengthen the idea that XP-C is not only a cancer-prone but also and age-related disease.

The importance of the tumor microenvironment (TME) and aging in cancer progression will be further discussed in the next chapter.

Chapter III: Tumor microenvironment "When a plant goes to seed, its seeds are carried in all directions; but they can only live and grow if they fall on congenial soil" -Paget, 1889

Over the last decades, the study of cancer cell mutagenesis allowed the identification of oncogenes and tumor-suppressor genes that actively participate in the tumorigenesis process.

Although cancer cells suffer modifications of their own that favor their growth and spread, their environment has also an influence in cancer progression and metastasis (Fidler 2003). In 1889, [START_REF] Paget | The distribution of secondary growths in cancer of the breast. 1889[END_REF]). This delicate theory set the basis for the study of the tumor microenvironment as an active part in the fate of cancer cells.

Hanahan and Weinberg were able to determine processes shared by many types of cancers.

They established these similarities as the "hallmarks of cancer" (Hanahan and[START_REF] Hanahan | The hallmarks of cancer[END_REF][START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF]. Of the ten hallmarks of cancer, sustainment of proliferative signals, evasion of growth suppressors, resistance to cell death, neo-angiogenesis, activation of invasion and metastasis, escape from immune destruction, and tumor-promoting inflammation, all involve the various contributions from TME cells (figure 14) [START_REF] Hanahan | Accessories to the crime: functions of cells recruited to the tumor microenvironment[END_REF].

Adapted from (Hanahan and[START_REF] Hanahan | The hallmarks of cancer[END_REF][START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF] Figure 14. The hallmarks of cancer.

Constituents of tumor microenvironment

Besides cancer cells, TME is composed of an ECM, and non-malignant cells embedded in blood vessels. The non-malignant cells are mainly fibroblasts, vascular and immune cells. In some cases, adipocytes can also be present (Junttila and de Sauvage 2013).

Fibroblasts associated to cancer

Fibroblasts account for the majority of non-tumor cells within the TME [START_REF] Kalluri | The biology and function of fibroblasts in cancer[END_REF]. As described in chapter I, normal fibroblasts have an important role in skin homeostasis, primarily via ECM upon development, maintenance, and remodeling. Fibroblasts are able to interact with various different cell types. Under conditions that require tissue remodeling, normal fibroblasts become "activated" and acquire morphological and functional specificities compared to non-activated, usually quiescent fibroblasts. Giulio Gabbiani was the first to identify this particular type of activated fibroblasts upon tissue wound healing. He showed that fibroblasts in wounds present a developed fibrillary system and surface similarities with smooth muscle cells [START_REF] Gabbiani | Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction[END_REF]). These so-called myofibroblasts present de novo expression of α-smooth muscle actin (α-SMA), contractile stress fibers, and splice variants of fibronectin (ED-A). Myofibroblasts are able to highly contract ECM through acto-myosin activation to bring closer the marges of the wound; myofibroblats secrete matrix proteins facilitating migration (and proliferation) of epithelial cells to complete the healing process [START_REF] Castor | Activation of lung connective tissue cells in vitro[END_REF][START_REF] Serini | The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1[END_REF][START_REF] Hinz | Alpha-smooth muscle actin expression upregulates fibroblast contractile activity[END_REF][START_REF] Tomasek | Myofibroblasts and mechano-regulation of connective tissue remodelling[END_REF]. As tumors have been considered as wounds that never heal, it became conceivable that fibroblasts associated to tumors were showing similarities to activated fibroblasts [START_REF] Dvorak | Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing[END_REF]. They both share expression of α-SMA and ED-A fibronectin but differ in the duration and the reversibility of the "activation". While in wounds, myofibroblasts are removed from the site after the healing process. In TME, fibroblasts stay in place and gain enhanced proliferative properties, becoming the irreversibly CAFs [START_REF] Desmouliere | Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar[END_REF][START_REF] Bizik | Cell-cell contacts trigger programmed necrosis and induce cyclooxygenase-2 expression[END_REF][START_REF] Bechtel | Methylation determines fibroblast activation and fibrogenesis in the kidney[END_REF].

CAFs are defined as a fibroblastic heterogeneous cell population. They can be identified by a panel of activation markers such as expression of α-SMA, FSP-1, fibroblast-activation protein α (FAP-α) and platelet-derived growth factor (PDGF) receptors-β but none of these markers seem exclusive of CAFs [START_REF] Garin-Chesa | Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers[END_REF][START_REF] Desmouliere | The stroma reaction myofibroblast: a key player in the control of tumor cell behavior[END_REF], Zhang, Chen et al. 2013[START_REF] Ostman | PDGF receptors in tumor stroma: Biological effects and associations with prognosis and response to treatment[END_REF]. Although fibroblasts and usually cells from mesodermal, are the main direct source of CAFs, stellate cells, bone marrow-derived cells, adipocytes, endothelial cells (from mesodermal origin) and epithelial cells (from ectodermal origin) have also been reported as possible source of CAFs [START_REF] Iwano | Evidence that fibroblasts derive from epithelium during tissue fibrosis[END_REF][START_REF] Ishii | Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction[END_REF][START_REF] Zeisberg | Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts[END_REF][START_REF] Kordes | Hepatic and pancreatic stellate cells in focus[END_REF][START_REF] Kidd | Origins of the tumor microenvironment: quantitative assessment of adipose-derived and bone marrow-derived stroma[END_REF]).

In the early stages of carcinogenesis, fibroblasts are able to inhibit cancer progression. It is presumed that the ability of fibroblasts to maintain the homeostasis of tissue architecture is essential to avoid cancer dissemination. Of note, the results presented in this manuscript also suggest a possible role of fibroblasts in the early immune response against cancer cell invasion. Once fibroblasts start changing their phenotype, the architecture and composition of the ECM change, and they become active actors in cancer cell growth and fate (Trimboli, Cantemir-Stone et al. 2009, Bissell and[START_REF] Bissell | Why don't we get more cancer? A proposed role of the microenvironment in restraining cancer progression[END_REF].

Vascular cells

Vascular cells are key players in the formation of new blood vessels in a process called angiogenesis. Angiogenesis is benefic to physiological processes like wound healing but when in a TME context, it contributes to tumor growth and metastasis. Briefly, the formation of new blood vessels within tumors allows the oxygenation of the tissue, supplies important metabolites and removes unimportant or detrimental ones [START_REF] Carmeliet | Molecular mechanisms and clinical applications of angiogenesis[END_REF]Jain 2011, Marcola and[START_REF] Marcola | Endothelial progenitor cells in tumor angiogenesis: another brick in the wall[END_REF]. This process is reminiscent of the vascularization process that happens during embryonic development. Endothelial progenitor cells (EPC) are recruited or already reside in the TME and, after maturation, start the formation of de novo vascular structures.

Asahara et al., were the first to identify these progenitor cells and determined that they originated from the bone marrow [START_REF] Asahara | Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization[END_REF]. Pericytes are the other vascular cell type important in angiogenesis, particularly during its initial stages. Activated pericytes lose their attachment to microvessels and starts developing cytoplasmic extensions into the tumor allowing endothelial cells to migrate into the perivascular space and create the migration tube [START_REF] Bergers | Tumorigenesis and the angiogenic switch[END_REF]Benjamin 2003, Raza, Franklin et al. 2010). Both cancer cells and CAFs are able to recruit and modulate endothelial precursor cells (EPC) in the TME [START_REF] Marcola | Endothelial progenitor cells in tumor angiogenesis: another brick in the wall[END_REF].

Immune cells

Different types of immune cells from both lymphoid and myeloid lineages can be found in TME.

They can interact with cancer cells both by direct contact and through chemokine and cytokine secretion and subsequent signaling. More importantly, they play dual roles in cancer progression, acting against or in favor of tumor growth and progression [START_REF] Zamarron | Dual roles of immune cells and their factors in cancer development and progression[END_REF]Chen 2011, Gajewski, Schreiber et al. 2013). Below are listed most of the lead actors in cancer immune microenvironment.

1.3.a. T cells

The different types of T cells found within the tumors can be divided in those associated with anti-tumor properties and those associated with tumor progression. The presence of CD8 + T cells and CD4 + T helper 1 (Th1) cells in TME correlates with good prognosis in many types of solid tumors [START_REF] Pages | Effector memory T cells, early metastasis, and survival in colorectal cancer[END_REF][START_REF] Fridman | The immune contexture in human tumours: impact on clinical outcome[END_REF]. T cells are thought to become activated through a process requiring antigen presentation by APCs including macrophages, B cells and conventional dendritic cells (cDC). APCs will uptake antigens in peripheral locations and then process and present antigens to naïve T cells in the lymphoid organs. In tumor context, cDC1, a specific type of cDC is of major importance as the most efficient APC that cross-present major histocompatibility complex class I (MHC-I) restricted antigens to CD8 + T cells. After priming, T cells will migrate and infiltrate TME [START_REF] Boon | Tumor antigens recognized by T lymphocytes[END_REF][START_REF] Hildner | Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity[END_REF][START_REF] Bottcher | NK Cells Stimulate Recruitment of cDC1 into the Tumor Microenvironment Promoting Cancer Immune Control[END_REF]). This process is summarized in figure 15.

Adapted from [START_REF] Rieth | Mechanisms of Intrinsic Tumor Resistance to Immunotherapy[END_REF] (1) Antigen presenting dendritic cells acquire cancer cell antigens and ( 2 APCs needed for the cytolytic functions of T cells and cytotoxic macrophages [START_REF] Shankaran | IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity[END_REF][START_REF] Mocikat | Natural killer cells activated by MHC class I(low) targets prime dendritic cells to induce protective CD8 T cell responses[END_REF]. IFN-γ can also prevent angiogenesis and is necessary for the induction of Th1 responses (Qin andBlankenstein 2000, Martin-Fontecha, Thomsen et al. 2004).

Not all T cells are classically considered as potent anti-tumor effectors. Regulatory T cells (Tregs) (a subpopulation of CD4 + T cells) are able to impair DC maturation therefore contributing to suppress the activation of effector T cells. They can also induce apoptosis and cytolysis of effector T cells [START_REF] Fallarino | Modulation of tryptophan catabolism by regulatory T cells[END_REF][START_REF] Vignali | How regulatory T cells work[END_REF]. The switch from Th1 to Th2 phenotype mediated by DC and CAFs also contributes to tumor Whatever the process, this leads to the balance between the different receptor engagements and consequently to the secretion of cytotoxic moleculesperforin and granzymesresponsible for the activity and targeting of NK-cell mediated killing. Perforins are capable of disrupting membrane proteins and, in turn, granzymes are proteolytic enzymes. (Voskoboinik, Whisstock et al. 2015, Morvan and[START_REF] Morvan | NK cells and cancer: you can teach innate cells new tricks[END_REF]. Antibody-dependent cell-mediated cytotoxicity is another mechanism allowing the elimination of cancer cells by NK cells. In the latter case, the Fc gamma receptor III CD16 on NK cells, recognizes antibodies that bound to specific antigens expressed at the surface of cancer cells, allowing the killing of the "antibody coated" malignant cells (figure 16) [START_REF] Morvan | NK cells and cancer: you can teach innate cells new tricks[END_REF]. Lastly, through an autocrine signaling cascade involving tumor necrosis factor (TNF) receptors family, NK cells induce the expression of "death ligands" FasL and TRAIL that promote apoptosis when bound to their receptors expressed in cancer cells [START_REF] Cretney | Increased susceptibility to tumor initiation and metastasis in TNF-related apoptosis-inducing ligand-deficient mice[END_REF][START_REF] Wallin | Regulation of perforin-independent NK cell-mediated cytotoxicity[END_REF].

Besides their killing capacities, NK cells can also contribute to the protection against cancer progression through secretion of IFN-γ and (TNF-α) [START_REF] Muller-Hermelink | TNFR1 signaling and IFN-gamma signaling determine whether T cells induce tumor dormancy or promote multistage carcinogenesis[END_REF].

The anti-tumor properties of IFN-γ has already been described. As for TNF-α most properties overlap with those from IFN-γ like inhibition of angiogenesis and tumor proliferation and induction of Th1 cell responses [START_REF] Stoelcker | Tumor necrosis factor induces tumor necrosis via tumor necrosis factor receptor type 1-expressing endothelial cells of the tumor vasculature[END_REF][START_REF] Muller-Hermelink | TNFR1 signaling and IFN-gamma signaling determine whether T cells induce tumor dormancy or promote multistage carcinogenesis[END_REF]).

1.3.d. Macrophages

Macrophages are classified in two functional polarized states -M1 and M2. While M1 macrophages contribute to anti-tumor immunity for instance through triggering apoptosis in tumor cells via TNF receptors, M2 macrophages promote angiogenesis, tumor invasion and metastasis [START_REF] Mantovani | Macrophage polarization: tumorassociated macrophages as a paradigm for polarized M2 mononuclear phagocytes[END_REF]. Tumor associated macrophages (TAMs) usually switch from an M1 to an M2 phenotype. In spite of the secretion of different cytokines, both M1 and M2 recruit pro-tumoral immune cells and contribute to the impairment of T cell priming [START_REF] Sica | Altered macrophage differentiation and immune dysfunction in tumor development[END_REF].

1.3.e. Neutrophils

Similarly to what happens with TAMs, tumor associated neutrophils (TANs) also switch from an anti-tumoral to a pro-tumoral phenotype. Their anti-tumoral activity is based on their ability to trigger cancer cell death and stimulate adaptive immune responses [START_REF] Stockmeyer | Polymorphonuclear granulocytes induce antibody-dependent apoptosis in human breast cancer cells[END_REF][START_REF] Mantovani | Neutrophils in the activation and regulation of innate and adaptive immunity[END_REF][START_REF] Martin | Tumor-derived granzyme B-expressing neutrophils acquire antitumor potential after lipid A treatment[END_REF]. Conversely, pro- ) and monocytes (HLA-DR + ) [START_REF] Bronte | Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards[END_REF]. As for PMN-MDSC and neutrophils, there is no really distinction between the two populations besides of their different densities [START_REF] Sagiv | Phenotypic diversity and plasticity in circulating neutrophil subpopulations in cancer[END_REF]. PMN-MDSC share the same phenotype with TAN and there is a debate whether they belong in fact to the same population [START_REF] Groth | Immunosuppression mediated by myeloid-derived suppressor cells (MDSCs) during tumour progression[END_REF].

Adipocytes

Adipocytes are one of cell types that can be at the origin of CAFs but infiltrating adipocytes can also be modulated by cancer cells into cancer associated adipocytes (CAA

The prison break of cancer cells

A complex crosstalk between the cellular components of the TME is needed for tumor progression. The way CAFs, cancer and immune cells communicate, activate with one another and play their role in cancer progression is the subject of this section.

Crosstalk between fibroblasts and cancer cells

Cancer cells are able to recruit nonmalignant cells and create conditions that promote the switch of ECM fibroblast to CAFs. One of the most known and well-studied tumor-cell derived factor affecting CAF activation is TGF-β. In a TGF-β rich microenvironment, fibroblasts start expressing factors involved in ECM production and remodeling and through a paracrine signaling cascade, they downregulate the gap junctions that are important to their intercellular communication [START_REF] Lieubeau | The role of transforming growth factor beta 1 in the fibroblastic reaction associated with rat colorectal tumor development[END_REF][START_REF] Lohr | Transforming growth factor-beta1 induces desmoplasia in an experimental model of human pancreatic carcinoma[END_REF][START_REF] Stuhlmann | Paracrine effect of TGF-beta1 on downregulation of gap junctional intercellular communication between human dermal fibroblasts[END_REF][START_REF] Hadj-Rabia | Unexpected extradermatological findings in 31 patients with xeroderma pigmentosum type C[END_REF]. TGF-β alone is enough to induce the expression of α-SMA in fibroblasts (Ronnov-Jessen and Petersen 1993). However, TGF-β is not required for the invasive properties of activated fibroblasts [START_REF] Albrengues | LIF mediates proinvasive activation of stromal fibroblasts in cancer[END_REF]. PDGF and basic fibroblast growth factor (bFGF) are two other growth factors highly expressed in cancer cells that stimulate proliferation and activation of CAFs [START_REF] Shao | Human breast carcinoma desmoplasia is PDGF initiated[END_REF][START_REF] Strutz | Basic fibroblast growth factor expression is increased in human renal fibrogenesis and may mediate autocrine fibroblast proliferation[END_REF]. After the switch, a positive feedback is established between CAFs and cancer cells (figure 17). While cancer cells maintain fibroblast activation, fibroblasts respond with secretion of growth factors, cytokines and proteases that sustain tumor progression. 2014). Finally, the overexpression of HGF by CAFs has been shown to promote proliferation of cancer cells and increases their invasive properties in 3D cultures through the paracrine activation of HGF/c-Met signaling pathway [START_REF] Jedeszko | Fibroblast hepatocyte growth factor promotes invasion of human mammary ductal carcinoma in situ[END_REF][START_REF] Ding | HGF-mediated crosstalk between cancer-associated fibroblasts and MET-unamplified gastric cancer cells activates coordinated tumorigenesis and metastasis[END_REF].

CAFs are also key players in the modulation of adhesive and proteolytic activities that are essential in cancer invasion and dissemination. Through the overexpression of matrix metalloproteinases (MMPs), CAFs are able to disrupt ECM by proteolysis, making it more prone to tumor expansion and invasion [START_REF] Chenard | Presence of high levels of MT1-MMP protein in fibroblastic cells of human invasive carcinomas[END_REF][START_REF] Foley | Matrix metalloprotease 1a deficiency suppresses tumor growth and angiogenesis[END_REF]. Furthermore, in pancreatic cancer cells, MMP-2 and MMP-9 are able to elicit an epithelial-mesenchymal transition (EMT) which promotes motility and migratory abilities [START_REF] Giannoni | Reciprocal activation of prostate cancer cells and cancer-associated fibroblasts stimulates epithelialmesenchymal transition and cancer stemness[END_REF]. IL-6 and CXCL12/SDF-1 also trigger cancer cell EMT through activation of JAK2-STAT3 and Hedgehog pathways [START_REF] Li | SDF-1/CXCR4 signaling induces pancreatic cancer cell invasion and epithelial-mesenchymal transition in vitro through non-canonical activation of Hedgehog pathway[END_REF], respectively.

In a mechanical process independent of MMPs activity, CAFs are able to disrupt the basement membrane and induce the formation of gaps through which cancer cells can migrate [START_REF] Glentis | Cancerassociated fibroblasts induce metalloprotease-independent cancer cell invasion of the basement membrane[END_REF].

One of the hallmarks of cancer is the acquisition/re-expression of stem-cell like plasticity by The "immunoediting theory" (figure 18) characterizes three different phases of interactions between immune and cancer cells i.e., i-) elimination, ii-) equilibrium and iii-) escape [START_REF] Dunn | Cancer immunoediting: from immunosurveillance to tumor escape[END_REF]. During these three phases, distinct subsets of immune cells are found in the TME. Here, I will pay more attention on the contribution of cancer cells and CAF's to the evolution of tumoral immune microenvironment upon the different phases of the immunoediting process. [START_REF] Schreiber | Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion[END_REF] 2.2.a. Elimination

The elimination phase is characterized by a major infiltration of T cells and NK cells in an attempt to mount an efficient anti-tumor immune response aiming at the elimination/eradication of cells recognized as "abnormal". Through the secretion of CXCL9 and CXCL10, cancer cells drive T-cell tumor infiltration [START_REF] Mikucki | Non-redundant requirement for CXCR3 signalling during tumoricidal T-cell trafficking across tumour vascular checkpoints[END_REF]. In such a way, T cells can also secrete type I interferon (IFN-α and IFN-β) which stimulates accumulation of DC in TME.

Accumulation of DC contributes to the presentation the "foreign" antigen to T cells and subsequently to T cell priming and tumor cell elimination [START_REF] Diamond | Type I interferon is selectively required by dendritic cells for immune rejection of tumors[END_REF].

Regarding NK cells, the signaling that drives their migration into solid tumors is not yet completely understood. If we stick to the theory that cancer is a wound that never heals, we may assume that the classic chemoattractant molecules that drive NK cells into the woundnamely IL-2, IL-12 and IFN-α, -are already present in the early steps of tumorigenesis [START_REF] Hodge | IL-2 and IL-12 alter NK cell responsiveness to IFN-gamma-inducible protein 10 by down-regulating CXCR3 expression[END_REF][START_REF] Hu | Long-term effect on natural killer cells by interferon-alpha therapy on the outcomes of HCV infection[END_REF][START_REF] Doersch | The contribution of interleukin-2 to effective wound healing[END_REF]). However, some reports have also suggested that specific signals expressed by cancer cells may also trigger NK cell infiltration. Among them, CXCL9 and CXCL10 promote triggering of NK cell migration to the TME [START_REF] Przewoznik | Recruitment of natural killer cells in advanced stages of endogenously arising B-cell lymphoma: implications for therapeutic cell transfer[END_REF]. These two cytokines have also been identified

as expressed in CAF's [START_REF] Ziani | Alteration of the Antitumor Immune Response by Cancer-Associated Fibroblasts[END_REF]).

Effector T cells and NK cells exhibit complementary roles in the killing process of foreign cells.

The cytotoxic capacities of effector T cells are dependent on the expression of MHC-I at the surface of cancer cells. MHC-I molecules are required for detection and subsequent destruction of malignant cells by T cells [START_REF] Atkins | MHC class I antigen processing pathway defects, ras mutations and disease stage in colorectal carcinoma[END_REF]). NK cells, however, are able to kill cells that lack MHC-I molecules when they overexpress NK activator receptors at their surface. Besides the canonical function of NK cells as cell killers, a recent paper from Böttcher et al. has shown that they can also have a role in the recruitment of antigen-presenting dendritic cells through the expression of XCL1 and CCL5 which leads to the infiltration of T cells [START_REF] Bottcher | NK Cells Stimulate Recruitment of cDC1 into the Tumor Microenvironment Promoting Cancer Immune Control[END_REF].

Other immune cells that can infiltrate the neoplastic structure and kill cancer cells are two specific subsets of T cells (NKT cells and γδ T cells), TAMs and TANs.

2.2.b. Equilibrium and Escape

After a first phase where cancer cells become highly immunogenic and can easily be eliminated, in the "equilibrium phase" some of them start eluding the immune system and escape killing. They may live for a quite long time while in an equilibrium with the infiltrated immune cells that still continue to prevent tumor growth and at the same time sculpt cancer cells immunogenicity. [START_REF] Koebel | Adaptive immunity maintains occult cancer in an equilibrium state[END_REF]). However, the immunosuppression of antitumor properties of immune cells, the infiltration of pro-tumoral immune cells and the newly acquired phenotypes that allow cancer cells to protect themselves against immune responses prompt cancer cells into the escape phase.

Modulation of effector T cell function

To dribble the ability of CD8 + cytotoxic T cells to recognize them, cancer cells acquired the capacity to downregulate MHC-I expression at their cell surface [START_REF] Atkins | MHC class I antigen processing pathway defects, ras mutations and disease stage in colorectal carcinoma[END_REF][START_REF] Garrido | Generation of MHC class I diversity in primary tumors and selection of the malignant phenotype[END_REF]. This is the first step of the "tumor editing" as it starts to shape cancer cells into a less immunogenic phenotype which allows them to escape the control of immune surveillance (figure 18). In addition, some cancer cells upregulate the non-classical MHC-I molecule HLA-E that interacts with the inhibitory receptor CD94/NKG2A induced on effector T cells and expressed by NK cells, leading to the impairment of their anti-tumor activities (Braud, Allan et al. 1998, Haanen and[START_REF] Haanen | NKG2A, a New Kid on the Immune Checkpoint Block[END_REF]. More generally, a number of inhibitory receptors are induced on tumor-reactive T cells that lead to "exhausted" T cells that no longer exert efficient anti-tumor activities [START_REF] Pardoll | The blockade of immune checkpoints in cancer immunotherapy[END_REF]. These interactions, so-called "immune checkpoints" are targeted in the newly developed immunotherapies for the treatment of cancer (see point 4.2 of this chapter).

TGF-β is expressed by both cancer cells and surrounding fibroblasts. TGFhas also an Alike what happens with T cells, TGB-β is also implicated in the modulation of NK phenotype.

TGF-β is able to decrease NK cell activation and cytotoxic activity by reducing the expression of some NK-activating receptors through both its soluble or membrane-bound form [START_REF] Castriconi | Transforming growth factor beta 1 inhibits expression of NKp30 and NKG2D receptors: consequences for the NK-mediated killing of dendritic cells[END_REF][START_REF] Park | Complex regulation of human NKG2D-DAP10 cell surface expression: opposing roles of the gammac cytokines and TGF-beta1[END_REF]. In fact, besides of the secretion of soluble TGF-β by cancer cells and CAFs, another important source of TGF-β are Treg. Treg cells express membrane-bound TGF-β at their surface which is directly responsible for the down regulation of the NK activating receptor NKG2D and activity restriction of the NK cell effector functions in vitro [START_REF] Ghiringhelli | CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor-beta-dependent manner[END_REF].

The secretion of prostaglandin 2 (PGE2) again by both CAFs, cancer cells and even by some infiltrated immune cells, is also able to impair NK cell (and T cell) infiltration, as well as their cytotoxic activity [START_REF] Li | Hepatocellular carcinoma-associated fibroblasts trigger NK cell dysfunction via PGE2 and IDO[END_REF][START_REF] Zelenay | Cyclooxygenase-Dependent Tumor Growth through Evasion of Immunity[END_REF]. PGE2 is the prostaglandin most commonly found in human cancer and its expression is associated with both in vitro and in vivo [START_REF] Brand | LDHA-Associated Lactic Acid Production Blunts Tumor Immunosurveillance by T and NK Cells[END_REF]).

Modulation of other immune cells

Finally, the modulation of dendritic cell activity and the transition of macrophages and neutrophils into pro-tumoral TAMs and TANs are also processes that contribute to the escape of cancer cells and are mostly regulated by cancer cells secreted factors.

The production of vascular endothelial growth factor (VEGF), IL-6, IL-10, TGF-β, macrophage colony-stimulating factor (M-CSF) and cyclooxygenase-2 (COX-2) by cancer cells is implicated in the impairment of differentiation, maturation and function of dendritic cells with direct consequences on the activation of effector T cells [START_REF] Zou | Immunosuppressive networks in the tumour environment and their therapeutic relevance[END_REF].

In TAMs, the activated M2 phenotype may arise due to the hypoxic conditions of the TME and to the presence of M-CSF, Granulocyte-macrophage colony-stimulating factor (GM-CSF) and TGF-β secreted by cancer cells or IL-4 secreted by CD4 + T cells [START_REF] Lin | The macrophage growth factor CSF-1 in mammary gland development and tumor progression[END_REF][START_REF] Gocheva | IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion[END_REF][START_REF] Su | A positive feedback loop between mesenchymal-like cancer cells and macrophages is essential to breast cancer metastasis[END_REF][START_REF] Zhang | TGF-beta induces M2-like macrophage polarization via SNAIL-mediated suppression of a proinflammatory phenotype[END_REF]. TANs' polarization into pro-tumoral N2 phenotype can be induced by TGF-β, G-CSF and tumor-derived exomes [START_REF] Coffelt | Neutrophils in cancer: neutral no more[END_REF][START_REF] Zou | IL-35 induces N2 phenotype of neutrophils to promote tumor growth[END_REF], Zhang, Shi et al. 2018).

To conclude, as a consequence of the immunoediting process, the TME is no longer efficiently anti-tumoral and allows tumor dissemination.

Senescent microenvironment drives tumorigenesis

As mentioned in chapter 1, aging is a risk factor for the development of cancer. Hayflick first described cellular senescence in 1965. He showed that primary cells have finite replicative lifespan and when they reach it, they are no longer able to reenter cell cycle yet remaining metabolically active [START_REF] Hayflick | THE LIMITED IN VITRO LIFETIME OF HUMAN DIPLOID CELL STRAINS[END_REF]. Senescent cells express different cell cycle inhibitors -like p16, p21 and p53a specific senescence associated β-galactosidase (SA-βGal) and exhibit a particular alteration of their secretory profile known as the senescent-associated secretory phenotype (SASP) [START_REF] Dimri | A biomarker that identifies senescent human cells in culture and in aging skin in vivo[END_REF][START_REF] Alcorta | Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts[END_REF][START_REF] Coppe | The senescence-associated secretory phenotype: the dark side of tumor suppression[END_REF]. In a tumor context, senescence plays a paradoxical role being both tumor promoter and tumor suppressor. This can partially depend on the cell in which senescence occurs. For instance, cancer cells presenting a senescent phenotype are able to trigger immune responses that lead to tumor clearance in mice [START_REF] Xue | Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas[END_REF][START_REF] Kang | Senescence surveillance of pre-malignant hepatocytes limits liver cancer development[END_REF]). On the other hand, senescent stromal cells within TME promote tumor growth [START_REF] Krtolica | Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging[END_REF]).

The possible implication of a senescent microenvironment in tumor progression was extensively studied in immune deficient or immune compromised systems. Senescent stromal cells are able to stimulate both epithelial and cancer cell proliferation through the expression of tumor promoting factors like HGF, FGF7 and CXCL12 [START_REF] Bavik | The gene expression program of prostate fibroblast senescence modulates neoplastic epithelial cell proliferation through paracrine mechanisms[END_REF].

Furthermore, through the secretion of MMPs, they are able to promote cancer cell EMT and ECM remodeling [START_REF] Begley | CXCL12 overexpression and secretion by aging fibroblasts enhance human prostate epithelial proliferation in vitro[END_REF][START_REF] Parrinello | Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation[END_REF]. This line of work points out the high similarity between SASP and CAFs associated phenotype. However, in contrast with the anti-tumor processes that can be triggered by senescent carcinogenic cells, a recent study shows that senescent stromal cells drive a tumor immunosuppressive microenvironment independently of tumor presence. Through the secretion of IL-6, senescent stromal cells recruit and accumulate MDSCs in the tissue which will suppress CD8 + T cells and in a tumor context, will promote tumor growth [START_REF] Ruhland | Stromal senescence establishes an immunosuppressive microenvironment that drives tumorigenesis[END_REF]. This paper was the first to demonstrate that a senescent healthy microenvironment can be considered as a protumoral immunosuppressive ME. After that, other studies have confirmed that MDSCs are in fact more present in a senescent tissue and can be considered as inducers of immunosenescence state [START_REF] Salminen | Immunosenescence: the potential role of myeloid-derived suppressor cells (MDSC) in age-related immune deficiency[END_REF]).

The at least partial impairment of the "elimination phase" by a MDSC rich senescent ME can have two paradoxical consequences: (i) it will more easily allow the progression of cancer from a pre-malignant lesion but (ii) it will also partially impair the immunoediting process and as a consequence, cancer cells stay more immunogenic which could translate into less aggressive and more easily treatable cancer cell phenotypes. Although until now no study prove these speculations, there are correlation studies that show that in elderly, tumors are more present but are less aggressive compared to younger population [START_REF] Berger | Cancer in the elderly[END_REF]).

Microenvironment as a target for cancer therapy

Chemotherapy often combined with surgery and radiotherapy are normally the first line of treatment for cancer. Despite the effectiveness of chemotherapy treatments, cancer cells are becoming multidrug resistant (MDR) which drives relapse and compromises the prognosis of the patients [START_REF] Wu | Multi-drug resistance in cancer chemotherapeutics: mechanisms and lab approaches[END_REF]. For a long time, cancer research has been focused on cancer cell itself but with all the body of evidence confirming the important role of TME in cancer progression and even in the MDR, new perspectives of therapies based on TME start to emerge [START_REF] Shain | Environmental-mediated drug resistance: a target for multiple myeloma therapy[END_REF]. Here, I will focus on the TME perspective and/or based therapies targeting fibroblasts and immune cells.

Targeting CAFs in cancer therapy

Many molecules are currently in different phases of clinical trials to prove their efficacy in targeting CAFs or CAFs' related functions. Based on solid in vitro evidence one of the candidates to target CAFs is the membrane protease FAP-α. FAP-α is one of CAFs well-known markers and in vitro, its depletion from fibroblast membranes impairs FAP-α-mediated migratory and invasive capacity [START_REF] Teichgraber | Specific inhibition of fibroblast activation protein (FAP)-alpha prevents tumor progression in vitro[END_REF]. However, the first trials using the sibrotuzumab, an antibody targeting FAP-α, or small molecule FAP-α inhibitors did not reveal any improvement in patient outcome [START_REF] Hofheinz | Stromal antigen targeting by a humanised monoclonal antibody: an early phase II trial of sibrotuzumab in patients with metastatic colorectal cancer[END_REF][START_REF] Narra | Phase II trial of single agent Val-boroPro (Talabostat) inhibiting Fibroblast Activation Protein in patients with metastatic colorectal cancer[END_REF]). Today, there are new ongoing clinical trials that use different methods to target FAP-α (U.S. National Library of Medicine 2019). Another recent approach aiming to deplete CAFs from the microenvironment is the targeting of NAD(P)H Oxidase-4 (NOX4). NOX4 is essential for the regulation of fibroblast to myofibroblast differentiation and its pharmacological inhibition on CAFs can abrogate their tumor-promoting function in vitro and in vivo [START_REF] Hanley | Targeting the Myofibroblastic Cancer-Associated Fibroblast Phenotype Through Inhibition of NOX4[END_REF]. Until now no clinical trial is reported.

Besides depleting CAFs from the TME, other possible therapeutic approaches are the impairment of CAFs' recruitment, activation and functions. PDGF expressed by cancer cells can recruit CAFs and promote growth and metastasis [START_REF] Tejada | Tumor-driven paracrine platelet-derived growth factor receptor alpha signaling is a key determinant of stromal cell recruitment in a model of human lung carcinoma[END_REF][START_REF] Anderberg | Paracrine signaling by platelet-derived growth factor-CC promotes tumor growth by recruitment of cancer-associated fibroblasts[END_REF]). There are many available PDGFR inhibitors but none of them is specific for PDGFR inhibition as they also target other tyrosine kinases like VEGFR. However, studies proved that PDGFR inhibition is the one responsible for the positive effects of imatinib, sunitinib and nilotinib -three PDGFR inhibitors -in the treatment of gastrointestinal stromal tumors and dermatofibrosarcoma protuberance [START_REF] Heldin | Targeting the PDGF signaling pathway in tumor treatment[END_REF]. To overcome the difficulty of the selectivity of the target, new specific antibodies were made but until now no clinical trial show a significant antitumor activity. However, there are clinical trials still ongoing (U.S. National Library of Medicine 2019).

TGF-β as one of the main factors that can drive a normal cell into a CAF is also a good target for cancer therapy. There are many different types of TGF-β targeting agents and some of them show good results in clinical trials. This is the case of galunisertib, a TGB-β receptor kinase inhibitor that shows anti-tumor responses in glioblastoma patients and in combination with cytotoxic gemcitabine, improves overall survival in pancreatic cancer patients [START_REF] Rodon | First-in-human dose study of the novel transforming growth factorbeta receptor I kinase inhibitor LY2157299 monohydrate in patients with advanced cancer and glioma[END_REF][START_REF] Colak | Targeting TGF-beta Signaling in Cancer[END_REF][START_REF] Melisi | Galunisertib plus gemcitabine vs. gemcitabine for first-line treatment of patients with unresectable pancreatic cancer[END_REF].

Because of the pleiotropic effects of TGF-β, it is difficult to say which mechanism is responsible for the therapeutic effect. Combining TGF-β targeting with other cancer therapies could help to determine the leading mechanism in TGF-β action in favor of cancer development.

Targeting the ECM composition is also a valid alternative in cancer therapy targeting microenvironment phenomena. One example is targeting ECM fibrosis with a Pirfenidone (PFD) treatment. PFD is a small molecule with anti-fibrotic properties that inhibit tumor fibrosis in vivo and when associated with the classic cytotoxic antineoplastic drug doxorubicin inhibits tumor growth and metastasis [START_REF] Takai | Targeting the cancer-associated fibroblasts as a treatment in triple-negative breast cancer[END_REF]. Phase I clinical trials are ongoing to approve the combination of pirfenidone with standard first line chemotherapy to treat non small lung cancer (NCT03177291)(U.S. National Library of Medicine 2019).

If at least some of all these clinical trials succeed, we will definitely enter in a new era of cancer treatment that will acknowledge the independent and important role of CAFs in cancer progression.

Targeting immune system in cancer therapy

The important role of the immune system in cancer progression pushed for the development of cancer immune based therapies far before what happened with fibroblasts. Besides the multiple clinical trials that are ongoing, today, different approaches regarding cancer immunotherapy are already used in clinic.

To maintain immune tolerance and avoid excessive systemic inflammation in the body, there are inhibitory pathways that act as regulators of the immune system called immune checkpoints. Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and programmed cell death protein 1 (PD-1) are two receptors expressed in T cells that triggers inhibitory cascades leading to impairment of T cell effector functions and immunosuppression. CTLA-4 is expressed on both CD4 + and CD8 + T cells upon activation [START_REF] Perkins | Regulation of CTLA-4 expression during T cell activation[END_REF]. CTLA-4 competes with the co-stimulatory CD28 receptor for the same ligands -CD80 and CD86.

However, CTLA-4 has higher affinity for both ligands and outcompete CD28. The engagement of CTLA-4 leads to T cells that are more resistant to activation, which reduces immune responses towards weak antigens such as self-and tumor antigens [START_REF] Rudd | CD28 and CTLA-4 coreceptor expression and signal transduction[END_REF][START_REF] Seidel | Anti-PD-1 and Anti-CTLA-4 Therapies in Cancer: Mechanisms of Action, Efficacy, and Limitations[END_REF]. PD-1 is expressed in T cells, also upon activation. In T cells, the expression of PD-1 is induced upon antigen recognition via T cell antigen receptor (TCR).The engagement of PD-1 with its ligands PD-L1 and PD-L2 leads to exhaustion and apoptosis of effector T cells [START_REF] Keir | PD-1 and its ligands in tolerance and immunity[END_REF]. Cancer cells are able to activate immune inhibitory checkpoints to avoid anti-tumor immune responses.

Approaches based on blocking ligand access to CTLA-4 and PD-1 are already used in the treatment of cancer. Ipilimumab and tremelimumab are two therapeutic anti-CTLA-4 antibodies that impair the binding of CTLA-4 ligands and show good efficacy in the treatment of numerous solid tumors [START_REF] Rowshanravan | CTLA-4: a moving target in immunotherapy[END_REF]. In clinic they are used for the treatment of metastatic melanoma and renal cell carcinomas (U.S. Food & Drug Administration 2019). The engagement of PD-1 by PD-L1 expressed in cancer cells makes them resistant to CD8 + T direct lysis and to apoptosis [START_REF] Azuma | B7-H1 is a ubiquitous antiapoptotic receptor on cancer cells[END_REF][START_REF] Kinter | The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands[END_REF].

Pembrolizumab, nivolumab and cemiplimab are three anti-PD-1 antibodies approved and currently used for the treatment of solid tumors. In addition, anti-PD-L1 antibodies were also designed and atelolizumab, avelumab and durvalumab are also suitable for clinical use(U.S. Besides from therapies targeting inhibitory checkpoints like the ones mentioned before, diverse strategies are used to enhance endogenous antitumor immune responses. As antigen presentation is one of the first step in generating immune response, therapeutic vaccines could augment adaptive immune responses. There are several therapeutic vaccines in clinical trials but until now, only Sipuleucel-T passed the cut off of FDA. Sipuleucel-T consists in autologous APC incubated ex vivo with a prostate antigen. It is an alternative treatment for prostate cancer [START_REF] Kantoff | Sipuleucel-T immunotherapy for castration-resistant prostate cancer[END_REF][START_REF] Gardner | Sipuleucel-T (Provenge) autologous vaccine approved for treatment of men with asymptomatic or minimally symptomatic castrate-resistant metastatic prostate cancer[END_REF].

Therapeutic modulation of the cytokine environment is also a way to enhance anti-tumor immune responses. IL-2 was the first cytokine to be considered and later approved for its efficacy in cancer immunotherapy [START_REF] Jiang | Role of IL-2 in cancer immunotherapy[END_REF]) It induces proliferation and enhances cytotoxicity of NK cells, activates T cells and promotes the differentiation of B cells [START_REF] Miyawaki | Interleukin-2 effects on human B cells activated in vivo[END_REF][START_REF] Sharma | IL-2 mediates NK cell proliferation but not hyperactivity[END_REF][START_REF] Tognarelli | Enhancing the Activation and Releasing the Brakes: A Double Hit Strategy to Improve NK Cell Cytotoxicity Against Multiple Myeloma[END_REF]. In metastatic melanoma and renal cell carcinoma, IL-2 therapy mediates tumor regression and provides clinical benefits to patients but at least monotherapy is not enough to improve patient's survival. Because (i) it also activates pro-tumoral Tregs expansion and (ii) it is associated with toxicity at high doses, its use in clinic remains restricted [START_REF] Fisher | Long-term survival update for high-dose recombinant interleukin-2 in patients with renal cell carcinoma[END_REF][START_REF] Saraceni | Clinical characteristics and treatment-related biomarkers associated with response to high-dose interleukin-2 in metastatic melanoma and renal cell carcinoma: retrospective analysis of an academic community hospital's experience[END_REF]. Another cytokine currently used as an adjuvant in cancer treatment is IFN-α. During clinical trials it proved to have a positive impact in recurrence free survival of metastatic melanoma [START_REF] Eggermont | Long-term results of the randomized phase III trial EORTC 18991 of adjuvant therapy with pegylated interferon alfa-2b versus observation in resected stage III melanoma[END_REF]. It is also approved for first line treatment of renal cell carcinoma -where its administration correlates with increased in cellular immune responses -and some forms of lymphoma and leukemia (Kosmidis, Baxevanis et al. 1992, Floros and[START_REF] Floros | Anticancer Cytokines: Biology and Clinical Effects of Interferonalpha2, Interleukin (IL)-2, IL-15, IL-21, and IL-12[END_REF].

Finally, similarly to the approach of targeting inhibitory signaling in T cells, targeting costimulatory receptors of effector cells are currently under development. Agonist antibodies for OX40 and CD40 -two costimulatory receptors expressed in T cells -and CD137 -an activating receptor of both T cells and NK cells -are currently being tested. In different phases of trials, the treatment with such antibodies promotes anti-tumor immune responses and induces tumor regression [START_REF] Vonderheide | Clinical activity and immune modulation in cancer patients treated with CP-870,893, a novel CD40 agonist monoclonal antibody[END_REF][START_REF] Redmond | The role of OX40-mediated co-stimulation in T-cell activation and survival[END_REF][START_REF] Kohrt | Stimulation of natural killer cells with a CD137-specific antibody enhances trastuzumab efficacy in xenotransplant models of breast cancer[END_REF]).

Chapter IV: NK cells and their receptors

It has been a long and astonishing journey for a cell that started its life as a background noise in a laboratory assay -Rolf Kiessling [START_REF] Kiessling | Natural Killer Cells: Basic Science and Clinical Application[END_REF] One of the first description of the existence of cells that are able to produce cytotoxicity only by contact with cancer-associated antigens was made by [START_REF] Rosenberg | Lymphocyte cytotoxicity reactions to leukemia-associated antigens in identical twins[END_REF][START_REF] Rosenberg | Lymphocyte cytotoxicity reactions to leukemia-associated antigens in identical twins[END_REF]. At that time, research efforts were focused on the study of T-cell immune reactivity to tumor antigens and on determining the best methods to evaluate this reactivity. First attempts to standardize the 51 Chromium release assay to measure T cell reactivity against murine Moloney Virus induced leukemia failed because of the "background lysis" of YAC-1 mouse lymphoma cell line in presence of cells from mouse spleen [START_REF] Kiessling | Genetic variation of in vitro cytolytic activity and in vivo rejection potential of non-immunized semi-syngeneic mice against a mouse lymphoma line[END_REF]. Researchers quickly understood that in fact this was not background noise but the effect of a different population of lymphocytes -the NK cells. The first characterizations of the NK cell phenotype were published in 1975. NK cells were described as a population with antitumor effector cells functions from non-tumor bearing mice that differed from T-or B-lymphocytes on the cell-surface markers (Herberman, Nunn et al. 1975). Authors suggested at the time that the lytic NK cell activity were triggered by murine endogenous type C viruses associated antigens (Herberman, Nunn et al. 1975).

However, despite this early discoveries, the mechanisms underlying this unique capacity to distinguish between healthy and infected or cancer cells remained unknown until the discovery in 1990 that NK cells were able to kill cells that fail to express the MHC class I molecules [START_REF] Ljunggren | In search of the 'missing self': MHC molecules and NK cell recognition[END_REF].

After that, the puzzle was rapidly solved with the identification of different NK inhibitory and activating receptors that clarified how NK cell functions are regulated. The unbalance between these two classes of receptors is at the basis of the regulation of NK cell cytotoxic activity.

NK receptors

The interaction between inhibitory receptors and MHC-I molecules is essential for NK cell development and maturation in the bone marrow. This is a process called NK cell education and will determine the threshold for activation in mature NK cells. Depending on the level of MHC-I inhibitory signals that they receive, NK cells are able to balance their activating receptors to adapt their phenotype to those of the host [START_REF] Raulet | Self-tolerance of natural killer cells[END_REF]. This dance of acquiring properties of self and missing-self recognition gives rise to a large NK heterogeneous population with 6000 to 30,000 different phenotypes within an individual [START_REF] Husain | Complex expression of natural killer receptor genes in single natural killer cells[END_REF][START_REF] Brodin | NK cell education: not an on-off switch but a tunable rheostat[END_REF][START_REF] Horowitz | Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry[END_REF].

The genes that encode for NK cell receptors are mostly clustered in two complexes at different sites of human genome: the natural killer complex (NKC) located on chromosome 12p12-p13 encoding for the C-type lectin-like molecules, and the leukocyte receptor complex (LRC), located on chromosome 19q13.4 encoding genes of the immunoglobulin superfamily (IgSF) receptors [START_REF] Trowsdale | Genetic and functional relationships between MHC and NK receptor genes[END_REF][START_REF] Kelley | Comparative genomics of natural killer cell receptor gene clusters[END_REF]. Importantly both activating and inhibitory receptors could be MHC-I dependent or independent.

LRC encodes for killer immunoglobulin-like receptors (KIR) that are NK receptors for MHC-I molecules and for one member of the natural cytotoxicity receptors (NCR), the NKp46, which is an activating receptor [START_REF] Wende | Organization of the leukocyte receptor cluster (LRC) on human chromosome 19q13.4[END_REF][START_REF] Hadad | NKp46 Clusters at the Immune Synapse and Regulates NK Cell Polarization[END_REF] [START_REF] Bartel | Modulation of NK cell function by genetically coupled C-type lectin-like receptor/ligand pairs encoded in the human natural killer gene complex[END_REF]. CTLR are structurally type II membrane-bound surface receptors with one extracellular C-type lectin-like domain (CTLD). The ones encoded in NKC complex belong to the C type lectin subfamily V that is characterized by the loss of a functional Ca 2+ -dependent ability to bind carbohydrates. They instead bind to protein ligands (Iizuka, Naidenko et al. 2003, Zelensky and[START_REF] Zelensky | The C-type lectin-like domain superfamily[END_REF]. KLR can be further divided in two subclasses: the ones that bind MHC-I like and non-classical MHC-I molecules, and the ones that do not bind MHC-I ligands. To the first class belong the well-studied NKG2D/KLRK1 activating receptor (their ligands MICA/B et ULBPs are not MHCI but MHC-I-like) and the subfamily CD94/NKG2 complex binding the non-classical MHCI molecule HLA-E [START_REF] Braud | HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C[END_REF][START_REF] Bauer | Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA[END_REF][START_REF] Borrego | The CD94/NKG2 family of receptors: from molecules and cells to clinical relevance[END_REF]). To the second class belong the members of the NK receptor protein subfamily 1 (NKRP1). NKRP1 subfamily is the topic of next section.

NKRP1 subfamily

In human there are three well described receptor/ligand couples among the NKRP1 subfamily:

KLRB1/CLEC2D, KLRF1/CLEC2B and KLRF2/CLEC2A.

1.1.a. KLRB1/CLEC2D

KLRB1 encodes for the NKRP1A/CD161 receptor that is expressed on both NK cells and T cells [START_REF] Lanier | Human NKR-P1A. A disulfide-linked homodimer of the C-type lectin superfamily expressed by a subset of NK and T lymphocytes[END_REF]. Its ligand is the lectin-like transcript-1 (LLT1) encoded by CLEC2D [START_REF] Aldemir | Cutting edge: lectin-like transcript 1 is a ligand for the CD161 receptor[END_REF][START_REF] Rosen | Cutting edge: lectin-like transcript-1 is a ligand for the inhibitory human NKR-P1A receptor[END_REF]. LLT1 is expressed on activated hematopoietic cells including dendritic cells (DC), plasmacytoid DC, B cells, T cells and NK cells [START_REF] Rosen | Functional consequences of interactions between human NKR-P1A and its ligand LLT1 expressed on activated dendritic cells and B cells[END_REF][START_REF] Germain | Characterization of alternatively spliced transcript variants of CLEC2D gene[END_REF][START_REF] Germain | Induction of lectin-like transcript 1 (LLT1) protein cell surface expression by pathogens and interferon-gamma contributes to modulate immune responses[END_REF]). In addition, IFN-is a key signal amplifying LLT1 induction by TLR or viral infections on DC and B cells [START_REF] Germain | Induction of lectin-like transcript 1 (LLT1) protein cell surface expression by pathogens and interferon-gamma contributes to modulate immune responses[END_REF].

The interaction between CD161 and LLT1 inhibits NK cell-mediated cytotoxicity and cytokine production. LLT1/CD161 interaction is therefore important in the crosstalk of antigen presenting cells with NK cells by conferring them protection against NK-cell mediated killing, independently of MHC-I molecules [START_REF] Aldemir | Cutting edge: lectin-like transcript 1 is a ligand for the CD161 receptor[END_REF][START_REF] Rosen | Cutting edge: lectin-like transcript-1 is a ligand for the inhibitory human NKR-P1A receptor[END_REF][START_REF] Germain | Induction of lectin-like transcript 1 (LLT1) protein cell surface expression by pathogens and interferon-gamma contributes to modulate immune responses[END_REF]). In addition, CD161 is a costimulatory receptor on T cells and is mainly expressed by Th17, Th1-like and MAIT cells [START_REF] Aldemir | Cutting edge: lectin-like transcript 1 is a ligand for the CD161 receptor[END_REF][START_REF] Fergusson | CD161-expressing human T cells[END_REF][START_REF] Germain | Induction of lectin-like transcript 1 (LLT1) protein cell surface expression by pathogens and interferon-gamma contributes to modulate immune responses[END_REF].

1.1.b. KLRF1/CLEC2B

Nkp80 is an activating receptor expressed by NK cells and some subsets of T cells that is encoded by KLRF1. Its ligand is the activation-induced C-type lectin (AICL) encoded by CLEC2B and expressed on myeloid cell lines and on tumor cell lines of hematopoietic and non-hematopoietic origins [START_REF] Vitale | Identification of NKp80, a novel triggering molecule expressed by human NK cells[END_REF][START_REF] Welte | Mutual activation of natural killer cells and monocytes mediated by NKp80-AICL interaction[END_REF][START_REF] Kuttruff | NKp80 defines and stimulates a reactive subset of CD8 T cells[END_REF][START_REF] Akatsuka | Tumor cells of non-hematopoietic and hematopoietic origins express activation-induced C-type lectin, the ligand for killer cell lectin-like receptor F1[END_REF]. The engagement of Nkp80 receptor by myeloid cells activate both myeloid and NK cells into releasing pro-inflammatory cytokines. Moreover, when the AICL-Nkp80 interaction is promoted by malignant myeloid cells, it triggers direct NK cell mediated cytolysis [START_REF] Welte | Mutual activation of natural killer cells and monocytes mediated by NKp80-AICL interaction[END_REF]. In an unexpected way, NK cells contain intracellular stores of AICL that through specific stimulation of IL-12 and IL-18 can become extracellular, whereas at the same time expression of NKp80 is downregulated. This now AICL + NK cells can be recognized and killed by autologous NK cells. The intra circuit Nkp80/AICL could be important to control NK cell responses during inflammation [START_REF] Klimosch | Genetically coupled receptor-ligand pair NKp80-AICL enables autonomous control of human NK cell responses[END_REF].

1.1.c. KLRF2/CLEC2A

The couple KLRF2/CLEC2A was the last to be identified (Spreu, Kienle et al. 2007). KLRF2

encodes for the Nkp65 receptor that until now, was only described in NK cell lines. There are however suspicions about its presence also in innate lymphoid cells (ILC) subsets [START_REF] Steinle | C-type lectin-like NKC-encoded immunoreceptors NKp80 and NKp65 are selectively expressed by human innate lymphocyte subsets, uniquely signal via hemITAMs and facilitate tissue-specific immunosurveillance via their genetically linked ligands AICL and KACL[END_REF]. Its ligand is encoded by CLEC2A and was described in keratinocytes (KACL), U937 monocytic cell line and more recently, our laboratory identified its expression on skin healthy fibroblasts (unpublished) (Spreu, Kienle et al. 2007, Spreu, Kuttruff et al. 2010).

Differently from other members of the CLEC2 family, CLEC2A is almost exclusively expressed on skin (Spreu, Kienle et al. 2007). CLEC2A is composed by five exons and four introns with a total length of 33.7kb. There are four CLEC2A variants described: CLEC2A1, CLEC2A2 and its spliced alternative variants that lack the exon coding for the transmembrane domain (figure 19). Only CLEC2A1 was identified as expressed at the cell surface. Interestingly the alignment of amino acid sequences of human CLEC2A1 with putative CLEC2A1 amino acid sequences from chimpanzee, rhesus macaque and cow revealed homologous conserved regions.

Besides the already mentioned species, CLEC2A gene is also homologous to murine Clec2f.

This indicates that CLEC2A is part of a stable conserved mechanism of the mammalian immune system (Spreu, Kienle et al. 2007[START_REF] Zhang | Mouse Nkrp1-Clr gene cluster sequence and expression analyses reveal conservation of tissue-specific MHC-independent immunosurveillance[END_REF], National Center for Biotechnology Information 2019). (Spreu, Kienle et al. 2007) Figure 19. CLEC2A transcript variants.

CLEC2A is a glycoprotein of approximately 32 kDa prior to the removal of glycosylated units (Spreu, Kienle et al. 2007, Spreu, Kuttruff et al. 2010). The engagement of Nkp65 by CLEC2Atransfected cells enhances the cytotoxic response of NK cells that effectively kill target cells and secrete IFN-γ. The cytolysis of NK cells towards U937 cells is also at least partially dependent on Nkp65 engagement. Furthermore, in co-culture experiments, freshly isolated keratinocytes partially mediate the degranulation of NK cells via CLEC2A-NKp65 binding (Spreu, Kuttruff et al. 2010).

The functional interaction between Nkp65 and CLEC2A is independent of CLEC2A glycosylation status (Bauer, Spreu et al. 2015). Structurally, 20 residues from loops L0, L3, L5, and L6 and strands β3 and β4 of NKp65 contact with 16 residues, also from loops L0, L3, L5, and L6 and strands β3 and β4 of CLEC2A (figure 20) (Li, Wang et al. 2013). (Li, Wang et al. 2013) β-strands and loops are cyan for CLEC2A (upper) and purple for NKp65 (lower); α-helices are red for both structures; carbohydrate chains (GluNAc-GluNAc-Man) attached to CLEC2A are yellow. Loops and strands involved in the contact are labeled.

The diversity of NK cell subsets in human skin

In healthy skin, the most prevalent NK cell population is part of the CD56 + CD16 -subset which contrasts to the circulating NK cell population (cNK) that is mainly CD56 + CD16 + . A CD16 + phenotype correlates with high cytolytic activity against infected or transformed cells whereas

CLEC2A

NKp65 a CD16 -phenotype has been associated with cytokine production and modulation of dendritic cell and T cell priming [START_REF] Moretta | NK cells at the interface between innate and adaptive immunity[END_REF]. The CD16 -subset is characterized by the absence of perforin expression which is also a characteristic of skin-related NK cells (srNK). However, differently from cNK, in skin, NK cells lack the expression of CCR7, important chemoattractant receptor which will interfere with migration properties [START_REF] Ebert | Homing and function of human skin gammadelta T cells and NK cells: relevance for tumor surveillance[END_REF].

Another difference between cNK and srNK cells is the expression of NKG2D. Although this activating receptor is wildely expressed in cNK cells, it is absent in freshly isolated cutaneous NK cells from healthy patients. However, its ligands MICA, MICB and ULBP are present in skin cells and NKG2D can be induced in srNK upon in vitro culture, suggesting it is downregulated in the skin upon binding with its ligands [START_REF] Zwirner | Differential surface expression of MICA by endothelial cells, fibroblasts, keratinocytes, and monocytes[END_REF][START_REF] Chalupny | ULBP4 is a novel ligand for human NKG2D[END_REF][START_REF] Ebert | Homing and function of human skin gammadelta T cells and NK cells: relevance for tumor surveillance[END_REF][START_REF] Knight | Conditional analysis identifies three novel major histocompatibility complex loci associated with psoriasis[END_REF]. Both MICA and MICB are upregulated in skin inflammatory diseases which could indicate that skin cells are able to modulate their crosstalk with NK cells depending on the health status of the tissue [START_REF] Molinero | Up-regulated expression of MICA and proinflammatory cytokines in skin biopsies from patients with seborrhoeic dermatitis[END_REF][START_REF] Knight | Conditional analysis identifies three novel major histocompatibility complex loci associated with psoriasis[END_REF].

Most studies of NK cell functions in skin are related to their implication in autoimmune and inflammatory diseases like psoriasis and dermatitis. Psoriasis is described as a chronic inflammation of the skin. NK cells isolated from psoriatic skin show significant differences from srNK of healthy individuals. They express high levels of CXCR3 and CCR5, two chemokine receptors that contribute to the NK cell homing when engaged with their ligands CXCL10 and CCL5 overexpressed by psoriatic keratinocytes [START_REF] Ottaviani | CD56brightCD16(-) NK cells accumulate in psoriatic skin in response to CXCL10 and CCL5 and exacerbate skin inflammation[END_REF][START_REF] Nestle | Psoriasis[END_REF]. CD16 + population is increased in NK cells associated with psoriatic lesions compared to srNK from healthy individuals. Furthermore, these alterations correlate with higher expression of NKG2A receptor and IFN-γ production upon IL-2 stimulation [START_REF] Batista | Skewed distribution of natural killer cells in psoriasis skin lesions[END_REF]. IFN-γ is able to induce in keratinocytes the appearance of ICAM-1 and HLA-DR, the overexpression of MHC-I molecules and the secretion of CXCL10, CCL5 [START_REF] Ottaviani | CD56brightCD16(-) NK cells accumulate in psoriatic skin in response to CXCL10 and CCL5 and exacerbate skin inflammation[END_REF]. This finding show that a modulation of NK phenotype occurs in psoriasis and suggest a role of NK-keratinocytes crosstalk in the chronic inflammatory process in psoriasis.

A specific NK cell subset was also identified in allergic contact dermatitis (ACD). This CD16 - NK cell population lacks CCR7 and like the one associated with psoriasis, highly expresses CXCR3. In ACD, NK cells play a role in the apoptosis-induced keratinocytes through the secretion of cytokines secreted upon T cell stimulation. This interaction between innate and adaptive immune responses is at least partially responsible for the exacerbation of allergic responses in ACD [START_REF] Carbone | CD56highCD16-CD62L-NK cells accumulate in allergic contact dermatitis and contribute to the expression of allergic responses[END_REF].

Thesis objectives

The susceptibility of XP patients to the development of skin cancers, and the propensity for SCC development in XP-C group set the bases for the different research axes of the laboratory.

In skin cancer, together with UV radiations that induce mutations in the keratinocytes, the microenvironment created by skin fibroblasts and immune cells is an important factor that leads to promotion of mutated cell invasion. When I arrived in the laboratory, the identification of genetic alterations induced by UV radiation and the role of XP-C dermal fibroblasts in XP carcinogenesis was extensively analyzed. However, another aspect concerning the implication of possible alterations in the immunosuppressive microenvironment of XP patients in cancer progression was not explored.

The 2) Using the expertise of the laboratory in reconstructing skin models in vitro, I evaluated the outcome of the cross-talk between fibroblasts and NK cells in the presence and absence of functional CLEC2A/NKp65 interaction. To do so, I developed a 3D organotypic NK immunocompetent model to measure SCC invasion in the presence and absence of NK cells and blocking anti-CLEC2A antibody. As the interaction between fibroblasts and NK cells in skin is relatively unexplored, I could therefore evaluate the impact of CLEC2A expression on SCC invasion in the dermis.

CLEC2A regulates natural killer cell and fibroblast-mediated control of cutaneous squamous cell carcinoma invasion.

Xeroderma pigmentosum patients from the complementation group C are prone to develop aggressive skin cancers. Dermal XP-C fibroblasts show pro-invasive properties in 3D organotypic cultures, suggesting that they have an active role in cancer progression. To identify deregulations in gene expression of XP-C fibroblasts that could favor the dissemination of cancer cells, we performed a comparative transcriptomic analysis of healthy and XP-C dermal fibroblasts cultured in three dimensional (3D) collagen matrices.

Our results revealed that CLEC2A, a ligand of the activating NK cell receptor NKp65, is present in healthy fibroblasts and absent in XP-C fibroblasts. Until now, CLEC2A had only been identified in differentiated keratinocytes and in U-937 monocytic cell line. The crosstalk between dermal fibroblasts and NK cells is largely unexplored.

In this scientific paper, we dissected the role that the dermal expression of CLEC2A in cancer progression. We first showed that the expression of CLEC2A is also absent in CAFs and that SCC cells are able to downregulate CLEC2A expression in healthy fibroblasts through secreted factors. We then developed 3D organotypic NK immunocompetent models, to measure SCC invasion in the presence and absence of NK cells and blocking anti-CLEC2A

antibody. We measured the invasion rate of SCC cells in WT, XP-C and CAF collagen matrices. In presence of NK cells, we only saw a decrease in the invasion rate in the WT condition, with fibroblasts expressing CLEC2A. When this interaction was blocked by anti-CLEC2A or did not occur because of loss of CLEC2A expression by XP-C fibroblasts or CAFs, SCC invasion rate was increased. Thus, we showed that CLEC2A/NKp65 interaction regulates NK cell-mediated control of SCC invasion by fibroblasts. The results of this article unveil a role for CLEC2A in skin immunosurveillance.

Introduction

Skin cancer is the most frequent cancer worldwide and its prevalence has highly increased over the last thirty years (Bray, Ferlay et al. 2018). Patients suffering from xeroderma pigmentosum (XP), a rare genetic autosomal recessive disease caused by mutations on complementation genes involved in the nucleotide repair of DNA lesions after a UV stress, exhibit a very high propensity to develop cutaneous cancers (Kraemer, Lee et al. 1987). They develop 10,000 more basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) and 2000 more melanomas than the general population. In addition, XP-C patients, the most frequent complementation group in Europe and US, are highly susceptible to aggressive (SCC)

skin cancers; however, mechanisms underlying the typology of XP-C skin cancer are not yet understood (Giglia, Dumaz et al. 1998).

Cancer progression depends on both mutations in cancer cells that favor their growth and spread and the characteristics of the surrounding environment and its cellular components (Fidler 2003). Within the tumor microenvironment (TME), fibroblasts represent the majority of non-tumor cells. There, they convert into cancer-associated fibroblasts (CAFs) [START_REF] Kalluri | Fibroblasts in cancer[END_REF]. CAFs are irreversibly activated fibroblasts that change the architecture and composition of TME and are active stimulators of cancer growth and progression leading poor survival. Immune cells also infiltrate tumors and can have anti-and pro-tumoral functions.

Natural Killer (NK) cells have been shown to interact with cancer cells both by direct contact and through chemokine and cytokine signaling. Their presence in the TME correlates with good prognosis in many types of cancers including cutaneous ones (Li, Simmons et al. 1998, Imai, Matsuyama et al. 2000, Ishigami, Natsugoe et al. 2000).

Interestingly, XP patients show specific phenotypes for both fibroblasts and immune cells that resemble those of CAFs and immunocompromised immune cells respectively (Norris, Limb et al. 1988, Mariani, Facchini et al. 1992[START_REF] Bernerd | Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro[END_REF]. XP-C fibroblasts, isolated from healthy non-photo exposed areas promote keratinocyte invasion in organotypic cultures. They express other similarities with CAFs, like the overexpression of the hepatocyte growth factor (HGF) (in preparation) and Matrix Metalloproteinase 1 (MMP1) implicated in ECM remodeling [START_REF] Bernerd | Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro[END_REF], Frechet, Warrick et al. 2008, Cui, Wang et al. 2018). Immune abnormalities have also been identified in XP patients. Several studies from the late 80s / early 90s reported that NK cells from XP patients present a deficiency in their lytic activity, suggesting that NK cell dysfunction may contribute to cancer progression in these patients (Norris, Limb et al. 1988, Norris, Limb et al. 1990, Mariani, Facchini et al. 1992).

These observations suggest that XP disease can be a good model to study development and progression of skin cancers. To address this question, we investigated the role of XP-C fibroblasts, first by performing a transcriptomic analysis of three dimensional (3D)-dermal cultures of fibroblasts from healthy (WT) and XP-C patients. Surprisingly, among the few differentially expressed genes, we identified the presence of CLEC2A, the ligand of the activating NK receptor NKp65, in healthy fibroblasts and its absence in XP-C fibroblasts. The modulation of CLEC2A expression in fibroblasts unveil an important crosstalk between fibroblasts and NK cells in healthy and cancerous tissues.

Results

CLEC2A differential gene expression in WT and XP-C fibroblasts from 3D-dermal

cultures. In an attempt to identify dermal components regulating the growth of skin cancers in XP-C patients, we first examined the genome wide expression profile of six primary independent fibroblast cell lines (3 WT, 3 XP-C) cultured in 3D-collagen matrices. Principal component analysis (PCA) showed that XP-C fibroblasts displayed a distinct transcriptomic profile and higher heterogeneity compared to WT fibroblasts (Fig. 1A). Twelve genes were found upregulated and ten downregulated in WT fibroblasts compared to XP-C fibroblasts (Fig. 1B and Table S1). Among these genes, we identified the C-type lectin domain family 2 member A (CLEC2A) as one of the genes that were the most significantly differentially expressed (Table S1). Two CLEC2A transcripts variants were found expressed in WT but not in XP-C fibroblasts (Fig. 1C) (Spreu, Kienle et al. 2007). CLEC2A variant 1 encodes for a cell surface molecule that interacts with the activating receptor, NKp65, expressed by NK cells (Spreu, Kuttruff et al. 2010). This finding suggested a putative cross-talk between fibroblasts and NK cells in the skin of healthy individuals which may dysfunction in XP-C patients.

Selective expression of CLEC2A at the cell surface of cutaneous primary fibroblasts from healthy individuals. To further document CLEC2A mRNA and protein expression in the skin, we analyzed a panel of WT and XP-C fibroblasts and keratinocytes that we isolated from healthy and XP-C skin biopsies (Otto, Riou et al. 1999). Using a set of primers that detects both CLEC2A1 together with CLEC2A2, we confirmed that CLEC2A transcripts only accumulated in WT fibroblasts and not in XP-C fibroblasts (Fig. 2A). In addition, when using primers specific for each variant, CLEC2A1 and CLEC2A2 mRNA were detected in WT fibroblasts and CLEC2A-expressing cell line U937 but not in XP-C fibroblasts (Fig. S1A) (Spreu, Kienle et al. 2007). Surprisingly, we could not detect CLEC2A transcripts in basal keratinocytes from both WT and XP-C patients (Fig. 2A). Likewise, when we monitored CLEC2A protein expression at the cell surface by flow cytometry, we found CLEC2A solely expressed on WT fibroblasts (Fig. 2 B andC). Using imaging flow cytometry, we found that CLEC2A was predominantly expressed at the cell surface (Fig. S1B). This suggested the accumulation of CLEC2A1 membrane related variant is predominant over the intracellular related variant CLEC2A2. Because XP-C patients bear deleterious mutation in the XPC gene, we considered the possibility of a direct implication of XPC protein in the regulation of CLEC2A expression. To test this hypothesis, we efficiently restored XPC expression in XP-C fibroblasts by retroviral transduction of a cassette containing XPC (Fig. S2A). We confirmed that transduced XP-C fibroblasts were able to perform DNA repair after UV radiation, using the measurement of unscheduled DNA synthesis (UDS) by incorporation of 5-ethynyl-2′deoxyuridine (EdU) labeled nucleotides into the newly DNA patches that are synthetized upon DNA repair (Fig. S2B). Under these circumstances, however, expression of CLEC2A was not restored in XP-C reverted fibroblasts (Fig. S2C). Altogether, these data highlight a default of CLEC2A expression in XP-C patients that is not directly reverted after XPC re-expression.

Downregulation of CLEC2A expression within SCC tumor microenvironment.

Because phenotypical similarities are observed between XP-C fibroblasts and CAFs, we investigated whether CLEC2A could be modulated in SCC microenvironment. We successfully isolated two different dermal CAF cell lines from human skin SCC that positively expressed α-SMA, consistent with a CAF phenotype (Fig. 3A). Contrary to control (WT) fibroblasts, CLEC2A transcripts and protein could not be detected in these two dermal CAFs (Fig. 3B andC). These data suggested that the expression of CLEC2A in skin fibroblasts could be downregulated during tumor development upon transformation into CAFs. To test this hypothesis, we generated conditioned-medium from an SSC12 tumor cell line that is able to convert WT fibroblasts into CAFs in vitro, as shown by the moderate induction of -SMA on SCC12conditioned medium (SSC12_CM)-treated WT fibroblast (Fig. S3A). We then studied the impact of factors secreted by SCC12 tumor cell line on CLEC2A expression. SCC12_CM was added to the culture medium of WT fibroblasts. After 5 days of incubation with SCC12_CM, the expression of CLEC2A in WT fibroblasts was found downregulated both at mRNA and protein level (Fig. 4 A and B). By contrast, no significant modulation in CLEC2A expression was seen after incubation of WT fibroblasts with conditioned-media from WT keratinocytes and fibroblasts (Fig. S3B andC). Furthermore, as TGF-β is one major driver of the transition of WT fibroblasts into CAFs, we checked the possible role of TGF-β in the modulation of CLEC2A.

Supplementation of the culture medium with TGF- did not decrease CLEC2A mRNA transcripts while it induced α-SMA expression (Fig. S4A andB). Moreover, neutralizing TGF- in SCC12_CM by addition of an anti-TGF- did not block CLEC2A downregulation triggered by SCC12_CM (Fig. S4A). We conclude that TGF-β is not the secreted factor responsible for the downregulation of CLEC2A expression in WT fibroblasts.

CLEC2A regulates SCC invasion through a cross-talk between fibroblasts and NK cells.

To determine whether the loss of CLEC2A has a real impact on cancer progression, we developed a 3D-immunocompetent organotypic culture model to measure SCC12 invasion in the presence or absence of NK cells. We generated a NK cell line that killed SCC12 and not the WT and XP-C fibroblasts that we used in the 3D-dermal cultures (Fig. S5A andB).

Interestingly, SCC12 cells, like WT and XP-C fibroblasts, express MHC-I molecules. Killing of SCC12 may therefore results from abundant expression of ligands of activating NK receptors, however, they do not express CLEC2A (Fig. S6B). Thanks to our newly developed software Epidepth, we were able to analyze SCC12 invasion rates in different conditions (Fig 5C). Our results showed that the presence of NK cells significantly reduced the levels of SCC12 invasion in a WT 3D-dermal culture but failed to limit SCC12 invasion rates in a XP-C 3D-dermal culture (Fig. 5 A andC). Furthermore, when we added the anti-CLEC2A blocking antibody OMA1 to the WT 3D-dermal immunocompetent culture, the levels of SCC12 invasion returned to those of the condition without NK cells (Fig 5 A andC). This was not the case when the isotype Ig was added (Fig 5 A andC). This revealed a direct and positive impact of CLEC2A expression on WT fibroblasts in controlling SCC12 invasion via NK cells. Additionally, the same experiment was performed with CAF 3D-dermal culture and results showed that, like what happened with XP-C dermal equivalents, the presence of NK cells had no impact in SCC12 invasion rates. These results indicate that the cross-talk between fibroblasts and NK via CLEC2A interaction with NKp65 plays a role in the regulation of SCC invasion and progression.

Discussion

Our findings uncover a key role for CLEC2A in immunosurveillance of the skin through a crosstalk between fibroblasts and NK cells. We show that CLEC2A/NKp65 interaction regulates NK cell-mediated control of SCC cancer invasion by WT fibroblasts. This regulation is disrupted in XP-C fibroblasts and sporadic CAFs. We propose loss of CLEC2A by CAFs as a novel tumor evasion mechanism.

The data described here show for the first time that CLEC2A is expressed in skin primary dermal fibroblasts of healthy individuals but is absent in skin primary dermal fibroblasts of cancer-prone XP-C patients. Previous RNA-seq analysis comparing skin WT and XP-C fibroblasts isolated by enzymatic digestion and cultured in classic two-dimension cultures didn't reveal a difference in CLEC2A expression [START_REF] Chacon-Solano | Fibroblast activation and abnormal extracellular matrix remodelling as common hallmarks in three cancer-prone genodermatoses[END_REF]. This suggests that spatial structure of fibroblasts interfere with the expression of genes as pointed by other studies (Duval, Grover et al. 2017, Tolle, Gaggioli et al. 2018).

We also did not find expression of CLEC2A in basal keratinocytes from both WT and XPC patients. Others, previously reported expression of CLEC2A in primary keratinocytes isolated from skin (Spreu, Kuttruff et al. 2010). Whether CLEC2A is specifically expressed at some stages of keratinocytes' differentiation deserves further studies.

Understanding the mechanisms through which CLEC2A is modulated in WT dermis is a key issue to decipher the consequences of its absence in XP-C dermis. Mutations in XPC gene are mostly deleterious (Stary and Sarasin 2002) and, beyond NER, XPC has been suggested

to have a role in the regulation of transcription in absence of genetic attack (Le May, Mota-Fernandes et al. 2010). We checked whether the lack of XPC could directly lead to loss of CLEC2A. Our results demonstrate that the absence of CLEC2A expression in XP-C fibroblasts is not directly (nor immediately) dependent on the expression of XPC. The evidences that XP-C fibroblasts behave like CAFs, enhancing epidermal invasion, even in absence of cancerous cells [START_REF] Bernerd | Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro[END_REF] prompted us to look into the expression of CLEC2A in human dermal skin CAFs. The absence of CLEC2A expression in dermal CAFs, similarly to XP-C fibroblasts strengthens relationships between the two and suggests that loss of CLEC2A facilitates somehow, the evasion of cancer cells. In agreement with this hypothesis, we were able to demonstrate that SCC cells can downregulate CLEC2A expression through still unidentified secreted factors. This data strongly supports that CLEC2A downregulation is part of the transitioning process that WT fibroblasts undergo to become CAFs.

Since the identification of CLEC2A expression in skin (Spreu, Kienle et al. 2007), studies have shown that CLEC2A is able to trigger NK cell cytotoxicity in vitro through the engagement of NKp65 receptor (Spreu, Kuttruff et al. 2010, Li, Wang et al. 2013, Bauer, Spreu et al. 2015).

However, no study until now explored the role of CLEC2A in a more general context of immunosurveillance in the skin. Our results suggest that CLEC2A could facilitate the elimination of cancer cells by NK cells at early tumorigenesis stages, when fibroblasts are still expressing CLEC2A and have not shifted their phenotype to a CAF phenotype.

Materials and Methods

Tissues and cells. Normal human skins were collected from mammary or abdominal plastic resection surgery. XP skin biopsies were collected from non-sun-exposed and normally pigmented sites. Skin SCC biopsies were collected from patients undergoing surgical excision.

All samples were obtained with the patient's or parents' informed consent according to the Declaration of Helsinki under local French ethic committee (CCPPRB: CSET935) or under institutional review board (Hôpital Pasteur, Centre Hospitalier Universitaire de Nice, France) approval. Human keratinocytes and fibroblasts from healthy, XP-C or cancer patients were obtained and cultured as described [START_REF] Rheinwald | Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells[END_REF]Green 1975, Otto, Riou et al. 1999). NK cell lines were generated from human blood purchased from the Etablissement Français du Sang (Marseille, France), as previously described (Fassy, Immunology, 2017).

Cells are described in detail in SI Table S2.

3D-skin cultures. For transcriptomic analysis, fibroblasts (1x10 6 ) were cultured in a tridimensional type I collagen matrix at a concentration of 2.5x10 5 cells/mL for 4 days. After that, the matrix was cultured 7 days in immersion and then 7 days at the air-liquid interface as described in [START_REF] Bernerd | Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro[END_REF]. For 3D immunocompetent models, fibroblasts

(5 × 10 5 ) were cultured for 24 h in a tridimensional type I collagen matrix at a concentration of 2.5x10 5 cells/mL Then SCC12 cells (2,5 × 10 5 ) were dissociated and then seeded on the top of the matrix and the organotypic construct were cultured 48h in immersion and then 5 days at air-liquid interface. To incorporate NK cells into the model, we cultured NK cells (5 x 10 5 ) in a type I collagen matrix at 5x10 5 cell/mL for 24h in a transwell insert (Corning) and then added the fibroblasts-SCC12 construct on the top at air-liquid interface allowing NK cells to migrate up to "dermal" compartment for 48 hours [START_REF] Bernerd | Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro[END_REF][START_REF] Valin | PTCH1 +/-dermal fibroblasts isolated from healthy skin of Gorlin syndrome patients exhibit features of carcinoma associated fibroblasts[END_REF].

RNA extraction. 3D-dermal cultures were frozen in liquid nitrogen, and then disrupted and solubilized in Qiazol reagent (Qiagen) using a gentleMACS TM Dissociator (Miltenyi Biotech)

with M tubes and the RNA_02 program recommended by the manufacturer (Miltenyi Biotech).

Total RNAs were extracted using miRNeasy Mini kit (Qiagen) (3D-dermal cultures) or mRNeasy Mini kit (Qiagen) (cells) and then purified using the RNA cleanup and concentration kit (Qiagen).

Whole genome microarray analysis. The integrity of the RNA was assessed using an Agilent BioAnalyzer 2100 (Agilent Technologies). Amplification and labeling was performed with the LowInput QuickAmp Labeling Kit (Agilent Technologies). cRNA were then hybridized on 8×60K high density SurePrint G3 human gene expression Agilent microarrays according to manufacturer's instructions (Agilent Technologies). The microarray experimental data were deposited in the NCBI GEO under the serial record number (to do). The data were quantile normalized using the Bioconductor package limma. Means of ratios from all comparisons were calculated and the moderated t-statistic of the limma package provided the per gene P values.

Quantitative real-time PCR. mRNA levels were determined by RT-PCR after DNase I digestion and reverse transcription using SuperScript II (Thermo Fisher Scientific). For quantitative RT-PCR, cDNA was amplified with SYBR Green PCR Master Mix (Applied Biosystems) using a StepOne RT-PCR thermal cycler (Applied Biosystems) following the manufacturer's instructions and data analysis was performed by ΔCT method for relative quantification. SB34 and 36B4 were both used for gene-expression data normalization. Each experimental condition was performed in triplicate. The following primers were used: CLEC2A (5′-ataccagaaattggacagcc-3′/5′-gaatgtggtgccatttgtcc-3′), 36B4 (5'-tgcatcagtaccccattcta-3'/5'aggcagatggatcagccaaga-3'), SB34 (5'-gcatcagtaccccattctatcat-3'/5'-aggtgtaatccgtctccacaga-

3').

Tissue histology. 3D organotypic tissues were fixed in Antigen-Fix reagent (Diapath) for 1 h at 4°C and incubated in 34% sucrose for 16 h at 4°C prior being embedded in OCT (Tissue-Tek) and immediately frozen in liquid nitrogen. Hematoxylin and eosin staining were performed by standard protocol in 5 µm thickness OCT sections.

Immunofluorescence staining. Cells were fixed in 3% paraformaldehyde for 20 min at RT and permeabilized with 0.5% Triton X-100 for 5 min. Following a blocking step, cells were incubated with purified Mo anti-α-SMA (1/400) (Sigma Aldrich) for 1 h at RT. After washing, antibody binding was detected with A488-conjugated anti-Mo IgG secondary antibody (1/400) (Thermo Fisher Scientific). The coverslips were mounted onto glass slides using mounting media containing DAPI (Southern Biotech) and images were acquired using a Zeiss Axio Observer.Z1 fluorescent microscope.

Flow Cytometry. Cells were first incubated in a blocking solution to saturate Fc receptors and then incubated with 10µg/mL purified Mo anti-CLEC2A (OMA1) kindly provided Prof. Dr.

Alexander Steinle (Frankfurt, Germany) or Mo IgG2a control isotype (BD Biosciences) for 30 min at 4°C. After washing, antibody binding was detected using A488-conjugated goat anti-Mo mAb (1/800) (BD Biosciences). Fluorescence staining was analyzed on a FACScalibur (BD Biosciences) unit using CellQuest Pro Software (BD Biosciences).

Conditioned media preparation. Cells were grown to 70% confluence, washed twice with PBS and then incubated in serum-free medium at 37°C. After 48 h, culture medium was collected, centrifuged at 5000g for 5 min to remove cell debris and the supernatant stored at -80°C. These conditioned media or control serum free medium were used to stimulate fibroblasts for five days. In some experiments, blocking antibodies were added in the last 48 h.

Epidepth software. We analyzed the images with Epidepth2.1, a program dedicated to skin sample analysis. Written in Matlab (The MathWorks, Inc.) with a user-friendly interface, it automatically segments each image between the dermis, the epidermis and the invasions (in contact or not with the epidermis). Then the program automatically extracts statistical parameters to quantify the image (see Sup Data for details) (to do). More specifically, we propose an "Invasion Rate" as:

𝐼𝑅 = 1 𝐿 𝑒𝑝𝑖 ∑ 𝑑𝑖𝑠𝑡 𝑒𝑝𝑖 (𝑗) 𝑗∈𝑖𝑛𝑣
where 𝐿 𝑒𝑝𝑖 is the epiderm length, 𝑑𝑖𝑠𝑡 𝑒𝑝𝑖 (𝑗) is the distance from the epiderm for all pixel "𝑗" in an invasion. Measured in µm², this index has the advantage to take into account both the invasion sizes and how deep they migrate in the dermis. The program also manages the image library, providing statistics per experiment and per conditions. For this project we analyzed 343 images and 4390 invasions (~1s per image). On request, this program is freely available (as an executable).

Statistical Analysis. Statistical analyses were performed using GraphPad Prism 8.0 software.

Each statistical test is specified in the text. A P value <0.05 was considered as evidence for statistical significance. TGF-β1 and α-TGF-β1,2,3 stimulations. Fibroblasts were stimulated with 2ng/ml TGFβ1 (Peprotech) for 48 h in serum-free medium or with 10µg/mL α-TGF-β1,2,3 neutralizing antibody (R&D) for 48 h in SCC12_CM.

Real-time NK cell cytotoxic assay. A real-time cytotoxic assay was performed as previously described (Fassy, Tsalkitzi et al. 2017, Fassy, Tsalkitzi et al. 2017). Briefly, target cells were labelled with 0.5 µM Calcein-AM (Molecular Probes) for 15 min at room temperature.

Fibroblasts FH84_WT and AS148_XP-C were additionally treated with 100 µM Indomethacin (Sigma Aldrich) to block multidrug-resistance transporters that expulse calcein. The inhibitor was maintained in the medium during the assay. Calcein-labelled targets were incubated with NK cell lines for 4 h at 37°C, 5% CO2 and real-time monitoring of NK cell killing was performed on a Cytation™ 5 (Biotek). Cell images were processed using GEN5 software (Biotek). The percentage of lysis from triplicates was calculated as follow: % lysis = {1-[(experimental well at t/experimental well at t0)/(control well at t/control well at t0)]} x 100.

Materials and Methods

Otherwise indicated, for cell culture conditions, cell descriptions, characteristics, Flow

A. Modulation of CLEC2A expression in aging and infection

As infection and aging are two "conditions" associated with changes in the immune system we decided to evaluate their impact in the expression of CLEC2A in healthy WT fibroblasts.

We started with infection. Because skin is an organ that is particularly exposed to microorganisms, we stimulated WT fibroblasts with lipopolysaccharide (LPS), a component present in the cell walls of Gram-negative bacteria. CLEC2A mRNA transcripts levels were transiently upregulated followed the LPS stimulation. However, by immunofluorescent analysis, we only detected intracellular levels of CLEC2A that transiently accumulate in small spots and disappeared. To determine the nature of these spots we performed electron microscopy analysis in WT fibroblasts with or without LPS stimulation and identified these spots as lysosomes. In parallel, we also performed the same electron microscopy experiment with XP-C fibroblasts. Our results reveal that XP-C fibroblasts do not accumulate lysosome after a LPS stimulation and, the presence of lipid droplets that we saw in their cytoplasm unveil a possible deregulation in XP-C lipid metabolism.

To investigate a possible modulation of CLEC2A during aging we looked into the expression of CLEC2A in three primary cell lines of WT fibroblasts during replicative senescence. Our results show a progressive decrease in the surface levels of CLEC2A during the consecutive propagation of fibroblasts. We stopped our experiment when saw decline in cellular replication of our cell lines and we confirmed the senescent state of the fibroblasts that expressed low levels of surface CLEC2A by analyzing the expression of senescent markers.

B. Expression of CLEC2A in other cancer-prone skin related diseases

Finally, because our previous experiments showed that XPC is not directly implicated in the absence of CLEC2A in XP-C fibroblasts we looked into the levels of CLEC2A expression first in fibroblasts from a different XP complementation group -XP-D, and after in fibroblasts from a different cancer-prone skin related disease -NBSCC.

Our results show that CLEC2A is also absent in fibroblasts from XP-C patients and CLEC2A mRNA transcripts levels are dramatically decreased in fibroblasts from NBSCC patients.

These results suggest that the absence of CLEC2A could be a characteristic of cancer-prone skin.

A.I. CLEC2A modulation during infection

The skin being the first barrier to microorganisms, we evaluated whether CLEC2A expression could be modulated in the context of infection. We did not perform an extensive analysis but restrained our study to a stimulation of fibroblasts expressing CLEC2A with lipopolysaccharide (LPS). LPS is a component present in the cell walls of Gram-negative bacteria that binds to the Toll-like receptor 4 (TLR4). The activation of TLR4 triggers the production of proinflammatory cytokines and chemokines, upregulation of costimulatory molecules and activation of antigen presentation by DC [START_REF] Chow | Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction[END_REF][START_REF] Akira | Toll-like receptor signalling[END_REF][START_REF] Wang | Toll-like receptors expressed by dermal fibroblasts contribute to hypertrophic scarring[END_REF].

Expression of CLEC2A mRNA was transiently upregulated in presence of LPS in WT dermal fibroblasts (figure 21A). We found that CLEC2A expression is enhanced 2/3 hours after LPS incubation and declines back to a baseline level 6 hours after treatment. However, the increase in CLEC2A mRNA levels didn't correlate with the increase in the levels of membrane associated CLEC2A that stayed unchanged with or without LPS stimulation (figure 21B).

We then looked into cytoplasmic levels of CLEC2A by immunofluorescence analysis and observed that in presence of LPS, WT fibroblasts overexpressed intracellular CLEC2A that transiently accumulates with time, thus correlating with mRNA data (figure 22). Moreover, the staining shows an accumulation of the signal in small spots that disappear between 8 and 24 hours after LPS stimulation. To determine the nature of these spots, we performed electron microscopy analysis in WT and XP-C fibroblasts after 6 hours of LPS incubation (figure 23 A and B). In WT fibroblasts, we identified a 5-fold increase in lysosome organelles in presence of LPS that could correlate with the spots that we saw in immunofluorescence assays (figure 23B). However, XP-C fibroblasts showed no change in lysosome numbers and we saw instead lipid droplets in XP-C fibroblasts cytoplasm that were present even in the absence of LPS stimulation (figure 23 A and B). Together, these data suggest that upon infection and stimulation with LPS, CLEC2A expression is stimulated but the protein remains intracellular and is degraded. This constitutes To assess the senescent state of the fibroblasts that expressed low levels of surface CLEC2A at late cumulative PD, expression of p21 and p16, two senescence markers, was analyzed in WT-fibroblasts at early and late cumulative PDs. Our results show that at late PD, fibroblasts overexpress p16 and p21 which is consistent with a typical phenotype of senescent cells (figure 25). Furthermore, we performed a senescence associated (SA) β-galactosidase staining that reveals the presence of high SA-β-gal activity in late PD fibroblasts (figure 26). These results confirmed the senescent state of WT fibroblasts at late PD.

In conclusion, our results show a decrease in CLEC2A expression during replicative senescence. This is in correlation with the loss of CLEC2A expression in CAFs (detailed in Scientific article, page 90).

B. CLEC2A expression in other skin related genetic diseases

We successfully transduced XP-C fibroblasts with a retroviral vector expressing the WT XPC and showed that the reintroduction of XPC in XP-C fibroblasts do not allow the novo expression of CLEC2A (detailed in Scientific article, page 90). This suggests that lack of CLEC2A in XP-C fibroblasts is not a direct consequence of the absence of XPC and prompted us to hypothesize that the absence of CLEC2A is not specific to XP-C fibroblasts. To prove our hypothesis, we first analyzed the expression of CLEC2A in fibroblasts from XP patients of another complementation group -XP-D. Using quantitative RT-PCR and flow cytometry analysis, we found that fibroblasts from XP-D patients present, like XP-C fibroblasts, a total absence of CLEC2A expression, both at the transcript and protein levels (figure 27 A and B).

We then analyzed the expression of CLEC2A by quantitative RT-PCR in two primary cell lines of fibroblasts from patients suffering from Nevoid Basal Cell Carcinoma Syndrome (NBCCS), an autosomal dominant disorder caused mainly by mutations in PTCH1 gene. This pathology is unrelated with changes in DNA repair mechanisms but is characterized by the development of multiple BCCs. Figure 28 shows a drastic and statistically significant decrease in the expression of CLEC2A mRNA in NBCCS fibroblasts compared to WT fibroblasts. Although they need clearly more investigations, these results are interesting because we show that CLEC2A expression is deregulated in the skin fibroblasts of different cutaneous cancer prone pathologies (i.e. XP and NBCCS). They suggest that the deregulation of CLEC2A may be a common feature in cancer-prone skin related diseases.

Discussion and Perspectives

Challenges in rare genetic diseases research "Rare diseases are diseases which affect a small number of people compared to the general population. In Europe, a disease is considered to be rare when it affects 1 person per 2000"from (Orphanet). Doing research in rare genetic diseases is a challenge for various reasons. First, "rare", means that there are small numbers of patients suffering from the disease and this can introduce serious difficulties to produce statistically robust results, at least as expected from conventional, large cohort studies. Xeroderma pigmentosum (XP) affects less than 3 people per million births in Europe [START_REF] Kleijer | Incidence of DNA repair deficiency disorders in western Europe: Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy[END_REF]. Because, along the past 20 years, our laboratory has accumulated a large collection of skin primary cells from XP patients, I wasn't directly confronted with the difficulty of not having enough samples from different patients.

Nevertheless, I have to acknowledge that the need for a consistent collection of cells limits the development of new research projects from laboratories that didn't have this resource. Second, research on rare diseases, particularly on curative therapies, tends to attract less investment compared to more common diseases due to the limited financial benefits for pharmaceutical companies and priorities of social security services. In spite of these pragmatic and disappointing considerations, genetic diseases can also be used as models to study physiological and mechanistic processes in human body. In fact, there are many examples of scientific breakthrough that were made thanks to the study of genetic diseases. A famous one is the discovery of Goldstein and Brown showing that mutations in the cell surface receptor of low density lipoprotein (LDL) lead to the familiar hypercholesterolemia disease [START_REF] Goldstein | Familial hypercholesterolemia: identification of a defect in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity associated with overproduction of cholesterol[END_REF]. LDL receptors are now indirect targets of the most used drugs (the statins) in the control of high levels of cholesterol and in the prevention of atherosclerosis [START_REF] Sirtori | The pharmacology of statins[END_REF]).

In the laboratory, research projects focus in (i) the development of safe curative approaches for xeroderma pigmentosum patients [START_REF] Warrick | Preclinical corrective gene transfer in xeroderma pigmentosum human skin stem cells[END_REF]) (ii) the use of XP-C keratinocytes to study cancer development under UV solar radiations; (iii) the use of XP-C fibroblasts as scaffold for the progression of cutaneous cancers. My thesis project derives from this third axe and is particularly focused on the interactions between skin fibroblasts and the immune system.

Fibroblasts cultures -2D vs 3D

Transcriptomic analysis of the mRNA expression profiles of WT and XP-C fibroblasts in conventional 2D cultures performed by the laboratory showed that almost three hundred genes were deregulated in XP-C fibroblasts compared to WT fibroblasts (unpublished results, please refer to page 182 for data). These data allowed the team to detail the role of HGF, surexpressed by XP-C fibroblasts, in cutaneous SCC invasion (Alqaraghuli et al., in preparation). The high amount of deregulated genes observed in 2D cell culture are implicated in numerous different physiologic pathways. We then were unable to clearly identify other important alterations that may explain the propensity of XP-C dermis to cancer cell invasion.

Aware that culture conditions of primary cells can change the expression of genes, we decided to perform transcriptomic analysis of WT and XP-C fibroblasts cultured in dermal equivalents (3D). This was for us a more relevant condition to study dermal fibroblasts in their dermal context, as suggested by several groups [START_REF] Ghosh | Three-dimensional culture of melanoma cells profoundly affects gene expression profile: a high density oligonucleotide array study[END_REF][START_REF] Griffith | Capturing complex 3D tissue physiology in vitro[END_REF], Duval, Grover et al. 2017). Two things immediately drove our attention after comparison of our transcriptomic analysis under 2D or 3D culture conditions: (i) only 22 genes came out as differentially expressed in 3D cultured cells compared to almost 300 in 2D cultures and (ii) only five genes, CLEC2A, PITX1, PRDM16, TBX5 and HOXC10 presented significant deregulation both in 2D and 3D approaches.

The paper of Tolle et al., 2018 compared protein expression in normal fibroblasts versus CAFs both in 2D and 3D cultures (Tolle, Gaggioli et al. 2018). This paper shows that in 2D culture, proteins involved in adherent junctions, cell-cell adhesion and wound healing are found differentially expressed between the two populations. In 3D cultures, a different protein expression profile emerges with an enrichment for differential expression in proteins involved in vacuolar and lysosomal transport, regulation of response to stress, multicellular organismal process and cellular development and differentiation. They furthermore compare the two sets of data with analysis of breast cancer biopsies and conclude that the 3D approach better mimic what happens in vivo. These recent data support the idea that 3D cultures better mimic fibroblasts behaviors in vivo in general.

What we found at mRNA level in our comparative transcriptomic analysis of WT and XP-C fibroblasts in 2D and 3D cultures, however, goes beyond the notion that culture conditions can change protein expression. Indeed, our results not only validate the notion that the expression of proteins/genes changes between 2D and 3D culture conditions, but they also unveil the dramatic decrease in the number of differentially expressed genes between two populations of dermal fibroblasts when cultured in 3D dermal equivalents. Our results indicate that at steady-state, fibroblasts from WT and XP-C patients cultured in 3D did not change drastically the expression of their genes as a 2D culture could prompt us to assume. We can hypothesize that the genes that show differential expression profiles both in 2D and 3D cultures could be major actors in the phenotypic differences observed between WT and XP-C fibroblasts. Based on these results, we recommend that further transcription analysis comparing fibroblasts from two different populations should be performed using 3D systems. 3D cultures could lead to a more precise and pertinent identification of potentially relevant genes.

Fibroblasts and NK cells

The importance of fibroblasts on skin homeostasis and cancer progression has been well described over the years and is summarized in the introductory chapters of this thesis.

However, the way they interact with immune cells and their potential role as an immune system component are much less explored.

A. CLEC2A role in cancer invasion

The discovery of CLEC2A, a NK ligand, as a differentially expressed gene in WT and XP-C fibroblasts following the 2D and 3D transcriptomic analysis immediately caught our attention.

It reminded us of work from the late 80s / early 90s that described modifications of the NK cell phenotype in XP patients and it suggests an implication of a deregulated immune system in the cancer invasion patterns associated with XP disease (Norris, Limb et al. 1988, Norris, Limb et al. 1990, Mariani, Facchini et al. 1992). Until our discovery, CLEC2A was only described as expressed in differentiated skin keratinocytes and its physiological role remains unclear (Spreu, Kuttruff et al. 2010). Our study shows for the first time that CLEC2A is also expressed in skin WT fibroblasts. We did not find expression of CLEC2A on basal WT keratinocytes suggesting that not all the different differentiation stages of keratinocytes are associated with CLEC2A expression. The total absence of CLEC2A in XP fibroblasts and CAFs and the downregulation of CLEC2A expression in WT fibroblasts by factor secreted by SCC cells prompted us to look into a possible protective role of CLEC2A against cancer invasion. Our results clearly show that the expression of CLEC2A in WT fibroblasts negatively affects the invasion rate of SCC cells in a 3D NK immunocompetent model. These results raise several questions. How fibroblasts interact with NK cells? Are fibroblasts able to activate NK cells via direct contact mechanisms? Do fibroblasts have a role in the immunosurveillance of the skin?

In the search for possible mechanisms that could answer some of these questions, we searched the literature for descriptions ok NK cell regulation when NK cells are activated by cells that are not NK's direct targets. Indeed, NK cells can be activated by either target cells or accessory cells like DC. After stimulation by stress signals, DC undergo maturation and overexpress cytokines and NK ligands responsible for NK cell activation. However, they are not targets of NK cells, probably because they express high levels of MHC-I molecules [START_REF] Monteiro | Role of interleukin-12 in primary influenza virus infection[END_REF][START_REF] Fernandez | Dendritic cells directly trigger NK cell functions: crosstalk relevant in innate anti-tumor immune responses in vivo[END_REF][START_REF] Pollara | Herpes simplex virus type-1-induced activation of myeloid dendritic cells: the roles of virus cell interaction and paracrine type I IFN secretion[END_REF][START_REF] Andrews | Cross-talk between dendritic cells and natural killer cells in viral infection[END_REF][START_REF] Barreira Da Silva | Natural killer cell activation by dendritic cells: balancing inhibitory and activating signals[END_REF]. Although it has been shown that DC activate NK cells mostly via cytokine expression, the direct contact alone is able to control NK cytotoxicity and in some cases mediate NK cell survival [START_REF] Poggi | NK cell activation by dendritic cells is dependent on LFA-1-mediated induction of calcium-calmodulin kinase II: inhibition by HIV-1 Tat C-terminal domain[END_REF][START_REF] Jinushi | Critical role of MHC class I-related chain A and B expression on IFN-alpha-stimulated dendritic cells in NK cell activation: impairment in chronic hepatitis C virus infection[END_REF][START_REF] Brilot | NK cell survival mediated through the regulatory synapse with human DCs requires IL-15Ralpha[END_REF]). In our experimental system, we show that NK cells are unable to kill WT and XP-C fibroblasts but contrary to what happens with DC, the interaction between NK and fibroblasts didn't reveal an increase in their expression of IFN-γ (data not shown). These data suggest that fibroblasts alone are not able to activate, nor change NK cell phenotype like DC. However, our cytotoxic experiments with fibroblasts were done at steady state and we cannot exclude that stimulated fibroblasts could at least partly, behave like mature DC, contributing therefore to the modulation of NK cell phenotype in the skin. Finally, the use of allogeneic IL-2 stimulated NK cells in our system skews the results as the use of already activated NK cells can hide the true role of fibroblasts in NK activation.

With our results, we propose that in a cancer system, NK cells are indeed mostly activated by cancer cells, but a direct contact with fibroblasts in early steps of cancer development may contribute to "drag" NK cells into the tissue and further contributes to their activation. We can hypothesize that in skin, CLEC2A has an important role in the regulation of the interaction between NK cells and fibroblasts. When this interaction is compromised, it may provide a less immunosuppressive microenvironment that will favor cancer cell invasion (figure 29). The absence of CLEC2A in other cancer-prone related skin diseases reinforce this hypothesis.

Considerations on CLEC2A expression in human skin cells

At first sight, our result showing the absence of CLEC2A on primary keratinocytes is contradictory to the previously published data that identify CLEC2A as expressed in keratinocytes (Spreu, Kienle et al. 2007). While Spreu et al. performed their analysis with freshly isolated keratinocytes from skin biopsies, we did it our study using primary keratinocytes in culture. This means that while they work with a heterogeneous pool of keratinocytes coming from all the differentiation layers of the epidermis, we work with a homogenous population of basal keratinocytes. We can then hypothesize that CLEC2A can be expressed in keratinocytes upon the differentiation process. Their immunofluorescence results of skin tissue samples, showing that the expression of CLEC2A is restricted to the stratum corneum, corroborate our hypothesis (Bauer, Spreu et al. 2015). To validate this hypothesis, cytometric analysis experiments in cells freshly isolated from skin are ongoing.

Another note to add is that in their experiments, both Spreu et al. and Bauer et al. first separate the epidermis from the dermis of skin biopsies and concentrate their attention only in the epidermal portion of the skin. This method prevented them to conclude on the expression of CLEC2A in fibroblasts as we did.

B. Modulation of CLEC2A expression in fibroblasts in a non-cancerous microenvironment

The increase of CLEC2A expression during infection and its decrease during replicative senescence experiments support our belief that the crosstalk between fibroblasts and NK cells has an important role in innate immune system besides the one played during cancer invasion.

Many studies before ours tested the effects of infection on fibroblast phenotype. During inflammation processes, fibroblasts undergo phenotypical changes including mechanisms involved in ECM remodeling. Besides this architectural capacity, some studies also showed that fibroblasts can regulate inflammatory processes through CD40-CD40 ligand (L) interactions or engagement of TLR receptors. The engagement between CD40 receptors expressed by fibroblasts and CD40L expressed by T lymphocytes enhances the inflammatory process by inducing the synthesis of cytokine mediators IL-1, IL-8, adhesion molecules ICAM-1 and VCAM and PGE2 by fibroblasts [START_REF] Yellin | Ligation of CD40 on fibroblasts induces CD54 (ICAM-1) and CD106 (VCAM-1) up-regulation and IL-6 production and proliferation[END_REF][START_REF] Zhang | CD40 engagement upregulates cyclooxygenase-2 expression and prostaglandin E2 production in human lung fibroblasts[END_REF][START_REF] Buckley | Fibroblasts regulate the switch from acute resolving to chronic persistent inflammation[END_REF]. More importantly, the engagement of fibroblasts' TLR4 receptors by LPS induces expression of pro-inflammatory cytokines such as IL-1α IL-1β, IL-6, IL-8, and TNF-α [START_REF] Chakravortty | Interaction of lipopolysaccharide with human small intestinal lamina propria fibroblasts favors neutrophil migration and peripheral blood mononuclear cell adhesion by the production of proinflammatory mediators and adhesion molecules[END_REF]. Our results add the information that LPS stimulation also induces the expression of a gene coding for a ligand of NK cell receptor (CLEC2A) and implies for the first time that fibroblasts should be considered as actors in NK cell activation during infection. The fact that we were unable to detect changes in surface expression of CLEC2A prompt us to think that other inflammatory signals are needed to translocate the newly synthetized CLEC2A to the surface membrane of fibroblasts.

Senescent stromal cells can drive a pro-tumoral immunosuppressive microenvironment, the recruitment of MDSC cells [START_REF] Ruhland | Stromal senescence establishes an immunosuppressive microenvironment that drives tumorigenesis[END_REF]. As for NK cells, several reports have shown lower infiltration of NK cells in murine senescent tissues and defective migration to lymph nodes upon viral infection [START_REF] Fang | Age-dependent susceptibility to a viral disease due to decreased natural killer cell numbers and trafficking[END_REF][START_REF] Beli | Natural killer cell function is altered during the primary response of aged mice to influenza infection[END_REF]. In young organisms, NK cells are able to kill senescent cells that overexpress activating ligands, and this is an important process that allows the regeneration of the tissues [START_REF] Krizhanovsky | Senescence of activated stellate cells limits liver fibrosis[END_REF][START_REF] Sagiv | NKG2D ligands mediate immunosurveillance of senescent cells[END_REF]. In old tissues, senescent cells do accumulate contributing to aging and age-associated pathologies [START_REF] Baker | Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan[END_REF]. A recent study proved that the accumulation of senescent cells in old tissues is caused by the lack of cytotoxic activity from both NK and T cells. However, the mechanisms underlying these defaults of cytotoxic activity are not fully understood [START_REF] Ovadya | Impaired immune surveillance accelerates accumulation of senescent cells and aging[END_REF]. Even if our results are only preliminary ones we can suppose that the loss of CLEC2A upon senescence could contribute to a decrease of the accumulation of functional NKs in skin. We tried to validate this hypothesis by including replicative senescent fibroblasts in our 3D immunocompetent model. However, senescent fibroblasts did not contract the collagen matrix in the same way that non senescent fibroblasts do, and we were unable to perform the experiment. As an alternative approach, it would be interesting to test "natural" senescent fibroblasts from old people in our system.

The low levels of NKp65 (receptor of CLEC2A) in NK cells extracted from human peripheral blood of different donors intrigued the authors that first described it. However, tissue-resident and recruited NK cells differ in the expression profile of their receptors from those that remain in circulation [START_REF] Bjorkstrom | Emerging insights into natural killer cells in human peripheral tissues[END_REF][START_REF] Shahrabi | Flip-flops of natural killer cells in autoimmune diseases versus cancers: Immunologic axis[END_REF]). As CLEC2A is almost exclusively expressed in skin, we cannot exclude that only NK cells present in skin tissue will acquire the capacity to enhance the expression of NKp65 (Spreu, Kienle et al. 2007).

If we take into account these considerations, we can imagine that in our 3D system the contact between fibroblasts and NK cells, or the different culture conditions (culture in suspension or in collagen matrix) may lead to an overexpression of Nkp65. Alternatively, the level of Nkp65 at cell surface may not need to be high as it has been reported a high affinity of CLEC2A-Nkp65 interaction (Li, Wang et al. 2013) We can speculate that the absence of CLEC2A in the two fibroblast populations that share similarities with XP-C fibroblasts (CAFs and senescent fibroblasts) is not a coincidence. Our results are consistent with the idea that a crosstalk between NK cells and skin fibroblasts via CLEC2A could have major impacts in cancer progression and perhaps also in inflammatory processes. XP patients could benefit from therapies enhancing NK cell activity. As for the general population, CLEC2A could be a good marker to predict skin cancer outcomes and could guide therapeutic choices. Further clarifications of the mechanisms underlying the regulation of CLEC2A expression may open the door to therapeutic approaches aiming to reestablish CLEC2A surface expression on skin fibroblasts.

XP-C fibroblasts: Thoughts on responses to infection and lipid metabolism

The activation of TLR4 receptors by LPS stimuli triggers the internalization of the ligandreceptor complex via the dimerization with surface membrane lipid rafts and directs the complex toward lysosomal degradation or endocytic recycling [START_REF] Latz | Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction[END_REF][START_REF] Triantafilou | Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation[END_REF][START_REF] Husebye | Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity[END_REF]. The degradation and recycling processes are important to regulate LPS inflammation and tolerance and are convincingly confirmed in WT fibroblasts in our electron microcopy analysis (page 129) [START_REF] Murase | Intravesicular Acidification Regulates Lipopolysaccharide Inflammation and Tolerance through TLR4 Trafficking[END_REF]). However, XP-C fibroblasts didn't show an increase in the number of lysosomal organelles after LPS stimuli which imply that XP-C fibroblasts' response to infection is deregulated. As the expression of TLR4 is not deregulated in XP-C fibroblasts compared to WT fibroblasts at steady state (transcriptomic results) we can suppose that there is an impairment, either in the engagement of the complex TLR4-LPS, or in the trafficking signaling pathway that follows internalization. Because we have no indication of XP-C patients presenting predisposition for chronic inflammatory processes, a default of engagement of TLR4 with LPS may be more likely. Furthermore, our observations of lipid droplets in the cytoplasm of XP-C fibroblasts are consistent with a deregulation in lipid metabolism. Lipids are involved in TLR4-LPS internalization.

In fact, our 2D transcriptomic analysis revealed moderate deregulation in the expression of many genes implicated in the lipid biosynthesis. The expression of genes implicated in the biosynthesis of cholesterol are downregulated in XP-C fibroblasts and genes implicated in the biosynthesis of triacylglycerides (TAG), phosphoglycerides and phosphoinositides are upregulated (figure 30). Cholesterol and phosphoglycerides are major structural components of biological membranes and TAG are mainly implicated in energy storage in the form of lipid droplets like the ones we saw in the cytoplasm of XP-C fibroblasts [START_REF] Baenke | Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development[END_REF]).

More importantly, cholesterol is one of the main components of lipid rafts directly implicated in the LPS-induced cell activation. Alterations in lipid rafts could impair LPS signaling and explains the lack of lysosomal accumulation that we saw in our electron microscopy analysis.

However, a lack of response against infection normally leads to a scenario of recurrent and/or chronic infection that again are not described in XP patients. Besides the important work that some research groups around the world are doing to contribute to the development of new therapies that ultimately improve the quality of life of XP patients, other nonscientific groups provide an enormous help to these patients and their families. They organize gatherings between the children and their families, scientific meetings, and the majority also work as a family support group. They raise funds for research projects and scientific collaborations to help these families acquire the expensive UV protective equipment and to organize activities with the children to avoid social exclusion. Their work is equally as important as ours and deserves to be mentioned in this manuscript. Below a list of the most active XP groups/associations/Charitable Trusts: XP Family Support Group: https://xpfamilysupport.org/

  Introduction ........................................................................................................................................... Chapter I: Skin ................................................................................................................................... 1. Structure of the skin .................................................................................................................. 1.1. Epidermis ............................................................................................................................ 1.1.a. The Basal layer (Stratum basale) ................................................................................. 1.1.b. Spinous layer or stratum spinosum ............................................................................. 1.1.c. The granular layers or stratum granulosum ................................................................ 1.1.d. Cornified layers or stratum corneum .......................................................................... 1.1.e. Other epithelial cells .................................................................................................... 1.2. Dermal-epidermal junction ................................................................................................ 1.3. Dermis ................................................................................................................................ 1.3.a. Fibroblasts ................................................................................................................... 1.3.b. Other dermal cells ....................................................................................................... 1.4. Hypodermis ........................................................................................................................ 1.5. Skin appendages ................................................................................................................. 1.5.a. Pilosebaceous units ..................................................................................................... 1.5.b. Glands .......................................................................................................................... 2. Function ..................................................................................................................................... 3. Skin cancers ............................................................................................................................... 3.1. Non melanoma skin cancers............................................................................................... 3.2. Melanoma skin cancer........................................................................................................ 3.3. Risk factors ......................................................................................................................... 3.3.a. UV exposure ................................................................................................................ 3.3.a. Pollution ....................................................................................................................... 3.3.b. Phototype .................................................................................................................... 3.3.c. Aging ............................................................................................................................ 3.3.d. Immunosupression ...................................................................................................... 3.3.e. Genetic disorders.........................................................................................................

Figure 1 .

 1 Figure 1. The skin and its appendages. ................................................................................................. Figure 2. Epidermal structure and keratinocyte differentiation ........................................................... Figure 3. Dermal extracellular matrix components. .............................................................................. Figure 4. Incidence rates of non-melanoma skin cancer in 2018 .......................................................... Figure 5. Incidence rates of melanoma in 2018. ................................................................................... Figure 6. UV light and the skin. ............................................................................................................. Figure 7. Structures of the UV-induced DNA lesions cyclobutane pyrimidine dimer (CPD) and (6-4) photoproducts ....................................................................................................................................... Figure 8. Age at onset of XP symptoms and cancers............................................................................. Figure 9. Nucleotide Excision Repair mechanism. ................................................................................. Figure 10. DNA repair pathways. ........................................................................................................... Figure 11. DNA Repair disorders. .......................................................................................................... Figure 12 XPC gene ................................................................................................................................ Figure 13. XPC protein. .......................................................................................................................... Figure 14. The hallmarks of cancer. ......................................................................................................Figure 15. Antigen presentation to T cells. ........................................................................................... Figure 16. NK cell killing......................................................................................................................... Figure 17. Afferent and efferent pathways in the crosstalk between CAFs and cancer cells. .............. Figure 18. Cancer immunoediting. ........................................................................................................ Figure 19. CLEC2A transcript variants. .................................................................................................. Figure 20. The NKp65-KACL binding structural interface. .................................................................... Figure 21. Kinetics of CLEC2A expression in WT fibroblasts (F-WT) after incubation with LPS. ......... Figure 22. CLEC2A immunofluorescence staining of FTM1_WT fibroblasts after incubation with 200 ng/mL LPS for 6, 7, 8 and 24 hours. ..................................................................................................... Figure 23. Lysosome quantification in WT and XP-C fibroblasts after stimulation with LPS for 6 hours. ............................................................................................................................................................. Figure 24. CLEC2A expression in WT fibroblasts over population doublings determined by flow cytometry. ........................................................................................................................................... Figure 25. Expression of p16 and p21 in three primary cell lines of WT fibroblasts at low and high PD as determined by Western blot. Anti-tubulin antibody was used as a loading control. ..................... Figure 26. Senescence associated β-galactosidase staining in two primary cell lines of WT fibroblasts at low and high PD. .............................................................................................................................. Figure 27. CLEC2A expression in primary WT and XP-D fibroblasts. ................................................... Figure 28. Relative CLEC2A mRNA transcripts levels in FH84_WT, AS537_NBCCS and AS_587_NBCCS fibroblasts determined by quantitative RT-PCR. ................................................................................. Figure 29. Crosstalk between fibroblasts and NK cells........................................................................ Figure 30. Schematic overview of the pathways involved in the different lipid classes biosynthesischolesterol, Fatty acids (FA), sphingolipids, eicosanoids, acylglycerides, phosphoinositides and phosphoglycerides...............................................................................................................................

Figure 15 .

 15 Figure 1. The skin and its appendages. ................................................................................................. Figure 2. Epidermal structure and keratinocyte differentiation ........................................................... Figure 3. Dermal extracellular matrix components. .............................................................................. Figure 4. Incidence rates of non-melanoma skin cancer in 2018 .......................................................... Figure 5. Incidence rates of melanoma in 2018. ................................................................................... Figure 6. UV light and the skin. ............................................................................................................. Figure 7. Structures of the UV-induced DNA lesions cyclobutane pyrimidine dimer (CPD) and (6-4) photoproducts ....................................................................................................................................... Figure 8. Age at onset of XP symptoms and cancers............................................................................. Figure 9. Nucleotide Excision Repair mechanism. ................................................................................. Figure 10. DNA repair pathways. ........................................................................................................... Figure 11. DNA Repair disorders. .......................................................................................................... Figure 12 XPC gene ................................................................................................................................ Figure 13. XPC protein. .......................................................................................................................... Figure 14. The hallmarks of cancer. ......................................................................................................Figure 15. Antigen presentation to T cells. ........................................................................................... Figure 16. NK cell killing......................................................................................................................... Figure 17. Afferent and efferent pathways in the crosstalk between CAFs and cancer cells. .............. Figure 18. Cancer immunoediting. ........................................................................................................ Figure 19. CLEC2A transcript variants. .................................................................................................. Figure 20. The NKp65-KACL binding structural interface. .................................................................... Figure 21. Kinetics of CLEC2A expression in WT fibroblasts (F-WT) after incubation with LPS. ......... Figure 22. CLEC2A immunofluorescence staining of FTM1_WT fibroblasts after incubation with 200 ng/mL LPS for 6, 7, 8 and 24 hours. ..................................................................................................... Figure 23. Lysosome quantification in WT and XP-C fibroblasts after stimulation with LPS for 6 hours. ............................................................................................................................................................. Figure 24. CLEC2A expression in WT fibroblasts over population doublings determined by flow cytometry. ........................................................................................................................................... Figure 25. Expression of p16 and p21 in three primary cell lines of WT fibroblasts at low and high PD as determined by Western blot. Anti-tubulin antibody was used as a loading control. ..................... Figure 26. Senescence associated β-galactosidase staining in two primary cell lines of WT fibroblasts at low and high PD. .............................................................................................................................. Figure 27. CLEC2A expression in primary WT and XP-D fibroblasts. ................................................... Figure 28. Relative CLEC2A mRNA transcripts levels in FH84_WT, AS537_NBCCS and AS_587_NBCCS fibroblasts determined by quantitative RT-PCR. ................................................................................. Figure 29. Crosstalk between fibroblasts and NK cells........................................................................ Figure 30. Schematic overview of the pathways involved in the different lipid classes biosynthesischolesterol, Fatty acids (FA), sphingolipids, eicosanoids, acylglycerides, phosphoinositides and phosphoglycerides...............................................................................................................................

  ..............................................................................................

Figure 2 .

 2 Figure 2. Epidermal structure and keratinocyte differentiation

  Melanocytes are DCs responsible for skin color. They derive from embryonic neural crest and are located either in hair follicles or in the basal layer of the epidermis where the ratio melanocytes : keratinocytes is 1: 10[START_REF] Cichorek | Skin melanocytes: biology and development[END_REF]. Melanocytes produce melanin pigments packed in cytoplasmic melanosome units that are responsible for the distribution of melanin to neighboring keratinocytes. Eumelanin and phaelomelanin are the two types of melanin produced in skin and hair. The different appearances of skin are not related to the number of melanocytes present in the epidermis but to the size of the melanosomes and the nature of the melanin. Melanin production is stimulated by ultraviolet (UV) radiation and contributes to skin protection against UV associated cell damage(McGrath and Uitto 2016, Yi, Su et al. 2018).

  Glycosaminoglycans (GAGs) (like hyaluran, chondroitin sulfate and dermatan sulfate) and proteoglycans (like versican, decorin, dermatopontin and lumican) are important to ensure dermis hydration. Normally proteoglycans bound to a GAG core protein. Matricellular proteins act as signaling molecules principally in injured skin.

Figure 3 .

 3 Figure 3. Dermal extracellular matrix components.

Figure 4 .

 4 Figure 4. Incidence rates of non-melanoma skin cancer in 2018

Figure 5 .

 5 Figure 5. Incidence rates of melanoma in 2018.

Figure 6 .

 6 Figure 6. UV light and the skin.

Figure 7 .

 7 Figure 7. Structures of the UV-induced DNA lesions cyclobutane pyrimidine dimer (CPD) and (6-4) photoproducts

Figure 8 .

 8 Figure 8. Age at onset of XP symptoms and cancers.

(

  [START_REF] Melis | The role of XPC: implications in cancer and oxidative DNA damage[END_REF] DNA damage can occur following the "attack" of different endogenous or exogenous factors. Cell machinery is equipped with different repair systems and is able to selectively trigger them depending on the type the DNA damage. NER and BER are the two excision repair pathways. As NER is the only one responsible of the removal of bulky DNA adducts caused by UV radiation (described in the main text), BER is mostly described for its role in recognition and excision of oxidative damaged bases like 8-oxoG. The key enzymes involved in the BER are DNA glycosylases responsible for the removal of damaged bases by cleavage of the Nglycosylic bonds of the nucleotide residues. The DNA glycosylase action is followed by an incision step, DNA synthesis, an excision step, and DNA ligation. The other repair pathways are the HR pathway that uses the homologous DNA strand as a template for re-synthesis when in presence of double strand breaks (DSB); MMR that is implicated in the removal of postreplicative DNA errors like mismatched nucleotides, insertions, deletions and NHEJ that is another pathway able to repair DSB by ligating the two DNA ends[START_REF] Hoeijmakers | Genome maintenance mechanisms for preventing cancer[END_REF].BER: Base ExcisionRepair); HR: Homologous Recombination; MMR: Mismatch Repair; NHEJ: Non-Homologous End-Joining 2.1.a. Genotype-phenotype relationships Products of the seven complementation genes have different roles in the NER and their mutations are associated with variable symptom severities, frequencies and residual levels of DNA repair (as measured by UDS) (table

  . Patients with XP/TTD overlap disorders were first described by Bernard C. Broughton et al in 2001. They exhibit features of both diseases but normally present milder symptoms with less prominent hair problems and lower cancer development frequency as compared to XP (Broughton, Berneburg et al. 2001).

Figure 11 .

 11 Figure 11. DNA Repair disorders.

Figure 12

 12 Figure 12 XPC gene

Figure 13 .

 13 Figure 13. XPC protein.

  of at least four DNA glycosylases -methylpurine-DNA glycosylase (MPG), thymine-DNA glycosylase (TDG), 8-oxo-guanine-DNA glycosylase 1 (OGG1) and single-strand-selective monofunctional uracil-DNA glycosilase 1 (SMUG1) -responsible for modified base removal in the BER mechanism[START_REF] Shimizu | Xeroderma pigmentosum group C protein interacts physically and functionally with thymine DNA glycosylase[END_REF], D'Errico, Parlanti et al. 2006[START_REF] Shimizu | Stimulation of DNA Glycosylase Activities by XPC Protein Complex: Roles of Protein-Protein Interactions[END_REF]. Moreover, XP-C keratinocytes and fibroblasts show reduced rates of BER and an accumulation of oxidizing bases in their DNA. D'Errico et al. speculate that the accumulation of oxidative DNA damage in XP-C cells could be related to the reports of internal cancers in these patients. They further support their theory with the results ofGiglia et al.

  vitro. XPC -/-HeLa cells have an attenuated response to retinoic acid induction corroborating the possible implication of XPC in the RARβ2 transcription. Le May et al. show that XPC not only binds to RARβ2 promoter but also assembles the NER pre incision complex in a transcription-dependent way without the presence of DNA damage. The pre-incision complex proved to be essential for chromatin remodeling via histone modifications and DNA demethylation at the activated gene sites (Le May, Mota-Fernandes et al. 2010, Le May, Fradin et al. 2012).

  Since early diagnosis is essential to minimize the symptoms of XP disease, the discovery of this founder mutation opens the possibility of prenatal newborn molecular diagnosis in the atrisk populations or families.Other founder mutations have been reported or suggested in different geographical areas. A study in a tertiary center in Nepal reported that all the fifteen XP-C patients in the center shared the same c.1243C>T mutation[START_REF] Espi | Clinical and genetic characteristics of xeroderma pigmentosum in Nepal[END_REF]. Recently, Ijaz et al. reported a prevalent founder mutation in XP-C patients from Pakistan and correlated the findings with a study reporting the same mutation in Comorian patients. The founder effect have an estimated age of about 800 years[START_REF] Cartault | A new XPC gene splicing mutation has lead to the highest worldwide prevalence of xeroderma pigmentosum in black Mahori patients[END_REF][START_REF] Ijaz | XPC gene mutations in families with xeroderma pigmentosum from Pakistan; prevalent founder effect[END_REF]) a period corresponding to the migration of Muslims in this region. Therefore, authors suggest that the ancestor was actually from the Middle East region.

  ) present the phagocytosed antigens to naïve T cells. Activated T cells (3) proliferate and (4) migrate into tumor site where they are able to (5) recognize and kill cancer cells.Activated CD8 + cytolytic T cells, helped by Th1, are able to kill cancer cells[START_REF] Shankaran | IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity[END_REF][START_REF] Dunn | A critical function for type I interferons in cancer immunoediting[END_REF][START_REF] Galon | Type, density, and location of immune cells within human colorectal tumors predict clinical outcome[END_REF][START_REF] Gajewski | Failure at the effector phase: immune barriers at the level of the melanoma tumor microenvironment[END_REF]. Moreover, both CD8 + T cells and Th1 cells express IFN-γ. IFNis known for its role as protector against tumor development. IFN-γ can be secreted by a large panel of cells and targets both tumor cells and hematopoietic cells. Multiple different tumor protective effects of IFN-γ have been published over the last few years. IFN-γ prevents primary tumor development and activates

Figure 15 .

 15 Figure 15. Antigen presentation to T cells.

(

  Morvan and Lanier 2016) (a) Balanced signals delivered by activating and inhibitory NK cells receptors are recognized as healthy and spared from NK cell-mediated lysis. NK cells recognize and kill tumour or damaged cells that (b) downregulate MHC class I molecules -'missing self' lysis; (c) upregulate stress-or damage related ligands that are recognized by activating NK cell receptors -'inducedself' lysis or (d) through antigen-specific antibodies that bind CD16 on NK cells resulting in antibody-dependent cell-mediated cytotoxicity (ADCC).

Figure 16 .

 16 Figure 16. NK cell killing.

tumoral

  TANs normally associated to a N2 polarization are implicated in dissemination and metastases, facilitating the bridge between cancer cells and the metastatic sites; these cells sustain the ECM remolding through the secretion of MMPs, in particular MMP9, and inhibit CD8 + T cell function[START_REF] Fridlender | Tumor-associated neutrophils: friend or foe?[END_REF].1.3.f. Myeloid-derived suppressor cells MDSC are generally defined as a heterogeneous population of immature myeloid cells.Contrary to TAMs and TANs, they only have immunosuppressive properties. Among these properties, the most relevant are the activation, expansion and recruitment of Treg cells and the impairment of T cell trafficking to-and from-lymph nodes[START_REF] Serafini | Myeloid-derived suppressor cells promote cross-tolerance in B-cell lymphoma by expanding regulatory T cells[END_REF][START_REF] Gabrilovich | Coordinated regulation of myeloid cells by tumours[END_REF][START_REF] Schlecker | Tumorinfiltrating monocytic myeloid-derived suppressor cells mediate CCR5-dependent recruitment of regulatory T cells favoring tumor growth[END_REF]. They are subdivided in two populations: monocytic MDSCs (M-MDSCs) and granulocytic or polymorphonuclear MDSCs (PMN-MDSCs). M-MDSC are phenotypically and morphologically similar to monocytes and PMN-MDSC are phenotypically and morphologically similar to neutrophils. HLA-DR expression patterns allows the distinction between M-MDSC (HLA-DR - /low

(

  [START_REF] Cirri | Cancer associated fibroblasts: the dark side of the coin[END_REF] Without being exhaustive, among CAFs' secreted factors, CXCL12 /SDF1α, CXCL14 and HGF have been proven to have a major role in cancer cell growth. CXCL12 binds to its CXCR4 receptor in cancer cells and promote both proliferation of cancer cells in vitro and tumor growth in vivo[START_REF] Orimo | Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion[END_REF]. Furthermore, Wang et al. reported that the inactivation of CXCL12/CXCR4 signaling in pancreatic cancer cells led to a partial inactivation of the Wnt signaling pathway which is known for its role in proliferation, invasion, metastasis, and angiogenesis[START_REF] Taketo | Wnt signaling and gastrointestinal tumorigenesis in mouse models[END_REF][START_REF] Wang | Blockade of SDF-1/CXCR4 signalling inhibits pancreatic cancer progression in vitro via inactivation of canonical Wnt pathway[END_REF]. CXCL14 autocrine signaling is activated in CAF following nitric oxide synthase 1 (NOS1) signaling. Induction of NOS1 is associated with oxidative stress and its inhibition reduces the ability of CXCL14-expressing fibroblasts to promote tumor formation in presence of breast and pancreatic cells(Augsten, Sjoberg et al.

Figure 17 .

 17 Figure 17. Afferent and efferent pathways in the crosstalk between CAFs and cancer cells.

  cancer cells. Numerous factors secreted by CAFs have been shown to be involved in the increase of the cancer stem cell (CSC) population. For instance, connective tissue growth factor (CTGF) increases the expression of multiple pluripotency genes in head and neck cancer cells. HGF seems to regulate CSC population through both activation of Wnt and c-Met signaling pathways (Vermeulen, De Sousa et al. 2010, Chang, Hsu et al. 2013, Li, Wang et al. 2019).2.2. Tumor immune microenvironmentDifferent types of immune cells exhibiting opposite functions in cancer progression are recruited to the TME and can be modulated by CAFs and cancer cells. The dual role of the immune system in tumor progression is the basis of the "immunoediting theory" first proposed by[START_REF] Dunn | Cancer immunoediting: from immunosurveillance to tumor escape[END_REF]

Figure 18 .

 18 Figure 18. Cancer immunoediting.

  important role in inhibiting T cell proliferation and cytotoxic properties. TGF-β mediates conversion into Tregs (T-Regulator lymphocytes) already describe as inhibitors of CD8 + effector T cells. TGF-promotes cell death and alteration of cytotoxic properties of CD8 + T cells by inhibiting the expression of the pro-survival protein Bcl-2 and inhibiting the expression of key genes involved in their cytotoxic activity, respectively[START_REF] Ahmadzadeh | TGF-beta 1 attenuates the acquisition and expression of effector function by tumor antigen-specific human memory CD8 T cells[END_REF][START_REF] Sanjabi | Opposing effects of TGF-beta and IL-15 cytokines control the number of short-lived effector CD8+ T cells[END_REF][START_REF] Hargadon | Dysregulation of TGFbeta1 Activity in Cancer and Its Influence on the Quality of Anti-Tumor Immunity[END_REF].Modulation of NK phenotypeBesides downregulation of MHC-I molecules, cancer cells have other ways to shuffle immune system through the modulation of their membrane receptors. They can protect themselves from NK cell killing by integrating in their membrane, vesicles containing MHC-I molecules previously released by platelets. This mechanism confers to cancer cells a "pseudo normal phenotype" allowing them to impair NK cytotoxicity[START_REF] Placke | Plateletderived MHC class I confers a pseudonormal phenotype to cancer cells that subverts the antitumor reactivity of natural killer immune cells[END_REF].Similarly to HLA-E discussed previously, another efficient way to negatively affects immune cell functions is the expression of the non-canonical MHC molecule, namely the human leukocyte antigen G (HLA-G). HLA-G is expressed in embryos and has an important role in protecting placenta from rejection[START_REF] Rizzo | The importance of HLA-G expression in embryos, trophoblast cells, and embryonic stem cells[END_REF]. The neo expression of HLA-G engages receptors from both NK cells and CD8 + effector T cells generating inhibitory signals that impair their activation. It also triggers pro-tumoral effects in DC, neutrophils and MDSCs[START_REF] Lin | Human Leukocyte Antigen-G (HLA-G) Expression in Cancers: Roles in Immune Evasion, Metastasis and Target for Therapy[END_REF].

  poor life span prognosis[START_REF] Wang | Eicosanoids and cancer[END_REF]. Although the mechanisms through which PGE2 modulates NK and T cell phenotypes are not yet fully understood, at least one of the signaling pathways was identified. Indeed, PGE2 leads to the downregulation of IFN-α levels within the TME[START_REF] Zelenay | Cyclooxygenase-Dependent Tumor Growth through Evasion of Immunity[END_REF]. Type I IFN (α/β) increases NK cell mediated killing, promotes the differentiation of CD8 + T cells and enhance the dendritic cell cross-presentation of tumor antigens to CD8 + T cells[START_REF] Nguyen | Coordinated and distinct roles for IFN-alpha beta, IL-12, and IL-15 regulation of NK cell responses to viral infection[END_REF][START_REF] Swann | Type I IFN contributes to NK cell homeostasis, activation, and antitumor function[END_REF][START_REF] Diamond | Type I interferon is selectively required by dendritic cells for immune rejection of tumors[END_REF][START_REF] Bhat | Interferon-gamma derived from cytotoxic lymphocytes directly enhances their motility and cytotoxicity[END_REF].Recently, a study from Ziani et al. reported that CAFs associated with melanoma could also modulate the activation of NK cells through the secretion of MMPs. MMPs are able to cleave MHC-I-related chain (MIC)-A and MIC-B, two ligands of the NK activating receptor NKG2D and, consequently, decrease the NKG2D-dependent cytotoxic activity of NK cells against melanoma tumor cells and their secretion of IFN-γ[START_REF] Ziani | Melanoma-associated fibroblasts decrease tumor cell susceptibility to NK cell-mediated killing through matrix-metalloproteinases secretion[END_REF]. Finally, the hypoxic tumor microenvironment leads to secretion of lactate by cancer cells. This phenomenon has been shown to reduce the numbers and activity of NK cells and CD8 + T cells

Food&

  Drug Administration 2019). Similar efforts are being made regarding the blocking of inhibitory receptors in NK cells. Lirilumab and monalizumab, two antagonist antibodies of NK inhibitory receptors are presently in clinical trials and first results show an enhancement of NK activity against tumor cell lines (Romagne,[START_REF] Romagne | Preclinical characterization of 1-7F9, a novel human anti-KIR receptor therapeutic antibody that augments natural killer-mediated killing of tumor cells[END_REF][START_REF] Vey | A phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission[END_REF][START_REF] Andre | Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells[END_REF]. Monalizumab, a humanized anti-NKG2A antibody also shows efficacy in early clinical trials, mostly by restoring anti-tumor activity of T cells and also NK cells[START_REF] Andre | Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells[END_REF][START_REF] Van Montfoort | NKG2A Blockade Potentiates CD8 T Cell Immunity Induced by Cancer Vaccines[END_REF]).

Figure 20 .

 20 Figure 20. The NKp65-KACL binding structural interface.

  main objective of my PhD work was therefore to assess the role of the skin microenvironment in XP-C patients on the development of squamous cell carcinomas (SCC) and to explore the cross-talk between fibroblasts and immune cells in immunosurveillance of the skin. Preliminary comparative transcriptomic analysis of WT and XP-C fibroblasts cultured in both two dimensional and three dimensional cultures, revealed that transcripts of CLEC2A, a ligand of the activating NK cell receptor NKp65, were present in WT fibroblasts and absent in XP-C fibroblasts. I therefore focused my research project on the study the role of CLEC2A expression in a healthy skin and the implications of its absence in XP-C dermis, more specifically in a context of SCC development. Towards this goal, I structured the research in two main axes: 1) Aiming to get hints on the function roles of CLEC2A in the skin, I (i) analyzed the expression of CLEC2A in skin cells of healthy individuals, XP-C patients, and from SCC areas and (ii) explored the regulation of CLEC2A expression in aging and in pathological conditions such as skin cancer and infection.

FiguresFig. 1 .Fig. 2 .

 12 Figures

Fig. 3 .

 3 Fig. 3. CLEC2A expression in dermal CAFs. (A) α-SMA and DAPI immunofluorescence staining of WT and XP-C fibroblasts and primary isolated dermal CAFs. Data are

Fig. 4 .

 4 Fig. 4. CLEC2A expression in WT fibroblasts stimulated by SCC12_conditioned medium (SCC12_CM) (A) Relative CLEC2A mRNA transcripts levels in WT fibroblasts stimulated by SCC12_CM for 5 days. Data represent mean ± SD of five different SCC12_CM. **p≤0,01 Mann-Whitney U-test. (B) Representative histograms of CLEC2A expression on WT fibroblasts stimulated or not by SCC12_CM as determined by flow cytometry. Three independent experiments were performed.

Fig. 5 .

 5 Fig. 5. 3D immunocompetent model that assess the impact of CLEC2A on the ability of NK cells to control invasion of SCC12 cells in WT, XP-C and CAF 3D-dermal cultures. (A) Representative images of hematoxylin and eosin (H&E) staining of SCC12 cell invasion in WT and XP-C 3D-dermal cultures in absence or presence of NK cells (first and second line), anti-CLEC2A blocking antibody (OMA1) (third line), and isotype Ig (fourth line). (B) Representative images of H&E staining of SCC12 cell invasion in CAF 3D-dermal culture in absence or presence of NK cells. (C) Quantification of SCC12 invasion rate in the aforementioned conditions in box plots. ***p≤0,001 Tukey's test in one-way ANOVA. Two independent experiments were performed.

  Figure 1

Fig. S1 .

 S1 Fig. S1. CLEC2A expression in fibroblasts. (A) CLEC2A transcript variants were amplified by RT-PCR in FH122_WT fibroblast (lane 1), U937 cell line (lane 2), AS433_XP-C fibroblast (lane 3), RT negative control (lane 4), PCR buffer negative control (lane 5). (B) CLEC2A expression in FH84_WT fibroblasts, determined by imaging flow cytometry. Data is representative of two independent experiments.

  Fig. S6. MHC-I and CLEC2A expression in WT, XP-C fibroblasts and SCC12 cells determined by flow cytometry. (A) MHC I cell surface expression measured using anti-MHC I antibodies, clone DX17 and W6/32 compared to isotype Ig controls. (B) CLEC2A expression on SCC12 cells using OMA1 antibody compared to isotype Ig control.

  (A) Relative CLEC2A mRNA transcripts levels in WT fibroblasts after incubation with 200 ng/mL LPS measured by quantitative RT-PCR. Data represent mean ± SD of three cell lines (FTM1, FTM5, FH122). ***p ≤0,001, *p≤0,05 Mann-Whitney U-test. (B) Representative histograms of CLEC2A expression in FTM1_WT fibroblasts after incubation with LPS for 3, 6 and 12 hours as determined by flow cytometry. Two independent cell lines were analyzed.

Figure 21 .

 21 Figure 21. Kinetics of CLEC2A expression in WT fibroblasts (F-WT) after incubation with LPS.

Figure 22 .

 22 Figure 22. CLEC2A immunofluorescence staining of FTM1_WT fibroblasts after incubation with 200 ng/mL LPS for 6, 7, 8 and 24 hours.

Figure 23 .

 23 Figure 23. Lysosome quantification in WT and XP-C fibroblasts after stimulation with LPS for 6 hours.

Figure 24 .

 24 Figure 24. CLEC2A expression in WT fibroblasts over population doublings determined by flow cytometry.

Figure 25 .

 25 Figure 25. Expression of p16 and p21 in three primary cell lines of WT fibroblasts at low and high PD as determined by Western blot. Anti-tubulin antibody was used as a loading control.

Figure 26 .

 26 Figure 26. Senescence associated β-galactosidase staining in two primary cell lines of WT fibroblasts at low and high PD.

(

  A) Relative CLEC2A mRNA transcripts levels in FTM1_WT and AS204_XP-D fibroblasts. Data represent mean ± SD of two independent experiments. ***p≤0,001 Mann-Whitney U-test. (B) Representative histograms of CLEC2A expression in FTM1_WT and AS204_XP-D fibroblasts determined by flow cytometry. Three independent experiments were performed.

Figure 27 .

 27 Figure 27. CLEC2A expression in primary WT and XP-D fibroblasts.

Figure 28 .

 28 Figure 28. Relative CLEC2A mRNA transcripts levels in FH84_WT, AS537_NBCCS and AS_587_NBCCS fibroblasts determined by quantitative RT-PCR.

  Left panel. (1) In early steps of cancer development, the presence of CLEC2A in skin WT fibroblasts is able to trigger NK cell cytotoxicity in vitro through the engagement of NKp65 receptor. (2) SCC cell secreted factors downregulate CLEC2A expression, possibly as part of the transitioning process that WT fibroblasts undergo to become CAFs. The loss of CLEC2A facilitates the evasion of cancer cells. Wright panel. In an XP-C skin, the absence of CLEC2A; weakens the skin immunosurveillance against cancer cells.

Figure 29 .

 29 Figure 29. Crosstalk between fibroblasts and NK cells.

FinallyFigure 30 .

 30 Figure 30. Schematic overview of the pathways involved in the different lipid classes biosynthesischolesterol, Fatty acids (FA), sphingolipids, eicosanoids, acylglycerides, phosphoinositides and phosphoglycerides.

Table 1

 1 Characteristics of Papillary and reticular dermis. ..................................................................... Table 2. The ABCDE criteria allows the self-diagnosis of melanoma. ................................................... Table 3. Xeroderma Pigmentosum complementation groups: insight in frequency, proneness to skin cancer development, neurological involvement and cellular repair capabilities. ................................

Table 4 . Patients and cell descriptions .

 4

Asymmetry One-half of lesion is different than the other half Border

		Irregular or poorly defined border
	Color	Varied from one area to another; different shades of tan, brown, black;
		sometimes red, white, or blue within the same lesion
	Diameter	Larger than 6 mm (bigger than the size of a pencil eraser)
	Evolving	A mole that looks different compared with surrounding moles ("ugly
		duckling" sign), or the mole is changing in size, shape, or color
	(Shenenberger 2012)

Table 2 .

 2 The ABCDE criteria allows the self-diagnosis of melanoma.

	3.3. Risk factors
	Predisposition to skin cancer include both exogenous and endogenous factors. Although the
	main environmental and modifiable risk factor is UV exposure, the combination between
	modifiable factors and non-modifiable ones, like chronological aging, can exacerbate the
	chances of developing cutaneous cancers.
	3.3.a. UV exposure
	Over the years, several studies reported a correlation between sun exposure and the
	development of cutaneous cancer. Even if recent data suggest that different sun exposure
	profiles are associated with different skin cancer subtypes, both NMSC and melanoma arises

  [START_REF] Khan | Xeroderma pigmentosum group C splice mutation associated with autism and hypoglycinemia[END_REF] added one more to the list and the list kept growing till today with nearly 50 described mutations

	c.1001C>A,	c.1103_1104delAA,	c.1643_1644delTG,	c.2092_2093insGTG,
	c.1643_1644delTG, c.2092_2093insGTG.

).

4.3. XP-C patients' mutation profile

Several different XPC mutations have been reported in XP-C patients over the last three decades. The majority are deletions, substitutions, splicing alterations, and insertions mainly resulting in premature stop codons. In 1993, Li et al. reported six different mutations -

  . Moreover, Schafer et al. proposed that extrinsic factors like the individual

	sun-exposure behavior are the greatest contributors to XP-C variable symptoms severity
	(Schafer, Hofmann et al. 2013).
	4.4 XP-C group as a model to study both non melanoma skin cancer and aging

XP-C patients are prone to develop aggressive cancers. One of the first evidences of this aggressive phenotype was published in 1998, after the study of the mutational spectra of the tumor supressor geneTP53 in XP-C patients. The molecular analysis of mutations TP53 from 58 XP-C skin biopsies showed two important things: first, SCC are more frequent than BCC

  microenvironment are preponderant to the fate of cancer progression[START_REF] Kalluri | The biology and function of fibroblasts in cancer[END_REF]. Fibroblasts from XP-C patients from non-photo exposed areas promote keratinocyte and SCC invasion in organotypic cultures and express other similarities with CAFs. Most notably, studies from the laboratory have indicated that the overexpression of the hepatocyte

	growth factor (HGF) and the Matrix Metalloproteinase 1 (MMP1) are implicated in ECM
	remodeling (Alqaraghuli, in preparation) (Bernerd, Asselineau et al. 2005, Frechet, Warrick et
	al. 2008, Cui, Wang et al. 2018). Unlike CAFs, XP-C fibroblasts show a pro-invasive phenotype
	at the steady state, i.e. even in the absence of activation by cancer cells. The identification of
	the molecular deregulations underlying the XP-C fibroblasts phenotype could help understand
	the very first steps of cancer cell invasion and the typology of cancer that XP-C patients
	preferentially develop, i.e. SCCs.

. Why XP-C patients develop more SCCs than BCCs is still not yet understood. There is evidence showing that in the general population, XPC is well expressed in BCCs and conversely is frequently inactivated in SCCs

[START_REF] De Feraudy | The DNA damage-binding protein XPC is a frequent target for inactivation in squamous cell carcinomas[END_REF][START_REF] Yang | Xeroderma pigmentosum complementation group C protein (XPC) expression in basal cell carcinoma[END_REF]

. To my knowledge, there are no studies that directly correlates TP53 mutations in BCC and SCC with expression of XPC in the general population. However, the difference in XPC expression in BCC vs SCC together with findings showing different hotspot mutations in TP53 from BCCs and SCCs could let us hypothesize that TP53 mutations associated with BCC, somehow, are not detrimental to the role of p53 in XPC induction

[START_REF] Giglia-Mari | TP53 mutations in human skin cancers[END_REF]

.

Although SCCs are naturally more aggressive than BCCs, tissue invasion and metastasis also depends on cancer cell microenvironment composed mainly by fibroblasts, dendritic cells, B lymphocytes, T lymphocytes and NK cells. Briefly, fibroblasts that are associated with cancer cells become "activated" (CAFs) and the crosstalk between CAFs and the other cells within the tumoral

  Paget, inspired by some previous notes from Fuchs, published the "seed and soil" hypothesis to explain different frequencies of metastasis in different organs. Making an analogy to what happened with plants, he proposed that cancer cells, alike seeds, are numerous and capable of development but they are only able to disseminate if they fall in fertile microenvironment (soil)

  ). CAA could support cancer growth and dissemination through different mechanisms. They (i) provide tumors with fatty acids and other adipocyte-related metabolites that fuel tumor growth, (ii) promote invasive properties of tumor cells and (iii) protects cancer against some cytotoxic therapies through the induction of pro-survival signals in cancer cells(Iyengar, Combs et al.

2003,

[START_REF] Nieman | Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth[END_REF][START_REF] Duong | The fat and the bad: Mature adipocytes, key actors in tumor progression and resistance[END_REF]

).

Table 4 . Patients and cell descriptions

 4 Cytometry, quantitative real-time RT-PCR, Western blot and statistical analysis, please refer to Material and Methods of the "Scientific Article" on page 90.

	Diagnosis	Patients Age at Biopsy	Ethnic origin Mutation
			(years)
	XP-D	AS204	15	Caucasian	XPD R683W
	NBCCS	AS537	42	Caucasian	PATCHED1	c.1762supG ;
					p.Val588GlyfsX39
		AS587	43	Caucasian	PATCHED1	c.1366del ;
					p.Thr456ProfsX35
	Lipopolysaccharide (LPS) stimulation. Fibroblasts cultured at 70% of confluence were
	treated with 200ng/mL of LPS (# 0111:B4, Sigma-Aldrich, Lyon, France) in 0,5% fibroblast
	DMEM 10% FBS cell culture medium for different periods of time.
	Indirect immunofluorescence analysis. Cells were grown on glass coverslips, fixed in 4%
	paraformaldehyde / 0.25% glutaraldehyde for 15 min at room temperature, rinsed twice in PBS
	and treated in PBS, 0.25% Triton X-100 for 5 min. Cells were next blocked in PBS, 3% BSA
	for 30 min and incubated for 1 h with mAb anti-CLEC2A OMA6 in blocking buffer (PBS / 0.5%
	BSA). After two washes in PBS, cells were incubated in the presence of Alexa Fluor 488-
	conjugated goat α-mouse secondary antibody for 1 h. Coverslips were then rinsed twice with
	PBS and mounted (Fluoromount-G, 0100-20, Southern Biotech). Images were acquired using
	a Zeiss Axio Observer Z1 fluorescent microscope.
	Ultrastructural analysis. Cells were fixed in 1.6% glutaraldehyde in 0.1 M PBS, rinsed in 0.1
	M cacodylate buffer and post-fixed for 1 h in 1% osmium tetroxide and 1% potassium
	ferrocyanide in 0.1 M cacodylate buffer. Cells were rinsed in distilled water, dehydrated in in
	increasing concentrations of alcohol and lastly embedded in epoxy resin. Contrasted ultrathin
	sections (70 nm) were analyzed under a JEOL 1400 transmission electron microscope
	mounted with a Morada Olympus CCD camera.
	Population doubling (PD) assay. Fibroblasts were seeded at a density of 6600 cells/cm2 in
	DMEM, 10% SVF. At 70-80 % confluence, cells were treated with trypsin / EDTA and the

: 1). Les CSC ont un potentiel métastatique plus élevé que les CBC. Des travaux précédents ont suggéré que la réponse immunitaire chez les patients XP pouvait être altérée, incluant un déficit de l'activité cytolytique des cellules Natural Killer (NK) et une diminution du nombre des lymphocytes T circulants. L'objectif central de cette thèse était, d'identifier des facteurs du microenvironnement impliqués dans la progression des cancer cutanées agressifs, en prenant comme modèle de susceptibilité au cancer, des cellules de patients XP-C. Une analyse transcriptomique comparant les fibroblastes WT et des patients XP-C a permis d'identifier que CLEC2A, un ligand activateur du récepteur NKp65 des cellules NK, est exprimé par les fibroblastes WT mais pas par les fibroblastes XP-C. Nos travaux ont pu montrer une diminution du niveau d'expression de CLEC2A au cours de la sénescence réplicative ; une absence dans les FAC et dans les CSC et que, des facteurs solubles secrétés para les CSC diminuent l'expression de CLEC2A. Ces résultats suggèrent que la perte de CLEC2A peut induire un déficit d'activation des cellules NK au sein du microenvironnement tumoral et dans les dermes des patients XP-C. Par la suite, nous avons élaboré un modèle de culture de peau 3D, dans lequel nous avons introduit des cellules NK, en présence ou absence d'anticorps bloquants CLEC2A. Ce modèle nous a permis de montrer que l'interaction CLEC2A/NKp65 régule l'invasion des CSC via un dialogue entre fibroblastes et cellules NK. Nos résultats suggèrent que l'expression de CLEC2A dans les fibroblastes WT contribue à la surveillance immunitaire dans la peau et que son absence, par des facteurs encore inconnus, favorise le développement des cancers agressifs chez les patients XP-C. CLEC2A peut être une cible dans le combat contre la progression des CSC. Mots clés : Xeroderma pigmentosum; carcinomes spino-cellulaires ; microenvironnement tumoral ; surveillance immunitaire ; CLEC2A

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Scientific article

References

Acknowledgements

Acknowledgements We thank Dr. O. Camuzard (Hôpital Pasteur, Centre Hospitalier Universitaire de Nice, France) for providing biopsies for CAF skin extractions, and F. Tessier (supported by Canceropôle PACA) for bioinformatics assistance. The authors acknowledge the IPCM's Genomic core facility and IRCAN's Cytometry (Cytomed), Microscopy (PICMI) Histology and Genomic core facilities. This work was supported by the French Government (National Research Agency, ANR; CNRS; INSERM) through the 'Investments for the Future' LABEX SIGNALIFE: program reference # ANR-11-LABX-0028-01; UCA (Université Côte d'Azur) and Association René Tourraine. MGM was supported by the Fondation de l'Avenir (PhD fellowship 2015) and LABEX SIGNALIFE; TM was supported by the Fondation ARC (SFI201212055859), the Fondation de l'Avenir, Société Française de Dermatologie, and The Institut National du Cancer. VMB was supported by Centre National de la Recherche Scientifique, Cancéropole PACA, Région Provence-Alpes-Côte d'Azur, Institut National du Cancer and Fondation ARC pour la recherche sur le Cancer.

Modulation of NK receptors during cancer was already described in chapter 3. In the skin context, the interaction between melanoma cells and NK cells leads to the downregulation of activating receptors NKG2D, NKp44, and DNAM-1 which render melanoma cells resistant to NK mediated killing [START_REF] Balsamo | Melanoma cells become resistant to NK-cell-mediated killing when exposed to NK-cell numbers compatible with NK-cell infiltration in the tumor[END_REF].

Together, all these data strongly suggest that the important modulation of NK receptors expression in skin can contribute to the active participation of NK cells in both immunosurveillance and physiopathology of skin diseases.

Altogether, these studies serve to assess whether CLEC2A could serve as a diagnostic tool and a therapeutic target in both XP patients and more generally in patients developing SCC.

Abstract

The ability of cancer cells to invade and disseminate can be affected by components of the surrounded microenvironment particularly fibroblasts and immune cells. Xeroderma pigmentosum (XP) disease is a good model to study the impact of the ME in cancer progression. XP patients are more prone to develop skin cancers compared to the general population. XP-C fibroblasts, by themselves, do not develop skin cancers, but show proinvasive phenotype similar to cancer associated fibroblasts (CAFs) from the general population. To identify dermal components regulating the growth of skin cancers, we performed transcriptomic analysis of 3D-dermal cultures of fibroblasts from healthy and XP-C patients. Interestingly, we identified the expression of CLEC2A, the ligand of the activating Natural Killer (NK) cell receptor NKp65, in healthy but not in XP-C fibroblasts. CLEC2A was also absent in dermal CAFs and was downregulated by secreted factors of squamous cell carcinomas (SCC), suggesting that loss of CLEC2A is part of the transitioning process that WT fibroblasts undergo to become CAFs. Furthermore, the development of 3D organotypic models containing NK cells showed for the first time that CLEC2A/NKp65 interaction regulates NK cellmediated control of SCC invasion by fibroblasts. Taken together, these data strongly suggest a major role of CLEC2A in skin immunosurveillance against cancer cells.

Significance statement

The influence of the interaction between fibroblasts and Natural Killer (NK) cells in cancer progression is poorly explored. In this report, we show that CLEC2A, a ligand of a NK activating receptor, is expressed in dermal fibroblasts from healthy patients and absent in fibroblasts from patients suffering from skin cancer-prone xeroderma pigmentosum disease and in sporadic cancer associated fibroblasts (CAFs). These findings indicate that CLEC2A expression decreases during the transition of fibroblasts to CAFs. Furthermore, using 3D NK immune competent organotypic cultures, we unveil a key regulation of SCC invasion by NK cells through a cross-talk with fibroblasts and a major role of CLEC2A in skin immunosurveillance against cancer cells. S1 to S2 References for SI reference citations

Supplementary Information Text

Epidepth. to do

Supplementary Materials and Methods

Cell lines. U937 (ATCC; CRL-1593.2) and K562 (ATCC; CCL-243).

Quantitative RT-PCR. Quantitative RT-PCR were performed using the following primers: CLEC2A1 (5'-tccatcggttagcgccttg-3'/5' acagaataagtgagaaagccact-3), CLEC2A2 (5'atgattaatccagagctgcgg-3'/5'-tctaaggggtcccagcag-3').

Flow cytometry and imaging flow cytometry. Cells were stained with purified Mo anti-MHCI (DX17, kindly provided by L. Lanier, UCSF, USA, and W6/32, BD Biosciences) followed by PE-conjugated goat anti-Mo mAb (1/50) (BD Biosciences). Fluorescence staining was analyzed on a FACScalibur (BD Biosciences) unit using CellQuest Pro Software (BD Biosciences). Imaging flow cytometry analysis were performed on an ImageStreamX MKII operated by INSPIRE software (Amnis Corporation). Cells were permeabilized using BD Cytofix/Cytoperm kit (BD Biosciences) and stained with purified Mo anti-CLEC2A (OMA1) followed by A488-conjugated goat anti-Mo (BD Biosciences). Data analysis was performed using the IDEAS software (Amnis Corporation).

Retroviral transduction and purification.

Fibroblasts were seeded at a density of 8,000 cells/cm 2 and were transduced with concentrated RV supernatants in serum-free medium for 16 h. As CD24 gene was present in the retroviral vector cassette, transduced cells were purified by flow cytometry cell sorting using FACSAria III (BD Biosciences) following staining with Mo anti-CD24-PE (1/10) (Beckman Coulter) [START_REF] Warrick | Preclinical corrective gene transfer in xeroderma pigmentosum human skin stem cells[END_REF].

Western blot analysis. Total proteins were extracted from fibroblasts in 8 mol/l urea buffer and protein concentrations were determined by Bradford assay (Bio-Rad). 30µg of protein for each condition were separated on 8% SDS-polyacrylamide gels and transferred onto a polyvinyl difluoride membrane (GE Healthcare). Membranes were blocked and incubated with rabbit polyclonal anti-XPC (dilution 1/1,000; Bethyl), or anti-GAPDH (dilution 1/2,000; Abcam).

After washing, membranes were stained with goat anti-rabbit HRP (1/5000; Bethyl) or rabbit anti-mouse (1/5000; Dako) HRP respectively. Blots were revealed using electrochemiluminescence reagents (GE Healthcare).

EdU incorporation. To access EdU incorporation 2 h after 500 J/cm 2 of UVB irradiation the EdU staining the Click-iT Alexa Fluor 488 Imaging Kit (Molecular Probes Invitrogen) was used following the manufacturer's instructions. EdU-stained cells were mounted in standard mounting media and images were acquired a Zeiss Axio Observer.Z1 fluorescent microscope.

Table S1. 22 differentially expressed genes between WT and XP-C fibroblasts

For RSPO3, GREB1L, PDLIM7 two probe sets for the same gene sit side by side. CLEC2A NM_001130711 corresponds to CLEC2A transcript variant 1; CLEC2A NM_207375 corresponds to CLEC2A transcript variant 2.