Linda Silina
email: linda.silina@curie.fr

Fatlinda Maksut

Isabelle Bernard-Pierrot
email: isabelle.bernard-pierrot@curie.fr.

François Radvanyi

Gilles Créhange

Frédérique Mégnin-Chanet

Pierre Verrelle
email: pierre.verrelle@curie.fr

Florent Dufour

Hélène Neyret-Kahn

Aura Moreno-Vega

Clémentine Krucker

Narjesse Karboul

Marion Dorland-Galliot

Pascale Maillé

Elodie Chapeaublanc

Yves Allory

Nicolas Stransky

Hélène Haegel

Thierry Menguy

Vanessa Duong

Review of Preclinical Studies to Improve Radiotherapy Response in Bladder Cancer: Comments and Perspectives

Keywords: Bladder cancer, radiosensitisation, tyrosine kinase receptors, TYRO3, urothelium

conversations in the Orsay office and for agreeing to be my mentor throughout my PhD and guiding me through all the difficulties. Further, I wish to acknowledge my colleague Florent Dufour, who gave me all the initial technical training with designing and performing TYRO3 downregulation and inhibition studies on bladder cancer cell lines. Thanks to him, I developed the understanding and built confidence to conduct my own work. I thank him warmly for inviting me to contribute to the publication establishing TYRO3 as a promising therapeutic target in bladder cancer. I thank him for giving me great examples of data organisation and analysis and giving me the independence to perform experiments, analyse, interpret my results, and put them in the context of previous experiments. I am deeply grateful to him for all the shared materials, publications and for the help to troubleshoot and get through any technical difficulties. I would also like to thank warmly other colleagues who contributed to the TYRO3 manuscript: Hélène N.K. Aura, Clémentine, Narjesse, Marion, Pascale, Elodie, Yves, Nicolas, Hélène H., Thierry and Vanessa.

I am very grateful to our collaborator Professor Jennifer Southgate who warmly welcomed me in her lab at the University of York and gave valuable feedback on my project. I thank her for her time to introduce me to the complexity of normal human urothelial development and urothelium homeostasis. I thank her making me feel like part of the team during the time I visited and including me in the charity walk; it was truly an enriching experience. I thank also Joanna Pearson who helped me to get familiar with all the lab techniques concerning normal human urothelium establishment and maintenance and I am grateful for the gym and running sessions we did together. I also thank the rest of the team members: Andrew, Claire, Debora, Jenny, Olivia, Rosalind, Simon, Sreemoti and Zhen who helped me to feel welcomed. I thank warmly to the director of CMIB U1196 unit at the Institut Curie site in Orsay, Marie-Paule Teulade-Fichou for including me in the unit meetings and giving the opportunity to present my work. I thank all the Chemistry lab of the CMIB unit, especially Oksana and Julie for great discussions. I thank the Biology lab of the CMIB unit, especially Florent Poyer for his helpful and supportive comments and always being able to find an answer to any question throughout these years. Many special thanks goes to Fatlinda Makust for the amazing technical support for various experiments, and for the collaborative work on the review and experimental paper. I thank you for being there for me and sharing the office with you has been just amazing! I thank also the other Orsay girls: Aafrin and Anouk for the wonderful support and for our 'presentation club'. I thank all the current and previous members of the Molecular Oncology team in Paris and for the opportunity to present my results in the lab meetings and for the valuable suggestions. I thank warmly to Helene, Virginia, Elodie for the valuable feedbacks and to Laure, Aura, Jacqueline, Jing, Mingjuin, and Daniela for sharing the PhD journey.

I am very grateful to Professor Anne Kiltie and Dr Céline Mirjolet for agreeing to review my thesis and to be part of my jury and for all their comments and valuable feedback which improved the quality of my work. I equally thank warmly Professor Eric Deutsch and Professor Catherine Durdux for accepting to be part of the thesis jury and for valuable discussion.

After, I want to thank to all my friends in Paris who helped me to relax and recharge. I especially thank the other students from the International IC-3i PhD program, especially Deep for keeping me company in Orsay, Sebastian and Jacopo for our dinners and meetings. I thank warmly to Lara for sharing the passion for a future career in medicine and helping to arrange the exciting volunteering and shadowing opportunities. I also thank to Ana, who has been great training partner and for always being always up for a meeting and a chat. I thank to Dr Ralf, who is my best friend in the world and has supported me during all my journey from highschool through the PhD. How lucky I have been to share these steps with him! I thank him for all the countries we have seen together and all the moments we have shared and all the kilometres we have run together! Even the lockdown with the endless conversations and cooking was great! I thank Ralf for being the person I am now and I am looking forward to what is ahead of us! I thank to Miss Latvia Liene, moments with you have been so precious and joyful and I cannot wait to meet you in Riga and learn some cooking skills from you! I thank my dearest friend Max for the regular supportive messages and all the fun travels we have had. I thank Constantin for the enthusiasm and always great time together during our travels. I thank my friend Ayham for the care and support throughout the years and being so encouraging and having genuine interest in my work and for always giving valuable insights. I equally thank all the training I gained at the University of Aberdeen prior my PhD journey: Dr Allison Carrington for encouraging me to apply to the various internships abroad, Dr Frank Ward and Dr Jason Holland for amazing internships I had under their supervision and the encouragement to pursue a PhD. I thank immensely to my dear Pierre who has been incredible support and who has brightened up my days in Paris with loads of walks, cycles, travels and discussions. It is wonderful to have such a kind and beautiful person by my side.

Last but not the least, I thank to my family. I thank to my late granny Erna who is my inspiration: who despite the difficult times and political situation made all her dreams come true, especially the one to go to the university. I thank my parents bringing me up with freedom and encouragement to pursue my passions. I thank them for guiding me through their passions of art and books and opening my mind to explore the world of the unknown. I thank to my granny Lūcija, with whom I have spent many memorable summer and winter holidays. I thank to my wonderful sister and brother for all the fun time we have whenever we manage to meet. I hope to see you all very soon. Table 1-

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ABSTRACT

Bladder cancer (BCa) is a major global health problem. It is the fourth most common cancer in men in industrialized countries. 25% of all diagnosed BCa are Muscle-invasive bladder cancers (MIBC) which have poor prognosis (60% of survival at 5 years in absence of metastasis). Cystectomy is the standard treatment for MIBC, but for patients with comorbidities it presents significant drawbacks including increased risk of infection and impacted quality of life. However, radiotherapy coupled with chemotherapy and tumor transurethral resection has emerged as a promising radiation-based bladder sparing treatment for limited disease. A significant proportion of patients are not eligible for cisplatin-based chemotherapies due to insufficient kidney function. In addition, both intravesical and systemic chemotherapy does not spare normal tissue and results in side effects. Therefore, it is of great interest to discover novel radiosensitisation strategies for bladder tumors. Selectively increasing bladder tumor sensitivity to radiation could improve the efficacy of radiotherapy, limiting its side effects and reducing the need for cystectomy. Such strategy targeting tyrosine kinase receptors (RTKs) in addition to radiation have already been successfully used in other types of cancer as for example targeting EGFR (a member of RTK family) for the treatment in head and neck squamous cell carcinoma. TYRO3 is a receptor tyrosine kinase of the TAM family (comprising TYRO3, AXL and MERTK) and is known to regulate diverse biological processes including immune regulation, clearance of apoptotic cells, platelet aggregation, cell survival and proliferation. TYRO3 is overexpressed in many types of cancer and promotes tumor cell proliferation, survival and resistance to chemotherapy. In addition, higher levels of TYRO3 expression have been associated with decreased overall survival in patients of diverse cancers. However, the role of TYRO3 in BCa has so far not been studied.

In this thesis, I investigated:

(1)

The role of TYRO3 in BCa; (2)

The radiosensitising effect of TYRO3 downregulation and inhibition in BCa cells;

The effect of TYRO3 downregulation and inhibition on normal human urothelial tissue.

We first analyzed the transcriptome of bladder cancer biopsy samples and demonstrated that TYRO3 is overexpressed in 50% of MIBCs. We demonstrated in vitro and in vivo that TYRO3 overexpression conferred a TYRO3-dependance to bladder tumor cells for cell growth and viability. Transcriptomic analysis of TYRO3-downregulated cells suggested that TYRO3 signaling controlled cell cycle and protected from apoptosis, which indicated a potential to improve radiation response. As radiotherapy is now as part of bladder preserving strategy, we then considered the role of TYRO3 in the radiation response. TYRO3 downregulation lead to a significantly increased radiosensitivity of BCa cells and conversely, TYRO3-overexpression induced radioresistance. In combination with radiotherapy, TYRO3 dowregulation lead to a cell cycle arrest and a long term persistence of Ionizing Radiation-Induced Foci (IRIF). These long term IRIF were not due to a differential repair of radiation-induced DNA damage assessed by the alkaline Comet assay, leading to a dysregulation of DNA damage signaling pathways or cell cycle checkpoints. Finally, I demonstrated that TYRO3 downregulation and inhibition did not impact viability of normal human bladder cells suggesting that inhibiting TYRO3 could improve radiotherapy efficiency while sparing normal surrounding tissues.

RÉSUMÉ

Le cancer de la vessie est un problème majeur de santé publique au niveau mondial. Il est le quatrième cancer le plus fréquent chez l'homme en termes d'incidence dans les pays industrialisés. 25% des cancers diagnostiqués sont des tumeurs envahissant le muscle (TVIM) présentant un mauvais pronostic (60 % de survie à 5 ans en absence de métastases). La cystectomie est le traitement standard de référence pour les TVIM, même si elle présente des inconvénients importants notamment un risque accru d'infections et impacte la qualité de vie. La radiothérapie, associée à une chimiothérapie et à une résection transurétrale de la tumeur, émerge comme traitement conservateur alternatif. La chimiothérapie systématique à base de platine est efficace, cependant une proportion non négligeable de patients ne sont pas éligibles à cette chimiothérapie du fait de leurs fonctions rénales altérées. De plus, qu'elle soit par voie systémique ou intravésicale, la chimiothérapie n'épargne pas les tissus sains et présentent de nombreux effets secondaires indésirables. Il est donc d'importance de découvrir de nouvelles stratégies de radiosensibilisation pour les tumeurs de la vessie. Ainsi, augmenter sélectivement la sensibilité d'une tumeur en réponse à la radiothérapie, permettrait d'améliorer l'efficacité du traitement, d'en limiter ses effets secondaires et de réduire le recours à la cystectomie.

Une telle stratégie a déjà été proposée dans d'autres types de cancer en ciblant les récepteurs à activité tyrosine kinase (RTK) en association avec des rayonnements. C'est le cas par exemple du ciblage de EGFR pour le traitement des carcinomes de la tête et du cou. TYRO3 est un récepteur à activité tyrosine kinase de la famille TAM (qui comprend TYRO3, AXL et MERTK), connu pour réguler divers processus biologiques, notamment la régulation de la réponse immunitaire, la clairance des cellules apoptotiques, l'agrégation plaquettaire ainsi que la survie et la prolifération cellulaires. TYRO3 est surexprimé dans de nombreux types de cancers et favorise la prolifération, la survie et la résistance des cellules tumorales à la chimiothérapie. De plus, la surexpression de TYRO3 a été associée à une diminution de la survie globale des patients. Cependant, le rôle de TYRO3 dans le cancer de la vessie n'a pas encore été étudié.

Dans cette thèse, je me suis intéressée :

(1) Au rôle de TYRO3 dans le cancer de la vessie ;

(2) A l'effet radiosensibilisant de la perte d'expression ou de l'inhibition de TYRO3 dans les cellules cancéreuses de la vessie ;

(3) A l'effet de l'inhibition ou de la perte d'expression de TYRO3 sur l'urothélium humain sain.

Dans un premier temps, nous avons analysé le transcriptome de divers échantillons issus de biopsies de cancer de la vessie et démontré que TYRO3 est surexprimé dans 50% des TVIM. De plus, nous avons mis en évidence, in vitro et in vivo, que les cellules tumorales de vessie surexprimant TYRO3 développaient une dépendance à ce récepteur pour leur survie et leur croissance. Les résultats des données transcriptomiques suggèrent également au niveau cellulaire que la perte d'expression de TYRO3 induit des modifications importantes dans le contrôle du cycle cellulaire et de l'apoptose, ce qui laisse supposer que TYRO3 pourrait augmenter la sensibilité de ces tumeurs à la radiothérapie. Etant donné que la radiothérapie est une stratégie en développement dans le traitement conservateur du cancer de la vessie, nous nous sommes intéressés au rôle de TYRO3 dans la réponse aux radiations ionisantes.

La perte d'expression ou l'inhibition de TYRO3 dans les cellules dérivées de tumeur de vessie induit une radiosensibilisation significative des cellules traitées. A l'inverse, la surexpression de TYRO3 par transfection plasmidique sur des cellules exprimant peu TYRO3 induit une radiorésistance. En association avec le rayonnement, la perte d'expression de TYRO3 conduit à un arrêt du cycle cellulaire et à une persistance à 24h des foyers de réparation (détectés par immunofluorescence). Cette persistance à 24h n'est pas due à un défaut de réparation des lésions de l'ADN (révélées par le test des Comètes alcalines) mais serait probablement le résultat d'une dérégulation des voies de signalisation des lésions de l'ADN ou des points de contrôles du cycle cellulaire. Enfin, les travaux sur les cellules dérivées de l'urothélium sain ont montré que la perte d'expression de TYRO3 n'affectait pas leur viabilité suggérant que le ciblage de TYRO3 pourrait améliorer l'efficacité de la radiothérapie tout en épargnant les tissus normaux environnants.

Mots clés : Cancer de la vessie, radiosensibilité, récepteur à activité tyrosine kinase, TYRO3, urothélium.

Urīnpūšļa vēzis ir nopietna veselības problēma pasaulē. Tas ir ceturtais izplatītākais vēzis vīriešiem augsti attīstītajās valstīs. 25% no visiem diagnosticētajiem gadījumiem ir muskuļus invazīva urīnpūšļa audzēji, ar kuriem ir sliktas izdzīvotības izredzes un augstas uzraudzības un ārstēšanas izmaksas. Cistektomija ir standarta terapija, taču tai ir nozīmīgi trūkumi, tostarp ar to saistītais augstais mirstības līmenis, paaugstināts infekcijas risks un pazeminātā dzīves kvalitāte. Tomēr staru terapija apvienojumā ar ķīmijterapiju un audzēja transuretrālu rezekciju ir kļuvusi par daudzsološu ārstēšnas metodi, kas pacientam ļauj saglabāt urīnpūsli. Sistemātiska uz platīnu balstīta ķīmijterapija ir efektīva, taču tā nelabvēlīgi ietekmē arī veselos audus. Tāpēc ir ļoti svarīgi atklāt jaunas stratēģijas, kas specifiski radiosensibilizētu urīnpūšļa vēža šūnas. Selektīvi palielinot urīnpūšļa audzēja jutību pret staru terapiju, varētu uzlabot tās efektivitāti, ierobežojot tās blakusparādības un samazinot nepieciešamību pēc cistektomijas.

Šāda stratēģija, kombinējot EGFR (tirozīnkināzes saimes receptora) deaktivizāciju un staru terapiju, tiek veiksmīgi lietota, lai ārstētu tādus vēža veidus, kā galvas un kakla plakanšūnu karcinomu. TYRO3 ir TAM saimes (kas ietver TYRO3, AXL un MERTK) tirozīnkināzes receptors, kas regulē dažādus svarīgus bioloģiskos procesus, ieskaitot imūnregulāciju, apoptotisko šūnu likvidēšanu, trombocītu agregāciju, šūnu izdzīvošanu un proliferāciju. TYRO3 ir augsti ekspresēts daudzu vēža veidu gadījumos un veicina audzēju šūnu proliferāciju, izdzīvošanu un izturību pret ķīmijterapiju. Turklāt augstāks TYRO3 ekspresijas līmenis ir saistīts ar samazinātu izdzīvošanas iespēju pacientiem ar dažādu tipu vēzi. Tomēr TYRO3 loma urīnpūšļa vēzī nav pētīta.

Šajā promocijas darbā es pētīju:

(1) TYRO3 lomu urīnpūšļa vēzī;

(2) TYRO3 deaktivizācijas radiosensibilizējošo iedarbību urīnpūšļa vēža šūnās;

(3) TYRO3 deaktivizācijas ietekmi uz cilvēka veseliem urotēlija audiem.

Mēs analizējām urīnpūšļa vēža biopsijas paraugu transkriptus un atklājām, ka TYRO3 ir augsti ekspresēts 50% muskuļus invazīva urīnpūšļa audzējumos. Mēs pierādījām in vitro un in vivo, ka TYRO3 augstā ekspresija piešķīra urīnpūšļa audzēja šūnām TYRO3 atkarību. Mūsu dati rāda, ka TYRO3 pazemināta regulēšana urīnpūšļa vēža šūnās ietekmē šūnu ciklu un DNS bojājumu atjaunošanas ceļu disregulāciju, kas liecina par potenciālu uzlabot staru terapijas efektus. TYRO3 pazemināta regulēšana noved pie ievērojami palielinātas urīnpūšļa vēža šūnu radiosensitivitātes. TYRO3 pazemināta regulācija samazina DNS bojājumu atjaunošanas efektivitāti pēc apstarošanas, kas izraisa DNS bojājumu uzkrāšanos un šūnu cikla apturēšanu. Un pretēji, TYRO3 pārmērīga ekspresija izraisa radiorezistenci un parāda paaugstinātu DNS bojājumu salabošanas efektivitāti pēc apstarošanas. Veselās cilvēka urīnpūšļa šūnās šis receptors nav būtisks izdzīvošanai, kas norāda, ka šādas ārstēšanas blakusparādības tiktu novērstas. TYRO3 pazeminātai regulācijai ir augsts potenciāls uzlabot staru terapijas efektivitāti urīnpūšļa vēža gadījumā, vienlaikus saudzējot apkārtējos apkārtējos audus.

Atslēgas vārdi: Urīnpūšļa vēzis, radiosensibilizācija, tirozīnkināzes receptori, TYRO3, urotēlijs.

ABBREVIATIONS Abbreviations

PREAMBLE

Bladder cancer (BCa) is a prevalent disease, and the Muscle-Invasive Bladder Cancer (MIBC) is mainly treated with bladder removal surgery (cystectomy), while bladder sparing alternative treatments still need be improved. Receptor tyrosine kinases (RTKs) play an essential role for a cell to communicate with its surroundings and are often overexpressed and/or overactivated in cancer. RTK family of TAMs (including TYRO3, AXL and MERTK) has been an attractive field of research due to their involvement in resistance of cancer therapies and immune response regulation in various cancers. Several TAM-targeting inhibitors have been approved for other cancer types. However, optimal treatment regimens need a full understanding of exact signaling pathways to appeal to personalized medical care and to overcome potentially acquired drug resistance. TAM receptors have never been explored in BCa.

Throughout my PhD research, I have been focusing to address the need of efficient targeted therapy in BCa, which could improve the current bladder-sparing therapy. When exploring the role of TAM receptors in BCa, it was found by my team that TYRO3 is overexpressed in BCa and I joined the lab at the time with the aim to work on understanding its role in the BCa. After identifying BCa cell dependency of TYRO3 activity, which I helped to investigate, we were interested to understand the underlying mechanisms. When analysing the transcriptomic data from the experiments of downregulation of TYRO3, we found indications that DNA damage response and repair pathways might be disrupted and I set-out to independtly investigate the potential of the combination of TYRO3 targeting therapy with radiotherapy in a collaborating laboratory in the site of Institut Curie in Orsay.

In order to present the results of my studies, in the chapter 1, I introduce clinical features of

. Bladder cancer

The urinary bladder is a hollow muscular organ that accumulates urine. It receives urine from the kidneys via the ureters, and releases it from the body via the urethra. A human bladder is composed of four layers (Figure 1-1). The innermost layer is transitional urothelium with stratified epithelial cells. The urothelium is followed by a lamina propria layer: connective tissue with interstitial cells and vascular system complemented by afferent nerves. Thereafter there is the muscularis propria with inner and outer longitudinal muscles. Finally, the fatty layer encapsulates the bladder and is constituted by fibroblasts, collagen and adipocytes. The transitional urothelium with stratified epithelial cells have sensory and signalling properties that allow the bladder to respond to different stimuli. These stimuli can be mechanical (filling and voiding), chemical (chemical mediators/peptides/transmitters released from the bladder nerves, which are in close proximity and within urothelium) or the presence of inflammatory cells [START_REF] Birder | Urothelial signaling[END_REF]. Bladder urothelium is quiescent with a low cell turnover rate (around 6 weeks in rodents and between 6 -12 months in humans) [START_REF] Cooper | The recovery of mouse bladder epithelium after injury by 4-ethylsulphonylnaphtha-lene-1-sulphonamide[END_REF][START_REF] Okajima | Effect of sex hormones on development of urinary bladder tumours in rats induced by N-butyl-N-(4-hydroxybutyl) nitrosamine[END_REF]. However, urothelium has a great regenerative capacity and in response to injuries it can enter into a highly proliferative state [START_REF] Kreft | Urothelial injuries and the early wound healing response: Tight junctions and urothelial cytodifferentiation[END_REF][START_REF] Balsara | Sleeping beauty: Awakening urothelium from its slumber[END_REF]. Bladder cancer (BCa) originates in the urothelium layer, can invade the underlying muscle and metastasize to other organs.

Epidemiology

BCa is the 6 th most common cancer in men and 10 th most common in both sexes. It is the second most frequent urologic malignancy after prostate cancer in men. In 2018, there were 549,393 new cases of BCa and 199,992 deaths worldwide. Of those there were 16 282 new cases in France [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF]. France is among seven countries showing a trend of rising incidence of BCa in the last decade, in particular in women [START_REF] Wong | The global epidemiology of bladder cancer: a joinpoint regression analysis of its incidence and mortality trends and projection[END_REF]. BCa incidence is increasing due to improved detection strategies and population aging. BCa is more prevalent in North America, and in Southern and Western Europe.

Risk factors

Being of male sex, increased age, cigarette smoking, occupational exposure to metalworking and industrial dye production, drinking water high in arsenic as well as infection by schistosomiasis are among the highest risk factors. Men are four times more likely to develop BCa than women due to reasons not yet fully elucidated (Dobruch et al., 2016)(IARC Publications Website -Cancer Today (powered by GLOBOCAN 2018); [START_REF] Krabbe | Bladder cancer risk: Use of the PLCO and NLST to identify a suitable screening cohort[END_REF][START_REF] Siegel | CA: A Cancer Journal for Clinicians[END_REF]. The male to female ratio of BCa seems to be higher in particular geographical areas. A study from 17 Spanish found a considerable 6.7 times increased risk of diagnosing BCa in males compared with females for both NMIBC and MIBC [START_REF] Puente | Gender-related differences in clinical and pathological characteristics and therapy of bladder cancer[END_REF]. An epidemiologic study of BCa in Russia found that men were 7 times more often diagnosed of BCa than females in 2004, although authors acknowledge that some inaccurate data recoding practices existed [START_REF] Pushkar | The epidemiology of bladder cancer in Russia[END_REF]. Differences in smoking habits are not sufficient to account for the different incidence (extensively reviewed by Dobruch et al., 2016. Several efforts to explore gender-related molecular differences have been made, identifying significant increase in histone lysine demethylase KDM6A mutations in female NIBC [START_REF] Hurst | Genomic Subtypes of Non-invasive Bladder Cancer with Distinct Metabolic Profile and Female Gender Bias in KDM6A Mutation Frequency[END_REF]. Dysregulated androgen receptor (AR)

signalling is thought to contribute. For example, the AR contributes to the bladder development and a lack of androgen can cause a fibrosis of the bladder and a decrease of bladder capacity [START_REF] Li | Androgen receptor signaling in bladder cancer[END_REF]. N-butyl-N-4-hydroxybutyl nitrosamine (BBN) is a chemical carcinogen commonly used in preclinical studies to generate BCa. It is long known to cause BCa more rapidly in male rodents than in females [START_REF] Okajima | Effect of sex hormones on development of urinary bladder tumours in rats induced by N-butyl-N-(4-hydroxybutyl) nitrosamine[END_REF]. BNN increases bladder tumour growth in female rodents treated with testosterone whereas males with synthetic oestrogens have less risks to develop the tumour [START_REF] Li | Androgen receptor signaling in bladder cancer[END_REF]. In addition, it has been found that AR alter the levels of circRNA-FNTA (circFNTA), which can increase BCa cell invasion and chemo-resistance. A clinical BCa sample survey showed that circFNTA expression is elevated in BCa tissues [START_REF] Chen | Androgen receptor-regulated circ FNTA activates KRAS signaling to promote bladder cancer invasion[END_REF].

Smoking

Cigarette smoking is the highest independent risk factor for BCa for both sexes, increasing the risk by 2-4-fold [START_REF] Freedman | Association between smoking and risk of bladder cancer among men and women[END_REF]. The whole smoker body is exposed to 60 diverse carcinogens such as beta-naphthylamine and polycyclic aromatic hydrocarbons [START_REF] Hecht | Tobacco carcinogens, their biomarkers and tobacco-induced cancer[END_REF].

Kidneys excrete the metabolic products of the chemicals and expose them to the inner linings of the bladder where they are in prolonged contact with urothelial cells. These molecules can induce DNA-adducts and generate genetic mutations, which in turn can activate oncogenes or suppress tumor-suppressor genes promoting carcinogenesis. There is a period of latency of 30 years between the beginning of smoking and the diagnosis of the disease [START_REF] Baris | A case-control study of smoking and bladder cancer risk: Emergent patterns over time[END_REF][START_REF] Freedman | Association between smoking and risk of bladder cancer among men and women[END_REF].

Bladder cancer is commonly considered as one of the cancers to be attributed to smoking.

This si supported by epidemiological data available regarding the smoking habits in population and incidence of smoking-related cancers (lung, lip, bladder among others). In countries,

where the smoking percentage of population is currently high (as for Greece (37%), France (36%) and Bulgaria (36%), also the attributable cases of cancer incidence are higher (Europe:

smoking rates, by country 2017| Statista, 2017;;[START_REF] Kulhánová | Tobacco-related cancers in Europe: The scale of the epidemic in 2018[END_REF]. For example, in France in 2017, there were totally of 36% of smokers and there was similar total percentage of cancer cases attributed to smoking (26% (men) and 10.2% (women)) [START_REF] Kulhánová | Tobacco-related cancers in Europe: The scale of the epidemic in 2018[END_REF]. Another study in France in 2015 found similar statistics (28% and 8% of cancer cases among men and women respectively were attributed to smoking [START_REF] Cao | Cancers attributable to tobacco smoking in France in 2015[END_REF]). In contrary, in the United Kingdom the smoking rate was estimdated to be around 18% in 2017

(Europe: smoking rates, by country 2017| Statista, 2017)). Correspondingly lower cases of cancer have been attributed in the UK to smoking as compared to France (22% in men and 18% in women in 2017) [START_REF] Kulhánová | Tobacco-related cancers in Europe: The scale of the epidemic in 2018[END_REF]. Another publication showed that 17.4%

(men) and 12.4% (women) of cancer cases were attributable to smoking in 2015 in the United Kingdom [START_REF] Brown | The fraction of cancer attributable to modifiable risk factors in England, Wales, Scotland, Northern Ireland, and the United Kingdom in 2015[END_REF]. In the UK, the absolute numbers of smokers are around 2 times lower than that of France and this could at least partially explain the corsepondingly lower cases of smoking-related cancers, especially in men. It has been suggested that more than 50% of all bladder cancer cases are at least in part linked to smoking [START_REF] Freedman | Association between smoking and risk of bladder cancer among men and women[END_REF].

Occupational exposure

Occupational exposure to carcinogens is among high risk factors mainly in industrial areas focused on processing paint, dye, metal and petroleum products. Exposure to aromatic amines (such as benzidine), polycyclic aromatic hydrocarbons (PAH) and heavy metals are thought to account for approximately 20% of all BCa incidence and only two years of exposure are shown to be sufficient to induce BCa development [START_REF] Burger | Epidemiology and risk factors of urothelial bladder cancer[END_REF].

Diet and drug intake

Diet can influence a risk to develop BCa. In fact, consumption of red meat is thought to increase the incidence of BCa by 17%. It has been shown that nitrosamines from nitrates in processed meats generate bladder tumors in rodents [START_REF] Bertram | Specific induction of bladder cancer in mice by butyl-(4hydroxybutyl)-nitrosamine and the effects of hormonal modifications on the sex difference in response[END_REF]. Furthermore, pre-obesity and obesity increased the risk of developing the disease of 7% and 10% correspondingly [START_REF] Sun | Obesity and risk of bladder cancer: A dose-response meta-analysis of 15 cohort studies[END_REF]. A study by Tuccori and colleagues revealed that antidiabetic drug pioglitazone is associated with significantly increased risk to develop BCa in type 2 diabetes patients [START_REF] Tuccori | Pioglitazone use and risk of bladder cancer: Population based cohort study[END_REF].

Arsenic

Arsenic is a naturally occurring metalloid in many minerals making up the Earth's crust. It can enter drinking-water sources in a dissolved state from the weathering of minerals and ores.

Arsenic in drinking water and contamination in food is a global health issue and has been linked with the risk of hypertension and several type of cancers, including BCa [START_REF] Saint-Jacques | Arsenic in drinking water and urinary tract cancers: A systematic review of 30 years of epidemiological evidence[END_REF]. Consuming water with high concentration of arsenic (greater than 50 μg/L) is associated with significant risk of developing BCa [START_REF] Christoforidou | Bladder cancer and arsenic through drinking water: A systematic review of epidemiologic evidence[END_REF]. In the past, also inhalation of arsenic used in the wallpaper production was associated with risk to develop BCa [START_REF] Hughes | Arsenic Exposure and Toxicology: A Historical Perspective[END_REF].

Urinary tract infections

Urinary tract infections (UTIs) caused by bacteria, parasite or virus are among risk factors for developing BCa. The majority of UTIs are caused by bacterial infections (mainly E.coli), and the infection can cause cystitis [START_REF] Flores-Mireles | Urinary tract infections: Epidemiology, mechanisms of infection and treatment options[END_REF]. Observational studies have showed that UTIs favors development of BCa [START_REF] Terlizzi | UroPathogenic Escherichia coli (UPEC) infections: Virulence factors, bladder responses, antibiotic, and non-antibiotic antimicrobial strategies[END_REF]. It is thought that chronic inflammation of the bladder may induce urinary retention and stasis, which can potentially increase exposure and absorption of carcinogens present in the urine. Bacterial flora in the urine may contribute to the production of nitrites that are converted to carcinogenic nitrosamines. However, controversial or even inverse results reported by others imply that UTI shows no association or even reduced risk in women patients for BCa due to the corresponding immune response triggered by infection [START_REF] Jiang | Urinary tract infections and reduced risk of bladder cancer in Los Angeles[END_REF][START_REF] Al-Badr | Recurrent urinary tract infections management in women: A review[END_REF]. Interestingly, all studies agree that the administration of antibiotics for UTI shows significant protective effect against BCa.

Infection by Schistosomiasis haematobium, a parasite responsible for schistosomiasis, the most prevalent parasitic infection in humans is also associated with significant risk of developing BCa. Indeed, in the Middle-East and Africa where this parasite is endemic, BCa is the second most common cancer [START_REF] Ross | Prevention and control of schistosomiasis: a current perspective[END_REF][START_REF] Saginala | Epidemiology of Bladder Cancer[END_REF]. The deposit of the parasite egg on the bladder creates an infection, which induces N-nitroso compound production (a carcinogen acting as a DNA-damaging free oxygen radical) [START_REF] Mostafa | Relationship between schistosomiasis and bladder cancer[END_REF]. Schistosomiasis causes chronic cystitis leading to squamous metaplasia of transitional epithelium, and subsequently development of squamous cell carcinoma.

Recent evidence suggests that the BK polyomavirus (BKV) reactivated infection, affecting organ transplant patients play a role in the development of BCa [START_REF] Starrett | BK polyomavirus is a cause of bladder cancer[END_REF]. Up to 90% of population is estimated to have contracted this infection in childhood and is latent asymptomatically in adult human urothelium, but can reactivate [START_REF] Yin | BK virus as a potential oncovirus for bladder cancer in a renal transplant patient[END_REF].

Radiation

Exposure to ionizing radiation (IR) has long been debated to be a potential cause of BCa [START_REF] Hall | Radiation-associated urinary bladder cancer[END_REF][START_REF] Ferrís | Environmental non-occupational risk factors associated with bladder cancer[END_REF]. Most of the data comes from the exposure of IR from atomic bombs, nuclear reactors and medical exposure.

Radiation from atomic bombs and nuclear power plants

In the most recent cancer incidence report on the Life Span Study (LSS) of atomic bomb survivors, the urinary bladder showed the highest excess relative risk (ERR) per unit dose of radiation of any of the solid cancers other than the female breast cancer [START_REF] Preston | Solid Cancer Incidence in Atomic Bomb Survivors: 1958-1998[END_REF][START_REF] Ozasa | Epidemiological research on radiation-induced cancer in atomic bomb survivors[END_REF]. At the cellular level, a comparative study of cell types absorbing energy from alpha particles originating from Plutonium radioactive nucleotides, found that bladder has the third highest absorption rate confirming the notion that bladder cells are sensitive to IR (just below blood vessels and bile ducts) [START_REF] Shichijo | Autoradiographic analysis of internal plutonium radiation exposure in Nagasaki atomic bomb victims[END_REF].

Studies after Chernobyl power plant explosion has showed increase in BCa incidence in the surrounding regions in Ukraine [START_REF] Romanenko | DNA damage repair in bladder urothelium after the Chernobyl accident in Ukraine[END_REF][START_REF] Romanenko | Urinary bladder lesions induced by persistent chronic low-dose ionizing radiation[END_REF]. However, recent re-analysis of the data has concluded that the concentration of 137 Cs levels found in urine (6.47 Bq/l) would be too low to cause any increase in bladder malignancy [START_REF] Jargin | Urological concern after nuclear accidents[END_REF].

There has been a debate on the potentially increased risk of developing certain types of cancers to workers at the nuclear power plants (NPP) and people living nearby. A recent study in France surveying people living in proximity of NNPs showed that BCa was the only cancer, whose incidence was significantly associated with the proximity to NNPs, however it might be explained by excess incidence in two areas where nuclear waste is being treated and results in environmental release of chemical contaminants such as arsenic, therefore not confirming the link [START_REF] Desbiolles | Cancer incidence in adults living in the vicinity of nuclear power plants in France, based on data from the French Network of Cancer Registries[END_REF]. Therefore, further studies investigating the extent of risks to develop BCa of workers at NPPs are needed.

Radiation from Radiotherapy

Radiotherapy for pelvic cancer treatments has been long associated with increased risk of developing BCa as a secondary cancer for patients undergoing radiotherapy for prostate or cervical cancer [START_REF] Kleinerman | Second cancers following radiotherapy for cervical cancer[END_REF][START_REF] Brenner | Second malignancies in prostate carcinoma patients after radiotherapy compared with surgery[END_REF][START_REF] Pickles | The risk of second malignancy in men with prostate cancer treated with or without radiation in British Columbia, 1984-2000[END_REF].

However, the absolute effect has been reported to be low as concluded by a recent metaanalysis of 15 studies encompassing 619,479 participants undergoing radiotherapy for prostate cancer [START_REF] Zhao | High prevalence of secondary bladder cancer in men on radiotherapy for prostate cancer: Evidence from a meta-analysis[END_REF]. Another study found that the risk to develop second cancer in patients after radiotherapy for prostate cancer was not clearly increased and was speculated to be highly likely to reflect advanced age and lifestyle habits and comorbidities [START_REF] Hegemann | Risk of second cancer following radiotherapy for prostate cancer: A population-based analysis[END_REF]. In contrast, recent analysis from Moschini and colleagues showed that prostate cancer patients treated with radiotherapy were at significantly increased risk of developing a second primary BCa, but not rectal cancer [START_REF] Moschini | External Beam Radiotherapy Increases the Risk of Bladder Cancer When Compared with Radical Prostatectomy in Patients Affected by Prostate Cancer: A Population-based Analysis[END_REF]. These results need to be validated in a well-designed randomized prospective trial. Therefore, there is still a controversy regarding the extent of IR exposure contributes to potential development of BCa, but nevertheless in clinics it is maintained an important consideration in the treatment planning.

Pathology and classification

Majority of urothelial tumors are urothelial carcinomas (90% to 95%), while less common histologic types such as sarcoma, signet ring cell carcinoma, squamous cell carcinoma, small cell carcinoma and adenocarcinoma exist. Urothelial carcinomas are staged using the 2009 consensus Tumour -Node -Metastasis (TNM) system, which describes the extent of invasion (Tis -T4), and is graded according to their cellular characteristics. Tumors presenting a T2-T4 stage are classified as muscle-invasive BCa (MIBCs) as opposed to superficial or non-muscleinvasive BCas (NMIBCs), including carcinomas in situ (CIS), Ta and T1 tumors. Tumor grade is determined upon microscopic examination of the biopsy. BCa grades were defined in 1973 by the World Health Organization (WHO) in the following way: grade 1 (G1, well differentiated), grade 2 (G2, moderately differentiated) or grade 3 (G3, poorly differentiated) [START_REF] Mostofi | Histological Typing of Urinary Bladder Tumours, Histological Typing of Urinary Bladder Tumours[END_REF]. In 2004, the grading system of BCa was improved and re-classified including urothelial papilloma, papillary urothelial neoplasms of low-malignant potential (PUNLMP), low-grade papillary urothelial carcinoma and high grade urothelial carcinoma [START_REF] Eble | Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs[END_REF]. The details of staging and grading are summarized in Figure 1-2. About 35% of PUNLMP and ~70% of low and high-grade Ta tumors reoccur after treatment [START_REF] Holmäng | Stage progression in Ta papillary urothelial tumors: Relationship to grade, immunohistochemical expression of tumor markers, mitotic frequency and DNA ploidy[END_REF]. Disease progression of PUNLMP have almost never been reported and low grade Ta tumors rarely progress (approximately 5%), whereas high grade Ta tumors progress in nearly one of four cases [START_REF] Holmäng | Stage progression in Ta papillary urothelial tumors: Relationship to grade, immunohistochemical expression of tumor markers, mitotic frequency and DNA ploidy[END_REF]. All MIBC tumors are systematically high-grade cancers. Finally, among NMIBCs, CIS are high-grade neoplasms. CIS have a more frequent progression rate than papillary tumors (40% -50% vs. 5%) and are often associated with MIBC tumors [START_REF] Eble | Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs[END_REF].

The high recurrence rate of Ta and T1 (80%) tumors requires regular cystoscopy and lifelong surveillance. Therefore, the cost per patient of BCa from diagnosis to death is the highest of all cancers (Botteman et al., 2003). The TNM status has a very significant impact on prognosis, with a five-year survival rate for NMIBC tumors of ~90%, while dropping from 85% for T2 to 25% for T4 MIBC tumors and a mean survival of six to twelve months for metastatic BCa (Knowles and Hurst, 2015).

Molecular subtypes of muscle-invasive BCa

It is now clearly established that MIBC is a heterogeneous disease with a recently attained consensus of 6 different subgroups: luminal papillary (LumP ), luminal nonspecified (LumNS), luminal unstable (LumU), stroma-rich, basal/squamous (Ba/Sq), neuroendocrine (NE) (Figure 1-3) (Kamoun et al., 2020). Sensitivity to radiotherapy of the subtypes remains yet to be investigated, but it has been suggested to be increased in two subtypes: NE-like and LumU,

showing elevated cell cycle activity and low hypoxia signals (Pawlik and Keyomarsi, 2004;Horsman and Overgaard, 2016). This remains to be validated in preclinical and clinical studies. A first retrospective study by Choi and colleagues indicated that patients with basal tumors have the highest benefit from neoadjuvant chemotherapy (Choi et al., 2014). This was further confirmed in a larger retrospective study by Seiler et al., where no difference was observed between cisplatin-based or alternative chemotherapy regimens [START_REF] Seiler | Impact of Molecular Subtypes in Muscle-invasive Bladder Cancer on Predicting Response and Survival after Neoadjuvant Chemotherapy [Figure presented[END_REF]. However, there was a lack of association between pathological response and overall survival (OS) observed in the basal tumors. Prospective validation in a larger cohort is required to address this issue. In addition, our group showed that basal tumors are sensitive to EGFR inhibition in preclinical models (Rebouissou et al., 2014). Choi and colleagues also identified a TP53

subtype which later was found to comprise both luminal and non-luminal tumors (Choi et al., 2014;[START_REF] Dadhania | Meta-Analysis of the Luminal and Basal Subtypes of Bladder Cancer and the Identification of Signature Immunohistochemical Markers for Clinical Use[END_REF][START_REF] Lerner | Bladder cancer molecular taxonomy: Summary from a consensus meeting[END_REF]. These tumors are enriched with extracellular matrix (ECM) biomarkers and are resistant to neoadjuvant cisplatin-based chemotherapy. This subtype has a very strong overlap with the recently established stroma-rich consensus subtype (Figure 1-3).

Prospective and retrospective assignment of subtypes should provide prognostic information and may help to identify subgroup(s) of patients with increased sensitivity to chemoradiotherapy for improved future treatments.

Clinical management

Treatment for NMIBC

Although NMIBCs account for 70-80% of BCas and represent a major health issue and an economic burden, much less studies have been devoted to NMIBCs than MIBCs. No consensus classification exists for NMIBCs and the relationships between NMIBCs and MIBCs subtypes have not been clarified.

Transurethral resection of bladder tumor (TURBT) is an effective treatment for NMIBC patients, although tumours have a high recurrence rate, they rarely progress to advanced stages [START_REF] Anastasiadis | Best practice in the treatment of nonmuscle invasive bladder cancer[END_REF]. Three to eighteen percent of Ta tumours are high grade, with an average of 6.9% [START_REF] Sylvester | Predicting recurrence and progression in individual patients with stage Ta T1 bladder cancer using EORTC risk tables: A combined analysis of 2596 patients from seven EORTC trials[END_REF], which is considered as a more important factor for progression than the stage [START_REF] Anastasiadis | Best practice in the treatment of nonmuscle invasive bladder cancer[END_REF]. In addition to surgery, intravesical chemotherapy (such as mitomycin C) shows significant delay of tumor recurrence [START_REF] Bosschieter | Value of an Immediate Intravesical Instillation of Mitomycin C in Patients with Non-muscle-invasive Bladder Cancer: A Prospective Multicentre Randomised Study in 2243 patients [Figure presented[END_REF]. Continual surveillance is managed by periodic cystoscopy and cytological examination of the urine sediment. The number of invasive procedures and hospitalisations required make BCa one of the most expensive cancer to manage and treat [START_REF] Svatek | The economics of bladder cancer: Costs and considerations of caring for this disease[END_REF].

When multiple and/or high-grade tumours or CIS are detected, a course of Bacillus Calmette -Guérin (BCG) may be used (a weakened form of the bacterium Mycobacterium bovis) after the initial TURBT. The working mechanism of BCG is not exactly understood. It is thought that BCG induces an immune reaction, which kills cancer cells and is therefore considered as a form of immunotherapy. The standard 6-week treatment is given (called induction therapy) and this is followed by maintenance therapy for a minimum of one year and can last up to two years [START_REF] Gandhi | Bacillus Calmette-Guérin immunotherapy for genitourinary cancer[END_REF]. More than 95% of patients including relatively older and frailer patients tolerate well the intravesical BCG treatment without significant morbidity [START_REF] Racioppi | The challenges of Bacillus of Calmette-Guerin (BCG) therapy for high risk non muscle invasive bladder cancer treatment in older patients[END_REF].

BCG intravesical instillation is considered to be effective in 50-60% of treated patients [START_REF] Babjuk | EAU Guidelines on Non-Muscle-invasive Urothelial Carcinoma of the Bladder: Update 2016[END_REF]. Considering the combination of BCG supply shortage and alternative bladdersparing approaches in patients with BCG-refractory disease, the development of hyperthermia-inducing device-assisted therapies have emerged as new options for NMIBC.

These techniques, such as radiofrequency induced thermochemotherapeutic effect (RITE), conductive hyperthermic chemotherapy, and electromotive drug administration (EMDA), have shown promising efficacy [START_REF] Tan | Intravesical device-assisted therapies for non-muscle-invasive bladder cancer[END_REF].

Treatment for MIBC

Around the World, radical cystectomy (RC) with bilateral pelvic lymphadenectomy (PLND) and [START_REF] Vos | Choosing chemotherapy in patients with advanced urothelial cell cancer who are unfit to receive cisplatin-based chemotherapy[END_REF]. It should be noted that after radical cystectomy and ureter-deviating surgery, the renal function is impaired in up to 17% of patients (Dash et al., 2006).

Although RC have increased risk of infection, extensive bleeding and affected sexual function and quality of life, it achieves oncologic control [START_REF] Aziz | Prediction of 90-day Mortality After Radical Cystectomy for Bladder Cancer in a Prospective European Multicenter Cohort[END_REF][START_REF] Donat | Potential Impact of Postoperative Early Complications on the Timing of Adjuvant Chemotherapy in Patients Undergoing Radical Cystectomy: A High-Volume Tertiary Cancer Center Experience[END_REF]. In the absence of prospective randomized studies, it is not possible to compare RC versus other forms of therapy [START_REF] Monteiro | Radical cystectomy is the best choice for most patients with muscle-invasive bladder cancer? Opinion: Yes[END_REF].

Bladder sparing treatment for MIBC

Multimodality organ-sparing treatment has become the standard of care for many malignancies. The combination of extensive transurethral resection, chemotherapy and radiation has proven beneficial, reducing the risk of morbidity associated to radical surgery in BCa (Jiang et al., 2020). Sufficient data now exist from many international prospective studies to demonstrate that the survival results from bladder preservation protocols using trimodality treatment strategies compare favourably to radical cystectomy [START_REF] Hagan | RTOG 97-06: initial report of a phase I-II trial of selective bladder conservation using TURBT, twice-daily accelerated irradiation sensitized with cisplatin, and adjuvant MCV combination chemotherapy[END_REF][START_REF] Maarouf | Bladder preservation multimodality therapy as an alternative to radical cystectomy for treatment of muscle invasive bladder cancer[END_REF]James et al., 2012;[START_REF] Song | Organ preservation in bladder cancer: an opportunity for truly personalized treatment[END_REF]. Bladder-preserving therapy has several benefits compared to cystectomy, including a better quality of life with intact bladder function (Jiang et al., 2020). The role of radiotherapy in bladder sparing treatment is further discussed in the next secton (Article 1).

Article 1: Review of Preclinical Studies to Improve Radiotherapy

Response in Bladder Cancer: Comments and Perspectives

Linda Silina 1,2,3,4* , Fatlinda Maksut 2,3 , Isabelle Bernard-Pierrot 1 , François Radvanyi 1 , Gilles

Créhange 5 , Frédérique Mégnin-Chanet 2,3,4 , Pierre Verrelle 2,3,4,5,6* Submitted for publication in Cancers

Abstract

Simple Summary: Bladder cancer is a major global health problem. Bladder removal surgery is the standard treatment for muscle-invasive bladder cancer (25% of all bladder cancer), but this treatment negatively affects the quality of life, especially for elderly and frail patients.

Tumour resection followed by combination of radiotherapy and chemotherapy has emerged as a promising bladder preserving strategy. However, this strategy is unable to avoid radiationrelated bladder side effects. Therefore, it is of great interest to discover novel strategies radiosensitising tumours while sparing normal bladder tissue. In this review, we analyzed the preclinical studies of radiosensitising strategies in bladder cancer and provided suggestions to improve forthcoming studies.

Abstract: Bladder cancer is among the top ten most common cancer types in the world.

Around 25% of all cases are Muscle-Invasive Bladder Cancer, for which the gold standard treatment in the absence of metastasis is the cystectomy. In the recent years, trimodality treatment associating maximal transurethral resection and radiotherapy combined with concurrent chemotherapy is increasingly used as an organ-preserving alternative. However, the use of this treatment is still limited by the lack of biomarkers predicting tumour response and by a lack of targeted radiosensitising drugs that can improve the therapeutic index, especially by limiting side effects such as bladder fibrosis. In order to improve the bladderpreserving treatment, preclinical studies addressing these main issues ought to be considered. for MIBC patients who were ineligible for cystectomy and observed high complete response and survival rates, which consequently encouraged further studies [START_REF] Shipley | Treatment of Invasive Bladder Cancer by Cisplatin and Radiation in Patients Unsuited for Surgery[END_REF].

Housset and colleagues first reported promising findings using 5-fluorouracil (5-FU) + cisplatin combination as a radiosensitiser in MIBC (Housset et al., 1993). Following further studies, it became evident that the concurrent chemoradiotherapy (CCRT) improves locoregional disease control in MIBC as compared to RT alone [START_REF] Coppin | Improved local control of invasive bladder cancer by concurrent cisplatin and preoperative or definitive radiation[END_REF]James et al., 2012;Jiang et al., 2020). But despite the existing volume of research, there remains no standard procedure of CCRT regimen. Although different chemotherapy (CT) agents have been investigated, most evidence exists for cisplatin [START_REF] Coppin | Improved local control of invasive bladder cancer by concurrent cisplatin and preoperative or definitive radiation[END_REF] or mitomycin C + 5-FU, (James et al., 2012) and more recently for gemcitabine [START_REF] Thompson | Tolerability of Concurrent Chemoradiation Therapy With Gemcitabine (GemX), With and Without Prior Neoadjuvant Chemotherapy, in Muscle Invasive Bladder Cancer[END_REF]. In addition, other approaches have been explored such as the use of nicotinamide and carbogen to fight hypoxia-related radioresistance [START_REF] Hoskin | Radiotherapy with concurrent carbogen and nicotinamide in bladder carcinoma[END_REF]. Mitomycin C + 5-FU is a very effective radiosensitising combination which has improved clinical outcomes in head and neck and anal cancers [START_REF] Budach | Hyperfractionated accelerated chemoradiation with concurrent fluorouracilmitomycin is more effective than dose-escalated hyperfractionated accelerated radiation therapy alone in locally advanced head and neck cancer: Final results of the Radiotherapy Cooperative Clinical Trials Group of the German Cancer Society 95-06 prospective randomized trial[END_REF][START_REF] Ajani | Fluorouracil, mitomycin, and radiotherapy vs fluorouracil, cisplatin, and radiotherapy for carcinoma of the anal canal: A randomized controlled trial[END_REF][START_REF] Forastiere | Long-term results of RTOG 91-11: A comparison of three nonsurgical treatment strategies to preserve the larynx in patients with locally advanced larynx cancer[END_REF] with RT. Although cisplatin + 5-FU is widely delivered, mitomycin C + 5-FU is also a common combination particularly for frailer and elderly bladder cancer (BCa) patients, given the absence of nephrotoxicity when compared to platinum drugs (James et al., 2012;[START_REF] El-Taji | Bladder Sparing Approaches for Muscle-Invasive Bladder Cancers[END_REF].

With the advances of the cystectomy techniques, radical cystectomy with pelvic lymphadenectomy and cisplatin-based CT has become the gold standard treatment for patients with MIBC. RT can be considered as an adjuvant therapy following radical cystectomy in patients with pathological high-risk of loco regional relapse (i.e. pT3-4, positive nodes, positive surgical margins), but the pelvic toxicity remains significant despite the advances in RT such as intensity-modulated radiation therapy. This management approach is supported by numerous renowned organizations, such as the National Comprehensive Cancer Network in the United States [START_REF] Flaig | NCCN Guidelines Updates: Management of Muscle-Invasive Bladder Cancer[END_REF], as well as by the European Association of Urology [START_REF] Witjes | European Association of Urology Guidelines on Muscle-invasive and Metastatic Bladder Cancer: Summary of the 2020 Guidelines[END_REF]. In fact, the latter has made strong recommendation to use cisplatin based neoadjuvant CT before radical cystectomy for treating MIBC (T2-T4aN0M0) and high-risk nonmuscle invasive bladder cancer. Level 1a evidence supports that neoadjuvant cisplatin-based

CT increases survival at 6 years by 8% [START_REF] Galsky | Comparative effectiveness of gemcitabine plus cisplatin versus methotrexate, vinblastine, doxorubicin, plus cisplatin as neoadjuvant therapy for muscle-invasive bladder cancer[END_REF][START_REF] Witjes | European Association of Urology Guidelines on Muscle-invasive and Metastatic Bladder Cancer: Summary of the 2020 Guidelines[END_REF]. Although postradical cystectomy history can be associated with increased risk of infection, extensive bleeding, affected sexual function and quality of life, it achieves locoregional control and results in 60% of overall 5-year survival [START_REF] Donat | Potential Impact of Postoperative Early Complications on the Timing of Adjuvant Chemotherapy in Patients Undergoing Radical Cystectomy: A High-Volume Tertiary Cancer Center Experience[END_REF][START_REF] Aziz | Prediction of 90-day Mortality After Radical Cystectomy for Bladder Cancer in a Prospective European Multicenter Cohort[END_REF]. The absence of prospective randomized studies has impeded the comparison of radical cystectomy versus other forms of therapy [START_REF] Monteiro | Radical cystectomy is the best choice for most patients with muscle-invasive bladder cancer? Opinion: Yes[END_REF]. The treatment choice for MIBC between radical cystectomy versus bladder preservation largely depends on the specialist expertise in the treatment centre and often varies among countries.

Although RT has been used in bladder cancer (BCa) treatment since the 1950s, there is a relatively low number of preclinical studies on the topic of radiosensitisers in BCa compared to other cancer types. The first study identified was in the 1979 [START_REF] Weldon | Combination radiotherapy and chemotherapy in murine bladder cancer[END_REF].

Altogether, we identified 85 studies investigating RT in BCa preclinical models published between 1979 and October 2020.

Radiotherapy as part of bladder preserving treatment in clinics

In the last decade, trimodality treatment consisting of maximal transurethral resection of bladder tumour (TURBT) coupled with CT has emerged as a bladder sparing treatment either driven by patients' choice or due to the patients' ineligibility for radical cystectomy. In most of the CCRT protocols, including the pioneering study of Housset et al. [2], following cystoscopic evaluation of the initial CCRT response, good responders complete the CCRT schedule. Bladder preservation outcomes heavily depend on tumour response to CCRT (reviewed by [START_REF] Ploussard | Critical analysis of bladder sparing with trimodal therapy in muscle-invasive bladder cancer: A systematic review[END_REF]) and in the case of poor response, radical cystectomy is planned [2]. Meta-analysis of studies has shown comparable survival outcomes and bladder preservation rates leading to the inclusion of trimodality treatment in the current MBIC treatment guidelines as an equally effective treatment for well selected patients [START_REF] Murali-Krishnan | Bladder-sparing treatment in MIBC: Where do we stand?[END_REF]. For RT, two schedules are in common use worldwide: a split-dose regimen with interim cystoscopy is used in the United States [START_REF] Efstathiou | Long-term outcomes of selective bladder preservation by combinedmodality therapy for invasive bladder cancer: The MGH experience[END_REF], whilst a single phase treatment is most widely used elsewhere (James et al., 2012). A standard RT schedule consists of external beam radiation therapy (EBRT) to the bladder and limited pelvic lymph nodes with an initial dose of 40-46 Gy, with a boost to the whole bladder of 14-20 Gy, with a total dose of 60-66 Gy [START_REF] Mak | Long-term outcomes in patients with muscle-invasive bladder cancer after selective bladder-preserving combined-modality therapy: A pooled analysis of radiation therapy oncology group protocols 8802, 8903[END_REF] with conventional fractionation. Partial bladder irradiation remains controversial [START_REF] Huddart | Randomized noninferiority trial of reduced high-dose volume versus standard volume radiation therapy for muscle-invasive bladder cancer: Results of the BC2001 Trial (CRUK/01/004)[END_REF] as well as a tumour dose escalation which is still investigational [START_REF] Murthy | Clinical outcomes with dose-escalated adaptive radiation therapy for urinary bladder cancer: A prospective study[END_REF]. Moderate hypofractionation is a welltolerated option for frailer and elderly patients, even in combination with CT (S. Zhang et al., 2015). In addition, RT has been recently successfully combined with several immunotherapy agents in clinical trials for metastatic BCa [START_REF] Daro-Faye | Combined radiotherapy and immunotherapy in urothelial bladder cancer: harnessing the full potential of the anti-tumor immune response[END_REF].

Limitations of use of RT in MIBC in clinics

There are three main limitations of using CCRT in MIBC. Firstly, there is a significant risk of pelvic recurrence (25%-50%) [17]. Secondly, CCRT treatment may create damage to the bladder wall resulting in undesirable toxicity. Bladder response to RT includes early toxicity that occurs within six months post-RT and late bladder toxicity that occurs after six months.

Late toxicity is characterized by replication of the injured vascular endothelial cells and connective tissue, but failure of regeneration, and may result in fibrosis, which can lead to the need of ultimate cystectomy [START_REF] Marks | The response of the urinary bladder, urethra, and ureter to radiation and chemotherapy[END_REF]. Presently, the underlying reasons of including whole bladder in the clinical target volume (CTV) are that the irradiation field is difficult to be adjusted to concentrate on the bulk tumour and due to the high risk of spread of bladder tumours within the urothelium layers. It is proving problematic to reliably and accurately define the CTV exact position for the RT delivery as the bladder volume is continuously changing with the level of urine and post-void residual volume [START_REF] Viswanathan | Radiation Dose-Volume Effects of the Urinary Bladder[END_REF]. Bladder wall and/or tumour movement of up to 4 cm and volume variations of up to 44% have been detected using repeated computed tomography scans during therapy volume, presenting significant challenge to spare surrounding tissue [START_REF] Hellebust | Inter fraction variations in rectum and bladder volumes and dose distributions during high dose rate brachytherapy treatment of the uterine cervix investigated by repetitive CT-examinations[END_REF][START_REF] Muren | Organ motion, set-up variation and treatment margins in radical radiotherapy of urinary bladder cancer[END_REF]. Nevertheless, it is important to emphasize that modern radiotherapy techniques such as image-guided radiation therapy, intensity-modulated radiation therapy and volumetric-modulated arc therapy have significantly advanced and improved the sparing of pelvic organs, especially small intestines, while better targeting delivery to the bladder (reviewed by (S. Zhang et al., 2015)).

Thirdly, at the present time there is a lack of validated biomarkers predicting tumour response to CCRT [START_REF] Forker | Biomarkers of Tumour Radiosensitivity and Predicting Benefit from Radiotherapy[END_REF][START_REF] Koga | Biomarkers for predicting clinical outcomes of chemoradiation-based bladder preservation therapy for muscle-invasive bladder cancer[END_REF][START_REF] Desai | The challenge of matching assays to biology in DNA damage response biomarkers for response to radiotherapy in bladder cancer[END_REF]. Several candidates from the DNA damage response (DDR) pathways have been investigated [START_REF] Choudhury | MRE11 expression is predictive of cause-specific survival following radical radiotherapy for muscle-invasive bladder cancer[END_REF][START_REF] Kawashima | Excision repair cross-complementing group 1 may predict the efficacy of chemoradiation therapy for muscle-invasive bladder cancer[END_REF][START_REF] Laurberg | Expression of TIP60 (tat-interactive protein) and MRE11 (meiotic recombination 11 homolog) predict treatment-specific outcome of localised invasive bladder cancer[END_REF].

Unfortunately, even the most promising biomarkers, such as double strand break repair nuclease MRE11 (MRE11), have failed to generate reproducible data. In the latest multicentre collaborative effort to validate MRE11 as a biomarker, the immunohistochemistry scoring results varied considerably and failed to attain a reliable dataset [START_REF] Walker | MRE11 as a Predictive Biomarker of Outcome After Radiation Therapy in Bladder Cancer[END_REF]. Finally, a complete or near complete response assessed by cystoscopy after 4-5 weeks induction phase remains as the only reliable predictor of treatment outcome.

MIBC molecular subtypes as biomarkers for CCRT response

BCa tumour subtypes

MIBC can be classified into molecular subtypes by transcriptome profiling, thus allowing patient stratification to consider different therapeutic options. However, MIBC subtyping is still not included in routine clinical practice due to several classifications existing simultaneously in the last decade.

In 2020, a consensus on MIBC subtypes has been reached (Kamoun et al., 2020), giving hope for a rapid translation into clinics. Furthermore, a single-sample classifier has been established enabling to assign a consensus class label to a tumour sample's transcriptome. This classifier can be applied to data from frozen as well as to paraffin embedded samples analyzed by different technologies (arrays, RNA-seq, including 3'RNA-seq). There are six biologically relevant consensus molecular classes, namely luminal papillary, luminal non-specified, luminal unstable, stroma-rich, basal/squamous and neuroendocrine-like (Kamoun et al., 2020).

Among the luminal subtypes the most represented is the luminal papillary subtype (24% of MIBC), the other two luminal subtypes representing 15% (for the luminal unstable subtype) and 8% (for the luminal non-specified subtype) of MIBC (Figure 1a). The other most frequent subtype is the basal/squamous subtype representing 33% of MIBC. Further, 15% of MIBC represent stroma-rich and 3% neuroendocrine-like subtype (Figure 1a) (Kamoun et al., 2020).

Several retrospective studies have highlighted the clinical significance of molecular stratification of MIBC suggesting that responses to treatment could be predicted by tumour subtyping [START_REF] Choi | Identification of Distinct Basal and Luminal Subtypes of Muscle-Invasive Bladder Cancer with Different Sensitivities to Frontline Chemotherapy[END_REF][START_REF] Rosenberg | Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: A singlearm, multicentre, phase 2 trial[END_REF][START_REF] Seiler | Impact of Molecular Subtypes in Muscle-invasive Bladder Cancer on Predicting Response and Survival after Neoadjuvant Chemotherapy [Figure presented[END_REF][START_REF] Mariathasan | TGFβ attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells[END_REF]. The first retrospective study by Choi et al. indicated that patients with basal tumours have the highest benefit from neoadjuvant CT [START_REF] Choi | Identification of Distinct Basal and Luminal Subtypes of Muscle-Invasive Bladder Cancer with Different Sensitivities to Frontline Chemotherapy[END_REF]. This was further confirmed in a larger retrospective study by Seiler et al., where no difference was observed between cisplatin-based or alternative CT regimens [START_REF] Seiler | Impact of Molecular Subtypes in Muscle-invasive Bladder Cancer on Predicting Response and Survival after Neoadjuvant Chemotherapy [Figure presented[END_REF]. However, there was a lack of association between pathological responses and overall survival for the patients having basal tumours. Prospective validation in a larger cohort is required to address this issue. In addition, our group showed that basal tumours are sensitive to epidermal growth factor receptor (EGFR) inhibition in preclinical models (Rebouissou et al., 2014). Sensitivity to RT of the subtypes remains yet to be investigated, but it has been suggested to be increased in two subtypes: neuroendocrine-like and luminal unstable, which show elevated cell cycle activity and low hypoxia signals (Pawlik and Keyomarsi, 2004;Horsman and Overgaard, 2016).

However, at the present time no significant difference has been found in local relapse-free survival between bladder tumour subtypes in MIBC patients treated by TURBT followed by CCRT (Efstathiou et al., no date) or RT alone [START_REF] Choudhury | A hypoxia transcriptomic signature to predict benefit from hypoxiamodifying treatment for muscle-invasive bladder cancer patients[END_REF].

BCa cell line molecular subtypes

There is no agreement yet on the molecular classification of BCa cell lines in particular with regards to the new consensus classification (Kamoun et al., 2020). Our group made a first dichotomy between basal and non-basal cell lines (Rebouissou et al., 2014). Then, Earl et al. assigned the subtypes to a series of 40 BCa cell lines (Earl et al., 2015). Our group has made more stringent classification [START_REF] Shi | Identification of new driver and passenger mutations within APOBEC-induced hotspot mutations in bladder cancer[END_REF]. For example, 5 cell lines discussed in this review were identified as 'basal' by Earl et al., while classified as 'non-luminal, non-basal' by Shi et al. (Shi et al., 2020). These non-luminal, non-basal cell lines expressed epithelium to mesenchyme transition markers and do not express E-cadherin. These cells could represent sarcomatoid tumours which is a rare entity in vivo. This phenotype could be present in the initial tumours or acquired in vitro. In vivo the sarcomatoid tumours are classified mainly in the basal subtype probably due to commonalities in their stroma. However, data from preclinical studies on radiosensitisation of relevant models representing different molecular subtypes are sparse. It is worth noting that none of the studies included in this review has discussed the relevance of a molecular subtype of the chosen preclinical models. It is in part due to the fact the consensus was only recently established and due to absence of classification of the cell lines until recently.

BCa preclinical models in RT studies

The preclinical models and study design should utilize the information available regarding the subtypes of the cell lines and also revisit the information available regarding the patient and the original tumour from which the cell line has been established. BCa preclinical models and their molecular features have been recently reviewed by Zuiverloon et al. [START_REF] Zuiverloon | Systematic Review: Characteristics and Preclinical Uses of Bladder Cancer Cell Lines[END_REF] and rodent models with relevant molecular subtypes have been described by [START_REF] Ruan | Mouse Models of Muscle-invasive Bladder Cancer: Key Considerations for Clinical Translation Based on Molecular Subtypes[END_REF]. Here we critically discuss the BCa preclinical models used in experimental studies in association with RT treatment.

BCa cell lines

Molecular subtypes

Selection of BCa cell lines with different mutation status and from different subtypes would better reflect the heterogeneity of MIBC cancer patients. We identified 29 different BCa cell lines used in the preclinical studies of RT. We assigned molecular subtypes to the human BCa using a classification recently proposed by our team [START_REF] Shi | Identification of new driver and passenger mutations within APOBEC-induced hotspot mutations in bladder cancer[END_REF] (Table 1, additional information regarding the mutational status and origin are available in Supplementary Table S1). We found that from all the BCa cell lines used in RT studies, 26% are of luminal subtype, which is in contrast to 47% of human tumours considered having luminal features (Figure 1a, 1b). 21% of cell lines used were of basal subtype (contrary to 33% of human tumours) and the largest part (40%) of BCa cell lines were classified neither luminal, nor basal (Figure 1b). These cells which express epithelial to mesenchyme transition markers and do not express Ecadherin are rarely found in vivo. They could represent sarcomatoid tumours or transient state of tumour cells representing only a fraction of tumours. This transient state could be of importance during the invasion or the metastatic process.

Regrettably, we found that 7% of all in vitro studies and 12% of all in vivo studies have used cell lines that have been identified as cross-contaminated or misidentified (Figure 1b and 1C, Supplementary Table S2 and Supplementary Table S3).

639-V CVCL_1048 J82 CVCL_0359 KK47 CVCL_8253 T24 CVCL_0554 TCC-SUP CVCL_1738 UMUC3 CVCL_1783 CAL29 CVCL_1808 n/c NTUB1 CVCL_RW29 n/a OBR n/a SW-800 CVCL_A684 UCRU-BL13 CVCL_M873 UCRU-BL17 CVCL_M007 UCRU-BL28 CVCL_4904 Mouse MB49 CVCL_7076 basal (mouse) 2 MB49-I CVCL_VL62 MBT2 CVCL_4660
Table 1. 1 RT4 cell line originates from re-occurring human transitional cell papilloma [START_REF] Rigby | A human tissue culture cell line from a transitional cell tumour of the urinary bladder: growth, chromosone pattern and ultrastructure[END_REF]); 2 Mouse cell lines have been classified by [START_REF] Ruan | Mouse Models of Muscle-invasive Bladder Cancer: Key Considerations for Clinical Translation Based on Molecular Subtypes[END_REF]. Abbreviations: n/a, information not available (classification have not been applied); n/c, not categorized (not coherent classification depending on the dataset used).

Gender

Given the difference incidence rates between men and women in BCa, it is important to exclude potential gender bias in the study design and include cell lines from both sexes. BCa is significantly more frequent in males, while the majority of studies (54 of 85 studies identified or 64%) have used RT112 and T24 cell lines which are of female origin (Supplementary Table S1, Supplementary Table S2). Androgen receptor (AR) signalling could be implicated in the gender disparity of BCa but it remains to be confirmed [START_REF] Zhang | Understanding the gender disparity in bladder cancer risk: The impact of sex hormones and liver on bladder susceptibility to carcinogens[END_REF]Dobruch et al., 2016).

Furthermore, AR singling has been recently shown to reduce radiosensitvity [START_REF] Ide | Androgen receptor signaling reduces radiosensitivity in bladder cancer[END_REF].

Intrinsic radiosensitivity

There is a lack of certainty when it comes to the intrinsic radiosensitivity of BCa cell lines as many have been used only in a single study and therefore obtained radiation-response curves has never been reproduced (Supplementary Table S1, Nr of studies). On the other hand, the wide range of different cell lines for the use of radiosensitisation studies offers opportunity to examine many potential factors influencing radiation response. Only a single dataset comparing intrinsic radiosensitivities of 19 BCa cell lines exists by Yard et al. [START_REF] Yard | A genetic basis for the variation in the vulnerability of cancer to DNA damage[END_REF]. Yard et al. used data derived from a single experimental platform and performed analysis using a rigorous statistical methodology. They studied genetic determinants influencing tumour response to DNA damage and influencing tumour survival as assessed by colony forming assays. The radiosensitivity was described by the area under the curve (AUC)

and scored from 0 (completely sensitive) to 7 (completely resistant) (Table 2). There was a high heterogeneity among the BCa cell lines (radiosenstivity varying from 1.883 (radiosensitive) to 5.228 (radioresistant)) [START_REF] Yard | A genetic basis for the variation in the vulnerability of cancer to DNA damage[END_REF] (Table 2). It is interesting to note that by associating with the molecular subtype, the three most radioresistant cell lines are of basal subtype (AUC≥=4.4). However, there were also two basal cell lines reported to be very radiosensitive (AUC<2.5). Luminal cell lines included in this study had a lower variation of the AUC (2.935-4.126) all being moderately radiosensitive/moderately radioresistant. However, this data set included only 5 luminal cell lines so more studies having stringent measurements of radiosensitivity are desirable. [START_REF] Rigby | A human tissue culture cell line from a transitional cell tumour of the urinary bladder: growth, chromosone pattern and ultrastructure[END_REF]. Abbreviations: n/c, not categorized (not coherent classification depending on the dataset used).

BCa xenografts

The majority of data generated from the RT studies in BCa are currently from BCa xenografts (Table 3), fewer studies have used syngeneic mouse models (Table 4), while none have used patient-derived xenografts (PDX). RT studies using 3D-BCa xenografts have the advantage to gather more information when compared to in vitro 2D-models, as radiosensitivity can be studied in 3D considering hypoxic areas. While interactions between tumour and its microenvironment can be limited by the species barrier. We found that 7 (29%) BCa xenografts used were of luminal subtype (Table 3, Figure 1c). While only 1 study (4%) used cell line of basal subtype (Table 3, Figure 1c). Clearly, the BCa cell lines of basal subtype has been less studied in the RT context. No study has compared the same treatment arms between xenografts of both main subtypes, thus not allowing to compare the CCRT response between these subtypes. We analysed the in vivo study design whereby different radiation schedules were employed (single larger radiation dose versus fractionated dose delivery schedule). R3

in vivo studies have used cell lines reported as cross-contaminated (Supplementary Table S3). [START_REF] Zhang | Combination with CD/5-FC gene therapy enhances killing of human bladdercancer cells by radiation[END_REF] Table 3. 1 The initial size of the tumour is defined as the size of the tumour at the start of the RT or combination treatment (Day 1); 2 The minimum follow-up for the non-treated control were used to compare the growth of the xenografts; 3 RT4 cell line originates from re-occurring human transitional cell papilloma [START_REF] Rigby | A human tissue culture cell line from a transitional cell tumour of the urinary bladder: growth, chromosone pattern and ultrastructure[END_REF];

Syngeneic models

Evidently, syngeneic mouse models are suitable choice to test immunotherapy agents in combination with radiation treatment. Furthermore, studies of interactions of tumour cells with endothelial and fibroblastic syngeneic cells are more relevant than in a xenogeneic model. We identified 7 studies using syngeneic mouse models (Table 4). The three cell lines used in these studies were all chemically induced (detailed in [START_REF] Zuiverloon | Systematic Review: Characteristics and Preclinical Uses of Bladder Cancer Cell Lines[END_REF]) and resemble the human basal/squamous subtype (Rebouissou et al., 2014;[START_REF] Ruan | Mouse Models of Muscle-invasive Bladder Cancer: Key Considerations for Clinical Translation Based on Molecular Subtypes[END_REF]. We noted that all of these models were heterotopic, excluding the assessment of treatmentinduced bladder toxicity. Results of most of the studies are discussed further. et al., 1979) Table 4. 1 The initial size of the tumour is defined as the size of the tumour at the start of the RT or combination treatment (Day 1); 2 The minimum follow-up for the non-treated control were used to compare the growth of the xenografts.

Use of CT agents in combination with RT in experimental studies for BCa treatment

Cisplatin

Cisplatin is currently the most widely used radiosensitizing agent in MIBC, supported by a randomized clinical trial [START_REF] Coppin | Improved local control of invasive bladder cancer by concurrent cisplatin and preoperative or definitive radiation[END_REF]. According to the Clinical Practice Guidelines in Oncology, the most comprehensive guidelines for treatment of oncological patients in the United States, the recommended radiosensitising regimen for locally advanced or metastatic BCa is a combination of cisplatin and gemcitabine. However, it should be emphasized that this combination has not been investigated in our identified preclinical studies.

In our literature analysis, we found only two in vitro and four in vivo (preclinical) studies investigating cisplatin and RT in BCa (Table 5). Weldon et al. for the first time used cisplatin in combination with RT in a murine BCa model and compared different administration schedules and included also two other drugs for comparison [START_REF] Weldon | Combination radiotherapy and chemotherapy in murine bladder cancer[END_REF]. They found that the concomitant administration of cisplatin and RT was toxic, but when cisplatin was used as adjuvant therapy after completion of RT, synergistic effect was produced, but another CT drug cyclophosphamide was more effective in terms of growth delay [START_REF] Weldon | Combination radiotherapy and chemotherapy in murine bladder cancer[END_REF]. Further, a study by Kyriazis et al. observed the most synergistic effect when cisplatin was given on days 3 and 6 post-radiation using human BCa xenograft model (SW-800, not classified cell line) [START_REF] Kyriazis | Time dependence of the radiation-modifying effect of cis-diamminedichloroplatinum II (cisplatin, DDP) on human urothelial cancer grown in nude mice[END_REF]. In an in vitro study, Bedford et al. demonstrated that radiation-resistant cell lines are more sensitive to cisplatin and radiation compared to wild type human BCa cell lines (Bedford et al., 1987). Kawashima and colleagues investigated whether CRT response can be predicted using expression of excision repair crosscomplementing group 1 (ERCC1) and found that its downregulation improved the effect of CRT, but not of cisplatin alone in vitro [START_REF] Kawashima | Excision repair cross-complementing group 1 may predict the efficacy of chemoradiation therapy for muscle-invasive bladder cancer[END_REF]. Two further studies have used mouse BCa xenografts. Yoshida and colleagues investigate the prospect to improve CRT response by using heat shock protein (HSp-90) inhibitors in non-luminal, non-basal mouse BCa xenograft (UMUC3) while evaluating the effect on normal human urothelial (NHU) cells in vitro [START_REF] Yoshida | Low-dose Hsp90 inhibitors tumor-selectively sensitize bladder cancer cells to chemoradiotherapy[END_REF]. They found greater BCa sensitisation to cisplatin-based CRT after lowdose Hsp90 inhibitor treatment than with the combination of trastuzumab (HER2 blocking antibody) or LY294002 (PI3K inhibitor). A little sensitizing effects of NHU to CRT were found [START_REF] Yoshida | Low-dose Hsp90 inhibitors tumor-selectively sensitize bladder cancer cells to chemoradiotherapy[END_REF]. Williams et al., investigated how to improve the efficiency of CDDP in BCa, taking into consideration hypoxia, which has been previously identified as a potential cause of therapy failure in BCa [START_REF] Hoskin | GLUT1 and CAIX as intrinsic markers of hypoxia in bladder cancer: Relationship with vascularity and proliferation as predictors of outcome of ARCON[END_REF]. Hypoxia-Activated ProDrug AQ4N increased the efficacy of RT alone and cisplatin-based CRT in vivo [START_REF] Hoskin | GLUT1 and CAIX as intrinsic markers of hypoxia in bladder cancer: Relationship with vascularity and proliferation as predictors of outcome of ARCON[END_REF].

Normal tissue toxicity

Yes (NHU in vitro)

---

Gemcitabine

Results from eight phase I-II trials concluded that there is a strong evidence that CCRT regimens with concurrent gemcitabine are feasible and well tolerated in BCa [START_REF] Caffo | Concurrent gemcitabine and radiotherapy for the treatment of muscle-invasive bladder cancer: A pooled individual data analysis of eight phase I-II trials[END_REF]. Prospective randomized controlled trials are ongoing to definitively assess the efficacy of gemcitabine-based CCRT for MIBC. From the preclinical studies identified in our literature search, two have made use of gemcitabine as a radiosensitiser in vitro, while two have used preclinical mouse models in vivo (Table 6).

There has been some conflicting results among the early studies of use of gemcitabine in BCa in vitro models. In 2003, Fechner and colleagues showed no effect of radiosensitivity of gemcitabine in four different BCa cell lines (RT112, RT4, T24 and TCC-SUP) with differing p53 status [START_REF] Fechner | Preclinical evaluation of a radiosensitizing effect of gemcitabine in p53 mutant and p53 wild type bladder cancer cells[END_REF]. In contrast, Pauwels et al. demonstrated correlation between gemcitabine-induced S phase block resulting and sensitization to RT of a BCa cell line ECV304 and cell lines from other cancers in vitro. It is worth to note that the cell line used has been recognized as being contaminated by another BCa cell line (T24, non-luminal, non-basal, Supplementary Table S2) [START_REF] Pauwels | Cell cycle effect of gemcitabine and its role in the radiosensitizing mechanism in vitro[END_REF]. These studies did not use colony forming assay to investigate radiosensitisation, which is considered the gold standard for assessing RT efficiency. Another study used colony forming assay to compare gemcitabine radiosensitising effect in related bladder cancer cell line MGHU1 and its radiosensitive subclone Sb40 [START_REF] Sangar | An evaluation of gemcitabines differential radiosensitising effect in related bladder cancer cell lines[END_REF]. They demonstrated that gemcitabine is an effective radiosensitiser in these BCa cell lines, with greater sensitisation in the radioresistant parental line [START_REF] Sangar | An evaluation of gemcitabines differential radiosensitising effect in related bladder cancer cell lines[END_REF].

Interestingly, MGHU1 cells did not show S-phase accumulation, which is the suggested radiosensitisation mechanism, but its subclone S40b did, despite both being radiosensitised by gemcitabine, indicating that S-phase accumulation is unlikely to be a major mechanism of radiosensitisation by gemcitabine [START_REF] Sangar | An evaluation of gemcitabines differential radiosensitising effect in related bladder cancer cell lines[END_REF]. However, also MGHU1 cell line has been reported as contaminated by T24 cell line (Supplementary Table S2) [START_REF] Capes-Davis | Check your cultures! A list of cross-contaminated or misidentified cell lines[END_REF].

Further, Choudhury et al., used gemcitabine in combination of another targeted agent (imatinib). Imatinib (inhibitor of c-ABL, c-KIT, and platelet-derived growth factor receptor (PDGFR) tyrosine kinases) was found to be a sensitizing agent to RT and gemcitabine-based CT treatment using RT112 luminal cell line in vitro (alongside a prostate cancer cell line in vitro and in vivo), concluding that imatinib can sensitize tumour cells to DNA damaging agents and induce mitotic catastrophe [START_REF] Choudhury | Targeting homologous recombination using imatinib results in enhanced tumor cell chemosensitivity and radiosensitivity[END_REF]. Two studies from Anne Kiltie team at the University of Oxford have investigated gemcitabine in vivo (Table 6) (Kerr et al., 2014;Groselj et al., 2018). In the first one, Kerr et al. demonstrated that gemcitabine-resistant Calgem heterotopic xenografts were responsive to the combination of gemcitabine and irradiation (Kerr et al., 2014). In the second study, Groselj et al. colleagues showed that gemcitabine + RT resulted in more acute and late intestinal toxicity than HDAC inhibitor panobinostat + RT (Groselj et al., 2018).

IR regimen in vivo

1x 10, 12, or 14 Gy (acute toxicity) 5 x 5 Gy (late toxicity) .

5x2 Gy

Gemcitabine dose

single 100 mg/kg injection single 100mg/kg injection

Normal tissue toxicity

Yes (intestinal) -

In vivo study reporting/design

We noted that the in vivo study design of studies using cisplatin and/or gemcitabine have been very few and heterogeneous. Different radiation schedules have been employed (single larger radiation dose vs fractionated, more clinically relevant schedule). The source of irradiation was different (X-rays and gamma rays) and different dose rates were reported. It has been shown that a variation of X-ray energy and the dose rate can impact the relative biological effectiveness (RBE) both in cells in vitro and in vivo [START_REF] Sangar | An evaluation of gemcitabines differential radiosensitising effect in related bladder cancer cell lines[END_REF]90]. From the studies using gemcitabine and cisplatin, three studies had not reported the dose rate. Different choice of the radiation delivery schedule will prevent a direct comparison of the studies.

Targeted agents to improve RT response in BCa

Following the studies of classical CT drugs, multiple agents have been tested as potential radiosensitisers since 2000. Nineteen studies using BCa xenografts (Table 7) and six studies of syngeneic mouse tumour models (Table 8) have reported a significant radiosensitisation.

These studies are very heterogeneous, testing diverse agents ranging from RTK inhibitors, epigenetic modifiers, hypoxia-or angiogenesis targeting molecules, among others. Below, we comment on the data of few selected studies.

Epidermal Growth Factor Receptor

Receptor tyrosine kinases (RTKs) are frequently differentially expressed between normal and cancerous tissue. They mediate pro-proliferative, pro-survival pathways as well as DNA repair pathways, the activation of which could ultimately protect cancer cells from radiation-induced cell death. Furthermore, radiation-induced activation of several RTKs has been reported and belongs to the earliest events in response to DNA damage ( [START_REF] Contessa | Ionizing radiation activates Erb-B receptor dependent Akt and p70 S6 kinase signaling in carcinoma cells[END_REF]; reviewed by (Schmidt-Ullrich et al., 2003)).

Epidermal Growth Factor Receptor (EGFR) overexpression has been found in up to 70% of BCa tumours [START_REF] Chaux | High epidermal growth factor receptor immunohistochemical expression in urothelial carcinoma of the bladder is not associated with EGFR mutations in exons 19 and 21: A study using formalin-fixed, paraffin-embedded archival tissues[END_REF]. The team of Francois Radvanyi has been studying role of several RTKs in BCa and has identified basal subtype BCa cell line dependency to EGFR when not mutated for a RAS family members (Colquhoun et al., 2007;Rebouissou et al., 2014). EGFR is the most-studied RTK in the field of radiation oncology as it was the first RTK to be shown to be activated with RT [START_REF] Contessa | Ionizing radiation activates Erb-B receptor dependent Akt and p70 S6 kinase signaling in carcinoma cells[END_REF]Schmidt-Ullrich et al., 2003). A clinical trial combining EGFR blocking monoclonal antibody with RT versus RT alone significantly improved overall survival in head and neck squamous cell carcinoma patients (Bonner et al., 2006).

In Further, Gefitinib + RT induced significant growth delay in non-luminal, non-basal J82 cell line xenografts compared to single treatment (Colquhoun et al., 2007).

Chromatin modifiers/Epigenetic regulators

High levels of histone deacetylases (HDACs) have been detected in BCa tumours [START_REF] Giannopoulou | Revisiting histone deacetylases in human tumorigenesis: The paradigm of urothelial bladder cancer[END_REF]. HDAC inhibitors have been tested alone in clinical trials in advanced solid tumours, including BCa, however high reported toxicities to normal tissue has impeded their progress to clinics for example of the HDAC inhibitor mocetinostat [START_REF] Grivas | Mocetinostat for patients with previously treated, locally advanced/metastatic urothelial carcinoma and inactivating alterations of acetyltransferase genes[END_REF]. The team of Anne Kiltie at Oxford University has been studying other HDAC inhibitors as potential radiosensitisers in BCa preclinical models and have found promising results using pan-HDAC inhibitor panobinostat (Groselj et al., 2018) and more selective HDAC inhibitor romidepsin [START_REF] Paillas | The Histone Deacetylase Inhibitor Romidepsin Spares Normal Tissues While Acting as an Effective Radiosensitizer in Bladder Tumors in vivo[END_REF]. In addition, no increase in acute or late toxicity following mouse pelvic irradiation has been reported in (Groselj et al., 2018).

Radio-Immunotherapy

In BCa preclinical models, immune checkpoint inhibitors have been used with RT only in one study using an anti-PD-L1 antibody (Wu et al., 2016a) (Table 8) Table 8. Abbreviations: EGFR: epidermal growth factor receptor, HER2: receptor tyrosine-protein kinase erbB-2; HMGB1: high mobility group box 1; PDGF-R: platelet-derived growth factor receptor; PD-L1: programmed death ligand 1; TKI: tyrosine kinase inhibitor.

HMGB1 inhibitor

Glycyrrhizin

Suggestions to improve the design of future preclinical BCa studies: New agents and relevant models

In order to improve RT, its therapeutic index (i.e. the ratio: antitumour efficiency/toxic effects on surrounding healthy bladder tissues) must be increased. Modern radiotherapy has advanced spatial targeting of clinical tumour volume, including whole bladder and pelvic nodes while reducing side effects to pelvic organs other than the bladder. However, since the whole bladder is included in the clinical target volume (CTV), only strategies aiming at radiosensitising tumour tissue selectively and not or to a lesser extent of the normal bladder wall, will improve therapeutic index of RT.

Molecular subtype consideration

Gemcitabine and cisplatin are effective radiosensitisers, but other agents have shown superior effect in the few comparative studies in vitro and in vivo (Table 5 and6

New targeted agents

In BCa, several experimental studies have shown the therapeutic efficacy of RTK inhibition other than EGFR. Fibroblast Growth Factor Receptor 3 (FGFR3) has been extensively studied in BCa due to its frequent mutations/translocations in the BCa, driving oncogenic dependency (more than 65% of NMIBCs and 15% of MIBCs) [START_REF] Hedegaard | Comprehensive Transcriptional Analysis of Early-Stage Urothelial Carcinoma[END_REF]Robertson et al., 2017;Mahe et al., 2018). Pan-FGFR inhibitor has been recently shown to radiosensitise tumours in head and neck squamous cell carcinoma xenografts and PDX [START_REF] Fisher | Fibroblast Growth Factor Receptors as Targets for Radiosensitization in Head and Neck Squamous Cell Carcinomas[END_REF]. In the context of BCa subtypes, 50% of luminal BCa tumours harbour an FGFR3 alteration and therefore could potentially benefit the most from anti-FGFR3+RT treatment.

Recently, TYRO3, a member of the TAM family of RTKs (comprising TYRO3, AXL and MERTK)

has been identified as a potential target in the BCa (Dufour et al., 2019). TYRO3 overexpression has been reported in 50% of MIBC and results in TYRO3-dependency for growth of BCa cancer cell lines (Dufour et al., 2019). However, FGFR3 and TYRO3 has never been explored as a target for radiosensitisation in BCa.

Using syngeneic models allow investigating the therapeutic index of different radiosensitising agents including effect on tumour cells but also on immune response and other interactions restricted by species barrier. For example, inhibition of the TAM receptors could improve RT efficacy by increasing directly radiation-induced tumour cell killing and also by promoting innate immunity [START_REF] Aguilera | Reprogramming the immunological microenvironment through radiation and targeting Axl[END_REF]Tormoen, Crittenden and Gough, 2017). Currently, in the field of radiation oncology there are many studies in syngeneic mouse models exploring different hypofractionated RT regimens in combination with immune checkpoint inhibitors, such as anti-PD-L1 and anti-T cell immunoreceptor with Ig and ITIM domains (TIGIT) in colorectal cancer [START_REF] Grapin | Optimized fractionated radiotherapy with anti-PD-L1 and anti-TIGIT: A promising new combination[END_REF]. The underlying interest is to combat PD-L1 expression which has been shown to be upregulated upon RT [START_REF] Dovedi | Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade[END_REF]. Further, also abscopal effect is being investigated and has been shown that RT can promote a responses of lung cancer to cytotoxic T-lymphocyte 4 (CTLA-4) blockade [START_REF] Formenti | Radiotherapy induces responses of lung cancer to CTLA-4 blockade[END_REF]. There is clinical evidence that basal subtype benefits from early aggressive management with CT agents and would benefit from T cell modulators (i.e., targeting CTLA-4) and EGFR, NFκB, Hif-1α/VEGF, and/or Stat-3-targeted agents will also be active within this subtype [START_REF] Choi | Intrinsic basal and luminal subtypes of muscle-invasive bladder cancer[END_REF]. Such preclinical studies are needed in BCa to improve the RT-immunotherapy modalities.

Pelvic toxisity assessment

Experimental devices dedicated to accurate mice irradiation are currently available, which was an in vitro study using normal human urothelium (NHU) cells [START_REF] Yoshida | Low-dose Hsp90 inhibitors tumor-selectively sensitize bladder cancer cells to chemoradiotherapy[END_REF]. NHU cultures are relevant models to study bladder toxicity in vitro (J [START_REF] Southgate | Normal human urothelial cells in vitro: proliferation and induction of stratification[END_REF]. NHU

cultures allow studying the impact on the normal urothelial cell proliferation from the RT or combined treatments as a first step before investigations in vivo. In addition, NHU can be differentiated into the non-proliferative phenotype, which is the physiological state of NHU cells in the human bladder (Varley et al., 2004;Cross et al., 2005). Differentiated NHU cell monolayers would be a more relevant model to study radiation-induced toxicity in vitro.

Use of orthotopic mice models

Currently, there is no preclinical published study using syngeneic or xenogeneic orthotopic graft in the field of BCa RT. However, Jäger et al., have developed a high-precision approach consisting in ultrasound-guided tumour cell inoculation within the bladder wall [START_REF] Jäger | Ultrasound-Guided Intramural Inoculation of Orthotopic Bladder Cancer Xenografts: A Novel High-Precision Approach[END_REF]. Another interesting model is the UPII-SV40T transgenic mouse model which expresses the SV40 large T antigen specifically in the urothelium and reliably develops BCa [START_REF] Zhang | Urothelium-specific expression of an oncogene in transgenic mice induced the formation of carcinoma in situ and invasive transitional cell carcinoma[END_REF]. In addition, there is a simple chemically induced BCa model, developed by daily exposure to BBN in drinking water (0.05%). Around 12 weeks of exposure to BBN result in development macroscopic lesions [START_REF] Vasoncelos-Nobrega | BBN as an Urothelial Carcinogen[END_REF]. At the present time, all of these orthotopic models have never been used in preclinical RT studies.

Humanised mouse models

In recent years, humanized mouse models have been more widely used in the field of immunology. For example NOD scid gamma (NSG) highly immunodeficient mouse model have no B-, T-, and natural killer (NK) cells, and therefore allow the engraftment of tumour and immune cells of human origin [START_REF] Shultz | Humanized mice in translational biomedical research[END_REF][START_REF] De La Rochere | Humanized Mice for the Study of Immuno-Oncology[END_REF].

Indeed, a recent study using this model established BCa xenografts in this humanized system and observed significant tumour growth delay using a pan-PI3K inhibitor in tumours bearing a PIK3CA mutation. Furthermore, pan-PI3K treated PIK3CA-mutated BCa tumours were sensitive to PD-1 blockade. These results showed potential of combination of PI3K inhibitors with immune checkpoint inhibitors to overcome resistance to immune checkpoint inhibitors [START_REF] Borcoman | Inhibition of PI3K pathway increases immune infiltrate in muscle-invasive bladder cancer[END_REF]. It would be interesting to include additional treatment arms combining such strategies with RT in the future.

Conclusion

Increasing the use of bladder preserving radiation-based treatment needs an improved therapeutic index leading to reduced side effects. Preclinical studies are needed to address this issue. This review first identified and analysed all preclinical investigations on concurrent combination of radiation with different agents in bladder cancer and then provide suggestions aiding the selection of appropriate cell lines, mouse models, radiosensitising agents and radiotherapy regimen to improve the design of future preclinical studies.

Methods

Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews [START_REF] Moher | Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement[END_REF], we conducted a literature search in PubMed. We used the following search string (((radiotherapy) OR (radiation therapy) OR S2. 3 Information from the https://web.expasy.org/.

Supplementary Data

Table S3. Preclinical studies of problematic/ cell lines in vivo.

RTKs as targets to improve therapeutic ratio of radiation therapy

Receptor tyrosine kinases (RTKs) are frequently differentially expressed between normal and cancerous tissue. They are also implicated in numerous cell-survival processes, therefore they are among most promising therapeutic targets for anti-cancer therapy and have a potential to increase the therapeutic index of radiation treatment efficiency (Figure 1234). RTK inhibition combined with radiation treatment has shown to be an effective treatment in preclinical models of different cancers [START_REF] Chinnaiyan | Mechanisms of enhanced radiation response following epidermal growth factor receptor signaling inhibition by erlotinib (Tarceva)[END_REF][START_REF] Dittmann | Radiation-induced epidermal growth factor receptor nuclear import is linked to activation of DNA-dependent protein kinase[END_REF][START_REF] Wang | Inhibition of Both EGFR and IGF1R Sensitized Prostate Cancer Cells to Radiation by Synergistic Suppression of DNA Homologous Recombination Repair[END_REF].

This lead to the first successful major clinical trial combining cetuximab (Epidermal Growth

Factor Receptor (EGFR)-targeting antibody and radiotherapy treatment for patients with squamous-cell carcinoma of the head and neck (Bonner et al., 2006).

There are 58 currently known RTKs in humans divided in twenty subfamilies [START_REF] Lemmon | Cell Signaling by Receptor Tyrosine Kinases[END_REF]. Several RTKs can be induced and upregulated upon ionizing radiation in several cancer types. They mediate pro-proliferative, pro-survival pathways as well as DNA repair pathways, the activation of which could ultimately protect cancer cells from radiationinduced cell death. RTKs are also driver of an oncogenic-dependency state in many cancer types and for the last 20 years are considered attractive targets for cancer therapy. A number of RTKs with relevance to radiation oncology have been identified including; EGFR, VEGFRs, IGF1R, MET, and HER2/ERBB2 (Nyati et al., 2004;Kim et al., 2006, Meyn et al., 2009;Higgins et al., 2016). Therefore, concomitant RTK inhibition using monoclonal antibodies or tyrosine kinase inhibitors (TKIs) and radiation therapy have been evaluated in preclinical and clinical studies to enhance tumour radiosensitivity for various types of cancer. Further investigations have addressed their roles in regulating the cellular response to radiation. In this context, only few studies has investigated RTKs targeting with radiation treatment in bladder cancer.

To our knowledge, only one study has investigated the pontentoial activation of RTKs after ionization radiation (IR) in bladder cancer (Tsai et al., 2015). The phosphorylation of 39 RTKs was evaluated after radiation using an RTK signalling array. This study highlighted that IR exposure of bladder cancer cells resulted in phosphorylation of EGFR, HER2 and the key downstream proteins (AKT, MAPK and S6 proteins). Based on these results, Tsai and colleagues first investigated the effects of EGFR inhibition on BCa cells with low baseline EGFR expression and a lack of EGFR activating mutations but were unable to successfully enhance their radiosensitivity. However, the in vitro and in vivo radiosensitizing effect of an EGFR/HER2 dual inhibitor was demonstrated to surpass that of an EGFR TKI. Interestingly, the EGFR TKI showed improved radiosensitization in BCa cells with downregulated HER2 expression as compared to control cells, indicating that EGFR is an important determinant of radioresponse in BCa cells, provided the associated pathways like HER2 are concomitantly blocked. Other reports had showed that EGFR-TKIs, including gefitinib and erlotinib only present modest radiosensitizing effects on bladder cancer cells (Dominguez-Escrig et al., 2004;[START_REF] Maddineni | Differential radiosensitisation by ZD1839 (Iressa), a highly selective epidermal growth factor receptor tyrosine kinase inhibitor in two related bladder cancer cell lines[END_REF]Colquhoun et al., 2007). It is however important to note that these studies were conducted without bladder cancer subtyping and that it is now suggested that mostly basal tumours are dependent on EGFR (Rebouissou et al., 2014).

The therapeutic index of radiotherapy can be defined as the difference between two sigmoidal curves relating the probability of effect (tumor control) with the radiation dose used on tumors (in red in Figure 1234) and probability of toxicity on normal tissues (in green in Figure 1- 4). The difference between both curves can be used to calculate the therapeutic index. High therapeutic index means that the tumor response is greater than that of surrounding normal tissue (by extending the therapeutic window). There is longstanding interest to increase the therapeutic index. Fractionated radiation dose delivery that results in preferential tumor cell killing over normal cells is among such strategies. Radioprotectors could be used to protect [START_REF]Fondation pour la Recherche Médicale (FRM): funding for the 4th year of PhD thesis from[END_REF]. AXL was discovered first in studies of genes that transform NIH 3 T3 cells [START_REF] Liu | Transforming genes in chronic myelogenous leukemia[END_REF][START_REF] O'bryan | axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase[END_REF]. MERTK was first identified as an oncogene from avian retroviruses and its similarity to AXL was recognized when its murine form was cloned [START_REF] O'bryan | axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase[END_REF]. TYRO3 was the last to be identified and added to the TAM family based on its structural similarity [START_REF] Polvi | The human TYROS gene and pseudogene are located in chromosome 15q14q25[END_REF]. High-resolution crystal structures have been determined for the TYRO3 [START_REF] Powell | Novel and selective spiroindoline-based inhibitors of sky kinase', Bioorganic and Medicinal Chemistry Letters[END_REF] and MERTK kinase domains [START_REF] Huang | Structural insights into the inhibited states of the Mer receptor tyrosine kinase[END_REF].

TAM receptors play an important role in promoting the growth, survival, and metastatic spread of several tumor types. In the recent years several studies have established their involvement in the resolution of inflammation, autoimmunity and tumor immunology (Lemke, 2013;[START_REF] Akalu | TAM receptor tyrosine kinases as emerging targets of innate immune checkpoint blockade for cancer therapy[END_REF].

TAM ligands

TAM receptors exist as monomers in the cell membrane, and in order to dimerise and transduce signals through trans-phosphorylation, a ligand binding is essential. Protein S (PROS1) and Growth arrest specific factor 6 (GAS6) have been the first identified TAM ligands.

Recently, three new ligands have been discovered, namely Tubby and galectin-3, which bind MERTK, and Tubby-like protein 1 (Tulp-1), which binds all three receptors [START_REF] Caberoy | Tubby and tubby-like protein 1 are new MerTK ligands for phagocytosis[END_REF][START_REF] Caberoy | Galectin-3 is a new MerTK-specific eat-me signal[END_REF][START_REF] Nomura | Activated Microglia Desialylate and Phagocytose Cells via Neuraminidase, Galectin-3, and Mer Tyrosine Kinase[END_REF]. However, yet not much is known regarding their function, while PROS1 and GAS6 gas been studied more extensively. Both PROS1 and GAS6 require vitamin K (Vit-K) dependent γ-carboxylation of their Gla domains to maintain their ability to bind to phosphatidylserine (PtdSer) (in blue in Figure 12345). Indeed, there are 11 highly conserved glutamic acid residues in the Gla domain of both ligands.

Biochemical binding studies have shown that non-y-carboxylated GAS6 can bind to TAMs, but does not result in activation [START_REF] Lew | Differential TAM receptor-ligand-phospholipid interactions delimit differential TAM bioactivities[END_REF][START_REF] Tsou | Receptor tyrosine kinases, TYRO3, AXL, and MER, demonstrate distinct patterns and complex regulation of ligand-induced activation[END_REF].

GAS6 and PROS1 share ~44% amino acid sequence homology, and have analogous domain organizations consisting of an N-terminal γ-carboxyl-glutamic acid (Gla) domain, four tandem Epidermal Growth Factor (EGF)-like repeats, and a C-terminal Sex Hormone-Binding Globulinlike region (SHBG), which is made up by two Laminin G (LG) repeats [START_REF] Mark | Characterization of Gas6, a member of the superfamily of G domaincontaining proteins, as a ligand for Rse and Axl[END_REF][START_REF] Anderson | Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells[END_REF][START_REF] Lew | Differential TAM receptor-ligand-phospholipid interactions delimit differential TAM bioactivities[END_REF]. Despite their homology and structural resemblances, both proteins exhibit distinct functions [START_REF] Davra | Ligand activation of TAM family receptors-implications for tumor biology and therapeutic response[END_REF].

Protein S

Protein S (PROS1) was unexpectedly discovered in 1977, while purifying Protein C from bovine plasma named Protein S after Seattle the city of its discovery [START_REF] Di Scipio | A Comparison of Human Prothrombin, Factor IX (Christmas Factor), Factor X (Stuart Factor), and Protein S[END_REF]. PROS1 is secreted primarily by endothelial cells, megakaryocytes, hepatocytes, and Leydig cells in the testicle. In the blood circulation, PROS1 is present at a concentration of 350nM [START_REF] Davra | Ligand activation of TAM family receptors-implications for tumor biology and therapeutic response[END_REF]. 60% of the circulating PROS1 is bound to complement protein C4b-binding protein (C4BP), while 40% is present in a free form. Both forms can act as anticoagulants, and their deficiency is associated with deep vein thrombosis and ischemic stroke [START_REF] Hayakawa | Mesenteric venous thrombosis in a child with type 2 protein S deficiency[END_REF][START_REF] Wang | Hereditary protein S deficiency leads to ischemic stroke[END_REF]. PROS1 knockout mice have been shown to result in embryonic lethality (Burstyn-Cohen, Heeb and [START_REF] Burstyn-Cohen | Lack of Protein S in mice causes embryonic lethal coagulopathy and vascular dysgenesis[END_REF]. In the amino terminus, PROS1 has carboxyglutamic acid (GLA) residues, which ensures an affinity for calcium ions and is especially important in the blood coagulation cascade. GLA is followed by a thrombin-sensitive loop region and the carboxy-terminal domain (in blue in Figure 12345) including four growth factor-like domains and two lamine G repeats with the sex hormone-binding globulin domain (SHBG). This carboxyterminal region is responsible for the binding to the TAM receptor and its phosphorylation.

PROS1 is specific to TYRO3 and MERTK, but its affinity with AXL has never been detected and is considered to be low ( Interestingly, PROS1 has attracted attention in the light of the recent COVID-19 pandemic.

Extensive blood coagulation and immune hyperactivity in large number of COVID-19 patients could be explained by the PROS1 activity. PROS1 its involved in immune regulation and also is an anticoagulant in the blood coagulation cascade. PROS1 levels in the blood of the COVID-19 patients has been proposed to be investigated in future studies [START_REF] Lemke | Blood clots and TAM receptor signalling in COVID-19 pathogenesis[END_REF].

GAS6

GAS6 was first identified in 1988 as an upregulated gene in growth-arrested fibroblasts [START_REF] Schneider | Genes specifically expressed at growth arrest of mammalian cells[END_REF]. In the GAS6 structure, instead of the thrombin-sensitive loop, there is a disulfide-bridged thumb loop. According to the Human Protein Atlas (https://www.proteinatlas.org/), the protein is present in the heart, kidneys and lungs and transcripts and expressed in epithelial cells, bone marrow and vascular smooth muscle cells.

GAS6 is upregulated in several cancers, including glioma, schwannoma [START_REF] Ammoun | Axl/Gas6/NFκB signalling in schwannoma pathological proliferation, adhesion and survival[END_REF], melanoma (Demarest et al., 2013), pancreatic ductal adenocarcinoma [START_REF] Wang | Overexpression of receptor tyrosine kinase Axl promotes tumor cell invasion and survival in pancreatic ductal adenocarcinoma[END_REF].

Recently, two studies have detected high GAS6 levels in BCa. Our group have described high GAS6 levels correlating to decreased survival in MIBC (Dufour et al., 2019). A study by Mao et colleagues demonstrated that GAS6 is highly expressed in both NMIBC and MIBC and is significantly associated with tumor grade, stage, and worse prognosis [START_REF] Mao | Overexpression of GAS6 promotes cell proliferation and invasion in bladder cancer by activation of the PI3K/AKT pathway[END_REF]. In their study, Mao and colleagues demonstrated that GAS6 downregulation resulted in decreased PI3K-AKT signaling and decreased proliferation and invasion of BCa cells. The potential mechanism of GAS6 upregulation in BCa could be explained by the activity of lncRNA GAS6-AS2 [START_REF] Rui | LncRNA GAS6-AS2 promotes bladder cancer proliferation and metastasis via GAS6-AS2/miR-298/CDK9 axis[END_REF].

GAS6 has different affinities with each of the TAM receptors in the following way:

AXL>>TYRO3>MERTK (Table 1-1) [START_REF] Davra | Ligand activation of TAM family receptors-implications for tumor biology and therapeutic response[END_REF].

TAM signaling

In most of the cell types, activation of TAM receptors results in activation of the downstream phosphoinositide 3 kinase (PI3K)/AKT pathway. Most of this PI3K signaling is mediated through a TAM-autophosphorylated Grb2-binding site, which is conserved in all three TAMs [START_REF] Fridell | Differential activation of the Ras/extracellular-signal-regulated protein kinase pathway is responsible for the biological consequences induced by the Axl receptor tyrosine kinase[END_REF][START_REF] Weinger | In brain, Axl recruits Grb2 and the p85 regulatory subunit of PI3 kinase; in vitro mutagenesis defines the requisite binding sites for downstream Akt activation[END_REF]. Coupling to phospholipase C, ERK1/2, Ras, and MAP kinase activation have also been described (Figure 1

Physiological functions of TAMs

Role in the immune system TAM receptor signaling negatively regulates the innate immune response and plays a role in the clearing of apoptotic cells or efferocytosis. AXL and MERTK are known to participate in these processes at a larger extent, while there is a lack of studies for TYRO3 [START_REF] Davra | Ligand activation of TAM family receptors-implications for tumor biology and therapeutic response[END_REF]. There is a functional redundancy between TYRO3, AXL and MERTK in the immune regulation as indicated by the normal phenotype of the single TYRO3 or AXL knockout mice in contrast to the severe autoimmunity observed in the TAM triple knockout mice (TYRO3 -/-, AXL - /-, MERTK -/-). TAM triple knockout mice present a normal immune system at birth, but later develop chronic inflammation due to hyperproliferation of B and T cells [START_REF] Lu | Homeostatic regulation of the immune system by receptor tyrosine kinases of the Tyro 3 family[END_REF].

TAM signalling negatively regulates immune response through influencing macrophage polarization (Figure 1234567). Macrophages are categorized depending on the activation signal.

The M1 phenotype produces a pro-inflammatory response specifically in the host defense against pathogens and also helps natural killer (NK) cells to identify and attack cancer cells. On the contrary, the M2 phenotype repairs damaged tissues by generating an anti-inflammatory response. Cancer cells are shown to upregulate TAM receptors and/or ligands to increase the M2 phenotype macrophages and thus escape the immune system attack. TAM receptors are thought as promising targets to re-activate the immune system [START_REF] Myers | Targeting Tyro3, Axl and MerTK (TAM receptors): implications for macrophages in the tumor microenvironment[END_REF].

TYRO3 expression

According to the Human Protein Atlas (https://www.proteinatlas.org/), TYRO3 is expressed in diverse cells. The levels of the TYRO3 transcript and protein are not highly correlated in several organs such as in the bladder, lung and lymphoid tissues, where low transcripts, but high protein levels are detected. TYRO3 is highly expressed in the central nervous system, lungs and kidneys at the protein level. TYRO3 at the protein level is moderately expressed in the bladder tissue, but has been detected highly overexpressed in bladder cancer (Dufour et al., 2019).

TYRO3 in Cancer

TYRO3 is overexpressed in multiple cancers, however, the mechanisms leading to the [START_REF] Smart | The emerging role of TYRO3 as a therapeutic target in cancer[END_REF]. Other mutations in the extracellular and transmembrane domains leading to a premature stop codon have been described in melanoma [START_REF] Easty | Novel and known protein tyrosine kinases and their abnormal expression in human melanoma[END_REF][START_REF] Krauthammer | Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma[END_REF] and lung cancer [START_REF] Ding | Somatic mutations affect key pathways in lung adenocarcinoma[END_REF].

TYRO3 targeting agents

TYRO3 overexpression and/or activity can be targeted by several approaches: using small molecule inhibitors blocking the kinase activity, using monoclonal antagonistic antibodies, using soluble TAM ecto-domains or vitamin k-antagonists. These different strategies can act directly on the tumor or indirectly via the activation of the suppressed immune system.

Several multi-kinase inhibitors at different stages of development have some activity against TAM family (Table 123). Several MERTK-targeting compounds such as UNC1062, UNC2025 and UNC569 for example show activity also to both TYRO3 and AXL at 10-50 fold higher concentrations. The VEGFR2 inhibitor Vandetanib and SRC/ABL kinase inhibitor Bosutinib are FDA approved agents.

Currently, there is no TYRO3-specific kinase inhibitor available. Multi-kinase inhibitors have great potential to kill tumour cells and avoid resistance, but also have a greater potential to generate undesirable side effects. As more severe phenotypes have been observed in mice lacking multiple family members, targeting multiple TAM kinases may result in increased side effects relative to agents that are selective for individual family members. Thus, development of agents that target TYRO3 with greater selectivity, particularly relative to the other TAM kinases, is desirable [START_REF] Smart | The emerging role of TYRO3 as a therapeutic target in cancer[END_REF], Hsu et al., 2019). Activation of AXL or MERTK on cancer cell results in suppression of antigen processing and presentation to T cells, while MERTK on tumor macrophages results in phagocytosis of dying cells to the exclusion of dendritic cell uptake. Cell uptake drives suppressive differentiation of macrophages, which can, in turn, suppress T cells in the tumor environment. Where TAM family members are absent, antigen is more available for uptake by dendritic cells that can expand naive T cells that can traffic to the tumor.

Tumor-infiltrating T cells find decreased macrophage suppression and increased antigen presentation, permitting increased control of tumors. From Tormoen, Crittenden and Gough, 2017.

In this context, AXL and MERTK, but not TYRO3 has been specifically targeted in combination with RT. AXL has been shown to downregulate DNA repair genes in breast, lung, and head and neck cancers and sensitize cancer cells to PARP therapy [START_REF] Balaji | AXL inhibition suppresses the DNA damage response and sensitizes cells to PARP inhibition in multiple cancers[END_REF]. A study by Brand and colleagues demonstrated that AXL signalling play a role in the resistance to EGFR-targeting monoclonal antibody Cetuximab treatment in HNSCC and non-small cell lung cancer (NSCLC) [START_REF] Brand | AXL mediates resistance to cetuximab therapy[END_REF]. A study from the same team demonstrated that tumor cell treatment with AXL-targeting TKI, R428 resulted of improved effect of radiotherapy, chemotherapy and cetuximab treatment in HNSCC (Brand et al., 2015). Significant increase in γ-H2AX fluorescent foci was observed in cells treated with R428 and radiation as compared with cells treated with radiation only explained decreased activity of DNA-PK and AKT levels as compared with cells treated with radiation alone. They also demonstrated that R428 did not augment radiationinduced DNA-PK or AKT activation in the AXL-negative HNSCC cell line. Collectively, these data suggested that AXL signaling mediates DNA DSB repair and therefore targeting AXL may enhance the efficacy of radiation therapy. The combination of R428 and Cetuximab treatment in HNSCC cells showed more significant decrease in proliferation compared to R428 single treatment, indicating that multiple-kinase inhibition as an effective anti-tumor strategy.

However, these studies had not evaluated the extent of TYRO3 inhibition with the inhibitor doses used, which also might contribute to the effectiveness of the treatment.

MERTK inhibition as radiosensitiser was evaluated in glioblastoma (GBM), where mouse GBM cell line treated with TAM inhibitor UNC2025 was found to synergise with radiation. However, the combination treatment with UNC2025 plus RT did not prolong median overall survial when compared to XRT alone in vivo [START_REF] Wu | MerTK as a therapeutic target in glioblastoma[END_REF]. The potential of TYRO3 to radisoensitise cells in TYRO3-dependant tumors remains to be investigated.

Urothelial cells in vitro

Isolation and culture conditions

Normal human urothelial (NHU) cells have the capacity to regenerate and it can be used in vitro to provide a valuable research tool. Southgate and colleagues have established isolation and propagation protocols from ureter or bladder tissue from surgical specimens [START_REF] Southgate | Normal human urothelial cells in vitro: proliferation and induction of stratification[END_REF], Southgate et al., 2002). Urothelium is dissociated from the basement membrane with an aid of calcium-chelating ethylenediaminetetraacetic acid (EDTA) to limit stromal contamination. Further, collagenase is used to obtain a single-cell suspension. Cultures are maintained in keratinocyte serum-free medium (KSFM) with a low Ca 2+ concentration (0.09 mM) to prevent the growth of non-epithelial cells. KFSM is supplemented with recombinant human epidermal growth factor (rhEGF), bovine pituitary extract (BPE) and cholera toxin (CT) in order to improve plating efficiency [START_REF] Southgate | Normal human urothelial cells in vitro: proliferation and induction of stratification[END_REF]. Primaria TM culture flasks are used for NHU culture due to the special coating layer aiding the cellular attachment (mixture of negative, positive, and nitrogen containing functional groups), which aid cellular attachment.

NHU cell phenotype

In serum-free, low calcium (0.09 mM) culture medium, NHU cells adopt a highly proliferative phenotype noted in wound healing processes, where they form non-stratified monolayers with a typical 'cobblestone' morphology cells undergo contact inhibited G1 growth arrest upon attaining confluence. Proliferating NHU cells express a non-differentiated, basal/intermediate cytokeratin profile with expression of CK7, CK8, CK17, CK18, CK19 and low levels of CK13 [START_REF] Southgate | Normal human urothelial cells in vitro: proliferation and induction of stratification[END_REF].

NHU growth and proliferation

In KSFMc, NHU cells display a high rate of proliferation with a mean population doubling time of 14.7 ± 1.8 hours [START_REF] Southgate | Normal human urothelial cells in vitro: proliferation and induction of stratification[END_REF]. NHU cells can be sub-cultured for ~12 passages before they undergo replicative senescence. Overexpression of telomerase (hTERT) can immortalise urothelial cells but results in compromised differentiation and functional capacity. Proliferation is an autonomous process driven primarily by autocrine epidermal growth factor (EGF) activation of EGFR and leads to transcription of genes that promote G1-S phase transition.

NHU cell differentiation

Cultured NHU cells retain their ability to undergo differentiation and two methods of NHU differentiation have been developed. In the first method, NHU cells are treated with a peroxisome proliferator activated receptor-γ (PPAR-γ) agonist, troglitazone (TZ), which induces differentiation but only when the EGFR pathway is concurrently blocked with an EGFR tyrosine kinase antagonist, PD153035 (TZ/PD differentiation protocol) (Varley et al., 2004).

TZ/PD treatment induces epithelial differentiation by heterodimerisation of activated PPARγ with retinoid X receptor  (RXRα) to form a transcription factor complex and transcription of genes with peroxisome proliferator response elements (PPRE) within their promoters. This method induces transcription of urothelial cytodifferentiation-associated genes, but does not result in organisation of a functional barrier.

In the second method, NHU cells are grown in KSFMc, pre-treated with serum and then subcultured in the presence of physiological calcium concentrations (hereafter referred to as ABS/Ca 2+ ). Increasing the extracellular calcium concentration from 0.09 mM to 2 mM induces stratification, desmosome formation and E-cadherin adherens junction formation. These cultures form a biomimetic urothelial tissue facsimile which displays many functional properties of the native urothelium including stratification, polarisation and barrier function.

Irrespective of method, induction of differentiation results in transcriptional upregulation of terminal differentiation-associated genes, such as the uroplakins, CK13 and CK20.

Radiation effects on the urothelium and the surrounding tissue

The use of radiotherapy for BCa treatment is limited by the pelvic toxicity, which is stemming from bowel, rectum and bladder toxicity and surrounding vasculature and tissues. Pelvic toxicity is known to encompass early (acute) and late effects. Different urothelium layers respond to irradiation and together with the response of surrounding tissues and vasculature constitute radiation-induced bladder injury (RIBI).

The radiation effects in the human bladder occurs in three phases (Figure 1-10) (Zwaans et al., 2016a). Firstly, a reversible acute phase starts at the beginning of the treatment and resolves in few weeks after the end of the therapy. Secondly, several months to several years there are no symptoms. The length of the symptom free phase depends on the radiation dose administered. Finally, a year up to 10 years after the treatment, a progressive and irreversible late response occurs manifesting in fibrosis, decreased bladder storage capacity and radiation cystitits. The corresponding urothelial changes in these three phases has been studied in mouse and rat preclinical studies. A reversible acute inflammatory phase is followed by a latent symptom-free phase, and a late chronic inflammatory phase which manifests in radiation cystitis. Each phase is characterized by specific pathologic changes that contribute to the symptoms associated with radiation cystitits. Abbreviations: D, detrusor; ECM, extracellular matrix; GAG, glycosaminoglycan; LP, lamina propria; UE, uroepithelium.

From (Zwaans et al., 2016b).

Changes in the urothelium

An early preclinical study explored the timing and histology of the urothelial damage and subsequent repair after irradiation. Stewart and colleagues used indirect measure of urothelium injury and documented increased urination frequency of the irradiated animals which correlated with appearance of fibrosis of the bladder at 12 months [START_REF] Stewart | Late radiation damage in the mouse bladder as measured by increased urination frequency[END_REF]. The mouse bladder received a single 10-40 Gy irradiation dose and the maximum increase in the incontinence was observed by 12 months. Histological examination reported the appearance of fibrosis of the deeper layers of the bladder after 12 months. At 18 months there was histological evidence of fibrosis after higher doses (30 to 40 Gy). These doses also resulted in sustained incontinence and a severe reduction in bladder capacity.

A similar study on rat urinary bladders showed that a single exposure of the bladder to a dose of 20 Gy of irradiation produced severe fibrosis of the bladder wall, resulted in smooth muscle degeneration and to induced carcinoma of the urothelium in 59% of treated animals within 20 months [START_REF] Antonakopoulos | Early and late morphological changes (including carcinoma of the urothelium) induced by irradiation of the rat urinary bladder[END_REF]. All tissues of the bladder wall were found to be affected by the irradiation. In the urothelium, initially the basal cells were damaged, then over time course it extended to intermediate cells and by 6 months post-irradiation also the focal cells were affected and was hyperplastic. In this study, the radiation effect on the blood vessels of the bladder wall was examined and endothelial cells appeared to be damaged.

Hydronephrosis (swelling of one or both kidney) was not reported in this study, but it was identified by another study in mice after bladder irradiation, with incidences of 10% after doses below 30 Gy and up to 60% after the use of higher doses [START_REF] Stewart | Repair during fractionated irradiation of the mouse bladder[END_REF].

A study by Jaal and Dörr compared cell numbers per unit length of uorothelium at regular intervals after 20 Gy of irradiation and found an early decrease of urothelial with the lowest cell number occurring at 1 month after irradiation. Subsequent restoration of the initial cell numbers (pre-radiation) was found 180 days after, while a hyperproliferation of urothelial cells was detected at 360 days after irradiation was detected. The increased cell number positively correlated with increased Ki-67 and EGFR expression. EGFR expression was the highest at 360 days after irradiation and indicated that EGFR signalling may play an important role in uorothelium regeneration after radiation-induced injury (Jaal and Dörr, 2010).

More serious clinical complications of the pelvic radiatiotherapy in humans develop between 1 and 10 years after irradiation and can include radiation cystitis and development of secondary BCa. Localisation of the maximal irradiation dose to particular regions of bladder/genitourinary system could possibly minimise the late toxicity. A study by Ghadjar and colleagues on human patients undergoing radiotherapy for prostate cancer found that by reducing the radiation dose delivered to the lower bladder and specifically to the bladder trigone appears to be associated with a reduction in late toxicity [START_REF] Ghadjar | Impact of dose to the bladder trigone on long-term urinary function after high-dose intensity modulated radiation therapy for localized prostate cancer[END_REF].

Radiation-induced BCa has been studied in vitro and in vivo. Neoplastic effects induced by a single 6 Gy irradiation dose were compared between immortalised normal human urothelial cells (SV-HUC) and a cell line from a low-grade mouse BCa tumor generated in the SV-HUC system (MC-T11). Both cell lines were irradiated or not, cultured in vitro for six weeks and subsequently inoculated in nude mice. While the irradiation did not induce SV-HUC neoplastic transformation, the irradiated MC-T11 produced higher grade, poorly differentiated tumors and exhibited adjacent tissue invasive properties. Furthermore, chromosome deletions, including losses of 4q, 11p, 13q and 18, were observed in these radiation-induced tumors (Pazzaglia et al., 1994). This study indicated that radiation alone, was not able to result in tumorigenicity, but when there was low tumor-generating potential, it was readily accelerated by the exposure of irradiation.

Important factor to predict acute bladder toxicity appears the T-stage of the tumor. Higher Tstage tumors seems to be associated with higher acute bladder toxicity [START_REF] Tonoli | Radical radiotherapy for bladder cancer: Retrospective analysis of a series of 459 patients treated in an Italian institution[END_REF][START_REF] Majewski | Acute and Late Toxicity in Radical Radiotherapy for Bladder Cancer[END_REF]. However, it should be considered that patients with these tumors are likely to have higher pre-treatment symptoms arising from repeated TURB and other treatments. There is lack of correlation between acute bladder toxicity and the dose intensity and it is thought to be due to the slow urothelial cell turnover time [START_REF] Hainau | Histology and cell proliferation in human bladder tumors. An autoradiographic study[END_REF][START_REF] Stewart | Mechanism of bladder damage and repair after treatment with radiation and cytostatic drugs[END_REF].

Bowel Toxicity

Compared to bladder tissues, bowel tissues are early-reacting and exhibit significant correlation between the acute bowel toxicity and dose intensity compared to bowel epithelial tissue. It is caused by the increased depopulation rate of the bowel epithelium which overcomes the regeneration rate. Some studies have reported unacceptable bowel toxicity when high irradiation doses have been used over short period of time, while acute bladder toxicity was not modified. Moonen and colleagues ran a feasibility study comparing the delivery of 66 Gy of total irradiation dose in 2 Gy fractions over 6.5, 5, or 4 weeks [START_REF] Moonen | A feasibility study of accelerated fractionation in radiotherapy of carcinoma of the urinary bladder[END_REF]. Two daily fractions of irradiation were delivered at least 6h apart. While the 5 weeks' schedule was well tolerated, the same dose delivered in 4 weeks resulted in highly increased acute bowel toxicity, while the bladder toxicity was not affected.

OBJECTIVES

Bladder cancer is still a major global health problem. 25% of all diagnosed BCa is Muscleinvasive bladder cancer (MIBC), which remains a lethal disease. Cystectomy is the standard treatment for MIBC, but presents significant drawbacks including associated increased risk of infection and impacted quality of life (Sanli et al., 2017). However, radiotherapy coupled with chemotherapy and tumor transurethral resection has emerged as a radiation-based bladder sparing treatment for limited disease (Leow et al., 2019). There is a limited number of studies investigating radiosensitisation strategies of bladder tumors, which have reached the clinical translation. Selectively increasing bladder tumor sensitivity to radiation could improve the efficacy of radiotherapy, limiting its side effects and reducing the need for cystectomy.

The TAM family of RTKs, consisting of TYRO3, AXL, and MERTK have recently gained attention as promising therapeutic targets in various types of cancers. These receptors have a varying expression pattern, but there is a great interest in using TAM inhibitors in cancer therapies.

Downregulation of TYRO3 has been shown to reduce cell proliferation in melanoma and colorectal cancer among others. AXL has been shown to be upregulated in response to EGFRtargeted therapies, while inhibition of both AXL and MERTK has been shown to improve the effects of radiotherapy in HNSCC and glioblastoma respectively (Brand et al., 2015;[START_REF] Wu | MerTK as a therapeutic target in glioblastoma[END_REF]. TAM inhibition has also been shown to potentiate the benefit from PD-L1 therapy when investigated in immunocompetent mice [START_REF] Yokoyama | Immuno-Oncological Efficacy of RXDX-106, a Novel Small Molecule Inhibitor of the TAM (TYRO3, AXL, MER) Family of Kinases Equal contribution # Corresponding Author Running Title: Immuno-oncological effect of RXDX-106, a TAM RTK inhibitor[END_REF]. Therefore, TAM targeting in combination with other treatment modalities such as radiation has shown improved antitumor effect. However, role of TAM receptors has not been explored in BCa.

We set out to investigate TAM expression in BCa and demonstrated that TYRO3 is overexpressed in 50% of MIBCs and that TYRO3 overexpression conferred a TYRO3dependance to bladder tumor cells (Dufour et al., 2019)

Introduction

Bladder cancer is the ninth most common cancer worldwide, with ~430,000 new cases diagnosed in 2012 and 165,000 deaths annually (Antoni et al., 2017) (growth arrest-specific 6), but ligand-independent activation has also been described (Taylor, Roy and Varmus, 1995). TAM receptors are involved in immune control, inflammation and homeostasis in several organs, including the nervous, reproductive and vascular systems [START_REF] Linger | TAM receptor tyrosine kinase: biological functions, signaling, and potential theraputics targeting in human cancer[END_REF]Lemke, 2013). Aberrant TAM receptor expression has been reported in diverse solid and hematopoietic tumours in humans, with effects on apoptosis, growth, metastasis and chemosensitivity, although the underlying mechanisms remain incompletely understood (Lemke, 2013;[START_REF] Graham | The TAM family: phosphatidylserine-sensing receptor tyrosine kinases gone awry in cancer[END_REF]Wu et al., 2017). AXL has been, so far, more extensively studied in cancers than either MERTK or TYRO3. In this study, we investigated TAM receptor's expression in two independent cohorts of bladder tumours and evaluated the influence of each receptor on the regulation of growth/survival in urothelial bladder cancerderived cell lines (UBC cell lines).

Results

TYRO3 is highly expressed in bladder carcinomas

We first investigated the role of TAM receptors in human bladder carcinoma, by analysing, using RT-qPCR, the levels of expression of AXL, MERTK, TYRO3 and GAS6 in our previously described CIT cohort of 169 bladder tumours encompassing 87 NMIBCs and 82 MIBCs (Biton et al., 2014;Rebouissou et al., 2014). Seven normal urothelium samples were used as controls (Fig. 1a). Expression levels for the three receptors and their ligand were heterogeneous in tumours, with only TYRO3 significantly elevated in NMIBCs and MIBCs relative to normal samples. GAS6 was more strongly expressed in MIBCs than in NMIBCs, suggesting that GAS6 might increase the autocrine or paracrine activation of TYRO3 in muscle-invasive tumours (Fig. 1a). Higher TYRO3 expression in MIBCs relative to normal samples was confirmed by an analysis of publicly available RNA-seq data from the TCGA cohort for 405 MIBCs (primary tumours only) and 14 normal samples (Robertson et al., 2017) (Supplementary Figure S1). As in the CIT cohort, no increased expression of AXL, MERTK or GAS6 was observed in MIBCs compared to normal samples but surprisingly in this data set a decrease of GAS6 and AXL mRNA levels could be observed. This discrepancy could be due to the difference of so-called normal samples in the two series: pure normal urothelium samples in the CIT cohort, versus peritumoural bladder tissue with a normal histological appearance in the TCGA cohort. The difference in the number of samples considered could also influence the results. We further analysed TAM receptor expression at the protein level, by western blots of a subset of tumours from our CIT cohort (Fig. 1b and Supplementary Figure S2). Consistent with the results of mRNA analysis, TYRO3 levels in almost all tumours were higher than those in normal urothelium and vesical muscle samples, whereas AXL levels were higher than those in the urothelium in only a few tumours and this protein was abundant in the vesical muscle.

However, strong MERTK expression was surprisingly detected in 8 of 21 tumours. Different migration profiles in immunoblotting assays were observed for TYRO3, possibly due to posttranslational modifications of the receptor (Fig. 1b). An analysis of the TYRO3 sequence with NetNGlyc1.0 software identified seven potent N-glycosylation sites (not shown). The treatment of two tumour protein samples with peptide Nglycanase F (PNGaseF), an enzyme that removes N-linked glycans, shifted the TYRO3 band to the expected theoretical molecular weight of 96 kDa, confirming the N-glycosylation of this receptor (Fig. 1c). We examined the protein expression pattern of TYRO3 in clinical samples from the CIT cohort by immunohistochemistry using an anti-TYRO3 antibody. We had validated the specificity of the antibody using paraffin-embedded 5637 cells expressing the three TAM receptors and that were transfected or not with TYRO3 siRNA (Supplementary Figure S3). No or weak TYRO3

staining was observed on urothelium from histologically normal tissue adjacent to the tumour (NAT), whereas TYRO3 was highly expressed by tumour epithelial cells and not by stromal cells in both NMIBCs and MIBCs (Fig. 1d). Strikingly, but as previously observed in colorectal tumour cells (Chien et al., 2016), TYRO3 staining was mainly detected in the cytoplasm of tumour cells and also at the plasma membrane. Overall, these results suggest that TYRO3 may play a role in bladder tumours.

TAM receptors are expressed in bladder cancer cell lines, but TYRO3 is the main TAM involved in modulating cell viability

We then investigated the protein expression of the TAM receptors by western blot in a panel of cell lines derived from human urothelial bladder cancer (UBC) (Fig. 2a). Consistent with our results for human bladder tumours (Fig. 1b), TYRO3 was the TAM receptor most frequently expressed in UBC cell lines (in 21 out of 25 cell lines), followed by AXL (13/25) and MERTK (7/25). Most of the UBC cell lines strongly expressed at least two TAM receptors, but MGH-U3 and BC3C expressed only TYRO3 (Fig. 2a). We then used a loss-of-function approach based on siRNA to investigate whether TAM receptors were required for cell growth/survival (Fig. 2b).

We investigated the potential redundancy and relative importance of the three receptors, by selecting one cell line expressing all three TAM receptors (VM-CUB-1), three cell lines expressing TYRO3 together with AXL or MERTK (RT112, UM-UC-5 and UM-UC-9) and one cell line expressing only TYRO3 (MGH-U3) (Fig. 2a,b). Western blot analysis showed that each targeted TAM was effectively silenced 72 h after transfection and that the knockdown of one TAM receptor had mostly no effect on the other TAM receptors with the exception of TYRO3 knockdown using siRNA#1 that reduced AXL and increased MERTK expression, particularly in RT112 (Supplementary Figure S4). TYRO3 knockdown significantly and strongly decreased cell viability in all the cell lines tested, regardless of the expression levels of other TAM receptors (by 60 to 75%, 96 h after transfection, depending on the cell line), demonstrating the high dependence for growth of bladder cancer cell lines on TYRO3 expression and the absence of redundancy with the other TAM receptors (Fig. 2b and Supplementary Table S2). Moreover, when TYRO3 was coexpressed with other TAM receptors, the effects of TYRO3 silencing were consistently more important than those of AXL or MERTK knockdown, highlighting the predominant role of TYRO3 in the modulation of bladder cancer cell viability and suggesting the potential for therapeutic targeting of TYRO3 (Fig. 2b). This inhibition of TYRO3 could be achieved using specific blocking antibodies as flow cytometry experiments highlighted TYRO3 expression at the cell surface of the TYRO3-dependent cell lines (MGH-U3, UM-UC-5 and RT112 (Supplementary Figure S5)). However, as no such blocking antibodies were comMERTKcially available, we carried out TYRO3 inhibition experiments using two pan TAM receptor inhibitors, UNC-2025 (IC50 values are 18 nM, 7.5 nM and 0.7 nM for TYRO3, AXL and MERTK, respectively) and BMS-777607 (IC50 values are 4.3 nM, 1.1 nM and 14 nM for TYRO3, AXL and MERTK, respectively) (Schroeder et al>, 2009;Graham>et al>, 2014;Zhang>et al>, 2014). These molecules are also described to inhibit with high affinity two other tyrosine kinase receptors, MET (IC50: 3.9 nM and 364 nM) and FLT3 (IC50; 16 nM and 0.35 nM); IC50

given for BMS-777607 and UNC-2025 respectively (Schroeder et al., 2009;Zhang et al., 2014).

Consistent with their affinity profile for the TAM receptors, treatment of VM-CUB-1 cells with 5 µM of BMS-777607 or UNC-2025 inhibited the phosphorylation of TYRO3, AXL and MERTK (Supplementary Figure S6). Pharmacological inhibition of TAM receptors with UNC-2025 and BMS-777607 significantly decreased in a dose-dependent manner the viability of RT112, MGH-U3, VM-CUB-1 and UM-UC-5 cells, whereas UMUC-9 cells were more resistant (Fig. 2c and Supplementary Table S3). In line with our TAM receptor depletion experimental results (Fig. 2b), these effects appeared to be more strongly linked to TYRO3 inhibition. Indeed, MGH-U3 cells, which express only TYRO3, and RT112, which do not express MERTK and are resistant to AXL silencing, were not only the most sensitive cell lines to TYRO3 depletion but also to the TAM receptor inhibitors. Although we cannot rule out that a double or a triple knockdown could have a greater impact on cell viability than a single knockdown, we did not observe a greater sensitivity of VM-CUB-1, which expresses TYRO3, AXL and MERTK, to the pan-TAM inhibitors than the cell lines expressing TYRO3 only or in combination with a second TAM receptor. In the other hand, we could not exclude that MET or FLT3 inhibition also contributed to the observed phenotype since the cells express both receptors (data not shown).

TYRO3 knockdown reduces anchorage-independent cell growth in vitro and tumour growth in vivo

We then investigated whether this identified role of TYRO3 in bladder cancer cells was relevant to clonogenic cells, by analysing the consequences of TYRO3 knockdown for the anchorageindependent growth of TYRO3-dependent bladder cancer cell lines in vitro and tumour growth in vivo. TYRO3 depletion significantly decreased the number of viable MGH-U3 and RT112 colonies in soft agar assays, by 80 and 60% respectively, demonstrating a role for TYRO3 in regulating the survival of clonogenic cancer cells (Fig. 3a). We also found that the treatment of established MGH-U3 xenografts in nude mice with intraperitoneal injections of TYRO3 siRNA reduced significantly the tumour growth as compared to in similar tumours treated with a scramble siRNA (≈354 mm3 versus 1090 mm3 at day 21, p < 0.0001) (Fig. 3b).

This inhibition of tumour growth was associated with significantly lower TYRO3 mRNA levels on RT-qPCR (Fig. 3c). These results indicate that the genetic depletion and pharmaceutical inhibition of TYRO3 lead to decreases in bladder cancer cell viability and tumour outgrowth ability, identifying TYRO3 as a potential therapeutic target.

TYRO3 depletion induces tumour suppressor pathways and apoptosis, while inhibiting proliferative pathways

We investigated the molecular mechanisms underlying the oncogenic dependency on TYRO3 expression in bladder carcinomas, by conducting a comprehensive gene expression analysis with Affymetrix U133 Plus 2.0 arrays, in MGH-U3, RT112 and UMUC-5 cells transfected with three different TYRO3 siRNAs. We identified 284 genes as differentially expressed after TYRO3 knockdown in these three cell lines (adjusted p value < 0.05, Ilog2(FC)I ≧ 0.58 (see "Methods" section); supplementary table S4). An analysis of this list of genes with Ingenuity Pathway Analysis (IPA) software identified key molecular and cellular functions (Fig. 4a, upper panel) and upstream regulators/ transcription factors presenting significant alterations upon TYRO3 knockdown (Fig. 4a, lower panel). TYRO3-regulated genes were highly significantly enriched in gene sets involved in the "cell cycle", "cell proliferation", "cell survival", "cell apoptosis" and "cell death" functions (Fig. 4a upper panel). The gene sets corresponding to each function are listed in supplementary table S5. Consistent with these findings, cell cycle and cell survival processes were significantly downregulated (negative z-score), whereas cell death and apoptosis, in particular, were upregulated (positive z-score) upon TYRO3 knockdown (Fig. 4a, upper panel). We also identified, among the top ten transcriptional regulators whose activities were predicted to be upregulated or downregulated by TYRO3 depletion, genes encoding important regulators of the cell cycle and cell survival such as MYC, FOXM1, E2F1, E2F2, E2F3 and CCND1, whose activity were predicted to be downregulated whereas the activities of the tumour suppressor transcription factors TP53 and RB1 were predicted to be upregulated (Fig. 4a, lower panel, table S6). Strikingly, CDKN2A was also predicted as upregulated upon TYRO3 depletion whereas cell lines harbour a CDKN2A homozygous gene deletion21 highlighting the need for prediction validation that can be biased if targeted genes used for a transcription regulator are also controlled by other regulators. Western blot analysis showed a downregulation of FOXM1 and CYCLIN D1 levels following TYRO3 depletion in MGH-U3, RT112

and UM-UC-5 cells, potentially accounting for the lower levels of activity for these transcription factors highlighted by our transcriptomic data analysis in these cell lines (Fig. 4a, b, lower panel). Although IPA predicted an inhibition of MYC, western blot analysis showed a decrease in c-MYC protein levels mainly in UMUC-5 and RT112. Conversely, during cell cycle progression, retinoblastoma protein (pRB) is sequentially phosphorylated by CYCLIN-CDK complexes, and CYCLIN D1-CDK4 specifically phosphorylates pRB on the Ser780 residue, leading to its inactivation and the release of E2F. 22 In accordance with our transcriptomic data showing that both CCND1 and CDK4 gene expression were downregulated upon TYRO3 depletion (supplementary table S4), we observed lower levels of pRB phosphorylation at Ser780 in the three cell lines studied after TYRO3 knockdown, which may, therefore, lead to pRB activation and E2F inactivation, as suggested by IPA analysis of our transcriptomic data (Fig. 4a, b, lower panel). Western blot analysis also confirmed, at the protein level, the downregulation of the AURORA A and AURORA B kinases involved in cell cycle entry and mitotic spindle assembly, and of SURVIVIN, for which mRNA levels had already been shown to be decreased by TYRO3 depletion (log2FC = -1.104; -1.159 and -1.298, respectively) (Fig. 4b).

Taken together, our data for the MGH-U3, RT112 and UM-UC-5 cell lines demonstrate that TYRO3 depletion inhibits proliferative pathways and activates tumour suppressor pathways.

These molecular mechanisms are undoubtedly of general importance, because all our western blot results (Fig. 4b) were confirmed in two other TYRO3-dependent cell lines, VM-CUB-1 and UM-UC-9 (Supplementary Figure S7). Given that the pRB pathway is frequently altered in bladder cancer, particularly through direct RB1 gene inactivation, we assessed the impact of TYRO3 depletion in the RB1 mutated bladder cancer cell lines, 5637 (pY325*), 647 V (p.Q383*) and HT1376 (p.Q383*) (Robertson et al., 2017). The cell viability of these RB1 mutant cells was significantly decreased upon TYRO3 silencing (Supplementary Figure S8). Although the impact of TYRO3 depletion was not as important as the one observed in RB1 wild type cells, these results suggest that the sensitivity to TYRO3 inhibition is not strictly dependent on RB1 gene status. SURVIVIN is not only involved in the cell cycle, in which it regulates the mitotic spindle checkpoint, but also in the inhibition of caspase-9, an initiator caspase involved in caspase-3 activation (Chen et al., 2016). Its loss is, thus, consistent with the induction of apoptosis upon TYRO3 silencing, through caspase activation (Fig. 4a, upper panel). Western blot analysis of lysates of TYRO3 siRNA-transfected RT112 cells showed a greater accumulation, over time, of the cleaved forms of the effector caspase-3 and the initiator caspase-8, resulting in the cleavage of PARP, one of the main targets of capase-3, thereby clearly demonstrating the activation of apoptosis by TYRO3 depletion (Fig. 4c, left panel). These results were confirmed 72 h after transfection with TYRO3 siRNA, in MGH-U3 and UM-UC-5 cells (Fig. 4c, right panel), and cleaved PARP was also identified in VMCUB-1 and UM-UC-9 cells after TYRO3 knockdown (Supplementary Figure S7). Moreover, inhibitors of either caspase-3/7 (z-IEVD-fmk) or caspase-8 (z-IETD-fmk) significantly reduced the impact of TYRO3 depletion on cell survival, demonstrating the crucial importance of apoptosis regulation for TYRO3 activity in bladder cancer cells (Fig. 4d). Finally, a TUNEL assay performed in our MGH-U3 xenograft mouse model (Fig. 3) clearly showed that apoptosis levels were significantly higher in TYRO3 siRNA-treated animals (Fig. 4e).

Discussion

One of the major aims of this study was to assess the expression of TAM receptors in bladder cancer. Our study showed that only TYRO3 expression levels were significantly increased in both NMIBCs and MIBCs relative to normal urothelium samples. The aberrant expression of TAM receptors is frequently observed in several cancers and has been associated with aggressive cancer phenotypes, overall poor patient survival and the emergence of drug resistance [START_REF] Graham | The TAM family: phosphatidylserine-sensing receptor tyrosine kinases gone awry in cancer[END_REF]. TYRO3 has been less studied than the other two TAM receptors, but several studies have reported results similar to ours, highlighting the contribution of this receptor in various types of cancer: leiomyosarcoma, melanoma, colorectal and thyroid cancers (Zhu et al., 2009;Avilla et al., 2011;El Sayadi et al., 2013;Chien et al., 2016). However, TYRO3 expression in bladder cancer is not correlated with disease stage (contrary to reports for colorectal cancer), suggesting a possible role for TYRO3 in the formation of bladder cancers, as well as in their progression. However, we can assume that TYRO3 plays a greater role in MIBCs than in NMIBCs, due to the stronger expression of its ligand, GAS6, in MIBCs than in NMIBCs. Nevertheless, ligand-independent activation has also been reported for strong TYRO3 expression (Taylor, Roy and Varmus, 1995), and we show here that TYRO3 is N-glycosylated in bladder tumours, which might lead to its ligand-independent activation in the presence of galectin, in a process similar to that described for N-glysosylated vascular endothelial growth factor receptor (Bellmunt, Powles and Vogelzang, 2017), which is activated by galectin-1 in the absence of ligand (Croci et al., 2014). No alteration of the TYRO3 copy number was detected (data not shown), and the mechanisms increasing the expression of TYRO3 in bladder tumour cells remain to be determined. TYRO3 was detected at the plasma membrane in several bladder cancer cell lines by flow cytometry, but IHC staining on human bladder tumour sections showed it to be present mostly in the cytoplasm and only weakly expressed at the plasma membrane of tumour cells. A similar RTK distribution has already been reported for TYRO3 and AXL in human colorectal cancer and pancreatic cancer, respectively (Leconet et al., 2014;Chien et al., 2016). Consistent with this general higher level of expression of TYRO3 in bladder tumours compared to normal samples, TYRO3 expression was detected in most of the UBC cell lines, whereas AXL and MERTK had more heterogeneous expression patterns in UBC cell lines. TYRO3 knockdown systematically resulted in a significant large decrease in cell growth/survival, in all the bladder cancer cell lines tested, regardless of AXL and MERTK expression levels, as previously reported for melanoma cells (Demarest et al., 2013). These results suggest that neither AXL nor MERTK could play a redundant role, rescuing the loss of TYRO3. The three TAM receptors therefore have different roles in bladder tumours.

Accordingly, the impact of TYRO3 knockdown on UBC cell proliferation was consistently greater than the impact of MERTK or AXL knockdown. Despite the preponderant role of TYRO3 in the control of bladder cancer cell growth/survival, we cannot rule out the possibility that AXL or MERTK knockdown affects other processes known to be regulated by TAM receptors, such as cell migration, and that long term inhibition of AXL or MERTK could impact tumour cell viability [START_REF] Rankin | The receptor tyrosine kinase AXL in cancer progression[END_REF]. Our transcriptomic analysis, with validation by immunoblotting, showed that TYRO3 silencing inhibited the cell cycle and cell survival processes, whilst inducing apoptosis. Such effects of TYRO3 knockdown have already been reported in other cancer types: melanoma, thyroid and breast cancer cells (Zhu et al., 2009;Avilla et al., 2011;[START_REF] Ekyalongo | TYRO3 as a potential therapeutic target in breast cancer[END_REF]. In breast cancer cells, as reported here for UBC cells, the regulation of these processes may involve a decrease in CYCLIN D1 and SURVIVIN levels induced by TYRO3 knockdown [START_REF] Ekyalongo | TYRO3 as a potential therapeutic target in breast cancer[END_REF]. Our transcriptomic analysis suggested a role for MITF regulation, as previously reported for melanoma (Zhu et al., 2009), but the involvement of MITF in bladder tumours has never been explored. Other pathways were identified as regulated in both studies, including proliferation, survival/growth and apoptosis, suggesting possible roles for TYRO3 in these processes and the existence of general features relating to TYRO3 activity in different cancer types. However, some cancer type-specific features may fine-tune TYRO3-mediated signalling pathways and their outcomes. Indeed, TYRO3 has been shown to stimulate not only cell proliferation, but also cell migration and EMT processes, via the regulation of SNAI1, in colorectal cancer (Chien et al., 2016), whereas our transcriptomic analysis detected no deregulation of cell migration or EMT processes following TYRO3 knockdown in three different UBC cell lines. In addition to this cell-autonomous effect in bladder cancer, TYRO3 may also be involved in cell-non-autonomous effects. Indeed, TAM receptors have been shown to favour the escape of tumour cells from immune surveillance by activating innate immune checkpoints and, in particular, by upregulating PD-L1 expression [START_REF] Akalu | TAM receptor tyrosine kinases as emerging targets of innate immune checkpoint blockade for cancer therapy[END_REF][START_REF] Kasikara | Phosphatidylserine sensing by TAM receptors regulates AKT-dependent chemoresistance and PD-L1 expression[END_REF]. Our transcriptomic analysis upon TYRO3 depletion revealed no modulation of CD274 (PD-L1) expression, but a significant increase in TNFSF9 (CD137L) expression. Given the current interest in agonistic anti-CD137

antibodies for cancer treatment [START_REF] Balaji | AXL inhibition suppresses the DNA damage response and sensitizes cells to PARP inhibition in multiple cancers[END_REF] [START_REF] Makkouk | Rationale for anti-CD137 cancer immunotherapy[END_REF], TYRO3

inhibition might be useful for both inhibiting tumour growth and boosting the antitumour immune response. In line with this hypothesis, a phase 1b clinical trial (NCT03170960) has recently begun to test the combination of a pan-TAM inhibitor (cabozantinib) with an anti-PD-L1 antibody (atezolizumab) in patients with advanced urothelial carcinoma (including bladder, renal pelvis, ureter, or urethra carcinoma) or renal cell carcinoma. Overall, our data suggests that TYRO3 is a promising potential therapeutic target for both muscle-invasive and nonmuscle invasive bladder cancers. Although our pharmaceutical inhibition experiments with TAM inhibitors reproduced the results obtained with TAM knockdown, we cannot rule out the possibility that other activities of UNC-2025 or BMS-777607 could affect tumour cell viability.

Given the preclinical evidence of the involvement of TYRO3 in cancer, it is important to develop potent small inhibitors or monoclonal antibodies specific against this tyrosine kinase receptor. Efforts in such direction are being made (Demarest et al., 2013;Chien et al., 2016).

were determined with the BioRad Bradford Protein Assay Kit (BioRad, Marnes-la-Coquette, France) and TAM protein levels were assessed by immunoblotting.

RNA extraction and real-time reverse transcription-quantitative PCR

RNA was isolated from cell lines and xenografts with RNeasy Mini kit (Qiagen, Courtaboeuf, France). Reverse transcription was performed with 1 µg of total RNA, and a high-capacity cDNA reverse transcription kit (ThermoFisher Scientific). A predesigned assay was used to quantify expression of the TATA-box binding protein (TBP) reference gene (ThermoFisher Scientific, Ref: 4326322E). Custom-designed assays were used to measure expression of the TYRO3, AXL, MERTK and GAS6 genes. Primers and probes were designed with Probe Finder software at the Universal Probe Library Assay Design Center (Roche). RT-qPCR settings were as described elsewhere.5 For each gene of interest, the amount of mRNA was normalised against the TBP reference gene by the 2-ΔΔCt method. TYRO3 (Roche Universal Probe Library

Cell viability after transfection with siRNA and treatment with small compounds

For siRNA transfection, cells were transfected with either 20 nM (MGH-U3, RT112, UM-UC-5, UM-UC-9, 5637 and 647 V) or 5 nM (VM-CUB-1) siRNA in the presence of Lipofectamine RNAi Max reagent (ThermoFisher Scientific), in accordance with the manufacturer's protocol.

siRNAs were purchased from Ambion and Qiagen. For the control siRNA, we used a Qiagen control targeting luciferase (Ref: SI03650353). The sequences of the siRNAs are described in Table S1. For small-compound inhibitor treatments, MGH-U3, RT112, UMUC-5, UM-UC-9 and VM-CUB-1 cells were used to seed 96-well plates with appropriate culture media supplemented with 10% FBS. After 24 h, the cells were treated with either UNC-2025 (Ref.

S7576, Euromedex, Souffelweyersheim, France) or BMS-777607 (Ref. HY-12076, CliniSciences, Nanterre, France). Cell viability was assessed with the CellTiter-Glo assay (Promega, Charbonnières-les-Bains, France).

Protein extraction and immunoblotting

Cell lysates were prepared and immunoblotting was carried out as previously described.13

Anti-TYRO3 (Ref.

Soft agar assay

MGH-U3 and RT112 cells, untransfected or transfected with siRNA, were used to seed 12-well plates containing DMEM supplemented with 10% FBS and 1% agar, in triplicate (20,000 cells/well). The medium was changed weekly. The plates were incubated for 14 days and colonies larger than 50 µm in diameter, as measured with a phase-contrast microscope equipped with a measuring grid, were counted.

DNA array

For the identification of genes displaying changes in expression after TYRO3 knockdown in MGH-U3, RT112 and UM-UC-5 cells, we transfected the cells for 40 h with TYRO3 siRNA#1, TYRO3 siRNA#2 or TYRO3 siRNA#3. mRNA was extracted and purified with RNeasy Mini kit (Qiagen). Total RNA (200 ng) from control and siRNA-treated MGH-U3, RT112 and UM-UC-5 cells was analysed with the Affymetrix U133 Plus 2.0 DNA array. Raw gene expression data were normalised and summarised by the RMA (robust multi-array averaging) method (R package affy) with a customised chip definition developed by Microarray Lab, BrainArray (HGU133Plus2_Hs_ENTREZG_v18) [START_REF] Bolstad | A comparison of normalization methods for high density oligonucleotide array data based on variance and bias[END_REF]Irizarry et al., 2003). One log2transformed signal value per gene was obtained. The microarray data described here are available from GEO (https:// www.ncbi.nlm.nih.gov/geo/) under accession number GSE100025. The LIMMA algorithm was used to identify genes differentially expressed between TYRO3 siRNA-treated (n = 9) and lipofectaminetreated control (n = 9) cells (Ritchie et al., 2015). The p values were adjusted for multiple testing by the Benjamini-Hochberg FDR procedure. Genes with |log2FC| ≧ 0.58, and with a FDR below 5% were considered to be differentially expressed.

Xenograft models

Six-week-old female Swiss nu/nu mice (Charles River Laboratories, Saint-Germain-Nuelles, Table S1. The details of siRNA used in the study.

Supplementary tables

DISCUSSION

In this chapter, we investigated TAM receptor expression in BCa and showed elevated expression of TYRO3 in both muscle-invasive and non-muscle-invasive BCa tumors compared to normal urothelium in two independent cohorts of bladder cancer patients. Several perspectives relating to this work are discussed below such as mechanisms of TYRO3 overexpression, activation and the need of specific inhibitors.

TYRO3 overexpression

Mechanisms leading to TYRO3 overexpression are still unclear. As discussed in Chapter 4, very low TYRO3 expression is present in normal human urothelium (NHU) cells and is not necessary for NHU survival. No gene alteration, such as mutation in TYRO3 has been identified in BCa. In our study, c-MYC was predicted as upstream regulator, however its decrease at protein level upon TYRO3 downregulation was varied among the cell lines. To elucidate the mechanism, mostly indirect studies have been conducted by others. One study has shown that treatment with stromal cell derived factor 1α (SDF-1α), which is a ligand for the chemokine receptor CXCR4, upregulated both TYRO3 and AXL mRNA and protein expression in thyroid cancer cells.

However, a treatment with a CXCR4 inhibitor did not reduce constitutive AXL expression, suggesting that its overexpression results from additional regulatory mechanisms (Avilla et al., 2011). Another study by Zheng and colleagues found that nerve growth factor (NGF) stimulation resulted in increased TYRO3 and AXL expression in rat neuronal cells (PC12).

Interestingly, they found that NGF binding to tropomyosin-related kinase receptor A (TrkA)

receptor was essential to result in this overexpression. TrkA co-localised and was also coimmunoprecipitated with TrkA suggesting a physical interaction and the possibility that an additional post-translational mechanism could increase TYRO3 expression in this context [START_REF] Zheng | NGF-induced Tyro3 and Axl function as survival factors for differentiating PC12 cells[END_REF]. It is likely that TYRO3 mRNA levels are indirectly regulated by these upstream signalling proteins and these mechanisms differ between tissues. Interestingly, the microRNA miR-7-5p (miR-7) has been identified as a direct regulator of TYRO3 gene expression in hepatocellular and colorectal carcinoma cells [START_REF] Kabir | A microRNA-7/growth arrest specific 6/TYRO3 axis regulates the growth and invasiveness of sorafenib-resistant cells in human hepatocellular carcinoma[END_REF]Qin and Qian, 2018).

Overexpression of miR-7 significantly decreased TYRO3 mRNA and protein levels and miR-7

was found to be binding to the 3′UTR TYRO3 sequence but not to the mutated sequence as found by reporter assay (Qin and Qian, 2018). Epigenetic regulation of TYRO3 expression could also be taken place. Previous study has demonstrated that AXL gene promoter must be unmethylated in order to be upregulated upon doxorubicin, cisplatin or VP16 chemotherapy agent stimulation in AML cell line [START_REF] Hong | Receptor tyrosine kinase AXL is induced by chemotherapy drugs and overexpression of AXL confers drug resistance in acute myeloid leukemia[END_REF]. This was confirmed by another study which demonstrated that AXL promoters are hypomethylated in AXL-high overexpressing cell lines of Kaposi sarcoma, while AXL-low cell lines had more promoter methylation [START_REF] Liu | Induction, regulation, and biologic function of Axl receptor tyrosine kinase in Kaposi sarcoma[END_REF]. Another potential mechanism regulating transcription of TAMs have been suggested by a study of Mitsiades and colleagues, showing that a histone deacetylase (HDAC) inhibitor (suberoylanilide hydroxamic acid (SAHA)) suppressed GAS6 expression in multiple myeloma cells [START_REF] Mitsiades | Transcriptional signature of histone deacetylase inhibition in multiple myeloma: Biological and clinical implications[END_REF]. Interestingly, from our transcriptomic data, HDAC was also predicted to be an upstream regulator and remains to be investigated.

TYRO3 activation

We detected presence of N-glycosylation of TYRO3 in bladder tumours and this might lead to its ligand-independent activation in the presence of galectin. High galectin-1 expression in BCa tumor tissue has been suggested to correlate with poor prognosis [START_REF] Wu | Galectin-1 dysregulation independently predicts disease specific survival in bladder urothelial carcinoma[END_REF]. Galectin-1 is encoded by LGALS1 gene and has been shown to be highly expressed in some BCa cell lines. A functional study downregulating Galectin-1 in two BCa cell lines by shRNA resulted in reduced cell proliferation, invasion and lower clonogenicity resulting from downregulated MAPK stimulation [START_REF] Shen | Role of galectin-1 in urinary bladder urothelial carcinoma cell invasion through the JNK pathway[END_REF]. Galectin-3 is a known ligand for MERTK [START_REF] Caberoy | Galectin-3 is a new MerTK-specific eat-me signal[END_REF] and has also been suggested to be a TYRO3 ligand in in human head and neck cancer cells, but remains to be confirmed [START_REF] Hafizi | Abstract 2638: Galectin-3 activates the receptor tyrosine kinase Tyro3 and stimulates ERK signaling, cell survival and migration in human head and neck cancer cells[END_REF].

TAMs can also be activated by direct ligand binding, but in this study, we did not investigate the extent of the two known TAM-ligand activation of TYRO3: GAS6 and PROS1. We detected that also GAS6 was significantly overexpressed in MIBC, but not in NMIBC. This was further confirmed by a recent study showing that GAS6 is highly expressed in BCa and was significantly associated with tumor stage and worse prognosis as assessed by IHC in BCa patient samples [START_REF] Mao | Overexpression of GAS6 promotes cell proliferation and invasion in bladder cancer by activation of the PI3K/AKT pathway[END_REF]. The authors also validated the increased expression of GAS6 in a panel of bladder cancer cell lines. They subsequently established that GAS6 depletion resulted in reduced proliferation, migration and survival according to our results, suggesting that the major signaling could be through GAS6/TYRO3 axis in BCa. The authors demonstrated that GAS6 downregulation impacted PI3K-AKT signaling pathway by showing that treatment of BCa cells with SC79, an AKT activator, partially restored the effect of GAS6 silencing on cell proliferation and invasion.

However, another study has shown that PROS1 has a higher potency to induce the phosphorylation of TYRO3 as compared to GAS6, using a TYRO3-only expressing bladder cancer cell line (MGHU3) [START_REF] Al Kafri | Tumour-secreted protein s (Pros1) activates a Tyro3-Erk signalling axis and protects cancer cells from apoptosis[END_REF]. This study equally demonstrated that both Erk1 and AKT are phosphorylated upon PROS1 ligands stimulation, however only AKT pathway was found to be activated following PROS1 stimulation in a SCC cell line co-expressing two TAM receptors (TYRO3 and AXL).

TAM redundancy

We observed that TYRO3 knockdown systematically resulted in a more significant decrease in cell growth/survival compared to AXL/MERTK knockout, in a panel of BCa cell lines, regardless of AXL and MERTK expression levels, suggesting that neither AXL nor MERTK play a redundant role, rescuing the loss of TYRO3. However, we noticed that one of the three siTYRO3 used constantly had more important impact on cell survival than the other siTYRO3 used and in one cell line (RT-112), and it also induced upregulation of MERTK. Therefore, it is possible that some degree of cooperation exists between TAM receptors in BCa and can be induced, especially when all three receptors are downregulated. This is an important consideration for potential treatment resistance studies in the future. It remains to be investigated whether long-term treatment with TYRO3-specific inhibitor would influence expression of other TAMs.

However, no TYRO3 specific inhibitor yet is available due to the high similarity between the TAM structures.

TYRO3 downstream signalling

We confirmed our transcriptomic analysis predictions by Western Blot analysis, showing that CYCLIN D1 and SURVIVIN levels are decreased, pointing that cell cycle and cell survival pathways are regulated. It was also confirmed by Mao and colleagues that GAS6 downregulation results in CYCLIN D1 downregulation in BCa cell lines. CYCLIN D1 downregulation has been shown to result in decreased DNA repair efficiency and result in radiosensitisation in prostate cancer and triple negative breast cancer cells [START_REF] Marampon | Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage[END_REF][START_REF] Choi | Cyclin D1 is associated with radiosensitivity of triple-negative breast cancer cells to proton beam irradiation[END_REF], suggesting that TYRO3 downregulation could radiosensitise BCa cells.

Overall, TYRO3 is significantly upregulated in BCa and its inhibition results in antitumor effect, therefore it is a promising target, which remains to be further explored.

Critical analysis of methods used

Soft agar assay

In this study, a soft agar clonogenic assays was used rather than a conventional plated one for the RT112 and MGHU3 cell lines. The reason for the choice of the soft agar assay is that we were interested to assess the ability of the siTYOR3 treatment to impede cellular anchorage-

In vivo experiment analysis

For the in vivo experiments non-parametrical statistical test was used to compare the size between the tumours. The test was selected due to some variation in the tumour sizes resulting in non-normal distribution of the data (which does not permet to use parametric test).

CHAPTER 3 TYRO3 targeting as a radiosensitizing strategy in bladder cancer

which indicates that normal bladder tissue would not be impacted from the anti-TYRO3 treatment.

Conclusion: TYRO3 is a potential target to improve radiotherapy efficiency in bladder cancer.

Introduction

Bladder cancer (BCa) is the fourth cancer in men in terms of incidence in industrialized countries (Antoni et al., 2017). It is also the most expensive cancer to treat per patient considering the relapses and life-long regular surveillance for Non muscle-invasive bladder cancer (NMIBC) [START_REF] Sievert | Economic aspects of bladder cancer: What are the benefits and costs?[END_REF]. 25% of all diagnosed BCa is Muscle-invasive bladder cancer (MIBC) which has abysmal long-term survival rates. Cystectomy is the standard treatment for MIBC, but presents significant drawbacks including associated high mortality rates, increased risk of infection and decreased quality of life (Sanli et al., 2017). Recently, radiotherapy coupled with chemotherapy and tumor transurethral resection has emerged as a radiation-based bladder sparing treatment for limited disease (Leow et al., 2019).

Alternative treatment strategies are also needed for frail patients who cannot tolerate cisplatin-based chemotherapy. Immunotherapy is recently used in the treatment of MIBC and metastatic disease. However, there are still no validated biomarkers to accurately predict response to immunotherapies. In addition, optimal sequencing and combination of immunotherapeutic agents with chemo/radiotherapy are still lacking [START_REF] Boegemann | Prospects and progress of immunotherapy for bladder cancer[END_REF].

Many international studies have shown that the survival data from bladder preservation protocols including radiotherapy have compared favourably to that of radical cystectomy (Leow et al., 2019). Selectively increasing bladder tumor sensitivity to radiation could improve the efficacy of such treatment, limiting its side effects and reducing the need for cystectomy.

However, there is a limited number of studies investigating radiosensitisation strategies of bladder tumors, which highlights the urgent need to research and optimize this approach.

TAM (including TYRO3, AXL and MERTK) receptor tyrosine kinase family play an important physiological role in the engulfment and phagocytic clearance of apoptotic cells (ACs) in adult tissues [START_REF] Lemke | Immunobiology of the TAM receptors[END_REF]. In addition, TAM signalling negatively regulates immune response through influencing macrophage polarization. TYRO3 specifically regulates myelination in the central nervous system [START_REF] Akkermann | The TAM receptor Tyro3 regulates myelination in the central nervous system[END_REF]. TAM receptors are frequently dysregulated in cancer and associated with resistance to therapies. Therefore, strategies targeting TAM receptors and their downstream pathways have been explored in many cancer types [START_REF] Wanigasooriya | Radiosensitising cancer using phosphatidylinositol-3-kinase (PI3K), protein kinase B (AKT) or mammalian target of rapamycin (mTOR) inhibitors[END_REF]. In particular, targeting AXL, a member of TAM receptor tyrosine kinase, successfully improved radiation response in head and neck squamous cell carcinoma (HNSCC) (Brand et al., 2015) and it has also been found to be upregulated and activated in induced radioresistant HNSCC cell lines [START_REF] Skinner | Integrative analysis identifies a novel AXL-PI3 kinase-PD-L1 signaling axis associated with radiation resistance in head and neck cancer[END_REF]. A Phase I clinical trial using an AXL inhibitor BGB324 is currently being tested in glioblastoma, which progressed after radiation and other combined treatments (NCT03965494).

We have recently demonstrated that TYRO3 is overexpressed in 50% of BCa and that TYRO3 overexpression conferred a TYRO3-dependance to bladder tumor cells both in vitro and in vivo. Thus far, no study has investigated the concurrent inhibition of TYRO3 and use of radiation treatment. Due to the high structural and sequence similarities of the three TAM receptors, it is difficult to develop specific inhibitors for a single TAM receptor. However, targeting multiple TAM receptors may be advantageous, as all three TAM receptors are known to stimulate macrophage polarization towards the cancer-promoting M2 phenotype, and therefore targeting multiple TAM receptors may have a stronger anti-cancer effect. UNC2025, a potent MERTK inhibitor, which has an off-target effect on TYRO3 has shown efficacy in leukemia preclinical models alone and in combination with chemotherapy (DeRyckere et al., 2017).

In this study, we first investigated the impact of TYRO3 modulation on BCa radiosensitivity. was partially downregulated (dilutions of siTYRO3 #1) in RT112 cell line, demonstrating in both cases a significant radiosensitisation (Fig. 1A). We found that 5 nM of siTYRO3 #1 did not allow sufficient cell survival without irradiation to assess the impact of irradiation. Therefore, siTYRO3#1 was serially diluted to be able to be combined with radiation treatment and we also noted by Western blot that its expression was not completely downregulated in the lowest doses of siTYRO3#1. AXL and MERTK levels were not impacted by siTYRO3 #4 and siTYRO3#801 (Supplementary Figure S2).

Conversely, when TYRO3 was over-expressed (O/E) in TYRO3-low expressing cell line (UM-UC-

3), the UM-UC-3-TYRO3 O/E cells were significantly more radioresistant than empty vectortransfected cells used as control (D10=6.6 Gy ±0.4 vs 5.5 Gy ±0.4, from three independent experiments) (Fig. 1B). These results were also supported by the analysis of the surviving fraction at 4 or 6Gy (SF4 or SF6) (Supplementary Figure S3).

The activation of membrane receptor Tyrosine kinases (RTKs) occurs usually within minutes after irradiation and belongs to the earliest events characterized next to the recognition of DNA damage (reviewed by Schmidt-Ullrich et al., 2003). Therefore, we examined the activation of TYRO3 following ionising radiation. No antibody to detect phospho-TYRO3 was commercially available. For this, we first immunoprecipitated total phospho-tyrosine residues, performed Western Blotting and probed the membrane using anti-TYRO3 antibody. We showed on 5637 cell line that after 2 Gy irradiation TYRO3 phosphorylation levels are significantly increased within 5 minutes following irradiation (Figure 1C).

TYRO3 modulation impacts long-term persistence of Ionising

Radiation-Induced Foci (IRIF)

In order to decipher the mechanisms underlying the observed effect of TYRO3 modulation on radiosensitivity of BCa, we first compared the H2AX foci between the treatments by immunofluorescence staining. We checked single (TYRO3 up-or down-regulation) treatment and the combination with radiation treatment at 30 min (initial damage) and 24 h (residual damage) after RT. For both cell lines undergoing the combined treatment including TYRO3 downregulation, between 40-50% cells still had more than 10 foci/nucleus after 24h of 2Gy irradiation, whereas for the single radiation treatment, almost all of the induced DNA foci had been repaired and only 5-9% of cells were still having more than 10 foci/nucleus (Fig. 2A andB). For RT112 cell line, the siTYRO3 #4 and #801 alone induced H2AX foci (around 20 foci/nucleus) already 30min after irradiation (corresponding to 48h post-transfection) (Supplementary Fig. S5), and at a lesser extent for 5637. Conversely, irradiated TYRO3 overexpressing cells had lower number of H2AX per nucleus as compared to the CTRL (23 vs 39%) 24 hours after 6 Gy radiation (Fig. 2C).

TYRO3 downregulation leads to higher levels of DNA damage 24h after radiation treatment

In order to discriminate whether H2AX foci are directly correlating with the extent of DNA lesions following TYRO3 downregulation and irradiation, we followed the DNA damage induction and repair using alkaline comet assay for RT112 and 5637 cell lines (Fig. 3A and3B).

It has been shown that data distribution of tail moments obtained from comets assays is generally non-Gaussian, which precludes the use of parametric tests for statistical analysis [START_REF] Duez | Statistics of the Comet assay: A key to discriminate between genotoxic effects[END_REF]. So accordingly, the comparison of samples was undertaken comparing the

TYRO3 downregulation affects cell cycle and causes cell accumulation in G2/M phase following irradiation

In order to investigate the effects of TYRO3 downregulation associated with RT on the DNA Damaging Response (DDR) signalling pathways, the BCa cell cycle distribution was explored in vitro after TYRO3 depletion using siRNAs and irradiation. BCa cell lines were treated with siLUC or siTYRO3 for 48h, then irradiated with 6Gy or mock-irradiated and analysed by flow cytometry 24h after. By that time, TYRO3 downregulation alone did not significantly affect the cell cycle distribution and cells remained largely in G0-G1 (55-65%) for RT112 (Fig. 4A) and in S phase (42-50%) for 5637 (Fig. 4B). Radiation alone induced moderate increase of percentage cells in G2/M phase in the siLUC transfected cells (by 8.6% and 7.0% in RT112 and 5637 cell lines respectively as expected in p53-mutated cell lines (Fig. 4A andB). However, when TYRO3 downregulation by different siRNAs was coupled with radiation, it resulted in significant increase of cells in G2/M phase (by 11.0 -26.3% for the RT112 and 30.1 -36.2% for the 5637 depending on the siTYRO3 used) (Fig. 4B) (Supplementary Table S4 andS5).

TYRO3 downregulation does not affect the expression levels or activation of DNA repair signalling proteins but disrupts mitosis regulation

As at 24h post-irradiation, more ionising radiation-induced foci and unrepaired DNA persisted in TYRO3-depleted cells, we investigated the potential molecular mechanism underlying this enhanced radiation response. For this, activation of key sensor proteins of the DNA damage signalling pathways was evaluated. Unaltered expression and phosphorylation levels of DNA damage response proteins of ATM, ATR, DNA-PK, Chk1, Chk2 after TYRO3 downregulation could be observed (Figure 5A). The significant increase in G2/M phase of the cell cycle in transfected cells 24h after irradiation let us to evaluate key proteins involved in the G2/M transition and mitotic spindle assembly. For this, the expression levels of AURORA A kinase, CDK1, phospho-CDK1, and CYCLIN B1 were examined (Figure 5B). The phosphorylation of CDK1 (Y15) and the expression levels of CYCLIN B1 increased 24h after irradiation, while that of AURORA A decreased using both siTYRO3 #4 and siTYRO3 #801 on RT112 and 5637 cell lines, leading to the conclusion that downregulation of TYRO3 might disrupt mitosis regulation through differential mitotic protein expression levels.

TYRO3 modulation does not affect proliferation of normal human urothelial cells

We showed previously that TYRO3 is overexpressed in BCa, while normal urothelium has lower levels of TYRO3 (Dufour et al., 2019). In order to investigate potential side effects of TYRO3 downregulation in normal urothelium, we used UNC2025, a pan-TAM inhibitor. UNC2025

impacted cell viability only when reaching 10 µM concentration (Fig. 6A) and the calculated Effective dose for 50% inhibition (ED50) was more than 70-times greater for two NHU explants tested as compared to RT112 and 5637 BCa cell lines (Fig. 6B).

Discussion

With increased use of bladder preserving therapies, there is growing interest to improve the current chemo-radiotherapy regiments by sensitizing BCa cells towards one of these modalities. The major aim of this study was to investigate the consequences of combining radiation therapy with TYRO3 downregulation in BCa.

TYRO3 belongs to the TAM (TYRO3, AXL and MERTK) family of RTKs. TYRO3 has been less frequently studied than the other two members of TAM family. Most recently, we demonstrated that TYRO3 is upregulated in 50% of MIBCs and that TYRO3 overexpression conferred a TYRO3-dependance to bladder tumor cells (Dufour et al., 2019). Despite of the wealth of literature and date implicating TYRO3 in cancers, there are no studies that have investigated TYRO3 therapeutic potential in combination with radiation treatment.

Receptor Tyrosine Kinases (RTKs) have been identified to play role in early cell response to radiation, inducing secondary activation of cytoplasmic protein kinase signalling cascades. This activation and their downstream effectors contribute to tumor cell resistance to treatments via either pro-proliferative or anti-apoptotic signals depending on the tumor type (Schmidt-Ullrich et al., 2003;Meyn et al., 2009). We found that as many other RTKs, TYRO3 is phosphorylated within minutes after irradiation. Downregulation of TYRO3 resulted in increased radiosensitivity of BCa cells, while overexpression resulted in increased radioresistance, demonstrating that TYRO3 plays a role in radiation response. Our results are the first report to identify TYRO3 as a mediator of radiation response in BCa. This observed radiosensitisation is supported by an excess of residual DNA damage and H2AX foci 24h after radiation. Similar observations have been made in Head and Neck Squamous Cell carcinoma (HNSCC) when AXL, another TAM receptor was downregulated or inhibited (Brand et al., 2015). However, in this study, AXL was found to regulate the DNA repair pathways via AKT and DNA-PK activity. Our results lead to different conclusions as the combined treatment of TYRO3 downregulation and irradiation did not modify the activation or expression levels of five different key DNA repair proteins including DNA-PK.

Modulation of cell cycle distribution is among mechanisms resulting to radiosensitisation (Maier et al., 2016;Wang et al., 2018). In our study, downregulation of TYRO3 alone did not modify the cell cycle distribution but the combination of TYRO3 downregulation and radiation resulted in remarkable increase in the G2/M phase of the cell cycle. This increase is supported by an increase of CYCLIN B1 levels in the combined treatment. It has been shown recently that BRCA1 interacts with CYCLIN B1 and that this interaction is modulated by DNA damage and cell cycle phase (Choi et al., 2018). BRCA1 plays essential roles in repair pathways as homology-directed repair, non-homologous end joining, nucleotide excision repair and homologous recombination (Deng and Wang, 2003). As homologous recombination (HR) pathway is restricted to late S or G2 phase of the cell cycle (Rothkamm et al., 2003), we can hypothetise that the radiosensitivity observed when TYRO3 is down-regulated and combined with radiation could be due to impaired HR pathway. Further experiments are needed to confirm this hypothesis. As G2/M is the most radiosensitive cell cycle phase [START_REF] Dillon | Selective Targeting of the G2/M Cell Cycle Checkpoint to Improve the Therapeutic Index of Radiotherapy[END_REF], this could be advantageous in clinical setting where fractionated radiation dose delivery is employed.

Due to the high structural similarities of the three TAM receptors, it is difficult to develop inhibitors specific for a single TAM receptor. However, targeting multiple TAM receptors may be advantageous, as all three TAM receptors stimulate macrophage polarization towards the cancer-promoting M2 phenotype, and therefore targeting multiple TAM receptors may have a stronger anti-cancer effect. In this study, UNC-2025, a potent MERTK inhibitor, which has an off-target effect to TYRO3 resulted in radiosensitisation of RT112 cell line, but not of 5637 cell line. This difference between the effects of siTYRO3 and the UNC-2025 could result from experimental design, where reduction of TYRO3 phosphorylation lower than 30% is possibly needed to induce downstream pathways leading to radiosensitization. However, a partial down-regulation of TYRO3 using the siTYRO3#1 could also radiosentize BCa cells, indicating that only the phosphorylation status of TYRO3 upon irradiation could not account for the differences in radiation response observed on BCa cells. We tested UNC-2025 on normal human urothelial cells and showed no toxicity at the doses for the radiosensitisation experiments (0.05-0.2 µM), indicating that normal bladder tissue would be spared using this inhibitor in combined treatments.

In summary, we report that TYRO3 downregulation in combination with radiation treatment is an effective treatment in BCa cells in vitro, resulting in decreased proliferation, accumulation of cells in G2/M phase, increased H2AX foci in the combined treatment and increased levels of DNA damage. We showed that TYRO3 overexpressing cell line is more resistant to radiation and have significantly less H2AX foci in the nucleus. This is a first study to implicate and demonstrate a prominent role of TYRO3 in radiation response in BCa indicating a promising sensitization marker that could be clinically employed in future treatments.

Materials and Methods

Cell lines

The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained

NHU Cell Culture

Urothelium from the ureter/renal pelvis of patients undergoing urological surgery was collected with National Health Service Research Ethics Committee (England) approval and required informed consent from urological procedures that excluded urothelial neoplasia.

Finite (nonimmortal, serially passaged) NHU cell lines were established and grown on Primaria™ plasticware (BD Biosciences, Wokingham, UK) in low calcium (0.09 mM) keratinocyte serum-free medium (KSFM) containing bovine pituitary extract and recombinant epidermal growth factor (Fisher Scientific UK Ltd, Loughborough, UK) supplemented with 30 ng/mL cholera toxin (KSFMc) (Cross et al., 2005). NHU cell lines were subcultured by trypsinization at just-confluence and used in experiments between passages 3-5.

Inhibitor treatment

5637 and RT112 cells were used to seed in 6-well plates for colony forming assays with RPMI culture media supplemented with 10% FBS. After 24 h, the cells were treated with UNC-2025 (Ref. S7576, Euromedex, Souffelweyersheim, France) for further 24h, then irradiated or shamirradiated without medium change and left to form colonies as detailed in clonogenic cell survival. Proliferaring NHU cells were seeded in 96-well plate, left to adhere for 24h, treated for 72h with inhibitor and cell viability was scored using Alamar Blue assay (AbD Serotec, Kidlington, UK) as detailed in [START_REF] Baker | Ketamine-Induced Apoptosis in Normal Human Urothelial Cells: A Direct, N-Methyl-D-Aspartate Receptor-Independent Pathway Characterized by Mitochondrial Stress[END_REF].

siRNA transfection

Cells in the exponential phase of growth were detached with accutase solution (Merck) and between 0.5 -1x10 6 cells per T25 flask were reverse-transfected with 5nM siRNA (or 0.6-5 nM for siTYRO3 #1) using Lipofectamine RNAi Max reagent (ThermoFisher Scientific), in accordance with the manufacturer's protocol. siRNAs targeting TYRO3 were purchased from Ambion and Qiagen. Then, the cells were seeded in 6-well plates (between 200-10 000 cells) for 48h to achieve complete downregulation of TYRO3. For the control siRNA, we used a Qiagen control targeting luciferase (siLUC, Ref: SI03650353). 3 different siRNAs were chosen and conducted in parallel targeting different regions of TYRO3 sequence (encoding Ig-like (siTYRO3 #1), intramembrane region (siTYRO3 #4) or kinase domain (siTYRO3 #801). siRNA sequences are listed in Supplementary Table S3. UM-UC-3 cell line was unable to form colonies. In this case, the control and the overexpressing cell lines were plated in 6-well dishes in duplicate at the density of 2x10 4 per plate.

Clonogenic

They were left to adhere for 8 hours, irradiated, and let to grow after irradiation for 5population doubling time (5 days). Cells were then trypsinized and counted. (SF) was calculated by dividing the number of cells in the irradiated vs non-irradiated cultures.

Each condition was repeated on at least 3 separate experiments. Survival curves were generated by fitting the surviving fraction to a linear-quadratic model: SF = exp (-αD -βD2), where SF is the surviving fraction and D is the dose, and α and β are the adjustable parameters characterizing the radiation response. For each experiment, the D10 (dose to achieve 10% cell survival) was calculated using these parameters.

Immunoprecipitation

Cells were cultured in 10 cm dishes, irradiated at 2Gy and lysed after 5 min using lysis buffer (50 mM Tris pH 7. The following day, 30 µl agarose protein G beads (1:2 diluted in lysis buffer) were added to the sample and incubated for 1h at +4°C on an orbital shaker. After 5 washes with lysis buffer, the supernatant was aspirated and 25 µl of warm loading buffer was added to the beads, vortexed and boiled for 10 minutes. 15 µl of sample was loaded in SDS-PAGE gel and electrophoresed. The sample was transfer to a nitrocellulose membrane and probed with TYRO3 antibody.

ELISA

PathScan® Phospho-Tyro3 (panTyr) Sandwich ELISA Kit was purchased from Cell Signalling Technologies (Ref. 53402). Cells were cultured in 10 cm dishes, irradiated at 2Gy and lysed after 5 min according to the manufacturer instructions.

Western Blot

Cell extracts were prepared as described previously (Mahe et al., 2018)

Immunofluorescence microscopy

The H2AX foci formation was evaluated at 30 min or 24 hours after irradiation of 2 or 6 Gy.

The cells were fixed with 4% paraformaldehyde for 15 minutes, treated with 0.5% Triton X-100 for 15 minutes, blocked with 5% BSA for 1 hour, and incubated with an anti-H2AX antibody (Merck, dilution 1:1000) for 1h. These cells were then labelled with Alexa Fluor antibody (Invitrogen) for 45 minutes and counterstained with ProLong Gold Antifade Mountant with DAPI (Invitrogen). Foci were visualized with a 3D-SIM immunofluorescence microscope and analyzed by ImageJ, using an optimized macro script. Over 300 cells were counted from more than three random fields photographed at the same exposure time under a ×60 objective for respective experiments, and the percentage of cells containing >10 fluorescent foci was calculated.

Flow cytometry

Cell cycle was evaluated using dual staining by 5-bromo-2′-deoxyuridine (BrdU) and propidium iodide (PI). siRNA transfected treated cells were exposed to radiation or mock treatment and 24h after were subjected to incubation with 100 uM BrdU for 30 minutes (#19-160, Milipore).

After, the medium was conserved and cells were washed with PBS and harvested by trypsin treatment. Cells were then either frozen in 10% DMSO or directly fixed in 70% EtOH, dropwise while vortexing. After fixation, cells were washed and rehydrated with PBS (supplementd with 5% FBS) and subjected to pepsin digestion. Hydrolysis with 2N HCL was performed and continued with permeabilisation by using 10mM HEPES, 0.5% Tween-20 and 5% Goat serum.

After wash, cells were stained with mouse anti-BrdU-fluorescein isothiocyanate (FITC) monoclonal antibody (BU20A Invitrogen/eBioscience, Ref. 11-5071-42). Cell cycle analysis was performed on a FACS Canto II cytometer (Beckton-Dickinson).

Comet assay

Single cell gel electrophoresis (SCGE) was carried out by Trevigen's comet assay kit (ref. 4250-050-K) with slight modification. Briefly, cell suspension (1.5 × 10 5 /ml) and molten LM agarose (at 37°C) were prepared in 1:10 (v/v) ratio. From there, instantly 50 μl was poured onto the comet slide (ref. 4250-200-03 and kept at 4°C in the dark for 10 min for agarose adhesion.

Slides have been irradiated for 6Gy (dose rate 1Gy/min) with gamma ray (137Cs, GSR D1). The slides were placed in an ice-cold lysis solution for 60 min at 4°C and then deeply immersed in freshly prepared alkaline unwinding solution (pH>13: 200 mM NaOH, 1 mM EDTA in 50 ml of dH2O) for 1 h at 4°C in the dark. Subsequently, the slides were placed in an electrophoresis chamber filled with alkaline buffer (200 mM NaOH, 1 mM EDTA) and run at 28 V for 30 min.

Slides were then immersed in a 70% ethanol solution for 5 min twice, dried for 10-15 minutes in incubator at +37°C and stored with desiccant prior to scoring. SYBR green staining was performed to visualize the comets through transmission light microscope. Randomly, 200 comets per slide were visualized and analyzed by scoring of various parameters such as head, tail and tail moment under light microscope with Comet Assay Metafer software (Metasystems). Olive tail moment was calculated as the product of the tail length and the fraction of total DNA in the tail for each comet and compared between conditions.

Statistical analysis

All functional experiments were carried out twice or three times, in triplicate. For the comparison of data between cell lines, two-tailed t-tests were used. The control siRNA (Luciferase GL2 siRNA) or DMSO was used as the reference group. Data are expressed as means ± SD, and differences with a p value < 0.05 were considered statistically significant. For

Comet assays, Kruskal-Wallis nonparametric tests with Multiple comparisons were used and medians compared. All statistical analyses were performed with Prism8.0 (GraphPad Software, Inc). Study supervision : Jennifer Southgate, Pierre Verrelle, Isabelle Bernard-Pierrot, François

Supplementary

Radvanyi, Frédérique Mégnin-Chanet.

In this chapter, I investigated the potential to combine TYRO3 downregulation with radiation treatment in BCa cell lines. This work is still remains to be finalised but consdierabla progress has been made.

From alkaline comet assay results for two TYRO3 high-expressing cell lines RT112 and 5637, we detected that TYRO3 downregulation alone does influence the efficiency of DNA Damage and sustains this delayed repair at 24h time point, it is especially prominent with siTYRO3 #801 in both cell lines. With the other siTYRO3#4 this effect is detected at lesser extent. It would be interesting to compare the effect of siTYRO3 with the one induced by pan-TAM inhibitor, thus allowing to evaluate effect of different doses. The accumulated damage could partially explain the persistent residual foci at 24h as detected by immunofluorescence and accumulation of cells in the G2/M cell cycle phase. In order to have more direct insight in the kinetics of the repair, homologous recombination (HR) assay could be performed. Also, it would be interesting to explore the type of the damage accumulating: alkaline comet assay is known to be more sensitive for smaller amounts of DNA damage, including alkali-labile sites, DNA-DNA or DNA-protein cross-linking, and DNA single-strand breaks, while also having the capability to visualise double-strand breaks [START_REF] Lu | Evaluating in vitro DNA damage using comet assay[END_REF]. Neutral comet assay would be a valuable to tool to explore specifically double stranded breaks.

In order to invstigate the underlying radiosensitising mechanism of the combined TYRO3 downregulation and radiation, western blots on DNA damage response (DDR) signalling proteins ATM, ATR, Chk1 and Chk2 revieled no significant change of these proteins or their phosphorylation at several time points following radiation. More detailed overview of downstream pathways affected by the combined treatment are planned to achieve by performing transcriptomic analysis in single and combined treatments in both TYRO3-high expressing cell lines using NanoString technology. The identified downstream candidates then could be confirmed by western blot.

Here I showed that TAM inhibitor does not influence normal human urothelial (NHU) cell proliferation, which indicates that side effects of TYRO3 downregualtion would not be anticipated from urothelium and I explored this further by performing single TAM downregulation experiments in chapter 4. However, it remains to be confirmed whether TYRO3 downregulation does not radiosensitise NHU.

While observing increased radiosensitivity in vitro, we are interested to investigate the combined treatment in vivo. In the systematic review (chapter 1), I concluded that very few in vivo studies with targeted agents as radiosensitisers have been performed in BCa. Therefore, I am interested to compare the combination versus single treatments in a cell line xenograft.

Although using xenograft has number or limitations, such as not having the possibility to investigate normal bladder toxicity, it would serve as the first approach to investigate the inhibitor and/or siTYRO3 treatment effciciency in combination with radiation in vivo.

Prologue

In order to improve the existing bladder-preserving treatment, we propose to target TYRO3, which specifically impacts BCa cell survival overexpressing TYRO3. However, to have some hints on the possible clinical side-effects of such treatment, the effect of TYRO3 inhibition on normal surrounding tissue must be studied. Side effects can result from the direct reaction of urothelial cells to radiation treatment or result from more complex response of the stroma or perturbed urothelium-stroma interactions. Limited life span (primary) normal human urothelial (NHU) cells are useful models to establish and verify the role of TAM receptors in the normal urothelium with a perspective to study direct cell response to radiation. More complex, physiological response to radiation cannot be explored in NHU culture. Up to now, only few studies have described the effects of radio-or chemo-therapy agents on NHU or immortalised urothelial cell lines (Groselj et al., 2018).

I hypothesized that targeting TYRO3, using a siRNA or pan-TAM inhibitor since no specific inhibitor is available up to now, in addition with radiation could improve the combined treatment outcome in BCa. However, TAMs are expressed in the normal bladder tissue and little is known about their interactions and physiological role so that side-effect of such treatment could not be anticipated.

In chapter 4, I address two questions:

1) What are the TAM expression levels in NHU?

2) What is the impact on NHU from TAM knock-down (and inhibition)? Cell viability of cells cultured from 2 donors are showed (NHU1 and NHU2). TAM receptors were downregulated using at least 2 different siRNAs for each receptor and normalised to that of siLUC.

Each experiment was carried out twice in triplicate. AXL is still expressed at the cell differentiated by TZ/PD protocol, but not in the ABS/Ca 2+ . This could be related to the differences in the phenotypes of the cells, where ABS/Ca 2+ differentiated cells exibit stronger barrier function. In the ABS/Ca 2+ differentiated cells there is a switch to decreased AXL expression and increased MERTK expression upon differentiation.

However, the confluency of the cells could also potentially be linked to the differential expression of TAM receptors since, the differentiated cells are near 100% confluency. Indeed, when a sample of quiescent cells was analysed (cells grown to confluence without initiating differentiation), MERTK expression was increased at equal level as the matched cells undergoing the differentiation (Figure 4-4, lane 10). This could be investigated by seeding cells at different confluencies and comparing the TAM expression levels.

TAM receptors transcript levels from the RNA-seq data showed similar trends as observed in the protein levels assessed by Western blot, however it would be important to have both information from the same samples due to donor by donor variation. Intrestingly from the RNA-seq data, GAS6, the TAM ligand is highly expressed proliferating but decreased upon the NHU differentiation. Also AXL levels were high at proliferating but decreased upon differentiation, however only RNA-seq data is available for comparison and it remains to measure the Gas6 expression at the protein level. It would be interesting to see whether GAS6/AXL singaling plays a role in sustaining the proliferation of NHU.

I also detected that downregulation of TAM receptors did not significantly impact the viability of proliferating NHU cells. Although, one of the siRNA, siTYRO3 #1 did significantly decrease proliferation. As shown in the article 2 of this thesis, this TYRO3 targeting oligonucleotide lacks specificity and downregulate also other TAM receptors. I did not attempt to study the impact of serially diluted siTYRO3#1 on the viability of NHU cells (as performed in the Chapter 3). It would be interesting to transfect cells with both siTYRO3 and siAXL/siMERTK, to see whether potential redundancy exist between the receptors.

Further, the different culture medium could account for the differences between NHU and BCa cell lines: NHU are growing in serum-free, EGF and pituitary gland extract-supplemented medium, while BCa cell lines are growing in 10% fetal bovine serum-supplemented medium (FBS), which is known to contain some concentration of TAM ligand PROS1. It is possible that due to the stimulated EGF-signalling, the NHU does not respond to TAM inhibiton. It would be important to gradually adapt the BCa cell lines to the growth in NHU medium, in this way, the potential NHU and or BCa cell line dependence on EGFR signalling for survival could be evaluated.

I would like to further investigate the role of TAM receptors in the NHU and assess migration and differentiation capability of proliferating cells after siRNA downregulation of TYRO3, AXL and MERTK. Since AXL is expressed at high levels in proliferating NHU, it would be interesting to see whether AXL knock down results in impacted migration capability. Proliferating NHU cells could be subjected to the differentiation protocols, in MERTK-knock down condition.

Then the differentiation could be followed through time while monitoring expression of differentiation markers by q-RT-PCR of Western Blots.

Apart from the questions of the general role of TAMs in the NHU cells, I would be interested ng/mL cholera toxin (KSFMc) (Cross et al., 2005). NHU cell lines were subcultured by trypsinization at just-confluence and used in experiments between passages 3-5. For this study, cell lines derived from 6 individuals.

NHU differentiation protocols

The differentiation assay was based on the optimised conditions for inducing uroplakin gene expression in NHU cell cultures as described previously. Control NHU cell lines were grown to 70% confluence and treated with or without 1 μM TZ for 24 hours followed by incubation with or without 1 μM PD153035 for up to 6 days. Due to the limited availability of cells from biopsyderived urothelial cell lines, experiments were constrained to a) 1 μM TZ for 24 hours followed by 1 μM PD153035 and b) no treatment control (0.01% v/v DMSO). Media were changed every 3 days and cells were harvested for RNA (day 3) or protein (day 6), as with the NHU cell controls. These time points were selected as optimal from previous work.

Conclusions

There is currently a need for new therapeutic targets in bladder cancer (BCa), especially in muscle-invasive BCa, which has poor prognosis, but where bladder-sparing approaches are emerging. It is equally important for eldery and frail patients who cannot tolerate cisplatinbased chemotherapy. There are currently no effective radiosensitising agents in the clinics specifically targeting BCa tumor cells and sparing normal tissue.

In the first chapter, in a form of systematic review I analysed the use of radiotherapy in BCa overexpression and activation are still not described in BCa. It would be important to identify potential transcriptional regulators and/or epigenetic modifiers, which could induce the TYRO3 overexpression in the BCa tissues. Further, impact of TAM ligand-binding and subsequent signaling must be better understood.

In the third chapter, I demonstrated that TYRO3 downregulation resulted in increased radiosensitivity in BCa cell lines. Current bladder-preserving treatment includes radiotherapy as part of the treatment and radiation treatment is used for eldery BCa patients. Therefore, it puts TYRO3 as a promising target to be combined with radiation in clinical setting of BCa treatment. This improved effect remains to be validated in vivo and I have started in vivo study combining TYRO3 downregulation and radiation treatment. I am currently leading this study, which is performed by Experimental Radiation Platform at the Institut Curie. In addition, also TAM inhibitor will be tested in combination with radiation in vivo (see Appendix, in vivo study plan, procedures 1-5). Currently, also a transcriptomic study is undergoing to better understand the underlying mechanism of the radiosensitisation observed.

In the fourth chapter, I investigated the expression levels of TYRO3, AXL and MERTK in normal urothelial tissues. I concluded that knockdown of each TAM receptor resulted in light impact on NHU cell viability, which would suggest that little side effects would result from anti-TAM therapy in urothelium. This remains to be investigated in the combination with radiation to confirm whether TAM downregulation does not impact radiosensitivity of NHU.

Perspectives

In vivo study

In ELISA test from protein extract of fresh frozen sections) (See Appendix, in vivo study plan, procedures 1-5). These tests will allow me to conclude about the expression and activation status of TYRO3 at the point of addition of radiation, which has been the set-up for the in vitro experiments, where cells were exposed to radiation when TYRO3 downregulation was complete. This in vivo study will allow me also to explore the feasibility to use a TAM inhibitor (UNC-2025) and compare it with the effects obtained with the siRNA treatment in combination with radiation. This inhibitor has not been previously used in vivo in solid cancer context. The xenograft model in nude mice has several imitations such as it will not be possible to investigate the role of immune cells in the treatment response. Further, the effect of radiation treatment on bladder surrounding vasculature, organs and tissue will not be possible to investigate, as the tumors will be engrafted in the leg of the mice.

Syngeneic mouse models could be used in the future follow-up in vivo study. That would allow to investigate the role of immune system in the treatment response, investigating the role of TAM receptors in the macrophage polarisation for example, by isolating and quantifying the different subgroups of these immune cells by flow cytometry. BCa can be chemically induced in the bladder by exposure to chemicals or a mouse bladder cancer cell line can be orthothopically implanted in the mouse bladder. This would allow to study the side-effects of the radiation treatment on the bladder, giving the opportunity to compare acute radiationinduced toxicity versurs long-term radiation-induced side effects such as fibrosis on the bladder. Alternatively, acute normal tissue effects could be investigated on the small intestine using the intestinal crypt assay, and later effects in small and large intestine by stool assessment and histologic examination (Groselj et al., 2018).

The mechanism of radiosensitisation

In order to decipher the exact molecular mechanisms underlying the radiosensitising effect of

Improvement of siTYRO3 stability/delivery strategies for in vivo use

Most of the experimental data of these thesis relies on the use of small interfering RNA (siRNA) molecules to downregulate the TYRO3 transcript. However, this approach has several disadvantages: such as potential off-target effects, the transient nature of the knock-down and the immune response which can be induced in immunocompetent animals. In this thesis, the possibility of off-target effects has been minimised by using several different siRNAs targeting TYRO3. However, there remains a possibility that other transcripts are downregulated and the readout is a result of cumulative effect of loss of function on several genes. The transient nature of the knock-down also means that long-term effects in the models where the TYRO3 is not essential are not possible. The use of small hairpin RNA (shRNA) could be a better approach, however, due to the very strong effects on the survival of the majority of the BCa cell lines, the transient knock-down by siRNA was decided to be the most appropriate tool. The main disadvantage of using siRNA, is the problematic translation of the use of siRNA to in vivo models and further to clinical setting due to known instability of siRNA molecules and the known unspecific immune response which siRNA treatment is known to induce. To minimise these effects, several siRNA chemical modifications and delivery strategies increasing siRNA half-life and specific delivey to cancer cells have been developed, such as siRNA strand chemical modification, lipoplexes, stable nucleic acid-lipid particles (SNALPs), poly-mer-based delivery, dendrimer-siRNA conjugate, conjugate siRNA delivery, aptamer-siRNA chimaeras, quantum dot complexed with siRNA, carbon nanotube-siRNA (Fig.

5-1).

Future experimental approaches could make the use of siTYRO3 in vivo, in immunocomptenent animals and could be adapted for a use in human patients in the future.

It is worth noting that from our experience with in vivo administration of siTYRO3 through intraperitoneal injection in immunocompromised mice we achieved effective downregulation of the TYRO3 at the end of the treatment (3 weeks of 3 injections per week). We did not record any adverse effects, but more throughout examination of retina, testis and other organs where TYRO3 plays important role would be important to examine, especially in longer-course studies. siRNA chimaeras, (h) quantum dot complexed with siRNA, (i) carbon nanotube-siRNA conjugate, (j) gold nanoparticles. From [START_REF] Singh | Advances in siRNA delivery in cancer therapy[END_REF].

Development of TYRO3 specific inhibitory strategies

Due to the high structural similarities of the three TAM receptors, it is difficult to develop inhibitors specific for a single TAM receptor. This should taken into consideration when using TAM-targeting agents. As single TYRO3 knockout mouse has relatively light phenotype suggesting immune hyperactivation, the double or triple TAM receptor knockout mice have very severe phenotype, such us lupus-like symptoms, sterility, platelet dysfunction and inflammatory brain damage. Blocking all three TAM receptors using inhibitors should be tightly controlled and loss or inhibition of only one of the TAM receptors should results in less systemic side effects. Specific TYRO3-inhibiting antibody is not currently available but would be a great tool to decipher TYRO3-specific effects.

On the other hand, targeting multiple TAM receptors may be advantageous in increasing the anti-tumor effect, as all three TAM receptors stimulate macrophage polarization towards the cancer-promoting M2 phenotype. However, it could result in severe side effects, such as visual impairement due to inefficient clearing of photoreceptor cells in the retina. So far no visual defects have been investigated in the several existing in vivo studies using pan-TAM inhibitors, such as UNC2025, a potent MERTK inhibitor, which has an off-target effect to TYRO3 has shown efficacy in leukemia preclinical models alone and in combination with chemotherapy (DeRyckere et al., 2017).

Combination of TYRO3 inhibition with other modalities

Immunotherapy

Considering the important physiological role of TYRO3 in the immune system, more specifically in the efferocytosis and the innate immunity activation, studies in immune competent animals would be essential to evaluate the role of TYRO3 in tumor microenvinronment and it's potential to increase the effect of currently used immunotherapies.

Immunotherapy principle relies on re-activating the immune system and produce anti-tumor response. Checkpoint molecules on the immune cells stimulated by their ligands maintain balance between self-tolerance and active immune response. For example, in physiological setting, PD-L1 expressed by antigen presenting cells (APCs) presenting self-antigen binds to PD-1 of T-cells to inhibit the immune activity and to avoid autoimmune disease or inflammation. Cancer cells often adapt to exploit the immune system by expressing PD-L1 to downregulate the immune response and generate favourable growth-conditions. Monoclonal antibodies (mAbs) targeting PD-1/PD-L1 have shown survival benefits in various advanced cancers, including melanoma [START_REF] Robert | Nivolumab in previously untreated melanoma without BRAF mutation[END_REF], lymphoma [START_REF] Ansell | PD-1 blockade with nivolumab in relapsed or refractory Hodgkin's lymphoma[END_REF], lung cancer [START_REF] Brahmer | Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer[END_REF], and BCa (Bellmunt et al., 2017). In metastatic BCa (after failure of first line therapy), pembrolizumab (a highly selective, humanized monoclonal antibody against PD1) was associated with significantly longer overall survival (3 months) and with a lower rate of treatment-related adverse events than chemotherapy for advanced BCa (KEYNOTE-045

ClinicalTrials.gov NCT02256436 (Bellmunt et al., 2017)).

Despite the encouraging results, prolonged survival and durable response has been achieved, only in a subset of patients. It is though that the poor treatment response in addition to genetic variation among individuals can be related to complex tumor microenvironment (TME). TMEs can activate, regulate and maintain adaptive immune system, mainly through stimulation of cytotoxic T cells (CD8 + ) and this has shown to favour respone to checkpoint inhibitors. It is recognised that innate immune cells such as macrophages and dendritic cells mediate the recruitment and activation of CD8+ T cells. TAM recpetors, including TYRO3 are often reffered to as immune checkpoints of the innate immune system as they can limit the engagement of the adaptive immunity. Therefore, targeting active TAM receptors on both: immune cells and cancer cells could release this break and re-activate adaptive immune system resulting in improved treatment outcomes.

TAM inhibition has already been shown to potentiate the benefit from PD-L1 therapy when investigated in immunocompetent mice [START_REF] Yokoyama | Immuno-Oncological Efficacy of RXDX-106, a Novel Small Molecule Inhibitor of the TAM (TYRO3, AXL, MER) Family of Kinases Equal contribution # Corresponding Author Running Title: Immuno-oncological effect of RXDX-106, a TAM RTK inhibitor[END_REF]. In this study, pan-TAM inhibitor treatment resulted in increased tumor-infiltrating leukocytes, M1-polarized intratumoral macrophages, and activation of natural killer cells in model of mouse colon carcinoma (CT26) and colon adenocarcinoma (MC-38). Interestingly, the same study concluded that pan-TAM inhibitor treatment does not result in tumor growth delay in immunocompromised mice, highlighting the importance of immune cell activity in the treatment response [START_REF] Yokoyama | Immuno-Oncological Efficacy of RXDX-106, a Novel Small Molecule Inhibitor of the TAM (TYRO3, AXL, MER) Family of Kinases Equal contribution # Corresponding Author Running Title: Immuno-oncological effect of RXDX-106, a TAM RTK inhibitor[END_REF]. Following these encouraging results in mouse models, it would be interesting to explore the possibility to combine for example pembrolizumab and anti-TAM inhibitor treatments in BCa and perform immunoprofiling of the infiltrated immune cells before and after the treatments.

Experimental design

I would first investigate the expression levels of TAM receptors in mouse bladder cancer cell line and their potential dependency on TYRO3 for their survival. In case of such a dependency was confirmed, I would implant this cell line in immune competent mice and establish tumors.

Further, I would treat the mice with TAM inhibitor/pembrolizumab or combination. Also addition of irradiation arm for these treatments culd be considered (discussed in the next paragraph). Then, I would collect the tumors, disassociate and perform immunophenotypical analysis of immune cells from the tumors of each treatment arm. I could do it by flow cytometry analysis, staining for markers of major immune cell markers and compare the percentage of different immune cells in each treatment. Alternatively, I could extract RNA from these tumors and analyse them using the NanoString Mouse PanCancer Immune profiling panel.

I could also benefit from the TYRO3 Knock-out mouse model (Jackson laboratory). It would allow me to directly evaluate of the effect of radiotherapy for syngenic tumor models expressing or not TYRO3.

TYRO3 inhibition with radio-immunotherapy

My work has not explored radiotherapy effects on the immune system but I would be very interested to explore this while also investigating the role of TAM receptors and their signalling in each of these modalities and in combination. It is recognised that radiotherapy can induce immunostimulatory effects. Irradiated cancer cells have been shown to release biologically active factors that act locally to promote the death of bystander cells (Narayanan et al., 1999a;[START_REF] Prise | Radiation-induced bystander signalling in cancer therapy[END_REF]. These factors include reactive oxygen and nitrogen species [START_REF] Lehnert | Extracellular factor(s) following exposure to alpha particles can cause sister chromatid exchanges in normal human cells[END_REF][START_REF] Shao | Nitric oxide-mediated bystander effect induced by heavy-ions in human salivary gland tumour cells[END_REF] as well as cytotoxic and immunomodulatory cytokines such as interleukin 6 (IL-6) [START_REF] Chou | Radiation-induced hepatitis B virus reactivation in liver mediated by the bystander effect from irradiated endothelial cells[END_REF], interleukin 8 (IL-8) (Narayanan et al., 1999b) and transforming growth factor b1 (TGFb1) [START_REF] Iyer | Factors underlying the cell growth-related bystander responses to alpha particles[END_REF][START_REF] Bouquet | TGFβ1 inhibition increases the radiosensitivity of breast cancer cells in vitro and promotes tumor control by radiation in vivo', Clinical Cancer Research[END_REF][START_REF] Vanpouille-Box | TGFβ is a master regulator of radiation therapy-induced antitumor immunity[END_REF][START_REF] Young | Tumor immune remodeling by TGFβ inhibition improves the efficacy of radiation therapy[END_REF]. Moreover, radiation therapy can stimulate anticancer immunity while inducing immunogenic cell death (ICD) which underlines the abscopal or out-of-field effect, i.e., the relatively rare but sometimes very pronounced clinical response to radiation therapy that can manifest in distant, non-irradiated lesions.

It would be very interesting to explore combinations of TAM inhibition and radioimmunotherapy. Moreover, it would be important to compare the benefit versus inhibiting one or all three TAM receptors with an aim to find the most effective treatment while minimising side effects. More generally, it would be important to survey the expression of TAM receptors on different immune cells and detect their activation mechanisms.

TYRO3 inhibition with other targeted therapies

Although other targeted treatments have been investigated in BCa and FGFR3 and EGFR inhbitors have shown some success in clinical trials, there are high proportion of patients which do not respond and develop resistanc to these drugs. There is a need to consider new treatment strategies and improve long-term outcomes in patients. RTK targeting therapies are known to result in treatment resistance; therefore, combination therapies are often used.

It would be important to conduct a longer time-course study of the anti-TYRO3 treatment to observe potential resistance mechanisms, which would allow using combination of agents. I would propose an in vitro approach where BCa cell lines would be treated winth increased doses of TYRO3-targeting inhibitor in order to establish TYRO3-resistant cell line. This model would allow studying the downstream signalling effects and attempt to re-establish the tumor cell dependency to TYRO3 targeting treatment.

TYRO3 autoantibodies in bladder tumours

The first autoantibodies were discovered decades ago [START_REF] Robbins | Complement Fixation with Cell Nuclei and DNA in Lupus Erythematosus[END_REF]. It is now becoming increasingly clear that autoantibodies play a pivotal role in the pathogenesis of many diseases. At the same time, autoantibodies became recognized as diagnostic features of many autoimmune diseases such as systemic lupus erythematosus and type 1 diabetes mullites [START_REF] Mackay | Travels and travails of autoimmunity: A historical journey from discovery to rediscovery[END_REF]. The molecular pathway underpinning the autoantibodies phenomenon is not clear. Whether it is the due to release of intracellular antigens due to and Protein Phosphatase 1 Catalytic Subunit Alpha (PPP1CA)-autoantibodies so far have been detected (Gumus et al., 2004;[START_REF] Minami | Analysis of Autoantibodies Related to Tumor Progression in Sera from Patients with High-grade Non-muscle-invasive Bladder Cancer[END_REF]. Interestingly, there was an association between the presence of serum anti-p53 antibodies and tumor p53 gene overexpression in the BCa tumors. Morover, serum anti-p53 antibody positive tumors had a worse prognosis than those with negative serum levels, regardless of the p53 status of the tumor (Gumus et al., 2004). It would be interesting to further explore autoantibody presence in the serum of BCa patients and also investigate whether anti-TYRO3 autobodies are present. This would of great clinical significance for the following reasons: autoantibodies can be utilised in the development of serology-based point-of-care assays which are minimally invasive, rapid and suitable for high throughput screening in order to monitor treatment, assess disease-stage and prognosis.

Experimental design

We could use mouse BCa model aberrantly expressing TYRO3. Blood samples could be collected regularly (two weeks intervals) and then used to blot TYRO3 recombinant protein.

The membranes will be probed with mouse anti-IgM, IgGs and IgA. Positive bands of expected size indicate the presence of autoantibodies. The next step will be to develop an ELISA assay to elucidate the autoantibodies kinetics which in turn will be used to determine assay performance. Of course, this assay has to be tested using human sera/plasma. Alternatively, exploratory study could be launched, where I could look for novel autoantibodies present in the serum of BCa patients.

In conclusion, TYRO3 receptors are attractive targets for bladder cancer therapy, especially in combination with radiation. This thesis established the important role of TYRO3 in the regulating the survival, cell cycle and DNA damage response in the BCa and encourage further studies of TAM receptor targeting therapies in BCa especially in combination with other treatment modalities in order to improve the outcome of BCa patients. TYRO3 is a receptor tyrosine kinase of the TAM family (comprising TYRO3, AXL and MERTK) and is known to regulate diverse biological. TYRO3 is overexpressed in many types of cancer and promotes tumor cell proliferation, survival and resistance to chemotherapy. In addition, higher levels of TYRO3 expression have been associated with decreased overall survival in patients of diverse cancers. However, the role of TYRO3 in BCa has so far not been studied. In this thesis, I investigated:

BIBLIOGRAPHY

(1)The role of TYRO3 in BCa; (2) The radiosensitising effect of TYRO3 downregulation and inhibition in BCa cells; (3) The effect of TYRO3 downregulation and inhibition on normal human urothelial tissue. We first demonstrated that TYRO3 is overexpressed in 50% of MIBCs. TYRO3 overexpression conferred a TYRO3-dependance to bladder tumor cells for cell growth and viability. Transcriptomic analysis of TYRO3downregulated cells suggested that TYRO3 signaling controlled cell cycle and protected from apoptosis, which indicated a potential to improve radiation response. TYRO3 downregulation lead to a significantly increased radiosensitivity of BCa cells and conversely, TYRO3overexpression induced radioresistance. In combination with radiotherapy, TYRO3 dowregulation lead to a cell cycle arrest and a long term persistence of Ionizing Radiation-Induced Foci (IRIF). Finally, I demonstrated that TYRO3 downregulation and inhibition did not impact viability of normal human bladder cells suggesting that inhibiting TYRO3 could improve radiotherapy efficiency while sparing normal surrounding tissues.

  ABSTRACT .............................................................................................................................19 RÉSUMÉ ...............................................................................................................................23 Review Registration in PROSPERO ................................................................................... 307 In vivo study plan ............................................................................................................ 315

Figure 1 - 1 .Figure 1 - 2 .Figure 1 - 3 .Figure 1 - 4 .Figure 1 - 5 .Figure 1 - 6 .Figure 1 -Figure 4 - 1 .Figure 4 - 2 .Figure 4 - 3 .Figure 4 - 4 .Figure 4 - 5 .Figure 4 - 6 .Figure 4 - 7 .Figure 5 - 1 .

 11121314151614142434445464751 Figure 1-1. Overview of a human urinary bladder. ................................................................41

1 .

 1 Properties of TAM ligands. ................................................................................. Table 1-2. TYRO3 expression and function in cancer. ......................................................... Table 1-3. Translational treatment targeting TYRO3. .........................................................

  BCa, describe epidemiology, risk factors and current treatments emphasizing the role of radiotherapy in the BCa treatment. Next, in a form of literature review, I discuss the current limitations of the radiation treatment and provide an overview of preclinical studies of BCa investigating radiation treatment and consider how they address clinical needs. Further, I introduce the TAM family of receptor tyrosine kinases and especially TYRO3 and their ligands and describe their physiological roles and roles in cancer. Then, I focus on the clinical perspectives by targeting TAM receptors in cancers. In the last introductory section, I provide an insight on the use of normal human urothelial cells (NHU) in preclinical studies.In the second chapter, I present a collaborative work establishing that TYRO3 is a promising therapeutic target due to its high level of expression in BCa tissue, where it is overexpressed in 50% of MIBC and highly expressed in most of the BCa cell lines. During the first years of my PhD, I significantly contributed with data to demonstrate that TYRO3 plays an important role in BCa, sustaining BCa cell survival and proliferation. I helped to identify that TYRO3 overexpression leads to TYRO3-dependency of BCa cell lines for survival and that TYRO3 downregulation results in BCa cell cycle disturbances and increases apoptosis in vitro and in tumor growth delay in vivo. In the third chapter, I present my independent work in the collaborating lab at the Insitut Curie site in Orsay where under supervision of Frederique Megning-Chanet, I have been able to demonstrate that TYRO3 downregulation results in increased radiosensitivity in BCa cell lines. I have been exploring the effects of the combination on cell cycle; DNA damage repair and DNA damage response signalling. I have gathered evidence which puts TYRO3 as a promising target to be combined with radiation in clinical setting and the combination is currently being explored in an in vivo model. In the fourth chapter, I describe my work resulting from a collaboration with Professor Jenifer Southgate at the University of York. I was able to visit the laboratory and perform series of experiments, learning to establish and culture normal human urothelial cells. I was very curious to investigate what role TYRO3 or other TAM receptors paly in normal tissue context. I investigated the expression levels of TYRO3, AXL and MERTK in normal urothelial tissues fromdifferent donors and explored their potential role in proliferating or differentiated cells. I conclude that knockdown of each TAM receptor results is light impact on NHU cell viability, which would suggest that no side effects on urothelium would result from anti-TAM therapy.

Figure 1 - 1 .

 11 Figure 1-1. Overview of a human urinary bladder. Two ureters connect the urinary bladder to the kidneys. Bladder wall is composed of four layers: a transitional epithelium, connective tissue, muscle and a fatty layer. Papillary or flat tumors can develop in the transitional epithelium. From Bladder Cancer Staging | Bladder Cancer Stages (https://www.cancer.org/).

Figure 1 - 2 .

 12 Figure 1-2. Overview of bladder tumors and their invasion of the bladder lining. NMIBC invades the urothelial and the lamina propria whereas MIBC invades the underlying smooth muscle and can spread into the fat layer and surrounding organs. BCa grading and staging is shown. a Staging of BCa according to the Tumour -Node -Metastasis (TNM) system. b Grading according to theWHO classifications of 1973 and 2004. Abbreviations: WHO, World Health Organization. From Knowles and Hurst, 2015.

A

  major separation based on gene expression results into luminal/basal and non-luminal, nonbasal groups. Luminal cancer type gene expression signature shares similarities with intermediate/superficial layer of normal urothelium, while basal cancer gene expression signature has similarities with the basal layer of normal urothelium. The non-luminal, non-basal tumors are heterogeneous comprising among others neuroendocrine tumors and tumors expressing EMT (epithelial-mesenchymal transition) markers.

Figure 1 - 3 .

 13 Figure 1-3. Summary of the main characteristics of the consensus classes. From top to bottom, the following characteristics are presented: proportion of consensus classes in 1750 tumor samples; consensus class names; schematic graphical representation of tumor cells and their microenvironments (immune cells, fibroblasts, and smooth muscle cells); differentiation-based color scale showing features associated with consensus classes, including a luminal-to-basal gradient and neuroendocrine differentiation; and a table displaying the dominant characteristics such as oncogenic mechanisms, mutations, stromal infiltrate, immune infiltrate, histology, clinical characteristics, and median overall survival. Abbreviations: Ba/Sq = basal/squamous; LumNS = luminal nonspecified; LumP = luminal papillary; LumU = luminal unstable; MIBC = muscle-invasive bladder cancer; NE = neuroendocrine; NK = natural killer(Kamoun et al., 2020).

Figure 1 .

 1 Figure 1. Comparison of MIBC subtype frequency in patient tumours and human BCa cell lines used in RT experimental studies in vitro and in vivo.

  (a) MIBC tumour subtypes (classified by (Kamoun et al., 2020)); (b) Cell line subtypes used in experimental studies of RT in BCa in vitro (classified by (Shi et al., 2020)); (c) Cell line subtypes used in preclinical studies of radiotherapy in BCa in vivo (mice xenografts) (classified by[START_REF] Shi | Identification of new driver and passenger mutations within APOBEC-induced hotspot mutations in bladder cancer[END_REF]).

  RT studies of BCa, Domniguez-Escrig et al. observed radiosensitising effect of the use of the EGFR inhibitor Gefitinib in vitro in two BCa cell lines. However, Gefitinib alone did not cause growth delay in the luminal RT112 xenograft in vivo, but was validated using the basal 253J B-V xenograft (Dominguez-Escrig et al., 2004). Colquhoun et al. demonstrated that radiation induced activation of EGFR and MAPK and Akt downstream effectors in two BCa cell lines.

demethoxygeldanamycin; 5 '

 5 -AdThd: 5'-amino-5'-deoxythymidine; AQ4N: 1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dio ne; AR: androgen receptor; HER2: Receptor tyrosine-protein kinase erbB-2, HMGB1: high mobility group box 1; HSA-MnO2-CQ: MnO2 and chloroquine in human serum albumin (HSA)-based nanoplatform; RNF8: ring finger protein 8; SQAP: sulfoquinovosylacylpropanediol; TKI: tyrosine kinase inhibitor; TUG1: taurine-upregulated gene 1.

  .Wu et al. demonstrated that RT upregulated PD-L1 expression in BCa tumour cells, correlating with radiation dose. Using heterotopic MB49 syngeneic mouse models, PD-L1 blockade induced a longer tumour growth delay following irradiation(Wu et al., 2016b). Bacillus Calmette-Guérin (BCG) bladder instillation is commonly used after local tumour resection for patients with superficial bladder cancer. Also BCG bacteria-induced immune response has been studied in BCa to improve response to RT[START_REF] Mc Cormick | Bacillus Calmette-Guerin improves local and systemic response to radiotherapy in invasive bladder cancer[END_REF]. Invasive murine BCa cell line MB49-I was cultured in monolayers in 2D, in spheroids in vitro in 3D and inoculated in vivo in the syngeneic mice. BCG pre-treatment radio-sensitized spheroids, while no effect was shown in monolayers. In vivo, BCG improved the local response to RT and decreased the presence of lung metastasis. The combined BCG+RT treatment also resulted in abscopal effect, where second tumour development in the opposite flank was completely rejected, compared to the untreated or RT only arms[START_REF] Mc Cormick | Bacillus Calmette-Guerin improves local and systemic response to radiotherapy in invasive bladder cancer[END_REF].

  ). Unfortunately, only one luminal cell line (RT112) and no basal cell line has been used in vivo to evaluate cisplatinor gemcitabine-based CRT, thus not allowing to study of the differential effect observed in clinics between tumour response to CT of basal and luminal subtypes (where basal tumours are shown to be better responders). It would be important to compare CCRT/RT response in models of different subtypes and study the underlying mechanisms. For that, patient-derived xenografts (PDX) are relevant models as they can conserve in mice the subtype of the original human tumour[START_REF] Inoue | Patient-derived xenografts as in vivo models for research in urological malignancies[END_REF]. Briefly, PDX establishment consists in engrafting a fragment of a patient tumour directly into an immunocompromised animal, and then maintaining it through passaging from animal to animal, avoiding the in vitro selection and allowing to conserve the initial histology. In the future studies, using a PDX model would eliminate the non-luminal non-basal subtypes which most of the BCa cell lines used in RT studies are representing, but which do not clearly represent human tumours. Despite the possible tumour loss of distinct molecular features over time, PDX models in BCa warrant future efforts to be used in RT studies.

  includes high resolution computerized tomography scanner for imaging, warmed beds to keep the model at a physiological temperature during longer irradiating sessions and possibility to target CTV more accurately. This allows studying the side effects of the RT on the bladder, giving the opportunity to compare acute radiation-induced toxicity versus long-term radiation-induced side effects such as fibrosis on the bladder. Although toxicity of normal tissue is one of the main limitations of use of RT in BCa, only two studies have considered pelvic toxicity. First study assessed pelvic irradiation-induced intestinal toxicity[56]. Second

(

  photodynamic therapy. After careful reviewing of each full text article, additional studies were

Figure 2 .

 2 Figure 2. Flow chart showing the identification, screening, evaluation of eligibility and inclusion criteria of publications.

  normal tissue or radiosensitisers to increase the sensitivity of tumor cells. Currently, tumour radiosensitization is most commonly achieved by combining radiotherapy with chemotherapeutics[START_REF] De Ruysscher | Radiotherapy toxicity[END_REF].

Figure 1 - 4 .

 14 Figure 1-4. The therapeutic index of radiation therapyTherepeutic index is the balance between probability of tumor control (in red) and probabiltiy of normal tissue complications (in green). Therapeutic index can be imrpoved by extending the therapeutic window by addition of radiosensitizers reduce the dose of radiation required for the same tumor control probability or by adding radio-productve drug. From[START_REF] De Ruysscher | Radiotherapy toxicity[END_REF]

Figure 1 - 5 .

 15 Figure 1-5. TAM receptors and ligands.TAM ligands (in blue) bind to apoptotic cells via Gla domain and to TAM receptors (in red) via SHBG domain. TAMs are widely expressed by cells of the mature immune, nervous, vascular, and reproductive systems. The carboxy-terminal SHBG domains of the ligands bind to the immunoglobulin (Ig) domains of the receptors, induce dimerization, and activate the TAM tyrosine kinases. When gcarboxylated in a vitamin-K-dependent reaction, the amino-terminal Gla domains of the dimeric ligands bind to the phospholipid phosphatidylserine expressed on the surface on an apposed apoptotic cell or enveloped virus. FromLemke, 2013.

  -6)[START_REF] Keating | Inhibition of Mer and Axl receptor tyrosine kinases in astrocytoma cells leads to increased apoptosis and improved chemosensitivity[END_REF][START_REF] Lijnen | Growth arrest-specific protein 6 receptor antagonism impairs adipocyte differentiation and adipose tissue development in mice[END_REF][START_REF] Ou | AXL regulates mesothelioma proliferation and invasiveness[END_REF].

Figure 1 - 6 .

 16 Figure 1-6. Overview of the TAM signaling cascade.Activated TAMs regulate cell survival, cell proliferation, inflammation and phagocytosis for TAMs. TAM receptor dimers can activate a conventional RTK signaling pathway that is driven by the phosphorylation and activation of Akt. The positions of major tyrosine autophosphorylation sites shared between TYRO3, AXL, and MERTK are indicated (P). MERTK activation has also been found to drive the downstream activation of PLC-γ, by a mechanism that is not delineated biochemically. FromLemke, 2013.

Figure 1 - 7 .

 17 Figure 1-7. TAM receptor signaling influences macrophage polarization. TAM receptor binding and downstream signaling dampens M1 polarization and promotes M2 polarization. Overall, this decreases anti-tumor M1-like phenotypes and functions and increases protumor M2-like phenotypes and functions. From Myers, Amend and Pienta, 2019.

Figure 1 -

 1 Figure 1-8. Comparison of TYRO3 RNA and protein expression in diverse human tissue. The RNA expression shows the consensus RNA-data based on normalized expression (NX). The protein expression shows the higher expression score found in each tissue from IHC. The gene expression level is estimated relative to the average normalised transcripts per million (TPM) value of all individual samples. The NX value represents the maximum level detected in each tissue. Color coding is based on tissue groups having common functional features. Adapted from Protein Atlas (https://www.proteinatlas.org/).

Figure 1 - 9 .

 19 Figure 1-9. Effect of TAM family members on the immune response to cancer cell death.

Figure 1 -

 1 Figure 1-10. Time course of urothelium response following radiation therapy treatment.

1 ) 2 ) 3 )

 123 (described in the second chapter of my thesis). AXL and MERTK inhibition has been shown to increase the benefit from radiotherapy in other cancers. However, no study has investigated the combination of radiation treatment and TYRO3 inhibition specifically. Radiation with chemotherapy agents are used as part of bladder preserving treatment, however no targeted agents are currently approved to use as radiosensitisers. Furthermore, no study has investigated the potential side effects of TYRO3 downregulation on normal human urothelium. Therefore, the objectives of my thesis are as follows: Describe the role of TYRO3 in BCa; Investigate the radiosensitising effect of TYRO3 downregulation or inhibition in BCa cells; Analyse the effect of TYRO3 downregulation or inhibition on normal human urothelial tissue. Prologue In this chapter, I describe my work with the postdoctoral fellow Florent Dufour identifying the role of TYRO3 in BCa. I joined the lab at the point when work on this manuscript had already begun. With an aim to investigate the TAM expression levels in BCa, transcriptomic analysis of publicly available data of BCa patient samples (The Cancer Genome Atlas (TCGA) were analysed revealing significantly higher expression of TYRO3, but not AXL or MERTK, in both NMIBCs and MIBCs, as compared to normal urothelium. The overexpression at protein level was then validated in an independent dataset of BCa tumors from French hospitals (CIT) and panel of BCa cell lines. At the time I joined the lab, I assisted with loss-of-function experiments in several BCa cell lines to establish the TYRO3-dependency of BCa-derived cells in vitro. This effect was also observed in a mouse xenograft model in vivo conducted by Isabelle Bernard-Pierrot. Further, Florent Dufour demonstrated efficiency of two pan-TAM inhibitors to decrease the cell viability of TYRO3-dependent cells. Finally, from transcriptomic analysis, we detected that growth inhibition upon TYRO3 depletion relies on cell cycle inhibition and induction of apoptosis associated, which we were able to validate by Western Blotting. Florent Dufour trained me thoroughly and following his example, I learned to complete experimental design and data analysis independently. Main experiments I contributed to this publication include downregulating TYRO3 in multiple bladder cancer cell lines, carrying out multiple repeats. I generated lysates and evaluated the extent of TYRO3 downregulation by Western Blotting and evaluated the effect on cell viability after these transfections at different time points. All the experiments I performed were clearly recorded, discussed with Florent Dufour to place them in context and prepared in form of reports. My contributions accounts for the data for the Figure 2b (5 cell lines) and Figure 4b (5 cell lines). Also, I contributed Figure S2, Figure S4, Figure S7, Table S2. The participation in the discussions throughout the article progression helped me to attain the independence needed for the work described in Chapter 3 where I carried out experiments independently in a collaborating lab at the Institut Curie site in Orsay. I would also like to acknowledge other people who contributed to this manuscript: Hélène Neyret-Kahn and Narjesse Karboul, Pascale Maillé, Yves Allory. This study was supervised by Hélène Haegel, Thierry Menguy, Vanessa Duong, François Radvanyi and Isabelle Bernard-Pierrot.

Figure 1 .

 1 Figure 1. TYRO3 is highly expressed in bladder cancer and is N-glycosylated. a TYRO3, AXL, MERTK and GAS6 mRNA levels, as measured by RT-qPCR, in the CIT cohort of nonmuscle-invasive bladder cancers (NMIBCs, n = 87) and muscle-invasive bladder cancers (MIBCs, n = 82) and in normal urothelium (NU, n = 7). The significance of differences was assessed by Mann-Whitney test, and the means and standard errors are shown. b Lysates from a subset of human tumours from the CIT cohort and from normal urothelium (NU) and normal vesical muscle (NM) were analysed by western blotting with antibodies targeting the indicated proteins. NMIBCs and MIBCs were identified by one and two asterisks, respectively. c TYRO3 immunoblots for tumour lysates (CIT.56 and CIT.190) treated with peptide-N-glycosidase F (PNGaseF). d Representative images of TYRO3 immunohistochemical staining for a panel of human bladder tumours (NMIBC and MIBC) and urothelium from histologically normal tissue adjacent to the tumour (NAT). Black bars represent 100 μm. IHC validation experiments are available in supplementary figure S3.

Figure 2 .a

 2 Figure 2. TAM knockdown and pharmaceutical inhibition studies in bladder cancer cell lines reveal the major contribution of TYRO3 to cell viability. a Lysates from a panel of human urothelial bladder cancer cell lines were analysed by western blotting for TAM expression. Alpha tubulin served as a loading control. b Knockdown of TYRO3, AXL and MERTK with three different siRNAs for each receptor, in a panel of bladder cancer cell lines. Cell survival was quantified by CellTiter-Glo at 72 h and 96 h. The TAM receptor(s) that is (are) expressed in each cell line is (are) indicated beneath their name. c Dose-response curves for bladder cancer cells treated with BMS-777607 and UNC-2025, two pan-TAM inhibitors. Cell viability was quantified by CellTiter-Glo at 72 h. The data shown are the means of three independent experiments, and error bars represent the SD. *p < 0.05. Statistical analyses based on unpaired t-tests with Welch's correction are shown in supplementary tables S2 and S3.

Figure 3 .

 3 Figure 3. TYRO3 depletion decreases colony-forming potential and tumourigenesis. a Impact of TYRO3 knockdown on the anchorage-independent growth of MGH-U3 and RT112 cells. Colonies in soft agar with a diameter larger than 50 μm were quantified 14 days after transfection with a control siRNA (siCTL) or with TYRO3 siRNA#1. Three experiments were performed, and the results shown are from a representative experiment conducted in triplicate. The data shown are means ± SD. Unpaired t-tests were used for statistical comparison. b MGH-U3 bladder cancer cells were injected subcutaneously into nude mice (n = 6 animals/group, two xenografts per animal (one in each flank)). When tumour volume reached 50( ± 5) mm3, tumour-bearing mice were treated three times weekly with intraperitoneal injections of 4 µg control scramble siRNA or TYRO3 siRNA#1 (the first injection corresponds to day 0). Tumour sizes were measured at the indicated time points and tumour volumes were calculated. Data are presented as means ± SEM. Tumour volumes were compared by Wilcoxon's test. c MGH-U3-derived xenograft tumours from mice treated with scramble siRNA or TYRO3 siRNA#1 were lysed and TYRO3 expression was measured by RT-qPCR. TBP was used as housekeeping gene. The data shown are means ± SD of two independent experiments performed in triplicate. Statistical analysis was performed with unpaired t-tests.

Figure 4 .

 4 Figure 4. TYRO3 silencing inhibits the cell cycle and cell proliferation and induces apoptosis. a Transcriptomic analysis upon TYRO3 knockdown in MGH-U3, RT112 and UM-UC-5 cells. Ingenuity Pathway Analysis (IPA) was performed for a list of 284 genes differentially expressed upon TYRO3 silencing (fold-change |log2| ≥ 0.58 and a p value < 0.05). Upper panel: Histograms showing the cellular functions predicted by IPA to be deregulated after TYRO3 knockdown. p values (hatched bar), and negative (white bar) and positive (black bar) absolute z-scores are indicated. Lower panel: Diagram showing the upstream regulators (transcription factors) significantly predicted by IPA to be involved in the regulation of gene expression observed after TYRO3 knockdown (p value < 1E-6). b, c Western blot analysis of TYRO3, AXL, MERTK, FOXM1, cMYC, CYCLIN D1, phospho-pRB (P-pRB S780) and total pRB, AURORA A and B, SURVIVIN levels in total cell lysates from MGH-U3, RT112 and UM-UC-5 cells transfected with a control siRNA (siCTL) or with TYRO3 siRNAs (siTYRO3#1, siTYRO3#2 and siTYRO3#3) for 72 h. c The activation of apoptosis was assessed at the indicated period of time by monitoring cleaved PARP, caspase-8 (CASP-8) levels, with detection of the profrom (CASP-8) and cleaved form (p43/41), and caspase-3 (CASP-3). Western blot results are representative of at least two independent experiments. d MGH-U3 and RT112 cells were transfected with either a control siRNA (siCTL) or with TYRO3 siRNA#1 for 24 h. They were then treated with DMSO, 50 μM Z-IETD-FMK (caspase-8 inhibitor) and 50 μM Z-DEVD-FMK (caspase-3/7 inhibitor) for 48 h. Cell viability was assessed by measuring MTT incorporation at 72 h. The results are expressed as the means ± SD of three experiments. An unpaired t-test was used for statistical analysis. e Sections of MGH-U3-derived xenograft tumours from mice treated with scramble siRNA or TYRO3 siRNA#1 were stained for DNA fragmentation with a TUNEL assay detection kit (n = 4 mice per group). Representative sections are shown (left panel, black bar = 100 μm) and the percentage of cells displaying apoptosis (right panel) was evaluated. The data shown are means ± SD (n = 4). Statistical analysis was performed with t-tests.

  (antisense strand) AXL (Roche Universal Probe Library probe ID: 76): 5′-AACCAGGACGACTCCATCC-3′ (sense strand) 5′-AGCTCTGACCTCGTGCAGAT-3′ (antisense strand) MERTK (Roche Universal Probe Library probe ID: 6): 5′-ATTGGAGACAGGACCAAAGC-3′ (sense strand) 5′-GGGCAATATCCACCATGAAC-3′ (antisense strand) GAS6 (Roche Universal Probe Library probe ID: 17): 5′-ATGGCATGTGGCAGACAAT-3′ (sense strand) 5′-CCCTGTTGACCTTGATGACC-3′ (antisense strand)ImmunohistochemistryFormalin-fixed, paraffin-embedded 3 µm tissue sections of tumours from the CIT-series were placed on poly-L-lysine coated slides. The paraffin was removed by immersion in xylene and the section was rehydrated by immersion in a graded series of alcohol concentrations.Antigens were retrieved by heating sections at 95 °C in 10 mM citrate buffer pH 9 (Microm Microtech France, Brignais, France) for 20 min. Endogenous peroxidase activity was inhibited by incubation in 3% H2O2. The sections were then incubated in Quanto Protein Block solution (Microm Microtech France) for 1 h to minimise nonspecific staining. The sections were then incubated with a rabbit polyclonal anti-TYRO3 antibody (Ref: HPA071245, Sigma-Aldrich, Saint-Quentin Fallavier, France) diluted 1:50 in antibody diluent solution (Diamond antibody diluent, Cell Marque, Rocklin, USA) for 1 h at 37 °C. Antibody binding was detected with N-Histofin® Simple staining and a DAB detection kit (Microm Microtech, France), according to the manufacturer's instructions. Antibody specificity for TYRO3 was assessed with formalinfixed paraffin-embedded 5637 bladder cancer cells, which express TYRO3, AXL and MERTK, after transfection with either control siRNA or TYRO3 siRNA#2 (Supplementary FigureS3). Flow cytometry Bladder cancer cells (1 × 10 6 cells/condition) were collected with Accutase (Sigma-Aldrich), washed in cold phosphate-buffered saline (PBS), and incubated with 1 µg of anti-TYRO3 clone 5B4 (Ref: GTX83459, GeneTex, Irvine, USA) or isotype control (Ref: MAB003 R&D Systems, Lille, France) antibody in PBS supplemented with 0.5% bovine serum albumin (BSA) for 45 min on ice. The cells were then washed three times in cold 0.5% BSA PBS and incubated for 30 min on ice with 2 μg/ml phycoerythrin-conjugated goat-antimouse Ig (Ref: 550589, BD Biosciences, Le Pont de Claix, France) in 0.5% BSA PBS. Cells were washed three times in 0.5% BSA PBS and the fluorescence signal was acquired with a BD LSR II flow cytometer (BD Biosciences).

  5585), AXL (Ref. 8661), MERTK (Ref. 4319), caspase-8 (Ref. 9746), cleaved caspase-3 (Ref. 9664), cleaved PARP (Ref. 5625), FOXM1 (Ref. 5436), pRB (Ref. 9309), phospho-pRB Ser780 (Ref. 9307), CYCLIN D1 (Ref. 2926), AURORA A (Ref. 14475), AURORA B (Ref. 3094), SURVIVIN (Ref. 2808), c-MYC (Ref. 9402), anti-mouse IgG, HRP-linked (Ref. 7076) and anti-rabbit IgG, HRP-linked (Ref. 7074) antibodies were purchased from Cell Signaling Technology (Ozyme, Montigny-leBretonneux, France). Anti-α-TUBULIN (Ref. T6199) and anti-βACTIN (Ref. A2228) antibodies were obtained from Sigma-Aldrich. For peptide-N-glycosidase F (PNGaseF), we digested 10 µg of protein with PNGaseF, according to the manufacturer's instructions (New England Biolabs, Evry, France).

France) were raised

  in the animal facilities of Institut Curie, in specific pathogen-free conditions. They were housed and cared for in accordance with the institutional guidelines of the French National Ethics Committee (Ministère de l'Agriculture et de la Forêt, Direction de la Santé et de la Protection Animale, Paris, France), under the supervision of authorised investigators. Mice received a subcutaneous injection, into each flank (dorsal region), of 5 × 106 MGH-U3 bladder cancer cells in 100 µl PBS. For each study, with each of the cell lines, mice were randomly separated into groups of six mice when tumours reached a volume of 50 mm3 (±5). The mice bearing MGH-U3 xenografts were treated three times weekly, for three weeks, with an intraperitoneal injection of 4 µg siRNA (TYRO3 siRNA#1 or corresponding scramble siRNA) or of vehicle (PBS). Tumour size was measured twice weekly with a calliper, and tumour volume in mm 3 was calculated as follows: π/6 × (largest diameter) × (shortest diameter)2 . The tumours were removed at the end of treatment. Part of the tumour was flashfrozen in liquid nitrogen for mRNA extraction. The rest was fixed in 4% formol and embedded in paraffin. TUNEL assay DNA fragmentation was evaluated with a TUNEL (deoxynucleotidyl transferase (Tdt)-mediated nick-end labelling) assay detection kit (Sigma-Aldrich), according to the manufacturer's instructions. Statistical analysis TAM expression in tumours was analysed with Mann-Whitney test. Linear Models for Microarray Data (LIMMA)(Ritchie et al., 2015) was used to analyse DNA array experiments involving simultaneous comparisons between large numbers of RNA targets. All functional experiments were carried out twice or three times, in triplicate. To compare data between cell lines, two-tailed t-tests were used. The control siRNA (Luciferase GL2 siRNA) or DMSO group was used as the reference group. Data are expressed as means ± SD, and differences with a p value < 0.05 were considered statistically significant. For the comparison of data between xenograft treatments, the nonparametric Mann-Whitney test was used, with the vehicle group as the reference group. All statistical analyses were performed with Prism7.0b (GraphPad Software, Inc).

Figure S2 .

 S2 Figure S2. Correlation of the TAM protein level expression to TAM mRNA level. A subset of tumors from CIT cohort where analysed for both TAM receptor expression at a protein level and transcript expression level for a TYRO3, b AXL, c MERTK. Protein levels from the dosimetry of the western blot (Fig. 1b) were normalised to the tubulin (loading control). mRNA levels were measured using RT-qPCR and normalised to that of TBP. The correlation factor R and P value denoting significance are displayed. Abbreviations: RT-qPCR: reverse transcription quantitative-polymerase chain reaction, TAM: TYRO3, AXL and MERTK receptors, TBP: TATA box binding protein.

Figure S3 .

 S3 Figure S3. Validation of TYRO3 antibody specifity. a TYRO3 was downregulated or not in 5637 cells expressing the three TAM receptors and effective downregulation specifically of TYRO3 was verified by Western Blot. b Immunohistochemistry staining of TYRO3 expression in 5637 cells with specific antibody (see Materials and Methods) embedded in paraffin showing effective reduction of TYRO3 expression in BCa cells.

Figure S4 .

 S4 Figure S4. Western blot analysis of TAM downregulation in 5 bladder cancer cell lines with differential TYRO3 expression. Potential redundancy and relative importance of the three TAM receptors were investigated by selecting one cell line expressing only TYRO3 (a MGH-U3), three cell lines expressing TYRO3 together with AXL or MERTK (b RT112, c UM-UC-5 and d UM-UC-9) and one cell line expressing all three TAM receptors (e VM-CUB-1). Western blot analysis showed that each targeted TAM was effectively silenced 72 h after transfection and that the knockdown of one TAM receptor had mostly no effect on the other TAM receptors. However, TYRO3 knockdown using siRNA#1 reduced AXL expression in (b RT112, c UM-UC-5 and e VM-CUB-1) and also increased MERTK expression in (b RT112).

Figure S5 .

 S5 Figure S5. Evaluation of TYRO3 expression at the cell surface. a Specifity of TYRO3 staining in Flow Cytometry was evaluated by comparing siCTL and siTYRO3#1 transfected UM-UC-5 cells showing effective downregulation of the protein expression from the cell surface. b TYRO3 expression was detected at the cell surface of untreated TYRO3-dependent cell lines MGH-U3, UM-UC-5 and RT112.

Figure S6 .

 S6 Figure S6. Evaluation of TAM inhibitor ability to inhibit phosphorylation of TAM receptors. Immunoprecipitation experiments were performed to evaluate the phosphorylation of TYRO3, AXL and MERTK with or without the treatment of VM-CUB-1 cells with 5 µM of BMS-777607 or UNC-2025. BMS-777607 inhibitor effectively inhibited phosphorylation of all three TAM receptors and UNC-2025 inhibited phosphorylation accordingly to its affinity profile for the TAM receptors (showing the following order of inhibition: MERTK>AXL>TYRO3).

Figure S7 .

 S7 Figure S7. Effects of TYRO3 downregulation of expression on panel of proteins involved in proliferation or tumor suppression in two TYRO3-dependant cell lines. TYRO3 was downregulated and downstream effects were evaluated by Western blotting in two TYRO3-dependent cell lines, VM-CUB-1 and UM-UC-9.

Figure S8 .

 S8 Figure S8. Effects of TYRO3 downregulation on cell viability of cell lines bearing RB1 mutation. TYRO3 was downregulated and verified by Western blotting for a 5637, b 647V and c HT1376 cell lines (upper panels). Cell viability was recorded 72h after transfection (lower panels).

  independent growth in vitro before considering in vivo experiments. The CellTiter Glo cell viability assay used in the other in vitro experiments assessed only the percentage of live cells based on the cellular ATP quantity and only indirectly allowed us to measure the cell proliferation. The soft agar method allowed us to validate that siTYRO3 treatment results in impeded cell growth in 3D. Conventional plated colony forming assay could have been considered in the place of the soft agar assay and it was performed in the future experiments in the Chapter 3.

  TYRO3 downregulation and inhibition lead to a significantly increased radiosensitivity of BCa cells and conversely, TYRO3-overexpression induced radioresistance. TYRO3 downregulation lead to a long-term persistence of Ionising Radiation-Induced Foci (IRIF) and a more pronounced cell cycle arrest in G2/M phase. These long-term IRIF were correlated with an accumulation of unrepaired DNA damage as assessed by the alkaline Comet assay. TYRO3 downregulation does not influence DNA damage response pathways but disrupts mitosis regulation through differential Cyclin B1 expression levels. Finally, we demonstrated that specific TYRO3 downregulation did not significantly affect viability of normal human bladder cells suggesting that inhibiting TYRO3 could improve radiotherapy efficiency while sparing normal surrounding tissues. significant decrease when TYRO3 was totally downregulated with 2 different siRNA (siTYRO3 #4 and #801) in both cell lines and demonstrated a dose dependent decrease when TYRO3

Fig. 1 TYRO3

 1 Fig. 1 TYRO3 modulation influences radiosensitivity of bladder cancer cell lines. A: Representative clonogenic survival curves of RT112 and 5637 cell lines (upper panel). 48h after transfection with siLUC (control, red), siTYRO3#4 (blue), siTYRO3#801 (green) or diluted concentrations of siRNA#1 (black) cells were exposed to graded doses of y-rays. In each case, downregulation was confirmed by Western-Blot at 48h after transfection. The corresponding D10 values were calculated after fitting the experimental data to the classical linear-quadratic equation (lower panel). B: Representative survival curves of UMUC3 control (empty plasmid, red) and TYRO3 over-expressed cell lines (TYRO3 encoding-plasmid, purple) (upper panel). The up-regulation was confirmed by Western-Blots. The corresponding D10 values were calculated after fitting the experimental data to the classical linear-quadratic equation (lower panel). C: Immunoprecipitation shows increased TYRO3 phosphorylation following irradiation. Data represents mean ± SD of 3 independent experiments. Unpaired t-test analysis: * p<0.05; ** p<0.005; *** p<0.0005; **** p<0.0005.

Fig. 2 .

 2 Fig. 2. TYRO3 downregulation leads to a long-term persistence of Ionizing Radiation-Induced Foci (IRIF).H2AX foci were visualised after 24h of 2Gy irradiation in TYRO3 downregulated RT112 and 5637 cell lines (A). H2AX foci were quantified and cells containing more than 10 foci were plotted at 24h

  medians through the Kruskal-Wallis test with multiple comparisons. Using either siTYRO3#4 or siTYRO3#801, non-irradiated transfected cells showed statically significant differences in the distribution of the olive tail moment both in RT112 and 5637 cell lines (Fig.3Aand 3B, left panels) showing increased DNA damage 48h after transfection. When data were normalized to each non-irradiated control, statistical excess DNA damage were highlighted 24h following irradiated siTYRO3 #801-transfected cells in RT112 and 5637 cells (Fig.3Aand 3B, right panels).

Fig. 3 .

 3 Fig. 3. TYRO3 modulation leads to higher DNA damage 24h after radiation treatment The comet assay was performed on TYRO3 downregulated RT112 (A) or 5637 cells (B) irradiated at 6Gy and resulting olive tail moments were compared before IR (NT) immediately after IR (0 min), 15 -60 min after or 24h after. Raw data is shown on left panels, while normalised data to that of NT is shown on right panel. The data shown here is from three experiments (RT112) and two experiments (5637), analysing 200 nucleus per condition per experiment and horizontal bars represent the median values. Kruskal-Wallis nonparametric tests with Multiple comparisons were used: * p<0.05; ** p<0.005; *** p<0.0005; **** p<0.0005.

Fig. 4 .

 4 Fig. 4. TYRO3 downregulation affects cell cycle following irradiation. A: TYRO3 was downregulated, irradiated and the cell cycle distribution was analysed 24h after. B: Percentage (%) of cells in G2/M phase were compared. The data shown for (A and B) is from three different experiments and error bars represent the SD. Unpaired t-test analysis: * p<0.05; ** p<0.005; *** p<0.0005.

Fig. 5 .

 5 Fig. 5. TYRO3 downregulation does not affect the expression levels or activation of DNA repair signalling proteins but disrupts mitosis regulation. TYRO3 was downregulated, irradiated at 6Gy and the lysates were prepared and analysed 0.5h-24h after. A: Western blot of DNA damage repair proteins (n=2). B: Western blot of cell cycle proteins (n=1).

Fig. 6 .

 6 Fig. 6. TYRO3 modulation does not affect proliferation of normal human urothelial cells. A: TYRO3 was downregulated in NHU from two different donors and irradiated after 48h. Resulting survival curves are shown from proliferation assays. B: D10 was calculated and compared between TYRO3-downregulated or control-transfected cells. C: Dose response curves of 2 BCa cell lines (in black) and NHU cells (in green) from two different donors. D: Comparison of calculated Median Effective Dose (ED50) among undifferentiated NHU (green) and BCa cell lines (black).

  from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC. UM-UC-3, UM-UC-5, UM-UC-9, VM-CUB-1 cells were cultured in DMEM (Life Technologies), whereas RT112 and 5637 cells were cultured in RPMI (Life Technologies). Media were supplemented with 10% foetal bovine serum (FBS). UM-UC-3 TYRO3-overexpressing cell line (UMUC3 O/E) was established by transfecting UM-UC-3 cells with plasmid containing TYRO3 coding sequence containing geneticin resistance gene and cultured in medium containing 1mg/ml geneticin (Gibco, Life Technologies). UM-UC-3 control cell line (UMUC3 CTRL) was established by transfecting UMM-UC-3 cells with an empty plasmid and was cultured in the same conditions. Cells were kept at 37°C, under an atmosphere containing 5% CO2. The identity of the cell lines used was checked by analysing genomic alterations on comparative genomic hybridization arrays (CGH arrays) and sequencing genes known to be mutated: RAS, TP53 FGFR3 and PIK3CA. The cells were routinely checked for mycoplasma contamination.

  Cell Survival or Cell proliferation Assay Following siRNA Transfection and Irradiation 48h after transfection, cells were irradiated with γ rays from a 137Cs source (GSR-D1) at a dose rate of 1 Gy/min. After culturing for 7-10 days, the colonies were washed with PBS, fixed with 100% EtOH and stained with a Coomassie R-250 solution (0.5%). Colonies formed by at least 50 cells were counted by microscopic inspection and the percentage of surviving cells (SF) was calculated by comparing the number of colonies formed in the irradiated vs nonirradiated control cultures.

Figure S 1 .

 1 Figure S 1. D10 values of 6 BCa cell lines (A) and TAM receptor expression at protein level in BCa cell lines (B).

Figure S 2 .

 2 Figure S 2. TAM expression levels following siTYRO3#4 and #801 downregulation.

Figure S 3 .

 3 Figure S 3. Surviving fraction comparison at 4 or 6Gy. Surviving fraction at 4 or 6Gy was normalised to non-irradiated siLUC (or non-irradaited for TYRO3 O/E) conditions and compared to irradiated TYRO3 knockdown or overexpression conditions. Data represents mean ± SD of 3 independent experiments. Unpaired t-test analysis: * p<0.05; ** p<0.005; *** p<0.0005; **** p<0.0005.

Figure S 4 .

 4 Figure S 4. UNC2025 effect on the radiosensitivity of BCa cell lines.RT112 and 5637 were treated with the displayed inhibitor doses, irradiated 24 after and their radiosensitivity was assessed by colony forming assays, from which D10 value was calculated (A and B). TYRO3 phosphorylation levels at the time of irradiation (after 24h exposure of UNC2025) was detected by pTYRO3 ELISA (C).

Figure S 5 .

 5 Figure S 5. H2AX foci 30 min after 2Gy or mock irradiation in RT112 and 5637 cell lines (representative experiment).

Figure 4 - 1 .

 41 Figure 4-1. Comparison of average mRNA levels of TAM receptors. mRNA levels of TAM receptors are compared. (NHU proliferating (A) corresponds to the proliferating NHU cells in the data set of ABS/Ca2+ (n=3); NHU proliferating (B) corresponds to the proliferating NHU cells from TZ/PD data set (n=5); no distinction between different time points), bladder cancer cell lines (n=30). Unequal variances t-test between cancer cell lines and proliferating NHU (A) determined TYRO3 to be significantly upregulated (p <0.0001), AXL, p =0.02.

Figure 4 - 2 .

 42 Figure 4-2. TAM receptor expression in proliferating NHU cells or differentiated using ABS/Ca2+ protocol. TYRO3, AXL and MERTK mRNA levels were measured in NHU samples from 3 different donors (named A, B and C), having proliferating and differentiated samples for each. TAM receptor expression is compared using transcripts per million (TPM) values.

Figure 4 - 3 .

 43 Figure 4-3. TAM receptor expression in proliferating NHU cells or differentiated using TZ/PD protocol.TYRO3, AXL and MERTK mRNA levels were measured in NHU samples from 3 different donors (named D, E and F), having samples from two proliferating (24h and 144h) and two differentiated cells (24h and 144h) for each. TAM receptor expression is compared using transcripts per million (TPM) values.

Figure 4 - 4 .

 44 Figure 4-4. TAM expression in NHU. Cells from 3 different donors were compared, 5637 BCa cell line (line 12) was used as a positive control.NHU cells were either at undifferentiated state (Undiff=proliferative) or differentiated using ABS/Ca2+ (n=3) or TZ/PD protocol (n=2, due to lack of material). Cells were harvested at the confluency not more than 70%, for the Undiff cells, while differentiated cells were at 95-100% confmuency. Quiescent cells (lane 10 were grown to confluency, but not differentiated).

4. 2 .

 2 TAM knock-down and inhibition has little impact on NHU viability siRNA transfection against TAM receptors results in effective knock-down in undifferentiated NHU after 48h as assessed by Western Blots (Figure4-5). As noted previously, siTYRO3 #1 did effectively downregulate all TAM receptors (right panel, Figure4-5) and resulted in the highest decrease of NHU survival. Interestingly, one siRNA designed for MERTK downregulation resulted also in TYRO3 downregulation in both NHU cells (siMERTK #74, line 7 (left panel) and line 8 (right panel) Figure4-5). However, siMERTK #74 did not impact the cell viability (Figure4-6). Overall, small impact on undifferentiated NHU cell viability (72h) was detected with the other siRNAs (Figure4-6) depending on the siRNA used. The NHU cell viability was not reduced for more than 20% for all siRNAs, except with siTYRO3 #1 (a decrease of AXL protein level is frequently observed in BCa cell lines with this siRNA). The more significant effect of siTYRO3#1 compared to other siTYRO3 on the viability of NHU cells 72h after siTYRO3 treatment was notable also in the cancer cell line (Figure4-7). Two of three siMERTK targeting siRNAs did not significantly reduce NHU cell viability, while siMERTK #72 reduced the cell viability by 30% (as detected by WB, siMERTK treatment slightly decreases TYRO3 protein levels). The impact on cell viability of TYRO3 downregulation was remarkably smaller than in BCa cell lines (Chapter 2 and 3).From the TAM ligands GAS6, ProS1 and TULP1, only GAS6 expression was high at the NHU, especially high at proliferating NHU and decreased with the differentiation using both protocols (Figure4-8, Figure4-9). PROS1 and TULP1 at the transcript level were low in the NHU and the expression of FLT3 (target for UNC2025 inhibitor) was less than 0.3 in all the samples (Figure4-8, Figure4-9).

Figure 4 - 5 .

 45 Figure 4-5. TAM downregulation in NHU by siRNAs targeting TYRO3. Cell lysates from cells of 2 donors are showed (NHU1 and NHU2). TAM receptors were downregulated using at least 2 different siRNAs for each TAM receptor for 48h. Line 9 (on right panel show effect of siTYRO3#1 on the TAM receptor expression: Not only TYRO3 but also AXL is completely downregulated). 5637 BCa cell line was used as positive CTRL for MERTK detection (+).

Figure 4 - 6 .

 46 Figure 4-6. NHU Cell viability at 72h following siRNA downregulation.

Figure 4 - 7 .

 47 Figure 4-7. Effect of siTYRO3 downregulation on VMCUB1 BCa cell line vs. NHU. Proliferating NHU cell phenotype were observed under light microscope transfection 72h after on VMCUB1 BCa cells (top panel) and NHU proliferating cells (bottom panel). Three different siRNA aiming to downregulate TYRO3 were used.

Figure 4 -

 4 Figure 4-8. TAM ligand GAS6, TULP1, PROS1 and UNC2025 target FLT3 expression in proliferating NHU cells or differentiated using ABS/Ca2+ protocol.TAM ligand GAS6, TULP1, PROS1 and UNC2025 target FLT3 mRNA levels were measured mRNA levels were measured in NHU samples from 3 different donors (named A, B and C), having proliferating and differentiated samples for each. TAM receptor expression is compared using transcripts per million (TPM) values.

Figure 4 -

 4 Figure 4-9. TAM ligand GAS6, TULP1, PROS1 and UNC2025 target FLT3 expression in proliferating NHU cells or differentiated using TZ/PD protocol.TAM ligand GAS6, TULP1, PROS1 and UNC2025 target FLT3 mRNA levels were measured in NHU samples from 3 different donors (named D, E and F), having samples from two proliferating (24h and 144h) and two differentiated cells (24h and 144h) for each. TAM receptor expression is compared using transcripts per million (TPM) values

  to investigate the effect of combined TYRO3-downregulation and radiation treatment on the NHU cells. Although the NHU model would not allow me to investigate the impact of radiation on the stroma, I could have an indication on the response of the combinatory treatment to the normal urothelial tissue. This can be done by downregulating TYRO3 in proliferating (undifferentiated) cells, irradiating at different doses of radiation and after 5 doubling times, comparing the resulting survival curves. Colony formation assay could not be performed, as the cells do not grow in colonies. However, other comparative tests could be done to verify whether TYRO3 knock down is impacting the cell processes such as DNA repair or cell cycle by analysing cell cycle distribution in response to different treatments. Differentiated cells could also be also irradiated in a monolayer and later collected to check in the total cell lysate whether TYRO3 downregulation has induced significant modifications in cell survival/DNA repair or cell cycle proteins. I could also test the hypothesis in vivo syngeneic mouse model by irradiating the normal baldder after the inhibition of TYRO3 and and compare the long-term effects such as fibrosis of the bladder.

  treatment and highlighted the unmet needs for this treatment, such as lack of biomarkers of response and lack of radiosensitising agents. I then investigated how preclinical studies could address these needs. I compared a number of targeted agents investigated as potential radiosensiters in BCa and concluded that despite encouraging results of some inhibitors, most of them have not been transferred to clinics. In order to accelerate targeted treatment transfer from bench to bedside in BCa, I proposed a step-by-step guide for future preclinical study design. In the future, it would be important to update the review with more studies comparing intrinsic radiosensitvites between cell lines and available in vivo models, thus allowing to correlate radisoensitivity and different receptor/target expression levels. It also remains to characterise the BCa subtype-specific responses to chemoradiation/radiation treatments.In the second chapter, I describe the identification of TYRO3 as a promising therapeutic target in BCa. TYRO3 is overexpressed in 50% of MIBC and in most of the BCa cell lines. I contributed to the work relieving that TYRO3 plays an important role in BCa, sustaining BCa cell survival and proliferation. TYRO3 overexpression in BCa cel lines resulted in TYRO3-dependency for survival. TYRO3 downregulation resulted in BCa cell cycle disturbances and increased apoptosis in vitro and resulted in tumor growth delay in vivo. Mechanisms of TYRO3

  order to confirm the in vitro results obtained in the Chapter 3, I am investigating the effects of the combination of TYRO3 downregulation and radiation treatment in vivo. I have decided to establish the proof of concept of the effectiveness of this combination first in nude mice BCa cell line xenografts (See Appendix, in vivo study plan). I selected a luminal cell line, RT-112, which has high tumorigenicity and readily grow in mice xenografts and shown improved effect in the combination in the in vitro studies described in the chapter 3. I have started the in vivo study (August 2020). First, I will be selecting the radiation dose to be used, evaluating the efficiency of TYRO3 downregulation (by qPCR on RNA isolated from fresh-frozen tumor sections) and evaluating efficiency of TAM-inhibitor, UNC2025 efficacy (by phospo-TYRO3

  siTYRO3 treatment, I plan to compare the changes at the transcriptome following the single or combined treatments explored in the Chapter 3. I have been awarded a grant from NanoString technologies where I proposed to use the Counter® Tumor Signaling 360 Panel and investigate the following 6 conditions in 2 BCa cell lines (luminal and basal): 1) IR only; 2) siTYRO3#4 treatment only; 3) siTYRO3#801 treatment only; 4) Combined IR+siTYRO3 treatment; 5) siCTRL treatment only; 6) Combined siCTRL+IR treatment. This study will allow me to detect the signalling pathways impacted by each single treatment and combination. This data will be included in the prepared manuscript. I have already gained an insight of the players involved in cell cycle dysregulation and DNA damage response signalling and aim to explore it further.

Figure 5 - 1 .

 51 Figure 5-1. Overview of different types of siRNA cancer therapeutics.

  excessive cell death or to ineffective clearance of apoptotic debris, modification of selfantigens during inflammatory responses, and aberrant overexpression and localisation of disease-markers contribute to the initiation of autoantibody production, remains to be elucidated. As I described previously, TYRO3 is involved in immune control, inflammation and homeostasis in several organs, including the nervous, reproductive and vascular systems. It is evident that TYRO3 expression can favour the escape of tumour cells from immune surveillance. Moreover, TRYO3 is aberrantly overexpressed in bladder tumours. With the important involvement of TAM receptors in the innate immunity regulation, it raises question whether we have complete knowledge on what happens upon TAM receptor upregulation and whether TYRO3 is detectable in the serum of BCa patients. It could be possible, that it could result in TYRO3 autoantibody generation. So far, only few studies has investigated autoantibodies in the serum of BCa patients and p53-

Table 4 -

 4 1. Total number (Nr) of different NHU cells used for RNA-seq and WB………………....249

Table 1 . BCa cell lines used in RT studies Cell line Cellosaurus accession nr (Cellosaurus -SIB Swiss Institute of Bioinformatics | Expasy, no date) Molecular subtype (Shi et al., 2020)

 1

		RT112	CVCL_1670
		SW780 UMUC5	CVCL_1728 CVCL_2750	luminal
		UMUC9	CVCL_2753
		RT4 1	CVCL_0036
		5637	CVCL_0126
		647V	CVCL_1049
		HT1197	CVCL_1291
		HT1376	CVCL_1292	basal
		KU19-19	CVCL_1344
		UMUC6	CVCL_2751
		VMCUB1	CVCL_1786
	Human	253J B-V	CVCL_7937

non-luminal, non basal

Table 2 . Intrinsic radiosensitivity of a panel of BCa cell lines reported by Yard et al., 2016 Cell line AUC 1 Yard et al., 2016 Molecular subtype Shi et al., 2020 HT1376

 2

		5.228	basal
	HT1197	4.449	basal
	VMCUB1	4.412	basal
	KMBC2	4.126	luminal
	TCCSUP	3.539	non-luminal/non-basal
	KU1919	3.503	basal
	647V	3.374	basal
	BC3C	3.362	basal
	UMUC1	3.346	luminal
	SW1710	3.309	n/c
	UMUC3	3.231	luminal
	J82	3.198	non-luminal/non-basal
	RT112	3.038	luminal
	RT4 2	2.935	luminal
	UBLC1	2.914	n/c
	JMSU1	2.792	non-luminal/non-basal
	5637	2.473	basal
	T24	2.366	non-luminal/non-basal
	SCABER	1.883	basal

Table 2 .

 2 1 AUC: Area Under the Curve; 2 RT4 cell line originates from re-occurring human transitional cell papilloma

Table 3 . Overview of human BCa cell line xenograft models used in radiosensitisation studies Subtype (Shi et al., 2020)

 3

						Mice	Initial	Study
		Cell line	IR regimen	Radiosensitising agent	Class	genetic backgroun	tumour size	follow-up	Ref.
						d (gender)	(mm 3 ) 1	(days) 2
			4 x 5 Gy	Panobinostat (vs. gemcitabine)	HDAC inhibitor	(Uknown strain) (F)	100	10-60	(Groselj et al., 2018)
					DNA intercalator
		RT112	2 x 5 Gy	AQ4N (banoxantrone) (vs. cisplatin)	inhibitor and Topoisomerase II	CBA (F)	240-280	10-60 (Williams et al., 2009)
			1 x 6 Gy	Romidepsin	HDAC inhibitor	CD1 (F)	50	25	(Paillas et al., 2020)
				Low-/soluble high-
	luminal		1 x 6 Gy	high-fibre diets /insoluble high-and mixed	Diet	CD1 ( F)	50	42	(Then et al., 2020)
		RT4 3	1 x 5 or 1 x 15 Gy	Photofrin II	Photosensitiser	(Uknown strain) (F)	2.6-3.0	15	(Schaffer et al., 2001)
			1 x 2 Gy	Caffeine	DNA Damage Response inhibitor	BALB/c (M) 30-75	0 4	(Z. W. Zhang et al., 2015)
		SW780 2 x 5 Gy	siTUG1	siRNA	(Uknown strain) (M)	100	21	(Jiang et al., 2017)
	basal	5637	2 x 2 Gy	Sulfoquinovosylacylpropan ediol	Synthetic sulfoglycolipid	BALB/c Slc (M)	100-300	33	(Yagisawa et al., 2016)
			1 x n/a Gy	shRNF8	shRNA	BALB/c (M)	100-150	30	(Zhao et al., 2016)
			1 x 6 Gy	Chloroquine	Other	BALB/c (F) ∼200	25	(Wang et al., 2018)
			1 x 6 Gy	Nanoparticles (chloroquine conjugated)	Nanoparticles	(Uknown (n/a) strain)	150	16	(Lin et al., 2020)
			1 x 6 Gy	LY294002	TKI	Ncr-nu/n (F)	300-400	40	(Gupta et al., 2003)
	Non luminal/		1 x 6 Gy	FTI-276 or L744832	Farnesyltransferas e inhibitors	Ncr-nu/n (n/a)	58	80	(Cohen-Jonathan et al., 2000)
	non basal		2 x 3 Gy	shHMGB1	shRNA	(Uknown strain) (F)	n/a	21	(Shrivastava et al., 2016)
		UMUC3	1 x 12 Gy	17-AAG or 17-4002 DMAG/Trastuzumab/LY29	Hsp90 inhibitors/ antibody/TKI monoclonal	BALB/c (M) 1000	12	(Yoshida et al., 2011)
			2 x 2 Gy	Flutamide/shAR	Antiandrogen/shR NA	NOD-SCID (M)	30	12	(Ide et al., 2018)
		J82	1 x 5 Gy Gefitinib ('Iressa', ZD1839)	TKI	BALB/c (n/a)	100	n/a (Colquhoun et al., 2007)
	n/a	KK47 1 x 4 Gy	Ad-RSV-CD+5-FC	A recombinant adenovirus vector	Balb/c (n/a)	n/a	n/a	(

Table 4 . Overview of mouse BCa syngeneic models used in radiosensitisation studies

 4

	Cell line	IR regimen	Radiosensitising agent	Class	Mouse background (gender)	Initial tumour (mm 3 ) 1 size	Study (days) 2 follow-up	Ref.
			PD-L1 blocking
	MB49	1 x 12 Gy	antibody	Immunotherapy	C57BL/6 (F)	500	27	(Wu et al., 2016a)
	MB49	2 × 5 Gy	Glycyrrhizin	HMGB1 inhibitor	C57BL/6 (M)	Once palpable	7	(Ayoub et al., 2019)
	MB49, MB49-I	6 × 3 Gy	Silybin	Flavonoid	C57BL/6J (n/a)	50	30	(Prack Mc Cormick et al., 2018)
	MB49-I	6 × 3 Gy	Bacillus Calmette-Guérin (BCG)	Immunotherapy	C57BL/6J (n/a)	50	21	(Prack Mc Cormick et al., 2017)
	MBT-2	1 x 15 Gy	Lapatinib	TKI	C3H/HeN (F)	162	21	(Mu and Sun, 2018)
	MBT-2	1 x 15 Gy	Afatinib	TKI	C3H/HeN (F)	162	21	(Tsai et al., 2013)
			Cisplatin,doxorubic
			in hydrochloride
	MBT-2	5 x 4 Gy	(adriamycin),	CT	CsH/Hej (n/a)	6	60	(Weldon
			cyclophosphamide

Table 5 .

 5 Overview of studies using Cisplatin in BCa in vivo in combination with RT.

	Yoshida

et al., 2011 Williams et al., 2009 Kyriazis et al., 1986 Weldon et al., 1979 Cell lines

		UMUC3 (non-luminal, non-basal)	RT112 (luminal)	SW-800 (not classified)	MBT-2
	Source / Dose rate (Gy/min.)	x-rays (225 V)/ 0.83 Gy/min	x-rays (230 kV)/ 2 Gy/min	x-rays (250 kV)/ 1.23 Gy/min	x-rays (250 kV)/ n/a
	IR dose and fractionation	5 x 2 Gy	5 × 2 Gy	1 × 10 Gy	5 × 4 Gy
				5 mg/kg once on
	Cisplatin	0 2 mg/kg in vivo	Cisplatin (2 mg/kg)	each specified day	3 mg/kg once a week
	dose	(administered once)	(administered once)	before or after	(3 weeks)
				radiation
	Treatment arms	Hsp90 inhibitors (17-AAG or 17-DMAG) Trastuzumab, LY294002	AQ4N (banoxantrone)	-	Doxorubicin hydrochloride (Adriamycin), cyclophosphamide

Table 6 . Overview of studies using Gemcitabine in BCa preclinical studies in combination with RT Groselj et al., 2018 Kerr et al., 2014 Cell lines

 6

		-	CALgem
	Source /	x-rays (220 kV) /	gamma ( 137 Cs)/
	Dose rate (Gy/min.)	n/a	1.7 Gy/min.

Table 7 . Targeted agents used as radiosensitisers in preclinical studies using BCa xenografts. Class Name Target Cell line subtype (according to Shi et al., 2020)

 7

	Year	Ref.

Table 7 .

 7 Abbreviations

: 17-AAG: 7-N-allylamino-17-demethoxygeldanamycin; 17-DMAG: 17-Dimethylaminoethylamino-17-

Table 8 . Targeted agents used as radiosensitisers in preclinical studies using syngeneic mice.

 8

				Cell line
	Class	Name	Target	(subtype according to Shi	Ref.
				et al., 2020)
	TKI	Afatinib Lapatinib	EGFR/HER2 PDGF-R	MBT-2, mice cell line (basal) MBT-2, mice cell line (basal)	(Tsai et al., 2013) (Mu and Sun, 2018)

Table S1 . Characteristics of BCa cell lines used in RT studies.

 S1

	UMUC5	CVCL_2750		0	F	n/a	n/a	n/a	wt	X	X	wt	(Shinohara et al., 1993)	X	-	basal-like	luminal
	VMCUB1	CVCL_1786		0	M	n/a	G2	n/a	wt	X	X	wt	(Fogh, 1978)	X	X	basal	basal
	Cell line KU19-19 UMUC6 T24 UMUC9 RT112 SW-800 5637 639-V RT4 3 MB49 UMUC3 MB49-I MBT2	Cellosaurus accession nr CVCL_1344 CVCL_2751 CVCL_0554 CVCL_2753 CVCL_1670 CVCL_A684 CVCL_0126 CVCL_1048 CVCL_0036 CVCL_7076 CVCL_1783 CVCL_VL62	In vitro 39 25 11 8 7 1 3	In vivo 0 0 0 0 0 0 0 2	Gen der M M F M F F M F M M M M F	Age 76Y n/a 82Y n/a n/a n/a 68Y n/a 63Y n/a n/a n/a n/a	Gra de n/a n/a G3 n/a G2 n/a G2 n/a G1-2 n/a n/a n/a n/a	Stage n/a n/a pTa n/a pTa n/a n/a n/a n/a 3 n/a pT2-T4 n/a n/a	Ra s M ut n/ a X n/ a wt n/ a wt n/ a wt n/ a X n/ a n/	p5 3 M ut X X X X X X X X X n/ a X n/ a n/	TERT-prom oter X n/a Mut X X m/r X wt X X n/a X n/a n/a	FGFR3 gene alterati on wt wt wt n/a m/r n/a wt n/a m/r n/a wt n/a n/a	Ref. (Fogh, 1978) (Grossman et al., 1986) (Bubeník et al., (Sabichi et al., 2006) 1973) (Marshall, Franks (Kerbel, Pross and Leibovitz, 1977) and Carbonell, 1977) (Fogh, Fogh and (Elliott et al., 1977) Orfeo, 1977) (Rigby and Franks, (Summerhayes and Franks, 1979) 1970a) (Lodillinsky et al., (Grossman et al., 2009) 1986)	Part of UBC-401 -X X X X X X X X --X -	Part of CCLE2 X -X -X -X X --X -	Molecular classification [47] n/a mixed basal Non-basal like luminal Non-basal like mixed Basal-like luminal basal Mouse BCa cell lines basal Molecular classification [48] basal luminal non-luminal, non-basal luminal n/a basal non-luminal, non-basal luminal non-luminal, non-basal
	HT1376	CVCL_1292 CVCL_4660	5		F	58Y	G3	>= pT2	wt a	X a	X	wt	(Rasheed et al., 1977) (Soloway, 1977)	X	X	mixed	basal
	647V	CVCL_1049	4		M	59Y	G2	pT2/3 a	wt	X	X	m/r	(Elliott et al., 1977)	-	X	basal	basal
	J82	CVCL_0359	3		M	58Y	G3	pT3	wt	X	X	m/r	(O'Toole et al., 1978)	X	X	basal	non-luminal, non-basal
	HT1197	CVCL_1291	3		M	44Y	G4	pT2	wt	m /r	X	X	(Rasheed et al., 1977)	X	X	mixed	basal
	KK47	CVCL_8253	1		M	50Y	G1	n/a	wt wt	wt	wt	(Taya et al., 1977)	X	-	n/a	non-luminal, non-basal
	SW780	CVCL_1728	1		F	80Y	G1	n/a	wt wt	X	m/r	(Kyriazis et al., 1984)	X	X	luminal	luminal
	CAL29	CVCL_1808	2		F	80Y	G4	pT2	wt	X	X	wt	(Cattan et al., 2001)	-	X	luminal	n/c
	253J B-V	CVCL_7937	2		M	53Y	G4	pT4	wt wt	m/r	wt	(Dinney et al., 1995)	X	X	basal	non-luminal, non-basal
	NTUB1	CVCL_RW29	1		F	70Y	n/a	n/a	wt wt	wt	wt	(Yu et al., 1992)	-	-	n/a	n/a
	UCRU-BL13	CVCL_M873	1		M	62Y	n/a	n/a	wt wt	wt	wt	(Russell et al., 1989)	-	-	n/a	n/a
	UCRU-BL17	CVCL_M007	1		F	69Y	n/a	n/a	wt	X	wt	wt	(Russell et al., 1988)	-	-	n/a	n/a
	UCRU-BL28	CVCL_4904	1		M	62Y	n/a	n/a	wt	X	wt	wt	(Russell et al., 1993)	-	-	n/a	n/a
	TCC-SUP	CVCL_1738	2		F	67Y	G4	n/a	wt	X	X	wt	(Nayak, O'Toole and Price, 1977)	X	X	basal	non-luminal, non-basal

Table S1 .

 S1

1

UBC-40, a comprehensive genomic characterization of 40 urothelial bladder carcinoma (UBC) cell lines

(Earl et al., 2015)

;

2 CCLE, Cancer Cell Line Encyclopedia (Barretina et al., 2012)

;

Table S2 . Characteristics of problematic human BCa cell lines used in RT studies.

 S2

			In
	Cell line	Cellosaurus accession nr	vitr	In vivo	Comment 3
			o
	MGHU-1 (EJ138)	CVCL_2443	8	0	T24 derivative
	S40b	n/a	4	0	Parent cell line (MGHU-1) has been shown to be a T24 derivative
	ECV304	CVCL_2029	3	0	T24 derivative
	EJ30	CVCL_2443	3	0	Parent cell line (Ej138) has been shown to be a T24 derivative.
	biu87	CVCL_6881	3	0	Contaminated by non-human cell
	KU7	CVCL_4714	1	1	HeLa derivative
	MGHU-2	CVCL_9826	1	0	T24 derivative
	TSGH 8301	CVCL_A342	1	1	Contaminated by a cervix cancer cell line ME-180
	Table

Table 1

 1

-1)

[START_REF] Van Der Meer | TAM receptors, Gas6, and protein S: Roles in inflammation and hemostasis[END_REF][START_REF] Davra | Ligand activation of TAM family receptors-implications for tumor biology and therapeutic response[END_REF]

.

Table 1 -1. Properties of TAM ligands.

 1

	Ligand Properties	GAS6	Protein S
	TAM Specificity	AXL >> TYRO3 > MERTK TYRO3 > MERTK; AXL (ND)
	TAM Specificity
	(In the presence of	TYRO3 = MERTK >> AXL TYRO3 = MERTK; AXL (ND)
	Phosphatidylserine)
	Vit. K dependency	√	√
	Plasma Concentration	0.16 to 0.28 nM/L	0.30 μM/L (~1000 times)

ND: not determined. From

[START_REF] Davra | Ligand activation of TAM family receptors-implications for tumor biology and therapeutic response[END_REF]

Table 1 -2. TYRO3 expression and function in cancer.

 1

		Expression				Functional Roles
	Cancer	Cell Lines	Patient Samples	Cell Proliferation and/or Survival Colony Formation	Xenograft Model	Migration and/or Invasion	EMT	Metastasis	Drug Resistance	Unfavorable Prognosis	Ref.
	Hepatocellula r carcinoma	OX +	OX OX OX	+ +			+			+ *	+	(Duan et al., 2016) (Uehara et al., 2017) (Kabir et al., 2018)
		+	OX	+					+	+	+	(Schmitz et al., 2016)
	Colorectal		+	+	+	+	+	+	+	+	+	(Chien et al., 2016)
		OX		+			+					(Qin and Qian, 2018a)
	Esophageal	+	OX									(Nemoto et al., 1997)
		+										(Easty et al., 1993)
		+	OX	+	+	+				+		(Zhu et al., 2009)
	Melanoma	+ +										(Easty et al., 2011) (Tworkoski et al., 2011)
		+										(Sensi et al., 2011)
		+	OX	+								(Demarest et al., 2013)
	Thyroid	Ectopi c		+								(Avilla et al., 2011)
		+										(A et al., 1999)
	Lung	+ +	+									(Rikova et al., 2007) (Linger et al., 2013)
								+				(Thomson et al., 2011)
	Prostate	OX +	OX						+			(Jansen et al., 2012) (Taichman et al., 2013)
	Bladder	OX	OX	+	+	+					+	(Dufour et al., 2019)
		OX										(Taylor et al., 1995)
	Breast	+ +		+ +						+		(Ekyalongo et al., 2014) (Kasikara et al., 2017)]
			+							+ #	+	(Alexander et al., 2017)
		+		+						+		(Lee, 2015)
	Ovarian	+								+		(Kim, Lee and Lee, 2015)
		+								+		(Suh et al., 2015)
	Endometrial Cancer		+									(Sun, Fujimoto and Tamaya, 2004)
	Leiomyosarco		OX									(El Sayadi et al., 2013a)
	ma	+	+	+	+
	Liposarcoma		+
	Pleomorphic Sarcoma		+									(Dantas-Barbosa et al., 2017)
	Synovial Sarcoma		+
	Schwanomma	OX	+									(Ammoun et al., 2010) (Ammoun et al., 2014)
	Multiple Myeloma	+										(De Vos et al., 2001)
		+	+									(Crosier et al., 1995)
	AML	+										(Shiozawa, Pedersen and Taichman, 2010)
		+		+								(Eryildiz and Tyner, 2016)
	ALL (B-cell &	Ectopi c										(Crosier et al., 1995)
	T-cell)	+										(Shiozawa, Pedersen and Taichman, 2010)
	CML	+										(Crosier et al., 1995)

CLL

OX

[START_REF] Sinha | Targeted Axl inhibition primes chronic lymphocytic leukemia B cells to apoptosis and shows synergistic/additive effects in combination with BTK inhibitors[END_REF]

Table 1 -2. TYRO3 expression and function in cancer.

 1

Abbreviations: ALL: Acute lymphocytic leukemia; CLL: Chronic lymphocytic leukemia; CML: Chronic myeloid leukaemia OX: overexpressed in tumor tissue/cells relative to normal tissue/cells; *: sorafenib resistance; #: trastuzumab resistance; EMT: epithelial-mesenchymal transition. Adapted from

[START_REF] Smart | The emerging role of TYRO3 as a therapeutic target in cancer[END_REF]

Table 1 -3. Translational treatment targeting TYRO3.

 1

Tyrosine kinase inhibitors with IC50 or Kd values less than 100 nM are shown. These values refer to activity in enzymatic assays. Adapted from

[START_REF] Smart | The emerging role of TYRO3 as a therapeutic target in cancer[END_REF] Hsu et al., 2019.

  . Muscle-invasive bladder cancers (MIBCs) account for 25% of bladder tumours at initial diagnosis and are lifethreatening. Indeed, despite radical cystectomy with cisplatin based neoadjuvant and/or adjuvant chemotherapy, the standard treatment for MIBC, overall survival is only about 50% at 5 years, and is as low as 5% in cases of distant metastasis. Immune checkpoint inhibitors can be beneficial in patients with bladder cancer(Bellmunt, Powles and Vogelzang, 2017), but

they are effective in only a limited number of patients (20%). New effective therapies targeting both MIBCs and non-muscle-invasive tumours (NMIBCs), which often recur, are therefore required. Efforts to decipher the molecular mechanisms involved in bladder carcinogenesis have led to the identification of a number of possible new treatment targets, such as mTOR in patients with TSC1 mutations, epidermal growth factor receptor 2 (HER2)/ERBB2 in HER2positive tumours, EGFR in basal-like tumours, and fibroblast growth factor receptors, particularly in patients harbouring mutations or gene fusions of FGFR3

(Rebouissou et al., 2014;[START_REF] Abbosh | Targeting signaling transduction pathways in bladder cancer[END_REF] Carneiro et al., 2015)

. The TAM family of receptor tyrosine kinases (RTKs), which includes TYRO3, AXL and MERTK, has emerged as new therapeutic target in many types of cancer, but their role in bladder cancer has not yet been determined. TAM receptors can be activated by the vitamin K-dependent protein GAS6

Table S1 : siRNA sequences siRNA Manufacturer Reference sense strand sequence antisense strand sequence

 S1

Table S2 : Mean values of unpaired t test with Welch's correction (Two-tailed) corresponding to Figure 2b a VM-CUB-1 (mean of 3 experiments)

 S2

	b RT112 (mean of 3 experiments).
	Cell Line	Treatment	Mean	Std. Deviation	P value (two tailed)
	RT112	siCTL -72h versus siTYRO3#2 -72h	52.1	15.97	<0.0001
	RT112	siCTL -72h versus siTYRO3#3 -72h	44.98 5.621	0.0026
	RT112	siCTL -96h versus siTYRO3#1 -96h	22.32 8.696	<0.0001
	RT112	siCTL -96h versus siTYRO3#2 -96h	32.76 13.75	<0.0001
	RT112	siCTL -96h versus siTYRO3#3 -96h	30.6	4.7	0.0004
	RT112	siCTL -72h versus siAXL#45 -72h	78.85 10.39	0.2129
	RT112	siCTL -72h versus siAXL#46 -72h	81.45 11.95	0.2721
	RT112	siCTL -72h versus siAXL#47 -72h	39.05 12.66	0.0928
	RT112	siCTL -96h versus siAXL#45 -96h	76.2	9.475	0.1747
	RT112	siCTL -96h versus siAXL#46 -96h	79.2	5.374	0.115
	RT112	siCTL -96h versus siAXL#47 -96h	30.35 23.55	0.1494
	c UM-
	Cell Line	Treatment	Mean	Std. Deviation	P value (two tailed)
	VM-CUB-1 siCTL -72h versus siTYRO3#1 -72h	31.13 8.301	0.0048
	VM-CUB-1 siCTL -72h versus siTYRO3#2 -72h	53.93 5.829	0.0053
	VM-CUB-1 siCTL -72h versus siTYRO3#3 -72h	38.9	2.512	0.0006
	VM-CUB-1 siCTL -96h versus siTYRO3#1 -96h	11.9	1.997	0.0002
	VM-CUB-1 siCTL -96h versus siTYRO3#2 -96h	31.97 3.57	0.0009
	VM-CUB-1 siCTL -96h versus siTYRO3#3 -96h	28.53 6.396	0.0027
	VM-CUB-1 siCTL -72h versus siAXL#45 -72h	80	2.8	0.0065
	VM-CUB-1 siCTL -72h versus siAXL#46 -72h	85.33 6.363	0.0574
	VM-CUB-1 siCTL -72h versus siAXL#47 -72h	48.73 1.06	0.0001
	VM-CUB-1 siCTL -96h versus siAXL#45 -96h	81.28 5.231	0.0056
	VM-CUB-1 siCTL -96h versus siAXL#46 -96h	95.55 5.307	0.1921
	VM-CUB-1 siCTL -96h versus siAXL#47 -96h	39.17 3.35	0.001
	VM-CUB-1 siCTL -72h versus siMERTK#72 -72h	59.17 12.92	0.0318
	VM-CUB-1 siCTL -72h versus siMERTK#73 -72h	73.3	11.71	0.0585
	VM-CUB-1 siCTL -72h versus siMERTK#74 -72h	75.83 9.501	0.0479
	VM-CUB-1 siCTL -96h versus siMERTK#72 -96h	40.6	5.295	0.0002
	VM-CUB-1 siCTL -96h versus siMERTK#73 -96h	65.65 9.248	0.005
	VM-CUB-1 siCTL -96h versus siMERTK#74 -96h	73.43 9.593	0.0116

UC-5 (mean of 3 experiments).

	Cell Line	Treatment	Mean	Std. Deviation	P value (two tailed)
	UM-UC-5	siCTL -72h versus siTYRO3#1 -72h	40.54 10.75	0.0002
	UM-UC-5	siCTL -72h versus siTYRO3#2 -72h	48.2	9.896	0.0003
	UM-UC-5	siCTL -72h versus siTYRO3#3 -72h	58.56 13.52	0.0024
	UM-UC-5	siCTL -96h versus siTYRO3#1 -96h	23.23 5.344	<0.0001
	UM-UC-5	siCTL -96h versus siTYRO3#2 -96h	34.23 7.221	0.0004
	UM-UC-5	siCTL -96h versus siTYRO3#3 -96h	51.13 13.08	0.005
	UM-UC-5	siCTL -72h versus siAXL#45 -72h	70.7	4.384	0.0671
	UM-UC-5	siCTL -72h versus siAXL#46 -72h	75.8	3.818	0.0707
	UM-UC-5	siCTL -72h versus siAXL#47 -72h	47	16.97	0.1418
	UM-UC-5	siCTL -96h versus siAXL#45 -96h	71.3	0.7071	0.0111
	UM-UC-5	siCTL -96h versus siAXL#46 -96h	75.85 0.7778	0.0145
	UM-UC-5	siCTL -96h versus siAXL#47 -96h	51.7	23.48	0.2107
	d

UM-UC-9 (mean of 3 experiments).

	e MGH-U3 (mean of 3 experiments).
	Cell Line	Treatment	Mean	Std. Deviation	P value (two tailed)
	MGH-U3	siCTL -72h versus siTYRO3#1 -72h	30.7	11.64	<0.0001
	MGH-U3	siCTL -72h versus siTYRO3#2 -72h	62.3	5.335	<0.0001
	MGH-U3	siCTL -72h versus siTYRO3#3 -72h	72.8	6.776	0.0009
	MGH-U3	siCTL -96h versus siTYRO3#1 -96h	10.67 4.737	<0.0001
	MGH-U3	siCTL -96h versus siTYRO3#2 -96h	41.3	5.62	<0.0001
	MGH-U3	siCTL -96h versus siTYRO3#3 -96h	48.67 10.05	<0.0001
	Cell Line	Treatment	Mean	Std. Deviation	P value (two tailed)
	UM-UC-9	siCTL -72h versus siTYRO3#1 -72h	64.28 1.823	<0.0001
	UM-UC-9	siCTL -72h versus siTYRO3#2 -72h	64.73 3.465	0.0003
	UM-UC-9	siCTL -72h versus siTYRO3#3 -72h	82.4	4.386	0.004
	UM-UC-9	siCTL -96h versus siTYRO3#1 -96h	47.25 4.481	0.0002
	UM-UC-9	siCTL -96h versus siTYRO3#2 -96h	52.6	4.158	0.0002
	UM-UC-9	siCTL -96h versus siTYRO3#3 -96h	75.58 3.69	0.0009
	UM-UC-9	siCTL -72h versus siMERTK#72 -72h	89.65 0.9192	0.0399
	UM-UC-9	siCTL -72h versus siMERTK#73 -72h	106.4 3.606	0.2431
	UM-UC-9	siCTL -72h versus siMERTK#74 -72h	108.3 7.142	0.3497
	UM-UC-9	siCTL -96h versus siMERTK#72 -96h	83.8	1.414	0.0392
	UM-UC-9	siCTL -96h versus siMERTK#73 -96h	97.9	6.081	0.7108
	UM-UC-9	siCTL -96h versus siMERTK#74 -96h	97.7	6.788	0.7155

Table S2 : Mean values of unpaired t test with Welch's correction (Two-tailed) corresponding to Figure 2b.

 S2 Mean percentage of viability was calculated from three independent experiments and further compared by t

test with Welch's correction (Two-tailed) for a VM-CUB-1, b RT112, c UM-UC-5, d UM-UC-9 and e MGH-U3 cell lines.

Table S3 : Unpaired t test results corresponding to figure 2C UNC2025 treatment

 S3

	Cell Line	Treatment	Mean	Std. Deviation	P value (two tailed)
	a VM-CUB-1 (mean of 3 experiments)
	VM-CUB-1	DMSO vs UNC2025 0.039 μM	101.8	3.885	0.43
	VM-CUB-1	DMSO vs UNC2025 0.078 μM	99.75	4.991	0.9265
	VM-CUB-1	DMSO vs UNC2025 0.156 μM	97.46	5.013	0.3859
	VM-CUB-1	DMSO vs UNC2025 0.312 μM	79.02	4.484	0.0026
	VM-CUB-1	DMSO vs UNC2025 0.625 μM	69.16	4.81	0.001
	VM-CUB-1	DMSO vs UNC2025 1.25 μM	53.42	0.9782	<0.0001
	VM-CUB-1	DMSO vs UNC2025 2.5 μM	34.16	2.34	<0.0001
	VM-CUB-1	DMSO vs UNC2025 5 μM	21.78	2.207	<0.0001
	VM-CUB-1	DMSO vs UNC2025 10 μM	2.128	1.227	<0.0001
	b RT112 (mean of 3 experiments)
	RT112	DMSO vs UNC2025 0.039 μM	97.14	3.881	0.2363
	RT112	DMSO vs UNC2025 0.078 μM	90.12	5.077	0.0301
	RT112	DMSO vs UNC2025 0.156 μM	74.88	8.984	0.0113
	RT112	DMSO vs UNC2025 0.312 μM	51	9.157	0.0017
	RT112	DMSO vs UNC2025 0.625 μM	28.93	10.92	0.001
	RT112	DMSO vs UNC2025 1.25 μM	8.645	4.298	<0.0001
	RT112	DMSO vs UNC2025 2.5 μM	3.758	2.181	<0.0001
	RT112	DMSO vs UNC2025 5 μM	2.135	1.021	<0.0001
	RT112	DMSO vs UNC2025 10 μM	0.21	0.09201	<0.0001
	c UM-UC-5 (mean of 3 experiments)
	UM-UC-5	DMSO vs UNC2025 0.039 μM	103.8	3.425	0.1133
	UM-UC-5	DMSO vs UNC2025 0.078 μM	104.6	3.041	0.0563
	UM-UC-5	DMSO vs UNC2025 0.156 μM	105.4	2.98	0.0359
	UM-UC-5	DMSO vs UNC2025 0.312 μM	96.8	3.951	0.2032
	UM-UC-5	DMSO vs UNC2025 0.625 μM	69.06	29.81	0.1295
	UM-UC-5	DMSO vs UNC2025 1.25 μM	70.07	24.87	0.0953
	UM-UC-5	DMSO vs UNC2025 2.5 μM	61.48	7.043	0.0016
	UM-UC-5	DMSO vs UNC2025 5 μM	45.84	3.291	<0.0001
	UM-UC-5	DMSO vs UNC2025 10 μM	0.1175 0.1744	<0.0001
	d

UM-UC-9 (mean of 3 experiments)

		BMS777607 treatment
	Cell Line	Treatment	Mean	Std. Deviation	P value (two tailed)
	a VM-CUB-1 (mean of 3 experiments)
	VM-CUB-1	DMSO vs BMS777607 0.039 μM	94.8	9.654	0.3605
	VM-CUB-1	DMSO vs BMS777607 0.078 μM	98.59	5.551	0.6459
	VM-CUB-1	DMSO vs BMS777607 0.156 μM	99.42	7.835	0.8922
	VM-CUB-1	DMSO vs BMS777607 0.312 μM	102	4.493	0.441
	VM-CUB-1	DMSO vs BMS777607 0.625 μM	105.6	5.382	0.1278
	VM-CUB-1	DMSO vs BMS777607 1.25 μM	108.2	5.122	0.0499
	VM-CUB-1	DMSO vs BMS777607 2.5 μM	109.6	8.416	0.1071
	VM-CUB-1	DMSO vs BMS777607 5 μM	100.5	18.26	0.9638
	VM-CUB-1	DMSO vs BMS777607 10 μM	67.05	11.72	0.0111
	b RT112 (mean of 3 experiments)
	RT112	DMSO vs BMS777607 0.039 μM	97.75	2.69	0.1926
	RT112	DMSO vs BMS777607 0.078 μM	98.14	2.049	0.1674
	RT112	DMSO vs BMS777607 0.156 μM	99.21	2.493	0.5712
	RT112	DMSO vs BMS777607 0.312 μM	97.98	3.504	0.3315
	RT112	DMSO vs BMS777607 0.625 μM	98.06	3.589	0.3583
	RT112	DMSO vs BMS777607 1.25 μM	95.04	1.434	0.0062
	RT112	DMSO vs BMS777607 2.5 μM	89.69	3.095	0.0069
	RT112	DMSO vs BMS777607 5 μM	74.77	12.21	0.0257
	RT112	DMSO vs BMS777607 10 μM	44.55	23.09	0.0172
	c UM-UC-5 (mean of 3 experiments)
	UM-UC-5	DMSO vs BMS777607 0.039 μM	99.56	3.942	0.8359
	UM-UC-5	DMSO vs BMS777607 0.078 μM	102.7	3.571	0.2337
	UM-UC-5	DMSO vs BMS777607 0.156 μM	104	4.166	0.1506
	UM-UC-5	DMSO vs BMS777607 0.312 μM	105.1	1.58	0.0075
	UM-UC-5	DMSO vs BMS777607 0.625 μM	107.3	5.799	0.0864
	UM-UC-5	DMSO vs BMS777607 1.25 μM	105.9	3.209	0.0344
	UM-UC-5	DMSO vs BMS777607 2.5 μM	103	7.5	0.4882
	UM-UC-5	DMSO vs BMS777607 5 μM	81.26	0.6364	0.0153
	UM-UC-5	DMSO vs BMS777607 10 μM	64.22	9.055	0.0042
	d
	UM-UC-9	DMSO vs UNC2025 0.039 μM	102.2	3.244	0.2746
	UM-UC-9	DMSO vs UNC2025 0.078 μM	102.1	4.18	0.3856
	UM-UC-9	DMSO vs UNC2025 0.156 μM	102.1	2.499	0.1903
	UM-UC-9	DMSO vs UNC2025 0.312 μM	94.87	4.307	0.0973
	UM-UC-9	DMSO vs UNC2025 0.625 μM	95.46	3.577	0.0849
	UM-UC-9	DMSO vs UNC2025 1.25 μM	94.17	3.478	0.044
	UM-UC-9	DMSO vs UNC2025 2.5 μM	91	1.828	0.0022
	UM-UC-9	DMSO vs UNC2025 5 μM	76.89	3.417	0.0009
	UM-UC-9	DMSO vs UNC2025 10 μM	41.74	8.42	0.0008
	e MGH-U3 (mean of 3 experiments)
	MGH-U3	DMSO vs UNC2025 0.039 μM	99.92	2.544	0.9428
	MGH-U3	DMSO vs UNC2025 0.078 μM	98.93	2.613	0.3598
	MGH-U3	DMSO vs UNC2025 0.156 μM	87.57	7.317	0.0088
	MGH-U3	DMSO vs UNC2025 0.312 μM	67.47	10.98	0.0008
	MGH-U3	DMSO vs UNC2025 0.625 μM	47.6	8.799	<0.0001
	MGH-U3	DMSO vs UNC2025 1.25 μM	42.96	8.625	<0.0001
	MGH-U3	DMSO vs UNC2025 2.5 μM	32.04	10.33	<0.0001
	MGH-U3	DMSO vs UNC2025 5 μM	16.38	15.03	<0.0001
	MGH-U3	DMSO vs UNC2025 10 μM	1.442	2.023	<0.0001

UM-UC-9 (mean of 3 experiments)

	UM-UC-9	DMSO vs BMS777607 0.039 μM	98.73	4.873	0.6389
	UM-UC-9	DMSO vs BMS777607 0.078 μM	102.5	2.618	0.1496
	UM-UC-9	DMSO vs BMS777607 0.156 μM	101.8	3.581	0.3854
	UM-UC-9	DMSO vs BMS777607 0.312 μM	102	3.45	0.3394
	UM-UC-9	DMSO vs BMS777607 0.625 μM	104.1	0.7276	0.0015
	UM-UC-9	DMSO vs BMS777607 1.25 μM	107.6	2.207	0.0063
	UM-UC-9	DMSO vs BMS777607 2.5 μM	111.6	1.381	0.0005
	UM-UC-9	DMSO vs BMS777607 5 μM	111.8	1.554	0.0006
	UM-UC-9	DMSO vs BMS777607 10 μM	111.5	2.329	0.0022
	e MGH-U3 (mean of 3 experiments)
	MGH-U3	DMSO vs BMS777607 0.039 μM	100.6	5.485	0.8043
	MGH-U3	DMSO vs BMS777607 0.078 μM	102.8	3.41	0.1031
	MGH-U3	DMSO vs BMS777607 0.156 μM	104	4.224	0.0666
	MGH-U3	DMSO vs BMS777607 0.312 μM	103.5	3.173	0.0411
	MGH-U3	DMSO vs BMS777607 0.625 μM	104.9	3.943	0.0293
	MGH-U3	DMSO vs BMS777607 1.25 μM	102.4	2.249	0.0488
	MGH-U3	DMSO vs BMS777607 2.5 μM	98.1	5.17	0.4082
	MGH-U3	DMSO vs BMS777607 5 μM	85.5	14.26	0.0551
	MGH-U3	DMSO vs BMS777607 10 μM	60.93	10.44	0.0003

Table S3 : Unpaired t test results corresponding to figure 2C. Range of doses of two TAM inhibitors were used on

 S3 VM-CUB-1, b RT112, c UM-UC-5, d UM-UC-9 and e MGH-U3 cell lines.

Table S4 : List of differentially expressed genes after TYRO3 knockdown (adjusted p-value < 0.05, Ilog2(FC)I ≧ 0.58)

 S4

	FIGNL1	-1.29694516448465	0.00940768359009906	IMPA2	-0.974223741265708	0.00152436800030135
	Gene Symbol Gene Symbol	Log2FC Log2FC	Adjusted p value Adjusted p value	Gene Symbol Gene Symbol	Log2FC Log2FC	Adjusted p value Adjusted p value
	Gene Symbol TYMS GINS2 CDCA7 RRM2 MAD2L1 DSCC1 MCM2 TRIP13 MCM5 MND1 ORC1 RFC5 PBK KIF15 POLE2 TYRO3 USP1 NEK2 NCAPG CDC20 ZWINT GINS1 CENPA HELLS CDCA5 EXO1 RMI2 FAM83D NEIL3 MCM3 HNRNPC AURKB WDR76 CCNA2 CDC45 RNASEH2A DLGAP5 CDK1 ARHGAP11A PARPBP DESI2 CENPM ASF1B DUT KIF4A BUB1B HIST1H4C STIL BLM HJURP FAM64A PRIM1 CENPU CDC7 SENP1 ATAD5 BORA ARHGAP19 BTG1 DNMT1 OSBPL1A CTDSPL2 SERINC1 SUV39H2 C5orf15 PSMC3IP MARCKS SSRP1 ZNF217 FAM111B NEK10 LYRM7 MPZL3 CCDC34 TM2D1 MTHFD1 LIMK2 LRRCC1 THRB MRPS27 PLK2 MIS18BP1 ZNF267 INCENP TSPAN31 PMCH ZNF277 ACD LAMC1 RECQL4 PPT2 RBL1 ABHD4 TICRR TMEM40 SAC3D1 FEZ2 DDX11 FKBP14 G2E3 ZBTB34 MLH1 KLHL29 POC1A GSN CTBP2 ITPKC RFWD3 RNASEL DEK STX3 POLD1 GBP1 SPDL1 KDM7A C14orf80 NMRK1 CENPJ TGFBR2 NDC1 PDCD4 CIT CROT HMMR-AS1 DNAJC16 POLD3 SECISBP2L ZGRF1 LMBR1L TTF2 DNAJB9 NASP KCTD21 PAQR4 ABCC5 EXOSC8 FRMD4B TDP1 EFNA1 PTTG3P BTBD3 PLK1 DUSP5 C19orf48 CFL2 CEP72 TNFSF9 DDX39A SC5D MCMBP TP53INP2 HAUS4 GATSL3 SGOL1 SOX9 CCDC150 LYPD3 CEP128 C3orf52 GSTCD C6orf120 TBC1D31 BTG2 CASP8AP2 IL6R	Log2FC -2.32633519874701 -2.25989515512308 -2.21812224913286 -2.15165489948141 -2.09998342815555 -1.99572659683288 -1.99110556760114 -1.9743742318283 -1.86123627892881 -1.8228859666659 -1.79387197803191 -1.78838092402732 -1.72899704439629 -1.72337139631725 -1.71356489898211 -1.70084977320961 -1.67641083041202 -1.65869645918555 -1.6521926023925 -1.64900643909533 -1.63090705112796 -1.62510871576045 -1.61631828411007 -1.61098094713699 -1.60633300664466 -1.58904506945797 -1.5849491983088 -1.56729567681065 -1.53996491112145 -1.52888558600266 -1.51310341073131 -1.50916853741347 -1.49565477982323 -1.49473107895524 -1.49157349421077 -1.46063262873921 -1.45984304227489 -1.44708551097963 -1.44484600426292 -1.43991142197805 -1.42825869024885 -1.42009672077511 -1.404317530559 -1.38319973355984 -1.37507818621123 -1.3700788505801 -1.3562048157385 -1.35266552985997 -1.33810169302243 -1.32402884455784 -1.31203734206177 -1.30874126908821 -1.30394458109878 -1.30053790856719 -0.729410443487602 0.0126349536618619 Adjusted p value 0.0352912237947859 0.0164424048117559 0.0291989358604571 0.0288085057016318 0.00105886143890613 0.0280679875443733 0.00872275718509133 0.0174553566454759 0.0369907752440196 0.0190771981890462 0.0120291454591872 0.00279890270037965 0.0120237201817505 0.00701766409455231 0.0492635780656493 0.0000129957283052645 GPSM2 Gene Symbol CDCA3 DEPDC1B FAM49B RAD54L FEN1 PLK4 CDCA8 SAP30 RAD54B MCM6 BRCA1 SKA3 ESPL1 SAPCD2 KIF2C 0.0161435973845789 POLA2 0.0167666910577561 GINS3 0.0407145710982809 KIF20B 0.0118717742924948 TRAIP 0.0110306993535686 RFC2 0.0369856777644402 VRK1 0.00810986903718914 CCDC109B 0.044691938664171 KIF22 0.0249321233178632 CENPW 0.021044453719113 KIFC1 0.00680590612049965 POLA1 0.0178375579916679 IFIT1 0.0189125031207068 C5orf34 0.0245650250728365 HMGB2 0.00680590612049965 TFAM 0.0275474294169825 CEP78 0.0156516726130169 CHAF1A 0.0267393138114839 MCM7 0.0397214875905346 AUNIP 0.0291989358604571 PTTG1 0.0373244532618172 CASC5 0.0497192478539408 MCM4 0.00152436800030135 UNG 0.0423913693488044 TIPIN 0.00764708782670369 CCT5 0.032786788608759 FLVCR1 0.00853929082957962 PCNA 0.00279890270037965 FANCG 0.0197796643553085 RHNO1 0.0197796643553085 ARHGEF39 0.0401917265939256 LIG1 0.0294420305238006 ANKRD32 0.0239151119257605 TMPO 0.0408199662152273 H2AFX 0.00239222212175116 SERTAD2 0.0408199662152273 NCAPD3 0.0336317797206165 SIVA1 0.012250181636814 KIAA1524 STYK1 -0.73899862280789 0.00279890270037965 ANKRD12 -0.966357898909892 0.0236548780405118 C1orf198 -0.958657845486179 0.0212968044386373 DAG1 0.607219090751096 0.048339924441887 GABARAPL1 -0.958161700840556 0.0120237201817505 FAM161A 0.610436567048069 0.0268889341270449 LURAP1L -0.947275884826401 0.00311130021352572 CLSPN 0.62160235652715 0.0226771806337481 YPEL5 -0.943198971864522 0.0254087048831078 H2AFZ 0.621904179597875 0.022483309615605 CYP1A1 -0.939414028086531 0.0204418878262815 SUZ12 0.62355749433903 0.0224278846867353 LIPH -0.935561333392183 0.0065568494012562 NEDD1 0.634210782253525 0.0248543621733866 CTGF -0.930190472392701 0.022483309615605 FAM122B 0.639939308957995 0.0257501864013222 -0.929375247653877 0.0286974866390726 TUBG1 0.648729825479646 0.0197796643553085 -0.924027065509199 0.0161435973845789 NUFIP1 0.652087183630766 0.044691938664171 -0.914941203122046 0.00701766409455231 SLC25A10 0.657150585435296 0.0197796643553085 -0.91456753307417 0.0208166720871371 TONSL 0.657418599402867 0.00140834320016116 -0.914186192064992 0.0456987025185162 PARP2 0.670539789298839 0.0378236031621732 -0.91215480443284 0.00659163259433179 NRM 0.671129573258574 0.032786788608759 -0.911296440418379 0.0292821329960885 FN3KRP 0.67333305691997 0.0179321305542266 -0.907037466620162 0.0362366875632673 AMD1 0.68048667341708 0.0172392931399768 -0.905634528749627 0.00370613747313495 CNOT6 0.682629302831849 0.00673561922218481 -0.902316113301801 0.0416222665187978 DIAPH3 0.69017395039156 0.0252923008631427 -0.896511816243652 0.000415883812997067 TSEN15 0.699150015907563 0.0224278846867353 -0.884370777799624 0.0118660818279311 HAUS1 0.70545017662154 0.021349437991459 -0.882365962492905 0.0118831792861475 NUP62 0.709479046116504 0.0238463682847262 -0.872470801169403 0.0310371201985745 LRRC8B 0.713551727698855 0.00527616460524262 -0.86847474616565 0.00384099721674659 HAUS2 0.738700239761737 0.0329765464263918 -0.866954014700935 0.00701766409455231 SAE1 0.740413917312753 0.00152436800030135 -0.865982920501658 0.0269574108113076 FANCB 0.74070278122668 0.0108085306894373 -0.865840246477207 0.0178963054902073 HSD17B6 0.743127301430306 0.00279890270037965 -0.859958071792201 0.0220136151446249 HAUS6 0.755539360178637 0.0498165788833688 -0.854207374998667 0.00769355093270947 FANCM 0.766823997032613 0.0180938761463507 -0.852957239424896 0.018408471406582 TMEM107 0.784054790094427 0.0445822340147917 -0.845623064831717 0.0197796643553085 LSM5 0.787407876814552 0.0105863363846317 -0.839811007405164 0.00823863916419177 XRCC4 0.795391816453176 0.0310443454163986 -0.836425223048815 0.0478527896592117 EML4 0.801970759489389 0.0153466908305699 -0.817810646629137 0.022483309615605 C16orf59 0.802826068409097 0.0153249925855015 -0.809114525129179 0.0197796643553085 HAUS7 0.806558929852818 0.00279890270037965 -0.806438747769767 0.0275474294169825 CDK4 0.814246071410616 0.022625865161838 -0.806396163690432 0.0266794158619404 SNRNP40 0.816395523078441 0.000415883812997067 -0.804215019043915 0.0487765254568019 BEND3 0.839724475165369 0.00401928771087991 -0.804038498029392 0.0073521785404644 XRCC3 0.842579496021301 0.0300873707418058 -0.792993590054676 0.0246987946836751 CIRH1A 0.852904021497776 0.00401928771087991 -0.792159129750882 0.0153249925855015 PGP 0.861034488755639 0.0161435973845789 -0.790749027723477 0.0120237201817505 PKMYT1 0.871816391818182 0.0332091216718961 -0.788862615772916 0.0174444873433772 NAP1L1 0.891032443228227 0.0310371201985745 -0.787341915769503 0.00701766409455231 OAS3 0.894500118117723 0.0439697921885657 -0.785754092259467 0.0212968044386373 FOXRED1 0.907727938144278 0.00384099721674659 -0.7696181669344 0.0276739389174627 APITD1 0.932446926239623 0.0263407657997964 -0.759059324967716 0.003435772796281 NUCKS1 0.973956783151589 0.0148217149360905 -0.757332455159508 0.0134744455932702 RNF11 0.976014862718409 0.00205032727746709 -0.753029536242773 0.022663377016292 PTPN11 0.986227305132056 0.0174444873433772 -0.74873350435378 0.0246987946836751 RAD18 0.992209436776778 0.0336317797206165 -0.744298215312228 0.0263407657997964 TUBB4B 0.995357408641802 0.0180616091631931 -0.743510278459651 0.00872275718509133 MDC1 1.04606665465404 0.00284806193343022 -0.742757269922545 0.0207959824211813 LACTB 1.07411066701686 0.0352912237947859 -0.741623207349638 0.0268889341270449 STX12 1.10057873982702 0.00279890270037965 -0.741337796684737 0.003435772796281 RBFOX2 1.20597538685838 0.00659163259433179 -0.740316920059631 0.0484566268167233 RAP2C 1.39918841206197 0.0329974884519565	Log2FC -1.29218917271615 -1.27818970384952 -1.27131387799394 -1.26944992777543 -1.26253149978589 -1.26218053947429 -1.2581399702575 -1.25068850307398 -1.23737660914296 -1.23413992685013 -1.22864021676027 -1.22863476667897 -1.22804450280035 -1.22629042818159 -1.22358866540339 -1.21612831323071 -1.20989692472587 -1.19537118679031 -1.19488709712392 -1.18501512089308 -1.17623490967163 -1.16227640393604 -1.16001978363742 -1.15806440041968 -1.15528716285062 -1.14665891775726 -1.14083340181758 -1.12102804390241 -1.12041582238406 -1.11721921755006 -1.10151825133906 -1.0967006166157 -1.09578638062121 -1.08138686886006 -1.07202503837188 -1.0688236808049 -1.06576446297139 -1.05106690038496 -1.04533705752015 -1.03448566073436 -1.02879543257971 -1.02587922131559 -1.0245515467369 -1.01647305733985 -1.01553182973422 -1.01397156271661 -1.00894648441598 -0.998015119633174 -0.997426038338157 -0.994933981419709 -0.992895472160304 -0.988218251606675 -0.986093818748857 -0.985734973871717 0.605609872943567 0.598772112811449 -0.724700428632388 0.0235105172126714 Adjusted p value 0.0212968044386373 0.0215168851973675 0.00660199160225694 0.024856474653568 0.0298838379216078 0.0197796643553085 0.0469166050198533 0.00152436800030135 0.0284167254926443 0.0283679213435287 0.0146289018948535 0.0286974866390726 0.0292930904594293 0.0210425582573731 0.0474162436730147 0.0477683033380899 0.013918915350938 0.0492635780656493 0.00758573857516864 0.00527616460524262 0.0263603413286981 0.00554802049391793 0.00636355025369637 0.0283679213435287 0.0161931390688985 0.0336317797206165 0.0263407657997964 0.0483472325581728 0.0105863363846317 0.0239151119257605 0.000572556384239827 0.0110306993535686 0.0116415104605114 0.0137708919603065 0.00572088987356591 0.0154000281956974 0.0248543621733866 0.0332091216718961 0.0120237201817505 0.0065568494012562 0.0103995933704089 0.00956279152669909 0.0336317797206165 0.0469166050198533 0.00380941487227665 0.0167666910577561 0.0353892516283588 0.0286974866390726 0.0263407657997964 0.00940768359009906 0.00133543036950184 0.0118717742924948 0.0284853829140019 0.0408199662152273 0.0378885137755604 0.0280679875443733 1.15063668421192 0.0286974866390726 -0.723789125373841 0.0352912237947859 1.16249094995923 0.0477683033380899 -0.723158926726832 0.0070176640945523 1.19340940350776 0.0226771806337481 -0.721683177257296 0.0167666910577561 1.40928575520158 0.014426586384064 -0.718510315956141 0.0485853345461125 -0.71441994953774 1.53501631453278 0.0266695874283275 0.0207959824211813 2.44991539018038 0.0020503272774670 -0.710292558376669 0.0382568076310581 -0.709056605069012 0.0157846850994489 -0.705411690978902 0.0268889341270449 -0.701935244346599 0.013918915350938 -0.699923020036208 0.0153249925855015 -0.699627241750275 0.0118831792861475 -0.699055140043086 0.0287551563976935 -0.69889167994903 0.0042544101520741 -0.698513226802991 0.0190771981890462 -0.698221650663541 0.0236548780405118 -0.695758681198315 0.0215168851973675 -0.69307882006046 0.012560490389218 -0.684936036585237 0.017087419211033 -0.684885211274653 0.0402534026097585 -0.683291146028172 0.0210425582573731 -0.681461026797255 0.0076935509327094 -0.681214412771801 0.026225315206653 -0.678598610533772 0.0038409972167465 -0.67738217621118 0.0167666910577561 -0.666605301109063 0.0226771806337481 -0.659136388747232 0.0153249925855015 -0.658857553596154 0.0197796643553085 -0.63977243784509 0.0294420305238006 -0.638670913375717 0.0154000281956974 -0.638286104965914 0.0432693689927907 -0.633636949973739 0.0153249925855015 -0.627374086112349 0.0267084016117157 -0.625475975480756 0.0492635780656493 -0.621199979880592 0.0016362953420204 -0.620112451419468 0.0010701354704005 -0.61678184777246 0.0252923008631427 -0.616095024546271 0.0065568494012562 -0.615650621238821 0.0317950368178618 -0.615118033388782 0.0273905998659249 -0.605379064516394 0.0359291253177696 -0.60485175216491 0.0020503272774670 -0.604278484464357 0.0492635780656493 -0.603119509778636 0.0263462052725117 -0.599938015017944 0.0070176640945523 -0.597093648931656 0.0087227571850913 -0.596979736848336 0.0266794158619404 -0.596009455221447 0.0052495848172129 -0.585642936994546 0.0110306993535686 -0.584975465606652 0.0065568494012562 -0.582503498793477 0.0224278846867353 0.580276500064896 0.0337954164529303 0.582121210872524 0.0476578582388808 0.584183929421506 0.0183199852364504 0.597293630548549 0.0292567247156871

Table S4 : List of differentially expressed genes after TYRO3 knockdown

 S4

(adjusted p-value < 0.05, Ilog2(FC)I ≧ 0.58).

Table S6 : List of upstream regulators impacted after TYRO3 knockdown (IPA Upstream regulator analysis)

 S6

	E2F1	-	3.34E-25 AURKB,BRCA1,CCNA2,CDC20,CDC45,CDK1,CDK4,DDX11,DNMT1,DUT,FEN
		3.723		1,GINS1,HELLS,HMGB2,LACTB,MAD2L1,MCM2,MCM3,MCM4,MCM5,MC
	Upstream	Activ	p-value	Target molecules in dataset M6,MCM7,MDC1,MLH1,MTHFD1,ORC1,PCNA,PLK2,POLA1,POLA2,POLD1
	Regulator	ation	of	,PPT2,RAD54L,RBFOX2,RBL1,RFC2,RFC5,RRM2,SERTAD2,SIVA1,TFAM,TNF
		z-	overlap	SF9,TYMS,UNG,VRK1
	FOXM1	score -	7.61E-08 AURKB,BUB1B,CCNA2,CDC20,CDCA8,CDK1,CENPA,NEK2,PCNA,PLK1,PLK4
	CDKN2A	5.330 2.24E-20 AMD1,ASF1B,AURKB,BLM,BTG2,BUB1B,CCNA2,CDCA5,CDK1,CHAF1A,CT 3.028 ,TGFBR2
	MYC	-	GF,DEK,EFNA1,FEN1,H2AFX,HAUS1,HMfGB2,KIFC1,MAD2L1,MCM4,MCM 2.37E-06 AMD1,AURKB,BRCA1,BUB1B,CCNA2,CDC20,CDCA7,CDK1,CDK4,DDX11,D
		3.018		5,MCM7,MCMBP,PCNA,PLK1,POLD3,RECQL4,RRM2,SAE1,SUZ12,TMPO,V NMT1,DUSP5,EXOSC8,H2AFZ,IFIT1,IMPA2,MAD2L1,MCM5,MCM6,MCM7
				RK1 ,MTHFD1,NAP1L1,PCNA,PDCD4,PLK1,POLD1,RRM2,SAE1,SOX9,SUZ12,TF
	NUPR1	5.191 7.40E-18 ABCC5,ARHGAP11A,ATAD5,BLM,BRCA1,BTG1,BUB1B,CCNA2,CDCA3,CDC AM,TGFBR2,TYMS,XRCC3
	E2F3	-	A8,CDK4,CROT,DUSP5,ESPL1,EXO1,FAM111B,FLVCR1,GINS1,GPSM2,HJU 7.27E-20 C1orf198,CCNA2,CDC45,CDCA3,CDK1,DAG1,HMGB2,MAD2L1,MCM2,MC
		2.840		RP,IL6R,KIF2C,KIFC1,KNL1,NEIL3,NUCKS1,PLK1,POLA2,POLE2,SERTAD2,SL M3,MCM4,MCM5,MCM6,MCM7,MLH1,NUCKS1,ORC1,PCNA,PLK1,POLA1
				F1,SPDL1,STIL,STX3,TICRR,TMPO,WDR76,ZBTB34 ,POLA2,PTTG1,RBL1,RRM2,TMPO
	TP53 E2F2	4.896 4.51E-20 ABHD4,ASF1B,AURKB,BRCA1,BTG1,BTG2,BUB1B,CCNA2,CDC20,CDC7,CD -7.01E-15 CCNA2,CDC45,CDK1,MCM2,MCM3,MCM4,MCM5,MCM6,MCM7,MLH1,O
		2.236		K1,CDK4,CTGF,CYP1A1,DEK,DLGAP5,DNMT1,DUSP5,DUT,EFNA1,ESPL1,EX RC1,PCNA,POLA1,RBL1,RRM2
				O1,FAM83D,FEN1,FIGNL1,GBP1,GSN,H2AFX,H2AFZ,HJURP,HMGB2,KIFC1,
				LIMK2,MAD2L1,MCM2,MCM3,MCM4,MCM5,MCM6,MCM7,MIS18BP1,M
				LH1,NCAPD3,NCAPG,NEK2,PARP2,PBK,PCNA,PLK1,PLK2,POLA1,POLD1,PO
				LE2,PRIM1,PTPN11,PTTG1,RAD54B,RBL1,RECQL4,RRM2,SIVA1,SPDL1,TFA
				M,TGFBR2,TNFSF9,TYMS,UNG,VRK1
	RB1	4.052 4.36E-21 ASF1B,AURKB,BLM,BRCA1,CCNA2,CDCA5,CDK1,CHAF1A,CTGF,DDX11,DE
				K,DNMT1,FEN1,HAUS1,HMGB2,LACTB,LIG1,MCM2,MCM3,MCM4,MCM5,
				MCM6,MCM7,MCMBP,ORC1,PCNA,PLK1,POLD3,PPT2,RBL1,RECQL4,RRM
				2,SUZ12,TMPO,TUBG1,TYMS,VRK1
	RBL1	3.411 4.47E-14 AURKB,CCNA2,CDK1,MCM2,MCM3,MCM5,MCM6,MCM7,NEK2,ORC1,PC
				NA,PLK1,RBL1,RRM2,TYMS
	HDAC1	3.000 5.68E-08 CCNA2,CDK1,GABARAPL1,H2AFX,LIG1,MCM3,MCM5,MCM7,NASP,PLK1,R
				AD54L,RBL1,RECQL4,RRM2,SOX9,TGFBR2,TYMS
	FOXP3	2.750 5.58E-06 BRCA1,CHAF1A,FEN1,LIG1,PDCD4,POLD1,POLD3,RAD54B,TONSL
	SMARCB1	2.709 9.75E-10 BTG1,CCNA2,CDC7,CDK1,CENPA,GSN,MCM2,MCM3,MCM4,MCM5,OAS3,
				PLK4,POLA1,PRIM1,RAD54B,RFC5
	E2F6	2.530 2.80E-11 BRCA1,CDC45,CDC7,DDX11,GINS2,LIG1,MCM2,MCM3,MCM5,POLA2,RR
				M2,UNG
	KDM5B	2.498 2.27E-06 BRCA1,BUB1B,CDCA3,CDK1,CTGF,DLGAP5,KIF2C,MCM2,MCM3,PBK,SOX9
	TBX2	-	3.84E-22 ASF1B,AURKB,CCNA2,CDCA3,CDCA5,CDK1,DNMT1,HAUS1,HELLS,LIG1,M
		4.583		AD2L1,MCM2,MCM4,MCM5,MCM6,MCM7,PKMYT1,PLK1,RBL1,SGO1,TIP
				IN
	RARA	-	1.24E-06 CCNA2,CDK1,CENPA,CENPM,CENPU,FAM64A,HELLS,HIST1H4C,IL6R,KNL1,
		4.123		MAD2L1,MND1,NCAPG,PBK,PLK4,PTTG1,STX3
	ESR1	-	3.58E-08 ABCC5,BRCA1,BUB1B,CASP8AP2,CCNA2,CDCA8,CDK1,CDK4,CENPA,CENP
		4.103		M,CENPU,CFL2,CIT,CLSPN,CYP1A1,DIAPH3,DUSP5,EFNA1,FAM64A,GPSM
				2,GSN,HELLS,HIST1H4C,INCENP,KNL1,MAD2L1,MARCKS,MND1,NCAPG,N
				DC1,PBK,PCNA,PDCD4,PLK4,POLA1,PTTG1,RBL1,SOX9,SPDL1,STX3,THRB,
				TMPO,TNFSF9
	CCND1	-	4.14E-20 BRCA1,CCNA2,CDC45,CDCA7,CDCA8,CDK4,CLSPN,DNMT1,FAM83D,HJUR
		4.054		P,KIAA1524,KIF20B,KIF2C,KIF4A,KNL1,MCM4,MCM7,PCNA,PDCD4,PLK1,P
				OLD3,PSMC3IP,RBL1,RFC5,RMI2,RRM2,TGFBR2,TP53INP2,TRAIP,TRIP13,T
				YMS
	MITF	-	1.96E-17 AURKB,BRCA1,CDCA3,CDCA8,CENPM,CHAF1A,ESPL1,FANCG,FRMD4B,IL6
		4.041		R,KIF4A,KIFC1,LIG1,MCM2,MCM4,MCM5,MCM6,MDC1,NCAPD3,PLK1,PO
				LD1,POLE2,PSMC3IP,RECQL4,RFC5,SOX9,XRCC3

  . Briefly, lyophilized proteins were solubilized in 1X Laemmli sample buffer and boiled for 10 minutes. Protein concentrations were determined with the Biorad Bradford Protein Assay Kit (BioRad, France) and protein levels were assessed by immunoblotting. The following antibodies were purchased from Cell Signalling Technologies (Ozyme, Montigny-leBretonneux, France):

	AURORA A (Ref. 14475), AXL (Ref. 8661), ATM (Ref. 2873), phospho-ATM (Ser1981) (Ref.
	4526), ATR (Ref. 2790), phospho-ATR (Ser428) (Ref. 2853), CDK1 (Ref. 9116), phospho-CDK1
	(Tyr15) (Ref. 9111), Chk1 (Ref. 2360), phospho-Chk1 (Ser345) (Ref. 2348), Cyclin B1 (Ref.
	4135), Cyclin D1 (Ref. 2926), Chk2 (Ref. 2662), phospho-Chk2 (Thr68) (Ref. 2197), DNA-PKc
	(Ref. 4602), H3 (Ref. 9717), MERTK (Ref. 4319), TYRO3 (Ref. 5585). Antibody against phospho-
	DNA PKc (Ser2056) (Ref. ab18192) was purchased from Abcam.

4.4. Materials and Methods NHU Cell Culture

	Urothelium from the uteter/renal pelvis of patients undergoing urological surgery was
	collected with National Health Service Research Ethics Committee approval and required
	informed consent from urological procedures that excluded urothelial neoplasia. Finite
	(nonimmortal, serially passaged) NHU cell lines were established in, NHU cells were grown on
	Primaria™ plasticware (BD Biosciences, Wokingham, UK) in low calcium [0.09 mM]
	keratinocyte serum-free medium (KSFM) containing bovine pituitary extract and recombinant
	epidermal growth factor (Fisher Scientific UK Ltd, Loughborough, UK) supplemented with 30

Titre : Cibler TYRO3 : une nouvelle stratégie de radio-sensibilisation des cellules cancéreuses de la vessie Mots clés :

  Cancer de la vessie, radiosensibilité, récepteur à activité tyrosine kinase, TYRO3, urothélium.Le cancer de la vessie est un problème majeur de santé publique. Il est le quatrième cancer le plus fréquent chez l'homme en termes d'incidence. 25% des cancers diagnostiqués sont des tumeurs envahissant le muscle (TVIM) présentant un mauvais pronostic. La cystectomie est le traitement standard de référence pour les TVIM, même si elle présente des inconvénients importants. La radiothérapie, associée à une chimiothérapie et à une résection transurétrale de la tumeur, émerge comme traitement conservateur alternatif. La chimiothérapie n'épargne pas les tissus sains et présentent de nombreux effets secondaires indésirables. Il est donc d'importance de découvrir de nouvelles stratégies de radiosensibilisation pour les tumeurs de la vessie. TYRO3 est un récepteur à activité tyrosine kinase de la famille TAM (qui comprend TYRO3, AXL et MERTK). TYRO3 est surexprimé dans de nombreux types de cancers et favorise la prolifération, la survie et la résistance des cellules tumorales à la chimiothérapie. De plus, la surexpression de TYRO3 a été associée à une diminution de la survie globale des patients. Cependant, le rôle de TYRO3 dans le cancer de la vessie n'a pas encore été étudié. Dans cette thèse, je me suis intéressée : (1) Au rôle de TYRO3 dans le cancer de la vessie; (2) A l'effet radiosensibilisant de la perte d'expression ou de l'inhibition de TYRO3 dans les cellules cancéreuses de la vessie;(3) A l'effet de l'inhibition ou de la perte d'expression de TYRO3 sur l'urothélium humain sain. Nous avons démontré que TYRO3 est surexprimé dans 50% des TVIM. De plus, nous avons mis en évidence que les cellules tumorales de vessie surexprimant TYRO3 développaient une dépendance à ce récepteur pour leur survie et leur croissance. Les résultats des données transcriptomiques suggèrent que la perte d'expression de TYRO3 induit des modifications importantes dans le contrôle du cycle cellulaire et de l'apoptose, ce qui laisse supposer que TYRO3 pourrait augmenter la sensibilité de ces tumeurs à la radiothérapie. La perte d'expression ou l'inhibition de TYRO3 dans les cellules dérivées de tumeur de vessie induit une radiosensibilisation significative des cellules traitées. A l'inverse, la surexpression de TYRO3 par transfection plasmidique sur des cellules exprimant peu TYRO3 induit une radiorésistance. En association avec le rayonnement, la perte d'expression de TYRO3 conduit à un arrêt du cycle cellulaire et à une persistance à 24h des foyers de réparation (détectés par immunofluorescence). Enfin, les travaux sur les cellules dérivées de l'urothélium sain ont montré que la perte d'expression de TYRO3 n'affectait pas leur viabilité suggérant que le ciblage de TYRO3 pourrait améliorer l'efficacité de la radiothérapie tout en épargnant les tissus normaux environnants. Bladder cancer, radiosensitisation, tyrosine kinase receptors, TYRO3, urothelium.Bladder cancer (BCa) is a major global health problem. It is the fourth most common cancer in men in industrialized countries. 25% of all diagnosed BCa are Muscle-invasive bladder cancers (MIBC) which have poor prognosis. Cystectomy is the standard treatment for MIBC, but for patients with comorbidities it presents significant drawbacks including increased risk of infection and impacted quality of life. Radiotherapy coupled with chemotherapy and tumor transurethral resection has emerged as a promising bladder sparing. Chemotherapy does not spare normal tissue and results in side effects. Therefore, it is of great interest to discover novel radiosensitisation strategies for bladder tumors.

Title : Targeting TYRO3: a Novel Strategy to Radiosensitise Bladder Cancer Cells

Keywords :

In this study, the mice were sacrified immediately after the treatment delivery. Abbreviations: n/a, information not available.

RT4 cell line originates from re-occurring human transitional cell papilloma[START_REF] Rigby | A human tissue culture cell line from a transitional cell tumour of the urinary bladder: growth, chromosone pattern and ultrastructure[END_REF]; abbreviations: n/a, information not available; n/c, not categorized (not coherent classification depending on the dataset used); m/r, mixed reports; wt, wild type; 3Information from the https://web.expasy.org/. Abbreviations: TKI, tyrosine kinase inhibitor; n/a, information not available. Information in this table has been collected and updated of that from Zuiverloon et al.[START_REF] Zuiverloon | Systematic Review: Characteristics and Preclinical Uses of Bladder Cancer Cell Lines[END_REF], CCLE, UBC-40.

Acknowledgements

Acknowledgements

First, I would like to thank all my supervisors who have guided me throughout my PhD: my thesis co-directors François Radvanyi and Pierre Verrelle for accepting me for this challenging PhD project carried out between two sites 20 km apart. I thank you for trusting me, giving guidance, but also for giving me the independence to grow as a scientist.

I thank François for always stepping in when needed and thank Pierre for his enthusiasm and for being my role model for a passionate doctor involved in multiple translational projects. I thank my thesis co-supervisor Isabelle Bernard-Pierrot for taking the time to have regular meetings and for the thoughtful suggestions to improve my work and keeping my reports and presentations on the point. I warmly thank Frédérique Mégnin-Chanet for welcoming me in the lab in Orsay and for allowing me to discover the fascinating field of radiobiology and giving the practical training and guidance. I thank Frédérique for all the encouragements and warm Acknowledgments: We thank Dr. Ayham Alnabulsi for valuable feedback on the manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.

Acknowledgements

We thank Jacqueline Fontugne (MD, PhD student) for the pathological examination of TYRO3 staining in normal urothelium samples and peritumoural tissue. We would like to thank David Gentien and Audrey Rapinat from the Translational Research Department of Institut Curie for performing transcriptome experiments. We thank the Cell and Tissue Imaging (PICT-IBiSA) and Nikon Imaging Centre, Institut Curie, a member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04). We also thank the Flow Cytometry Core Facility of Institut Curie.

Acknowledgments

We thank to Laetitia Besse from the Institut Curie Microscopy platform for creating the macro for the yH2AX foci counting, Charlene Lasgi from Institut Curie Flow Cytometry platform for the help of the cell cycle analysis and Cristophe Roulin from the Institut Curie RadEx platform for his help of performing the Comet Assay. This project received funding from the LabEx CelTisPhyBio grants (ANR-11-LABX-0038, ANR-10-IDEX-0001-02) and the European Union's Horizon 2020 research and innovation program, under Marie Skłodowska-Curie grant agreement No. 666003, Fondation pour la recherche Médicale (FRM) (grant agreement FDT201904008237) and a grant from Comité scientique/Comite de radioprotection from Électricité de France (EDF) (D4008/10.11.16/532).

Funding: This study has been supported by a Fondation pour la Recherche Médicale (FRM) grant (agreement FDT201904008237) and a grant from Comité scientique/Comité de radioprotection from Électricité de France (EDF) (D4008/10.11.16/532) and by SIRIC-Curie INCa-DGOS-4654. This project received funding from the European Union's Horizon 2020 research and innovation program, under Marie Skłodowska-Curie grant agreement No. 666003. Funding This work was supported by La Ligue Nationale Contre le Cancer, through the funding of an accredited team (FR, IBP, FD, LS, HNK, AMV, CK, NK, MDG, EC, YA, NS, Equipe labellisée), by ANR Emergence Bio and by ElsaLys Biotech. This project also received funding from the European Union's Horizon 2020 research and innovation program, under Marie Skłodowska-Curie grant agreement No. 666003. Data availability: The microarray data are available from GEO (https://www.ncbi.nlm. nih.gov/geo/) under accession number GSE100025. Note: This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

In vivo study plan

Appendix: In vivo study

Author Contributions: Conceptualization, L.S., F.M. P.V.; methodology, L.S., P.V.; resources, L.S. and F.M.; writing-original draft preparation, L.S., F.M., P.V; writing-review and editing, L.S., F.M., G.C., I.B.P, F.R., F.M.C, P.V; visualization, L.S. and F.M.; supervision, P.V. All authors have read and agreed to the published version of the manuscript.

Targeting TAMs in combination with radiation therapy

The use of radiotherapy (RT) aims to kill cancer cells in the treatment field. However, evidence exist that RT can also direct immune responses, which can reduce tumor growth outside the treatment field. Cancer cell death plays an important role in activating these mechanisms.

TAMs are implicated in clearance of dying cancer cells and mitigating innate immunresponse, therefore, TAMs could be excellent targets in combination with RT (Figure 1-9) (Tormoen, Crittenden and Gough, 2017).

Additional information

Supplementary information is available for this paper at https://doi.org/10.1038/ s41416-019-0397-6.

Competing interests

Materials and methods

Cell lines

The human bladder cancer-derived cell lines RT112, UM-UC-5, UMUC-9, VM-CUB-1, 5637, 647

V and HT1376 were obtained from DSMZ (Heidelberg, Germany). MGH-U3 cells were kindly provided by Dr. Francisco X. Real (CNIO, Madrid). MGH-U3, UM-UC-5, UMUC-9, VM-CUB-1, 647 V and HT1376 cells were cultured in DMEM, whereas RT112, and 5637, cells were cultured in RPMI. Media were supplemented with 10% foetal bovine serum (FBS) (ThermoFisher Scientific, Courtaboeuf, France). Cells were kept at 37 °C, under an atmosphere containing 5%

CO2. The identity of the cell lines used was checked by analysing genomic alterations on comparative genomic hybridization arrays (CGH array) and sequencing genes known to be mutated: RAS, TP53, FGFR3 and PIK3CA. The cells were routinely checked for mycoplasma contamination.

Human bladder samples

We assessed expression of the TYRO3, AXL, MERTK and GAS6 genes by RT-qPCR, using 169 bladder tumour samples (87 NMIBCs and 82 MIBCs) from the previously described CIT-series cohort ("Carte d'Identité des Tumeurs" or "Tumour identity card") of bladder tumours (Biton et al., 2014;Rebouissou et al., 2014). Seven normal urothelial samples were obtained from fresh urothelial cells scraped from the normal bladder wall and dissected from the lamina propria during organ procurement from a cadaveric donor for transplantation. RNA, DNA and protein were extracted from the surgical samples by cesium chloride density centrifugation, as previously described (Calderaro et al., 2014;Rebouissou et al., 2014). We used protein extracted from 21 human bladder tumours from the CIT-series (4 NMIBCs and 17 MIBCs) for western blot analysis (Calderaro et al., 2014;Rebouissou et al., 2014). Lyophilized proteins were solubilised in 1X Laemmli sample buffer and boiled for 10 min. Protein concentrations

Prologue

Our findings described in chapter 2 identifying the important role of TYRO3 in BCa survival, prompted us to investigate further investigate the downstream mechanisms of TYRO3 downregulation. Specifically, we decided to investigate the potential to combine TYRO3 downregulation with radiation treatment due to the following reasons:

(1) There is a need to develop new radiosensitisers to improve the treatment of elderly BCa patients who cannot tolerate combined chemotherapy and radiotherapy treatment and receive only radiotherapy. There is also lack of targeted therapies in BCa. We aimed to further investigate whether TYRO3 has a promise to be translated as a clinical target for BCa, by combining its targeting with radiation treatment in order to potentially improve the radiation-based treatment.

(2) In the in vivo experiment, we observed, that tumor growth was delayed effectively following TYRO3-targeting siRNA injections, however, the tumor growth recurred, when the treatment was stopped, suggesting that combined treatment could improve the outcome;

(3) In chapter 2, I describe that TYRO3 downregulation, results in impaired cell cycle regulation and DNA repair mechanisms that indicated that radiation-induced damage could not be effectively repaired and thus might result in effective combination with radiation.

In this chapter, I have gathered evidences that combination of TYRO3 downregulation with radiation shows superior therapeutic effect compared to each treatment alone. We further investigated the effect of the combination on the extent of DNA damage signaling and the cell cycle. The results are in preparation for submission (Article 3), however, still a few experiments are ongoing to strengthen the conclusions made.

Article 3: TYRO3 targeting as a radiosensitising strategy in bladder cancer

TYRO3 targeting as a radiosensitising strategy in bladder cancer

Linda Silina 1,2,3 , Fatlinda Maksut 2,3 , Florent Dufour 1 , Jennifer Southgate 4 , Pierre Verrelle 2,3,5,6 , François Radvanyi 1 , Isabelle Bernard-Pierrot 1 and Frédérique Mégnin-Chanet 2,3,# In preparation for submission Article 3

TYRO3 targeting as a radiosensitizing strategy in bladder cancer

Linda Silina 1,2,3 , Fatlinda Maksut Results: TYRO3 is phosphorylated within 5 minutes following 2 Gy irradiation. We demonstrated that TYRO3 downregulation leads to a significantly increased radiosensitivity of BCa cell lines. Moreover, TYRO3 downregulation leads to accumulation of DNA damage 24h after irradiation with more persistent yH2AX foci and a significant cell cycle arrest in G2/M phase. Conversely, we showed that TYRO3-overexpression induces radioresistance with less persistent yH2AX foci following irradiation. TYRO3 downregulation does not influence DNA damage response pathways but disrupts mitosis regulation through differential Cyclin B1 expression levels. In normal human bladder cells this receptor is not essential for survival,

Results

TYRO3 modulation influences radiosensitivity of bladder cancer cell lines

It has been shown that several RTKs can modulate radiation response (Meyn et al., 2009). The potential role of TYRO3 RTK in radiation response has never been investigated. Using six BCa cell lines with different TYRO3 expression protein levels, we evaluated the intrinsic radiosensitivity of each cell line (Supplementary Figure S1). For this, cells were exposed to increasing doses of radiation using colony forming (RT112, UM-UC-5, VM-CUB-1, 5637 and UM-UC-9) or proliferation assay after five-population doubling time following irradiation (UM-UC-3). Surviving fractions were plotted, fitted with the classical linear quadratic function and the D10 values were compared. We showed that the D10 ranged between 4.7Gy ± 0.43 -7.2Gy

±0.33 (Supplementary Figure S1A). There was no correlation between the expression level of TYRO3 and the intrinsic radiation response (Supplementary Figure S1A,B). We thus selected three BCa cell lines, two TYRO3-high expressing (luminal RT112 and basal 5637 cell line) and a TYRO3-low expressing UM-UC-3 cell line (non-luminal/non-basal) for further investigation of the impact of the modulation of TYRO3 expression on cell sensitivity to radiation. In the two TYRO3-high expressing RT112 and 5637 cell lines, TYRO3 was downregulated using 3 different siRNAs targeting sequences encoding different TYRO3 regions (encoding Ig-like (siTYRO3#1), intra-membrane region (siTYRO3#4) or kinase domain (siTYRO3#801) (sequences are listed in Supplementary Table S1). Transfection with siLUC (non-specific siRNA) was used as a control.

48 h after transfection, the expression of TYRO3 was efficiently downregulated (Fig 1 .A). The cells were then irradiated with increasing doses of radiation (2)(3)(4)[START_REF]Fondation pour la Recherche Médicale (FRM): funding for the 4th year of PhD thesis from[END_REF][START_REF]Winner of the NanoString Technologies grant competition: award to utilise nCounter® Tumor Signaling 360 Panel[END_REF][START_REF] References Antoni | Bladder cancer incidence and mortality: a global overview and recent trends[END_REF](8) and survival fractions assessed by clonogenic assay (Fig. 1A, radiobiological parameters are available at Supplementary Tables S1 andS2). As showed in Fig. 1B, the D10 showed a statistically Table S3. Sequences of siRNAs used in the study.

Competing Interests

IBP and FR hold a patent describing TYRO3 as a therapeutic target for the treatment of cancer (patent publication WO/2010/031828), which has been licensed to ElsaLys Biotech. FD, FR and IBP hold a patent describing anti-TYRO3 antibodies (patent publication WO/2016/166348).

The other authors have no potential conflicts of interest to disclose.

Authors' Contributions

Conception and design: Linda Silina, Florent Dufour, Pierre Verrelle, François Radvanyi, Isabelle Bernard-Pierrot, Frédérique Mégnin-Chanet.

Acquisition of data: Linda Silina, Fatlinda Maksut, Florent Dufour.