Dr Sophie Tartare-Deckert

Marie-Dominique Galibert

Dr Sophie Acton

Pr Colin Goding

Dr Fatima Mechta-Grigoriou

Vuyo, Thato, Michelle, Abid, Mohsin, Steph, Claire, Rehana Vicky Saif

Arul, Shanel, Suparna, Supratim, Thato, Phillip Jenna Hapi

Dr Amaal Abrahams

Professor Denver Hendricks

Professor Malcom Collins

Minja Amy Hazel

Netanya, Dale, Robyn Lauren

Ian Andrew

Grant, Rowan, Max, Rich, Nick, Cam Tiffany Aubery

Nikos Chuck

Marissa Juan

Serge Robin

cancer au sein du microenvironnement tumoral des mélanomes

Keywords: cancer, melanoma, cancer associated fibroblasts

which is accompanied by enhanced myofibroblast activity and is important for ECM remodelling in the TME. Furthermore, this ECM remodelling is contributing to the aggressive melanoma behaviour. Future work will investigate the downstream targets of FAPα to shed light on its role in the melanoma TME.

Resumé en Français

Aux stades avancés, le mélanome est le cancer de la peau le plus agressif en raison de son fort potentiel métastatique et sa résistance aux traitements. Malgré les progrès récents réalisés dans sa prise en charge, environ 50 % des patients atteints de mélanome avancé sont en impasse thérapeutique. Une stratégie prometteuse consiste à cibler les fibroblastes associés au mélanome (MAFs) présents dans le microenvironnement tumoral (TME), car les fibroblastes activés par le mélanome sont reconnus comme des acteurs clés dans la tumorigenèse, les métastases et la résistance thérapeutique. La compréhension du ou des mécanismes moléculaires responsables de la transition phénotypique des fibroblastes en MAFs permettra d'élaborer de nouvelles stratégies afin de les inhiber et améliorer le taux de guérison des stades avancés. La majorité des MAFs présentent des propriétés myofibroblastiques et expriment la protéine d'activation des fibroblastes α (FAPα).

Cependant, le rôle de FAPα dans la transition et le maintien du phénotype MAF ainsi que les processus tumoraux contrôlant son expression sont très peu connus. Mon travail repose sur l'étude de la régulation et du rôle de FAPα dans le TME du mélanome. Dans ce contexte, nous avons investigué les molécules de signalisation qui règlent le passage phénotypique des fibroblastes cutanés en MAFs et le rôle de FAPα dans le phénotype MAF et la progression du mélanome. Nos résultats montrent que le milieu conditionné (CM) provenant d'un mélanome invasif conduit les fibroblastes à adopter un phénotype tumoral avec une augmentation des marqueurs moléculaires de MAFs comme αSMA, intégrine β1 et FAPα et pro-invasifs comme YAP. De plus, les fibroblastes traités avec le CM de mélanome invasif présentent des caractéristiques myofibroblastiques, comme la capacité à produire une matrice extracellulaire (ECM) de fibres de collagène et de fibronectine alignées. L'inhibition pharmacologique de la voie de signalisation du TGFβ1 montre que le TGFβ1 est le principal facteur sécrété par les cellules invasives, qui induit la transition phénotypique en MAF. Fait important, les propriétés myofibroblastiques des MAFs dépendent de l'expression de FAPα. L'inhibition de FAPα dans les MAFs, diminue l'expression des marqueurs moléculaires proinvasifs et supprime leur capacité à produire une matrice fibrillaire alignée. Nous avons par la suite établi un modèle cellulaire dans lequel FAPα est surexprimée dans les fibroblastes normaux. La surexpression de FAPα favorise l'expression de marqueurs de MAFs, les propriétés contractiles et le dépôt de fibres d'ECM alignées. De plus, la culture de cellules de mélanome sur des matrices 3D dérivées de fibroblastes sur-exprimant FAPα diminue leur prolifération et augmente leur migration.

Des observations inverses sont obtenues lorsque les cellules de mélanome sont cultivées sur des matrices dérivées de MAFs éteints pour FAPα. En conclusion, nous montrons que TGFβ1 sécrété par des cellules de mélanome invasives est responsable de l'activation des fibroblastes cutanés en un phénotype MAF exprimant FAPα, qui s'accompagne d'une activité myofibroblastique accrue qui est importante pour le remodelage matriciel du stroma tumoral. En outre, ce remodelage de l'ECM contribue au comportement invasif et agressif du mélanome. Les travaux futurs porteront sur les cibles en aval de FAPα afin de faire la lumière sur son rôle dans le TME des mélanomes.

Mots clés: cancer, mélanome, fibroblastes associés au cancer

Abstract in English

Advanced melanoma is the most aggressive skin cancer and is notorious for its tendency to metastasise. Approximately 50% of patients with advanced melanoma experience relapse due to its intrinsic resistance to current therapies. A promising therapeutic strategy is targeting melanoma associated fibroblasts (MAFs) in the melanoma tumour microenvironment (TME) because MAFs, have been implicated as key players in melanoma progression, metastasis and drug resistance. An understanding of the molecular mechanism(s) responsible for the phenotypic switching of skin fibroblasts to MAFs will enable more diverse ways of inhibiting them. In this regard, we are interested in observations that MAFs exhibit myofibroblast properties and that they express fibroblast activation protein α (FAPα). However, whether FAPα is a key player in driving the MAF phenotype and what regulates its expression are poorly understood. To address this, we investigated: 1) the signalling molecules that regulate the phenotypic switching of skin fibroblasts to MAFs and 2) the role of FAPα in the MAF phenotype and melanoma progression. Our results show that conditioned medium (CM) from invasive melanoma stimulated skin fibroblasts to adopt a MAF phenotype, as we observed an increase in mRNA and protein levels of MAF molecular markers αSMA, β1integrin and FAPα. Moreover, skin fibroblasts treated with invasive melanoma CM exhibited myofibroblast characteristics, including the deposition of aligned ECM fibres (fibronectin and collagen) and an increase in protein expression of proinvasive molecular markers such as fibronectin, collagen I and YAP. Importantly, chemical inhibition of transforming growth factor β1 (TGFβ1) in the melanoma CM demonstrated that TGFβ1 is the key signalling molecule secreted by invasive melanoma cells, which is inducing the phenotypic switching of skin fibroblasts to MAFs. Importantly, we demonstrated that MAF myofibroblast properties are dependent on FAPα expression. Our data suggest that silencing FAPα in MAFs, significantly decreased CAF molecular and proinvasive marker expression and abolished their ability to deposit aligned ECM fibres. To further investigate the role of FAPα in the MAF phenotype, we established a cell culture model in which FAPα was overexpressed in skin fibroblasts. FAPα overexpression in skin fibroblasts promoted protein expression of several MAF and proinvasive markers, contractile capacity and the deposition of aligned ECM fibres.

Interestingly, we observed a decrease in melanoma cell proliferation and an increase in their migration on aligned 3D matrices derived from FAPα overexpressing skin fibroblasts and the opposite occurred when melanoma cells were cultured on matrices derived from FAPα-silenced MAFs. We therefore concluded that TGFβ1 secreted by invasive melanoma cells is responsible for activating skin fibroblasts to a FAPα-expressing MAF phenotype,

Plagiarism declaration

I, Ana Popovic, hereby declare that the work on which this thesis is based is my original work (except where indicated with acknowledgements) and that neither the whole nor any part of it has been, is being, or is submitted for another degree in this or any other university, except the University of Côte d'Azur in Nice, France, which has a formal joint award degree agreement for this PhD with the University of Cape Town.

I authorise the University of Cape Town to reproduce either the whole or any part of the contents for research purposes in any manner whatsoever. of melanoma patients present with metastatic lesions at the lymph nodes and 15% of patients experience metastasis at distant organs including the lungs, liver, brain or bone (3,[START_REF] Kozar | Many ways to resistance: How melanoma cells evade targeted therapies[END_REF]. Advanced malignant melanoma is currently treated with chemo-and more recently, with immuno-and targeted therapies, however it is often recalcitrant to treatment regimens (3).

March 2021

Table of Contents

Prognosis is poor for patients with late-stage melanoma, as approximately 50-60% of these patients acquire drug resistance and go into therapeutic failure [START_REF] Kozar | Many ways to resistance: How melanoma cells evade targeted therapies[END_REF][START_REF] Robert | Five-year outcomes with dabrafenib plus trametinib in metastatic melanoma[END_REF]. A possible reason for this, is that melanomas are highly heterogenous both within single tumours but also amongst different tumours and amongst metastases [START_REF] Grzywa | Intratumor and Intertumor Heterogeneity in Melanoma[END_REF]. This heterogeneity is in part caused by the notoriously high mutation rate observed in tumours that are commonly induced by ultraviolet. Furthermore, non-genetic mechanisms can drive melanoma cells to readily switch between different phenotypic states [START_REF] Arozarena | Phenotype plasticity as enabler of melanoma progression and therapy resistance[END_REF][START_REF] Kemper | Phenotype switching: Tumor cell plasticity as a resistance mechanism and target for therapy[END_REF]. The tumour microenvironment (TME) also critically affects malignant progression at both the primary and secondary site and has been shown to contribute to therapeutic resistance [START_REF] Quail | Microenvironmental regulation of tumor progression and metastasis[END_REF][START_REF] Farc | An overview of the tumor microenvironment, from cells to complex networks (Review)[END_REF] The TME, which has been recognized as a hallmark of cancer, is a complex and evolving milieu that consists of not only the tumour cells but also of non-malignant cell types, such as fibroblasts and immune cells as well as the extracellular matrix (ECM) [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF]. Thus, the crosstalk between melanoma cells and their cellular and non-cellular surroundings is playing a key role in disease progression and is impacting the efficacy of anti-cancer therapies [START_REF] Arozarena | Phenotype plasticity as enabler of melanoma progression and therapy resistance[END_REF]. The following section will introduce several topics on melanoma which are relevant for this PhD project, including melanoma progression and classification, the genetic and non-genetic heterogeneity of melanoma, current therapies for melanoma treatment and the melanoma tumour microenvironment.

Melanoma progression and classification

Cutaneous melanoma arises from neural-crest derived pigment producing melanocytes in the epidermis that have undergone genetic alterations, leading to aberrant growth and clonal expansion [START_REF] Gesbert | Le mélanome cutané[END_REF]. While some melanomas arise from pre-existing naevi, the majority of melanoma appears de novo [START_REF] Busse | Role of TGF-β in melanoma[END_REF]. The clinical-histopathological classification of melanoma gives rise to 4 different subtypes: superficial spreading melanoma (SSM), nodular melanoma (NM), acral-lentiginous melanoma (ALM) and lentigo maligna melanoma (LMM) which are depicted by the Clark model of melanoma progression [START_REF] Greenwald | Superficial spreading and nodular melanoma are distinct biological entities: A challenge to the linear progression model[END_REF][START_REF] Greene | A study of tumor progression: The precursor lesions of superficial spreading and nodular melanoma[END_REF]. The Clark model (Figure 1.1) of cutaneous melanoma progression is a classical and well accepted model based on the histopathological analyses of melanoma tissue samples at various stages of progression [START_REF] Greene | A study of tumor progression: The precursor lesions of superficial spreading and nodular melanoma[END_REF]. This model begins with aggregated melanocytes forming a benign nevus in the epidermis that experience abnormal growth to form a dysplastic nevus, which can progress upon clonal expansion to the radial growth phase (RGP), where the melanoma cells are confined to the epidermis [START_REF] Gesbert | Le mélanome cutané[END_REF][START_REF] Miller | Mechanisms of Disease Melanoma[END_REF]. Should the melanoma cells proliferate further and breach the basement membrane, they progress to the vertical growth phase (VGP), as the melanoma cells are able to invade the dermis [START_REF] Gesbert | Le mélanome cutané[END_REF][START_REF] Greene | A study of tumor progression: The precursor lesions of superficial spreading and nodular melanoma[END_REF]. At the molecular level, the benign nevus is driven to dysplasia as a result of driver mutations primarily in the BRAF, NRAS or NF1 genes that occur in approximately 50%, 30% and 15% of melanoma patients, respectively [START_REF] Akbani | Genomic Classification of Cutaneous Melanoma[END_REF]. Progression from a dysplastic nevus to the RGP requires further unchecked mutations and epigenetic changes in cell cycle and cell survival regulators including PTEN, NF1, CDKN2A, and CCND1 that allow the melanoma cells to bypass senescence (Figure 1.1) [START_REF] Gesbert | Le mélanome cutané[END_REF][START_REF] Bittner | Molecular classification of cutaneous malignant melanoma by gene expression profiling[END_REF]. Additionally, approximately 15% of melanomas occur due to a familial genetic predisposition, resulting in germline mutations in CDKN2A (most common) CDK4, TERT, ACD, TERF2IP, POT1, MITF, MC1R, and BAP1 [START_REF] Toussi | Germline mutations predisposing to melanoma[END_REF]. The transition from the RGP to the VGP is characterized by an upregulation of mesenchymal markers such as N-cadherin, SPARC and β-catenin and a downregulation of the cell-cell adhesion molecule E-cadherin (Figure 1.1) [START_REF] Gesbert | Le mélanome cutané[END_REF]. Once the melanoma cells have invaded the dermis they can disseminate through lymphatic or blood vessels and metastasise to distant organs, which is the metastatic phase [START_REF] Gesbert | Le mélanome cutané[END_REF][START_REF] Greene | A study of tumor progression: The precursor lesions of superficial spreading and nodular melanoma[END_REF][START_REF] Arrangoiz | Melanoma Review: Epidemiology, Risk Factors, Diagnosis and Staging[END_REF].

Although the Clark model has often been used as a reference for melanoma progression in studies investigating the molecular mechanisms that are driving melanoma progression, it is not routinely used by clinicians for melanoma diagnosis and classification in patients [START_REF] Gesbert | Le mélanome cutané[END_REF].

The first melanoma classification used by clinicians is the Breslow index which measures the thickness of the melanoma tumour, as a direct correlation exists between the depth of the melanoma in the skin and patient survival [START_REF] Breslow | Thickness, cross-sectional areas and depth of invasion in the prognosis of cutaneous melanoma[END_REF]. Furthermore, the American Joint Committee on Cancer (AJCC) has recommended the use of the TNM (Tumour, Node, Metastasis) classification (stages 0 -IV) which grades the melanoma according to the thickness, the presence of ulceration in biopsies and any evidence of metastatic dissemination or lesions [START_REF] Balch | Final version of 2009 AJCC melanoma staging and classification[END_REF]. More recently following the analysis of RNA, DNA and protein in 333 primary or metastatic samples from 331 melanoma patients, The Cancer Genome Atlas Network has proposed the classification of melanoma according to genetic mutations into 4 categories: BRAF, NRAS, NF-1 and triple negative (wild type) [START_REF] Akbani | Genomic Classification of Cutaneous Melanoma[END_REF]. The data indicated that mutations in BRAF are the most prevalent in melanoma (50%) with the BRAF V600E being the most common, followed by the BRAF V600K and the most rare mutations being BRAF V600R

and BRAF K601E [START_REF] Akbani | Genomic Classification of Cutaneous Melanoma[END_REF]. NRAS mutations occurred in approximately 30% of melanomas and were identified as hotspot mutations where glutamine at position 61 (Q61) was replaced by an arginine (Q61R) (90%), lysine (Q61K) (35%) and less often with leucine (Q61L) or histidine (Q61H). Furthermore, 5% of NRAS mutations also affected glycine at position 12 (G12R / D / A). NF1 which encodes neurofibromin 1 a negative regulator of RAS, was mutated in 14% of the melanomas and the mutations were predicted to be loss of function events that resulted in deleterious alterations [START_REF] Akbani | Genomic Classification of Cutaneous Melanoma[END_REF]. Lastly, the triple negative (wild type) category was evident in approximately 15% of melanomas which did not harbour somatic mutations in BRAF, NRAS or NF1 [START_REF] Akbani | Genomic Classification of Cutaneous Melanoma[END_REF]. It is important to note that the genetic classification of melanomas has become particularly important to determine whether a patient is eligible for immuno-or targeted therapy, as the administration of either immuno-or targeted therapy depends on the BRAF status (3). If the patient has wild type BRAF, they are treated with immune check-point inhibitors and if a V600E/K mutation is identified in BRAF, they can be treated with BRAF and MEK inhibitors or with immunotherapy (3,[START_REF] Luke | Targeted agents and immunotherapies: Optimizing outcomes in melanoma[END_REF].

Current therapies for malignant melanoma

targeted therapies (3). The most commonly used chemotherapeutic drug used for melanoma treatment is Dacarbazine which was initially approved for melanoma in the 1970s [START_REF] Eggermont | Re-evaluating the role of dacarbazine in metastatic melanoma: What have we learned in 30 years?[END_REF]. However, as a monotherapy it was shown to have minimal efficacy in treating malignant melanoma, with the average response rate in patients being less than 5% and the 5 year survival of patients was suggested to be less than 6% [START_REF] Eggermont | Re-evaluating the role of dacarbazine in metastatic melanoma: What have we learned in 30 years?[END_REF][START_REF] Shtivelman | Pathways and therapeutic targets in melanoma[END_REF][START_REF] Fujimura | Treatment of advanced melanoma: Past, present and future[END_REF]. Furthermore, the combination of Dacarbazine treatment with earlier immunotherapies such as Interferon -2b

and Interleukin-2 showed very little improvement in patient survival (3,[START_REF] Fujimura | Treatment of advanced melanoma: Past, present and future[END_REF]. Notably, the landscape of malignant melanoma treatment has changed drastically in the last decade, following the FDA-approval of immune checkpoint / blockade inhibitors and targeted therapies [START_REF] Robert | Five-year outcomes with dabrafenib plus trametinib in metastatic melanoma[END_REF][START_REF] Larkin | Five-Year Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma[END_REF]. The immune checkpoint inhibitors are antibodies against cytotoxic Tlymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1)

receptor /PD-1 ligand (PD-L1) (3,[START_REF] Luke | Targeted agents and immunotherapies: Optimizing outcomes in melanoma[END_REF]. The targeted therapies, BRAF and MEK inhibitors, target the mitogen-activated protein kinase (MAPK) pathway which is constitutively active in melanoma (3,[START_REF] Luke | Targeted agents and immunotherapies: Optimizing outcomes in melanoma[END_REF]. Seeing as these immuno-and targeted therapies significantly improved overall response in approximately 40% of melanoma patients, they are fast becoming the standard of care for unresectable or metastatic melanoma in many countries including the United Kingdom, the United States, Australia and Europe (3,[START_REF] Luke | Targeted agents and immunotherapies: Optimizing outcomes in melanoma[END_REF]. This next section will therefore discuss anti-CTLA-4, anti-PD-1/PD-L1 and BRAF and MAP/ERK kinase inhibitors for the treatment of malignant melanoma.

Cutaneous melanoma is a highly immunogenic tumour and yet melanoma cells are effectively able to evade detection by immune cells [START_REF] Ganguly | Cancerassociated fibroblasts: Versatile players in the tumor microenvironment[END_REF]. To address this, the anti-CTLA-4

antibody binds to the inhibitory checkpoint CTLA-4 receptor that is present on the surface of regulatory T-cells, which prohibits the interaction of T-cells with antigen presenting cells that express the CTLA-4 ligand, B7 co-stimulatory molecule. [START_REF] Ganguly | Cancerassociated fibroblasts: Versatile players in the tumor microenvironment[END_REF][START_REF] Sullivan | An update on the Society for Immunotherapy of Cancer consensus statement on tumor immunotherapy for the treatment of cutaneous melanoma: Version 2.0[END_REF]. The binding of anti-CTLA-4 antibodies to CTLA-4 promotes the activation of T-cells and the inhibition of the immune checkpoint blockade (Figure 1.2 A) [START_REF] Luke | Targeted agents and immunotherapies: Optimizing outcomes in melanoma[END_REF][START_REF] Ganguly | Cancerassociated fibroblasts: Versatile players in the tumor microenvironment[END_REF]. Activated T-cells are then able to illicit an immune response via cytokine secretion, clonal expansion and infiltration into the tumour leading to tumour regression [START_REF] Ganguly | Cancerassociated fibroblasts: Versatile players in the tumor microenvironment[END_REF][START_REF] Marzagalli | Unraveling the crosstalk between melanoma and immune cells in the tumor microenvironment[END_REF]. To date, Ipilimumab is the only FDA approved anti-CTLA-4 antibody for the treatment of malignant melanoma and is used in clinics as a monotherapy or in combination with the anti-PD-1 antibody Nivolumab (Figure 1.2) (3,[START_REF] Luke | Targeted agents and immunotherapies: Optimizing outcomes in melanoma[END_REF] .

T-cell activation can also be supressed is via another receptor-ligand interaction, where ligands PD-L1 and PD-L2 can bind the PD-1 receptor, resulting in a co-inhibitory effect (3,[START_REF] Luke | Targeted agents and immunotherapies: Optimizing outcomes in melanoma[END_REF]. The anti-PD-L1 antibody prevents the binding of PD-L1 or PDL-2 to the PD-1 receptor, thereby promoting T-cell activation (Figure 1.2 A) [START_REF] Mahoney | The next immune-checkpoint inhibitors: Pd-1/pd-l1 blockade in melanoma[END_REF]. The PD-1 receptor is expressed on the surface of immune cells including T-cells, melanoma cells express the ligands PD-L1 and PD-L2 and fibroblasts present in the TME were shown to express the ligand PD-L2 [START_REF] Yi | Regulation of PD-L1 expression in the tumor microenvironment[END_REF][START_REF] Mhaidly | Fibroblast heterogeneity in tumor microenvironment: Role in immunosuppression and new therapies[END_REF][START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF]. In 2014, Nivolumab became the first FDA-approved anti-PD-1 antibody for the treatment of unresectable or metastatic melanoma and subsequently, Pembrolizumab was also approved by the FDA for malignant melanoma treatment in 2015 (3,[START_REF] Luke | Targeted agents and immunotherapies: Optimizing outcomes in melanoma[END_REF][START_REF] Fujimura | Treatment of advanced melanoma: Past, present and future[END_REF]. Currently, Nivolumab and Pembrolizumab are administered as monotherapies or

Ipilimumab can be administered in combination Nivolumab with as a combo-therapy (3,[START_REF] Fujimura | Treatment of advanced melanoma: Past, present and future[END_REF][START_REF] Babacan | Treatment Options for Advanced Melanoma After Anti-PD-1 Therapy[END_REF]. Immunotherapies have improved overall and relapse free survival in patients with late stage (stage III and IV) melanoma, however, immune-related adverse effects are particularly common in these patients including diarrhoea, fatigue, skin rash, nausea and headaches and joint pain (3,[START_REF] Sullivan | An update on the Society for Immunotherapy of Cancer consensus statement on tumor immunotherapy for the treatment of cutaneous melanoma: Version 2.0[END_REF] The administration of ant-CTLA4 and anti-PD-1 immunotherapies does not depend on the melanoma genetic profile, whereas the use of BRAF and MAP/ERK kinase inhibitors is currently used to treat patients with melanoma harbouring the BRAF V600E/K mutation [START_REF] Fujimura | Treatment of advanced melanoma: Past, present and future[END_REF][START_REF] Alqathama | BRAF in malignant melanoma progression and metastasis: potentials and challenges[END_REF].

(Figure 1.2 C)
The molecular consequence of the BRAF V600 mutation is the constitutive activation of the MAPK pathway [START_REF] Poulikakos | Mutant BRAF melanomas-dependence and resistance[END_REF]. BRAF encodes the rapidly accelerated fibrosarcoma (RAF) serine / threonine kinases (isoforms: ARAF, BRAF and CRAF) that constitutively dimerise due to the V600E mutation [START_REF] Alqathama | BRAF in malignant melanoma progression and metastasis: potentials and challenges[END_REF][START_REF] Poulikakos | Mutant BRAF melanomas-dependence and resistance[END_REF]. Seeing as BRAF and its isoforms are upstream of the MAPK signalling cascade they further induce its constitutive activation [START_REF] Poulikakos | Mutant BRAF melanomas-dependence and resistance[END_REF][START_REF] Eroglu | Combination therapy with BRAF and MEK inhibitors for melanoma: Latest evidence and place in therapy[END_REF]. The MAPK signalling cascade has been shown to play a role in key processes of melanomagenesis such as differentiation, survival, proliferation, metastasis, invasion and angiogenesis [START_REF] Alqathama | BRAF in malignant melanoma progression and metastasis: potentials and challenges[END_REF].

Briefly, this signalling pathway (Figure 1.2 B) is activated by extracellular signals binding to G-coupled receptors or receptor tyrosine kinase (RTK) at the cell membrane, which then activates intracellular GTPase NRAS (also KRAS and HRAS) to NRAS-GTP that is able to further activate all 3 isoforms of the RAF protein including BRAF [START_REF] Alqathama | BRAF in malignant melanoma progression and metastasis: potentials and challenges[END_REF][START_REF] Poulikakos | Mutant BRAF melanomas-dependence and resistance[END_REF]. This results in the phosphorylation of downstream MAP/ERK kinase and the initiation of the MAPK signalling cascade by further phosphorylation of ERK1/2 which can induce transcriptional and translational signalling [START_REF] Alqathama | BRAF in malignant melanoma progression and metastasis: potentials and challenges[END_REF][START_REF] Poulikakos | Mutant BRAF melanomas-dependence and resistance[END_REF]. Vemurafenib was the first selective oral BRAF small molecule inhibitor approved in 2011 by the FDA for advanced melanoma treatment and in 2013 a second selective BRAF inhibitor, Dabrafenib, was approved by the FDA (Figure 1.2 B) (3,[START_REF] Kakadia | Mechanisms of resistance to BRAF and MEK inhibitors and clinical update of us food and drug administration-approved targeted therapy in advanced melanoma[END_REF]. Furthermore, the MAP/ERK kinase inhibitor Trametinib was FDA-approved in 2013 and compared to BRAF inhibitors Trametinib was to be partially successful in treating melanomas harbouring NRAS mutations in clinical trials (Figure 1.2 B) (3,[START_REF] Ascierto | MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF mutations: A non-randomised, open-label phase 2 study[END_REF].

Vemurafenib, Dabrafenib and Trametinib can all be administered as monotherapies and Dabrafenib and Trametinib can also be administered as a BRAF and MAP/ERK kinase inhibitor combination therapy (3,[START_REF] Robert | Five-year outcomes with dabrafenib plus trametinib in metastatic melanoma[END_REF]. Furthermore, in 2015, the FDA approved the administration of the combo-therapy with the oral MAP/ERK kinase inhibitors Cobimetinib and Vemurafenib (Figure 1.2 C) (3,[START_REF] Eroglu | Combination therapy with BRAF and MEK inhibitors for melanoma: Latest evidence and place in therapy[END_REF].

It is important to note that there is currently no ideal therapeutic strategy for advanced or metastatic melanoma. Therefore, clinical trials with various immune checkpoint inhibitors (e.g., Durvalumab, CK-301, Avelumab, Atezolizumab and Resiquimob) and targeted therapies which predominantly include inhibitors targeting the cKIT/P13K/AKT signalling pathway (e.g. Imatinib, Nilotinib, Dasatinib, Sunitinib, PI-103, BKM120, GSK2636771, INCB050465, IPI-549, MK2206, ASN003) are currently in progress, as well as clinical trials with different combination therapies using traditional chemo-and immuno-therapies (3).

Unfortunately, approximately 50-60% of melanoma patients treated with the current FDAapproved immunno-and target therapies respond transiently but often progress to develop therapeutic resistance [START_REF] Robert | Five-year outcomes with dabrafenib plus trametinib in metastatic melanoma[END_REF][START_REF] Larkin | Five-Year Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma[END_REF]. The resistant mechanisms employed my melanoma cells to circumvent these therapies are major research area in the field of melanoma and the genetic and non-genetic heterogeneity of melanoma have been described as key contributors to melanoma therapeutic resistance [START_REF] Kozar | Many ways to resistance: How melanoma cells evade targeted therapies[END_REF][START_REF] Rambow | Melanoma plasticity and phenotypic diversity: Therapeutic barriers and opportunities[END_REF].

A) B) C)

Genetic and non-genetic heterogeneity of melanoma

A key feature that makes malignant melanoma highly aggressive is heterogeneity within the same tumour and between primary and metastatic sites, as well as among metastases.

Differences between primary tumour and metastatic tumours and among metastases constitute intra-patient intertumoral heterogeneity [START_REF] Grzywa | Intratumor and Intertumor Heterogeneity in Melanoma[END_REF]. The coexistence of tumour cells with distinct phenotypic and molecular characteristics within a tumour is known as intratumour heterogeneity, which hinders accurate diagnosis and effective treatment [START_REF] Arozarena | Phenotype plasticity as enabler of melanoma progression and therapy resistance[END_REF]. It has been postulated that several subclone populations exist within the tumour which can experience varied genetic mutations in a number of different genes [START_REF] Grzywa | Intratumor and Intertumor Heterogeneity in Melanoma[END_REF][START_REF] Andor | Pan-cancer analysis of the extent and consequences of intratumor heterogeneity[END_REF]. Indeed, it has been reported that in comparison to all cancer types, melanoma has the highest mean mutation rate [START_REF] Akbani | Genomic Classification of Cutaneous Melanoma[END_REF][START_REF] Hodis | A landscape of driver mutations in melanoma[END_REF]. This has been depicted by the various possible somatic mutations in the oncogenes BRAF, NRAS and NF1 [START_REF] Akbani | Genomic Classification of Cutaneous Melanoma[END_REF]. Furthermore, somatic mutations in melanoma can occur in genes involved in RTK/PI3KT/AKT cascade, including KIT, ERBB4, PDGFRα, PIK3CA, PTEN, PIK3R1, AKT, RAC1 and CDKN2A as well as downstream targets of this pathway such as MDM2, TP53, MITF and [START_REF] Grzywa | Intratumor and Intertumor Heterogeneity in Melanoma[END_REF][START_REF] Kakadia | Mechanisms of resistance to BRAF and MEK inhibitors and clinical update of us food and drug administration-approved targeted therapy in advanced melanoma[END_REF][START_REF] Todd | The MAPK pathway functions as a redundant survival signal that reinforces the PI3K cascade in c-Kit mutant melanoma[END_REF] The above mentioned genes undergo mutations as a result of genetic events, including amplification, alternative splicing, impaired DNA damage repair, errors in replication, defects in chromosome segregation, mutagenesis induced by ultraviolet and attenuation of telomeres [START_REF] Kemper | Phenotype switching: Tumor cell plasticity as a resistance mechanism and target for therapy[END_REF][START_REF] Kakadia | Mechanisms of resistance to BRAF and MEK inhibitors and clinical update of us food and drug administration-approved targeted therapy in advanced melanoma[END_REF][START_REF] Rambow | Melanoma plasticity and phenotypic diversity: Therapeutic barriers and opportunities[END_REF]. One of the consequences of these somatic mutations is the constitutive activation of MAPK signalling.

Importantly, the activation of the RTK/PI3K/AKT pathway has been described to compensate for MAPK inhibition induced by targeted therapies, thereby promoting melanoma cell proliferation and survival [START_REF] Kemper | Phenotype switching: Tumor cell plasticity as a resistance mechanism and target for therapy[END_REF][START_REF] Kakadia | Mechanisms of resistance to BRAF and MEK inhibitors and clinical update of us food and drug administration-approved targeted therapy in advanced melanoma[END_REF].

Seeing as MAPK signalling is critical for melanoma cell proliferation and survival, it is of great concern that approximately 51-58% of patients may develop secondary genetic mutations in response to treatment with the BRAF inhibitors, Vemurafenib and Dabrafenib [START_REF] Kemper | Phenotype switching: Tumor cell plasticity as a resistance mechanism and target for therapy[END_REF]. As a resistant mechanism in response to BRAF inhibition, secondary genetic mutations arise in melanomas in order to reactivate the MAPK pathway [START_REF] Poulikakos | Mutant BRAF melanomas-dependence and resistance[END_REF][START_REF] Kakadia | Mechanisms of resistance to BRAF and MEK inhibitors and clinical update of us food and drug administration-approved targeted therapy in advanced melanoma[END_REF][START_REF] Todd | The MAPK pathway functions as a redundant survival signal that reinforces the PI3K cascade in c-Kit mutant melanoma[END_REF]. These secondary mutations occur most often in BRAF ( frequency of 25%) and NRAS (frequency of 22%)

and can also occur in other genes that are involved in MAPK signalling including NF1, MAPK 1/2 and ERK1/2 [START_REF] Grzywa | Intratumor and Intertumor Heterogeneity in Melanoma[END_REF][START_REF] Kakadia | Mechanisms of resistance to BRAF and MEK inhibitors and clinical update of us food and drug administration-approved targeted therapy in advanced melanoma[END_REF]. Although genetic heterogeneity provides a plausible explanation for the resistance mechanisms employed by melanomas, which primarily involve MAPK and AKT signalling, melanomas also display non-genetic heterogeneity which can also contribute to therapeutic resistance [START_REF] Kemper | Phenotype switching: Tumor cell plasticity as a resistance mechanism and target for therapy[END_REF]. The genetic mechanisms that melanoma employs as resistance mechanisms against targeted therapies are summarised graphically in Figure 1.4.

In addition to genetic heterogeneity, it is fast becoming accepted that non-genetic heterogeneity is also a key feature of melanomas that contributes to tumour progression and therapeutic resistance [START_REF] Kozar | Many ways to resistance: How melanoma cells evade targeted therapies[END_REF][START_REF] Arozarena | Phenotype plasticity as enabler of melanoma progression and therapy resistance[END_REF][START_REF] Rambow | Melanoma plasticity and phenotypic diversity: Therapeutic barriers and opportunities[END_REF]. Non-genetic heterogeneity is characterised by melanoma cell plasticity, as it has been shown that within the tumour, melanoma cells exist in different phenotypic states and can readily switch between distinct transcriptional programs to adapt to the tumour microenvironment pressure and conditions [START_REF] Kozar | Many ways to resistance: How melanoma cells evade targeted therapies[END_REF][START_REF] Arozarena | Phenotype plasticity as enabler of melanoma progression and therapy resistance[END_REF][START_REF] Rambow | Melanoma plasticity and phenotypic diversity: Therapeutic barriers and opportunities[END_REF]. For example, some melanoma cells might be highly differentiated and melanocytic, whereas other cells might be proliferating faster than others, as some cells might be in a low-cycling dormant-like, resembling a more undifferentiated mesenchymal-like state [START_REF] Rambow | Melanoma plasticity and phenotypic diversity: Therapeutic barriers and opportunities[END_REF]. Phenotypic switching of melanoma cells has been shown to occur as a result of reversible epigenetic changes caused by but not limited to chromatin remodelling, microRNAs, long non-coding RNAs and histone modifications [START_REF] Kozar | Many ways to resistance: How melanoma cells evade targeted therapies[END_REF][START_REF] Rambow | Melanoma plasticity and phenotypic diversity: Therapeutic barriers and opportunities[END_REF]. These epigenetic changes are thought to occur in melanoma cells as an adaptive response to microenvironmental cues such as hypoxia, glucose or amino acid deprivation, ECM-derived mechanical cues, secreted and inflammatory factors, including transforming growth factor β (TGFβ) and tumour necrosis factor α (TNFα) and in response to immuno-and target therapies [START_REF] Kozar | Many ways to resistance: How melanoma cells evade targeted therapies[END_REF][START_REF] Kemper | Phenotype switching: Tumor cell plasticity as a resistance mechanism and target for therapy[END_REF][START_REF] Rambow | Melanoma plasticity and phenotypic diversity: Therapeutic barriers and opportunities[END_REF][START_REF] Friedl | Review Cancer Invasion and the Microenvironment : Plasticity and Reciprocity[END_REF]. In response to these cues, melanoma cells are able to temporarily change their phenotype with regards to their proliferation, cell cycling, metabolic status and motility [START_REF] Kemper | Phenotype switching: Tumor cell plasticity as a resistance mechanism and target for therapy[END_REF][START_REF] Rambow | Melanoma plasticity and phenotypic diversity: Therapeutic barriers and opportunities[END_REF].

To date melanoma cells have been described to exist in several phenotypic states including the proliferative, invasive, melanocytic, differentiated, undifferentiated, neural crest stem cell-like, starved as well as transitory states (Figure 1.3) [START_REF] Hoek | Metastatic potential of melanomas defined by specific gene expression profiles with no BRAF signature[END_REF][START_REF] Hoek | In vivo switching of human melanoma cells between proliferative and invasive states[END_REF][START_REF] Rambow | Toward Minimal Residual Disease-Directed Therapy in Melanoma[END_REF][START_REF] Wouters | Robust gene expression programs underlie recurrent cell states and phenotype switching in melanoma[END_REF][START_REF] Tsoi | Multi-stage Differentiation Defines Melanoma Subtypes with Differential Vulnerability to Drug-Induced Iron-Dependent Oxidative Stress[END_REF]. Importantly, the proliferative and invasive states have been identified in patient tumours using RNA sequencing [START_REF] Hoek | In vivo switching of human melanoma cells between proliferative and invasive states[END_REF]. Single-cell RNA sequencing further identified the differentiated, starved, neural crest-like states, as well as the proliferative and invasive states in primary melanoma cells that were isolated from biopsies of melanoma patients, who were treated with BRAF or MAP/ERK kinase inhibitors [START_REF] Rambow | Toward Minimal Residual Disease-Directed Therapy in Melanoma[END_REF][START_REF] Tirosh | Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNAseq[END_REF]. These phenotypic states have been linked to distinct transcriptomic profiles and gene signatures in melanoma cells [START_REF] Hoek | Metastatic potential of melanomas defined by specific gene expression profiles with no BRAF signature[END_REF][START_REF] Hoek | In vivo switching of human melanoma cells between proliferative and invasive states[END_REF][START_REF] Rambow | Toward Minimal Residual Disease-Directed Therapy in Melanoma[END_REF][START_REF] Tsoi | Multi-stage Differentiation Defines Melanoma Subtypes with Differential Vulnerability to Drug-Induced Iron-Dependent Oxidative Stress[END_REF][START_REF] Carreira | Mitf regulation of Dia1 controls melanoma proliferation and invasiveness[END_REF][START_REF] Müller | Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma[END_REF]. More specifically, changes in the expression of the master regulator microphthalmia-associated transcription factor (MITF) has been associated with melanoma phenotypic switching [START_REF] Rambow | Melanoma plasticity and phenotypic diversity: Therapeutic barriers and opportunities[END_REF][START_REF] Carreira | Mitf regulation of Dia1 controls melanoma proliferation and invasiveness[END_REF][START_REF] Wellbrock | Elevated expression of MITF counteracts B-RAF-stimulated melanocyte and melanoma cell proliferation[END_REF]. MITF is a key transcription factor that regulates melanocyte development and melanoma progression and has been implicated in cellular processes such as differentiation, survival, cell cycle regulation, senescence bypass, autophagy and lysosomal production and regulation [START_REF] Goding | Mitf-the first 25 years[END_REF]. Furthermore MITF plays a role in genetic processes such as DNA damage repair and chromosome stability [START_REF] Goding | Mitf-the first 25 years[END_REF]. With regards to melanoma phenotypic states, MITF high expressing melanoma cells have been associated

with a more proliferative and differentiated state, whereas MITF low expressing melanoma cells have been characterised as more aggressive, undifferentiated and mesenchymal-like cells that are associated with invasion and therapeutic resistance [START_REF] Hoek | In vivo switching of human melanoma cells between proliferative and invasive states[END_REF][START_REF] Carreira | Mitf regulation of Dia1 controls melanoma proliferation and invasiveness[END_REF][START_REF] Wellbrock | Elevated expression of MITF counteracts B-RAF-stimulated melanocyte and melanoma cell proliferation[END_REF].

A second transcriptomic signature that has been associated with the melanoma cell phenotypic switch, which is inversely correlated to MITF expression is the expression of the receptor tyrosine kinase AXL [START_REF] Müller | Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma[END_REF][START_REF] Sensi | Human cutaneous melanomas lacking MITF and melanocyte differentiation antigens express a functional Axl receptor kinase[END_REF]. This receptor is expressed by cancer cells including melanoma cells as well as immune cells [START_REF] Zhu | AXL receptor tyrosine kinase as a promising anti-cancer approach: Functions, molecular mechanisms and clinical applications[END_REF]. The binding of AXL and its ligand growth arrest-specific protein 6 (GAS6) induces intracellular signalling that can affect cancer progression, metastasis and drug resistance [START_REF] Zhu | AXL receptor tyrosine kinase as a promising anti-cancer approach: Functions, molecular mechanisms and clinical applications[END_REF]. In melanoma, compared to the MITF hgih AXL low signature which promotes a more proliferative and drug sensitive phenotype, the MITF low AXL high signature is specifically associated with an invasive and drug resistant phenotype [START_REF] Müller | Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma[END_REF][START_REF] Sensi | Human cutaneous melanomas lacking MITF and melanocyte differentiation antigens express a functional Axl receptor kinase[END_REF]. Interestingly, MITF low AXL high melanoma cells that acquired resistance to targeted BRAF therapy display myofibroblast-like features with the ability to produce and remodel a drug-tolerant ECM [START_REF] Girard | A feed-forward mechanosignaling loop confers resistance to therapies targeting the MAPK pathway in BRAF-mutant melanoma[END_REF]. Importantly, the MITF low AXL high expression was shown to be common in BRAF mutated melanoma and has been identified in patients who relapsed following BRAF and MAP/ERK kinase inhibitor treatment [START_REF] Müller | Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma[END_REF].

Melanoma cells that have the MITF low AXL high signature have also been associated with a more undifferentiated phenotype which has been shown in part, to occur as a result of the epithelial to mesenchymal transition (EMT) marker expression [START_REF] Kozar | Many ways to resistance: How melanoma cells evade targeted therapies[END_REF][START_REF] Rambow | Melanoma plasticity and phenotypic diversity: Therapeutic barriers and opportunities[END_REF]. It is important to note that melanoma cells cannot undergo EMT as they arise from the neuroectoderm but an association MITF/AXL expression was shown to associated with SOX10 and SOX9 which promote the epithelial-like and mesenchymal-like phenotypes respectively [START_REF] Rambow | Melanoma plasticity and phenotypic diversity: Therapeutic barriers and opportunities[END_REF]. Therefore, MITF low AXL high expressing melanoma cells have been associated with SOX10 low SOX9 high expression and the opposite was shown for MITF hgih AXL low expressing cells. Furthermore, lower levels of SOX10 have been shown to contribute to therapeutic resistance in part via the transcription factors JUN and/or AP-1 and TEAD [START_REF] Tsoi | Multi-stage Differentiation Defines Melanoma Subtypes with Differential Vulnerability to Drug-Induced Iron-Dependent Oxidative Stress[END_REF][START_REF] Shaffer | Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance[END_REF]. The SOX10 low SOX9 high signature has been shown to induce a mesenchymal-like phenotype in melanoma cells with the increased expression of mesenchymal markers including vimentin and platelet derived growth factor receptor β (PDGFRβ) [START_REF] Diazzi | Bad neighborhood: Fibrotic stroma as a new player in melanoma resistance to targeted therapies[END_REF]. This signature has also been associated with melanoma therapeutic resistance [START_REF] Girard | A feed-forward mechanosignaling loop confers resistance to therapies targeting the MAPK pathway in BRAF-mutant melanoma[END_REF][START_REF] Diazzi | Bad neighborhood: Fibrotic stroma as a new player in melanoma resistance to targeted therapies[END_REF]. Additionally, the HIPPO pathway has also been implicated in the invasive mesenchymal-like phenotype, as there is some evidence for the nuclear accumulation of transcription co-factors YAP/TAZ in melanoma cells promotes resistance to BRAF inhibitors. Pharmacological inhibition of YAP function in melanoma cells decreased ERK1/2 signalling and tumour growth, and enhances BRAF inhibitor efficacy [START_REF] Girard | A feed-forward mechanosignaling loop confers resistance to therapies targeting the MAPK pathway in BRAF-mutant melanoma[END_REF][START_REF] Wang | Verteporfin inhibits YAP function through up-regulating 14-3-3σ sequestering YAP in the cytoplasm[END_REF]. Interestingly, melanoma cells in a highly differentiated and melanocytic state characterised by MITF high expression have also been shown to play a role in resistance to BRAF and MAP/ERK kinase targeted therapies, via the rheostatic (on/off) regulation of the transcription factors, PAX3 and BRN2 [START_REF] Wellbrock | Elevated expression of MITF counteracts B-RAF-stimulated melanocyte and melanoma cell proliferation[END_REF][START_REF] Smith | A PAX3/BRN2 rheostat controls the dynamics of BRAF mediated MITF regulation in MITFhigh/AXLlow melanoma[END_REF]. On the other hand, BRAF inhibitor treatment was also shown to enrich a small population of melanoma cells in a dedifferentiated state, termed BRAF inhibitor persister cells. These persister cells constitutively expressed the Aryl hydrocarbon Receptor (AhR) transcription factor and were shown to promote relapse [START_REF] Toussi | Germline mutations predisposing to melanoma[END_REF].

The different pathways and expression patterns that have been described above, to play a role in non-genetic resistance to targeted therapy in melanoma are summarised graphically in Figure 1.4.

In light of the various and complex signalling pathways likely to be involved in melanoma phenotypic switching, an updated nomenclature has been proposed for 6 distinct phenotypic states: hyper-differentiated, melanocytic, intermediate, starved, undifferentiated and neural crest stem cell-like [START_REF] Rambow | Melanoma plasticity and phenotypic diversity: Therapeutic barriers and opportunities[END_REF]. To this end the evidence for the role of phenotypic switching of melanoma cells as an adaptive process involved in non-genetic resistance is convincing but the investigation into other transitory phenotypic states and the factors that influence and regulate this phenotypic switching is ongoing. Given that melanoma cells can switch between these different phenotypic states in response to external cues from the TME, it is critical to understand the paracrine signalling between melanoma cells and the different cellular and non-cellular components of the TME.

The melanoma tumour microenvironment 1.2.1 An overview of the melanoma tumour microenvironment

Melanomas, like all solid tumours coexist with their tumour microenvironment (TME) which comprises of the vasculature and the stroma. The stroma is a dynamic and plastic milieu consisting of the extracellular matrix (ECM) and several non-cancerous cells types including cancer associated fibroblasts (CAFs), endothelial cells, tumour associated macrophages, regulatory T-cells, myeloid-derived suppressor cells, tumour associated neutrophils, cluster differentiation (CD) lymphocytes (CD4+ and CD8+), dendritic cells, lymphatic cells and nerve fibers [START_REF] Quail | Microenvironmental regulation of tumor progression and metastasis[END_REF][START_REF] Villanueva | Melanoma and the tumor microenvironment[END_REF][START_REF] Falcone | Tumor Microenvironment: Implications in Melanoma Resistance to Targeted Therapy and Immunotherapy[END_REF]. Importantly, the TME has been shown to contribute to all aspects of melanoma progression including tumour growth, metastasis, angiogenesis, resistance to immuno-and targeted therapies and more recently the TME has also been shown to promote melanoma cell phenotypic switching [START_REF] Girard | A feed-forward mechanosignaling loop confers resistance to therapies targeting the MAPK pathway in BRAF-mutant melanoma[END_REF][START_REF] Villanueva | Melanoma and the tumor microenvironment[END_REF][START_REF] Ju | The role of melanoma cell-stroma interaction in cell motility, invasion, and metastasis[END_REF][START_REF] Hirata | Tumor microenvironment and differential responses to therapy[END_REF][START_REF] Henke | Extracellular Matrix in the Tumor Microenvironment and Its Impact on Cancer Therapy[END_REF][START_REF] Miskolczi | Collagen abundance controls melanoma phenotypes through lineage-specific microenvironment sensing[END_REF]. Melanoma cells interact continuously with the TME in a paracrine fashion via secreted cytokines, chemokines, growth factors, extracellular vesicles, direct cell to cell interactions and via mechanical cues, especially with the ECM [START_REF] Villanueva | Melanoma and the tumor microenvironment[END_REF][START_REF] Brandner | Melanoma's connections to the tumour microenvironment[END_REF][START_REF] Kai | The Extracellular Matrix Modulates the Metastatic Journey[END_REF]. Furthermore, the cells present in the TME also interact in a paracrine manner with one another and the ECM, forming complex networks thus further promoting melanoma progression and drug resistance [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF][START_REF] Ju | The role of melanoma cell-stroma interaction in cell motility, invasion, and metastasis[END_REF]. The following section will introduce CAFs with regards to their properties and functions, discuss ECM remodelling by CAFs and give details about the activation of CAFs in the TME.

The properties and functions of cancer associated fibroblasts

CAFs play several important roles in the TME such as contributing to tumour growth, invasion, metastasis, immune suppression and angiogenesis [START_REF] Sahai | A framework for advancing our understanding of cancer-associated fibroblasts[END_REF]. CAFs execute these roles by remodelling the ECM, secreting soluble factors, cytokines and chemokines and by exchanging or depleting metabolites [START_REF] Sahai | A framework for advancing our understanding of cancer-associated fibroblasts[END_REF]. In the melanoma TME, melanoma associated fibroblasts (MAFs) have been identified as key players in melanoma progression and therapeutic resistance by secreting cytokines and chemokines that influence proliferation (e.g. chemokine C-X-C motif ligand 1 (CXCL1)), metastasis (e.g. chemokine C-C motif ligand 2 (CCL2) , interleukin 1 (IL), secreted frizzled-related protein 2 (sFRP2)) and angiogenesis (e.g. CXCL14, vascular endothelial growth factor (VEGF), sFRP2) and drug resistance (e.g. hepatocyte growth factor, IL-6, IL-8, and CXCL1) [START_REF] Zhou | Perspective of Targeting Cancer-Associated Fibroblasts in Melanoma[END_REF][START_REF] Kaur | SFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance[END_REF]. Furthermore, ECM remodelling by MAFs has been described to play a role in melanoma cell metastasis, invasion and resistance to targeted therapies [START_REF] Goetz | Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis[END_REF][START_REF] Hirata | Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin β1/FAK Signaling[END_REF][START_REF] Hutchenreuther | Why target the tumor stroma in melanoma?[END_REF]. Furthermore, specific CAF clusters which were distinguished according to ECM and TGFβ signalling signatures have been associated with primary resistance to immunotherapies in melanoma [START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF].

All activated fibroblasts in the TME are recognized as CAFs and most likely originated from resident fibroblasts but may also arise from mesenchymal stem cells (MSCs) (bone marrow or adipose derived), endothelial cells and epithelial cancer cells that have undergone EMT [START_REF] Sahai | A framework for advancing our understanding of cancer-associated fibroblasts[END_REF]. To date, there is no unique marker by which CAFs can be identified, as CAFs express a combination of markers including α-smooth muscle actin (αSMA), fibroblast activation protein-α (FAPα), β1integrin (CD29), fibroblast specific protein 1 (FSP1), caveolin 1 (CAV1), podoplanin (PDPN) and platelet derived growth factor receptor β (PDGFRβ) [START_REF] Sahai | A framework for advancing our understanding of cancer-associated fibroblasts[END_REF][START_REF] Kalluri | Fibroblasts in cancer[END_REF].

Furthermore, CAFs can express various combinations of these markers and at various levels [START_REF] Mhaidly | Fibroblast heterogeneity in tumor microenvironment: Role in immunosuppression and new therapies[END_REF]. Given the different cells of origin of CAFs and their expression of various markers, it is not surprising that CAFs are extremely heterogenous [START_REF] Sahai | A framework for advancing our understanding of cancer-associated fibroblasts[END_REF]. The heterogeneity of CAFs in the breast and ovarian cancer TME was demonstrated by 2 studies from the same group that identified 4 different CAF subpopulations [START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF][START_REF] Givel | MiR200regulated CXCL12β promotes fibroblast heterogeneity and immunosuppression in ovarian cancers[END_REF]. The authors characterized the 4 CAF subpopulations according to the expression levels (negative, lownegative, low, medium, medium-high or high) of known CAF markers: FAPα, αSMA, β1intergin, FSP1 and CAV1 [START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF][START_REF] Givel | MiR200regulated CXCL12β promotes fibroblast heterogeneity and immunosuppression in ovarian cancers[END_REF]. The CAF subpopulations were distinguished as follows: the CAF-S1 population expressed high levels of FAPα, medium levels of αSMA, medium to high levels of β1integrin, medium levels of FSP1, medium to high levels PDGFRβ and low levels of CAV1 . (Figure 1.5 A). The CAF-S2 population expressed low levels of β1integrin, negative to low levels of FSP1 and did not express αSMA, PDGFRβ or CAV1 (Figure 1.5 A). The CAF-S3 population expressed medium levels of β1integrin, medium to high levels of αSMA, medium levels of PDGFRβ and did not express FAPα, or CAV1 (Figure 1.5 A). Lastly, the CAF-S4 population expressed low to negative levels of FAPα, high levels of β1integrin and αSMA, low to medium levels of FSP1 and PDGFRβ and low levels of CAV1 (Figure 1.5 A) [START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF][START_REF] Givel | MiR200regulated CXCL12β promotes fibroblast heterogeneity and immunosuppression in ovarian cancers[END_REF]. The CAF-S1 and CAF-S4 populations have further been identified in the TME of several cancers including melanoma [START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF].

The CAF-S1 and the CAF-S4 subsets were identified in primary tumours and metastatic lesions of the lymph node and were found to contribute to invasion by exhibiting myofibroblastic properties [START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF][START_REF] Pelon | Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms[END_REF][START_REF] Bonneau | A subset of activated fibroblasts is associated with distant relapse in early luminal breast cancer[END_REF]. However, the CAF-S1 and CAF-S4 populations were found to have slightly different functions. The CAF-S1 population was associated with ECM remodelling, adhesion and wound healing properties, whereas the CAF-S4 population was

shown to be perivascular and highly contractile [START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF][START_REF] Givel | MiR200regulated CXCL12β promotes fibroblast heterogeneity and immunosuppression in ovarian cancers[END_REF][START_REF] Pelon | Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms[END_REF][START_REF] Bonneau | A subset of activated fibroblasts is associated with distant relapse in early luminal breast cancer[END_REF]. Furthermore another key difference between these two CAF populations is the difference in FAPα expression levels as CAF-S1 express high and CAF-S4 express low to negative FAPα levels, respectively [START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF][START_REF] Givel | MiR200regulated CXCL12β promotes fibroblast heterogeneity and immunosuppression in ovarian cancers[END_REF][START_REF] Pelon | Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms[END_REF][START_REF] Bonneau | A subset of activated fibroblasts is associated with distant relapse in early luminal breast cancer[END_REF]. At the metastatic lymph node site, the CAF-S1 subset was further found to contribute to the proliferation and EMT of carcinoma cells and the CAF-S4 subset promoted carcinoma cell invasion in a 3D assay [START_REF] Pelon | Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms[END_REF]. Furthermore, these two populations were found to promote carcinoma invasion via complementary transforming growth factor β (TGFβ), CXCL12 and NOTCH signalling [START_REF] Pelon | Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms[END_REF]. Furthermore, using the specific CAF-S1 and CAF-S4 molecular signatures, analysis of bulk or single cell data identified populations resembling the CAF-S1 and CAF-S4 in the TME across various carcinomas (breast, ovarian, lung, head and neck and pancreatic), colorectal cancer and melanoma [START_REF] Pelon | Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms[END_REF][START_REF] Li | Reference component analysis of single-cell transcriptomes elucidates cellular heterogeneity in human colorectal tumors[END_REF][START_REF] Bartoschek | Spatially and functionally distinct subclasses of breast cancer-associated fibroblasts revealed by single cell RNA sequencing[END_REF][START_REF] Biffi | Il1-induced Jak/STAT signaling is antagonized by TGFβ to shape CAF heterogeneity in pancreatic ductal adenocarcinoma[END_REF][START_REF] Elyada | Crossspecies single-cell analysis of pancreatic ductal adenocarcinoma reveals antigenpresenting cancer-associated fibroblasts[END_REF][START_REF] Dominguez | Single-cell RNA sequencing reveals stromal evolution into LRRC15+ myofibroblasts as a determinant of patient response to cancer immunotherapy[END_REF][START_REF] Lambrechts | Phenotype molding of stromal cells in the lung tumor microenvironment[END_REF][START_REF] Puram | Single-Cell Transcriptomic Analysis of Primary and Metastatic Tumor Ecosystems in Head and Neck Cancer[END_REF]. It can therefore be anticipated that the roles of the CAF-S1 and CAF-S4 populations and how they functionally contribute to these roles might be similar across different cancer types [START_REF] Mhaidly | Fibroblast heterogeneity in tumor microenvironment: Role in immunosuppression and new therapies[END_REF].

CAF heterogeneity does not only exist at a sub population level but also within each sub population, as distinct phenotypic clusters have been described in CAF-S1 and CAF-S4 populations [START_REF] Mhaidly | Fibroblast heterogeneity in tumor microenvironment: Role in immunosuppression and new therapies[END_REF]. For example, in the pancreatic adenocarcinoma TME, myofibroblastic (myCAFs) and inflammatory (iCAFs) phenotypes were identified in the CAF-S1 population [START_REF] Öhlund | Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer[END_REF]. More specifically, myCAFs preferentially produced and remodelled the ECM and expressed high levels of αSMA, whereas iCAFs secreted immunomodulatory factors such as IL-6 [START_REF] Öhlund | Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer[END_REF]. Furthermore, myCAFs were found to be located in the vicinity of cancer cells, however, iCAFs had a more distal location, which suggests that these clusters may not only have distinct functions but might be involved in different signalling networks [START_REF] Mhaidly | Fibroblast heterogeneity in tumor microenvironment: Role in immunosuppression and new therapies[END_REF][START_REF] Öhlund | Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer[END_REF].

Indeed, a recent study performed single cell RNA-sequencing of the CAF-S1 population and identified 5 and 3 different clusters in the myCAF and iCAF phenotypes respectively [START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF]. The 5 myCAF clusters identified according to molecular signalling signatures were: ECM proteins (ecm-myCAF), TGFβ-dependent pathway (TGFβ-myCAF), wound-healing signalling (wound-myCAF), interferon αβ (IFNαβ)-related pathway (IFNαβ-myCAF) and acto-myosin-signalling (acto-myCAF) (Figure 1.5 B) [START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF]. On the other hand the iCAF clusters were identified as detoxification pathway (detox-iCAF), interleukinsignalling (IL-iCAF) and IFNγ-related pathway (IFNγ-iCAF) (Figure 1.5 B) [START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF].

(Figure 1.5 B)
Furthermore, PDPN expression also differed in the CAF-S1 population as cells could be distinguished into FAPα + PDPN + or FAPα + PDPN -groups [START_REF] Cremasco | FAP delineates heterogeneous and functionally divergent stromal cells in immune-excluded breast tumors[END_REF]. Interestingly, both groups were associated with the ECM remodelling phenotype but FAPα + PDPN + CAFs were associated with higher expression of genes related to TGFβ signalling and fibrosis, compared to FAPα + PDPN -CAFs [START_REF] Cremasco | FAP delineates heterogeneous and functionally divergent stromal cells in immune-excluded breast tumors[END_REF]. Interestingly, in the context of breast and lung cancer, CAFs which expressed FAPα , αSMA and two cell surface molecules cluster of differentiation 10 (CD10) and G protein-coupled receptor 77 (GPR77), were shown to be resistant to chemotherapy and induced chemotherapeutic resistance by providing a niche to support cancer stem cells [START_REF] Su | CD10+GPR77+ Cancer-Associated Fibroblasts Promote Cancer Formation and Chemoresistance by Sustaining Cancer Stemness[END_REF]. These studies therefore provide an explanation as to why it is so challenging to target these cells therapeutically and that the mechanisms by which CAFs are contributing to primary tumour growth, metastatic evolution and therapeutic resistance might be multifaceted.

To date, there is evidence for the contribution of the CAF-S1 population to immune suppression and resistance to immunotherapies [START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF][START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF]. Interestingly, specific myCAF clusters (ecm-myCAF and TGFβ-myCAF) were shown to have a direct effect on the expression levels of PD-1 and CTLA4 on the surface of regulatory T-cells [START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF]. Seeing as the anti-PD1 antibody Pembrolizumab is currently used to treat melanoma patients, the authors performed gene set enrichment analysis of CAF-S1 cluster-specific signatures on publicly available data from patients with metastatic melanoma. These patients had been treated with Pembrolizumab and were classified according to evident progressive disease as non-responders and those that showed partial or total response were classified as responders [START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF]. Their data showed that compared to responders, non-responders had significantly higher expression of genes associated with ecm-myCAF, TGFβ-myCAF and wound-myCAF clusters in melanoma tumours at the point of diagnosis [START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF]. Furthermore, this study showed that a collagen-rich stroma was identified in primary tumours from several cancers including melanoma, which is known to negatively impact the interaction between tumour cells and T-cells and therefore also promotes resistance to immunotherapy [START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF].

This study is therefore a perfect example of how CAFs and the ECM contribute to therapeutic resistance in tumours.

A)

The expression levels of CAF markers for each CAF subpopulation B)

Extracellular matrix remodelling by cancer associated fibroblasts

The extracellular matrix (ECM) is critical for maintaining tissue homeostasis and is becoming increasingly acknowledged as an important feature of tumours which contributes to all the hallmarks of cancer via biomechanical and biochemical properties [START_REF] Pickup | The extracellular matrix modulates the hallmarks of cancer[END_REF][START_REF] Hynes | Overview of the matrisome-An inventory of extracellular matrix constituents and functions[END_REF]. In the context of melanoma, the ECM was shown to promote metastasis, invasion, phenotypic switching and therapeutic resistance [START_REF] Girard | A feed-forward mechanosignaling loop confers resistance to therapies targeting the MAPK pathway in BRAF-mutant melanoma[END_REF][START_REF] Miskolczi | Collagen abundance controls melanoma phenotypes through lineage-specific microenvironment sensing[END_REF][START_REF] Goetz | Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis[END_REF][START_REF] Hirata | Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin β1/FAK Signaling[END_REF]. The ECM is protein-rich and the proteins constituting the ECM, known as the core matrisome are collagens (e.g. collagen I, collagen IV), glycoproteins (e.g. fibronectin, laminin), proteoglycans which are glycoproteins with attached glycosaminoglycans (e.g. perlecan), ECM-bound growth and secreted factors (e.g. (VEGF), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF) and matricellular proteins (e.g. SPARC) [START_REF] Hynes | Overview of the matrisome-An inventory of extracellular matrix constituents and functions[END_REF][START_REF] Socovich | The cancer matrisome: From comprehensive characterization to biomarker discovery[END_REF]. The ECM in the TME is predominantly produced and remodelled by CAFs, more specifically by myofibroblastic CAFs (myCAFs)

belonging to the CAF-S1 population [START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF][START_REF] Pelon | Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms[END_REF]. Furthermore the Tartare-Deckert laboratory and others have found that MITF low /AXL high mesenchymal melanoma cells can deposit and remodel the stromal ECM [START_REF] Girard | A feed-forward mechanosignaling loop confers resistance to therapies targeting the MAPK pathway in BRAF-mutant melanoma[END_REF][START_REF] Miskolczi | Collagen abundance controls melanoma phenotypes through lineage-specific microenvironment sensing[END_REF]. Importantly, the presence of specific ECM proteins, the alignment of ECM fibres and the mechanical stiffness of the ECM have all been shown to play a role in melanoma progression and resistance to targeted therapies, as melanoma cells interact directly with the ECM via cell surface receptors [START_REF] Arozarena | Phenotype plasticity as enabler of melanoma progression and therapy resistance[END_REF][START_REF] Kemper | Phenotype switching: Tumor cell plasticity as a resistance mechanism and target for therapy[END_REF][START_REF] Greenwald | Superficial spreading and nodular melanoma are distinct biological entities: A challenge to the linear progression model[END_REF][START_REF] Greene | A study of tumor progression: The precursor lesions of superficial spreading and nodular melanoma[END_REF].

ECM remodelling leads to alterations in ECM composition and organization that can alter many aspects of tumour cell behaviour [START_REF] Pickup | The extracellular matrix modulates the hallmarks of cancer[END_REF]. ECM remodelling involves 3 key mechanisms: where further post translational modifications can occur [START_REF] Winkler | Concepts of extracellular matrix remodelling in tumour progression and metastasis[END_REF]. For example, collagen is first synthesized as pre-collagen and is post translationally modified in the Golgi to a procollagen α-chain by glycosylation, pro-peptide alignment, disulphide bond formation and hydroxylation [START_REF] Winkler | Concepts of extracellular matrix remodelling in tumour progression and metastasis[END_REF]. Hydroxylation of the procollagen α-chain by lysyl hydroxylases results in triple helix formation of procollagen α-chains, which is followed by the secretion of procollagen α-helices into the extracellular space. In the extracellular space collagen fibrils are formed by proteolytic cleavage of pro-peptides at the C and N terminals [START_REF] Winkler | Concepts of extracellular matrix remodelling in tumour progression and metastasis[END_REF].

Thereafter, the collagen fibrils are assembled via covalent cross-linking by lysyl oxidase (LOX) family members [START_REF] Winkler | Concepts of extracellular matrix remodelling in tumour progression and metastasis[END_REF]. The cross linking of collagen fibrils is important for collagen fibre assembly and also increases the tensile strength and stiffness of the ECM [START_REF] Winkler | Concepts of extracellular matrix remodelling in tumour progression and metastasis[END_REF].

Furthermore, collagen fibres are also cross linked to other ECM proteins such as fibronectin by tissue transglutaminase 2, as the deposition of collagen I and III is dependent on the presence and stability of deposited fibronectin [START_REF] Winkler | Concepts of extracellular matrix remodelling in tumour progression and metastasis[END_REF].

ECM composition has been shown to be important for cancer cell invasion [START_REF] Pickup | The extracellular matrix modulates the hallmarks of cancer[END_REF][START_REF] Hynes | Overview of the matrisome-An inventory of extracellular matrix constituents and functions[END_REF][START_REF] Winkler | Concepts of extracellular matrix remodelling in tumour progression and metastasis[END_REF].

Indeed, the presence of collagen IV in the ECM was shown to promote melanoma cells spreading and motility via the activation of matrix metalloproteases (MMPs) [START_REF] Pasco | Control of melanoma cell invasion by type IV collagen[END_REF].

Furthermore, fibronectin was also found to be an essential ECM adhesive component required for the directional migration of prostatic and head and neck squamous carcinoma cells [START_REF] Erdogan | Cancer-associated fibroblasts modulate growth factor signaling and extracellular matrix remodeling to regulate tumor metastasis[END_REF]. The fibronectin monomer consists of 15 domains and two extra domains. The extra domain A (EDA) has been implicated in the activation of TGFβ signalling and its presence in tumours is associated with increased ECM remodelling, MMP expression and the actin cytoskeleton re-organization [START_REF] Su | CD10+GPR77+ Cancer-Associated Fibroblasts Promote Cancer Formation and Chemoresistance by Sustaining Cancer Stemness[END_REF]. Interestingly, compared to younger CAFs, aged CAFs were also found to express lower amount of the proteins hyaluronan and proteoglycan link protein 1 which decreased the integrity of the lymphatic vasculature and promoted melanoma metastasis [START_REF] Kaur | Remodeling of the collagen matrix in aging skin promotes melanoma metastasis and affects immune cell motility[END_REF]. Furthermore, the stiffness of the ECM, induced by the cross-linking of collagen by LOX family members, was also shown to contribute to the motility and invasion of breast cancer cells [START_REF] Leight | Extracellular Matrix Remodeling and Stiffening Modulate Tumor Phenotype and Treatment Response[END_REF]. Therefore, the ECM composition and stiffness contributes to metastasis and invasion of cancer cells [START_REF] Winkler | Concepts of extracellular matrix remodelling in tumour progression and metastasis[END_REF][START_REF] Leight | Extracellular Matrix Remodeling and Stiffening Modulate Tumor Phenotype and Treatment Response[END_REF].

The second mechanism by which the ECM is modified is degradation (Figure 1.6) which involves the cleavage of ECM components by proteases such as MMPs which promote the release of ECM fragments growth factors and cytokines in the ECM and allows for the migration of cells (eg. immune cells) in the stroma [START_REF] Winkler | Concepts of extracellular matrix remodelling in tumour progression and metastasis[END_REF]. This is important for the cross talk between the ECM and immune cells as well as with melanoma cells and CAFs [START_REF] Pickup | The extracellular matrix modulates the hallmarks of cancer[END_REF].

The third mechanism by which the ECM is remodelled is by force-mediated modification Different ECM remodelling mechanisms are tightly regulated during tumourigenesis, metastasis and drug resistance [START_REF] Pickup | The extracellular matrix modulates the hallmarks of cancer[END_REF]. Furthermore, melanoma cells can interact directly with the ECM via transmembrane receptors including integrins, syndecans and discoidin domain receptors (DDRs). Melanoma cells are therefore directly influenced by the composition, stiffness and alignment of the ECM which can induce downstream intracellular signalling cascades [START_REF] Pickup | The extracellular matrix modulates the hallmarks of cancer[END_REF][START_REF] Winkler | Concepts of extracellular matrix remodelling in tumour progression and metastasis[END_REF][START_REF] Leight | Extracellular Matrix Remodeling and Stiffening Modulate Tumor Phenotype and Treatment Response[END_REF]. Interestingly, the abundance of collagen and the effect of collagen stiffness was shown to play a role in promoting melanoma cell adhesion and the nuclear localisation of the mechanosensory and transcription co-factor Yes-associated protein (YAP) [START_REF] Miskolczi | Collagen abundance controls melanoma phenotypes through lineage-specific microenvironment sensing[END_REF]. YAP was shown to regulate melanoma cell pigmentation and differentiation through the expression of MITF in response to collagen abundance [START_REF] Arozarena | Phenotype plasticity as enabler of melanoma progression and therapy resistance[END_REF]. This study therefore, provided some evidence that ECM composition and stiffness plays a role in melanoma phenotypic switching [START_REF] Miskolczi | Collagen abundance controls melanoma phenotypes through lineage-specific microenvironment sensing[END_REF]. Furthermore, the authors also analysed mRNA expression in melanoma patient tumours and found that low levels of MITF and high levels of collagen expression correlated to poorer patient survival [START_REF] Miskolczi | Collagen abundance controls melanoma phenotypes through lineage-specific microenvironment sensing[END_REF]. This therefore suggests that ECM composition and stiffness influences therapeutic response of melanoma patients.

(
Furthermore, a recent publication by the Tartare-Deckert laboratory found that stiffer collagen substrates induce mechanosensitivity via the mechanosensors YAP and myocardin-related transcription factor (MRTF) in undifferentiated, invasive mesenchymallike melanoma cells, in response to BRAF inhibitor treatment [START_REF] Girard | A feed-forward mechanosignaling loop confers resistance to therapies targeting the MAPK pathway in BRAF-mutant melanoma[END_REF]. Furthermore, this mechanosensitivity promoted melanoma cells to deposit a stiff, organized and drugprotective ECM and these observations were further confirmed in vivo [START_REF] Girard | A feed-forward mechanosignaling loop confers resistance to therapies targeting the MAPK pathway in BRAF-mutant melanoma[END_REF]. These studies therefore suggest that ECM composition and collagen stiffness plays a role in drug adaptive responses of melanoma cells.

Interestingly, another study showed that BRAF inhibitor treatment can activate melanoma associated fibroblasts (MAFs) in the melanoma TME to remodel the ECM, making it stiffer, which induced drug tolerance in melanoma cells [START_REF] Hirata | Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin β1/FAK Signaling[END_REF]. Furthermore, the direct interaction of melanoma cells via β1integrin with the MAF deposited ECM, induced intracellular FAK signalling that consequently re-activated the ERK signalling pathway, following BRAF inhibitor treatment [START_REF] Hirata | Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin β1/FAK Signaling[END_REF]. Furthermore, a fibroblastic and remodelled ECM was observed in the tumoral stroma of melanoma tissue from Vemurafenib (BRAF inhibitor) treated patients [START_REF] Hirata | Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin β1/FAK Signaling[END_REF]. This study provides evidence that the role of MAFs in the melanoma TME requires further investigation and that effect of targeted and immunotherapies on MAFs are poorly understood.

Given the emerging importance of MAFs in melanoma progression and therapeutic resistance an obvious strategy to counteract this effect is to target MAFs. However, it was reported that ablating CAFs in pancreatic ductal adenocarcinoma (PDAC) mouse models resulted in invasive, undifferentiated and highly hypoxic tumours, showing evidence of EMT and cancer stem cells, which resulted in poor survival and immune suppression.

Furthermore, the study also reported that lower myofibroblast number in PDAC patient tumours correlated with reduced survival (102). Another study found that targeting iCAFs by JAK inhibition shifted them to a myofibroblastic phenotype in vivo [START_REF] Biffi | Il1-induced Jak/STAT signaling is antagonized by TGFβ to shape CAF heterogeneity in pancreatic ductal adenocarcinoma[END_REF]. Therefore, the current opinion in the field is to rather achieve normalization in the TME than completely ablate CAFs (103,104). This approach focuses on restoring homeostasis in the TME, in particular with regards to the ECM, to resemble a non-tumourgenic normal tissue-like state (103,104). One possible way to achieve this could be to reprogram CAFs to a normal fibroblast phenotype however, this requires improved understanding of the mechanisms underpinning the activation of CAFs in the TME (71).

The activation of melanoma associated fibroblasts

Given the contribution of MAFs (fibroblasts in the melanoma TME) to melanoma progression, immunosuppression and therapeutic resistance, MAFs are attractive targets for novel anti-cancer strategies [START_REF] Mhaidly | Fibroblast heterogeneity in tumor microenvironment: Role in immunosuppression and new therapies[END_REF][START_REF] Hirata | Tumor microenvironment and differential responses to therapy[END_REF][START_REF] Sahai | A framework for advancing our understanding of cancer-associated fibroblasts[END_REF]. However, the heterogeneity of MAFs and the activation of MAFs in the TME upon BRAF inhibitor treatment has made targeting MAFs a challenge [START_REF] Mhaidly | Fibroblast heterogeneity in tumor microenvironment: Role in immunosuppression and new therapies[END_REF][START_REF] Hirata | Tumor microenvironment and differential responses to therapy[END_REF][START_REF] Sahai | A framework for advancing our understanding of cancer-associated fibroblasts[END_REF]. One strategy might be to target the signalling mechanism(s Several studies have also used a gain or loss of function approach to study the phenotypic conversion of normal fibroblasts to CAFs (114-117). One study showed that the knockdown of p53 in CAFs increased their contractile, migratory and invasive capabilities and affected their secretome. p53 levels were also found to be lower in CAFs compared to skin fibroblasts (114). Similarly, another study showed that the down regulation of the neurogenic locus notch homolog protein (Notch) effector CBF1 suppressor of hairless lag-1 (CSL) and consequent loss of p53 expression resulted in a CAF phenotype in dermal fibroblasts (115). Interestingly, the depletion of Snail-1 in mouse embryonic fibroblasts that were activated with TGFβ1 decreased their myofibroblastic properties. Furthermore, the authors found that increased Snail-1 expression in CAFs correlated to increased ECM remodelling (116). Another study also found that the overexpression of mechanosensor and transcription co-factor YAP in normal mouse fibroblasts induced a CAF phenotype via the regulation proto-oncogene tyrosine-protein kinase src (SRC) which resulted in the activation of actin and cytoskeletal proteins (117). These studies suggest that diverse intracellular signalling mechanisms are able to activate normal fibroblasts to a CAF phenotype.

)
The TME has also been shown to a play a key role in the activation of CAFs (118-120).

One group showed that YAP function was required for the mechanical remodelling of the ECM by CAFs. Furthermore, the stiff ECM deposited by CAFs was found to activate YAP signalling in CAFs via a feed-forward loop (118). Furthermore, YAP signalling was shown to interact directly with another mechanosensors MRTF to regulate the cytoskeletal dynamics of CAFs (119). Interestingly, YAP signalling was shown to be dependent on CAF contraction and MRTF signalling was shown to be dependent on TGFβ signalling (119).

Furthermore, a recent study showed that the negative regulator of β-catenin, Dickkopf-3 (DKK3) which is downstream of heat shock factor 1 (HSF1) was shown to concomitantly activate YAP and β-catenin signalling (120). This promoted proinvasive CAF functions such as contraction and ECM remodelling via canonical Wnt signalling (120). These studies therefore suggest the importance of the ECM in the activation of CAFs.

TGFβ signalling has also been described as one of the key drivers of the CAF phenotype, as it is readily secreted by cancer cells and it is well accepted that TGFβ signalling activates a myofibroblast phenotype during wound healing and fibrosis [START_REF] Sahai | A framework for advancing our understanding of cancer-associated fibroblasts[END_REF]121). Indeed in vitro studies have shown that treatment with rhTGFβ1 induces myofibroblastic CAF properties such as actomyosin-dependent contractility and ECM remodelling in fibroblasts (111,116,122,123). Furthermore, the ecm-myCAF cluster identified in the CAF-S1 subset was associated with molecular signatures related to TGFβ signalling [START_REF] Mhaidly | Fibroblast heterogeneity in tumor microenvironment: Role in immunosuppression and new therapies[END_REF][START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF]. Moreover, it was suggested that TGFβ signalling can antagonize IL-1-induced JAK/STAT activation of iCAFs and drive them to a myCAF phenotype [START_REF] Biffi | Il1-induced Jak/STAT signaling is antagonized by TGFβ to shape CAF heterogeneity in pancreatic ductal adenocarcinoma[END_REF]. TGFβ signalling therefore plays an important role in driving the myCAF phenotype of the CAF-S1 sub population. Although it is well described that melanoma cells secrete TGFβ, whether melanoma-derived TGFβ drives dermal fibroblasts to a CAF-S1 (high FAPα expression) phenotype has not been confirmed.

Transforming growth factor β

TGFβ signalling plays a crucial role in the melanoma TME as it is involved in the crosstalk between melanoma cells and CAFs as well as between CAFs and the stromal cells (124).

The TGFβ superfamily consists of different cytokines including the 3 isoforms of TGFβ (TGFβ1, TGFβ2 and TGFβ3), activin, nodal, inhibins and bone morphogenetic proteins [START_REF] Busse | Role of TGF-β in melanoma[END_REF]125). TGFβ signalling plays a key role in cellular processes including proliferation, differentiation, migration, cell adhesion, cell death and homeostasis [START_REF] Busse | Role of TGF-β in melanoma[END_REF]125). In cancer,

Fibroblast Activation Protein α (FAPα) 1.3.1. An overview of FAPα

Fibroblast activation proteinα (FAPα) is a widely accepted CAF marker which was recently highlighted as a key marker that is highly expressed by the CAF-S1 subpopulation identified in the TME of carcinomas [START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF][START_REF] Pelon | Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms[END_REF]. Furthermore, FAPα expressing MAFs have been The inactive FAPα monomer is 95 kDa and has 5 potential N-glycosylation sites. Upon glycosylation, the FAPα monomers assemble, forming a 170kDa active homodimer (144,147). The FAPα homodimer binds DPPIV forming a complex at the cell membrane and the formation of this complex is required for FAPα enzymatic activity (147). It has further been demonstrated that the FAPα-DPPIV complex binds to α3β1integrin which docks this complex at the invadopodia (cell membrane protrusions) of migrating and invading cells (148). In addition it has been suggested that α3β1integrin binds to urokinase receptor (uPA)

FAPα biochemical properties and localisation

and that this interaction is important for the adhesion of cells to the ECM (149). It has furthermore been demonstrated that FAPα might be interacting with uPA in vitro (142). It is therefore postulated that FAPα resides in the plasma cell membrane in a complex with DPPIV, α3β1integrin and uPA (Figure 1.3.2.A) (138). Furthermore, at the plasma cell membrane, FAPα has also been shown to interact with caveolin-1 and stomatin that are known to localise to lipid rafts, which suggests that the interaction of FAPα with these two proteins is important for its localization and anchoring at the plasma cell membrane

. . FAPα enzy ati fun tion

To exude enzymatic activity, the FAPα homodimer has to form a complex at cell membrane invadopodia with DPPIV and α3β1integrin (138). FAPα has been described to exhibit both endo-and ecto-peptidase activity and therefore has dual functions where it functions as a gelatinase and a post-prolyl amino peptidase (146,147). The gelatinase function of FAPα has been studied mostly using kinetic studies and zymography which have indicated that

. . he role of FAPα in the tu our i roenviron ent

The most well understood role of FAPα in the TME is its contribution to immunosuppression which has been demonstrated in vitro, in vivo and in patient tumours [START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF][START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF]142,160). To this end, Yang et al, 2016, showed that the FAPα-positive CAF subpopulation, which was isolated from a murine hepatoma model, secreted significantly higher levels of the inflammatory chemokine CCL2 compared to the FAPα-negative CAF subpopulation (142). Furthermore, pancreatic and lung cancer cells decreased their motility in co-culture with both cell types that had FAPα depleted from the cell surface (163). This study therefore suggested that the presence of FAPα at the cell surface of CAFs is required for their protumourigenic functions. Additionally, there is some evidence that FAPα might be playing a role in the remodelling of the ECM in the TME, as one study showed that mouse embryonic fibroblasts (MEFs) which over express FAPα deposit organized and aligned ECM fibres (164). Furthermore, the authors showed that pancreatic cancer cells significantly increased their migration velocity and direction, when they were cultured on ECM derived from FAPα overexpressing MEFs (164). These in vitro studies therefore suggest that FAPα expressing

CAFs might be playing a key role in cancer progression and resistance to chemotherapeutic drugs.

Notably, a recent publication by Pelon et al, 2020, characterised the role of the CAF-S1 population derived from breast cancer tissue, which express high levels of FAPα [START_REF] Pelon | Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms[END_REF]. The CAF-S1 subpopulation was identified in patient breast cancer metastatic lymph node lesions and these cells were co-cultured with breast cancer cells (MCF7). Co-culture experiments showed that the CAF-S1 population contributed to breast cancer proliferation, EMT transition and migration but the effect on migration was observed to a greater degree in 2D compared to 3D assays. Furthermore, the authors suggest that the observed effects of the CAF-S1 population on breast cancer cells were as a result of secreted CXCL12 and TGFβ signalling [START_REF] Pelon | Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms[END_REF]. Interestingly, there is some evidence that FAPα might be affecting the proteins secreted by CAFs, also referred to as the CAF secretome, which was described in a study by Koczorowska et al, 2016 (159). The authors performed mass spectrometry analysis of CM derived from CAFs that were isolated from the colon cancer TME in mice, in which FAPα was either overexpressed, knocked down or rendered enzymatically inactive. Their data revealed that FAPα expression and activity enriched the secretion of proteins involved in TGFβ signalling, angiogenesis and ECM remodelling (159).

Furthermore, the presence of proteins involved in these processes were validated in the CM including uPA, pigment epithelium-derived factor, vascular endothelial growth factor C, mature form TGFβ, latency associated peptide, CCL2, MMP1, and osteopontin. In additional biological assays, including the collagen gel contraction assay and co-culture with endothelial cell spheroids, FAPα expression in CAFs was shown to contribute to their capacity to remodel the ECM and promote angiogenesis (159). These two studies therefore imply that FAPα might be important for the proinvasive behaviour of CAFs.

Despite the potentially important roles that FAPα might be involved in, in the TME, as well as in CAFs, not much is known about the signalling pathways that regulate FAPα in CAFs because most studies have studied the regulation of FAPα in cancer cells and its regulation seems to be cell and context dependent (141). Interestingly, treatment with recombinant TGFβ1 of mouse embryonic fibroblasts, bone marrow derived mesenchymal stem cells and melanoma cells resulted in an increase in FAPα expression in these different cell types (165-167). Furthermore, there is evidence that the canonical TGFβ pathway directly regulates FAPα transcription in human melanoma cells (167).However, the signalling pathway(s) involved in promoting this phenotype and the upstream and downstream signalling mechanisms by which FAPα might be executing its functions are not fully elucidated. Furthermore, the role of FAPα in MAFs and the melanoma TME is yet to be understood.

Aims

While it is well established that melanoma cells secrete growth factors into the TME, which can promote the activation dermal fibroblasts to MAFs, the recent discovery of the immunosuppressive FAPα-expressing CAF-S1 subset in the melanoma TME warrants further investigation into the mechanism(s) activating dermal fibroblasts to a FAPα expressing MAF phenotype. To this end, this PhD project addresses the following aims:

1) To confirm whether melanoma secreted-factors and their signalling pathways drive the phenotypic conversion of dermal fibroblasts to FAPα-expressing MAFs.

2) To investigate the role of FAPα in the MAF phenotype.

3) To determine the contribution of FAPα expressing MAFs on aggressive melanoma behaviour.

Chapter 2

Materials and Methods

Cell Culture

Isolation of skin fibroblasts

Skin fibroblasts were isolated from skin tissue samples as previously described (168).

Briefly, the dermis and epidermis were separated from small pre-cut pieces that had been submerged overnight in 5mg/ml of dispase solution (#D693, Sigma-Aldrich, Germany) at 4ºC. Thereafter, the dermal pieces were placed underneath a glass coverslip (Marienfeld Laboratory Glassware, Germany) in a 35mm tissue culture dish (Greiner Bio-one, Germany) containing Dulbecco's modified Eagle's medium (DMEM) (#31966-021, Gibco, United Kingdom), supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS) (Hyclone™, Thermo Fisher Scientific, USA) and 1% (v/v) penicillin / streptomycin (#151400-122, Gibco, USA). Following approximately 10 days, skin fibroblasts migrated spontaneously from the dermal samples and adhered to the bottom of the tissue culture dish and the coverslips were removed. These adherent cells were considered passage 0 and further propagated for 2 to 3 weeks to obtain a pure fibroblast population. The passage number of the skin fibroblasts used in all experiments remained below passage 10.

Isolation of melanoma associated fibroblasts

Melanoma associated fibroblasts (MAFs) were isolated from human melanoma tissue.

Written informed consent was obtained from melanoma patients at the University Hospital Centre in Nice, France. The ethics approval to obtain metastatic melanoma clinical samples was obtained from the University Côte d'Azur and the University Hospital Centre in Nice,

France, in accordance with the declaration of Helsinki. Melanoma tissue sample was cut into smaller pieces and digested using collagenase and dispase. To obtain a cell pellet, debris was filtered using serial centrifugations and the supernatant was discarded. The cells were then re-suspended in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin and plated in a tissue culture dish (Corning, USA). MAFs adhered to the tissue culture dish within 30min and the medium containing floating cells was removed. MAFs were stimulated to grow using Fibroblast Medium (ScienceCell, USA), supplemented with 1% fibroblast growth supplement (ScienceCell, USA), 1% penicillin / streptomycin and 10% FBS. The MAF population was confirmed using fluorescent activated cell sorting of cells expressing well described MAF markers including αsmooth muscle actin (αSMA), platelet derived growth factor receptor β (PDGFRβ), vimentin, Podoplanin (PDPN) and β1integrin.

MAFs were used until passage 12 for all experiments.

Cell maintenance

he undifferentiated human melanoma cell line which displays an invasive phenotype, 1205Lu, was obtained from ATCC (ATCC® CRL2812™ To maintain the authenticity of primary patient-derived cells, cells were frozen at low passages and used within 3 months of thawing for experiments. Cell morphology and phenotypic markers were closely monitored with increasing passages and regular mycoplasma testing was routinely performed using polymerase chain reaction (PCR), thereby ensuring that all cells used in experiments were mycoplasma negative.

Conditioned medium treatment

Melanoma cells, at 70-80% confluency, were washed with 1x PBS (#14190, Gibco, United Kingdom) and incubated at 37°C in serum-free DMEM supplemented with 1% penicillin / streptomycin. Following 24h, the CM was collected and centrifuged at 5000xg for 5 min to remove any cell debris and the supernatant was harvested. CM was aliquoted and stored immediately at -80°C. CM aliquots were thawed once prior to immediate single use and used within 3 months of freezing. Skin fibroblasts and MSCs were plated as required for specific assays and allowed to adhere overnight. The following day, cells were treated with CM supplemented with10% (skin fibroblasts) or 20% (MSCs) FBS. The first day of treatment was recorded as day 0 and thereafter cells were treated every 48h with fresh CM for a period of 6 days.

Treatment with factors and inhibitors

Treatment with factors

Recombinant human factors including TGFβ1 (#11343160, ImmunoTools, Germany), LIF (#300-05, PeproTech.Inc, France), IL6 (#14806962, eBiosciences, USA) and PDGF (#AF-10014B, PeproTech.Inc, France) were reconstituted according to the manufacturer's instructions. Skin fibroblasts were treated with a final concentration of 2ng/ml for each factor diluted in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. The initial treatment was recoded as day 0 and the cells were treated every 48h for a period of 6 days.

Inhibition of TGFβ1 in M

Skin fibroblasts and MSCs were stimulated with CM as described in 2.2.2. with or without the chemical TGFβ1 inhibitor SB-431542 (#S1067, Selleckchem, USA), which inhibits TGFβ intracellular signalling by competitively binding to the TGFβ receptor I (172).

SB431542 was reconstituted as per manufacturer's instructions and 10µM of SB431542 was added to the CM. CM containing SB431542 was replaced every 48h for the duration of the experiment.

Inhibition of FAPα enzy ati a tivity

FAPα enzymatic activity was inhibited using the PAN dipeptidyl peptidase enzymatic inhibitor PT100 (Talabostat mesylate) which has been shown to inhibit FAPα enzymatic in vivo (173). PT100 (HY-13233A, MCE MedChemExpress) was reconstituted according to the manufacturer's instructions to make a 5mM stock solution. Skin fibroblasts and MAFs were treated with 1µM and 5µM, respectively, every 2 days for the duration if the experiment.

Growth curves

Skin fibroblasts were starved for 1 week in DMEM supplemented with 0.5% FBS and 1% penicillin / streptomycin. Thereafter, cells were plated in culture medium at 50% confluency, (5x10 4 per well) in duplicate, in 12 well tissue culture plates (Corning, USA). The following day, cells were treated with CM as described in section 2.2. and counted on days 0, 2,4 and 6. The negative control was represented by skin fibroblasts treated with complete DMEM (culture medium) and the positive control included the cells treated with 2ng/ml TGFβ1.

Fibroblast derived matrices (FDMs)

3D Extracellular matrix (ECM) deposition assay

The ECM deposition assay is an in vitro assay that is used to model the ECM remodelling properties of CAFs [START_REF] Goetz | Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis[END_REF]101,116). Cell culture plates were coated with gelatin as previously described (174). Briefly, wells were coated with a 0.2% gelatin solution (#G1393, Sigma-Aldrich, Germany, Appendix A) for 1h at 37°C. Thereafter, the gelatin was removed and stabilised via cross-linking with 1% glutaraldehyde (#16120, Electron Microscopy Sciences, USA) (Appendix A), at room temperature for 30min. To neutralise the glutaraldehyde, wells were then washed thrice with 1x PBS and further incubated with 1M ethanolamine (#E958, Sigma-Aldrich, Germany, Appendix A), at room temperature. Following a 30min incubation, wells were washed thrice with 1x PBS and 1x with medium. Cells were then plated in coated wells at a 100% confluency (1x10 5 cells per well in a 24 well plate; 1.5x10 5 cells per well in a 12 well plate) in culture medium. The following day, cells were treated with 50µg/ml ascorbic acid (#A4544, Sigma-Aldrich, Germany, Appendix A) diluted in complete medium and this day was considered as day 0. Cells were treated with ascorbic acid in fresh medium every 2 days for 6 days.

Immunofluorescence staining of FDMs

Glass coverslips (VWR, Germany) were coated with a 0.01% (MW 150 000 -300 000) polylysine solution (#P4832, Sigma-Aldrich, USA, Appendix B) for 10min at room temperature. Once the wells were coated with gelatin, cells were plated and treated with ascorbic acid, as described in 2.5.1. On the 6th day, the medium was removed, and wells were washed with 1 x PBS. Cells were lysed off the 3D-fibroblast derived matrices (FDM) using pre-warmed extraction buffer (Appendix B) for 5min at 37°C. Thereafter. the FDM were washed thrice very gently with 1x PBS and fixed with 3% paraformaldehyde (#15712, Electron Microscopy Sciences, USA) for 30min. Following the fixation, coverslips were washed thrice with 1x PBS and blocked for 1h at room temperature in 1% BSA-PBS blocking solution (Appendix B). The primary antibodies against fibronectin I (#sc59824, Santa Cruz Biotechnology, USA) and collagen I (#ab3470, Abcam, United Kingdom) were prepared in blocking solution at a 1:200 dilution and incubation was carried out overnight at 4°C. The following day, coverslips were washed with thrice with 1x PBS and incubated for 1h at room temperature in blocking buffer containing Alexa-Flour conjugated secondary antibodies at a dilution of 1:200. The following secondary antibodies were purchased from ThermoFisher Scientifc: goat anti-mouse, Alexa Fluor 488 (#A-11029) and the goat antirabbit, Alexa Fluor 594 (#A-32740). Coverslips were then wash thrice with 1 x PBS and mounted in ProLong diamond antifade mounting fluid (#P36961, ThermoFisher Scientific) onto glass slides (Ghaasel, Germany). Images were taken using the 40X objective lens of the wide-field Leica DM5500B microscope. Alignment of fibronectin and collagen fibres was determined using ImageJ analysis software.

Live video microscopy of melanoma cells on top of FDMs

The ECM deposition assay was carried out using skin fibroblasts, as described in section 2.5.1. The cells were carefully lysed off the FDMs carefully as stipulated in 2.5.2. and the live 3D FDMs kept submerged in 1x PBS to prevent drying out. Melanoma cells (MEL501 and 1205Lu) were stably transduced with the Incucyte® NucLight Red Lentivirus Reagent (#4625, Essen Bioscience, USA) which fluorescently labels cell nuclei and allows for them to be monitored in real time. To monitor melanoma cell proliferation, 1x10 4 melanoma cells were plated on top of the FDMs per well in a 24 well plate, in triplicate and allowed to adhere overnight. The following day, cells were subjected to the IncuCyte ZOOM® system for livecell Imaging and analysis. The IncuCyte ZOOM® system acquired 9 images per well every 2h for a period of 72h. The cell proliferation was calculated by counting the number of red fluorescent nuclei using the IncuCyte ZOOM® analysis software. This data was further analysed using GraphPad Prism version 6.0 software and represented as the mean and ±SEM for each timepoint. To assess melanoma cell motility, 5x10 4 1205Lu or 501MEL melanoma cells were plated on top of FDMs per well in a 12 well plate, in duplicate and allowed to adhere overnight. Cells were subjected to live cell video microscopy the next day, using the MultiColor TIRF (Total Internal Reflection Fluorescence) microscope from Leica Microsystems. This microscope acquired 10 images from 10 fixed positions per well, every 10mins for a period of 24h. The average area displaced by each cell at each time point was determined using the cell tracking application of FIJI ImageJ analysis software.

Overexpression and kno kdown of FAPα .6 .1. DNA onstru ts used for the sta le overex ression of FAPα in skin fibroblasts

The FAPα overexpression construct (pCMV6-FAPα) was purchased from Origene, USA (RC204692, Appendix C). The pCMV6 empty vector was generated from the pCMV6Cers1 construct (RC205167, Origene, USA) which has an identical vector structure as the pCMV6-FAPα construct, apart from the open reading frame that contains the Cers1 insert.

The Cers1 insert was excised with a restriction enzyme double digest, using the BamH1-HF (#R3136S, New England Biolabs, USA) and MluI1-HF (#R3198S, New England Biolabs, USA) restriction enzymes. One unit of each enzyme, 1µg of construct DNA and 5µl of 10x

CutSmart® buffer (B7204S, New England Biolabs, USA) were mixed in a total volume of 50µl with RNAase / Dnase free water and incubated for 15min at 37⁰C. The digested product was then run on a 1% agarose gel (Appendix C) and the bands were visualised under a UV light. The band corresponding to the size of the vector was cut out of the gel and the DNA was cleaned up using the Wizard® SV Gel and PCR Clean-Up System (#A9281, Promega, USA). Thereafter, the DNA was ligated overnight at 4⁰C, with the T4 DNA ligase (#M02020T, New England Biolabs, USA). This reaction included 100ng of the digested DNA, 2µl of the T4 DNA ligase and 4µl of the 10x T4 DNA Ligase Buffer (B0202S, New England Biolabs, USA). The following day, the ligation reaction was heat inactivated at 65°C for 10mins. Both vectors were prepared and amplified according to standard techniques (175). DNA quality and concentration were confirmed using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA) and the molecular mass and integrity of both constructs was confirmed by running these constructs on a 1% agarose gel.

G418 dose response curve

To establish the ideal concentration for antibiotic selection of successfully transfected skin fibroblasts, a dose curve was performed using the neomycin analogue G418 (V7983, Promega, USA, Appendix C). Cells were plated at 50-60% confluency in a 6 well plate (Corning, USA) and allowed to adhere overnight. Cells were treated with increasing doses of G418 including 100µg, 150µg/ml, 200µg/ml, 250µg/ml, 300 µg/ml, 500µg/ml, 800µg/ml and 1000µg/ml every 2 days for a period of 10 days. Cell viability and number were monitored daily using light microscopy. The lowest dose of 200µg/ml was determined as the lowest, lethal dose which was used for selection and maintenance of successfully transfected overexpressing and empty vector clones.

Generation of stable FAPα overexpressing skin fibroblasts

Skin fibroblasts were plated at 60% confluency in a 6 well plate in complete medium. The following day, the cells were transfected with 0.5µg of construct DNA using the TransFectin™ Lipid Reagent (#170-3350, BIORAD, USA), according to the manufacturer's instructions. Briefly, DNA and 5µl of TransFectin™ were diluted separately in serum and antibiotic free DMEM, to make a total volume of 250µl. Once mixed, the DNA and TransFectin™ were incubated for 20min at room temperature and thereafter 250µl of the DNA-Tranfectin complexes were added dropwise to each well. Following a 6h incubation at 37°C, the medium was replaced with fresh complete medium. The cells were lifted after 48h and plated in 100mm tissue culture dishes at a 1:3 or 2:3 dilution. Antibiotic selection commenced the following day, as the transfected cells were treated every 2 days with 200µg/ml of G418. Initial colonies were observed after 13 days of antibiotic selection and separate colonies were picked when they were confluent in the centre and had defined borders. Each picked colony was plated into a 24 well as a separate FAPα overexpressing clone. Once the clone was 80-90% confluent the cells were lifted and plated into a 35mm tissue culture dish and further expanded prior to freezing. FAPα overexpression was confirmed using western blotting. Clones were maintained under selection during culture as G418 was included in the culture medium when the medium was changed as required, however G418 was absent from media during experiments.

.

Kno kdown of FAPα in MAFs

MAFs were plated as described in 2.5.1 in gelatin coated 12 well plates and the following day, the medium was changed to antibiotic free DMEM containing 10% FBS. FAPα was transiently knocked down in these cells using 2 different small interfering RNA sequences specific to FAPα mRNA. Cells were transfected with 100nM of siFAPα #1 (SASI_Hs01_00227289) and siFAPα #2 (SASI_Hs02_00337654, Sigma-Aldrich, Germany) or the siMISSION® universal, negative, off target sicontrol (#SiCOO1, Sigma-Aldrich, Germany), using the Lullaby transfection reagent (#LL71000, OZ Biosciences, France). siRNAs and the transfection reagent (5µl per well) were diluted separately in Opti-MEM™ I reduced serum medium (# 31985070, Gibco, United Kingdom), prior to mixing and incubating for 20min at room temperature. Thereafter, 500µl of siRNA-transfection reagent complexes were added dropwise to each well. Following 6 hours of incubation at 37°C, the medium was replaced with complete medium. FAPα knockdown was confirmed using western blotting.

Collagen / matrigel gel contraction assay

The gel contraction assay was used to model the contractile capacity of skin fibroblasts and MAFs and was carried out as previously described (176,177). Briefly, 7.5x10 4 cells were resuspended in 90µl of complete medium and mixed on ice with 6µl of 1M Hepes pH 7.5

(#15630056, Gibco) 60µl Matrigel (#E1270, Sigma), 120 µl Collagen Type I (#354249, Corning) and 24µl of 5X DMEM solution (Appendix D). This mixture was mixed slowly for several min to ensure a homogeneous mixture and 30µl were plated per well in an uncharged glass-bottomed 96 well plate (#P96G-1.5-5-F, MatTek). Different conditions were plated in quadruplicates and the gels were incubated at 37°C for 1h to polymerise.

Once the gels had polymerised, 100µl of medium was added onto of each gel and medium was changed every 2 days. Gels were photographed every 2 days and the gel area was measured using ImageJ software. Contraction of the gel was measured using the following formula: (total area of wellarea of gel) / 100 x total area.

Western blotting

Proteins were harvested by scraping the cells from the dish on ice in 1X RIPA lysis buffer (#30004854, Merck, Millipore, USA) supplemented with protease and phosphatase inhibitor (Pierce™ Protease and phosphatase inhibitor mini tablets, #A32959, Thermo Fisher Scientific). Cells were lysed on ice for 1h and the protein lysate was separated from the cell debris during a 20 min centrifugation at 13 000 rpm. The supernatant was then collected and the protein concentration was quantified using the bicinchoninic acid (BCA), DC ™ Protein Assay Kit II (# 5000112 BioRad, USA) and bovine serum albumin (BSA) (#23208, Thermo Fisher Scientific) as the standard, according to the manufacturer's instructions.

Equal amounts of protein for each condition were prepared in reducing Laemmli SDS sample Buffer, (Alfa Aesar™, Thermo Fisher Scientific) and then boiled for 5 mins at 95°C.

Protein samples were then separated on sodium dodecyl sulphate (SDS) polyacrylamide gels and transferred onto Amersham™ Hybond™, polyvinylidene difluoride (PVDF) membranes (GE Healthcare Lifesciences, United Kingdom). Once the protein had been transferred, the PVDF membrane was blocked for 1h at room temperature in 3% BSA-TBS-Tween 20. Immunoblotting was then carried out by incubating the membrane in the primary antibody overnight at 4°C. The details of the poly-and mono-clonal antibody dilutions and their references are listed in the table below (Table 2.1). The following day, membranes were washed 3 times in washing buffer (Appendix E) for a total of 30 mins and incubated for 1h at room temperature with an anti-mouse (#7076S, Cell Signalling Technology), antirabbit (#7074S, Cell Signalling Technology) or anti-goat secondary (#sc-2354, Santa Cruz Biotechnology) antibody at a dilution of 1:3000. All secondary antibodies were conjugated to the horseradish peroxidase enzyme, allowing for the chemiluminescent visualization of proteins using the Amersham™ ECL Western Blotting Detection Reagents (#RPN2106, GE Healthcare Lifesciences), with the Fujifilm LAS-3000 Imager. Densitometry of the detected bands was performed using Fiji ImageJ software (National Institute of Health, Bethesda, USA) and normalised to the loading control. Recipes for polyacrylamide gels and buffers are described in detail in Appendix E.

Enzyme-linked immunosorbent assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) was carried out to measure the concentration of secreted TGFβ1 in CM derived from melanoma cells, using the TGFβ1 uncoated ELISA kit (#88-8350, Invitrogen). Briefly, a 96 well plated was coated overnight at 4°C with a purified antibody against human TGFβ1. The following day, the wells were washed with washing buffer (1x PBS containing 0.05% Tween 20) and blocked for 1h at room temperature with 1x diluent buffer (provided in kit). Thereafter, the wells were washed and reconstituted human TGFβ1 standards were added in duplicate, ranging from 7.8 to 1000pg/ml. Prior to plating the CM samples in the ELISA plate, the latent TGFβ1 in the CM was activated to the detectable active form with the addition of 1N HCL for 10min and the reaction was neutralised with 1N NaOH. Samples and TGFβ1 standards were incubated at 4°C overnight. The next day, wells were washed with washing buffer and then incubated for 1h at room temperature with the biotin-conjugated detection antibody against human TGFβ1. The wells were then washed with washing buffer and incubated for 30mins at room temperature with Avidin-HRP, washed with washing buffer and further incubated for 15mins

with tetramethylbenzidine substrate solution. The calometric reaction was then stopped using H2SO4 stop solution and the absorbance was read at 450nm. The concentration of TGFβ1 was measured as an average of 3 independently collected CM samples from each melanoma cell line.

Statistical analysis

Data are represented as the mean and standard error mean (SEM) of 3 independent experiments, unless otherwise described. Statistical analyses were carried out using GraphPad Prism, version 6.0 software. A one way or two way ANOVA and the Bonferroni multiple comparison test were performed to the statistical difference between different groups and the statistical significance was accepted at p< 0.05, p< 0.01 and 0.001 denotated as *, ** and *** respectively.

Chapter 3

Results

Introduction

MAFs have been shown to contribute to the growth of primary melanoma, metastatic progression, and drug resistance and they therefore represent attractive targets for novel melanoma therapies (107,178). One possible approach to do this is to target the molecular pathways that are responsible for the phenotypic conversion of resident skin fibroblasts to MAFs. In this regard, the TGFβ signalling pathway is of interest because it is well known for its role in the activation of skin fibroblasts to a myofibroblast phenotype in contexts such as wound healing and fibrosis (125). It is also well described that melanoma cells secrete growth factors and cytokines, including TGFβ1 into the melanoma TME (134). Furthermore, preliminary data in the Tartare-Deckert laboratory have revealed that patient-derived MAFs, isolated from melanomas in the skin, have similar contractile capacity and ECM deposition patterns to skin fibroblasts treated with recombinant TGFβ1 (rhTGFβ1). Whether melanoma secreted TGFβ1 is in fact driving the MAF phenotype has not been fully described, as the signalling mechanism(s) by which skin fibroblasts might be activated to adopt a MAF phenotype are not entirely understood.

Given the inherent heterogeneity of melanomas, it can be expected that melanoma cells in .1 Fβ1 is the redo inant se reted fa tor in onditioned ediu derived fro invasive elano a ells that drives a FAPαpositive MAF phenotype.

different

Secreted factors from invasive melanoma cells increase the expression of FAPα and several AF-S1 population markers in skin fibroblasts

To establish whether FAPα expression is indeed higher in MAFs compared to skin fibroblasts, endogenous FAPα mRNA and protein levels were compared between skin fibroblasts and two primary MAF cell cultures (MAF #1 and MAF #2). qRT-PCR analysis revealed that, compared to skin fibroblasts, the relative FAPα mRNA levels were 3.

B) Endogenous FAPα protein expression was determined by subjecting skin fibroblast and MAF

lysates to western blotting. Protein densitometry is represented as the mean of two independent experiments and was determined using Fiji ImageJ software, relative to the HSP60 loading control.

To model the activation of resident skin fibroblasts to MAFs by melanoma secreted factors in the TME, skin fibroblasts were treated with conditioned medium (CM) derived from melanoma cells in a differentiated, melanocytic-like and proliferative (PRO) or dedifferentiated, mesenchymal-like and invasive (INV) state. To accomplish this, it first needed to be confirmed that the melanoma cells from which CM was to be collected, did indeed display PRO or INV like-phenotypes, as previously described [START_REF] Rambow | Toward Minimal Residual Disease-Directed Therapy in Melanoma[END_REF][START_REF] Wouters | Robust gene expression programs underlie recurrent cell states and phenotype switching in melanoma[END_REF][START_REF] Girard | A feed-forward mechanosignaling loop confers resistance to therapies targeting the MAPK pathway in BRAF-mutant melanoma[END_REF]170).

Briefly, total protein was prepared from two PRO (501MEL and MM074

Secreted factors from invasive melanoma cells stimulate skin fibroblasts to exhibit myofibroblast activity

One of the key features of the CAF-S1 subpopulation is myofibroblast activity which has been shown to contribute to ECM remodelling and CAF contraction in tumour tissue [START_REF] Sahai | A framework for advancing our understanding of cancer-associated fibroblasts[END_REF]121). The effect of CM derived from PRO or INV melanoma cells on the myofibroblast properties of skin fibroblasts was assessed using the ECM deposition assay. The ECM deposition assay is an in vitro assay that is used to model the ECM remodelling properties of CAFs [START_REF] Goetz | Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis[END_REF]101,116). Briefly, the assay was carried out for skin fibroblasts treated with ascorbic acid, which stimulates cells to deposit ECM and CM from PRO or INV melanoma cells every 2 days for a period of 6 days. Thereafter, deposited fibroblast derived matrices (FDMs) were decellularized, fixed and subjected to immunohistochemistry using antibodies against fibronectin and collagen I. Immunofluorescent staining of deposited fibronectin and collagen fibres showed that when skin fibroblasts were treated with CM derived from INV melanoma cells there was a change in their organization to a more linear and aligned

FAPα overexpression in skin fibroblasts drives them to a MAF phenotype.

To investigate whether FAPα is driving the MAF phenotype, it was stably overexpressed in skin fibroblasts using the pCMV6FAP expression construct. Transfection with the pCMV6 empty vector was used as a negative control. Forty-eight hours after transfection the cells were grown in the presence of G418 (200 μg/ml) for a minimum of 13 days and a number of G418-resistant clones were isolated and analysed for FAPα expression using western blotting. were carried out on skin fibroblasts treated every 2 days for a period of 6 days with 2ng/ml rhTGFβ1 in the presence or absence of 1µM PT100. Immunofluorescence images from these assays revealed that PT100 inhibited the ability of rhTGFβ1 to induce skin fibroblasts from depositing aligned fibronectin and collagen I fibres (Figure 3.12 A). This result was further confirmed with quantification, which showed a significant decrease of 10% and 8% in the number of deposited fibronectin (Figure 3.12 B) and collagen (Figure 3.12 C) fibres respectively. These results suggest that FAPα enzymatic activity is crucial for the acquired ability of skin fibroblasts treated with rhTGFβ1 to deposit and organized ECM. Western blotting further showed that in these experiments, the enzymatic activity of FAPα was inhibited by PT100 and did not result from decreased FAPα expression and was not associated with changes in levels of β1integrin, which is one of the proteins that resides in a complex with FAPα at the plasma cell membrane (Figure 3.12 D). Furthermore, compared to skin fibroblasts treated with rhTGFβ1 alone, skin fibroblasts treated with rhTGFβ1 and PT100 expressed lower levels of the myofibroblast marker αSMA, ECM remodelling markers fibronectin, collagen I and YAP and pSTAT3 which is important for activating and maintaining a contractile myofibroblast phenotype (Figure 3.12 D). These

E)

Cell lysates were subjected to western blotting using antibodies against FAPα, β1integrin and fibronectin. Protein densitometry was calculated using FIJI ImageJ analysis software and normalised to the loading control HSP60. Statistical significance was calculated using a two-way ANOVA and Bonferroni multiple comparison's post-test (** p < 0.01, *** p < 0.001). Notably, CM derived from PRO melanoma cells did not result in concomitant increase in FAPα, β1integrin and αSMA protein levels in skin fibroblasts and did not affect the ECM remodelling abilities of skin fibroblasts. However, compared to the untreated control, an increase in FAPα protein levels, but not mRNA levels were observed in skin fibroblasts treated with CM derived from PRO melanoma cells. It is therefore possible that secreted factors from PRO melanoma cells might be promoting an inflammatory MAF phenotype (iCAF), which was one of the clusters identified the CAF-S1 subset [START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF]. In this regard, one limitation of this study is that immunomodulatory functions of skin fibroblasts treated with CM derived from PRO and INV melanoma cells were not assessed. Future work should therefore investigate whether CM derived from PRO melanoma cells might be inducing an iCAF phenotype in skin fibroblasts. Furthermore, a surprising result showed that compared to treating skin fibroblasts with CM derived from PRO melanoma cells alone, the presence of the chemical TGFβRI inhibitor in PRO melanoma CM, increased FAPα expression in skin fibroblasts. This suggests that inhibiting TGFβ signalling in the melanoma TME might drive some skin fibroblasts to assume a FAPα expressing MAF phenotype, as both PRO and INV melanoma cells would be present in the tumour. Interestingly, a recent study found that in PDAC tumours, TGFβ signalling can act as an antagonist between the iCAF and myCAF phenotypes [START_REF] Biffi | Il1-induced Jak/STAT signaling is antagonized by TGFβ to shape CAF heterogeneity in pancreatic ductal adenocarcinoma[END_REF]. It therefore might be interesting to explore the different implications of inhibiting and promoting TGFβ signalling on phenotypically different MAF clusters. These findings therefore give important insight into the crosstalk between melanoma cells in different phenotypic states and dermal fibroblasts and how this cross talk might be influencing the phenotypic conversion of dermal fibroblasts to MAFs and melanoma intra-tumoural heterogeneity.

The ECM produced by

The CAF-1 henoty e is de endent on FAPα ex ression and enzymatic activity.

To investigate the role of FAPα in the MAF phenotype, FAPα was knocked down in two MAF cell cultures derived from patient melanoma skin tissue. The data obtained revealed that following FAPα knockdown in MAFs, MAFs were no longer able to deposit aligned ECM. Furthermore, results showed that FAPα knock down in MAFs resulted in decreased expression of the CAF-S1 population markers β1integrin, αSMA and PDGFRβ and ECM proteins fibronectin and collagen I. These results therefore stipulate that FAPα expression is required for the myCAF and ECM remodelling MAF phenotype. These findings are in agreement with a previous study by Koczorowska et al, 2016 (159). The authors showed that FAPα knock down in CAFs derived from murine colon cancer, decreased αSMA expression and proteomic analysis showed a decrease in secreted proteins involved in ECM processing (159). Results also showed that a decrease in YAP and pSTAT3 protein expression, following FAPα knock down in MAFs which suggested that the maintenance of the myCAF ECM remodelling MAF phenotype is dependent on FAPα expression.

Importantly, preliminary qRT-PCR data also showed that FAPα knock down in MAFs resulted in decreased relative mRNA levels of CAF-S1 markers (FAPα, β1integrin, αSMA, PDGFRβ and FSP1), inflammatory markers (CXCL12, CD44), ECM remodelling markers (fibronectin and LOXL2) and YAP target genes (CYR61, SERPINE 1, Thrombospondin 1, AMOTL2 and ANLN). These data further support that FAPα expression is required for the myofibroblast ECM remodelling MAF phenotype and suggest that FAPα might be upstream of YAP signalling. However, future work is required to confirm at the protein level the differential expression of these markers, YAP localization and actomyosin contractility in FAPα-silenced MAFs. Moreover, results showed that stable overexpression of FAPα in skin fibroblasts promoted the phenotypic conversion of skin fibroblasts to a myofibroblastic, ECM remodelling MAF phenotype. This result corroborated with previous findings where the overexpression of FAPα in mouse embryonic fibroblasts promoted aligned ECM deposition (164). Taken together these data suggest that the myCAF and ECM remodelling MAF phenotype is dependent of FAPα expression.

To assess whether the MAF phenotype was also dependent on FAPα enzymatic activity, FAPα enzymatic activity was inhibited in MAFs using a dipeptidyl peptidase PAN inhibitor, which decreased the ECM remodelling and contractile capacities of MAFs. This decrease in myofibroblast activity was also confirmed with a decrease in key ECM proteins involved in ECM remodelling, fibronectin and β1integrin. These results are in agreement with a study which showed that the overexpression of an enzymatically inactive form of FAPα in mouse embryonic fibroblasts resulted in disorganized ECM deposition, compared to the aligned ECM deposited by mouse embryonic fibroblasts that overexpressed the enzymatically active form of FAPα (164). Furthermore, another study also showed that enzymatic The ECM has been described to contribute to melanoma metastasis, invasion, plasticity and therapeutic resistance [START_REF] Girard | A feed-forward mechanosignaling loop confers resistance to therapies targeting the MAPK pathway in BRAF-mutant melanoma[END_REF][START_REF] Miskolczi | Collagen abundance controls melanoma phenotypes through lineage-specific microenvironment sensing[END_REF][START_REF] Goetz | Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis[END_REF][START_REF] Hirata | Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin β1/FAK Signaling[END_REF]. Seeing as MAFs are key cells that remodel the ECM in the melanoma TME, the effects of the ECM deposited by FAPα expressing MAFs were investigated. Data in this study showed that skin fibroblasts overexpressing FAPα Finally, analysis of melanoma patient TCGA data showed that FAPα expression was negatively related to the overall patient survival rate. Interestingly, Miskolczi et al, 2018

showed that collagen abundance in melanoma was associated with poor patient survival and analysis of TCGA data showed that FAP associated with an MITF low signature in melanoma patients [START_REF] Miskolczi | Collagen abundance controls melanoma phenotypes through lineage-specific microenvironment sensing[END_REF]. Therefore, the role of the ECM remodelled by FAPα expressing MAFs on melanoma cell phenotypic switching warrants further investigation.

Implications of findings and future directions

The The findings in this study provide evidence that FAPα expression and activity are important for the myCAF ECM remodelling MAF phenotype. However, the regulation of FAPα in MAFs is poorly described. Interestingly, one study showed that TGFβ signalling transcriptionally regulated FAPα in melanoma cells (167). This study described 9 putative Smad binding elements on the FAPα promoter and confirmed using ChIP that the Smad complex is able to bind one of the putative Smad binding sites (167). However, whether TGFβ transcriptionally regulates FAPα at this same Smad site in MAFs has yet to be confirmed.

In this study, the inhibition of FAPα enzymatic activity in MAFs impaired their abilities to deposit aligned ECM. However, it is not possible for FAPα cleave full length collagen or fibronectin (138,147). Instead, FAPα is a serine protease that executes its enzymatic function by cleaving the proline of peptide bonds preceding any amino acid on the Nterminal of peptides (141,147,153). Furthermore, one study has suggested that FAPα might be able to proteolytically cleave uPA which promoted the activation of the CAF phenotype via p-FAK, p-c-Src, p-JAK signalling in CAFs (142). Therefore, it can be speculated that FAPα might be proteolytically cleaving proteins that are involved in ECM remodelling. One such protein might be LOXL2 as preliminary qRT-PCR results showed that LOXL2 mRNA levels decreased significantly upon FAPα knockdown in MAFs. LOXL2 can be secreted by

CAFs and is able to crosslink collagen and elastin fibres in the ECM (185). Furthermore, it has also been described that the proteolytically cleavage of LOXL2 by serine proteases in the extracellular space is required for it to execute its function in the ECM (185,186).

Interestingly, a recent study described that the enzymatic activity of LOXL2 in prostate cancer CAFs, decreased their ability to deposit aligned ECM (187). However, whether In conclusion, this study confirmed that the phenotypic conversion of skin fibroblasts to MAFs is mediated by TGFβ1, which is preferentially secreted by dedifferentiated mesenchymal-like melanoma cells. Furthermore, this study highlights the important role of FAPα expression and enzymatic activity in MAFs and provided preliminary evidence that the ECM remodelled by FAPα expressing MAFs affects melanoma cell behaviour.

Therefore, the regulation of FAPα and its downstream targets as well as the role of FAPα expressing MAFs in melanoma cell plasticity warrants further investigation.

Figure 1 . 1 Figure 1 . 2 Figure 1 . 3 Figure 1 . 4 Figure 1 . 5 Figure 1 . 6

 111213141516 Figure 1.1 The Clark model depicting melanoma progression Figure 1.2 Current FDA-approved immuno-and targeted therapies approved for malignant melanoma treatment Figure 1.3 The different phenotypic states that have been proposed for melanoma cells Figure 1.4 Diagram depicting the different genetic and non-genetic resistance mechanisms that melanoma cells employ during therapeutic resistance to targeted therapies Figure 1.5 Different CAF subpopulations (CAF-S1, CAF-S2, CAF-S3 and CAF-S4) Figure 1.6 Diagram of the 3 mechanisms of ECM remodelling in the melanoma TME.

Figure 1 . 7 Figure 1 . 8

 1718 Figure 1.7 Diagram of the canonical TGFβ signalling pathway Figure 1.8 Protein structure of FAPα.

Figure 1 . 9

 19 Figure 1.9 FAPα localisation in a complex at invadopodia of the plasma cell membrane

Figure 3 . 1

 31 Figure 3.1 Endogenous FAPα levels are higher in MAFs compared to skin fibroblasts.

Figure 3 . 2

 32 Figure 3.2 Treatment with conditioned medium derived from invasive melanoma cells increases the expression of FAPα and CAF-S1 markers in skin fibroblasts.

Figure 3 . 3 Figure 3 . 4 Figure 3 . 5

 333435 Figure 3.3 Conditioned medium derived from invasive melanoma cells stimulates skin fibroblasts to exhibit myofibroblast activity and deposit aligned ECM Figure 3.4 Treatment with conditioned medium derived from invasive melanoma cells and rhTGFβ1 stimulate MSCs to increase their expression of FAPα and deposit aligned ECMFigure 3.5 TGFβ1 is the predominant secreted factor in conditioned medium derived from invasive melanoma cells that is upregulating FAPα expression in skin fibroblasts.

Figure 3 . 6

 36 Figure 3.6 The presence of TGFβ1 in conditioned medium derived from invasive melanoma is required for the activation of skin fibroblasts myofibroblastic activity.

Figure 3 . 7

 37 Figure 3.7 The expression of CAF-S1 markers is dependent on FAPα expression in MAFs.

Figure 3 . 8

 38 Figure 3.8 FAPα-silenced MAFs lose their ability to deposit aligned ECM.

Figure 3 . 9

 39 Figure 3.9 FAPα-silenced MAFs decrease their expression of ECM remodelling and CAF-activation markers.

Figure 3 .

 3 Figure 3.10 FAPα-silenced MAFs have decreased mRNA levels of CAF-S1, ECM remodelling and inflammatory markers and YAP target genes

Figure 3 . 11

 311 Figure 3.11 FAPα overexpression in skin fibroblasts drives them to a MAF phenotype.

Figure 3 . 12 Figure 3 . 13

 312313 Figure 3.12 Inhibition of FAPα enzymatic activity in skin fibroblasts treated with rhTGFβ1 decreases expression of myofibroblast and CAF-S1 markers Figure 3.13 Inhibition of FAPα enzymatic activity partially impairs the myofibroblast properties of MAFs.

Figure 3 . 14 Figure 3 . 15

 314315 Figure 3.14 Melanoma cells decreased their proliferation and increase their motility on aligned fibroblast derived matrices deposited by FAPα expressing MAFs Figure 3.15 FAPα high expression is associated with poor overall survival in melanoma patients

Figure 1 . 1

 11 Figure 1.1 The Clark model depicting melanoma progression. Diagram adapted from Gesbert and Larue (12).

Figure 1 . 2

 12 Figure 1.2 Current FDA-approved immuno-and targeted therapies approved for malignant melanoma treatment.

Figure 1 . 3

 13 Figure 1.3 The different phenotypic states that have been proposed for melanoma cells.Table taken from Rambow et al, 2018 (41).

Figure 1 . 4

 14 Figure 1.4 Diagram depicting the different genetic and non-genetic resistance mechanisms that melanoma cells employ during therapeutic resistance to targeted therapies. Figure taken from Diazzi et al, 2020 (60).

Figure 1 . 5

 15 Figure 1.5 Different CAF subpopulations (CAF-S1, CAF-S2, CAF-S3 and CAF-S4)

Figure 1 . 6 )

 16 (a) ECM deposition and post translational modification, (b) ECM degradation and (c) forcemediated ECM modification ((95). During deposition and post translational modification, ECM proteins are synthesised and translocated to the Golgi in the cytoplasm,

Figure 1 . 6 )

 16 , which is caused by the binding of cell surface integrins to ECM molecules, thereby applying a mechanical force to these molecules. This mechanical force can result in confirmational changes that expose binding sites on the ECM molecules and can then self-assemble into fibrils and create aligned fibres[START_REF] Winkler | Concepts of extracellular matrix remodelling in tumour progression and metastasis[END_REF]. The alignment of collagen and fibronectin fibres was shown to be important for metastasis and invasion by promoting the directional migration of carcinoma cells (100,101). These 3 mechanisms of ECM remodelling are summarised in Figure1.6.

Figure 1 . 6

 16 Figure 1.6 Diagram of the 3 mechanisms of ECM remodelling in the melanoma TME. ECM deposition and post-translational modification (PTM), degradation and force-mediated modification, using collagen as the example of an ECM protein. Diagram adapted from Winkler et al, 2020 (95).

  TGFβ signalling has been shown to play both a tumour promoter and a tumour suppressor role(124,126,127). Furthermore, in the context of melanoma TGFβ1 signalling has been shown to contribute to metastasis, invasion, inflammation, immune suppression and ECM remodelling (124,128,129). Importantly, both melanoma cells and MAFs secrete TGFβ1, resulting in paracrine signalling between melanoma cells, MAFs, the ECM and immune cells[START_REF] Busse | Role of TGF-β in melanoma[END_REF][START_REF] Villanueva | Melanoma and the tumor microenvironment[END_REF][START_REF] Brandner | Melanoma's connections to the tumour microenvironment[END_REF]130,131). TGFβ is secreted in its latent form, which upon activation can initiate the canonical TGFβ signalling pathway. The canonical TGFβ pathway (Figure 1.7) involves the binding of an active TGFβ dimer to a family of transmembrane serine-threonine kinases known as TGFβ receptors Type I and Type II. This allows TGFβ type II receptor to cross-phosphorylate the TGFβ Type I receptor, resulting in its activation and the phosphorylation of receptoractivated Smad proteins (R-Smads: Smad2 and Smad3) (125,126). The pSmad2/3 then binds to the co-Smad, Smad4 which allows the heteromeric Smad2/3/4 complex to translocate into the nucleus (125,126). Inside the nucleus the Smad complex can bind to the Smad binding element (SBE) on the promoter region of target genes (e.g. SK1, FN1, COL1α, FAP, TMEPA, TGFBIP, IGFBP7, TSP-1, MMP2, TGM2, ITGAV, SNAI1, TWIST1) and associate with other co-factors (e.g. YAP, TAZ) (125-127,132). Besides stimulation of the canonical signalling pathway, ligand-activated TGFβ receptors can also activate additional non-canonical intracellular pathways that do not involve the Smad proteins. Noncanonical TGFβ signalling includes the mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K)/AKT, and Rho-like GTPase signalling pathways. It is now well accepted that a combination of the canonical Smad-mediated and non-canonical (non-Smad-mediated) functions determine the complex cellular outcomes of the TGFβ1 response in melanoma.Although TGFβ signalling has been described to be involved in CAF activation and TGFβ signalling in the TME is associated with metastasis, invasion and ECM remodelling, whether melanoma secreted TGFβ1 might be driving a FAPα-positive MAF phenotype remains to be investigated (133,134).

Figure 1 . 7

 17 Figure 1.7 Diagram of the canonical TGFβ signalling pathway

FAPα

  is a type II transmembrane glycoprotein comprised of 761 amino acids, of which the majority amino acids (734) constitute the extracellular domain (143). The extracellular domain contains the α/β hydrolase catalytic domain (amino acids 500-761) and the 8 bladed β-propeller domain (amino acids 99-499) (143). Resolution of the FAPα protein structure revealed that the extracellular FAPα domain is critical for its enzymatic activity because the active site known as the catalytic triad is highly conserved and is located in the catalytic domain, containing a catalytic serine (S624), aspartate (D702) and histidine (H734) (143-146). Furthermore, the 8 bladed β-propeller domain serves to select appropriate substrates and direct them to the active site either via a flexible central pore formed by the propeller blades or by forming a channel between the two domains (144). FAPα is anchored in the plasma cell membrane by a hydrophobic transmembrane domain (amino acids 7-25) that is connected to a short intracellular domain, consisting of 6 amino acids(143,145,146). The details of the FAPα protein structure are illustrated in Figure 1.3.1.

Figure 1 . 8 Figure 1 . 9

 1819 Figure 1.8 Protein structure of FAPα. Images adapted fromKelly, 2005 (138)

FAPα shows substrate specificity

  and cleaves gelatin and denatured collagen type I and IV(146). Seeing as gelatin does not exist in the ECM and FAPα cannot cleave full length collagen I and IV, it has been suggested that in the context of cancer, FAPα might be digesting collagen I and III fragments which were previously degraded by matrix metalloproteases (MMP) (152).Given the limited substrate specificity of FAPα's gelatinase activity it has therefore been accepted that FAPα is predominantly functioning as post-prolyl aminopeptidase and is able to cleave the proline of peptide bonds preceding any amino acid (141,147,153) This type of proteolytic activity has been shown to be important in cell signalling, particularly in inflammation and cancer because proteins can be modified by enzymatic cleavage, which can affect their cellular functions. To date, several neuropeptides have been identified as FAPα substrates, including neuropeptide Y, brain natriuretic peptide, substance P and peptide YY (154,155). Additionally FAPα has been shown to cleave and consequently inactivate Fibroblast Growth Factor 21 which plays a key role in glucose and lipid metabolism (156-158). In the context of the TME, FAPα was shown to cleave the basement membrane protein perlecan which had been pre-cleaved by MMP7. The cleavage of perlecan by FAPα was demonstrated to be important for prostate cancer transwell invasion (158). This suggests that FAPα enzymatic activity in the TME might play a contributing role to cancer cell metastasis and invasion. Furthermore, another study investigated putative FAPα substrates in the secretome of CAFs from the colon cancer TME. FAPα was either overexpressed, knocked down or rendered enzymatically in active in CAFs and the conditioned medium from these cells lines was subjected to proteomic analysis by Terminal Amine Isotopic Labelling of Substrates (TAILS), to identify any the N-terminal peptide fragments that may have been proteolytically cleaved. The authors found putative cleavage sites in the N-terminals of ADAM15, interleukin 6, fibrillin-2, matrillin-3, serine protease 23, testican-1, and TGFβ-induced protein (159). However, these putative FAPα substrateshave not yet been validated in vitro or in vivo. Given that there have only been 2 studies to date that have investigated FAPα substrates in the context of the TME, there is a need for future studies to investigate the substrates of FAPα which is predominantly expressed by CAFs in the TME because this may elucidate the signalling pathways and biological implications of FAPα.

Furthermore, the increased

  secretion of CCL2 was dependent on increased pSTAT3 expression in FAPα-positive CAFs. Co-immunoprecipitation experiments further suggested that the pSTAT3-CCL2 signalling axis was induced by the possible interaction of FAPα and uPA, resulting in a consequential downstream cascade, that was indicated by elevated levels of p-FAK, p-c-Src, p-JAK. This signalling pathway was shown to upregulate pSTAT3 and promote the secretion of CCL2, in FAPα-positive CAFs. Interestingly, the authors also found that co-injection of hepatoma cells with FAPα-negative CAFs or with STAT3-slilenced CAFs did not promote tumour formation in mice and an impaired infiltration of myeloidderived suppressor cells was observed, whereas the opposite occurred when hepatoma cells were co-injected with FAPα-expressing CAFs (142). These findings were further supported by another study which demonstrated that FAPα expressing CAFs promote immunosuppression and promote resistance to immunotherapy (anti-PD1 treatment), in a colorectal cancer mouse model, as a result of upregulated CCL2 secretion (160).Furthermore, analysis of human colorectal cancer tissue samples depicted a correlation between stromal FAPα expression, myeloid cell number and poor patient prognosis. However, FAPα expression was inversely proportional to the number of T-cells which further provided evidence for FAPα-mediated immunosuppression in the colorectal cancer TME (160). Subsequently, the immunosuppressive and inflammatory role of FAPα expressing CAFs and their contribution to immunotherapy resistance has been confirmed in several patient derived tumours including breast cancer, non-small lung carcinoma and head and neck squamous cell carcinoma[START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF][START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF].Apart from the immunosuppressive role of FAPα in the TME, a few studies have suggested that FAPα expressing CAFs might be promoting cancer cell proliferation, migration, invasion and chemotherapeutic resistance by investigating the paracrine signalling between FAPα expressing CAFs and cancer cells (161-163). To model this paracrine signalling in vitro, cancer cells were either treated with exogenous (recombinant) FAPα or co-cultured with various fibroblast cell lines overexpressing FAPα (161-163). One study demonstrated that exogenous FAPα treatment increased the proliferation of ovarian epithelial cancer cells and decreased their sensitivity to Cisplatin treatment (161). Another study demonstrated that exogenous FAPα treatment increased gastric cancer cell proliferation, migration and protein expression of EMT markers, in a dose dependent manner (162). This study further showed that co-culturing gastric cancer cells with human embryonic lung fibroblasts that overexpressed FAPα, increased gastric cancer cell proliferation, migration, invasion and promoted EMT via the Wnt / βcatenin pathway (162).Similarly, Teichgräber et al, 2015, found that murine pancreatic and lung cancer cells increased their proliferation, migration and invasion, when they were co-cultured with FAPα expressing murine fibroblasts that had been isolated from the lung or pancreas of C57BL/6 mice (163). Furthermore, this study also investigated the effect of FAPα depletion from the cell surface. Primary murine fibroblasts or human embryonic kidney cells which overexpressed FAPα, were treated with an engineered FAPα specific IgG1 antibody (ESC11), resulting in the cells internalizing FAPα, which hampered their motility.

  phenotypic states might influence the conversion of skin fibroblasts to MAFs via different signalling pathways, thereby contributing to the heterogeneity of the MAF population in the TME. To this end, data generated by a previous PhD student in the Tartare-Deckert laboratory suggested that conditioned medium (CM) derived from melanoma cells in the more differentiated, melanocytic-like and proliferative state (PRO) had a different effect on the phenotype of lymph node fibroblast reticular cells, compared to CM derived from melanoma cells in the more undifferentiated, mesenchymal-like and invasive state (INV). It can therefore be hypothesised that melanoma cells in different phenotypic states might be secreting different factors and thereby activating skin fibroblasts to different MAF-subpopulations. The CAF-S1 population has been associated with TGFβ1 signalling, myofibroblastic properties and high expression levels of FAPα (78). However, whether FAPα is driving the phenotype of this CAF sub-population requires further investigation. The data generated during this PhD project, which are described in the following chapter, are currently in preparation for publication. The findings give novel insight into the role of factors secreted by PRO and INV melanoma cells on the phenotypic conversion of skin fibroblasts to MAFs. Furthermore, the data shed light on the role that FAPα plays in the conversion of skin fibroblasts to MAFs and the contribution of FAPα expressing MAFs to the melanoma TME.

7 and 2 . 8 -Figure 3 . 1

 2831 Figure 3.1 Endogenous FAPα levels are higher in MAFs compared to skin fibroblasts.

Figure 3 . 2

 32 Figure 3.2 Treatment with conditioned medium derived from invasive melanoma cells increases the expression of FAPα and CAF-S1 markers in skin fibroblasts.

pattern (Figure 3 . 3 A(Figure 3 . 3 EFigure 3 . 3 4 CFigure 3 . 4 1 .(Figure 3 . 5 CFigure 3 . 5

 33333343413535 Figure 3.3 Conditioned medium derived from invasive melanoma cells stimulates skin fibroblasts to exhibit myofibroblast activity and deposit aligned ECM

Figure 3 . 6

 36 Figure 3.6 The presence of TGFβ1 in conditioned medium derived from invasive melanoma is required for the activation of skin fibroblast myofibroblastic activity.

3. 2 .. . 1 .

 21 The CAF-S1 phenotype is dependent on FAPα expression and enzymatic activity. FAPα is required for the ex ression of CAF-S1 markers in MAFs and for their myofibroblast propertiesTo investigate the role of FAPα in the MAF phenotype, FAPα was knocked down in two MAF cell cultures, derived from patient melanoma skin tissue, using a siRNA approach.The successful knockdown of FAPα in both MAFs (MAF #1 and MAF #2), using two different siFAPα sequences was confirmed using western blotting (Figure3.7). FAPα knockdown was accompanied by a decrease in protein expression of CAF-S1 markers, β1integrin, αSMA and PDGFRβ (Figure 3.7). To assess the role of FAPα in the myofibroblast properties of MAFs, the ECM deposition assay was carried out, following siFAPα transfection of the 2 MAF populations. Immunofluorescent images illustrated that FAPα knockdown hampered the ability of MAFs to deposit organized and aligned ECM fibres (Figure 3.8 A). This result was further confirmed with the alignment quantification of fibronectin and collagen I fibres deposited by FAPα-silenced MAF #1 cells, which showed a significant 10% decrease in fibronectin alignment (Figure 3.8 B) and a significant 4-8% decrease in collagen fibre alignment (Figure 3.8 C). Data for the alignment of fibronectin and collagen fibres deposited by MAF #2 cells transfected with siFAPα displayed a similar trend with a significant 17-18% decrease in fibronectin (Figure 3.8 B) and a significant 4-5% decrease in collagen alignment (Figure 3.8 C). These data therefore suggest that FAPα expression in MAFs is, in part, required for CAF-S1 marker expression and myofibroblast activity.

Figure 3 . 7 Figure 3 . 8 Figure 3 . 9 Figure 3 . 10 A(Figure 3 .Figure 3 .

 37383931033 Figure 3.7 The expression of CAF-S1 markers is dependent on FAPα expression in MAFs.FAPα was knocked down using 100nM of two different siRNA sequences targeting FAPα (siFAPα #1; siFAPα #2) or an-off target siControl in two patient MAF cell cultures derived from human melanoma skin tissue denotated as A) MAF #1 and B) MAF #2. Western blotting was used to confirm FAPα knockdown and protein expression of CAF-S1 markers was determined using antibodies against β1integrin, PDGFRβ, αSMA. Densitometry of western blots was calculated as a mean of two independent experiments using FIJI ImageJ software and normalized to the loading control HSP60.

Figure 3 . 11 A 11 DFigure 3 . 3 . 2 . 3 .

 311113323 Figure 3.11 FAPα overexpression in skin fibroblasts drives them to a MAF phenotype.

Figure 3 .Figure 3 .

 33 Figure 3.12 Inhibition of FAPα enzymatic activity in skin fibroblasts treated with rhTGFβ1 decreases expression of myofibroblast and CAF-S1 markers.

FAPα expressing MAFs decreases melanoma cell proliferation and promotes melanoma cell migration .Figure 3 .

 migration3 Figure 3.14 Melanoma cells decreased their proliferation and increase their motility on aligned fibroblast derived matrices deposited by FAPα expressing MAFs PRO (501MEL) and INV (1205Lu) melanoma cells were stably transduced with the Incucyte® NucLight Red Lentivirus Reagent and cultured on top of 3D-fibroblast derived matrices (FDMs). The IncuCyte ZOOM® system for live-cell Imaging and analysis was used to monitor proliferation for 72h of melanoma cells cultured on FDMs from A) Skin fibroblast FAPα clone 1, the EV control or MAFs and B) MAFs transfected with 100nM of Dharmacon SMARTtpool siFAPα or the off target siControl.The number of red fluorescent nuclei were counted using the IncuCyte ZOOM® analysis software.Data are represented as the mean and ±SEM of 9 images acquired per well every 2 hours with each condition plated in triplicate. C) Melanoma cell motility was assessed using live video microscopy of 501MEL or 1205Lu melanoma cells cultured on top of FDMs derived from the FAPα clone 1 or the EV control for a period of 24h.The distance displaced by each cell was tracked using FIJI ImageJ analysis software. Data represent the mean and ±SEM of ten image per well that were acquired every 10 min with every condition plated in duplicate. Statistical significance was calculated using the twoway ANOVA and Bonferroni's post-test (***p < 0.001).

Figure 3 .

 3 Figure 3.15 FAPα high expression is associated with poor overall survival in melanoma patients

  inhibition of FAPα in CAFs isolated from murine colon cancer influenced the CAF secretome and decreased CAF secretion of factors involved in ECM remodelling (159). The obtained results in this study therefore further confirm that FAPα enzymatic activity is also required for the myCAF ECM remodelling MAF phenotype. To further investigate whether the activation of skin fibroblasts by rhTGFβ1 to a MAF phenotype was dependent on FAPα enzymatically activity, skin fibroblasts that were treated with rhTGFβ1 and the enzymatic FAPα inhibitor. Interestingly, when skin fibroblasts were treated with rhTGFβ1 and the enzymatic FAPα inhibitor they could longer deposit aligned ECM and decreased their expression of αSMA, fibronectin, collagen I, YAP and pSTAT3. These results show for the first time that the activation of skin fibroblasts by TGFβ1 is partly dependent on FAPα enzymatic activity. Therefore, take together these results provide evidence that 1) myCAF ECM remodelling MAF phenotype is dependent on FAPα expression and enzymatic activity and 2) the phenotypic conversion of skin fibroblasts by TGFβ1 to a MAF phenotype is in part dependent on FAPα enzymatic function. Future work should further investigate how TGFβ signalling might be regulating FAPα expression and enzymatic activity in MAFs. he M rodu ed y FAPα ex ressing MAFs de reases elano a ell roliferation and promotes melanoma cell migration.

  deposited aligned ECM compared to empty vector control cells and FAPα-silenced MAFs deposited disorganized ECM, compared to the aligned ECM deposited by FAPα expressing MAFs. To this end, live video microscopy was used to survey melanoma cells which were plated on top of FDMs. The results showed that both PRO and INV melanoma cells decreased their proliferation on aligned FDMs from FAPα overexpressing skin fibroblasts or FAPα expressing MAFs. This result suggests that biomechanical and biochemical properties of an aligned ECM might affect the cell cycle status of melanoma cells, which has not previously been observed. Future work is therefore required to investigate how ECM alignment might affect the cell cycle status of PRO and INV melanoma cells as well as expression of phenotypic and differentiation markers. Importantly, results showed that melanoma cells increased their motility on aligned FDMs from FAPα overexpressing skin fibroblasts compared to when melanoma cells were cultured on top of the disorganized FDMs from empty vector control cells. A possible explanation for this observation is that fibronectin has been described to promote directional migration of carcinoma cells and aligned fibronectin fibres were identified in FDMs from FAPα overexpressing skin fibroblasts and FAPα expressing MAFs (184). Furthermore, another study showed that aligned organization of fibronectin-rich matrices deposited by prostate cancer CAFs promoted prostate cancer cell elongation and migration (101). Future work should confirm the whether the migration of melanoma cells on aligned FDMs from FAPα expressing MAFs is directional and whether there is evidence of melanoma cell elongation and invasion into the FDMs. Taken together, the decrease in proliferation and increase in migration of melanoma cells cultured on top of FDMS from FAPα expressing MAFs is interesting, as it further supports that the ECM might contribute to melanoma cell phenotypic switching, because INV melanoma cells have also been identified to have a slow cycling phenotype. The expression of markers such as MIF, AXL, SOX9 and SOX10 in PRO and INV melanoma cells cultured on top of the above mentioned FDMs therefore warrants further investigation.

  findings in this study show that TGFβ1 is the predominant factor secreted by INV melanoma cells that activates skin fibroblasts to a FAPα expressing myCAF ECM remodelling MAF phenotype. Interestingly, secreted factors from PRO melanoma cells did not have this same effect on the phenotype of skin fibroblasts. This study therefore provides some evidence that PRO and INV melanoma cells might be engaging in different crosstalk with dermal fibroblasts and thereby activating them to adopt a MAF phenotype via different signalling pathways. Comparative proteomic analyses of factors present in CM derived from PRO and INV melanoma cells might reveal which other potential signalling pathways could be activating skin fibroblasts to MAFs. This might also shed some light on the heterogeneity of MAFs in the melanoma TME. One limitation of this study is that single cell analysis of skin fibroblasts treated with PRO or INV melanoma CM was not performed. Single cell and RNA-sequencing analyses of skin fibroblasts subjected to CM derived from PRO or INV melanoma cells might give insight into whether particular secreted factors from PRO or INV melanoma cells preferentially activate skin fibroblasts to a particular phenotype, as 8 different phenotypic clusters have been identified in the FAPα-positive CAF subset (34). Further investigation is therefore required into the contribution of melanoma cells in different phenotypic state on the heterogeneity of MAFs. Given the paracrine signalling between melanoma cells and MAFs in the TME, it would also be interesting to investigate the effect of secreted factors from skin fibroblasts that have been activated by CM derived from PRO or INV melanoma cells on melanoma cell behaviour. Furthermore, whether melanoma cells in different phenotypic states might influence the MAF secretome has not yet been shown which might have important implications on melanoma progression and therapeutic resistance. It may also be interesting to assess whether skin fibroblasts that are activated by secreted factors from PRO or INV melanoma cells are able to initiate a MAF phenotype in unactivated skin fibroblasts in a feed-forward mechanism either, via secreted factors or via deposited and remodelled ECM.

FAPα

  is proteolytically cleaving LOXL2 which in turn promotes aligned ECM deposition by MAFs currently unknow and requires further interrogation. Furthermore, it might be interesting to compare the matrisome of FAPα expressing MAFs compared to FAPαsilenced MAFs and MAFs with inhibited FAPα activity as this may also give some insight into potential ECM remodelling proteins that could be downstream of FAPα in MAFs.The results in this study also show some insight into the role of FAPα expressing MAFs in the melanoma TME, as PRO and INV melanoma cells decreased their proliferation and increased their migration on FDMs from FAPα expressing MAFS and FAPα overexpressing skin fibroblasts. This finding suggests that the FDMs are directly affecting the behaviour of melanoma cells. However, further investigation into whether PRO melanoma cells might be acquiring a more INV phenotype on FDMs from FAPα expressing MAFs is required. Given the emerging link between ECM signalling and cellular metabolism, it is possible that melanoma cells reprogram their metabolic status when cultured on MAF-derived ECM , which might induces phenotypic changes associated with metastasis in melanoma cells (188). However, this requires further investigation. Seeing as ECM remodelling by MAFs and INV melanoma cells was shown to contribute to BRAF inhibitor resistance in melanoma, it would be interesting to evaluate whether the FDMs from FAPα expressing MAFs provide protection against BRAF-targeted therapies[START_REF] Girard | A feed-forward mechanosignaling loop confers resistance to therapies targeting the MAPK pathway in BRAF-mutant melanoma[END_REF][START_REF] Hirata | Tumor microenvironment and differential responses to therapy[END_REF]. Furthermore, in light of the current opinion to rather normalise the ECM than ablate CAFs in the TME, it might be worthwhile to investigate whether the repurposing of anti-fibrotic drugs used to treat idiopathic pulmonary fibrosis such as the TGFβ regulator Pirfenidone in combination with current therapies might interfere with MAF and ECM-mediated resistance (188).

Table 2 . 1

 21 Antibodies used for western blotting

Table 2 . 2

 22 Sequences of forward and reverse primers

1.1.1 An overview of cutaneous melanoma

	Malignant cutaneous melanoma is the most aggressive skin cancer and the World Health
	Organization reported that 287 723 melanoma cases had occurred globally in 2018 which
	were responsible for 60 712 deaths (1). Early-stage melanoma (clinical stage I -II) that
	has not presented with nodal or distant metastasis is effectively treated with surgery (2).
	However, late-stage melanoma (clinical stages III and IV) can present with microsatellite
	and in transit metastatic lesions (Stage III) or metastatic dissemination can occur to lymph
	nodes and distant organs (stage IV) (2). It is therefore concerning that approximately 60%

  ). B) Mechanism of action of BRAF and MAP/ERK (MEK) kinase inhibitors which target the MAPK signalling pathway in melanoma cells. Diagram taken from Hepner et al, 2018 (29). C) A summary table adapted from Domingues et al, 2018, describing the clinical use of FDA approved immuno-and target therapies for melanoma treatment (3).

  that drive MAF conversion in the melanoma TME. In this regard, resident fibroblasts are an obvious source of CAFs and in the context of melanoma dermal fibroblasts are in close proximately with melanoma cells, especially during the VGP, where melanoma cells have invaded the dermis[START_REF] Zhou | Perspective of Targeting Cancer-Associated Fibroblasts in Melanoma[END_REF]105,106). Studies have reported that TGFβ signalling, physiological stress, , which resulted in the downstream activation of JAK/STAT signalling (111). LIF was shown to promote the proinvasive activation of normal skin fibroblasts characterised by increased contractile capacity and ECM remodelling, in a TGFβ-

	dependent manner (111). LIF induced the activation of JAK1/STAT3 which further activated
	RhoA/ROCK, which increased actomyosin contractility, ECM remodelling and the secretion
	of TGFβ by skin fibroblasts, resulting in an autocrine feedback loop (111). These results
	corroborated with a previous study that showed cooperative JAK1 and ROCK signalling
	was regulating CAF (CAFs isolated from oral squamous cell carcinoma TME) contractile
	capacity downstream of IL6 signalling (112). Furthermore, IL1 secreted by PDAC cells was
	found to activate normal fibroblasts to CAFs upstream of JAK/STAT signalling (84).
	Additionally, IL6 secreted by gastric cancer cells activated normal gastric fibroblasts to
	CAFs by increasing Twist1 expression which resulted in the phosphorylation of STAT3
	phosphorylation and therefore activation of JAK1 (109). Furthermore, DNA
	methyltransferase 1 (DNMT1) was found to maintain constitutive JAK1/STAT3 signalling
	(109). Another group found that co-culturing carcinoma CAFs with pancreatic ductal
	adenocarcinoma cells (PDAC) resulted in DNA methylation by DNMT1 of suppressor of
	cytokine signalling 1 (SOCS1) in carcinoma CAFs and activated the STAT3 signalling
	pathway (110). These studies suggest that epigenetic modifications in normal fibroblasts
	occur which activate them to become CAFs.

inflammatory signals, DNA damage, direct cell to cell contact with cancer cells, receptor tyrosine kinase ligands and the ECM can all promote CAF behaviour in normal fibroblasts

[START_REF] Sahai | A framework for advancing our understanding of cancer-associated fibroblasts[END_REF]107)

. However, the signalling mechanism(s) that might drive the phenotypic conversion of dermal fibroblasts to MAFs is poorly understood. It is thought that normal fibroblasts undergo a phenotypic conversion to become CAFs because CAFs display increased contractile capacity, ECM production and mechanical remodelling capabilities as well as secretion of proinflammatory markers, compared to normal fibroblasts (71,107,108) Furthermore, distinct epigenetic changes have been reported in CAFs which result in the constitutive activation of the Janus kinase / signal transducers and activators of transcription (JAK/STAT) signalling pathway, that results in the maintenance of the CAF phenotype (109,110). One study found that leukaemia inhibitory factor (LIF) secreted by carcinoma cells, initiates epigenetic changes in normal fibroblasts via the acetylation of STAT3 which then activates the DNMT3b DNA methyltransferase (109). DNMT3b was shown to methylate CpG sites on the SHP-1 promoter, to silence SHP-1 expression. The silencing of SHP-1 resulted in the constitutive Several secreted factors by cancer cells including TGFβ, LIF, interleukin 1 (IL 1) and IL6 have been shown to activate normal fibroblasts to CAFs by various signalling mechanisms in different cancers(113). These studies therefore suggest that sustained activation of JAK/STAT signalling drives normal fibroblast conversion to a CAF phenotype.

  melanoma cells, representing the differentiated and more proliferative state (MM074) and the undifferentiated more invasive state (MM099) were kindly provided by Dr.Jean Christophe Marine (VIB Centre for the Biology of Disease, Belgium) (170). Melanoma cells, skin fibroblasts and MAFs were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. Adipose derived human mesenchymal stem cells (MSCs), A10021501, were kindly gifted by Professor Michael Pepper (University of Pretoria, South Africa), which were previously fully characterised according to the criteria stipulated by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (171). MSCs were cultured in DMEM supplemented with 20% FBS and 1% penicillin / streptomycin. All cell lines were maintained in a humidified incubator at 37°C, containing a 95% air and 5% CO2. Media was changed 3 -4 times per week as required.

). The differentiated human melanoma cell line that displays a proliferative phenotype, 501MEL, was kindly gifted by Dr. Ruth Halaban (Yale University School of Medicine, United States) (169). Short term patient-derived

Table 2 .1: Antibodies used for western blotting Proteins recognized by monoclonal antibody

 2

		Dilution	Isotype	Reference
	AXL	1:1000	Rabbit	4560, Cell Signalling Technology
	1integrin	1:1000	Mouse	610468, BD Biosciences
	Collagen I	1:500	Mouse	C2456, Sigma-Aldrich
	FAPα	1:500	Mouse	sc-65398, Santa Cruz Biotechnology
	Fibronectin	1:500	Mouse	sc-8422 Santa Cruz Biotechnology
	HSP90	1:1000	Mouse	sc-13119, Santa Cruz Biotechnology
	MITF	1;200	Mouse	MA5-14146, ThermoFisher Scientific
	PDGFRβ	1:1000	Rabbit	4664, Cell Signalling Technology
	Total Smad 1/2/3	1:500	Mouse	sc-7960 Cell Signalling technology
	pSmad2 (Ser465/467)	1:1000	Rabbit	3108, Cell Signalling Technology
	SOX9	1:500	Mouse	Sc-166505,	Santa	Cruz
				Biotechnology

2.10 Quantitative Real-Time PCR (qRT-PCR)

using Graphpad Prism 6 software, depicting the mean and SEM. All primers were purchased from Invitrogen (ThermoFischer Scientific) and the sequences of forward and reverse primers are listed in table 2.2.

Table 2 .2: Sequences of forward and reverse primers Gene Sequence of forward primer Sequence of reverse primer

 2

	ACTA2	CTATGCCTCTGGACGCACAACT	CAGATCCAGACGCATGATGGCA
	ANLN	CAGACAGTTCCATCCAAGGGAG	CTTGACAACGCTCTCCAAAGCG
	AMOTL2	GCGACTGTCAGAACAACTGC	GCACCTTTAACCTGCTTTCCA
	INTB1	GGATTCTCCAGAAGGTGGTTTCG	TGCCACCAAGTTTCCCATCTCC
	CD44	CCAGAAGGAACAGTGGTTTGGC	ACTGTCCTCTGGGCTTGGTGTT
	CYR61	TGAAGCGGCTCCCTGTTTT	TGAAGCGGCTCCCTGTTTT
	CXCL12	CTCAACACTCCAAACTGTGCCC	CTCCAGGTACTCCTGAATCCAC
	FAP	TCTAAGGAAAGAAAGGTGCCAA	GATCAGTGCGTCCATCATGAAG
	FN1	ACAACACCGAGGTGACTGAGAC	GGACACAACGATGCTTCCTGAG
	FSP1	CAGAACTAAAGGAGCTGCTGACC	CTTGGAAGTCCACCTCGTTGTC
	LOXL2	TGACTGCAAGCACACGGAGGAT	TCCGAATGTCCTCCACCTGGAT
	PDGFRβ	TGCAGACATCGAGTCCTCCAAC	GCTTAGCACTGGAGACTCGTTG
	RPL32	CCTTGTGAAGCCCAAGATCG	TGCCGGATGAACTTCTTGGT
	SERPINE1	CTCATCAGCCACTGGAAAGGCA	GACTCGTGAAGTCAGCCTGAAAC
	THBS1	GCTGGAAATGTGGTGCTTGTCC	CTCCATTGTGGTTGAAGCAGGC

is the redo inant se reted fa tor in onditioned ediu derived fro invasive melanoma cells that drives a FAPα-positive MAF phenotype.

  ,92,95,96,98). Furthermore, it is of great concern that the BRAF and MEK inhibitors which are a currently used to treat melanoma patients harbouring the BRAF oncogenic mutation, have been shown to promote a drug tolerant ECM, by activating ECM reprogramming in MAFs and melanoma cells[START_REF] Girard | A feed-forward mechanosignaling loop confers resistance to therapies targeting the MAPK pathway in BRAF-mutant melanoma[END_REF][START_REF] Miskolczi | Collagen abundance controls melanoma phenotypes through lineage-specific microenvironment sensing[END_REF][START_REF] Hirata | Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin β1/FAK Signaling[END_REF]. MAFs are the key cells in the TME that contribute to ECM remodelling and are presumed to be highly heterogenous, displaying different phenotypes and functions. In this regard, different CAF subpopulations have been identified in carcinoma and melanoma tissues[START_REF] Kieffer | Single-cell analysis reveals fibroblast clusters linked to immunotherapy resistance in cancer[END_REF][START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF][START_REF] Pelon | Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms[END_REF]. More specifically, a cluster of the This study aimed to confirm whether melanoma secreted factors could drive the conversion of dermal fibroblasts to a MAF phenotype. Prior to addressing this aim, endogenous FAPα levels were conformed to be significantly lower in skin fibroblasts compared to MAFs, isolated from the skin. Furthermore, a previous student in the Tartare-Deckert laboratory observed that unlike skin fibroblasts, MAFs readily remodel the ECM in vitro and deposit aligned ECM (182). It can therefore be postulated that skin fibroblasts are not the same phenotype as MAFs. Although the epigenetic status of skin fibroblasts was not compared to MAFs in this study, it is widely accepted that resident fibroblasts are a source of CAFs and have different epigenetic signatures[START_REF] Sahai | A framework for advancing our understanding of cancer-associated fibroblasts[END_REF]109,110). Skin fibroblasts therefore serve as a good model for the MAF cell of origin, when investigating the mechanism(s) that might be activating MAFs in the melanoma TME. Melanomas are heterogenous tumours, in part due to the high plasticity of melanoma cells which can exist and switch between different phenotypic states[START_REF] Rambow | Melanoma plasticity and phenotypic diversity: Therapeutic barriers and opportunities[END_REF]. To model the paracrine activation of dermal fibroblasts by Interestingly, the observed myofibroblast properties in skin fibroblasts treated with INV CM did not correlate to fibronectin and collagen I expression but correlated with increased YAP expression. This observed increase in YAP expression was not surprising as YAP has been previously described to regulate CAF activation and ECM remodelling (118). Seeing as YAP is a transcriptional co-factor, future work is required to confirm its translocation into the nucleus and activation in skin fibroblasts treated with INV melanoma CM compared to PRO melanoma CM. This would shed light on whether YAP signalling might be regulating the myofibroblast activity induced in skin fibroblasts by INV melanoma secreted factors. Furthermore, preliminary results showed that CM derived from INV melanoma cells resulted in increased FAPα expression in mesenchymal stem cells (MSCs) and stimulated MSCs to deposit aligned ECM. This suggests that secreted factors from INV melanoma cells promote a myCAF phenotype both dermal fibroblasts and MSCs. However, compared to skin fibroblasts the ECM deposited by MSCs appeared to be less dense suggesting that skin fibroblasts might exude enhanced ECM remodelling capabilities compared to MSCs. To confirm this, the thickness and stiffness of ECM deposited by skin fibroblasts and MSCs treated with INV melanoma CM requires further investigation, using techniques such as confocal and atomic force microscopy. In comparison to skin fibroblasts treated with INV melanoma CM, YAP activation and actomyosin-contractile activity should also be analysed in MSCs treated with secreted factors from INV melanoma cells. Furthermore, treatment of skin fibroblasts and MSCs with The difference in the levels of secreted TGFβ1 by PRO and INV melanoma cells might be accounted for by the known tumour promoting and tumor suppressor rolesof TGFβ signalling in melanoma, with higher levels of secreted TGFβ1 being associated with melanoma metastasis and invasion[START_REF] Busse | Role of TGF-β in melanoma[END_REF]127,129,130).

	Chapter 4 Discussion and Concluding Remarks Melanoma associated fibroblasts (MAFs) have been shown to promote melanoma progression, metastasis and therapeutic resistance by exhibiting myofibroblastic properties, resulting in ECM remodelling (63,71,75,106,122). The ECM is a key component of the TME which has been shown to contribute to melanoma metastasis, invasion, phenotypic switching and drug resistance via biomechanical and biochemical mechanisms (58,68FAPα-expressing CAF-S1 subset (myCAF) which is associated with myofibroblastic properties, was identified in tissues from melanoma patients responded poorly to immunotherapy (34,78,80). Novel therapeutic approaches are therefore needed to target the ECM remodelling effects of MAFs in the TME, in order to re-establish ECM normalization. One possible way to achieve this might be to reprogram MAFs to a resident rhTGFβ1 stimulated both cell lines to increase FAPα expression and to deposit aligned ECM. Treatment of skin fibroblasts with rhTGFβ1 also increased the expression of CAF-S1 TGFβ1 phase increased upon treatment with melanoma CM (68). However, using single cell markers β1integrin and αSMA. Therefore, to confirm whether TGFβ1 was the predominant
	fibroblast phenotype in primary the tumour and metastatic sites. However, this requires an analysis, a different study found that CAFs in the breast cancer TME, with a myCAF tumour-derived factor from INV melanoma cells which was inducing a myCAF CAF-S1-like
	improved understanding of the mechanisms underpinning the activation of normal phenotype accumulated in the G1 phase (83). These observational differences might be as phenotype in skin fibroblasts, TGFβ signalling was inhibited in melanoma CM using a
	fibroblasts to MAFs. To this end, this study sought to first confirm whether factors secreted a result of different techniques used to evaluate cell proliferation and cycle. Future work chemical TGFβRI inhibitor. Indeed, data showed that TGFβ1 is the predominant factor in
	by melanoma cells can drive the phenotypic conversion of dermal fibroblasts to FAPα-should investigate the cell cycle parameters by cytometry and expression of markers such INV melanoma CM that is upregulating FAPα expression in skin fibroblasts, as the presence
	expressing MAFs, second to investigate the role of FAPα in MAFs and third to elucidate of TGFβ1 in INV melanoma CM was required for the activation of skin fibroblasts as p21, p27, Cyclin D, pRb, and p53 in skin fibroblasts treated with melanoma CM, to further how this MAF phenotype might be contributing to melanoma cell behaviour. Results myofibroblastic activity and CAF-S1 marker expression. Furthermore, data showed that explain the observed decrease in cell number. Nevertheless, this result is consistent with obtained during this study 1) confirmed that TGFβ1 is the predominant factor secreted by compared to treatment with other factors secreted by melanoma cells such as IL6, LIF and the general notion that cells reduce their proliferative potential as they differentiate. undifferentiated, invasive and mesenchymal-like (termed INV) melanoma cells that drives PDGF, treatment of skin fibroblasts with rhTGFβ1 resulted in the highest fold increase in
	a FAPα-positive MAF phenotype 2) showed that the CAF-S1 phenotype observed in MAFs Interestingly, results in this study show for the first time, that treatment of skin fibroblasts FAPα expression. These data therefore corroborated previous studies that have associated
	is dependent on FAPα expression and enzymatic activity and 3) showed that the ECM with CM derived from INV melanoma cells resulted in a concomitant increase in the TGFβ signalling signatures with the ECM remodelling capabilities of CAFs
	produced by FAPα expressing MAFs decreases melanoma cell proliferation and promotes expression of CAF-S1 markers FAPα, β1integrin and αSMA. Furthermore, INV melanoma (34,68,80,81,84,85,183). In addition, ELISA results confirmed higher levels of secreted
	melanoma cell migration. These findings are discussed in detail below. CM stimulated skin fibroblasts to exhibit myofibroblast activity, characterised by aligned TGFβ1 in CM derived from INV melanoma cells, compared to CM derived from PRO
	melanoma cells.

melanoma cells in different phenotypic states, skin fibroblasts were treated with conditioned medium (CM) derived from either more differentiated, melanocytic and more proliferative (PRO) melanoma cells or from more undifferentiated, invasive and mesenchymal-like (INV) melanoma cells. Results showed that CM from both PRO and INV melanoma cells decreased skin fibroblast number. This result was contrary to what was previously observed in a study by Miskolczi et al, 2018 which showed that the number of skin fibroblasts in S-ECM deposition. This result was in agreement with data previously described by Miskolczi et al, 2018, which also showed that CM derived from INV cells stimulated skin fibroblasts to deposit organized ECM (68).

Acknowledgements

Acknowledgements ........................................................................................................... Table of Contents ...........................................................................................................

and the Université Côte d'under the formal agreement for a joint awarded degree both incredible mentors and I hold you in the highest regard as role models of Women in Science who are in leadership positions. I would like to thank all the generous funders who have supported me financially throughout my PhD studies. Thank-you to the National Research Foundation of South Africa, for granting me a PhD scholarship from 2015 -2017. Thank you to the University of Cape Town for awarding me an International Travel Scholarship in July 2018 which allowed me to visit and spend time in Sophie Tartare-Deckert's laboratory in Nice, France. Thank you to the French Embassy in South Africa and Campus France for awarding me a PhD mobility scholarship from that allowed me to finish all the necessary experiments required to complete my PhD degree in Sophie Tartare-Deckert's laboratory.

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