Masayuki Fujii

Aurelie Chiche
email: aurelie.chiche@pasteur.fr

Jakub Sumbal

Elsa Charifou

Zuzana Koledova
email: koledova@med.muni.cz

Han Li
email: han.li@pasteur.fr

• Gunnhildur

As Traustadottir

Mohamed Bentires-Alj

Beatrice Howard

Alexandra Van Keymeulen
email: alexandra.van.keymeulen@ulb.be

Primary Mammary Organoid Model of Lactation and Involution

Keywords: 3D culture, fibroblast growth factor 2, involution, lactation, mammary gland, milk production, organoid, prolactin BOM, basal organoid medium, Csn2, Casein2-b-casein gene, ECM, extracellular matrix, EGF, epidermal growth factor, FGF2, 7, or 10, fibroblast growth factor 2, 7, or 10, LM, lactation medium, Mmp, matrix metalloproteinase, TGFa, transforming growth factor-a, TGFb, transforming growth factor-b, Wap, whey acidic protein Mammary gland, Breast cancer, Breast development, Branching, Organoids, Patient-derived xenografts, Circulating tumor cells, Metastasis, Tumor dormancy, Tumor microenvironment, Signaling, Premetastatic niche, In vivo live imaging, Lineage tracing, Transcriptomics, Resistance to therapy

Cellular senescence is a biological stress response characterized by a stable cell cycle arrest. Nonetheless, cells remain metabolically active and acquire a senescence-associated secretory phenotype (SASP), a complex secretome composed of cytokines, chemokines, growth factors, and extracellular matrix remodeling modulators. Senescence is associated with various pathological processes, such as tumorigenesis and aging. However, it is unknown when, where and how senescence contributes to physiological processes. To answer this question, we took advantage of the mammary gland (MG), an organ with

If your dreams do not scare you, they are not big enough.

Mike Horn, world explorer I would like to thank all the members of my Ph.D. jury. First, Dr. Felicity Davis and Dr. Serge Adnot for putting their expertise in the mammary gland and the senescence fields to review my work. Thanks also to Dr. Bethan Lloyd-Lewis, Dr. Charhazade El Amri, Dr. Dmitry Bulavin, and Dr. Louis Gervais for accepting to participate in my Ph.D. defense. It would be an honor to discuss my work with you all.

Louis, merci également de m'avoir suivie tout au long de ces quatre années. Merci aux autres membres de mon comité de thèse, Dr. Silvia Fre et Dr. Sandrine Etienne-Manneville, pour vos conseils sur ce projet.

Thanks to my thesis director, Dr. Han Li, for the opportunity to do a Ph.D. in your lab. After four challenging years, I feel proud of the work we achieved together. Thanks for the budget allocated to this project, especially all the (expensive) in vivo experiments we tried. Thank you also for giving me multiple opportunities to present my data, at the departmental retreats or the Dev'Club. I'll carry away your advice to improve my storytelling capacity, as a major skill for my future career.

À mon équipe de folie: Aurélie, ma dealeuse d'oeufs, mon grain de café, mon petit Kévin, mon éleveuse de chèvres, ma (contrefaçon de) Céline Dion, mon arnaqueuse de tredlnik, ma reine des crop. C'est simple, sans toi je n'aurais jamais écrit ces lignes… Merci de m'avoir retourné le cerveau pour faire une thèse, je ne regrette finalement pas (enfin je crois) ! Merci de m'avoir prise sous ton aile pour me transmettre ton savoir et ton amour des glandes mammaires. Merci d'avoir porté et partagé ce projet avec moi. Et merci d'avoir été là tous les jours pour que j'y arrive. Dans les moments compliqués, avec notre ''confessionnal'' en pièce de culture. Mais surtout dans les moments de joie. Et mon Dieu (arrête de jurer !), ils ont été nombreux !! Lamia, merci d'avoir été une étudiante aussi appliquée et impliquée, ton aide a été précieuse. Merci pour ton attitude vaillante qui m'a motivée quand j'en avais le plus besoin. Merci aussi d'avoir dévoilé ton côté vulnérable et de m'avoir fait confiance. Et enfin, merci pour tous les fous rires qu'on a pu avoir à la cantine ou à Prague ! Laurianne, à l'héritière de mon bureau et de mes pipettes. Je garde en tête nos moments d'extase au microscope devant les adipocytes différenciés en forme de fleur, ou les sublimes organoides au confocal… qui étaient parfois quand même bien difficiles à trouver ! Merci de m'avoir suivie dans tous mes vices culinaires pour combler mes pulsions rédactionnelles. Il va falloir souscrire à uber eats premium quand tu rédigeras ta thèse ;) Cheng, thanks for training me to all the techniques of mice injections and optimizing the program for automatic quantification! But more importantly, thanks for being the mom of the lab and for comforting me anytime I needed. Thanks also for trusting me with your daughter and for launching my teaching career. Marielle, reine de la cytométrie, merci pour toutes les fois où tu t'es étalée sur la porte de mon bureau pour me dire au revoir. En remerciement de ce petit rituel, je te lègue mon bureau au calme ;) merci aussi pour tous les moments bavardages/thé qu'on a partagé, tu as été très bonne conseillère. Et également une super coloc' sur l'île d'Oléron ! Jérémy, mon petit poulain, aussi connu comme l'homme au bonnet, aka le roi de l'informatique au pull rose à froufrous, aka l'estomac sur pattes. J'ai été ravie de te voir t'épanouir à nos côtés pendant ta première année de thèse. Face aux difficultés à venir, pense à notre Cémantix en 6 coups : tout est possible ! Finally, thanks to Shaoxiang for your help in the bioinformatic analyses. Good luck with the learning of both English and French! Merci à mes prédécesseurs Mathieu et Coralie pour avoir posé quelques pavés pour adoucir ce chemin semé d'embûches. Merci aussi pour vos manuscrits de thèse qui m'ont guidée lors de ma rédaction. Thanks to all the students who came to our lab during the last four years giving it a nice international vibe. Especially thanks to Victor, for sharing my office during these three months of intense labor and for giving me a coffee addiction. Daryna, thanks for our discussions, your strength and your way to see positivity in every situation. Quentin, merci pour les lundis passés ici et nos innombrables fous rires à la cantine. Lucia, thanks for your kindness and for listening to me in this tough time. Yanis, merci pour toutes tes blagues (enfin pour celles qui étaient drôles :D), ça m'a permis de garder le moral jusqu'au bout ! Un grand merci à tous les gens rencontrés à Pasteur qui ont été d'une grande aide. Tout d'abord, Tara, notre assistante qui a tout fait pour nous faciliter la vie, et avec le sourire. Un énorme merci pour le travail effectué pour mettre en place ma soutenance. Pierre-Henri, merci pour les nombreux tris sur la plateforme de cytométrie. Merci à l'équipe de l'animalerie Monod et en particulier à Mathieu et Sylvain pour avoir pris soin de mes souris et m'avoir aidée à veiller sur la bonne lactation de mes portées. Clémire, merci pour m'avoir appris à dompter le microtome et le cryostat pour faire de sublimes tranches de glandes mammaires. Merci également pour ta bonne humeur, tu as été mon rayon de soleil du 7 ème étage. Thanks to Alina and Clari for your positivity and your precious help in establishing my flow cytometry panels. Merci à nos voisins de labo, Pauline pour tes encouragements qui m'ont donné confiance pendant l'écriture ; David, pour toutes les fois où tu t'es moqué de mon « writing mood » claquettes/chaussettes/cache-oreilles… alors qu'au fond tu rêvais de porter la même chose, je le sais ! Marco, thanks for always taking the time to talk and laugh with me. I send you all my strength directly to Roma, you'll succeed! Et Sabine pour ton sourire permanent. Je n'oublierai pas tous les compliments mutuels qu'on a pu se faire sur nos baskets ou nos boucles d'oreilles ;) Olinda, merci pour ton écoute attentive et tes précieux conseils qui m'ont aidée à naviguer en eaux troubles. Merci à Marc, tes encouragements dans les couloirs de Pasteur ou au parc des Princes ont été à chaque fois un vrai coup de boost au moral. Carole, merci pour m'avoir mis le pied à l'étrier lors de la rédaction et pour tous les shows que j'ai pu voir grâce à toi ! Thanks to Jakub and Zuzana for the collaboration we established on organoids. It was a really motivating period of my Ph.D. Thank you also for inviting me to your wedding in Czech Republic. I felt privileged to witness so much love.

Merci à toute ma team Vis Ma Vie De Chercheur. Rémy, merci pour le temps que tu as passé à m'écouter, me conseiller et me booster avec tes blagues. Tu as été un soutien de taille, merci pour toutes les belles paroles pour me redonner confiance en moi quand j'en avais besoin ; Helena, ma mexicaine préférée, merci pour ton écoute et tes conseils avisés ; Yoann, mon compagnon de galère, on peut être fiers du chemin parcouru depuis le MEG ; Ségolène, tu représentes à mes yeux une femme forte, merci de m'avoir inspirée et motivée à me dépasser ; Antoine, mon petit Patrick, merci d'avoir été un super binôme pour enseigner ; Flaminia, merci pour ta douceur et ta vulnérabilité. J'ai vécu certains des moments les plus euphorisants de ma thèse à vos côtés, à encadrer de nombreux lycéens et à (je l'espère) créer des vocations.

To my international IP girls squad, thank you for all the moments (and the amazing food!) we shared together. Miriam my German top model, thanks for your party spirit and for always coming to my office to cheer me up. I will remember our Coldplay concert as a highlight of the last four years! Ralitza, my best Bulgarian girl, thanks for your kindness, your generous smile, your strength and your adventurous mindset (except for the chilli pepper!). Your faith in me during the writing was a source of appeasement. And Tanya, my Canadian friend soul mate, thanks for simply being you. You made me feel so comfortable in such a short amount of time. I'm sure we were best friends in a former life! Thanks also for our self-defense classes/food trip nights/bubble teas, it helped me to survive this crazy writing time.

Et enfin à Laure, ou devrais-je dire Laure Bernard, Ph.D., (et future MD !). Tu as été mon bras droit dans cette aventure. Merci pour nos pauses thés qui ont rythmé nos trois années à Pasteur. Merci pour tes précieux conseils sur mon projet, et pour le brainstorming sur la discussion de ma thèse. Tes messages de soutien ont aussi été d'un grand réconfort. Tu resteras mon modèle de réussite en tant que doctorante. J'espère que la date de ma soutenance me portera chance pour marcher sur tes traces ! A mes amis les plus proches qui m'ont toujours soutenue, merci pour tous ces moments qui m'ont rappelé qu'il n'y avait pas que la thèse qui comptait ! Merci à Yassin pour nos incroyables aventures à travers le monde. Tu m'as toujours poussée à aller plus loin. Merci aussi pour ta positivité et ta folie légendaires. Grâce à toi, (et à nos soirées poké), j'ai toujours réussi à dédramatiser et voir le bon côté des choses. Tu as été un soutien vraiment précieux pendant toutes ces années. Mélody, ma biquette du sud, le yin qui équilibre mon yang depuis maintenant 14 ans… Merci d'avoir toujours cru en moi et de m'avoir rassurée quand j'en avais le plus besoin. Ton bouquet de fleurs a été fidèle à ton image : touchant et réconfortant. Mickaël, tu as été le premier (bien avant moi-même) à croire qu'un jour je ferais une thèse… je suis heureuse de t'avoir à mes côtés depuis tout ce temps. Merci pour ta motivation, pour nos soirées jeux de société et nos dimanches matin piscine avec Céline ;) Merci à Léa et Marina, mes ''Memphis roommates'' pour la vie. Qui aurait pu croire qu'on en arriverait là, après 3 thèses, de nombreuses soirées de folie, du poulet frit et… un bébé! Merci à mes danseuses orientales, Naïra, Sirine, Nadine, Claire, Marion, Malika, Adeline, Orianne, Vanessa, Zineb et Margot. Danser à vos côtés pendant trois ans a été une vraie bouffée d'oxygène. C'est grâce aux longues heures passées au microscope que j'ai pu améliorer mes isolations : bouger le bassin sans bouger les yeux de l'objectif ! Merci à ma famille adoptive normande, Sylvie, Mimoun, Yamna, Mynka, Kalany, Mehdi, Hafida, Barbara, et Gilles. Pour toutes les fois où vous m'avez prouvé que loin des yeux, cela ne signifiait pas loin du coeur.

Enfin, merci à ma famille sans qui je n'en serai pas arrivée là : à ma Maman, merci pour ton accueil. Tu as tout fait pour me rendre la vie plus facile et que je puisse me concentrer sur ma thèse. Merci pour nos discussions quotidiennes et ton envie d'écouter mes aventures. Tu es depuis toujours ma plus grande fan, merci pour ton soutien inconditionnel. Merci à ma cousine Kim, pour ton amour, ta joie de vivre, nos fous rires. Tu m'as toujours soutenue dans les moments difficiles. Merci pour ta confiance et la place que tu m'as donnée dans ta vie. Merci à ma soeur Evelyne, pour m'avoir transmis ta passion pour les études et pour les sacrifices que tu as fait pour que j'y arrive. Merci à mon frère Christophe pour ton soutien, tes blagues et nos soirées « mille borne » pendant la rédaction. Aux amours de ma vie, mes neveux et nièces Alicia, Nathanaël, Giulia, et Lucas. Pour toute la douceur, les rires et les câlins dont Tatie Zou avait grandement besoin. A ma cousine Romina, l'original Dr. Charifou. Merci pour tous les 'Ph.D. students memes' dans lesquels je me suis reconnue un peu trop souvent. A mes proches, tata Marie-Ange, tata Farida, tonton Rahim, pour le soutien moral.

Et enfin à mon Papa, moi qui pensais que la thèse serait l'épreuve la plus dure que j'aurais à traverser… J'espère que de là-haut, tu es fier de ce que j'ai pu accomplir. Et comme tu me l'as si bien dit, après la thèse, il est temps maintenant « de trouver un vrai travail, ma fille » :) Parce que ce manuscrit n'aurait jamais pu voir le jour sans toutes les personnes bienveillantes qui m'ont aidée, encouragée, soutenue, et fait rire pendant ces quatre années. Je pourrais écrire à nouveau plus de 200 pages pour vous exprimer ma gratitude. Mais pour m'éviter une belle tendinite, je vais m'arrêter sur un dernier mot. Simple. Mais comme on le dit si bien aux enfants, un mot magique… Merci.

remarkable plasticity throughout postnatal development. The MG involution is one of the major mammalian cell death and tissue remodeling events, when milkproducing epithelial cells are removed, and the MG returns to its near pregestation state, resting for further pregnancy. During my Ph.D., we showed that senescence was transiently induced during the irreversible phase of involution. The senescent program occurred specifically in the alveolar milk-producing luminal cells and correlated with the expression of the cell cycle inhibitor p16. In parallel, we established a novel organoid system to mimic MG gestation, lactation, and involution. In this ex-vivo model, we also highlighted the presence of senescent cells strictly during the involution-like process. To assess the biological impact of senescence in vivo, we used a teat sealing method to uncouple the reversible and irreversible phases of involution. We unveiled a close association between the withdrawal of lactogenic hormones occurring in the second phase of involution and the induction of the senescence program. To further define the physiological roles of senescence during involution, we treated mice with ABT-263, a senolytic compound inducing apoptosis of senescent cells. Interestingly, we observed an impaired tissue remodeling upon senescence elimination, with larger remaining alveolar structures and delayed adipocyte refilling. Moreover, in organoids from transgenic p16-3MR mice, we successfully removed senescent cells with ganciclovir and delayed the involution-like process. Taken together, both in vivo and ex-vivo models suggest an essential role of senescence in modulating the tissue remodeling phase of MG involution. Importantly, the involution process is intimately associated with postpartum breast cancer (PPBC), a cancer diagnosed within 10 years following delivery with a poor prognosis. Investigating how senescence impacts the microenvironment during the involution process might provide major insights to understand PPBC.

Résumé

La sénescence est une réponse à un stress biologique, caractérisée par un arrêt stable du cycle cellulaire. Néanmoins, les cellules restent métabolliquement actives et acquièrent un phenotype sécrétoire associé à la sénescence, avec la production d'un sécrétome complexe composé de cytokines, chémiokines, facteurs de croissance et modulateurs du remodelage de la matrice extracellulaire. La sénescence est associée à de nombreux processus pathologiques, comme la tumorigénèse et le vieillessement. Cependant, où, quand et comment la sénescence contribue aux processus physiologiques reste méconnu. Pour répondre à cette question, nous avons tiré profit de la glande mammaire (GM), un organe avec une plasticité remarquable pendant le développement post-natal. L'involution de la GM est l'un des évenements majeurs de mort cellulaire et de remodelage tissulaire chez les mammifères, lorsque les cellules épithéliales produisant le lait sont éliminées et que la GM retourne à un état similaire à celui pré-grossesse, attendant la prochaine gestation. Au cours de ma thèse, nous avons montré que la sénescence était induite transitoirement pendant la phase irréversible de l'involution. De plus, le programme de sénescence apparaissait spécifiquement dans les cellules luminales productrices de lait et corrélait à l'expression de l'inhibiteur du cycle cellulaire p16. En parallèle, nous avons établi un nouveau modèle d'organoides pour mimer la gestation, la lactation et l'involution de la GM. Dans ce modèle exvivo, nous avons aussi relever la présence de cellules sénescentes strictement lors du processus d'involution. Pour évaluer l'impact biologique de la sénescence in vivo, nous avons utilisé une méthode de scellement des mamelons pour découpler les phases réversible et irréversible de l'involution. Nous avons dévoilé une association étroite entre le sevrage des hormones lactogéniques qui a lieu lors de la seconde phase d'involution, et l'induction du programme de sénescence. Pour mieux définir les rôles physiologiques de la sénescence pendant l'involution, nous avons traités des souris avec de l'ABT-263, un composé sénolytique induisant l'apoptose des cellules sénescentes. Nous avons observé une altération du remodelage tissulaire suite à l'élimination des cellules sénescentes, avec des alvéoles résiduelles plus larges et un remplissage adipocytaire retardé. De plus, dans des organoides provenant de souris transgéniques p16-3MR, nous avons éliminé les cellules sénescentes avec succès grâce à l'administration de ganciclovir, ce qui a retardé le processus d'involution. Dans leur ensemble, les modèles in vivo et ex-vivo suggèrent un rôle important de la sénescence pour moduler la phase de remodelage tissulaire dans l'involution de la GM. Enfin, le processus d'involution est intiment lié avec le cancer du sein post-partum, un cancer diagnostiqué dans les 10 ans suivant une grossesse et associé à un mauvais pronostic. Explorer comment la sénescence impacte le microenvironnement lors de l'involution pourrait ainsi fournir de nouvelles connaissances pour mieux comprendre le cancer du sein post-partum. Introduction 20

I.

Cellular senescence A.

General introduction

Senescence is a term deriving from the latin root "senex", which can be translated by the adjective "old". Cellular senescence has been historically

described by Hayflick and Moorhead in the early 60s as the failure of nontransformed cells to divide indefinitely [START_REF] Hayflick | The serial cultivation of human diploid cell strains[END_REF]. They showed that human fetal lung fibroblasts could proliferate in vitro up to a defined number of divisions, termed the "Hayflick's limit". Independently of the origin of the human tissues, cells stopped replicating after 50±10 mitosis. This pioneering work refuted Carrel's long-standing dogma of cell immortality, based on chicken embryonic heart cells, which were supposedly grown in vitro for more than 34 years [START_REF] Carrel | On the Permanent Life of Tissues Outside of the Organism[END_REF]. After identifying technical issues in Carrel's methodology and reproducing Hayflick's observations worldwide for six decades, it is now widely accepted that cells have a limited replicative lifespan, after which they enter a state of cellular senescence.

Following this groundbreaking discovery and 60 years of broad research in this field, cellular senescence is now commonly defined as a permanent proliferative arrest due to the increased expression of cyclin-dependent kinase inhibitors (e.g., p16 INK4A ; p21 CIP1 ). A plethora of biological stresses can induce the irreversible cell cycle arrest (e.g., telomere exhaustion, DNA damage, oncogene activation, oxidative stress…). Depending on the cell type of origin or on the senescence-inducing signal, senescent cells do not exhibit a unique common biological barcode but a wide range of non-exclusive biomarkers (e.g., senescence-associated beta-galactosidase (SAβGal) histological staining, overexpression of cell cycle inhibitors, lack of proliferation hallmarks, enlarged morphology, or chromatin rearrangements). Although senescent cells are nonproliferative, they are resistant to apoptosis and remain metabolically active, acquiring secretive phenotypes called Senescence-Associated Secretory Phenotypes (SASPs). SASPs are heterogeneous and are composed of a variety of key cell players (e.g., inflammatory cytokines, chemokines; matrix remodeling factors, growth modulators…) [START_REF] Campisi | Cellular senescence: when bad things happen to good cells[END_REF].

The heterogeneity of senescent features is reflected in diverse in vivo roles of senescence. The senescence program has been conserved through evolution as an intrinsic defense mechanism to prevent the proliferation of damaged cells.

Therefore, senescence is a potent tumor suppression mechanism, avoiding uncontrolled replication of preneoplastic cells. The counterpart of this senescence-induced growth arrest is the exhaustion of stem and progenitor proliferative cells, contributing to aging [START_REF] Rodier | Four faces of cellular senescence[END_REF].

Senescence also has a major extrinsic role in tissues. Through the secretion of SASPs, senescent cells interact with neighboring cells and modify the tissue microenvironment. Transient induction of senescence has been recently shown to be beneficial in different contexts (e.g., wound healing, cellular plasticity upon tissue damage, or embryonic development). Whereas the inability to eliminate senescent cells leading to their accumulation is associated with well-exemplified detrimental effects (e.g., cancer cell proliferation, age-related diseases, chronic inflammation, or fibrosis) (Munoz-Espin and Serrano 2014).

Focus has been mainly placed on the deleterious dark side of senescence, leading to the flourishing of senotherapies [START_REF] Childs | Senescent cells: an emerging target for diseases of ageing[END_REF]). These newly developed therapeutic strategies aim to eliminate senescent cells or to modulate SASP functions, to extend healthy lifespan and improve age-related diseases.

However, senescence contribution and regulation in in vivo physiological processes still remains elusive. Better understanding the spatio-temporal regulation of physiological in vivo senescence is therefore essential to comprehend its pathological outcomes and its clinical relevance.

B. Main inducers of cellular senescence

Entrance into the senescence cell state can be triggered by a wide range of intrinsic and extrinsic signals, such as telomere dysfunction, persistent DNA damage, oncogenic activation, oxidative stress, or cytotoxic drugs (Figure 1). The developed senescence phenotype depends on the type of trigger signals, leading to the production of stimuli-specific SASP. Therefore, the senescence program can be categorized according to inducer stimuli, most of them being deeply characterized in vitro [START_REF] Collado | The power and the promise of oncogeneinduced senescence markers[END_REF].

Figure 1. Inducers of cellular senescence. The senescence program can be triggered by various stresses such as telomere erosion, persistent DNA damages, or oncogene activation. The cells interpret these signals as sub-lethal stresses and respond by entering an irreversible cell cycle arrest. Senescence can therefore be classified regarding its inducing stressor in replicative senescence, DNA damage-induced senescence, or oncogene-induced senescence. Adapted from [START_REF] Ngoi | The redox-senescence axis and its therapeutic targeting[END_REF]).

Replicative senescence

Replicative senescence is the first type of senescence historically identified by

Hayflick and is triggered by telomere attrition (Figure 1). Telomeres are repetitive sequences capping the end of linear chromosomes and function as a molecular clock, keeping track of the number of cell divisions. These sequences of 10-15kb are replicated by the telomerase, an enzyme competent only in embryonic stem cells but inactive in somatic stem cells. During each somatic cell division, normal DNA polymerases fail to replicate DNA ends, and as telomerases are inactive, each chromosome loses 50-200bp per S phase. Telomeres are shortened until reaching a critical size, which triggers the DNA damage response (DDR) machinery, and permanent cell cycle arrest [START_REF] Allsopp | Telomere length predicts replicative capacity of human fibroblasts[END_REF]).

DNA damage-induced senescence

Various stressors such as metabolites, chemical reagents, or physical agents also induce DNA damage and trigger DDR (Figure 1) [START_REF] Jackson | The DNA-damage response in human biology and disease[END_REF].

When DDR attempts to repair DNA fail, and damages remain unresolved, cells can either enter apoptosis or senescence. Events leading towards one or the other outcome are still not well defined, but it has been proposed that severe short-term DNA damages activate apoptosis, whereas mild DNA damages tend to promote senescence [START_REF] Petrova | Small molecule compounds that induce cellular senescence[END_REF].

Reactive oxygen species (ROS) accumulations are perceived as an oxidative stress and can lead to mutations in nuclear and mitochondrial DNA (Figure 1) [START_REF] Busuttil | Oxygen accelerates the accumulation of mutations during the senescence and immortalization of murine cells in culture[END_REF]. For example, an increase of ROS due to UVB exposure has been shown to induce senescence in keratinocytes [START_REF] Lewis | UVB-induced senescence in human keratinocytes requires a functional insulin-like growth factor-1 receptor and p53[END_REF].

Chemotherapies using ionizing irradiation or chemotherapeutic agents such as doxorubicin are also proposed to induce senescence through the formation of double-strand breaks [START_REF] Suzuki | Stress-induced premature senescence (SIPS)--influence of SIPS on radiotherapy[END_REF][START_REF] Roberson | Escape from therapy-induced accelerated cellular senescence in p53-null lung cancer cells and in human lung cancers[END_REF].

Oncogene-induced senescence

Oncogene-induced senescence (OIS) is a cell cycle arrest triggered by abnormal oncogene overexpression (Figure 1). OIS was described for the first time in response to Ras overexpression in human and mouse fibroblasts [START_REF] Serrano | Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a[END_REF]). Upon oncogene activation, cells enter into a burst of proliferation, perceived as a replicative stress signal triggering premature senescence.

Nowadays, OIS induction is reported after the deregulation of one of more than 50 genes classified as oncogenes. Therefore, OIS is a robust tumor suppression mechanism [START_REF] Gorgoulis | Oncogene-induced senescence: the bright and dark side of the response[END_REF].

C. Biomarkers of senescent cells

Cell cycle arrest

One prominent feature of senescent cells is their stable cell cycle arrest(Figure 2). Therefore, senescent cells lack proliferative markers, typically incorporation of 5-bromodeoxyuridine (BrdU) in vitro or Ki-67 immunostaining in vivo.

However, stable cell cycle arrest is not exclusive to senescent cells, as quiescent and differentiated post-mitotic cells are also not replicating (Figure 3). Unlike senescent cells, quiescent cells are in a temporary arrest, called the G0 phase, with low metabolism and can re-enter into the G1 phase upon adequate mitogen stimuli [START_REF] Pardee | G1 events and regulation of cell proliferation[END_REF] Quiescence, differentiation, and senescence are three cellular states with a withdrawal from the cell cycle, but they can be separated based on the reversibility of the cell cycle arrest, the presence of macromolecular damages, the activated signaling pathways, and the production of a secretome.

Arrows figure the interconnection between the states; once senescent, cells cannot reverse their cell fate. From [START_REF] Gorgoulis | Cellular Senescence: Defining a Path Forward[END_REF]. encoding for p16 and ARF (p19 ARF in mice, p14 ARF in humans) and CDKN2B

Expression of cyclin-dependent kinase (CDK) inhibitors

encoding for p15 (Figure 4).

CDKN2A locus is epigenetically repressed in normal proliferative cells [START_REF] Gil | Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all[END_REF]. Thus, Cyclin D-CDK4/6 can phosphorylate Rb, dissociating from a complex with the transcription factor E2F. Free E2F enters the nucleus and transactivates genes involved in the progression of the cell cycle (Figure 4). Upon senescence induction, epigenetic de-repression of CDKN2A locus, higher promoter accessibility, and increased protein stability are combined strategies to express p16 (Hernandez-Segura, Nehme, and Demaria 2018). p16 binds to CDK4/6, preventing its kinase activity. Rb is maintained in a hypophosphorylated state and sequestrates E2F, preventing transcription of proliferative genes and leading to G1 cell cycle arrest (Figure 4) [START_REF] Serrano | The tumor suppressor protein p16INK4a[END_REF].

Therefore, p16 expression is a robust and powerful biomarker of in vivo senescence (Figure 2) [START_REF] Krishnamurthy | Ink4a/Arf expression is a biomarker of aging[END_REF]).

b)

p53/p21 CIP1 pathway p21 CIP1 , or p21, is also a critical cyclin-dependent kinase inhibitor transcriptionally induced by p53 activity. p21 inhibits the kinase activity of Cyclin E-CDK2, leading to hypo-phosphorylation of Rb, sequestration of E2F, and inhibition of cell cycle progression (Figure 4) [START_REF] Deng | Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control[END_REF].

Morphological changes a) Nucleus

Lamin B1 is a protein present on the nucleoplasmic side of the inner nuclear membrane, necessary for nuclear 3D structure and chromatin organization [START_REF] Gerace | Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution[END_REF]. In many types of senescence, Lamin B1 is down-regulated, destabilizing the nuclear integrity [START_REF] Freund | Lamin B1 loss is a senescence-associated biomarker[END_REF]. This decline induces large-scale chromatin rearrangements, as lamina-associated domains spatially cluster within the nucleus and lose their condensation state.

Senescence-associated heterochromatin foci (SAHFs) enriched in repressive heterochromatin marks are proposed to be a compensatory mechanism to maintain heterochromatin status in senescent cells [START_REF] Chandra | Chromosome organisation during ageing and senescence[END_REF]. SAHFs heterochromatinization specifically represses cell cycle genes normally activated transcriptionally by E2F [START_REF] Narita | Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence[END_REF]) (see "Expression of cyclin-dependent kinase inhibitors" for further details). Loss of Lamin B1 and appearance of SAHFs are found in different types of senescence but are not universal markers (Figure 2).

b) Cytoplasm

In vitro, senescent cells as fibroblasts present an enlarged size, with a flattened morphology, mainly due to cytoskeleton rearrangements [START_REF] Wang | Increased organization of cytoskeleton accompanying the aging of human fibroblasts in vitro[END_REF]. However, in vivo senescent cells preserve their 3D structure, presumably due to constraints applied by the tissue architecture (Figure 2) [START_REF] Herranz | Mechanisms and functions of cellular senescence[END_REF].

Senescence-associated !-galactosidase (SA!Gal)

Senescent cells exhibit an expansion of lysosomes in number and size. This extended lysosomal content is correlated with overexpression and higher activity of a lysosomal enzyme named !-galactosidase (Figure 2) [START_REF] Kurz | Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells[END_REF]. This enzyme is not required for a functional senescence program, as patients deficient for the !-galactosidase encoding gene GLB1 do not show any impairment in establishing or maintaining a senescent phenotype [START_REF] Lee | Senescence-associated beta-galactosidase is lysosomal beta-galactosidase[END_REF]). However, !-galactosidase activity is a predominant marker of senescence.

This enzyme is active at acidic pH 4.0 in lysosomes of normal cells under physiological conditions. In senescent cells, reflecting the expanded lysosomal compartment, this enzyme is active under sub-optimal pH 6.0. This pH-linked property is widely used in the classical cytochemical assay termed "SA!Gal staining", in which the chromogenic substrate X-Gal (5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside) is cleaved by SA-!-galactosidase, forming a blue-dyed precipitate observed in bright-field [START_REF] Dimri | A biomarker that identifies senescent human cells in culture and in aging skin in vivo[END_REF]. A newly developed fluorogenic compound, C12FDG (5-Dodecanoylaminofluorescein di-β-Dgalactopyranoside) [START_REF] Cahu | A sensitive method to quantify senescent cancer cells[END_REF], can replace X-Gal substrate for fluorescent analyses as fluorescence-activated cell sorting (FACS).

SA!Gal staining is a powerful labeling of expanded lysosomal content, which is a characteristic non-exclusive to senescent cells. Indeed, other cell types with autophagy properties as macrophages can be positive for SA!Gal staining [START_REF] Bursuker | Beta-galactosidase--an indicator of the maturational stage of mouse and human mononuclear phagocytes[END_REF], rising the need to combine this widely used assay with additional senescent markers.

Resistance to apoptosis

Apoptosis is an extreme stress-response inducing a tightly controlled cell death, destroying cellular components and leading to clearance by engulfing cells [START_REF] Kerr | Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics[END_REF]. Apoptosis and senescence are described as alternative cell fates for damaged cells beyond repair. Decision to undergo one or the other program is not fully understood yet, especially as the same stressors can trigger both pathways. However, once engaged in senescence, cells lock this decision by mechanisms to resist the alternative fate.

Compared with normal proliferating cells, senescent cells are more resistant to various pro-apoptotic stimuli in vitro, such as UV damage, withdrawal of serum in culture, oxidative stress, or addition of cytotoxic compounds (Soto-Gamez, Quax, and Demaria 2019).

More specifically, senescent cells are proposed to become resistant to apoptosis by either up-regulating transcription or increasing transcripts stability of BCL-2 anti-apoptotic genes [START_REF] Ryu | Failure of stress-induced downregulation of Bcl-2 contributes to apoptosis resistance in senescent human diploid fibroblasts[END_REF]) [START_REF] Yosef | Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL[END_REF]. Senescent cells can also epigenetically repress the pro-apoptotic gene Bax [START_REF] Sanders | Histone modifications in senescence-associated resistance to apoptosis by oxidative stress[END_REF].

Therefore, resistance to apoptosis is an interesting property of certain senescent cells (Figure 2). Furthermore, chemical compounds specifically targeting the apoptosis resistance/increased survival pathways is a newly developed strategy to specifically eliminate senescent cells (Figure 13) (van Deursen 2019) (see "Senolytics" for further details).

Acquisition of Senescent Associated Secretory Phenotype (SASP)

Senescent cells do not proliferate but remain metabolically active and secrete a set of molecules termed senescent-associated secretory phenotype (SASP). The composition of this secretome is cell type-, stress-, and time-dependent and is a highly heterogeneous combination of cytokines, growth factors, and extracellular matrix proteases [START_REF] Coppe | The senescenceassociated secretory phenotype: the dark side of tumor suppression[END_REF]. Moreover, SASP functions depend on the nature of the secreted factors and the responsiveness of the microenvironment (see "SASP" for further details on SASP composition and functions).

Consequently, the utility of SASP as a biomarker to identify senescent cells in vivo is limited (Figure 2) [START_REF] Herranz | Mechanisms and functions of cellular senescence[END_REF].

D. Senescence Associated Secretory Phenotype (SASP)

1.

Heterogeneity of SASP composition

Senescent cells can influence their microenvironment through the secretion of a combination of molecules termed Senescence Associated Secretory Phenotype (SASP) (Figure 5). Large-scale transcriptomic studies have been performed to attempt to define a widely common core of SASP. But SASP is dynamically regulated and is highly heterogeneous depending on the tissue of origin and the and IL-8 [START_REF] Chien | Control of the senescence-associated secretory phenotype by NF-kappaB promotes senescence and enhances chemosensitivity[END_REF][START_REF] Kuilman | Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network[END_REF].

Mechanistic regulation of SASP

SASP

Autocrine/paracrine reinforcement of senescence

SASP functions are pleiotropic and depend not only on SASP composition but also on the response of surrounding cells exposed to this senescence-messaging secretome.

SASPs factors, such as IL-1α, Il-6, and IL-8, are proposed to reinforce senescence growth arrest in an autocrine manner. Via a positive feedback loop, they enhance NF-κB and C/EBPβ activity, amplifying SASP signaling [START_REF] Orjalo | Cell surface-bound IL-1alpha is an upstream regulator of the senescence-associated IL-6/IL-8 cytokine network[END_REF]).

SASP secreted from senescent cells also propagates the senescence growth arrest to surrounding proliferative cells, a phenomenon termed paracrine senescence or secondary senescence (Figure 5). Both in vitro treatment of nonsenescent cells with conditioned medium from senescent cells and in vivo injections of senescent cells in recipient mice induce a complete senescence response of neighboring cells. As revealed by unbiased quantitative transcriptomic analysis, paracrine senescence is mediated mainly by various members of the TGF-β family, VEGF, and chemokines [START_REF] Acosta | A complex secretory program orchestrated by the inflammasome controls paracrine senescence[END_REF].

Mediation of senescence to neighboring cells can also be done via Notch signaling in a juxtacrine manner [START_REF] Kirschner | Functional heterogeneity in senescence[END_REF]. Interestingly, as primary and secondary senescent cells can activate different senescent pathways, they might have a distinct gene expression profile, adding complexity to the heterogeneity of the senescent phenotype [START_REF] Kirschner | Functional heterogeneity in senescence[END_REF].

Interaction with the immune system

One of the major roles of the immune system is to remove dying cells to preserve the integrity of the body. Communication between senescent cells and immune cells is very complex and poorly defined but proposed to occur via SASP production (Figure 5). Indeed, many SASPs are revealed to be pro-inflammatory as IL-6, IL-8, monocyte chemoattractant proteins (MCPs), macrophage inflammatory proteins (MIPs), or TGFβ. Through their SASP, it is proposed that senescent cells could promote inflammation, chemoattract immune cells, and favor immune cell migration by remodeling the extracellular matrix. Then, activated immune cells would help for the specific removal of senescent cells while leaving surrounding healthy cells intact [START_REF] Prata | Senescent cell clearance by the immune system: Emerging therapeutic opportunities[END_REF]).

Among immune cells, senescent cells might preferentially interact via the SASP with neutrophils, macrophages, natural killer (NK) cells, and T lymphocytes to help for their clearance [START_REF] Xue | Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas[END_REF]).

a) Neutrophils

Via the sensing of chemoattractant IL-8, neutrophils are generally the first actors to be recruited on the inflammation site. They perform a cytotoxic process known as NETosis, with the formation of extracellular traps by releasing of chromosomal DNA into the extracellular environment. Neutrophils might contribute to senescence surveillance as neutrophil depletion using antibodies impairs senescence clearance from liver [START_REF] Kang | Senescence surveillance of pre-malignant hepatocytes limits liver cancer development[END_REF]).

b) Macrophages

Multiple studies showed a strong correlation between macrophages, professional phagocytic actors, and senescence clearance. For example, in vitro, macrophages migrate in response to senescent conditioned medium from human fat progenitor cells [START_REF] Xu | JAK inhibition alleviates the cellular senescence-associated secretory phenotype and frailty in old age[END_REF]. In vivo, macrophages are proposed to participate in the clearance of senescent cells during salamander limb regeneration, as depletion of macrophages induces accumulation of senescent cells [START_REF] Yun | Recurrent turnover of senescent cells during regeneration of a complex structure[END_REF]. How macrophages trigger senescent cells is not fully elucidated, but proposed mechanisms involve the secretion of cytotoxic factors (M1 subtype) and phagocytosis (M2 subtype).

c) NK cells

NK cells might also be involved in senescence clearance. In vitro, senescence was induced in lung fibroblasts IMR90 cells, and both senescent and proliferating cells were incubated with NK cells. Interactions in the co-culture system were followed using time-lapse imaging and showed a preferential killing of senescent cells mediated by NK cells [START_REF] Krizhanovsky | Senescence of activated stellate cells limits liver fibrosis[END_REF]. Moreover, impairing NK functions correlates with levels of senescent cells. Indeed, in a model of liver fibrosis, treatment with NK neutralizing antibodies revealed an impaired clearance of senescent hepatic stellate cells, whereas stimulation of NK cell activity correlated with a decreased number of senescent cells [START_REF] Krizhanovsky | Senescence of activated stellate cells limits liver fibrosis[END_REF].

d) T lymphocytes

Correlative studies suggest that T lymphocytes might also participate in senescence surveillance as well as macrophages and NK cells. IL-6, one of the major SASP components, is a cytokine known to activate two populations of lymphocytes, CD8+ cytotoxic T lymphocytes (T-CD8+) and T-CD4+ helper cells (T-CD4+) [START_REF] Bettelli | Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells[END_REF]. T-CD8+ express the receptor NKG2D, a receptor expressed in NK cells and stimulated for their cytotoxicity against senescent cells in vitro [START_REF] Hu | Regulation of NKG2D(+)CD8(+) T-cellmediated antitumor immune surveillance: Identification of a novel CD28 activation-mediated, STAT3 phosphorylation-dependent mechanism[END_REF][START_REF] Sagiv | NKG2D ligands mediate immunosurveillance of senescent cells[END_REF]. T-CD4+ helpers might also be involved in senescence surveillance. Indeed, patients with HIV infection, which depletes the pool of T-CD4+ helper, present an accumulation of senescent cells induced by cirrhosis [START_REF] Kang | Senescence surveillance of pre-malignant hepatocytes limits liver cancer development[END_REF].

To summarize, it is proposed that senescent cells could interact via their SASP with many key immune system players for their clearance. Therefore, impairment of immune surveillance could be a central mechanism leading to the accumulation of senescent cells.

E.

In vivo roles of senescence

Senescence is a complex mechanism influencing a plethora of in vivo processes.

Depending on cell types, secreted factors, and spatio-temporal dynamic, senescent cells exhibit beneficial or detrimental effects that are detailed thereafter (Figure 6). Senescent secretome can influence the microenvironment through different aspects, such as cell proliferation and differentiation, matrix remodeling, or immune reaction. Therefore, senescence is involved in many processes and plays beneficial roles as in embryogenesis, tissue repair upon injury, and tumor suppression. However, senescence is also associated with detrimental processes, promoting tumor progression, chronic inflammation, and age-related diseases. This balance between beneficial and detrimental effects might be time-dependent, as transient SASP is proposed to be salutary, whereas persistent SASP creates a deleterious microenvironment. From (Paramos-de-Carvalho, Jacinto, and Saude 2021).

Senescence and cancer

Senescence has dichotomous roles in cancer (Figure 7). In essence, senescence is a potent tumor mechanism. Indeed, senescent cells are irreversibly cell cycle arrested and are not responding to mitotic signals, which are intrinsic obstacles to oncogenesis. Cells mutated for p53 or p16 proteins fail to senesce, and corresponding p53 or p16 knockout mice show a premature tumor development due to unrestrained proliferative capacity [START_REF] Ghebranious | Mouse models in tumor suppression[END_REF].

Moreover, when exposed to oncogene overexpression, cells preferentially enter into oncogene-induced senescence (OIS) state, which restricts their tumorforming capacity [START_REF] O'brien | Suppression of tumor growth by senescence in virally transformed human fibroblasts[END_REF].

Anti-tumor effect of senescence is mainly attributed to the intrinsic cell cycle arrest, but senescent cells are also anti-tumorigenic via their secretome. Many SASPs factors induce senescence in a paracrine manner (see "Autocrine/paracrine reinforcement of senescence" for further details) and therefore could prevent excessive proliferation of surrounding precancerous cells during early tumorigenesis (Perez-Mancera, Young, and Narita 2014).

Moreover, through their SASPs, senescent cells might recruit and stimulate the immune system (see "Interaction with the immune system" for further details), and this immune surveillance contributes to tumor clearance [START_REF] Swann | Immune surveillance of tumors[END_REF]. In p53 deficient hepatocellular carcinomas, reactivation of p53 was sufficient to induce senescence, production of inflammatory cytokines, and activation of an immune response against cancerous cells, reducing tumor size [START_REF] Xue | Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas[END_REF].

However, senescent cells can also display potent antagonistic pro-tumorigenic effects in a SASP-dependent manner (Figure 7). First of all, senescent cells can specifically stimulate the proliferation of premalignant and malignant cells, which are more sensitive to SASP, notably composed of growth factors. Via their SASPs, senescent cells promoted the growth of S1 human mammary epithelial cells co-cultured in transwells without affecting normal epithelial cells. Coinjection of epithelial cells with senescent cells also favored tumorigenesis in recipient mice [START_REF] Krtolica | Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging[END_REF]. Secondly, senescent cells can promote angiogenesis and tumor vascularization, notably via secretion of pro-angiogenic factors such as VEGF, increasing the size and number of blood vessels [START_REF] Coppe | Secretion of vascular endothelial growth factor by primary human fibroblasts at senescence[END_REF]. Thirdly, senescent cells secrete a variety of proteases as MMP-2 and MMP3, degrading the extracellular matrix and disrupting tissue structures. This permissive microenvironment supports cancer cell dissemination in vivo.

Moreover, senescent fibroblasts can promote epithelial-mesenchymal transition (EMT), thus enhancing cell migration and invasion [START_REF] Coppe | Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor[END_REF].

Therefore, senescence can inhibit or enhance tumor progression. It has been proposed that this balance between anti-and pro-tumorigenic effects depends on the heterogeneous SASP composition and the time senescent cells have to modulate their microenvironment. Transient presence of senescent cells might be beneficial to limit tumor development, whereas persistence of senescent cells might favor tumorigenesis (See "Acute versus chronic senescence" for further details) [START_REF] Yang | The Paradoxical Role of Cellular Senescence in Cancer[END_REF]).

Figure 7. The paradoxical roles of senescence in cancer development.

Senescence is a major tumor suppression mechanism, as the main characteristic of senescence is the irreversibility of the cell cycle arrest, limiting the proliferation of pre-cancerous cells. Moreover, through SASP, senescent cells stimulate immune surveillance, promoting the elimination of tumor cells. However, senescence can also promote tumorigenesis in different ways. SASP can help with the maintenance of stem cell features and increases cellular plasticity. The growth of cancerous cells is also supported via angiogenesis and the recruitment of immature immunosuppressive cells, impairing immune surveillance. SASP also favors the dispersion of tumor cells by promoting the epithelial-to-mesenchymal transition (EMT) and tissue remodeling of the microenvironment. Finally, senescence can confer drug resistance, as chemotherapies classically target hyperproliferative cells. Senescent cancerous cells can stay dormant, prone to events to escape senescence, such as mutation of cell cycle inhibitors, inducing tumor relapse. From [START_REF] Yang | The Paradoxical Role of Cellular Senescence in Cancer[END_REF]).

Senescence and aging / age-related pathologies

It has been postulated for decades that senescence is linked to aging and agerelated pathologies. Senescent cells accumulate in aged mice and in various tissues such as the lung, liver, or gut [START_REF] Wang | DNA damage response and cellular senescence in tissues of aging mice[END_REF]). In primates, senescent cells even represent up to 15% of total cells in old animals [START_REF] Herbig | Cellular senescence in aging primates[END_REF].

Removing senescent cells in vivo throughout life in the INK-ATTAC model extended the median lifespan of naturally aged mice and demonstrated the causative link between senescence and aging [START_REF] Baker | Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan[END_REF]) (see "Strategies to target senescent cells" for further details on INK-ATTAC mouse model).

The presence of senescent cells also correlates with the development of numerous age-related pathologies such as Alzheimer's disease or chronic obstructive pulmonary disease (COPD) [START_REF] Bhat | Astrocyte senescence as a component of Alzheimer's disease[END_REF][START_REF] Adnot | Telomere Dysfunction and Cell Senescence in Chronic Lung Diseases: Therapeutic Potential[END_REF].

Moreover, senescence abolition using INK-ATTAC also alleviated age-associated disorders in multiple organs such as skeletal muscle or adipose tissue, extending healthspan [START_REF] Baker | Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders[END_REF].

Age-associated impaired tissue regenerative capacity is possibly due to stem cell exhaustion. Indeed, to avoid pre-neoplastic transformation, stem cells are prone to senescence induction, limiting their self-renewal capacity [START_REF] Oh | Stem cell aging: mechanisms, regulators and therapeutic opportunities[END_REF]. In aged muscle, satellite muscle stem cells switch from their quiescence status to a senescent phenotype, impairing regenerative muscle functions. The microenvironment also regulates cell stemness, and aging of the niche also leads to an impairment of their stem cell functions [START_REF] Fujita | Stem cell aging and the implications for stem cell-based therapies for aging-related diseases and aged tissues[END_REF]. Overall, stem cell aging is partially accountable for loss of tissue regeneration [START_REF] Sousa-Victor | Geriatric muscle stem cells switch reversible quiescence into senescence[END_REF].

Accumulation of senescent cells has been mainly described as detrimental in cancer and age-related pathologies. However, artificial induction of senescence might also have beneficial effects on chronic diseases. Nutlin-3a is an inhibitor of murine double minute 2 (MDM2), preventing the interaction between MDM2 and p53 and related p53 degradation. Interestingly, administration of Nutlin-3a induced p21-p53 senescent associated cell cycle arrest in pulmonary artery smooth muscle cells (PA-SMCs). This senescent inducer treatment showed therapeutic effects as it partially reverted pulmonary hypertension [START_REF] Mouraret | Activation of lung p53 by Nutlin-3a prevents and reverses experimental pulmonary hypertension[END_REF]).

Senescence and tissue repair

Emerging evidence suggests that senescence has beneficial roles in tissue repair.

Senescent cells can curb fibrosis, notably through over-secretion of enzymes responsible for extracellular matrix degradation and stimulation of immune surveillance. Indeed, upon liver damage, induced senescent hepatic stellate cells create a fibrotic scar, whose elimination by the immune system, in turn limits liver fibrosis. In mice deficient for p16 and p53, hepatic stellate cells bypass senescence and proliferate abnormally, leading to exacerbated liver fibrosis [START_REF] Krizhanovsky | Senescence of activated stellate cells limits liver fibrosis[END_REF]. During wound closure, skin senescent fibroblasts also secrete proteins from the extracellular matrix as CCN1, which induces anti-fibrotic gene expression. CCN1 deficient mice present excessive fibrosis, a phenotype restored with topical application of purified CCN1 protein [START_REF] Jun | The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing[END_REF].

A transgenic mouse model allowing targeted elimination of p16 + cells has been used to confirm the beneficial role of senescence in wound healing, as removal of senescent cells delayed the proper kinetic of wound closure. Secretion of SASP component PDGF-AA during early wound healing has been proposed to drive differentiation of myofibroblasts, thus optimizing wound closure. Topical application of recombinant PDGF-AA on the skin of these transgenic mice, treated to eliminate senescent cells, was sufficient to restore a correct wound closure [START_REF] Demaria | An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA[END_REF].

Tissue repair has also been studied in salamanders, an organism with a remarkable capacity for regenerating complex structures such as sections of its nervous system, portions of its heart, and the totality of its limbs and tail [START_REF] Joven | Model systems for regeneration: salamanders[END_REF]. Upon limb ablation, senescence is rapidly triggered at the blastema, where progenitors are generated and proliferate. Senescent cells disappear in the fully regrown limb, emphasizing the potential beneficial role of senescence in tissue regeneration [START_REF] Yun | Recurrent turnover of senescent cells during regeneration of a complex structure[END_REF].

Senescence and embryogenesis

Senescence has been mainly studied as a response to a stress signal. Surprisingly, a growing number of studies fuel the hypothesis that senescence might have a role in programmed development. Indeed, senescent cells were spotted in embryos from a wide variety of species, including human, mouse, chicken, or zebrafish [START_REF] Munoz-Espin | Programmed cell senescence during mammalian embryonic development[END_REF][START_REF] Storer | Senescence is a developmental mechanism that contributes to embryonic growth and patterning[END_REF][START_REF] Villiard | Senescence gives insights into the morphogenetic evolution of anamniotes[END_REF].

Interestingly, developmental senescence seemed to be induced preferentially in transient tissues such as the mesonephros and structures intensively remodeled as the apical ectodermal ridge (AER), region of limb outgrowth, and patterning.

In mice and humans, embryonic senescent cells were positive for p21 but independent of other common markers such as p16, ARF, or DNA damage. In mice embryos genetically ablated for p21, mesonephros regression was delayed but finally caught up by a compensatory apoptosis program, resulting in no significant developmental defects at birth [START_REF] Munoz-Espin | Programmed cell senescence during mammalian embryonic development[END_REF][START_REF] Storer | Senescence is a developmental mechanism that contributes to embryonic growth and patterning[END_REF]. Interestingly, senescence appears tightly regulated in time and space during embryonic development, as well-defined senescent patterns were identical in each studied embryo. Therefore, these studies suggested for the first time that senescence was not only a stochastic response to cellular damage but might play a beneficial role in programmed physiological events. Especially, the senescence program might contribute to the removal of transient structures and to the precise tissue remodeling associated with embryonic development [START_REF] Rhinn | Cellular senescence in development, regeneration and disease[END_REF]. Senescent cells have also been detected in the placenta, an organ of fetal origin, allowing exchanges between the mother and the fetus. Senescence is proposed to be a stress-response to the cell fusion at the origin of the formation of the syncytiotrophoblast, where it prevents cell proliferation [START_REF] Chuprin | Cell fusion induced by ERVWE1 or measles virus causes cellular senescence[END_REF].

Interestingly, programmed physiological senescence has been mainly documented in embryonic tissues but remains poorly defined in adult homeostasis. Recently, it has been shown that senescent cells appear in the human uterus. To favor embryo implantation, endometrial stromal cells specialize in secretory decidual cells, which promote the formation of the placenta. From the mid to the late luteal phase of the menstrual cycle, differentiation in the decidual lineage was associated with a cell cycle arrest, and some decidual cells exhibited a senescent phenotype, with SA!gal and p16 + cells noticed in the uterus. It is proposed that SASP secreted by decidual senescent cells created a pro-inflammatory microenvironment promoting tissue remodeling, which makes the uterus prone to embryo implantation [START_REF] Brighton | Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium[END_REF]. This process might be evolutionary conserved, as senescent cells have also been detected in the murine uterus [START_REF] Egashira | F4/80+ Macrophages Contribute to Clearance of Senescent Cells in the Mouse Postpartum Uterus[END_REF].

Acute versus chronic senescence

In conclusion, senescence is dichotomic and triggers beneficial or detrimental effects depending on the context (Figure 8). Currently, the proposed paradigm defines acute senescence as beneficial. Proliferation arrest and transient SASP

F. Strategies for senescent cells identification or elimination

Genetically modified mouse models

As previously discussed, a common marker solely expressed in all senescent cell types has not been discovered yet. However, as many senescent cells express the cell cyclin-dependent kinase inhibitor p16, we propose to review the genetic approaches to visualize or eliminate senescent cells based on p16 increased expression.

a) INK4A/ARF knockout

INK4A/ARF locus is a genomic sequence containing the gene Cdkn2a, encoding for two overlapping cyclin-dependent inhibitors, p16 and p19/Arf (Figure 9).

Both proteins originate from two different transcripts, sharing common exons 2 and 3 but with an alternative reading frame in the first exon. Therefore, p16 and p19/Arf proteins do not share a sequences homology and have distinct effects.

INK4A/ARF knockout (KO) mice were generated by homologous recombination of exons 2 and 3, depleting both p16 and p19 [START_REF] Stone | Complex structure and regulation of the P16 (MTS1) locus[END_REF] [START_REF] Serrano | Role of the INK4a locus in tumor suppression and cell mortality[END_REF].

b) INK-ATTAC

INK-apoptosis through targeted activation of caspase (ATTAC) is a transgenic mouse model inducing specific cell death of p16 positive cells (Figure 10). The ATTAC transgene allows the expression of an intracellular membrane-bound myristoylated FK506-binding-protein-caspase 8 (FKBP-Casp8). Upon administration of a synthetic drug, AP20187, FKBP-Casp8 forms an active dimer inducing apoptosis. The ATTAC transgene was firstly described in FAT-ATTAC mouse model, in which expression was regulated under a Fabp4 promoter, therefore promoting apoptosis specifically in adipocytes [START_REF] Pajvani | Fat apoptosis through targeted activation of caspase 8: a new mouse model of inducible and reversible lipoatrophy[END_REF] [START_REF] Baker | Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders[END_REF]). However, some senescent cells from various tissues such as liver or colon, are not efficiently triggered by the INK-ATTAC system [START_REF] Baker | Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan[END_REF]. This tissueselective elimination of senescent cells remains unclear but has raised concerns about the truncated p16 promoter sequence that might not fully resemble endogenous p16 expression.

c) p16-3MR

p16-trimodality reporter (3MR) is a transgenic mouse model taking advantage of the artificial protein LUC/mRFP/HSV-TK containing fused functional domains of Renilla luciferase (LUC), monomeric red fluorescent protein (mRFP), and truncated herpes simplex virus 1 thymidine kinase (HSV-TK) (Figure 11) [START_REF] Ray | Imaging tri-fusion multimodality reporter gene expression in living subjects[END_REF]). In p16-3MR, 3MR fused protein was inserted in a bacterial artificial chromosome (BAC) containing 50kb of p16 INK4 locus, with inactivated adjacent p16 INK4 and p19 ARF genes, ensuring that the full promoter of p16 drove expression of 3MR fusion. p16-3MR mice were described as an effective model to detect p16 + senescent cells by luciferase luminescence; to sort them using mRFP fluorescent signaling; and to kill them using the synthetic drug ganciclovir (GCV).

GCV has a low affinity for endogenous cellular TK but is metabolized specifically by HSV-TK. HSV-TK converts the drug into a toxic DNA chain terminator, which fragments mitochondrial DNA. This cell death signal triggers apoptosis in p16 + senescent cells. In vivo elimination of p16 + senescent cells in p16-3MR mice demonstrated for the first time a beneficial role of SASP in wound healing [START_REF] Demaria | An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA[END_REF]. However, the 3MR cassette was not highly expressed in vivo due to a low level of p16 mRNA expression. By correlation, this model failed to efficiently track all p16 + cells due to a low p16-driven level of mRFP fluorescence [START_REF] Grosse | Defined p16(High) Senescent Cell Types Are Indispensable for Mouse Healthspan[END_REF]. e) p16-Cre/R26-mTmG and p16-Cre/DTA

In parallel, two complementary mouse models were developed to trace and ablate p16+ cells efficiently. Three elements, a constitutive CRE recombinase, a thymidine kinase, and a fluorescent dtTomato reporter, were fused at the end of the third exon of p16. This knock-in p16-Cre mouse line was then crossed with two different reporter mouse models, p16-Cre/R26-mTmG, to trace p16 + cells and p16-Cre/DTA to eliminate constitutively p16 + cells without the use of an exogenic compound. These models were used to define a population of senescent vascular endothelial cells in liver sinusoids of aged animals. Depleting this p16 + population disrupted the blood-liver barrier, leading to health deterioration [START_REF] Grosse | Defined p16(High) Senescent Cell Types Are Indispensable for Mouse Healthspan[END_REF]).

Senolytics

Senolytics are chemicals able to target and kill senescent cells. As senescent cells are resistant to apoptosis, the primary hypothesis-driven strategy to kill senescent cells is to find compounds that could break down pro-survival barriers (Figure 13) [START_REF] Kirkland | Senolytic drugs: from discovery to translation[END_REF].

a) Dasatinib and Quercetin

Dasatinib and quercetin were the first senolytics identified to induce senescent cell death [START_REF] Zhu | The Achilles' heel of senescent cells: from transcriptome to senolytic drugs[END_REF]. Dasatinib is an approved anti-cancer tyrosine kinase inhibitor targeting Src kinase [START_REF] Araujo | Dasatinib: a potent SRC inhibitor in clinical development for the treatment of solid tumors[END_REF]. Quercetin is a natural flavonoid, which can impair glycolysis and inhibit anti-apoptotic Bcl-2

and Bcl-XL proteins [START_REF] Wu | Quercetin Inhibits the Proliferation of Glycolysis-Addicted HCC Cells by Reducing Hexokinase 2 and Akt-mTOR Pathway[END_REF][START_REF] Primikyri | Direct binding of Bcl-2 family proteins by quercetin triggers its pro-apoptotic activity[END_REF]. Dasatinib and quercetin have senolytic activities against senescent human primary adipocyte progenitors and senescent human umbilical vein endothelial primary cells respectively. But the combination of dasatinib + quercetin (D+Q) synergizes their senolytic effects and in vivo administration of the combination D+Q extends healthspan and lifespan of treated mice [START_REF] Xu | Senolytics improve physical function and increase lifespan in old age[END_REF]. Co-administration of D+Q has been used in clinical trials to improve the health conditions of patients with idiopathic pulmonary fibrosis and diabetic kidney disease [START_REF] Hickson | Senolytics decrease senescent cells in humans: Preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals with diabetic kidney disease[END_REF]). However, the mechanisms of action of combined drugs remain unclear.

Moreover, their senolytic activity remains restrained to specific cell types. As the senescent phenotype is heterogeneous, there is a need to identify senolytics with broad-spectrum activity to target larger populations of senescent cells.

b) ABT-263

To discover new potent senolytics, a wide in vitro screen of compounds was performed. Senescence was triggered in WI-38 fibroblasts by an extensive range of inducers such as irradiation, replicative stress, or oncogene overexpression.

The survival of these senescent fibroblasts was then compared to the survival of non-senescent human WI-38 fibroblasts upon treatment with different chemicals. This study identified ABT-263, a compound also known as Navitoclax, as a potent senolytic [START_REF] Chang | Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice[END_REF].

ABT-263 is an orally bioavailable inhibitor of anti-apoptotic proteins Bcl-2, Bcl-XL, and Bcl-W, which are upregulated in various senescent cell types. Bcl-2 family proteins sequestrate pro-apoptotic molecules and prevent senescent cell death. ABT-263 is a Bcl-2 homology domain 3 (BH3) mimetic, which binds to BCL-2 anti-apoptotic proteins. These interactions prevent the sequestration of pro-apoptotic proteins as BIM, which freed acts to induce apoptosis [START_REF] Tse | ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor[END_REF].

ABT-263 drug has a very powerful in vitro senolytic activity species-cell typeand senescence-inducing stress signal-independent. It exhibits cytotoxicity against senescent mouse embryonic fibroblasts, senescent human fibroblasts, and senescent human renal epithelial cells [START_REF] Chang | Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice[END_REF]. ABT-263 has also been used in vivo to target senescent hematopoietic stem cells (HSCs) induced by ionizing irradiation, allowing the expansion of normal HSCs and the rejuvenation of the hematopoietic system [START_REF] Chang | Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice[END_REF]. Despite its potent senolytic activity, ABT-263 induces long-term severe side effects such as thrombocytopenia [START_REF] Kaefer | Mechanism-based pharmacokinetic/pharmacodynamic meta-analysis of navitoclax (ABT-263) induced thrombocytopenia[END_REF]. Indeed, platelets overexpress Bcl-XL for their survival, making them an off-target for ABT-263 cytotoxicity. Cell surface biomarkers can also be targeted to eliminate senescent cells specifically. A new extracellular epitope, B2M, has been recently identified in some senescent cells, allowing their isolation by FACS [START_REF] Althubiti | Characterization of novel markers of senescence and their prognostic potential in cancer[END_REF].

Commercially available antibodies recognizing B2M epitope are internalized after binding at the surface of senescent cells. This allows the design of senolytic drugs coupled to antibodies anti-B2M through a cleavable enzymatic linker, digested into lysosomes once internalized, releasing active senolytics specifically into B2M-expressing senescent cells [START_REF] Poblocka | Targeted clearance of senescent cells using an antibody-drug conjugate against a specific membrane marker[END_REF]. Chimeric antigen receptor T (CAR-T) cells are T cells genetically designed to express an artificial T cell receptor. This chimeric receptor recognizes a chosen epitope of interest and activates the cytotoxic function of the T cell [START_REF] Kuwana | Expression of chimeric receptor composed of immunoglobulin-derived V regions and T-cell receptor-derived C regions[END_REF]). After identifying a new cell surface protein upregulated in some senescent cells, the urokinase-type plasminogen activator receptor (uPAR), CAR-T cells were designed to specifically target uPAR-expressing senescent cells. These CAR-T cells were efficient in vitro and in vivo in eliminating senescent cells and extending the lifespan of mice with lung adenocarcinoma [START_REF] Amor | Senolytic CAR T cells reverse senescence-associated pathologies[END_REF].

These two approaches are promising to target senescent cells specifically and reduce toxicity toward non-senescent cells. However, both strategies are based on cell surface markers, which are not commonly expressed by all senescent cell types, reducing the range of targeted senescent cells.

Undoubtedly, the field of senolytics is expanding fast, and new strategies will continue to emerge, increasing the promising possibility of targeting all types of senescent cells without off-targets.

II. Mammary Gland

A.

General introduction of mammary gland

The mammary gland (MG), from the Latin root mamma or "breast", is a highly specialized exocrine gland whose main function has been selected through evolution to produce milk to feed the offspring [START_REF] Oftedal | The mammary gland and its origin during synapsid evolution[END_REF].

Moreover, various molecules are conveyed through milk secretion, which contributes to the newborn's immune protection by transmitting antibodies against infection and colonization of the gut microbiota (Camacho-Morales et al.

2021

). Finally, breastfeeding promotes mother-infant bonding in numerous species [START_REF] Nagasawa | Oxytocin and mutual communication in mother-infant bonding[END_REF]. The presence of MG is one of the main characteristics defining the taxon of mammals, with a slight heterogeneity of MG structure amongst mammalian species. Indeed, ruminants exhibit an udder, a single massive MG with multiple teats, whereas primates and rodents have breasts, where each MG ends with its own nipple. Moreover, breasts usually operate by pair, but number of MGs depends on the anatomy of the specie and the mean litter size. In humans, only one pectoral pair of glands is developed, while mice have five pairs symmetrically localized all along the ventral area, from the neck to the perianal zone (Figure 14). As discussed later, a few other discrepancies between humans and mice are noticed, especially in the stromal composition of the mammary tissue and some slightly different developmental behaviors [START_REF] Mcnally | Overview of Mammary Gland Development: A Comparison of Mouse and Human[END_REF]. and MGs are remodeled to a near pre-gestation state, ready for a subsequent pregnancy (Richert et al. 2000).

MG is a potent model for developmental studies as most of its morphogenesis occurs post-natally. Moreover, MG is the organ of origin of breast cancer, the most prevalent cancer, with more than 2.3 million women diagnosed and 685.000 deaths worldwide in 2020 alone (Source: World Health Organization, July 2022). Understanding the physiological development of MG is a major health concern in unraveling underlying mechanisms leading to tumorigenesis.

B. Cellular organization of mammary tissue

Mammary tissue comprises many cell types contributing to its development and structure. Epithelial cells are surrounded by a stroma rich in fibroblasts and immune cells, all enclosed in a vascularized and innerved fat pad (Figure 15).

However, the main focus has been done on the epithelial compartment, primarily because it undergoes massive alterations during morphogenesis and is critical for the mammary function of milk production [START_REF] Inman | Mammary gland development: cell fate specification, stem cells and the microenvironment[END_REF].

Epithelium

The mammary epithelium consists of two functionally different entities, alveoli, and ducts (Figure 15). Mammary alveoli are the secretory lobules producing milk during lactogenesis, whereas lactiferous ducts connect the secretory units to the nipple to transport milk [START_REF] Visvader | Mammary stem cells and the differentiation hierarchy: current status and perspectives[END_REF]. Both structures are composed of two different cell types, luminal and basal cells, surrounded by a basement membrane. In ducts, the epithelium is stratified, with cuboidal luminal cells forming an inner lumen and a layer of spindle-shaped basal cells on top. In alveoli, the epithelium is pseudostratified, with each star-shaped contractile basal cell in contact with multiple milk-producing luminal cells underneath.

Basal and luminal cells have distinct functions and specific markers for their identification. Thanks to the specific cytoskeletal structural components expressed either by basal or luminal cells, the two populations can be detected in vivo by immunostaining. Anti-K5, anti-K14, or anti-SMA antibodies allow the identification of basal cells, and anti-K8 or anti-K18 antibodies specifically mark luminal cells [START_REF] Mikaelian | Expression of terminal differentiation proteins defines stages of mouse mammary gland development[END_REF]. Moreover, flow cytometry can separate isolated basal and luminal cells from each other and from the stromal compartment. Indeed, CD24 and α6 stainings segregate mammary cells into three populations: CD24 -, α6 -non-epithelial cells; CD24 low , α6 high basal cells; and CD24 high , α6 low luminal cells [START_REF] Sleeman | CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells[END_REF][START_REF] Asselin-Labat | Steroid hormone receptor status of mouse mammary stem cells[END_REF].

a) Basal cells

Basal cells inherit their name due to their position in contact with the basement membrane (Figure 15). They exhibit an epithelial phenotype with expressions of intermediate filaments, more specifically cytokeratin 5 (K5) and cytokeratin 14 (K14) [START_REF] Deugnier | The importance of being a myoepithelial cell[END_REF]. In contrast with various epithelia involved in fluid transport, such as in the epididymis or the bladder, mammary basal cells are not encapsulated in a smooth muscle cell layer. Indeed, more than 90% of mammary basal cells in the adult MG also exhibit a smooth muscle-like phenotype via smooth muscle actin (SMA) expression, giving them contractility properties during pregnancy and lactation [START_REF] Deugnier | Myoepithelial cell differentiation in the developing mammary gland: progressive acquisition of smooth muscle phenotype[END_REF]. Thus, basal cells can also be commonly termed as myoepithelial cells in the post-natal MG. However, whether these SMA + cells are truly capable to generate contractile forces in nonpregnant MG remains ambiguous [START_REF] Gieniec | Mammary basal cells: Stars of the show[END_REF]. The term "basal" is therefore privileged here to refer to the whole cell population, from embryonic development to adulthood.

The primary function of alveolar basal cells is to contract to help milk expulsion.

Basal cells respond to pituitary oxytocin, released into the systemic circulation after suckling stimulation [START_REF] Gimpl | The oxytocin receptor system: structure, function, and regulation[END_REF]. Contractions of basal cells act on the release of lipids droplets from luminal cells and on the propulsion of milk out of the lumen of alveoli and all along ducts, helping maintain a continuous milk flow [START_REF] Gieniec | Mammary basal cells: Stars of the show[END_REF]. Moreover, in ducts, basal cells form a continuous layer on top of the basement membrane, separating the connective tissue from the luminal layer and mediating their interactions.

b) Luminal cells

Mouse luminal cells lie on the basal cell layer, on their basal pole while their apical pole faces the mammary lumen (Figure 15). Luminal cells commonly express cytokeratins K8 and K18, with an intensity of expression allowing the subdivision into two subtypes of luminal cells, K8 low and K8 high [START_REF] Davis | Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny[END_REF]. The functional relevance of this differential expression remains to be elucidated but questions the reliability of K8 reporters to faithfully trace luminal cells [START_REF] Davis | Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny[END_REF].

Luminal cells can be classified by their function and position in the mammary tissue. Indeed, the main function of ductal luminal cells is to line the inner face of ducts, whereas alveolar luminal cells produce and secrete milk. ~15% of luminal cells also express hormonal estrogen (ER) and progesterone receptors (PR) [START_REF] Clarke | Dissociation between steroid receptor expression and cell proliferation in the human breast[END_REF]. ER + , PR + double positive cells can be found only in ducts, whereas alveolar luminal cells are ER -, PR - [START_REF] Clarke | Dissociation between steroid receptor expression and cell proliferation in the human breast[END_REF]. Double immunostainings showed a co-expression of both ER and PR in 96% of cells positive for at least one receptor [START_REF] Clarke | Dissociation between steroid receptor expression and cell proliferation in the human breast[END_REF]. ER activation regulates pubertal elongation of the ductal network, whereas PR activation is required during pregnancy to form tertiary ducts [START_REF] Tanos | ER and PR signaling nodes during mammary gland development[END_REF]. Mechanistically, in their inactive form, ER and PR are bound to heat shock proteins (HSP). Upon hormonal stimulations, receptors dissociate from HSP, form homo-or hetero-dimers, and enter the nucleus, where they induce changes in the transcription levels of various genes notably involved in cell proliferation (Brisken and O'Malley 2010). ER and PR can also mediate non-genomic crosstalks with major signaling pathways such as EGFR [START_REF] Acconcia | Signaling regulation of genomic and nongenomic functions of estrogen receptors[END_REF]. As only a tiny fraction of luminal cells is hormone-sensitive, it is proposed that activation of surrounding ER -, PR -cells is mediated by paracrine signals, efficiently coordinating multiple cell types [START_REF] Tanos | ER and PR signaling nodes during mammary gland development[END_REF]).

c) Mammary stem cells

The mammary epithelium is a dynamic tissue, which undergoes many cycles of development and regeneration during the reproductive lifetime. The existence of mammary stem cells to explain this plasticity was proposed in the late 50s.

Epithelial fragments were transplanted into a cleared fat pad and were sufficient to regenerate a fully branched epithelium, itself capable of regeneration after a second serial transplantation [START_REF] Deome | Development of mammary tumors from hyperplastic alveolar nodules transplanted into glandfree mammary fat pads of female C3H mice[END_REF][START_REF] Daniel | The in vivo life span of normal and preneoplastic mouse mammary glands: a serial transplantation study[END_REF]). Mammary stem cell characterization was then on standby for fifty years until the rise of flow cytometry and the identification of cell surface markers to differentiate basal and luminal lineages. Interestingly, by transplanting a single cell into the mammary fat pad, it has been shown that only single cells isolated from the basal compartment can regenerate a full bi-layered mammary epithelium, with ducts and alveolar structures [START_REF] Shackleton | Generation of a functional mammary gland from a single stem cell[END_REF][START_REF] Stingl | Purification and unique properties of mammary epithelial stem cells[END_REF]). However, this bi-potent capacity of mammary stem cells has been identified in transplantation assays, which do not mimic physiological development. Using genetic-lineage tracing, K5 + , K14 + multipotent stem cells giving rise to both basal and luminal lineages were identified during embryonic development (Van Keymeulen et al.

2011).

There is an active debate on the potency of mammary stem cells during post-natal development (Figure 16). Some bi-potent mammary stem cells in the basal compartment could give rise to both basal and luminal lineages during postnatal morphogenesis and adult homeostasis [START_REF] Rios | In situ identification of bipotent stem cells in the mammary gland[END_REF]. Or two distinct types of unipotent stem cells, basal stem cells, and luminal stem cells, could respectively give rise to basal and luminal lineages and maintain mammary gland homeostasis [START_REF] Taddei | Beta1 integrin deletion from the basal compartment of the mammary epithelium affects stem cells[END_REF][START_REF] Van Keymeulen | Distinct stem cells contribute to mammary gland development and maintenance[END_REF].

This discrepancy might be due to technical difficulties in individually tracing each epithelial population. Using an unbiased lineage tracing approach, limiting the number of tracked cells drastically, single cells were stochastically labeled and traced in virgin and lactating mice. Interestingly, labeled clones were lineage-restricted to basal or luminal layers during both ductal and alveolar morphogenesis, promoting the theory of unipotent stem cells to maintain MG homeostasis [START_REF] Davis | Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny[END_REF].

Stroma

The main difference between human and murine MG resides in the stromal compartment, especially its composition and its architecture. Indeed, in mice, the fibrous tissue remains a minority, and the stromal compartment is mainly composed of fat tissue, enclosing the epithelium directly. In contrast, in humans, fibrous tissue surrounds the epithelium forming lobules, enclosed by an interstitial stroma and a fat tissue less extended (Ingthorsson et al. 2016).

a) Adipocytes

Virgin mouse mammary epithelium is encapsulated in a stromal fat pad mainly composed of white adipocytes (Figure 15). This mammary fat tissue serves as massive energy reserve thanks to lipid storage and is highly plastic during the MG reproductive cycle [START_REF] Colleluori | Mammary gland adipocytes in lactation cycle, obesity and breast cancer[END_REF]. Two decades ago, it has been proposed that adipocytes could transdifferentiate into epithelial cells during pregnancy and lactation and could reversely transdifferentiate back to adipocytes during involution [START_REF] Morroni | Reversible transdifferentiation of secretory epithelial cells into adipocytes in the mammary gland[END_REF]). However, the Cre-mouse model used in this study was based on the expression of adipocyte fatty acid binding protein (AFABP, also known as aP2), which is now known to be a fatderived circulating protein [START_REF] Kralisch | Adipocyte fatty acid binding protein: a novel adipokine involved in the pathogenesis of metabolic and vascular disease?[END_REF]. Using a specific tracking system of mature adipocytes, it is now proposed that, during pregnancy and lactation, mature adipocytes de-differentiate into adipocyte precursors positive for platelet-derived growth factor receptor α (PDGFRα + ). Then, they redifferentiate into mature adipocytes upon involution [START_REF] Wang | Reversible De-differentiation of Mature White Adipocytes into Preadipocyte-like Precursors during Lactation[END_REF].

Adipocyte involution-associated hypertrophy is facilitated via lipid refilling derived from epithelial-produced milk (Zwick et al. 2018).

b) Fibroblasts

Fibroblasts are essential components of the mouse mammary stroma (Figure 15). In MG, periductal fibroblasts are localized in the fat pad, in close proximity to the epithelial basement membrane, and create a sheath separating epithelial ducts from surrounding adipocytes. Interstitial and perivascular fibroblasts are, for their part, disseminated between adipocytes [START_REF] Sumbal | Fibroblasts: The grey eminence of mammary gland development[END_REF]. There is no unique marker to label fibroblasts. However, there is a consensus, and fibroblasts are usually characterized as PDGFRα + or vimentin + cells, with a characteristic spindle shape and a stromal localization [START_REF] Sahai | A framework for advancing our understanding of cancer-associated fibroblasts[END_REF].

Fibroblasts are key mediators of the crosstalk between stroma and epithelial cells to support MG development, from rudimentary embryonic morphogenesis to pubertal branching [START_REF] Wiseman | Stromal effects on mammary gland development and breast cancer[END_REF]. Fibroblasts are also critical during the reproductive cycle, especially during MG involution, where they secrete metalloproteinases and contribute to the deposition of a fibrotic-like extracellular matrix (ECM). This ECM has been proposed to promote epithelial cell death and reorganization of the mammary tissue [START_REF] Schedin | Mammary ECM composition and function are altered by reproductive state[END_REF]Simian et al. 2001).

c) Macrophages

Macrophages are major components of the mammary immune landscape and are present at all the mammary developmental stages, as visualized using a colony stimulating factor 1 receptor (Csf1R)-EGFP reporter mouse model (Stewart et al. 2019). Macrophages occupy a stromal position in close proximity with epithelial ducts but can also be located inside the epithelial compartment, between the basal and the luminal cell layers, in ducts and in the alveolar structures (Stewart et al. 2019;[START_REF] Dawson | Tissueresident ductal macrophages survey the mammary epithelium and facilitate tissue remodelling[END_REF]). These tissue resident macrophages, closely associated to the epithelium, are also called ductal macrophages and are proposed to monitor the epithelial network through dendritic movements, allowing for a rapid response [START_REF] Dawson | Tissueresident ductal macrophages survey the mammary epithelium and facilitate tissue remodelling[END_REF]. As macrophages are professional phagocytes, they are recruited during pubertal morphogenesis to assist epithelial cells in the fat pad invasion (Gouon-Evans, Rothenberg, and Pollard 2000). Moreover, macrophages have also been linked to successful alveogenesis during pregnancy, as Csf1 deficient mice exhibited an impaired development of alveolar structures resulting in an incapacity to lactate their progeny [START_REF] Pollard | Colony stimulating factor 1 is required for mammary gland development during pregnancy[END_REF]. Finally, massive increase of macrophages number has been observed during the irreversible phase of involution (Hughes et al. 2012). It has been proposed that macrophages play a critical role in involution to promote epithelial cell clearance and tissue remodeling, as macrophages depletion using the Mafia mouse model, in which Csf1R + cells can be conditionally deleted, impaired the proper regression of the alveolar structures [START_REF] O'brien | Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution[END_REF].

C. Mammary gland morphogenesis

1.

Pre-natal development and puberty

Embryonic morphogenesis is succinct and is limited from E10.

Estrus cycle

After puberty, MG is considered quiescent in adult virgin mice. However, mammary tissue is dynamic, and there is a cyclic turnover of epithelial cells in response to hormonal variations caused by the 4-day estrous cycle in rodents.

Estrogen and progesterone lead to the proliferation of a subset of cells to form secondary ducts and budding alveoli, which then enter into apoptosis at the end of the estrus cycle [START_REF] Fata | Cellular turnover in the mammary gland is correlated with systemic levels of progesterone and not 17beta-estradiol during the estrous cycle[END_REF].

Reproductive cycle

During pregnancy, MGs undergo massive changes to mature into a functional milk-producing gland for lactation (Figure 17). Under progesterone action, ductal basal and luminal progenitors will go through substantial proliferation to establish tertiary ductal branches and then form alveolar buds at the extremities of ducts (Hennighausen and Robinson 2005). As shown with mutant mice for progesterone and prolactin receptors, once developed, alveoli require a hormonal combination of progesterone and prolactin to mature into secretory bodies (Ormandy et al. 1997;[START_REF] Brisken | A paracrine role for the epithelial progesterone receptor in mammary gland development[END_REF]. To promote epithelial development, the stromal compartment is also remodeled during pregnancy with de-differentiation and shrinkage of adipocyte lipid content, in parallel with extensive angiogenesis [START_REF] Vernon | Adaptations of maternal adipose tissue to lactation[END_REF][START_REF] Zygmunt | Angiogenesis and vasculogenesis in pregnancy[END_REF]. At the end of pregnancy, most of the fat pad is filled with mature lobulo-alveolar structures.

After parturition, due to progesterone drop concomitant to a high level of prolactin, lactation is initiated with the formation of the colostrum. Alveolar luminal cells are fully differentiated into their secretory phenotype, producing and ejecting milk into the lumen; and basal cells contract in response to oxytocin and calcium oscillations to synchronize and favor milk expulsion [START_REF] Soloff | Oxytocin receptors and mammary myoepithelial cells[END_REF][START_REF] Stevenson | Multiscale imaging of basal cell dynamics in the functionally mature mammary gland[END_REF]. Suckling stimulation further promotes alveolar proliferation and maturation and increases milk production gene expression [START_REF] Neville | Hormonal regulation of mammary differentiation and milk secretion[END_REF]. Milk production requires much energy to breastfeeding mothers. Therefore, after weaning, when milk production is dispensable, MGs undergo a tightly controlled process of involution to return to a near pre-pregnant resting state (Figure 17). differentiated, giving MG its milk-producing organ functionality. After weaning, a process of involution leads to the disappearance of the milk-secreting alveoli and allows MG to return to a resting stage resembling its pre-pregnancy state, ready for a further gestation. MG development is under hormonal regulation. E: estrogen; Pg: progesterone; Prl: prolactin. Adapted from [START_REF] Fu | Stem Cells and the Differentiation Hierarchy in Mammary Gland Development[END_REF].

D.

Involution: a chaos carefully-orchestrated

Overview

The MG involution is one of the main cell death and tissue-remodeling events in the adult mammalian organism. This process occurs naturally after suckling arrest when pups diversify their food intake and do not consume milk anymore.

Therefore, involution is critical for removing milk-producing epithelial cells, which are no longer needed (Watson 2006). To study mechanisms underlying the involution process, it can also be synchronized by the physical separation of the progeny from the mother at the peak of lactation at 10 days post-delivery, also termed involution day 0 (Inv0) (Lloyd- [START_REF] Lloyd-Lewis | Analysis of the Involuting Mouse Mammary Gland: An In Vivo Model for Cell Death[END_REF]). This strategy allows temporally defining involution, commonly divided into two distinct morphological phases (Lund et al. 1996). During the first stage of involution, programmed cell death occurs, but the global architecture of the gland is not impaired. In contrast, a major tissue remodeling of mammary tissue characterized the second phase of involution. At the end of involution, around 14 days after the initiation, MGs resemble their pre-pregnant morphology and return to the quiescent state, ready for a subsequent pregnancy (Watson 2006). correspond respectively to involution day 0 (physical pups removal), or 1, 3, 7, or 14 days after the initiation of involution. Adapted from [START_REF] Hitchcock | Dynamic architectural interplay between leucocytes and mammary epithelial cells[END_REF][START_REF] Sutherland | c-myc as a mediator of accelerated apoptosis and involution in mammary glands lacking Socs3[END_REF][START_REF] Kreuzaler | Stat3 controls lysosomal-mediated cell death in vivo[END_REF]).

The first reversible phase of involution

Mouse MG involution is characterized by two phases of involution based on the reversibility of the process (Figure 18). During the first 48h of the process, involution is reversible. Indeed, as pups are placed back with the mother and resume the suckling stimulation, involution is halted, and lactation can be reinitiated [START_REF] Li | Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution[END_REF]. This reversibility has been evolutionary conserved, as it is crucial that pups can be nursed after a long absence of the mother in search of food [START_REF] Watson | Remodeling mechanisms of the mammary gland during involution[END_REF].

This first involution phase starts as early as 12h after the last feed and is closely linked to the stagnation of milk in the alveolar structures. Sealing of mammary nipples with veterinary glue is a creative approach to study involution inducers by uncoupling the influence of the build-up of local factors versus the response to lactogenic hormones. Indeed, some MGs can be physically sealed, whereas other nipples from the same animal are left untouched. Suckling on unsealed nipples preserves lactation and maintains the production of lactogenic hormones. Interestingly, the teat sealing leads to a regular milk accumulation and does not disturb the early involution phase, emphasizing the role of milk stasis in the process [START_REF] Li | Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution[END_REF].

It has been proposed that cells perceive both chemical and mechanical signals due to milk stasis and respond by cell death [START_REF] Quaglino | Mechanical strain induces involution-associated events in mammary epithelial cells[END_REF]). Apoptosis was proposed as a major mechanism during this first cell death wave. However, apoptotic cells have a typical morphology: the chromatin is condensed, and the plasma membrane is fragmented into apoptotic bodies [START_REF] Saraste | Morphologic and biochemical hallmarks of apoptosis[END_REF]. During the first phase of involution, epithelial cells detached and discharged into the lumen do not exhibit this classical apoptosis-associated morphology but instead have a swollen shape, and their membrane is intact without blebbing. Moreover, cells are not positive for TdT-mediated dUTP nick end label (TUNEL), a classical apoptotic staining [START_REF] Watson | Remodeling mechanisms of the mammary gland during involution[END_REF].

Instead of apoptosis, lysosomal membrane permeabilization (LMP) has been

shown to occur in the mammary epithelium during the first phase of involution [START_REF] Kreuzaler | Stat3 controls lysosomal-mediated cell death in vivo[END_REF]. Lysosomes are important acid organelles containing enzymes, which primary function is to degrade cellular macromolecules. In LMP, lysosomal proteins as cathepsins leak from lysosomes and remain active in the cytosol, where they replace caspases in their role of death executing-proteases, inducing cell death.

Mechanistically, milk stasis induces a wide range of stimuli with the build-up of local factors as an overload of intracellular Ca 2+ and mechanical stretching of the epithelial cells [START_REF] Stewart | Mammary mechanobiology -investigating roles for mechanically activated ion channels in lactation and involution[END_REF]. In response, epithelial cells up-regulate the expression of numerous cytokines as LIF and TGFβ3, linked to the phosphorylation of the master regulator of involution, the transcription factor Stat3 (Kritikou et al. 2003;Nguyen and Pollard 2000). Activated pStat3 modifies the lysosomal content by up-regulation of lyosomal proteases cathepsins B and L and down-regulation of the endogenous cathepsins inhibitor Spi2A. While the lysosomal membrane is dismantled, by a down-regulation of LAMP, a major lysosomal membrane structural protein [START_REF] Kreuzaler | Stat3 controls lysosomal-mediated cell death in vivo[END_REF]). In early involution, the leakage of lysosomal content in the cytoplasm is then responsible for the LMP cell death mediated by Stat3. Importantly, mice genetically KO for Stat3 in the mammary epithelium exhibit a dramatic delay of involution [START_REF] Humphreys | Deletion of Stat3 blocks mammary gland involution and extends functional competence of the secretory epithelium in the absence of lactogenic stimuli[END_REF]. Activation of Stat3 in its phosphorylated form is concomitant with the dephosphorylation of Stat5, which is a crucial survival factor implicated in the proliferation and differentiation of epithelial cells in the alveolar lobules, during pregnancy and lactation [START_REF] Barash | Stat5 in the mammary gland: controlling normal development and cancer[END_REF]). pStat5 dephosphorylation is required to initiate cell death, as constitutive activation of Stat5 delays MG involution in transgenic mice by the maintenance of prosurvival signals [START_REF] Iavnilovitch | Overexpression and forced activation of stat5 in mammary gland of transgenic mice promotes cellular proliferation, enhances differentiation, and delays postlactational apoptosis[END_REF].

During the first phase of involution, the global architecture of the gland is not impaired as there is no remodeling of the stroma concomitant with the epithelial cell death. Indeed, tissue inhibitors of metalloproteinases (TIMPs) are highly expressed during the first 48h, allowing to maintain the reversibility of the involution process. More specifically, tightly controlled TIMP-3 and TIMP-1 expressions are crucial during the first involution phase. Indeed, MGs from mice KO for TIMP-3 exhibited an accelerated cell death and remodeling, whereas, surprisingly, TIMP-1 overexpression promoted a faster adipogenic redifferentiation (Fata et al. 2001;[START_REF] Alexander | Stromelysin-1 regulates adipogenesis during mammary gland involution[END_REF]).

The second irreversible phase of involution

The second involution phase starts 48h after the removal of the litter and is irreversible (Figure 18). Indeed, when pups are placed back with the mother and resume the suckling stimulation after 48 hours, lactation cannot be re-initiated, and involution continues until the end of the process [START_REF] Li | Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution[END_REF].

While the first phase of involution was defined by cell death without changes in the MG structure, the second phase is characterized by massive tissue remodeling. Briefly, the alveolar structures are collapsed; the extracellular matrix (ECM) is profoundly restructured notably due to the high expression of metalloproteinases (MMPs) and the deposition of new ECM fibers as fibrillar collagen; immune cells are recruited to help in the clearance of the tissue; and redifferentiation of adipocytes occurs in the stroma for the refilling of the mammary fat pad. After two weeks of involution, the majority of the MG resembles its near pre-gestation state and returns back to the quiescent state [START_REF] Watson | Remodeling mechanisms of the mammary gland during involution[END_REF].

Signals to switch from the first to the second phase of involution are not clearly defined. However, it has been proposed that it depends on lactogenic hormones regulated at the systemic level. Indeed, teat sealing does not affect the progression of the early phase of involution. However, maintenance of lactogenic hormones completely disturbed the entrance in the second phase of involution, with an absence of tissue remodeling persistent at Inv6 [START_REF] Li | Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution[END_REF].

Moreover, implantation in the MG of pellets releasing exogenous hormones such as glucocorticoids was sufficient to impair the entrance in the second phase of involution [START_REF] Feng | Glucocorticoid and progesterone inhibit involution and programmed cell death in the mouse mammary gland[END_REF].

Mechanistically, a decrease of lactogenic systemic hormones might induce the expression of metalloproteinases (MMPs), such as MMP-2 (gelatinase-A), or MMP-3 (stromelysin-1), concomitant to a decrease in expression of MMPs inhibitors TIMP-1 and TIMP-3 [START_REF] Talhouk | Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution[END_REF]. MMPs are broad-spectrum proteases and use their catalytic domains to cleave components of the extracellular matrix (ECM) as collagens of the basement membrane, fibronectin, or laminin [START_REF] Uria | Matrix metalloproteinases and their expression in mammary gland[END_REF].

The degradation of the ECM might be sensed as a stress signal favoring apoptotic cell death, characteristic of the beginning of the second phase of involution [START_REF] Watson | Remodeling mechanisms of the mammary gland during involution[END_REF]. Indeed, mitochondria-mediated intrinsic cell death, also known as the "classical apoptosis" is induced during the early irreversible phase, marked by the presence of cleaved-caspase 3 (CC3) as well as TUNEL positive cells in alveoli walls [START_REF] Marti | Mouse mammary gland involution is associated with cytochrome c release and caspase activation[END_REF]. Apoptosis is regulated through the balanced expression of pro-and anti-apoptotic proteins from the Bcl2 family [START_REF] Hardwick | Multiple functions of BCL-2 family proteins[END_REF]. During the first phase and the early second phases of involution, when cell death is induced via LMP or mitochondria-mediated apoptosis, there is an overexpression of pro-apoptotic proteins Bax, Bak, and Bad; whereas anti-apoptotic proteins such as Bcl-2, Bcl-X and Bcl-W are down-regulated [START_REF] Metcalfe | Developmental regulation of Bcl-2 family protein expression in the involuting mammary gland[END_REF]). This correlates with the transgenic overexpression of Bcl-2 under WAP promoter, specific to alveolar luminal cells, which delays mammary epithelial cell death during in vivo involution [START_REF] Jager | Overexpression of Bcl-2 inhibits alveolar cell apoptosis during involution and accelerates c-myc-induced tumorigenesis of the mammary gland in transgenic mice[END_REF]. Surprisingly, the expression of anti-apoptotic proteins Bcl-2, Bcl-X, and Bcl-W increases as early as Inv3 up to Inv12 [START_REF] Metcalfe | Developmental regulation of Bcl-2 family protein expression in the involuting mammary gland[END_REF].

Immune cells contribute significantly to the mammary involution process. In early involution, the immune response is limited to the recruitment of neutrophils via the expression of the neutrophil chemoattractant CXCL1 at Inv1 To conclude, the second phase of involution displays many similarities with the tissue remodeling associated with wound-healing, such as removal of damaged epithelium; proliferation of mesenchymal cells; inflammatory response; and restructuration of the ECM with collagen deposition [START_REF] Gosain | Aging and wound healing[END_REF].

Back to the future

MG involution is completed around two weeks after induction, with the removal of alveolar structures and the refilling of the fat pad by adipocytes. MGs remain resting until a further pregnancy. Interestingly, how epithelial stem cells are protected from pro-death signals and conserve their plasticity through multiple reproductive cycles remains unclear [START_REF] Watson | Remodeling mechanisms of the mammary gland during involution[END_REF].

Classical or "post-lactational" involution should be differentiated from lobular involution, where mammary epithelial gradually disappears with age progression. Indeed, lobular involution is characterized by reduced size and complexity of the ductal network during menopause [START_REF] Radisky | Mammary involution and breast cancer risk: transgenic models and clinical studies[END_REF]. Interestingly, it is proposed that removing epithelial tissue after the end of the reproductive life span is a mechanism protective against breast cancer [START_REF] Milanese | Age-related lobular involution and risk of breast cancer[END_REF]. In contrast, post-lactational involution is associated with an increased transient risk of pregnancy-associated breast cancer (Schedin 2006).

In conclusion, post-lactational MG involution is a very complex process, extensively studied for more than two decades. However, essential questions remain open in the field. What are the precise signals associated with milk stasis initiating the first involution phase? How is the switch from the reversible to the irreversible phases of involution controlled? How are the multiple cell types involved in the tissue remodeling phase coordinated to restructure the tissue?

Are there other mechanisms regulating the elaborate changes of the involuting microenvironment? Unraveling this physiological process is crucial to understanding why the post-lactational involution is associated with protumorigenic development.

Involution and postpartum-associated breast cancer

Epidemiologic studies suggest that pregnancy at an early age is protective against general forms of breast cancer, supposedly due to the maturation of epithelial cells during pregnancy and the reduction of the total number of menstrual cycles [START_REF] Rosner | Reproductive risk factors in a prospective study of breast cancer: the Nurses' Health Study[END_REF]. However, there is a transiently increased risk of developing breast cancer after pregnancy, and diagnosis of breast cancer within 10 years after delivery is categorized as postpartum breast cancer (PPBC) [START_REF] Albrektsen | Breast cancer risk by age at birth, time since birth and time intervals between births: exploring interaction effects[END_REF]. PPBC is correlated with a worse prognosis, as women diagnosed with PPBC have a 40% increased risk of breast-cancer-associated death within 5 years compared to breast cancer in nulliparous women, principally due to the development of metastasis [START_REF] Daling | The relation of reproductive factors to mortality from breast cancer[END_REF].

Involuting MG microenvironment has intrinsic characteristics that can promote tumorigenesis (Figure 19) [START_REF] Watson | Remodeling mechanisms of the mammary gland during involution[END_REF]. Indeed, during involution, mammary tissue is pro-inflammatory with the recruitment of immune cells, the intense remodeling of the ECM, and the impairment of the integrity of the basement membrane due to increased metalloproteinases activity. In vitro, ECM from involuting MGs stimulated tumor cell invasion. In vivo, co-injection of mammary tumor cells with involution ECM in mammary fat pad promoted invasiveness and metastasis in various organs, as well as angiogenesis [START_REF] Mcdaniel | Remodeling of the mammary microenvironment after lactation promotes breast tumor cell metastasis[END_REF]). Therefore, it has been proposed that involution creates a microenvironment permissive for tumorigenesis and metastasis, promoting PPBC (Schedin 2006).

E.

In vitro 3D models to recapitulate MG development MG development has been extensively studied in vivo, but three-dimensional (3D) culture provides many advantages regarding optical observation and live imaging, or genetic manipulations while conserving a complex tissue architecture missing in 2D cultures (Shamir and Ewald 2014).

Spheroids are 3D cell aggregates, but they do not fully recapitulate either the complex 3D organization or the function of the mammary tissue. Organoids are 3D assays intrinsically different from spheroids, as they can form complex 3D structures with a spatial organization of multiple cell lineages, mimicking organ functions [START_REF] Gilazieva | Promising Applications of Tumor Spheroids and Organoids for Personalized Medicine[END_REF]. Various mammary organoids have been developed to mimic MG morphogenesis and understand the crosstalk between organoids derived from epithelial cells and the ECM or the fibroblasts population from the stromal compartment (Figure 20) (Ewald et al. 2008;Simian et al. 2001;Sumbal and Koledova 2019). Some organoids were derived from single basal cells and reproduced a functional bilayer of basal and milk-producing luminal cells (Jamieson et al. 2017). However, prior to our study (see Annexes (Sumbal et al. 2020) (Charifou et al. 2021)), no organoid system resuming the three key stages of mammary gland reproductive cycle, gestation-lactationinvolution, was characterized yet [START_REF] Srivastava | Organoid models for mammary gland dynamics and breast cancer[END_REF]. Cellular senescence is a form of stress response characterized by a stable cellcycle arrest and acquisition of a robust secretome, termed SASP. The permanent growth arrest is a potent cell-intrinsic tumor suppression mechanism comparable to apoptosis, whereas SASP paracrine signaling is important for optimized wound healing and tissue regeneration. Meanwhile, senescent cells accumulate during aging, contributing to aging and age-related diseases.

Importantly, the elimination of senescent cells can improve a variety of agingdependent pathologies, including cancer, and enhance tissue regeneration.

Although it has become increasingly clear that senescence plays multifaceted roles during many physiological and pathological processes, its involvement in most physiological processes remains largely unknown. Where and when does senescence occur during a lifetime? How does senescence contribute to different physiological processes? Addressing these questions is crucial for our understanding of the pleiotropic roles of senescence and for providing necessary precision in specifically targeting the detrimental effects of senescence. To start answering these relevant questions, we took advantage of the MG system. The MG is an organ with remarkable plasticity and its development mainly occurs postnatally. Importantly, it is also the organ of origin for breast cancer.

This Ph.D. project was divided in three main aims to define the potential contribution of senescence during MG development:

I/ First, we aimed to assess whether senescence was induced during MG development and, if so, in which specific developmental stage(s) II/ Second, as we detected senescence specifically during the MG involution, we aimed to deeply characterize the involution-associated senescence program, from both in vivo murine model and novel ex vivo mammary organoid system.

III/ Third, we aimed to assess the role of senescence in the physiological process of involution, both in vivo and ex vivo using techniques impairing the proper involution process, senolytic treatments and transgenic mouse models.

Results

Cellular senescence is induced during mammary gland involution associated with the onset of tissue remodeling

To assess whether senescence occurs during mammary gland (MG) postnatal development and reproductive cycle, we collected murine MGs at different developmental stages, including mature young virgin (8 weeks), pregnancy (D18.5), and lactation (L10). After 10 days of lactation (L10/Inv0), we physically removed the pups to induce synchronized involution and harvested involuting MGs (Lloyd- [START_REF] Lloyd-Lewis | Analysis of the Involuting Mouse Mammary Gland: An In Vivo Model for Cell Death[END_REF]). In addition, we also collected MGs from old females, both multiparous (10 months) and nulliparous (18 months) (Figure 1A). We use senescence-associated beta-galactosidase (SAβGal) staining, a widely used assay [START_REF] Cahu | A sensitive method to quantify senescent cancer cells[END_REF] as the primary method to detect senescent cells in the mammary gland sections. Of note, in most time points collected except involution, we failed to detect any SAβGal positive cells, including the MGs from old females (Supplementary Figure 1A).

Involution is characterized by the massive cell death of the secretory alveolar

Transcriptomic analysis of involuting mammary gland

To confirm the SAβGal staining results, we performed bulk RNA sequencing on whole MG at different time points during involution. Principlecomponent analysis (PCA) revealed that samples from the same time point cluster together (Supplementary Figure 1B). To gain an unbiased global view on which biological pathways/responses occurred during involution process, we compared our datasets at different time points with hallmark gene set.

Comparing Inv7 vs Inv0 or Inv3 vs Inv0, we found that similar pathways are significantly enriched or depleted, with more changes detected in Inv7 than in Inv3 (Supplementary Figure 1C).

Next, we created our customized mouse senescence gene set based on the SeneQuest (http://Senequest.net) [START_REF] Gorgoulis | Cellular Senescence: Defining a Path Forward[END_REF]. We considered a gene as associated with senescence when there were at least 5 consistent reports in one direction (upregulation or downregulation). We generated multiple gene sets separated in two categories: SENESCENCE-UP and SENESCENCE-DOWN, based on how many consistent reports where found in the Senequest.net. The higher is the reports number, the stronger is the association to senescence. Next, we performed gene set enrichment analysis (GSEA) using the customized gene sets (SENESCENCE-UP5) and our transcriptomic datasets. We found a significant enrichment of senescence up-regulation in Inv3 and Inv7 comparing to Inv0 (Figure 1C). We also detailed a list of senescence-associated genes significantly up-regulated at both Inv3 and Inv7 (Figure 1D). Interestingly, CDKN2a and

Trp53 were also significantly up-regulated in Inv7 (Supplementary Figure 1D).

Taken together, the transcriptomic data analysis further confirmed the induction of senescence program in the involuting MG, specifically at Inv3 and Inv7.

Milk-producing alveolar luminal cells are the main senescent cell type during mammary gland involution

To define cell types that were becoming senescent in the mammary tissue during involution, we first performed SAβgal staining, co-stained either with Keratin 5 (Krt5), marker of basal cells (Figure 2A); or Keratin 8 (Krt8), marker of luminal cells (Figure 2B), using histological sections of MGs collected at Inv3, a timepoint for which the epithelial structure of the alveoli is still well preserved. We found that the majority of SAβGal + cells were alveolar luminal cells while few Krt5 + basal cells were also positive for SAβGal staining.

To further define senescent cell types within involuting MG and gain a quantitative idea on the tissue level, we optimized a fluorescent staining of senescent cells using C12FDG. Briefly, we harvested MGs from mice at Inv0 and Inv5. We chose Inv0 as a clear negative control without any senescent cell, whereas Inv5 was a potent time point, compromising between a very intense senescent signal and still a high proportion of remaining epithelial cells. After cell dissociation, we incubated mammary cells with C12FDG and performed a cytometry analysis to separate stromal cells (CD31 -, CD45 -, CD24 -, α6 -); basal cells (CD31 -, CD45 -, CD24 low and α6 high ); and luminal cells (CD31 -, CD45 -, CD24 high and α6 low ) (Supplementary Figure 2A-B). In every cell population, we then counted the number of cells positive for C12FDG-GFP. Only ~6% of stromal cells were positive at Inv5 (Supplementary Figure 2C). Interestingly, in the epithelial compartment, ~15% of basal cells were C12FDG-GFP + at Inv5 compared to Inv0 (Supplementary Figure 2C). And more than ~52% of luminal cells were positive for C12FDG-GFP at Inv5, suggesting that luminal cells were the main cell type to become senescent (Supplementary Figure 2C).

It is well established that alveolar luminal cells are removed via programmed cell death during involution [START_REF] Kreuzaler | Stat3 controls lysosomal-mediated cell death in vivo[END_REF], while a senescent cell does not undergo cell death. Therefore, we performed cleaved caspase 3 (CC3) and SAβGal co-staining on Inv3 to further confirm SAβGal + luminal cells were distinct from dying cells. We could detect many CC3 + cells consistent with the literature.

Interestingly, most of SAβGal + cells were negative for CC3 staining (Figure 2C).

Moreover, these SAβGal + cells were also non-proliferating, as they were negative for proliferative maker, Ki67 (Figure 2D). Therefore, SAβGal+ cells were nonapoptotic and non-proliferating, two mutually exclusive cell states from senescence.

Mammary involution-associated senescence might be mediated by p16 in vivo and ex vivo

Senescence cell cycle arrest can be mediated by two major cyclin-dependent kinase inhibitors, either p21 or p16 [START_REF] Kumari | Mechanisms of Cellular Senescence: Cell Cycle Arrest and Senescence Associated Secretory Phenotype[END_REF]. The bulk RNAseq analysis of the whole MG at different time point revealed that CDKN2a but not CDKN1a was significantly upregulated at Inv7 vs Inv0 (Supplementary Figure 1C). To confirm the bioinformatic analysis result, we took advantage of a new p16-reporter mouse model (https://doi.org/10.1101/2020.06.10.142893) (BioRXiv reference) known as INKBRITE, with a GFP cassette under the regulation of endogenous p16 promoter, to detect p16 protein expression pattern during involution. At Inv0, the p16 expression was rather heterogenous.

There were few GFP + cells located in the stromal compartment, outside of the alveolar structures marked by Krt5 (Figure 2E, upper panel). Within the mammary epithelium, we found some alveolar structures without any GFP + cells (Figure 2E, upper panel, asterisk), while some alveolar structures did contain GFP + cells (Figure 2E, upper panel, zoom-in). Interestingly, we detected many more GFP + cells at Inv3 compared to Inv0. A good proportion of the GFP + cells were located within the alveolar structures facing the lumen (Figure 2E, lower panel). Therefore, p16 expression correlated with senescence induction during involution, and some alveolar luminal cells were p16 + at Inv3.

Previously, we established a mammary organoid model to mimick pregnancy, lactation, and involution ex vivo (Sumbal et al. 2020). We wondered whether senescence was also induced during involution-like process in this organoid model. Therefore, we cultured primary organoids in FGF2 for 6 days to mimic pregnancy-associated proliferation, followed by 4 days in prolactin and hydrocortisone to induce lactation-like process. Then, we induce involution-like process by removing hormonal stimulation (basal organoid medium, BOM). We used organoids maintained in a lactation-like state (LM) for additional 8 days as control (Figure 2F, experimental scheme, upper panel). We harvested organoids on the indicated days and performed whole mount SAβGal staining. The organoids from 6 days of FGF2, or in the lactation-like state after 4 more days in LM, were not positive for SAβgal staining (Supplementary Figure 2D). However, we noticed a significant increased intensity of SAβgal staining in organoids from D14 and D18, during involution-like process (Supplementary Figure 2E). When we examined the histological sections from whole mount organoids, we found that involuting-like (BOM) organoids were much smaller than lactating-like (LM) organoids consistent to our previous findings (Figure 2F). Moreover, there were many SAβGal + cells in the involuting-like (BOM) organoids. This was specific to involuting organoids and not due to a long period in culture, as organoids maintained in lactation-like state were not positive for SAβgal staining (Figure 2F). Next, we extracted RNA from organoids at D10 of culture, or in involutionlike (BOM D18) or maintained lactation-like (LM D18) states and performed qPCR analysis. p16 was up-regulated by 16-fold in involution-like organoids compared to D10 (Figure 2H). In contrast, p21 expression was not significantly changed between D10 and D18 BOM (Figure 2H). Taken together, we showed that organoids undergoing involution-like process contain senescent cells and this was coincident with the upregulation of p16 but not p21, consistent with our in vivo observations. Therefore, organoids can be used as a simplified model to study involution-associated senescence.

Next, we wondered whether involution-associated senescence was triggered by DNA damage, a common inducer of senescence. There were many γH2AX positive cells in Inv3 and most of them were alveolar luminal cells, while we did not detect any at the Inv0 (Figure 2G), suggesting DNA damage response was induced during involution process. Interestingly, GFP+ and γH2AX+ cells were mutually exclusive (Figure 2G), which was further confirmed by SAβGal and γH2AX co-staining in WT involuting MG (Supplementary Figure 2E). Taken together, these results suggest involution-associated senescent cells did not contain a significant amount of DNA damage.

Persistence of lactogenic hormones prevents the involution-associated senescence induction

We showed that senescence was specifically induced on Inv3, coinciding with the beginning of the irreversible phase of involution. Next, we wondered whether entering to the irreversible phase of involution was important for the senescence induction. We took advantage of the nipple sealing method, where some nipples are bonded with veterinary glue to mimic involution by preventing their accessibility while other nipples are still in lactation [START_REF] Li | Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution[END_REF]). This approach enables the sealed MG to enter the first phase of involution caused by milk stasis. However, due to maintenance of systemic lactogenic hormones, entrance to the irreversible phase of involution is blocked and massive tissue remodeling does not occur [START_REF] Li | Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution[END_REF]).

We sealed nipples from one side of lactating female and collected MGs 2 or 3 days after sealing. We compared sealed MGs to control MGs going through regular involution, at Inv2 and Inv3 (Figure 3A). First, we confirmed the correct sealing of MGs during tissue harvesting (Supplementary Figure 3A). Lactating-MGs exhibited an enlarged morphology, without milk stasis as alveoli were regularly emptied. Sealed contralateral MGs were comparable in size as lactating

MGs, but milk stasis gave them a characteristic yellow color. Involuting-MGs were smaller (Supplementary Figure 3B). To further characterize MGs morphology, we stained involuting and sealed MGs with Carmine. The first phase of involution is characterized by cell death without regression of alveolar structures, and MGs kept their typical "bunch of grapes" shape as seen in Inv2

and sealed day 2 MGs (Supplementary Figure 3C). At Inv3, we noticed a regression of alveoli size, exposing ducts underneath in the involuting-MGs, while sealed-MGs maintained similar morphology (Figure 3B-left panel).

Moreover, on tissue sections, we observed many dying cells shredded into the lumen of alveolar structures in both involuting-MGs and sealed-MGs at day 2, indicating that the first phase of involution was initiated (Supplementary Figure 3C-right panel, black arrow head). On day 3, involuting-MGs exhibited hallmarks of the onset of tissue remodeling events, with easily identifiable shrinking alveoli and refilling of the adipocytes, while sealed-MGs remain the same morphology as day 2 (Figure 3B, right panel). Moreover, we confirmed the correct sealing of MGs by qPCR analysis. Indeed, sealed MGs stopped their milk production, as seen by a decreased of CSN2 and WAP expressions, as involuting MGs. However, adipocyte refilling was impaired, as seen with Adipoq level at day 3, which was only upregulated in involuting MGs (Figure 3D). Taken together, these data showed that we succeeded to correctly seal MGs while maintaining lactogenic stimulation in mice. Sealing enabled MGs to start the involution process without entering the irreversible remodeling phase.

To test whether senescence program was impaired in sealed MGs, we determined the number of senescent cells using SAβgal staining. In accordance with our previous results, we showed the appearance of SAβgal positive cells at Inv3 (Figure 3B &C). Surprisingly, in sealed-MGs, we did not detect any SAβgal positive cell on day 3 (Figure 3B &C). We also collected MGs on day 5 and we could start to see a few SAβGal + cells in the sealed-MG (Supplementary Figure 3D). Of note, we also observed a delayed adipocyte refilling in the same sections (Supplementary Figure 3D, asterisk), suggesting sealing significantly delays the tissue remodeling phase of involution on day 5. We further confirm the impairment of senescence program by qPCR analysis. Indeed, from day 2 (D2) to day 3 (D3), p16 gene was only significantly increased in the involuting MGs (Figure 3D). Taken together, we confirmed that using teat sealing to maintain lactogenic hormones was sufficient to block entrance into the irreversible phase of MG involution. More importantly, we showed that teat sealing impaired the senescence induction. Therefore, senescence is closely associated to the irreversible remodeling phase of involution.

Removing senescent cells with senolytic ABT-263 delays mammary gland involution in vivo

To determine the functional link of senescence in the involution process, we specifically eliminated senescent cells using a senolytic compound, ABT-263 [START_REF] Chang | Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice[END_REF] ). First, we showed by qPCR analysis that 3MR cassette was expressed during in vivo involution process, with a ~10-fold increase in Inv5 compared to Inv0 (Supplementary Figure 5B). Then, we treated mice with 50mg/kg ganciclovir (GCV) by intraperitoneal injection every day from Inv0 to Inv4 and collected glands on Inv5 (Supplementary Figure 5C).

However, we did not observe a significant reduction of senescent cells, judged by the SAβGal staining, on the treated samples compared to controls (Supplementary Figure 5D). Consistently, the ratio of luminal cells remains the same in GCV treated mice at Inv5 compared to the controls (Supplementary Figure 5E). There are several potential explanations for this negative result (see Discussion).

Next, we wondered whether we could use mammary organoid, the simplified ex vivo system, to examine the direct consequence of removing p16 + senescent epithelial cells during involution-like process. Therefore, we generated mammary organoids from p16-3MR mice and treated them with GCV in vitro during the involution-like process (Figure 5A). First, we confirmed by qPCR that 3MR cassette was induced by a ~15-fold from Inv0 to Inv8 (Figure 5B).

Moreover, upon GCV treatment, there was a significant decrease of the 3MR cassette expression (Figure 5B). Importantly, there was a significant reduction of endogenous p16 expression in the GCV treated organoids compared to the controls (Figure 5C). Consistently, we observed a reduction of SAβGal + cells in the GCV treated organoids (Figure 5D). Therefore, the GCV treatment could reduce the senescence induction in the involution-like organoids ex vivo.

Next, we evaluated the impact of removing senescent cells on the involution-like process using time-lapse imaging. At Inv0, organoids were branched forming a complex 3D structure. Consistent with our previously observation, during the involution-like process, organoids progressively lost their branched shape, recovering a circular conformation at the end of the involution process (Figure .   5E, upper panel) (Sumbal et al. 2020). However, upon GCV treatment, the morphological changes of the organoids were delayed, evidenced by persistent branching (Figure 5E-5G), suggesting a slower regression of the mammary epithelium. Taken together, we showed that removing p16 + senescent mammary epithelial cells directly hindered the involution-like process in the ex vivo organoid system. Overall, our study revealed that senescence is induced during MG physiological involution, coincident with the onset of tissue remodeling phase. The alveolar luminal cells are the major cell type becoming senescent, which is correlated with the up-regulation of p16 expression and is independent of DNA damage.

Importantly, removing senescent cells by ABT-263 treatment delays tissue remodeling process during involution. Consistently, targeting p16 + senescent cells hinders the involution-like process in an ex vivo organoid system.

Collectively, our study demonstrates, for the first time, that senescence plays a relevant role during a postnatal physiological process.

standard deviation. Two-way ANOVA (normality and equality of variances verified; significant interaction) was performed to analyze the effect of ABT-263 treatment with time on the number of SAβGal + cells, followed by Tukey adjustments for comparisons. p-values **<0.01.

Cellular senescence is a major mechanism of cell cycle arrest associated with functional alterations, notably the acquisition of secretory-associated phenotype (SASP) [START_REF] Coppe | The senescenceassociated secretory phenotype: the dark side of tumor suppression[END_REF]. Prior to our study, senescence was associated mainly with pathological processes such as aging or tumor progression, and the involvement of senescence in physiological processes remained largely unknown [START_REF] Herranz | Mechanisms and functions of cellular senescence[END_REF]. Understanding when, where, and how senescence contributes to physiological processes are crucial questions to address. Indeed, senescence exhibits a plethora of roles in vivo that remain to be fully

A. Characterization of the senescence program

Kinetic of MG development using SAβgal staining

We used SAβGal staining to mark senescent cells during whole MG development.

Interestingly, we showed the presence of SAβGal + cells, appearing transiently during the irreversible phase of involution. We confirmed the senescent phenotype of these SAβGal + cells, which were non-proliferative and nonapoptotic.

Detecting senescent cells in vivo remains challenging due to the lack of exclusive markers. SAβGal staining is the most widely used assay to detect senescent cells based on their extended lysosomal content. It allows the identification of the senescent cells in their native environment, which is particularly important for in vivo study. However, other cells with a high level of lysosomal activity, such as macrophages, can be SAβGal + without being senescent. Thus, we showed that only a few SAβGal cells were positive for pan-macrophage marker F4/80 (Annexes Figure 1).

Moreover, another caveat regarding the SAβGal staining is that mammary cells undergo massive lysosomal changes during the involution process and could therefore be false positive for SAβGal staining [START_REF] Kreuzaler | Stat3 controls lysosomal-mediated cell death in vivo[END_REF]). However, lysosomal membrane permeabilization is described during the first reversible phase of involution, which does not correlate in time with the presence of SAβGal + cells, appearing only during the irreversible phase.

Therefore, SAβGal staining remains a powerful technique to detect senescent cells in the MG and has been combined with other analyses to truly identify a new senescent population during MG involution.

C12FDG optimization and identification of senescent cell types

We optimized C12FDG staining in the mammary tissue to define precisely the senescent cell populations. As detailed in the established C12FDG staining protocol, we used bafilomycin, an inhibitor of V-ATPase, which blocks the entrance of H + into lysosomes, to decrease their acidity [START_REF] Cahu | A sensitive method to quantify senescent cancer cells[END_REF].

Thus, we confirmed that only cells with high lysosomal content as senescent cells hydrolyzed the C12FDG compound, and bafilomycin incubation efficiently reduced the false C12FDG-positive mammary cells (Annexes Figure 2).

We combined C12FDG staining with cell surface markers to identify different mammary cell populations via flow cytometry. As expected, in the CD31 + ; CD45 + population containing endothelial and immune cells as macrophages, we detected some C12FDG + , representing ~43% of the CD31 + ; CD45 + population (Annexes Figure 1). But more importantly, in the CD31 -; CD45 -population comprising epithelial and remaining stromal cells, we detected some C12FDG + cells, especially in luminal (~52%) and basal (~15%) compartments. We used SAβGal staining to confirm that luminal and basal cells were also SAβGal + in their native environment with intact tissue architecture. Interestingly, we found a discrepancy between the number of luminal C12FDG + (52%) and SAβGal + (91%) cells. Indeed, C12FDG + is a prominent technique to precisely define cell types in combination with cell surface markers. However, the panel of antibodies used to identify luminal cells did not distinguish between the two subpopulations of luminal cells, from ducts or from alveoli. Therefore, negative ductal luminal cells diluted the percentage of positive alveolar luminal cells. In contrast, we did not observe SAβGal + ducts in the SAβGal assay. As we manually counted the number of double-positive cells over the number of total cells in the alveolar structures, it increased the ratio of positive luminal cells. To conclude, SAβGal and C12FDG

were outstanding techniques that, once combined, allowed us to identify senescent cells in the epithelial compartment, mostly in the alveolar luminal cell population. Interestingly, these populations are the ones mainly removed during mammary gland involution.

p16 expression correlated to involution-associated senescence

p16 and p21 are two key mediators of senescence and are often used as prominent markers to support the senescent phenotype of a cell. To assess the dynamic of senescence mediators, we performed an RNA sequencing analysis on sorted epithelial cells. We decided to analyze first the whole MG, as we planned to perform later a deep characterization of each cell population using single-cell RNA sequencing. We showed that p21 expression was increased at both RNA and protein levels at Inv1 during the first reversible phase of MG involution and did not correlate with SAβGal and C12FDG stainings (Annexes Figure 3).

In contrast, p16 RNA expression was up-regulated during the second phase of MG involution, especially at Inv7. Confirming p16 expression at the protein level was more challenging, as efficient anti-p16 antibodies were not available at the time of our study. We tried to take advantage of the p16-3MR mouse model to visualize and isolate p16 + cells during involution [START_REF] Demaria | An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA[END_REF]. This system contains a cassette expressing luciferase, mRFP, and HSV-TK under p16 regulation, and we showed an increased expression of the cassette at the mRNA level during MG involution. Unfortunately, the reporter cassette was not sensitive enough to detect p16 + cells, as the mRFP level remained undetectable in dissociated involuting MGs (Annexes Figure 4). Indeed, induction of p16associated mRFP during MG involution might not be as strong as in the various pathological settings in which the p16-3MR model was previously described.

Therefore, we used the super sensitive reporter mouse model INKBRITE, with a GFP reporter cassette under the endogenous p16 promoter regulation, to follow p16 expression. We detected the presence of a few p16 + cells in the stroma of lactating MGs. Importantly, we noticed an increase in p16 protein from Inv0 to Inv3 and Inv7, especially in luminal cells, which correlates with SAβgal and C12FDG stainings. Therefore, we showed a strong correlation between p16 expression and the senescence program. However, it also confirmed that there is no unique marker for senescence, and multiple biomarkers should be combined to label senescent cells correctly.

Deep characterization using single-cell RNA sequencing

To deeply characterize the involution-associated senescence program, we performed a single-cell RNA seq on luminal and basal populations at different time points of involution: Inv0, Inv1, Inv3, Inv7, and Inv28. We aimed to identify inducers of senescence, define new senescent biomarkers, and detect specific SASPs up-regulated in senescent cells to understand their roles in the mammary tissue. We used a MARS-seq approach, which comports more than sixty steps for sample processing. Unfortunately, we had technical issues with the quality of cDNA and their barcoding before pooling. We plan to try single-cell RNA sequencing again, this time with the 10x Genomics technique, which is much easier to use.

B. Teat sealing and senescence induction

To characterize senescence induction, we used the previously described technique of sealing [START_REF] Li | Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution[END_REF]. This strategy offers the possibility to uncouple the first reversible phase of involution, triggered by milk stasis, from the second irreversible phase, induced by withdrawal of lactogenic hormones.

Indeed, MGs from sealed nipples entered the first phase of involution characterized by cell death, whereas the second phase was not initiated, with the absence of tissue remodeling. We previously described the appearance of senescent cells three days after physical pups weaning. Interestingly, we did not observe senescent cells in sealed tissue on day 3, when lactogenic hormones are maintained in the system, and tissue remodeling is inhibited. Therefore, initiation of the senescence program might be closely linked to the withdrawal of lactogenic hormones and the onset of tissue remodeling. This in vivo-driven hypothesis is supported by the ex vivo organoid model developed in our laboratory (Sumbal et al. 2020;[START_REF] Charifou | A Robust Mammary Organoid System to Model Lactation and Involution-like Processes[END_REF]) (Charifou et al. 2021). Indeed, after branching with FGF2 and lactation using prolactin, we removed lactogenic stimulations to induce the involution of organoids. As controls, we maintained organoids in culture with prolactin stimulation.

Interestingly, in organoids, milk is not expulsed from the system and accumulates with days in culture. This milk stasis and continuous lactogenic stimulation artificially mimic the sealing strategy ex vivo. Interestingly, in this system with the maintenance of lactogenic stimuli, we did not observe senescence induction, as in sealed MGs. Both systems support the idea that senescence induction is closely linked to the tissue remodeling phase thanks to the withdrawal of lactogenic hormones. Therefore, we aimed to find an efficient strategy to remove senescent cells during MG involution, to assess whether the senescence program was crucial for tissue remodeling.

C. Targeting of senescent cells

Constitutive senescence-free mouse model Cdkn2a; Cdkn1a KO

We illustrated that senescent cells were present during the MG involution.

However, the physiological impact of senescence on MG homeostasis remains unknown. We took advantage of a genetic "senescence-free" mouse model available in our laboratory, constitutively knockout for Cdkn2a and Cdkn1a loci (abbreviated DKO thereafter), thus depleting for p16, Arf, and p21 proteins. To decipher the role of these senescence mediators in MG involution, we induced involution in the DKO mice, and we did not observe any difference in the kinetic of involution between DKO and WT (Annexes Figure 5A). We showed previously that p21 expression during involution was not associated with senescence and might be more related to apoptosis activation. We proposed that pleiotropic functions of p21, lacking in this DKO mouse model, might hide potential p16dependent defects. Another possibility is that constitutive knock-out of these cell cycle inhibitors might be bypassed by activating redundant senescent mediators to maintain viability from early embryonic development. This hypothesis is supported by SAβGal + cells still observed during the involution of MGs from DKO mice (Annexes Figure 5B). Therefore, instead of a constitutive depletion of senescence, we switched strategy by targeting the senescence program only during the MG involution process.

Senolytic treatment with ABT-263 during involution

To further study and characterize the role of senescent cells, we established a loss-of-function system by treating mice with ABT-263 during MG involution.

This senolytic is an inhibitor of anti-apoptotic Bcl-2 family proteins Bcl2, Bcl-W, and Bcl-XL and induces apoptosis of senescent cells both in vitro and in vivo [START_REF] Chang | Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice[END_REF]. We showed that depleting senescent cells during the irreversible phase impaired the kinetic of involution. More specifically, tissue remodeling was affected by the removal of senescent cells, as assessed with larger remaining alveoli and delayed adipocyte refilling. This loss-of-function approach confirmed the central role of senescence in MG tissue remodeling.

However, as the mechanism of action of ABT-263 is based on inhibiting antiapoptotic Bcl2 family proteins, ABT-263 administration might induce harmful effects by depleting other cells expressing high levels of anti-apoptotic proteins, such as platelets [START_REF] Kaefer | Mechanism-based pharmacokinetic/pharmacodynamic meta-analysis of navitoclax (ABT-263) induced thrombocytopenia[END_REF]. Therefore, we attempted to confirm our results on the impact of senescence on tissue remodeling with a complementary strategy, using the p16-3MR mouse model.

Ganciclovir treatment in p16-3MR mice/organoids

As we showed a strong correlation between p16 expression and involutionassociated senescence, we decided to take advantage of the p16-3MR genetic mouse model available in the laboratory. Indeed, this system induces apoptosis of p16 + cells upon ganciclovir administration [START_REF] Demaria | An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA[END_REF].

Interestingly, as p16 was expressed during involution, we detected an associated increased expression of the 3MR cassette. However, we failed to target p16 + cells efficiently with this system (Supplementary Figure 5). This can be explained by the non-optimal concentration of GCV or unsuitable ways of administration to target MG. Therefore, we would like to continue to explore the p16-3MR mouse model and find an efficient method to clear senescent cells in vivo. However, using the reporter INKBRITE mouse model, we showed that some non-senescent cells were also p16 +, especially in the stromal compartment. It raises the concern that eliminating p16 + cells in vivo might also trigger non-senescent cells and interfere in biological processes independent of senescence. To overcome this constraint, we decided to produce organoids from p16-3MR virgin mice, as organoids originate purely from the epithelial compartment, and to treat them with GCV ex vivo during the organoid involution. We showed a delay in the involution-like process when senescent cells were depleted from the system, with more remaining buddings. This phenotype consolidated our hypothesis that senescence was primordial for proper tissue remodeling. Moreover, whereas previous data established a strong correlation between p16 expression and senescent epithelial cells, this system confirmed that senescence was p16dependent ex vivo.

Long-term effects of senescence removal

Strikingly, when we administrated ABT-263 for a short period of only three days, we saw a delay in the kinetic of involution. We also tried to assess the impact of long-term ABT-263 treatment by drug administration from Inv3 to Inv9 and MG harvest at Inv10. The delayed phenotype observed at Inv5 was not noticeable anymore and treated MGs recovered to exhibit a completed process at the end of the involution (Annexes Figure 6). This result was not surprising, as the MG involution process is very dynamic. In many knockout mouse models, depletion of key mediators of involution delayed MG involution, but compensatory mechanisms restored a normal phenotype at the end of the process [START_REF] Radisky | Mammary involution and breast cancer risk: transgenic models and clinical studies[END_REF]. We are now interested in the consequences of removing senescent cells during involution in subsequent pregnancies. Indeed, even if the morphology of the MG might not be impaired at the end of the involution process after senescent cell removal, the plasticity of the whole tissue might be reduced and impact the development of the gland in the next reproductive cycle. This hypothesis could also be assessed in vitro by multiple re-seeding of GCV-treated organoids to mimic subsequent pregnancy-lactation-involution cycles and evaluate the remodeling while senescent c ells are removed.

Senolytics versus senomorphics and senoinducers

We targeted senescent cells using two strategies: senolytic ABT-263 and GCV administration in p16-3MR mice. However, both approaches are similar conceptually, as they induce senescent cell apoptosis. This massive cell death induction could be detrimental to the tissue. Indeed, as lots of cells are removed at once, it can be detected as critical stress and might impair tissue integrity.

Therefore, instead of using senolytics, it can be interesting to use senomorphics.

These drugs target the effects of senescent cells via the inhibition of their SASP, without killing them and disrupting the organization of the tissue.

Another interesting approach would be to do a gain-of-function system.

Senoinducers such as Palbociclib, an inhibitor of cyclin-dependent kinases CDK4 and CDK6, are compounds with the ability to induce senescence [START_REF] Vanarsdale | Molecular Pathways: Targeting the Cyclin D-CDK4/6 Axis for Cancer Treatment[END_REF]. As senescent cell removal delayed MG involution both in vivo and ex vivo, we would like to increase the number of senescent cells and assess the kinetic of involution. From this experiment, we might observe a diversity of phenotypes. Indeed, in opposition to the senescence removal experiments, an increased number of senescent cells might accelerate the kinetic of regression, which could be measured by smaller alveoli and accelerated adipocyte refiling.

However, the number of senescent cells is already high at Inv3. This level might already be optimal, and adding more senescent cells might not affect the effective progression of involution. Another interesting approach would be to induce senescent cells when they are not present yet in the tissue. We showed that senescence was closely associated with tissue remodeling during the second phase of involution. By using senoinducers during the first phase of involution, the presence of senescence might be sufficient to initiate remodeling, reinforcing the causative link between senescence and tissue remodeling.

D. Proposed roles of senescence in MG

We demonstrated a close link between senescence and tissue remodeling during mammary gland involution. However, this tissue-remodeling phase is a sum of various intricate processes, such as removing epithelial cells concomitant with adipocyte refilling, restructuring the extracellular matrix, and recruiting immune cells for clearance of cells and debris.

By depleting senescent cells with ABT-263, we showed a delay of involution with more remaining epithelial cells, which correlated with slower adipocyte refilling.

Adipocyte refilling can be either a passive phenomenon to fill emptied space in the fat pad or an active phenomenon helping alveoli shrinkage. We proposed that senescent cells might secrete SASPs promoting adipocyte refilling. To test this hypothesis, we successfully established an in vitro model of adipogenic differentiation using sorted mammary Pdgfr-α + cells, precursors of fibroblasts, and adipocytes (Annexes Figure 7). GFP + cells were isolated from a Pdgfr-α-GFP mouse model, and as previously described, we induced differentiation using an adipogenic cocktail of dexamethasone, IBMX, and insulin [START_REF] Joshi | PDGFRalpha(+) stromal adipocyte progenitors transition into epithelial cells during lobulo-alveologenesis in the murine mammary gland[END_REF]. We would like to test the effect of conditioned medium collected from non-senescent or senescent cells to assess whether SASPs could promote adipogenic maturation and accelerate the filing of lipids.

During the tissue-remodeling phase of mammary involution, numerous compounds are secreted to degrade the extracellular matrix (ECM), such as matrix metalloproteinases (MMPs). It is also well exemplified that in the SASPs factors produced by senescent cells, many regulate the composition and the organization of the extracellular matrix [START_REF] Mavrogonatou | Extracellular matrix alterations in senescent cells and their significance in tissue homeostasis[END_REF]. Therefore, we wondered whether senescent cells are helping the remodeling of mammary tissue via ECM reorganization. As we detected senescent cells in the epithelial compartment, specifically during the second phase of involution, we performed RNA sequencing on epithelial cells at Inv1 and Inv7. Interestingly, we showed a significant up-regulation of tissue remodeling and matrix disassembly gene ontology biological processes (GOBPs) (Annexes Figure 8). We are now interested in finding in our RNA seq data specific candidates secreted by senescent cells, which could explain part of their impact on tissue remodeling during MG involution.

SASP factors are also usually composed of various cytokines and chemokines [START_REF] Coppe | The senescenceassociated secretory phenotype: the dark side of tumor suppression[END_REF]. During the tissue-remodeling phase of involution, there is an influx of immune cells recruited to remove dead cells and debris. Therefore, we hypothesized that senescent cells present during MG involution might promote immune cell recruitment. In MGs treated with ABT-263, we assessed the number of different types of immune cells. Interestingly, we showed a decrease in the number of total mammary macrophages, and ductal macrophages in MGs depleted for senescent cells (Annexes Figure 9). This phenomenon could be due to delayed involution, where immune cell influx might be passively lagged.

However, it could also reflect the active recruitment of immune cells by senescent cells, lacking in ABT-263 treated glands. To assess this question, we would like to test whether sorted mammary senescent cells can chemoattract macrophages in vitro. Moreover, we could use compounds to induce senescence during the first phase of involution and evaluate if the presence of senescent cells is sufficient to recruit immune cells earlier (See "Senolytics versus senomorphics and senoinducers" for further details).

How mammary stem cells maintain their stemness through reproductive cycles is not fully understood. It has been shown recently that senescence, via ectopic expression of Rank in the luminal cells of virgin MG, promotes stemness properties of both basal and luminal populations [START_REF] Benitez | RANK links senescence to stemness in the mammary epithelia, delaying tumor onset but increasing tumor aggressiveness[END_REF]. Therefore, we could assume that physiological senescence associated with involution might protect mammary stem cells by increasing their stemness before a new development cycle. To test this hypothesis, we would like to take advantage of the mammosphere assay, an in vitro technique to quantify stem cells in the mammary tissue and evaluate their self-renewal capacity [START_REF] Shaw | A detailed mammosphere assay protocol for the quantification of breast stem cell activity[END_REF]). We propose to collect conditioned medium from sorted luminal cells at Inv0 (nonsenescent) and Inv7 (senescent), culture mammospheres in both conditions, and assess any difference in the number of mammary stem cells or their stemness when cultured in senescent conditioned medium.

E. Fate of senescent cells

At the end of the involution process, we did not detect SAβGal + cells suggesting that senescence induction was transient. It correlated with the fact that shortterm senescence is associated with beneficial processes, as in embryonic development. In contrast, the persistence of senescent cells in tissues is associated with pathological conditions. However, how senescent cells are removed from the tissues remains unclear. One assumption could be that senescent cells are removed through apoptosis. However, it is well described that senescent cells up-regulate intrinsic pathways to resist apoptosis [START_REF] Childs | Senescence and apoptosis: dueling or complementary cell fates?[END_REF]). Another hypothesis is that senescent cells might be eliminated through immune cell clearance, primarily via macrophages. Indeed, evidence

shows that immune cell-mediated response is critical in senescent cell clearance. To validate this hypothesis, we proposed to deplete macrophages and quantify the number of senescent cells not cleared from the tissue. We successfully established a model of macrophage depletion during involution using clodronate liposomes (Van Rooijen and Sanders 1994). As previously described using a transgenic MAFIA mouse model inducing macrophages apoptosis, we also showed a delay of involution upon macrophages depletion with clodronate liposomes (Annexes Figure 10). We would like to measure senescence levels in MGs depleted for macrophages. Moreover, we will take advantage of the in vitro system to analyze interactions between sorted senescent cells and immune cells (See "Proposed roles of senescence in the mammary tissue" for further details) to see if chemoattracted macrophages can engulf senescent cells.

F. Pathological perspectives of involution-associated senescence

It is well established that pregnancy and involution transiently increase the risk of breast cancer. Postpartum breast cancer (PPBC), defined as breast cancer diagnosed within ten years of most recent childbirth, accounts for over half of all breast cancers diagnosed in women under age 40. Notably, PPBC exhibits increased rates of metastasis and poorer long-term survival compared to non-PPBC, highlighting a cancer-promotional microenvironment explicitly associated with the postpartum period (Schedin 2006). Concomitantly, it is well described that senescent cells promote tumorigenesis in a paracrine manner via their SASP [START_REF] Faget | Unmasking senescence: contextdependent effects of SASP in cancer[END_REF]. Therefore, we hypothesized that the involutionassociated senescence might promote initiation, progression, and metastasis of PPBC. Indeed, in a paracrine manner, senescent cells might secrete SASPs factors during involution, contributing to the pro-inflammatory microenvironment and promoting proliferation and plasticity of pre-existing pro-tumoral cells.

Interestingly, postlactational involution, in which the senescence program is crucial, is associated with an increased risk of PPBC. In contrast, lobular involution related to aging of the reproductive capacity, in which we did not detect senescent cells, correlates with a decreased risk of developing breast cancer [START_REF] Radisky | Mammary involution and breast cancer risk: transgenic models and clinical studies[END_REF]. This exciting pathological perspective of involution-associated senescence is explored in another project in our laboratory. Using a breast cancer mouse model and our ex vivo 3D organoid culture system, we aim to investigate how senescent cells might induce plasticity of existing pre-cancerous cells. Moreover, we established a partnership with Paris Curie Institute to obtain samples from PPBC patients, to measure the number of senescent cells in cancerous mammary tissue. Linking involution-associated senescence characterized in this Ph.D. project to PPBC might unravel new mechanisms involved in PPBC emergence and enlarge therapeutic options for women affected by PPBC.

Annexes Figures Discussion

Annexes Figure

Material and Methods

A.

Mice models and sample processing PDGFRα EGFP (Pdgfra tm11(EGFP)Sor ) mouse model was obtained from The Jackson Laboratory (JAX mouse number #007669) [START_REF] Hamilton | Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms[END_REF].

Genotyping methods

Gene

Standardization and synchronization of involution

Induction of MG involution and processing of the tissues have been extensively reviewed previously (Lloyd- [START_REF] Lloyd-Lewis | Analysis of the Involuting Mouse Mammary Gland: An In Vivo Model for Cell Death[END_REF]. Briefly, adult virgin females were mated between 8 to 10 weeks old. To homogenize lactation stimuli, litters were evenly distributed to 6 pups per female after birth and fed by the mother for 10 days. Then, pups were physically separated to allow a synchronized involution of MGs. The day of removal of the progeny was x35 cycles x34 cycles considered as the beginning of involution, also called involution day 0 or Inv0.

The involution process was let to progress from Inv0, for which mice were euthanized right after offspring removal, to 1,2,3,4,5,or 7 days of involution (respectively named Inv1,Inv2,Inv3,Inv4,Inv5,Inv7) up to the end of the process 14 days after the initiation (Inv14).

To ensure reproducibility of the study, MGs studied for involution were harvested after a unique pregnancy and a successful lactation (Lloyd- [START_REF] Lloyd-Lewis | Analysis of the Involuting Mouse Mammary Gland: An In Vivo Model for Cell Death[END_REF]. MGs from virgin mice were either collected at 8 weeks old (virgin) or 18 months old (old nulliparous); MGs from pregnant mice were collected at p18.5; MGs from multiparous mice were collected at the resting stage from females coming from breeding cages, after around 8 rounds of pregnancy. For involution studies, males were removed from mating cages around 1 week prior to delivery, avoiding a new fertilization upon delivery, and limiting the stress associated to male removal close to the delivery. For precise pregnancy time point, male and female were placed together for the night and female was manually checked early next morning for the presence of a vaginal plug [START_REF] Behringer | Selecting Female Mice in Estrus and Checking Plugs[END_REF].

Mammary glands harvesting and processing

Mice were euthanized by cervical dislocation, and specific mammary glands were collected, respectively: MG #2L for RNA; MG #3L for wholemount SAβGal staining; MG #3R for OCT cryopreservation; MG #4L for formalin fixation; MG #4R for methacarn fixation.

a) OCT cryopreservation

MGs were embedded in OCT to obtain cryosections. Tissues were placed in OCT cassettes right after harvesting, covered by tissue freezing medium [Leica #14020108926] and kept at -80°C until cutting. Frozen samples were cut using a cryostat and slices of 10μm were spread on SuperFrost glass slides and kept at -80°C for further stainings.

b) Formalin fixation

MGs were spread on glass slides and fixed overnight in a solution of formalin 10%. After 16h at room temperature, tissues were briefly washed in Phosphate-Buffered Saline (PBS) and dehydrated with ethanol (successive 45 min baths, increasing concentration of ethanol, 50%; 70%; 95% and twice 100%). Samples were then transferred in cassettes and disposed into two baths of 1h of xylenes before a mix of 50/50 xylenes/paraffin during 1h at 65°C. Then, the cassettes were placed in a first solution of 100% paraffin for 1h at 65°C, and in a second bath of pure paraffin for an overnight at the same temperature. The next day, samples were embedded in paraffin and kept at room temperature to solidify.

Embedded tissues were cut using a microtome and slices of 5μm were disposed on SuperFrost glass slides for further stainings [START_REF] Tucker | Sectioning Mammary Gland Whole Mounts for Lesion Identification[END_REF].

c) Methacarn fixation

MGs were spread on glass slides and fixed overnight in a solution of methacarn (60% methanol; 30% chloroform; 10% acetic acid) before being processed for Carmine coloration (see Whole mount Carmine). Tissues were then washed and dehydrated as described for formalin fixed samples and embedded in paraffin [START_REF] Tucker | Sectioning Mammary Gland Whole Mounts for Lesion Identification[END_REF].

B. Histological analysis

Whole mount Carmine

Straight away after harvesting, mammary glands #4 were fixed overnight in methacarn. After 16h of incubation, MGs were washed twice in EtOH 70%, once in H2O, and incubated overnight in Carmine Alum staining [Stem Cell

Technologies #07070] at room temperature, protected from light in aluminum covers [START_REF] Tolg | Mouse Mammary Gland Whole Mount Preparation and Analysis[END_REF]. MGs were then washed twice in EtOH 70% and processed as Formalin samples to be embedded in paraffin. During the second clearing with xylenes, pictures of whole mounts stained MGs were taken using a binocular magnifier (zoom-in x0.7 and x5) before the embedding to obtain a global histology of MGs.

Deparaffinization

Formalin or methacarn fixed samples were embedded in paraffin and sliced at 5μm as described. Paraffin was removed prior to any staining by using successive 5 min baths of: xylenes twice, and decreasing concentration of ethanol from EtOH 100% to EtOH 50%. Finally, slides were washed in H2O and PBS [START_REF] Thompson | Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology[END_REF].

Hematoxylin and Eosin H&E

After deparaffinization, sections were stained in Hematoxylin for 5 to 10 min and washed with tap water for 5 min to mark nuclei in purple. Sections were then stained with eosin for 2 to 5 min and rinsed in PBS to mark cytoplasm and extracellular matrix in pink. The intensity of each staining was checked under the microscope before washing. Tissues were finally dehydrated with one 5 min bath of EtOH 95%, two 5 min baths of EtOH 100% and dried for 30 sec under a laminar flow hood before mounting with Eukitt [START_REF] Thompson | Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology[END_REF].

C. Immunohistochemistry and immunofluorescence

After a step of deparaffinization, slides were processed for antigen retrieval when specified. Briefly, 10x buffer for acid retrieval pH 6.0 [Electron Microscopy Sciences #62706-10] was resupended to 1x with ultrapure Milli-Q water prior to use. Slides were then immerged in the retrieval buffer and placed in a retriever [Electron Microscopy Sciences #2100] for a 2.5h cycle (20min heat followed by a 2h gentle cooling).

For immunochemistry only, an inhibition of endogenous peroxidase was performed using H2O2 1% during 10min prior to a 1h blocking step using PBS + 1% Bovine Serum Albumin (BSA), followed by an overnight incubation at 4°C of primary antibody, in PBS +1%BSA. After corresponding secondary antibody incubation for 1h, revelation was made using DAB for 1min. Tissue sections were then counterstained with Hematoxylin, dehydrated using increasing concentrations of ethanol and mounted with Eukitt.

For immunofluorescence, after blocking, overnight primary incubation and corresponding secondary antibody incubation, nuclei were stained during 15min

with DAPI (1µg/mL in H2O) in PBS +1%BSA, washed in PBS and mounted with Immu-Mount [START_REF] Thompson | Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology[END_REF].

Primary antibody

SAβGal staining on sections

SA!Gal staining solution for MG staining was prepared as previously described for muscle samples [START_REF] Cazin | Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle[END_REF]

OilRedO staining

OilRedO staining solution for MG staining was prepared as previously described [START_REF] Joshi | PDGFRalpha(+) stromal adipocyte progenitors transition into epithelial cells during lobulo-alveologenesis in the murine mammary gland[END_REF] For Oil RedO staining on plated cells, wells were washed in PBS before fixation.

Cells were then processed as tissue sections, and imaged after the last washing in PBS.

D. Flow cytometry

Isolation of mouse mammary cells

Preparation of isolated mammary cell solution was performed as previously described [START_REF] Taddei | Beta1 integrin deletion from the basal compartment of the mammary epithelium affects stem cells[END_REF]. Briefly, for each mouse, MGs #2, #3, #4 and #5

were collected and minced before an enzymatic digestion for 1h30 at 37°C, and immediately centrifuged. After a resuspension in CO2-independent medium + 5% FBS, cell solution was filtered through a nylon mesh cell strainer with 40μm pores before immunolabeling and cell sorting.

C12FDG staining

C12FDG staining was performed in isolated mammary cell suspension following the C12FDG protocol previously described to stain senescent cells [START_REF] Cahu | A sensitive method to quantify senescent cancer cells[END_REF].

Stainings of isolated mammary cells for FACS

Freshly dissociated mammary cells were incubated at 4°C for 30min, out of direct light, with a combination of antibodies directly conjugated for flow cytometry. After incubation, cells were washed twice with CO2-independent medium. Labeled cells were sorted using a MoFlo Astrios EQ [Beckman Coulter] and data were analyzed with FlowJo software.

Antibodies used for flow cytometry analysis to identify mammary cells as previously described [START_REF] Shackleton | Generation of a functional mammary gland from a single stem cell[END_REF] For immune cells identification, the combination of antibodies was selected in accordance with a previous description of the mammary immune landscape

E. RNA extraction and RT-qPCR

For RNA extraction from whole MG, MG tissue pieces were snap-frozen and kept at -80°C until processing. Samples were maintained on dry ice until adding 1ml

of TRIzol [Invitrogen #15596026] increase rate of 0,11°C/s; Cooling to reach an end temperature of 40°C) and corresponding primers. For each sample, all values were obtained at least in duplicates and in a total of at least two independent assays. Calculations for the values were done as previously described using the ΔΔCt method [START_REF] Yuan | Statistical analysis of realtime PCR data[END_REF]).

F. Organoids

Organoids culture

Mammary organoids isolation, culture, passage, process and time lapse imaging were extensively described in (Charifou et al. 2021).

Ex vivo treatment

Ganciclovir treatment on organoids was optimized from previous studies targeting p16-3MR cells in in vitro 2D-cell cultures [START_REF] Demaria | An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA[END_REF][START_REF] Kohli | A novel suicide gene therapy for the treatment of p16(Ink4a)-overexpressing tumors[END_REF]. Ganciclovir [Medchemexpress #HY-13637] was resuspended in DMSO, at 50mg/ml and stored at -20°C in single-use aliquotes.

After 6 days of 2.5nM FGF2 and 4 days of 1µg/ml prolactin + 1µg/ml hydrocortisone, organoids from p16-3MR mice were treated during involutionlike process with either 50µg/ml ganciclovir in BOM (DMEM/F12, Glutamax, Pen/Strep, Insulin-Transferrin-Selenium) or BOM+DMSO, from Inv0 to Inv8, with refreshing of media every 2 days.

G. RNA sequencing on isolated epithelial cells

RNA preparation

Pieces of MG #2 were harvested from mice at Inv0, Inv3 and Inv7 in 4 biological replicates for each time point and were stored at -80°C until RNAs extraction using Trizol. 1000ng of each sample were sent to the Beijing Genomics Institute for RNA sequencing.

Bioinformatic analysis

The potential biological pathways with differences between the experimental

H. Adipogenic differentiation

Mammary glands from Pdgfrα EGFP mice were processed (see isolation of mammary cells) to isolate Pdgfrα-GFP + by FACS. GFP + cells were seeded after sorting in 12-well plates at a density of 200,000 cells in SVF medium (DMEM/F12 [Gibco #21331-020], Glutamax, Pen/Strep, and 10% FBS).

Adipogenic differentiation was performed as previously described [START_REF] Joshi | PDGFRalpha(+) stromal adipocyte progenitors transition into epithelial cells during lobulo-alveologenesis in the murine mammary gland[END_REF]. Briefly, SVF medium was renewed the day after the sort to remove dead cells. Attached cells were allowed to grow to confluence (approximately 1 day)

and were held confluent for 2 days without changing of the media. Cells were then incubated with SVF medium supplemented with an adipogenic cocktail of 1μM dexamethasone [Sigma-Aldrich #D4902]; 0,5mM IBMX [Sigma-Aldrich #I7018]; and 1μg/ml insulin [Sigma-Aldrich #I7018]; for 3 days; before switching back to fresh SVF medium. Adipocytes were visible 2 days after withdrawal of the adipogenic cocktail, with a peak of differentiation between 2-4 days after adipogenic cocktail removal. Cells were either collected in RLT buffer for RNA extraction, or stained for OilRedO staining.

I.

In vivo experiments

In vivo sealing of mammary nipples

Teat sealing was performed as previously described [START_REF] Li | Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution[END_REF]. Briefly, after delivery, females were allowed to lactate for 10 days. Hair remover was applied to clean the nipple area from mammary glands #3L and #4L before sealing. After

In vivo treatment with ABT-263

The

J. Softwares and statistical analysis

Fluorescent images were captured using the inverted fluorescent microscope Olympus IX83 with a Hamamatsu C11440 camera and Zen Blue imaging software; scanned brighfield images were captured using the virtual microscope Olympus V5120 with a Hamamatsu C13440 camera and Zen Blue imaging

INTRODUCTION

Lactation, the production of milk to feed progeny, is achieved by the mammary gland. This hallmark organ of mammals mainly develops postnatally and is highly dynamic (Macias and Hinck, 2012). With each pregnancy, mammary epithelium undergoes massive proliferation, tertiary branching of the mammary ductal system, and alveoli di erentiation to prepare the epithelium for proper lactation (Brisken and Rajaram, 2006;Sternlicht, 2006). After parturition, mammary epithelium fully transforms into a milk-producing factory. Alveoli expand and take up space of regressing mammary stromal adipocytes, thereby multiplying epithelial volume many times (Macias and Hinck, 2012). After weaning, when milk production is no longer required, milkproducing epithelial cells are removed, and mammary gland is remodeled into a prepregnancy state. This process is called involution, which includes programmed cell death of the epithelium, ECM remodeling, and redi erentiation of adipocytes (Hughes and Watson, 2012;Macias and Hinck, 2012;Zwick et al., 2018;Jena et al., 2019). By the end of involution, mammary gland is ready for a new cycle of pregnancy-associated growth, lactation, and subsequent involution, which can be repeated throughout the reproductive lifespan. During these changes, mammary epithelium retains its bilayered architecture with lumen-facing luminal cells and basally situated myoepithelial cells, which is essential for proper function of the organ [START_REF] Adriance | Myoepithelial cells: good fences make good neighbors[END_REF][START_REF] Haaksma | Myoepithelial cell contraction and milk ejection are impaired in mammary glands of mice lacking smooth muscle alpha-actin[END_REF]Macias and Hinck, 2012).

Endocrine signaling is a crucial regulator of mammary morphogenesis during pregnancy. Ovarian hormones estrogen and especially progesterone govern growth and morphogenesis of epithelium via induction of paracrine signaling between mammary stroma and epithelium, involving members of several growth factor families (Hennighausen and Robinson, 2005;Brisken and O'Malley, 2010). Pituitary hormone prolactin, on the other hand, acts directly on prolactin receptor on luminal cells and triggers alveoli maturation and lactogenic di erentiation (Hennighausen and Robinson, 2005;Brisken and Rajaram, 2006). Involution is linked to cessation of hormonal stimuli and increase in inflammatory cytokines (Watson, 2006;[START_REF] Stein | Mammary gland involution as a multi-step process[END_REF].

To study various aspects of mammary gland biology, threedimensional (3D) cell culture models have been widely used for decades (Koledova, 2017a). They combine the advantages of easy manipulation of 2D cellular systems with providing complex cell-cell and cell-ECM interactions, thereby mimicking physiological conditions of in vivo experiments more faithfully (Shamir and Ewald, 2014;Huch and Koo, 2015;Koledova, 2017a;Artegiani and Clevers, 2018). Among the 3D culture models, primary mammary organoids have played a major role in understanding mechanisms of mammary branching morphogenesis (Ewald et al., 2008;Huebner et al., 2016;Neumann et al., 2018), including the role of ECM (Simian et al., 2001) and stromal cells (Sumbal and Koledova, 2019). Furthermore, spheroids produced from mammary cell lines were used to study tissue response to growth factors (Xian et al., 2005); organoids grown from sorted single primary mammary epithelial cells were used to study developmental potential of mammary epithelial cells (Linnemann et al., 2015;Jamieson et al., 2017), and di erentiation of mammary-like organoids was achieved from induced pluripotent stem cells (Qu et al., 2017).

Despite these advances in 3D cell culture models of mammary gland, systems faithfully modeling pregnancy-associated morphogenesis and lactation have been spare. In some studies, b-casein or milk protein expression was used as a read-out of mammary epithelial functionality (Mroue et al., 2015;Jamieson et al., 2017). Several aspects of lactation and involution were captured in a coculture of mammary epithelial and preadipocyte cell lines (Campbell et al., 2014) or in hormone-treated breast cancer cell spheroids [START_REF] Ackland | Extracellular matrix induces formation of organoids and changes in cell surface morphology in cultured human breast carcinoma cells PMC42-LA[END_REF]Freestone et al., 2014). However, a system modeling lactation and involution in primary mammary organoids with proper architecture of bilayered epithelium with myoepithelial cell layer has not been characterized.

Here, we report on a mammary 3D culture system for studying induction and maintenance of lactation using easily accessible and physiologically relevant murine primary mammary organoids cultured in Matrigel. Upon prolactin stimulation, the organoids produce milk for at least 14 days and maintain a histologically normal architecture with a functional contractile myoepithelial layer. Moreover, upon prolactin signal withdrawal, our system recapitulates several aspects of involution. Altogether, we describe a robust, consistent, and easy-to-do system for modeling crucial aspects of pregnancyassociated mammary gland morphogenesis and lactation.

MATERIALS AND METHODS

Isolation of Primary Mammary Epithelial Organoids

Primary mammary organoids were prepared from 7-to 10week-old female mice (ICR or C57/BL6) as previously described [START_REF] Koledova | 3D coculture of mammary organoids with fibrospheres: a model for studying epithelial-stromal interactions during mammary branching morphogenesis[END_REF]; Supplementary Figure 1A). ICR strain was used for the branching morphogenesis and time-lapse imaging, cell viability and replating assays, and confocal imaging. C57/BL6 strain was used for the rest of the experiments. The animals were obtained from the Central Animal Facility of the Institut Pasteur and the Laboratory Animal Breeding and Experimental Facility of the Faculty of Medicine, Masaryk University. Experiments involving animals were approved in accordance with French legislation in compliance with European Communities Council Directives (A 75-15-01-3), the regulations of Institut Pasteur Animal Care Committees (CETEA), the Ministry of Agriculture of the Czech Republic, and the Expert Committee for Laboratory Animal Welfare at the Faculty of Medicine, Masaryk University. The study was performed by certified individuals (AC, JS, EC, and ZK) and carried out in accordance with the principles of the Basel Declaration.

Briefly, the mice were euthanized by cervical dislocation, the thoracic and inguinal mammary glands were collected, visible lymph nodes were excised, and the pooled mammary glands were finely chopped to approximately 1-mm 3 pieces and digested in a solution of collagenase and trypsin [2 mg/mL collagenase (Roche, Switzerland or Sigma, United States), 2 mg/mL trypsin ( ⇤ Dutscher Dominique, France or Sigma, United States), 5 µg/mL insulin (Sigma, United States), 50 µg/mL gentamicin (Sigma, United States), 5% fetal bovine serum (Hyclone/GE Healthcare, United States) Dulbecco's in modified Eagle medium (DMEM)/F12 (Thermo Fisher Scientific, United States)] for 30 min at 37 C with shaking at 100 rpm. Next, the tissue suspension was treated with 20 U/mL DNase I (Sigma, United States) and 0.5 mg/mL dispase II (Roche, Switzerland) and exposed to five rounds of di erential centrifugation at 450 ⇥ g for 10 s, which resulted in separation of epithelial (organoid) and stromal fractions (Supplementary Figure 1A). The organoids were resuspended in basal organoid medium [BOM; 1⇥ insulintransferrin-selenium supplement, 100 U/mL of penicillin, and 100 µg/mL of streptomycin, in DMEM/F12 (all from Thermo Fisher Scientific, United States)] and kept on ice up to 2 h before seeding for 3D culture.

3D Culture of Mammary Organoids

Freshly isolated primary mammary organoids were mixed with growth factor reduced Matrigel (Corning, United States) and plated in domes in 24-well culture plate (one dome per well, 70 µL of Matrigel per dome). 200, 400, or 1000 organoids per dome were seeded for histology, gene expression, and Western blot analysis, respectively. After setting the Matrigel for 45-60 min at 37 C, the 3D organoid cultures were overlaid with cell culture medium according to the experiment and incubated at 37 C in a humidified atmosphere with 5% CO 2 (Supplementary Figure 1B). The media used were as follows: growth factor medium [BOM supplemented with di erent growth factors: 2.5 nM FGF2 (Peprotech, United States or Thermo Fisher Scientific, United States), 2.5 nM FGF7, 2.5 nM FGF10, 50 ng/mL EGF (all from Peprotech, United States), 5 nM TGFa (Sigma, United States), or a combination of 10 ng/mL WNT3A and 50 ng/mL R-spondin 1 (W3/R1, both from Peprotech, United States)] and lactation medium {LM; 1 µg/mL prolactin [mouse recombinant prolactin for quantitative polymerase chain reaction (qPCR), Western blot, immunohistochemistry and contraction experiments (Sigma, United States or Peprotech, United States), and sheep pituitary prolactin for confocal and time-lapse imaging, including contraction experiments (Sigma, United States)], and 1 µg/mL hydrocortisone (Sigma, United States) in BOM}. Media containing growth factors were changed every 3 days; LM was changed every 2 days. To induce contraction of lactation organoids grown with mouse recombinant prolactin, 40 µg/mL recombinant oxytocin (Sigma, United States) was used. For time-lapse imaging experiments, organoid cultures were incubated in a humidified atmosphere of 5% CO 2 at 37 C on Olympus IX81 microscope equipped with Hamamatsu camera and CellR system for time-lapse imaging. For morphological analysis of organoid development, the organoids were photographed from days 8 to 17 of culture; one image per organoid was taken every hour. The images were exported and analyzed using ImageJ (NIH, United States). For analysis of organoid contraction, the organoids were photographed from days 6 to 20 of culture. On each imaging day, the photographs were taken every second for 120 s. The images were exported to video at 10 frames per second using xCellence software (Olympus, Japan).

Replating of Organoids

To replate organoids, 3D cultures were rinsed with phosphatebu ered saline (PBS) and disintegrated by pipetting up and down in ice-cold PBS with a 1000 µL pipette. Successful disintegration of Matrigel was checked under a microscope. Organoid suspensions were centrifuged at 450 ⇥ g for 3 min. Organoid pellets were resuspended in fresh Matrigel and plated as described above. Organoids were maintained in BOM or in BOM supplemented with 2.5 nM FGF2; the medium was changed every 3 days. Organoid area was measured in ImageJ.

Cell Viability Assay

To asses cell viability in organoids treated with LM or LM-BOM, on the 20th day of culture, the media were changed with fresh BOM, and then resazurin (Merck, Germany) was added to the medium to the final concentration of 10 µg/mL. The plates were incubated for 6 h. Resorufin fluorescence (excitation at 560 nm, emission at 590 nm) was measured using Synergy H4 Hybrid multimode microplate reader (BioTek, United States) in technical triplicates. As a positive control of dying cells, organoids in LM-BOM conditions were treated from day 16 with 40 µM taxol (Sigma, United States) or killed on day 20 by treatment with 70% ethanol for 5 min.

Histology and Immunostaining Analysis

For histological analysis, organoids were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, United States) for 30 min and embedded in 3% low gelling temperature agarose (Supplementary Figure 1C). After solidification, samples were dehydrated and para n embedded and cut in 5-µm sections, which were dewaxed for hematoxylin and eosin staining or immunostaining. For localization of prolactin receptor expressing cells, 10-µm cryosections of mammary glands from Prlr-IRES-Cre;ROSA26-CAGS-GFP mice [START_REF] Aoki | Widespread Cell-specific prolactin receptor expression in multiple murine organs[END_REF] were labeled with antibodies and counterstained with 0.5 µg/mL DAPI, mounted with Vectashield (Vector Labs, United States), and images were taken on LSM800 microscope (Zeiss, Germany). The following primary antibodies were used: goat anti-GFP (Origene, United States, R1091P, 1:200), rabbit polyclonal anti-keratin 5 (BioLegend, United States, 905501, 1:200), mouse monoclonal anti-keratin 8 (BioLegend, United States, 904801, 1:200), mouse monoclonal anti-b-casein (Santa Cruz, United States, sc-166530, 1:250), and rabbit anti-mouse milk proteins ( ⇤ Accurate Chemical, United States, YNRMTM, 1:500). Corresponding secondary antibodies were used: donkey anti-rabbit Dylight 488 (Immuno Reagents, United States, DkxRb-003-D594NHSX, 1:200) and donkey anti-mouse Dylight 594 (Immuno Reagents, United States, DkxMu-003-D488NHSX, 1:200), together with 1 µg/mL of Hoechst-33342 (Thermo Fisher Scientific, United States) for immunofluorescence labeling, or anti-mouse/anti-rabbit horseradish peroxidase (HRP)-associated secondary antibodies (Dako, United States).

Whole Mount Staining of Mammary Organoids

Organoids were fixed with 10% formalin for 30 min, washed with PBS and 70% ethanol, and incubated with oil red O solution [0.3% (wt/vol) oil red O (Sigma, United States) in 70% (vol/vol) ethanol [START_REF] Koopman | Optimisation of oil red O staining permits combination with immunofluorescence and automated quantification of lipids[END_REF][START_REF] Kim | A post-developmental genetic screen for zebrafish models of inherited liver disease[END_REF]] for 30 min in the dark. Next, organoids were washed with 70% ethanol and PBS and incubated with 0.5 µg/mL DAPI and 2 units/sample phalloidin-AlexaFluor488 (Thermo Fisher Scientific, United States) in PBS for 1 h at room temperature (RT) in the dark. Subsequently, organoids were washed and transferred to coverslip-bottom 35-mm dishes (ibidi) covered with 1% low gelling temperature agarose (Sigma, United States) and overlaid with PBS. Images were acquired using LSM800 confocal microscope (Zeiss, Germany, Supplementary Figure 1D) and analyzed using ZEN blue software (Zeiss, Germany).

RNA Isolation and Real-Time qPCR

Total RNA was extracted from organoid samples using RNeasy Micro Kit (Qiagen, Germany) following the manufacturer's instructions. Reverse transcription was performed using high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, United States). Quantitative real-time PCR was performed using 5 ng cDNA, 5 pmol of the forward and reverse gene-specific primers each in Light Cycler SYBR Green I Master mix (Roche, Switzerland) on LightCycler 480 II (Roche, Switzerland). All reactions were performed at least in duplicates and in a total of at least two independent assays. Relative gene expression was calculated using the 11Ct method, and the values were normalized to housekeeping gene Gapdh. The primers of following sequences (5 0 -3 0 ) were used: Csn2-forward (F): CCTCTGAGACTGATAGTATTT, Csn2reverse (R): TGGATGCTGGAGTGAACTTTA; Wap-F: TT GAGGGCACAGAGTGTATC, Wap-R: TTTGCGGGTCCTACC ACAG; Mmp3-F: CCTGATGTTGGTGGCTTCA, Mmp3-R: TC CTGTAGGTGATGTGGGATTTC; Mmp13-F: ACTTCTACCCA TTTGATGGACCTT, Mmp13-R: AAGCTCATGGGCAGCAA CA; Gapdh-F: TTCACCACCATGGAGAAGGC, Gapdh-R: CC CTTTTGGCTCCACCCT. All primers were purchased from Sigma, United States.

Western Blot

Three-dimensional cultures were dissociated by repetitive pipetting in ice-cold PBS supplied with phosphatase inhibitor cocktail II (Merck, Germany; 2 mM imidazole, 1 mM sodium fluoride, 1.15 mM sodium molybdate, 1 mM sodium orthovanadate, 4 mM sodium tartrate dihydrate), followed by centrifugation at 450 ⇥ g for 3 min at 4 C. Supernatant was discarded, and pellets were lysed in ready-to-use RIPA bu er [Merck, Germany; 150 mM NaCl, 1.0% IGEPAL R CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris, pH 8.0] supplied with protease inhibitor cocktail I (Merck, Germany; 500 µM AEBSF hydrochloride, 150 nM aprotinin, 1 µM protease inhibitor E-64, 0.5 mM EDTA, 1 µM leupeptin hemisulfate) and phosphatase inhibitor cocktail II. After vortexing and sonication, protein lysates were cleared by centrifugation, and protein concentration was measured using Coomassie reagent (Merck, Germany). Denatured, reduced samples were resolved on 12.5% SDSpolyacrylamide electrophoresis (Bio-Rad, United States) and blotted onto nitrocellulose membranes by Trans-blot Turbo transfer system (Bio-Rad, United States). After blotting, the membranes were blocked with 2% bovine serum albumin in PBS with 0.1% Tween-20 (Merck, Germany; blocking bu er) and incubated with primary antibodies diluted in blocking bu er overnight at 4 C. After washing in PBS with 0.05% Tween-20, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at RT. Signal was developed using an ECL substrate (Thermo Fisher Scientific, United States) and imaged with ChemiDoc MP imaging system (Bio-Rad, United States), and band density was analyzed in ImageJ. The following antibodies were used for immunoblotting: mouse monoclonal anti-b-casein (Santa Cruz, United States, sc-166530, 1:1000), mouse monoclonal anti-a-tubulin (Santa Cruz, United States, sc-5286, 1:1000), and anti-mouse secondary antibody (Merck, NA931, 1:1000).

Statistical Analysis

Statistical analysis was performed using the Prism software (GraphPad, United States); statistical test used is specified in figure legends. ⇤ p < 0.05, ⇤⇤ p < 0.01, ⇤⇤⇤ p < 0.001, ⇤⇤⇤⇤ p < 0.0001. The number of independent biological replicates is indicated as n.

RESULTS

FGF2 Pretreatment Enhances Lactogenic Differentiation of Mammary Epithelium

During mammary gland morphogenesis, lactation is preceded by excessive branching of epithelial ducts. We hypothesized that epithelial expansion by branching morphogenesis might be required for lactogenic di erentiation in vitro. Therefore, we first tested the impact of several growth factors on mammary epithelial morphogenesis. The primary mammary epithelial organoids were treated with FGF2, FGF7, FGF10, EGF, TGFa, or a combination of WNT3A and R-spondin 1 (W3/R1) for 7 days. Interestingly, only FGF2, a potent mammary epithelium branching-inducing factor (Ewald et al., 2008), induced extensively branched morphology (Supplementary Figures 2A-D).

Next, we tested if FGF2-induced epithelial expansion facilitated lactogenic di erentiation. To this end, the primary mammary epithelial organoids were either treated only with LM (containing prolactin and hydrocortisone) for 4 days, or they were treated with FGF2 for 6 days and followed by 4 days of LM (Figure 1A). To detect lactogenic di erentiation, we measured the expression of Csn2 and Wap by RT-qPCR. Our results revealed that treatment of freshly isolated organoids with LM induced only expression of Csn2 (Figure 1B). However, when organoids were pretreated with FGF2, the expressions of both Csn2 and Wap were significantly increased (Figure 1B). These data suggest that mammary epithelial expansion, induced by branching morphogenesis, could enhance the lactogenic ability of mammary epithelium.

Lactation Medium Induces Production of Milk Proteins and Secretion of Lipid Droplets

Next, we compared the morphology of organoids treated with either FGF2 only or FGF2 and LM (FGF2-LM) to further characterize the phenotype of lactation organoids. On bright-field micrographs, we noticed that FGF2-LM organoids appeared to have a darker lumen, possibly due to the milk accumulation (Figure 1C). Interestingly, we also observed bubble-like structures at the apical site of epithelium in the same organoids, which potentially represented lipid droplets (Figure 1C). To further characterize these droplets, we stained the organoids for F-actin (with phalloidin), a cytoskeletal protein, or with oil red O. Confocal microscopy revealed that the droplets were negative for F-actin and strongly positive for oil red O, confirming the droplets were lipid (Figures 1C,D).

Next, we assessed the expression of milk proteins in the organoids. First, we detected a significant increase in Csn2 by four orders in FGF2-LM-treated organoids compared to FGF2 alone by RT-qPCR (Figure 1E). Consistently, in FGF2-LM-treated organoids, we detected up-regulation of b-casein on the protein level by Western blot (Figure 1F) and a strong cytoplasmic signal by immunohistochemistry (Figure 1G), which was further confirmed by antibody against milk proteins (Supplementary Figures 3A-C). Taken together, these data demonstrate that mammary primary organoids are capable of milk production after prolactin treatment, which could be greatly enhanced by branching morphogenesis.

Morphology Maintenance in Long-Term Lactating Organoids

After successful induction of lactation in the primary mammary organoids with the FGF2-LM protocol, we went on to investigate the lactation-associated phenotype in long-term organoid culture. After 6 days of FGF2 treatment, the organoids were either cultured continuously with LM (FGF2-LM) or switched to BOM after 4 days of LM treatment (FGF2-LM-BOM) (Figure 2A). The morphogenesis of the organoids was recorded using time-lapse microscopy for 20 days. Interestingly, FGF2-LM-BOM cultured organoids regressed both in size and the complexity of the shape, whereas the organoids in FGF2-LM maintained the size and only partially lost the branched phenotype (Figures 2B,C and Supplementary Figures 4A,B).

In contrast, continuous treatment with FGF2 for 20 days maintained the organoid branched morphology (Supplementary Figures 4A,B). In addition, unlike the organoids in FGF2-LM-BOM, the organoids in FGF2-LM retained the darker appearance, possibly due to the milk accumulation (Figures 2B,D and Supplementary Figure 4A). Morphologically, FGF2-LM-treated organoids exhibited complex architecture with multiple lumens filled with dense eosinophilic material, which was maintained throughout the experiment (Figure 2E, upper panel). However, upon LM withdrawal, the complex architecture was lost rapidly, and organoids involuted into small spheroids with much simpler structures (Figure 2E, lower panel).

Milk Production in Long-Term Lactating Organoids

Of note, we detected strong b-casein signal in the intraluminal of long-term lactating organoids by immunohistochemistry. Closer observation revealed that cytoplasmic b-casein signal was sustained in long-term LM culture (Figure 3A, upper panel), but lost after LM withdrawal (Figure 3A, lower panel). In addition, RT-qPCR revealed that FGF2-LM-treated organoids maintained a high level of Csn2 expression, which was dramatically reduced by four to five orders of magnitude in FGF2-LM-BOM-treated organoids (Figure 3B). The same change was confirmed in the protein level by Western blot (Figure 3C). Therefore, the production of b-casein depended on the prolactin signaling. Altogether, these data suggest that these organoids have a proper epithelial architecture and the capacity to maintain milk production over prolonged culture time in response to the prolactin signaling.

Lactating Organoids Retain Functional Myoepithelial Layer With Contractility

Next, we co-stained the lactating organoids for keratin 5 and keratin 8, markers of myoepithelial and luminal cells, respectively, to confirm that the organoids contain proper bilayer epithelial architecture. We found that FGF2-LM-treated organoids contained a continuous layer of myoepithelial cells, similar to FGF2-treated organoids (Figure 4A). Moreover, the myoepithelial cell layer was retained during the long-term culture in LM treatment, as well as after LM withdrawal (Figure 4B), suggesting the luminal-myoepithelial cell homeostasis was stable during long-term culture.

Importantly, FGF2 treatment induced stratification of the luminal layer, which is in agreement with published work (Figure 4A; Ewald et al., 2008). Upon LM treatment, the organoids showed resolution of the stratified epithelium to a predominantly bilayer structure, with luminal cells (keratin 8 positive) lining the luminal space (Figures 4A,B), which is important for producing milk. Remarkably, we observed the LMtreated organoids could contract periodically (Supplementary Movie 2). In comparison, organoids never treated with LM showed relatively static structures (Supplementary Movie 1). Of note, the contracting phenotype maintained during the longterm LM treatment and quickly ceased after LM withdrawal (Figure 4C). This result is somewhat puzzling because prolactin receptor is present only in the luminal cells (Supplementary Figure 5A). Of note, the prolactin used in our contraction experiments was isolated from sheep pituitary, which contains oxytocin [START_REF] Vorherr | Contamination of prolactin preparations by antidiuretic hormone and oxytocin[END_REF]. To test whether the contraction of myoepithelial cells is a direct e ect of prolactin signaling, we compared contraction induction upon LM containing either sheep pituitary prolactin or mouse recombinant prolactin. Interestingly, only sheep pituitary prolactin induced organoid contraction; mouse recombinant prolactin did not induce contraction (Supplementary Figure 5B and Supplementary Movie 3). However, when the organoids cultured with mouse recombinant prolactin were treated with recombinant oxytocin, they did contract (Supplementary Movie 4), demonstrating that oxytocin is required for myoepithelial cell contraction. Taken together, these results demonstrate that myoepithelial layer is present in the lactating organoids. And more importantly, these myoepithelial cells can contract in response to LM treatment, suggesting they are functionally similar to the in vivo counterpart.

LM Withdrawal Triggers Involution-Like Phenotype in Lactating Organoids

Involution is characterized by the regression of the lactating epithelium through programmed cell death and remodeling of the mammary gland, which is induced upon weaning of the pups (Jena et al., 2019). Interestingly, withdrawal of LM from lactating organoids also induced a size regression and loss of the branched morphology with luminal architecture (Figures 2B-E). Using cell viability assay that is based on conversion of nonfluorescent resazurin to fluorescent resorufin by viable cells, we found that lactating organoids upon LM withdrawal (FGF2-LM-BOM) showed reduced viability in comparison to lactating organoids in LM (FGF2-LM) (Figure 5A), most likely due to increased cell death in response to LM withdrawal, which is a characteristic of involution. Yet the viability of organoids upon LM withdrawal was higher than that of organoids undergoing taxol-or ethanol-induced cell death (Figure 5A). Furthermore, replating of the involution-like organoids (FGF2-LM-BOM) to fresh Matrigel and FGF2 treatment reversed the size regression (Figure 5B) and, more importantly, induced branching morphogenesis (Figures 5C,D). This demonstrates that involuting organoids are viable and that the morphological changes induced upon LM withdrawal are reversible. Furthermore, cessation of milk production and ECM remodeling are two hallmarks of involution. Consistently, we detected a reduced b-casein signal (Figures 3A,C) and Csn2 expression (Figure 3B) in the organoids upon LM withdrawal. Interestingly, we also found that the expression of Mmp2 and Mmp13, two important Mmps for the ECM remodeling process during involution, was up-regulated in organoids after LM withdrawal (Figures 5E,F). Together, these results demonstrate that upon withdrawal of hormonal stimulation lactating organoids stop milk production and enter an involution-like process, thereby mimicking the in vivo situation upon weaning.

DISCUSSION

In this work, we described the use of primary mammary epithelial organoids to model pregnancy-associated morphogenesis and lactation. In our 3D culture system, primary mammary organoids exposed to LM with prolactin recapitulated several aspects of lactation process. Upon LM withdrawal, organoids regressed in a manner similar to the involution process in vivo.

Our data showed that FGF2 primes mammary epithelium for lactation. This is consistent with in vivo studies that noted morphological abnormalities in pregnancy-associated tertiary branching of mammary epithelium with attenuated FGF receptor signaling [START_REF] Lu | Genetic mosaic analysis reveals FGF receptor 2 function in terminal end buds during mammary gland branching morphogenesis[END_REF][START_REF] Parsa | Terminal end bud maintenance in mammary gland is dependent upon FGFR2b signaling[END_REF]. However, it remains to be elucidated what of the FGF2-mediated processes, including epithelial expansion, branching, and maturation, are essential contributors to milk production e ciency.

While several previous studies reported lactation induction in mammary epithelial organoids in response to prolactin in vitro, they did so only at a single time point (Mroue et al., 2015;Jamieson et al., 2017). Long-term lactation in organoid cultures has not been reported before. In this study, we documented milk production maintenance and stable morphology of lactating organoids over 14 days' culture period. Physiological lactation in mouse lasts for circa 3 weeks [START_REF] König | Maternal care in house mice[END_REF], and milk composition and production rate vary during the lactation period to accommodate the needs of the o spring [START_REF] Knight | Milk yield and composition in mice: e ects of litter size and lactation number[END_REF]. We propose that our model would be suitable to study factors that influence dynamic changes in milk composition and quantity in the long term. Among others, insulin is used in our model to support cell survival and growth and has been implicated in milk production (Nommsen-Rivers, 2016) both in rodent and human. Our model could help to further elucidate how insulin signaling impacts on milk production. Moreover, while previous studies used sampledestructive methods to detect lactation, such as organoid fixation and immunodetection of milk proteins (Mroue et al., 2015;Jamieson et al., 2017), we propose approaches for observing changes in milk production in the same organoid over time. They include morphological changes accompanying lactation in organoids, namely, appearance of lipid droplets in luminal space, increase in organoid darkness (integrated density), and the intriguing contraction of myoepithelial cells, which are easily observable by light microscopy and traceable by timelapse imaging.

Myoepithelial cells form a layer of mammary epithelium that is situated basally to the luminal cells (Macias and Hinck, 2012). Besides the recently elucidated role in keeping epithelial homeostasis and integrity [START_REF] Adriance | Myoepithelial cells: good fences make good neighbors[END_REF][START_REF] Goodwin | Myoepithelial crowd control of cancer cells[END_REF][START_REF] Sirka | Myoepithelial cells are a dynamic barrier to epithelial dissemination[END_REF], the key function of myoepithelial cells is to enable milk ejection by contraction when pups are suckling [START_REF] Haaksma | Myoepithelial cell contraction and milk ejection are impaired in mammary glands of mice lacking smooth muscle alpha-actin[END_REF]. In response to tactile stimuli, oxytocin is released from pituitary, and it binds to oxytocin receptor on myoepithelial cell to induce contraction [START_REF] Nishimori | Oxytocin is required for nursing but is not essential for parturition or reproductive behavior[END_REF][START_REF] Froemke | Chapter 13 -oxytocin and brain plasticity[END_REF]. Therefore, oxytocin was used to induce myoepithelial contraction in single cells [START_REF] Raymond | Control of mammary myoepithelial cell contractile function by a3b1 integrin signalling[END_REF], as well as in an organoid system (Mroue et al., 2015). However, organoid contraction was shown only as a decrease in organoid area over 20 min (Mroue et al., 2015). In contrary, we observed that contraction of a lactating organoid is a very fast process, and the dynamic changes in organoid shape and size are visible to human eye. From videos of contracting organoids, recorded at the rate of one frame per second, we calculated that the frequency is about one contraction per 10 s, which is very similar to the recently reported alveoli warping frequency of lactating mammary tissue upon oxytocin stimulation (Stewart et al., 2019). Therefore, our model provides a suitable in vitro system for studying the regulation of the contractile function of myoepithelial cells.

Upon LM withdrawal, lactating organoids underwent involution-like process: regression in size and complexity, which is reversible by FGF2 treatment upon reseeding; and up-regulation of the expression of MMPs, the proteases typically found in mammary gland during involution (Lund et al., 1996;[START_REF] Green | ECM degrading proteases and tissue remodelling in the mammary gland[END_REF]. Involution-like morphological changes upon prolactin withdrawal were documented also in the 3D coculture model of lactation using mammary epithelial and preadipocyte cell lines. However, epithelial cells cultured without preadipocytes were not reported (Campbell et al., 2014). Thus, for the first time in organoid culture, we show that involution-like regression of epithelium occurs, at least in part, in an epithelium-intrinsic manner. Our observations do not contradict the crucial role of paracrine signaling required for proper involution, including the leukemia inhibitory factor and TGFb signaling that activate STAT3mediated regression of epithelium (Nguyen and Pollard, 2000;Kritikou et al., 2003;Hughes and Watson, 2012). Our results point to the existence of epithelial-intrinsic mechanisms of involution, for study of which our epithelialonly organoid model could be advantageous. Certainly, more work is required to establish this model as a valid system for studying physiological involution. In this study, we did not evaluate the onset of programmed cell death and its regulation. In addition, optimization of the culture conditions with cytokine cocktail would be required to further mimic physiological involution.

Several human diseases, developmental defects, or insu ciencies in mammary epithelial tissue are linked to lactation and involution period. Among others, inadequate milk production a ects many women after giving birth, especially after premature deliveries and with obese mothers [START_REF] Olsen | Breast disorders in nursing mothers[END_REF][START_REF] Kent | Principles for Maintaining or Increasing Breast Milk Production[END_REF][START_REF] Nommsen-Rivers | Does insulin explain the relation between maternal obesity and poor lactation outcomes? an overview of the literature[END_REF]. We propose that human breast tissue, gained from reduction mammoplasties, could be utilized to isolate primary human breast organoids for an analogous lactation assay. Furthermore, findings from murine organoids could be translated into human organoids to identify physiological barriers for lactation, which will provide valuable information for developing novel interventions to support lactation success and provide health benefit across two generations. Moreover, our organoid model could be used to investigate mechanisms of pregnancy-associated breast cancer, an aggressive form of breast cancer with peak of incidence within 5 years after delivery (Schedin, 2006). Mammary organoids isolated from genetic mouse models, such as animals carrying mutations in oncogenes or tumor suppressors, or organoids exposed to carcinogens could be used in our lactation model to unveil mechanisms and signaling pathways leading to epithelial cell carcinogenesis.

Masaryk University. We also thank the Revive Consortium for funding the exchange program. We acknowledge the core facility CELLIM of CEITEC supported by the Czech-BioImaging large RI project (LM2015062 funded by MEYS CR) for their support with obtaining scientific data presented in this manuscript.

www.bio-protocol.org/e3996

Bio-protocol 11(08): e3996. DOI :10.21769/BioProtoc.3996 Please cite this article as: Charifou et al., (2021). A Robust Mammary Organoid System to Model Lactation and Involution-like Processes. Bioprotocol 11( 8 Please cite this article as: Charifou et al., (2021). A Robust Mammary Organoid System to Model Lactation and Involution-like Processes. Bioprotocol 11(8): e3996. DOI: 10.21769/BioProtoc.3996.

[Background] The primary function of the mammary gland is to provide nutrition to newborns via milk production. The development of the mammary gland is a highly dynamic process that occurs mainly after birth and is regulated by several factors including hormones and growth factors (Brisken and Rajaram, 2006;Sternlicht, 2006). During puberty, hormones and growth factors regulate ductal morphogenesis from a rudimentary embryonic ductal tree (Brisken and O'Malley, 2010). During each pregnancy, the mammary gland begins a new morphogenetic step initiated by hormonal stimulation, which is characterized by massive proliferation for epithelial expansion and alveolar development accompanied by adipocyte regression (Brisken and O'Malley, 2010). Importantly, prolactin signaling plays a crucial role in the terminal differentiation of luminal cells to enable milk production (Ormandy et al., 1997). At the end of lactation after weaning of the progeny, the mammary gland enters the involution stage characterized by programmed cell death, tissue remodeling, and redifferentiation of adipocytes (Hughes and Watson, 2012;Macias and Hinck, 2012;Zwick et al., 2018;Jena et al., 2019).

Histologically, the mammary gland is composed of a bilayered epithelium consisting of an inner layer of luminal cells (keratin 8+) and an outer layer of contractile basal cells (keratin 5+). Luminal cells are responsible for milk production during lactation, while basal cells aid milk ejection. The epithelium is surrounded by a stromal fat pad that comprises fibroblasts, nerves, vasculature, lymphatics, immune cells, adipocytes, and extracellular matrix (ECM) (Richert et al., 2000).

Over the past decade, organoids of various tissues, such as stomach, colon, lung, and pancreas, have been developed (Huch and Koo, 2015), offering many exciting possibilities for the study of tissue development and disease. The organoid system is a powerful tool that combines the advantages of a 2D culture (easy manipulation, precise control of cell composition and microenvironment, live imaging) with the opportunity to study complex cell-cell and cell-ECM interactions in a more controlled ex vivo manner (Huch and Koo, 2015;[START_REF] Shamir | Adhesion in mammary development: novel roles for Ecadherin in individual and collective cell migration[END_REF]Koledova, 2017;Artegiani and Clevers, 2018).

Several models have been developed to study the mechanisms of mammary branching morphogenesis in primary mammary epithelium using different protocols (Ewald et al., 2008;Huebner et al., 2016;Neumann et al., 2018), cell lines (Xian et al., 2005), sorted cells (Jamieson et al., 2017;Linnemann et al., 2015), or induced pluripotent stem cells (Qu et al., 2017). However, an organoid system modeling key aspects of the late postnatal developmental stages of the mammary gland has remained challenging to establish.

Previously, there have been several attempts to model lactation in 3D culture: spheroids of a breast adenoma cell line were used to study copper secretion into milk (Freestone et al., 2014); organoids of primary epithelium were shown to produce milk following the administration of a lactogenic stimulus (Mroue et al., 2015;Jamieson et al., 2017); and co-culture of breast epithelium and pre-adipocyte cell lines was shown to initiate an involution-like process (Campbell et al., 2014). However, in-depth characterization of milk production and involution or the proper bilayered architecture of mammary epithelium remained to be carried out.

Recently, we developed a model of lactation and involution of mammary epithelium based on organoids of primary mammary gland tissue cultured in 3D Matrigel ® (Sumbal et al., 2020b). Under lactogenic stimuli, primary organoids maintain long-term milk production, retain the contractile www.bio-protocol.org/e3996 a. Euthanize the donor mouse using an ethically approved method (e.g., cervical dislocation) and immediately proceed to mammary gland collection. Notes: i. Cervical dislocation is a common method for animal euthanasia and provides a fast and painless death. With this method, cell/tissue survival in culture is not altered if collected immediately.

ii. In the case of processing multiple mice, euthanize one animal and collect the glands immediately, then proceed to the next animal.

b. Sanitize the ventral side of the animal by spraying 70% EtOH on the skin.

Note: After disinfection, work inside a laminar flow hood to maintain aseptic conditions. antibiotic supplements (gentamicin in digestion solution; penicillin and streptomycin in culture medium) will prevent the occurrence of contamination.

c. Pin the mouse by its four paws to a dissection board, with the abdomen facing upward (see Figure 1A, pins 1-4).

d. Using forceps, tightly grasp the skin of the lower part of the abdomen at half the width (see Figure 1A, point A).

e. Using surgical scissors, make the first incision in the skin at point A.

Note: Be careful to incise only the skin and not rupture the underlying peritoneum.

f. Continue to incise the skin cranially to the throat of the animal (see Figure 1A, from point A to point B).

g. From this median line, use forceps to grasp the skin and cut toward each of the four paws (see Figure 1A, incise to join the middle line to points C, D, E or F, respectively).

h. Using forceps and a cotton swab, gently separate the skin from the peritoneum on one side of the animal. Attach the skin to the dissection board with three pins (see Figure 1B, pins 5-7).

i. Repeat step 8 on the other side of the animal (see Figure 1B, pins 8-10). The mammary glands are now exposed. j. Identify the lymph node of the mammary gland #4 (a small dense structure, round in shape;

see Figure 1B, surrounded). Remove the lymph node from both glands using forceps and scissors and discard.

k. Proceed to the harvest of the mammary glands #3 and #4. Using curved forceps, grasp the mammary glands and gently separate them from the skin and other tissues with scissors. Note: Carefully separate the mammary glands #3 (whitish and shiny) from the muscles (light brown ribbed structure) since this protocol does not prevent muscle contamination.

l. Place all the collected glands in the same sterile Petri dish containing cold PBS (approximately 3 ml, previously stored at 4°C) for washing prior to tissue processing.

m. Properly dispose of the animal corpse and continue with mechanical and enzymatic dissociation of the mammary glands.

Mechanical and enzymatic dissociation

Reminder: Work inside a laminar flow hood to maintain aseptic conditions.

a. Freshly prepare 10 ml dissociation solution for the four glands collected from one mouse, pass through a 0.2-μm filter, and pre-heat at 37°C. Note: Do not exceed the maximum 30 ml dissociation solution in a 50-ml tube to ensure correct dissociation.

b. Transfer the freshly collected mammary glands to a new sterile Petri dish.

c. Use three scalpels to finely chop the mammary glands and obtain a homogeneous mince of 1-mm 3 mammary fragments (see Figure 1C).

d. Transfer the mince to a 50-ml tube containing the pre-warmed dissociation solution.

e. Place the tube in a shaking incubator for 30 min at 37°C, 100 rpm.

Notes:

i. All the following steps are performed at room temperature except incubation with dispase.

ii. From here on, pre-coat all the pipettes, tips, and tubes with 2.5% BSA solution. Prepare the BSA solution in a 50-ml tube and aspirate/remove from every consumable following coating; this will prevent stickiness and loss of organoids. The BSA solution can then be filtered, stored at 4°C, and re-used.

f. After incubation, resuspend the dissociated mammary glands by performing ten up-anddown motions with a 10-ml pipette. Centrifuge for 10 min at 400 × g.

g. After centrifugation, handle the 50-ml tube carefully to prevent disturbance of the three separated layers (see Figure 1C). Keep the epithelial pellet intact and transfer the middle aqueous phase and the top fatty layer into a clean 15-ml tube.

h. Resuspend the epithelial pellet in 5 ml DMEM/F12 and set it aside.

i. Focus on the fatty and aqueous solutions in the 15-ml tube: resuspend by performing ten up-and-down motions with a 10-ml pipette. Centrifuge for 10 min at 400 × g.

Note: This step allows recovery of epithelial fragments trapped in the fatty layer.

j. Again, handle the 15-ml tube carefully to avoid disturbing the three separated layers.

Discard the fatty and aqueous layers.

k. Take the 5 ml resuspended pellet from the 50-ml tube to resuspend the pellet in the 15-ml tube.

l. Wash the 50-ml tube with 5 ml DMEM/F12, pool with the suspension in the 15-ml tube, and mix.

m. Centrifuge for 10 min at 400 × g.

n. Discard the supernatant. Use 4 ml DMEM/F12 to resuspend the pellet. Subsequently, add 80 μl DNAse I at 100 μg/ml and agitate for 5 min by hand or on an orbital shaker at 100 rpm.

o. Add 6 ml DMEM/F12 and resuspend the solution by performing 5 up-and-down motions with a 10-ml pipette.

p. Centrifuge for 10 min at 400 × g.

q. Discard the supernatant. Use 4 ml DMEM/F12 to resuspend the pellet. Subsequently, add 150 μl dispase II at 0.5 mg/ml and incubate for 5 min at 37°C. Note: Set the time on the centrifuge to 1 min. Once a speed of 400 × g is reached, time 15 s precisely and stop the centrifuge manually.

v. Repeat the previous step (t) 4 times, for a total of 5 differential centrifugations, to efficiently remove stromal contamination.

w. Resuspend the final pellet in 1 ml basal organoid medium (BOM) and place on ice. The organoids are now ready to be counted and cultured. Note: Adjust the volume of resuspension according to pellet size. From a pool of 2-3 mice, the final pellet was resuspended in 1 ml basal organoid medium, for an expected range of d. Count the organoids under the microscope at 4× magnification (see Figure 1D). Notes:

i. Take each quarter of the cross as a landmark to avoid double-counting of the same organoid.

ii. Organoids appear as rounded structures with a smooth perimeter. Occasionally and unavoidably, nerves and endothelium are also present. The nerves appear as rope-like structures and can be organized in bundles (see Figure 1D). g. Remove the 24-well plate from the cell incubator and place on the 37°C heating plate.

h. In each Matrigel ® -precoated well, cautiously seed the suspension of organoids (in Matrigel ® ) as a dome on top of the solidified Matrigel ® patch.

i. Place the 24-well plate back in the cell incubator (5% CO2) for 30 min at 37°C to solidify the Matrigel ® (see Figure 1D). j. In the meantime, pre-warm BOM at 37°C. k. Following incubation, carefully add 1 ml pre-heated BOM to each well and culture in the cell incubator at 37°C, 5% CO2. Notes:

i. Add medium against the edges of the well to avoid disruption of the dome.

ii. Characterization of the organoids can be performed using regular histological stains (e.g., hematoxylin & eosin) or immunostaining on day 1 post-recovery in BOM (see Figure 1E and Step B2 of the procedure).

Morphogenesis with FGF2

Reminder: Work inside a laminar flow hood to maintain aseptic conditions.

Note: Overnight recovery is optimal for organoid culture; however, FGF2 treatment can be administered immediately after plating the organoids. d. Renew all medium with fresh morphogenesis medium every 3 days, for a total of 6 days of treatment.

3. Lactogenic differentiation with prolactin a. Pre-heat the BOM at 37°C.

b. Add 1 μg/ml prolactin and 1 μg/ml hydrocortisone to the pre-heated BOM to obtain the lactation medium.

c. Aspirate the medium from the wells without touching the Matrigel ® dome and replace with 800 μl fresh lactation medium.

d. Renew all medium with fresh lactation medium every two days, for a total of 4 days of treatment.

4. Myoepithelial cell contraction with oxytocin a. Prepare fresh lactation medium, filter, and pre-heat at 37°C.

b. Add 40 μg/ml recombinant oxytocin to the lactation medium.

c. Aspirate the medium from the wells without touching the Matrigel ® dome and replace with 800 μl fresh lactation medium supplemented with oxytocin.

d. Using live cell imaging, record contraction images every second for 120 s. c. Renew all medium with BOM every two days, for a total of 8 days of treatment.

Replating

Note: Use tips pre-coated with 2.5% BSA.

a. Aspirate the supernatant and wash the wells twice with 800 μl cold PBS.

b. Add 1 ml cold PBS and disrupt the Matrigel ® dome using an up-and-down motion with a P1000 pipette.

c. Check for successful disintegration of the Matrigel ® under a microscope.

d. Transfer the suspension to a 15-ml tube and add cold PBS to a total volume of 10 ml.

e. Centrifuge for 3 min at 400 × g.

f. Carefully remove the supernatant, resuspend the organoid pellet in fresh Matrigel ® and plate as described in B1. versus d18), or at the morphological level by the progressive disappearance of branching (see Figure 2B and Figure 3B). f. Vortex the samples, cool on ice, and repeat the sonication according to Step C2e.

g. Centrifuge for 20 min at >10,000 × g, 4°C.

h. Transfer the supernatant to a clean 1.5-ml tube.

i. Measure the protein concentration using a Coomassie Protein Assay Kit.

Introduction

The European network of breast development and cancer (ENBDC) organizes an annual workshop on methods in mammary gland biology and breast cancer. This threeday meeting has been hosted every year until now in the charming small town Weggis in Switzerland [1], but the twelfth annual meeting took place virtually on April 22 and 23, 2021. The virtual format allowed many more scientists to attend the meeting and we reached over 100 participants from many different countries, while the ENBDC meeting on site usually gathers around 65 participants [1]. The meeting schedule was rearranged to fit in two full days, with regular breaks, and more relaxed poster sessions in the afternoon. This first virtual meeting was a great success, with high quality talks on very hot topics and discussions after each talk. Poster sessions enabled all attendees to present their work to a smaller audience. The conviviality and friendly interactions we usually enjoy during breaks and meals in Weggis were however missed by most of us, and we are all looking forward to gather next year in Weggis on April 28-30, 2022.

Meeting Report

The meeting started with the Keynote speaker Nicola circulating tumor cells (CTC), some of which will eventually lead to metastasis. He started his presentation by explaining the size-based, antigen-independent microfluidic technique used to trap CTC which are bigger and less deformable than blood cells [2]. Nicola Aceto then described the presence of CTC clusters [3] and their characteristics and showed how these CTC clusters in patients are associated with a worse prognosis. Using mice engrafted with tagged mammary tumor cells, he demonstrated that these CTC clusters had a 50-fold increased metastatic potential compared to single CTC [4][5][6]. He went further in the comparison of single and clustered CTC and showed that the latter bear hypomethylated DNA regions and featured stem and proliferative-like programs, expressing OCT4, SOX2 and NANOG. He then presented an elegant idea to screen for drugs able to dissociate clustered CTC without killing the cells, and found that a Na + K + ATPase inhibitor presented these characteristics. Validation in vivo in mice showed that this inhibitor dissociated CTC clusters without killing single CTC and decreased the metastatic index [7]. Ongoing effort is focused on translating this work to human patients in ongoing clinical trials. Another characteristic of clustered CTC is that some of them include immune cells. He showed that breast cancer patient survival was worst when they present CTC white blood cell (WBC) clusters. Using single cell RNA sequencing of patient samples, he found that in 90% of the cases these WBC were neutrophils. In mice, he showed that CTC-neutrophil clusters were the most metastasis-competent CTC subpopulation [8]. He concluded this talk presenting his work on hypoxia, which he showed is not restricted to the center of the tumor. Intratumor hypoxia leads to cell-cell junction upregulation. Clustered CTC were usually hypoxic, while single CTC were normoxic. Anti-angiogenic therapy reduced tumor size, but increases hypoxia, CTC clusters and metastasis [9]. This observation will be important to keep in mind when testing anti-cancer therapies.

The first session focused on breast cancer metastasis and was chaired by Mohamed Bentires-Alj from University of Basel, Switzerland. The first speaker of the session was Ilaria Malanchi form the Francis Crick Institute in London, UK. Her work focuses on the local interaction of metastatic cells within the tissue. To study this technically challenging question, she set up a new method called Cherry-niche which enables cells expressing a fluorescent protein to label surrounding cells [10,11]. In particular, she focused her study on the metastatic niche in the mouse lung, using injection of 4T1 mouse mammary metastatic cells. Using the Cherry-niche system, cancer cells express GFP and a liposoluble red fluorescent tag and appear yellow, while neighboring cells, which constitute the niche, are tagged with the red fluorescent compound and the rest of the lung is not labelled. With this elegant approach, she showed the presence of cancer associated parenchymal cells with stem cell-like features and expression of lung progenitor markers, as well as self-renewal and differentiation potential [10].

Eva Gonzalez-Suarez, from the Spanish National Cancer Research Center (CNIO) in Spain, was the second speaker of this session, and presented her work on RANK and RANKL. She showed that loss of RANK signaling in MMTV-PyMT mouse tumor cells increased the number of lymphocytes, leukocytes and CD8 + T cells, and reduced macrophage and neutrophil infiltration, clearly demonstrating an effect of RANK pathway on mammary tumor immune surveillance. She showed that RANKL inhibition increased the effect of immunotherapy in mammary gland tumors. This immunemodulatory effect of RANK signaling (increased TILS and CD8 + T cells) was confirmed in pre-menopausal early breast cancer patients treated with denosumab in the D-BEYOND trial and is being further investigated in an ongoing clinical trial (D-BIOMARK) treating pre-and post-menopausal women with early breast cancer with neoadjuvant denosumab. Her findings indicate that tumor cells use RANK pathway to evade immune surveillance and support the use of RANK pathway inhibitors to increase response to immunotherapy in luminal breast cancer [12]. She presented new results showing that RANK expression unexpectedly delayed mammary tumor latency in two oncogene-driven mouse models, (MMTV-PyMT and MMTV-Neu). The mechanism behind this observation is that activation of RANK signaling induced senescence through P16/P19. This RANK-induced senescence increased stemness properties and promoted tumor growth and metastasis. This work shows that while RANK induces senescence and stemness, which delays tumor initiation, it eventually increases tumor aggressiveness [13].

Roger Gomis, from the Institute for Research in Biomedicine (IRB) Barcelona in Spain, presented a short talk on metastasis latency and bone metastasis in breast cancer. His work demonstrated that gain in a chromosomal region coding for the transcription factor MAF in primary breast tumors is associated with metastasis in bone but not to other organs [14]. MAF is therefore a potential biomarker to select patients at risk of bone metastasis for bisphosphonate adjuvant therapy. To go further on this important observation, a clinical trial was performed and concluded that MAF amplification status predicts likelihood of benefit from adjuvant zoledronic acid [14,15]. Roger Gomis is now focusing on the MAF-mediated metastasis mechanisms and is studying MAF interactome as well as the MAF-mediated chromatin opening and transcriptional program.

Ana Luisa Correia, a postdoc from Mohamed Bentires-Alj lab at the University of Basel in Switzerland, closed this session on breast tumor dormancy and metastasis with a short talk on her recent findings. Her work focuses on dormant disseminated tumor cells (DTC). In order to study the differences between cycling and quiescent DTCs, she used mouse models of metastasis consisting of xenografting human MDA-MB-231 cells into the mammary glands of immunocompromised NOD-SCID mice, followed by primary tumor resection. The novelty of her experimental approach is in using tumor cells co-expressing a Tomato reporter and a mutant reporter of P27 which identifies quiescent cells. Analysis of the distribution of DTCs amongst different metastatic organs showed that the liver, often associated with poor prognosis, mainly harbored DTCs in dormant state. She showed that there was a selective increase in natural killer (NK) cells in the dormant microenvironment. She then showed in mice that immunotherapy ensured a pool of NK cells that sustained dormancy through interferon gamma signaling, which appeared to be a master switch between dormancy and metastasis in the liver. Lastly, Ana found that liver injury limits NK cell expansion. Using a model of chemically-induced liver injury, she showed that hepatic stellate cells become activated and secrete an inhibitor of NK cell proliferation, CXCL12, through its cognate receptor CXCR4. This study is very promising as it suggests that therapies aiming at normalizing NK cell density could halt dormant DTCs from awakening and prevent metastatic disease [16].

We were pleased to listen to John Stingl next, a former member of the ENBDC committee now working at STEM-CELL Technologies Inc. in Canada, for the Meet the Expert session. STEMCELL is commercializing media for growing organoids from a wide variety of tissues, including the mouse mammary gland. John shared with us his protocols and advice on how to culture mammary gland organoids in Matrigel domes with the MammoCult Organoid Growth Medium (OGM) developed at STEMCELL. An important trait of the MammoCult OGM is that it is serum-, estrogen-, progesterone-and phenol red-free. Different combinations of supplements are used, depending on whether branched multilineage or luminal-restricted organoids are desired. John recommended that for maintaining organoid lines they were passaged as fragments since this would help maintain a normal karyotype, however seeding cells as single cells was recommended for quantitative experiments since there would be less variability in organoid numbers between technical replicates. Branched organoids are multilineage, with basal cells expressing K14 and SMA, and luminal cells expressing K8 and PR. These organoids were able to synthesize casein in response to lactogenic stimulation. He also presented data on EpiCult Plus Medium, which is a serum-free medium that promotes robust long-term expansion of mouse mammary cells in 2D culture in the absence of feeders, while still retaining organoid-forming potential. This medium could be useful for gene editing prior to organoid culture.

The second day, we moved on with the branching and mammary development session, chaired by Beatrice Howard from The Institute of Cancer Research, UK. Our first speaker was Thorarinn Gudjonsson from the University of Iceland, Iceland, with his talk on epithelial to mesenchymal transition (EMT) in breast morphogenesis and cancer. He presented the generation of the D492 cell line by isolating MUCIN 1 negative, EPCAM positive cells from primary cultures of breast cells obtained from reduction mammoplasties. This cell line has epithelial characteristics as well as stem cell properties and is able to form branched 3D structures resembling terminal duct lobular units [17,18]. By co-culturing D492 cells and primary breast endothelial cells in Matrigel, he showed that endothelial cells induced branching morphogenesis, highlighting the importance of endothelial cells in branching morphogenesis, but also that some colonies showed spindle shape with EMT characteristics. A new cell line, named D492M, was derived from these spindle colonies, and showed mesenchymal traits [19]. The two cell lines, D492 with epithelial properties and D492M with mesenchymal properties, allowed to study EMT and mesenchymal to epithelial transition (MET). Comparison of microRNA profiling of the two cell lines indicated that miRNA 200c/miRNA 141 and miRNA 203 were down-regulated in D492 cells and that overexpression of these miRNA in D492M cell line reinduced epithelial traits, demonstrating their role in EMT and MET. In particular, overexpression of miRNA 200c/miRNA 141 combined with p63 was able to completely reverse the mesenchymal phenotype to a fully branched epithelial phenotype [20]. The D492 cell line was also used to generate a tumorigenic cell line expressing HER2 for use in breast cancer studies [21].

The next speaker was Bethan Lloyd-Lewis, who recently established her lab at the University of Bristol, UK. She presented her previous postdoctoral work where she used intravital imaging to investigate mammary epithelial cell fate dynamics during normal and pathological mammary morphogenesis, alongside developing a flexible and suture-less, silicone-based imaging window for rodent intravital microscopy. This low-cost, suture-less device is suitable for many anatomical locations, representing a substantial advance the field [22]. Using intravital imaging in NOTCH1-CREERT2 and SMA-CREERT2 lines crossed to mT/mG fluorescent reporter mice, she traced luminal and basal mammary cell behaviors during ductal development, and in response to mutagenic beta-catenin activation. This approach revealed that, regardless of the mammary lineage targeted, mutant beta-catenin stabilisation leads to cellular rearrangements that precipitate the formation of hyperplastic lesions that undergo squamous transdifferentiation [23].

Satu-Marja Myllymäki, a postdoc from Marja Mikkola's lab in Helsinki Institute of Life Science (HiLIFE), Institute of Biotechnology in Helsinki, Finland, presented a short talk entitled "From cells to branches-Insights into mammary branch formation". While there are two mechanisms leading to branching: tip bifurcation and side branching, there are many behaviors that can drive branching like localized cell proliferation, oriented cell division, directional migration, cell rearrangement, cell shape change or adhesion remodeling. She focused her work on the contribution of localized cell proliferation and directional migration. Using the Fuc-ci2a mice [24], in which cells in M/G2/S phase are green and cells in G1/GO are red, she showed that luminal cells were more frequently detected in M/G2/S phase than basal cells, and that luminal cells in tips were more frequently detected in M/G2/S phase than luminal cells in duct and branch points. Mikkola lab used an ex vivo culture system of embryonic mammary glands to study branching events and quantified the proportion of lateral branching, bifurcations and trifurcations. She presented her data on how cell proliferation and cell motility contributed to branching. Her data suggest that establishment of a new branch point during bifurcation may require cell immobilization and inhibition of cell cycle activity and that proliferation becomes restricted to the daughter tips and cells assume a new direction of movement.

Zuzana Koledova from Masaryk University in Czech Republic closed this session with a short talk on her current work on the role of fibroblasts on mammary gland morphogenesis. Fibroblasts are known to interact with the mammary gland epithelium through paracrine signaling as well as through extracellular matrix production and remodeling [25]. Zuzana Koledova team currently investigates the potential involvement of mechanical forces exerted by fibroblasts in mammary epithelial branching using mouse models, co-cultures of fibroblast and mammary gland epithelium [26] and time lapse visualization of organoid dynamics and branching.

The student/postdoc session chairs of this year were Elsa Charifou from Institut Pasteur in France and Gunnhildur Asta Traustadottir from University of Iceland in Iceland. They chose the topic of the session, tumor microenvironment, as well as the speakers for their session.

The first talk was given by Jayakumar Vadakekolathu from Nottingham Trent University in UK. His work is focused on the EMT as one of the key steps in the metastatic process. He modelled EMT transition in a cell line model from which clonal progenies with different epithelial and mesenchymal characteristics have been derived [27]. Comparison of expression profiles and proteomics between the different clonal progenies revealed that NNMT is the most upregulated gene in the most mesenchymal clone. Data mining and analysis as well as in vitro experiments showed that NNMT seemed to be a key regulator of EMT and that NNMT expression was associated with tumor grade level in breast cancer. High expression of NNMT was correlated with poor relapse free survival, indicating its probable role in promoting metastatic spreading. His findings suggest that inhibition of NNMT might be an ideal candidate to target to reduce triple negative breast cancer progression.

The last talk of this session, presented by Ingunn Holen from the University of Sheffield, UK, was focused on the role of the bone microenvironment in regulating breast cancer metastasis. Studies have shown that up to 60% of patients with detectable disseminated tumor cells (DTC) in bone marrow remain relapse-free 6 years later. This implies that tumor cells reaching bone is not in itself sufficient to develop metastasis and that events at the secondary site are key to tumor progression. To study the effect of the bone microenvironment on DTC and metastasis, her team used a mouse model where intracardiac injection of labelled human breast cancer MDA-MB-231 cells formed tumors in the bone and DTCs in bone can then be studied using 2-photon microscopy. She showed that tumor cell homing was not uniform, with the majority of breast cancer cells located in the trabecular areas of the bone. Her work also showed that there may be a limited number of suitable niches available in bone for DTCs to colonise. When hematopoietic stem cells (HSC) were mobilized from their bone niches to the circulation (via injection of AMD3100, an inhibitor of CXCR4) prior to the injection of tumor cells, the number of DTC in trabecular regions increased, suggesting that the HSC and the bone metastatic niche overlap [28]. She presented a model of tumor dormancy in bone, allowing comparison of conditions in which DTC will develop in metastasis or remain dormant based simply on the age of mice (which impacts the bone microenvironment) at the onset of the experiment. By expanding the osteoblast number with parathyroid hormone prior to tumor cell injection, she showed that the osteoblasts were important for tumor cell survival and progression but not for DTC homing [29]. She also described studies of the effect of osteoclast activity on DTC by ovariectomizing (OVX) mice. OVX induced rapid bone loss and increased osteoclast activity, which triggered a large increase in growth of DTC to form new colonies in bone, compared to a sham operation. The OVX-induced growth of DTCs was inhibited with anti-resorptive agents, demonstrating tumor growth is driven by osteoclast-mediated mechanisms [30].

In conclusion, the virtual 2021 workshop was a success, with a very wide range of techniques described to obtain highly relevant new observations for breast cancer patient benefit and mammary gland fundamental comprehension. The DeOme prize for the best short talk presentation was awarded to Ana Luisa Correia from Mohamed Bentires-Alj lab in University of Basel, Switzerland. The three poster prizes were awarded to Johanna Englund, from the University of Helsinki, for her study of the role of laminin alpha 5 in the mammary gland, Guillaume Jacquemin, from Silvia Fre's lab in Institut Curie in Paris, for his work on improving windows for in vivo imaging of the mouse mammary gland, and Hannah Harrison, from University of Manchester, for her work describing a novel explant culture model for studying normal human breast tissue.

The next ENBDC workshop will take place in Weggis, Switzerland on April 28-30, 2022 and will be chaired by Beatrice Howard from the Institute of Cancer Research in London, UK, co-chaired by Jos Jonkers, from the Netherlands Cancer Institute in Amsterdam. The student/postdoc chairs will be Jakub Sumbal, from Masaryk University in Czech Republic, and Hannah Harrison, from University of Manchester.

Résumé

La sénescence est une réponse à un stress biologique, caractérisée par un arrêt stable du cycle cellulaire. Néanmoins, les cellules restent métabolliquement actives et acquièrent un phenotype sécrétoire associé à la sénescence, avec la production d'un sécrétome complexe composé de cytokines, chémiokines, facteurs de croissance et modulateurs du remodelage de la matrice extracellulaire. La sénescence est associée à de nombreux processus pathologiques, comme la tumorigénèse et le vieillessement. Cependant, où, quand et comment la sénescence contribue aux processus physiologiques reste méconnu. Pour répondre à cette question, nous avons tiré profit de la glande mammaire (GM), un organe avec une plasticité remarquable pendant le développement post-natal. L'involution de la GM est l'un des évenements majeurs de mort cellulaire et de remodelage tissulaire chez les mammifères, lorsque les cellules épithéliales produisant le lait sont éliminées et que la GM retourne à à un état similaire à celui pré-grossesse, attendant la prochaine gestation. Au cours de ma thèse, nous avons montré que la sénescence était induite transitoirement pendant la phase irréversible de l'involution. De plus, le programme de sénescence apparaissait spécifiquement dans les cellules luminales productrices de lait et corrélait à l'expression de l'inhibiteur du cycle cellulaire p16. En parallèle, nous avons établi un nouveau modèle d'organoides pour mimer la gestation, la lactation et l'involution de la GM. Dans ce modèle exvivo, nous avons aussi relever la présence de cellules sénescentes strictement lors du processus d'involution. Pour évaluer l'impact biologique de la sénescence in vivo, nous avons utilisé une méthode de scellement des mamelons pour découpler les phases réversible et irréversible de l'involution. Nous avons dévoilé une association étroite entre le sevrage des hormones lactogéniques qui a lieu lors de la seconde phase d'involution, et l'induction du programme de sénescence. Pour mieux définir les rôles physiologiques de la sénescence pendant l'involution, nous avons traités des souris avec de l'ABT-263, un composé sénolytique induisant l'apoptose des cellules sénescentes. Nous avons observé une altération du remodelage tissulaire suite à l'élimination des cellules sénescentes, avec des alvéoles résiduelles plus larges et un remplissage adipocytaire retardé. De plus, dans des organoides provenant de souris transgéniques p16-3MR, nous avons éliminé les cellules sénescentes avec succès grâce à l'administration de ganciclovir, ce qui a retardé le processus d'involution. Dans leur ensemble, les modèles in vivo et ex-vivo suggèrent un rôle important de la sénescence pour moduler la phase de remodelage tissulaire dans l'involution de la GM. Enfin, le processus d'involution est intiment lié avec le cancer du sein post-partum, un cancer diagnostiqué dans les 10 ans suivant une grossesse et associé à un mauvais pronostic. Explorer comment la sénescence impacte le microenvironnement lors de l'involution pourrait ainsi fournir de nouvelles connaissances pour mieux comprendre le cancer du sein post-partum.

  Senescent cells are highly heterogeneous, especially in vivo, due to the variety of inducing signals and the cell type. Moreover, senescent cells exhibit a heterogeneous transcriptional profile if they respond directly to the cellular stress (primary senescence) or if they respond to a paracrine signal from a cell already senescent (secondary senescence)[START_REF] Kirschner | Functional heterogeneity in senescence[END_REF]) (see "Autocrine/Paracrine reinforcement of senescence" for further details). Finally, most of the described characteristics to define a senescent cell have been identified in vitro, and some, but not all, are valuable indicators of in vivo senescence. Therefore, it remains challenging to characterize senescence by a unique signature, and a set of combined features is required to define a senescent cell in a tissue (Figure2)[START_REF] Di Micco | Cellular senescence in ageing: from mechanisms to therapeutic opportunities[END_REF]).

Figure 2 .

 2 Figure 2. Markers of senescent cells. There is no unique but a combination of biomarkers used to identify senescent cells, most of them characterized in vitro. Senescent cells are in an irreversible cell cycle arrest and overexpress cell cycle inhibitors such as p16 and p21. They remain metabolically active and acquire a secretory phenotype (SASP), composed of various cytokines, chemokines, growth factors, and extracellular matrix remodelers. Due to their extended lysosomal content, senescent cells have increased !-galactosidase activity and are positive for SA!Gal staining. They are resistant to apoptosis with an upregulation of Bcl2 family proteins. Morphologically, senescent cells have an enlarged phenotype, and remodeling of the heterochromatin leads to the appearance of senescence-associated heterochromatin foci (SAHF). From (Di Micco et al. 2021).

  . Regarding post-mitotic cells, precursors differentiate into specialized cells upon a defined developmental program distinct from stress response inducing senescence. Thus, effectors mobilized for this permanent cell cycle arrest are different between terminally differentiated cells (p18 INK4c , p27 KIP1 ) and senescent cells (p16 INK4a , p21 CIP1 , ARF, p53, and RB) (Figures 2 and 3) (He and Sharpless 2017).

Figure 3 .

 3 Figure3. Differences in cell cycle arrest in quiescent, differentiated, and senescent cells. Quiescence, differentiation, and senescence are three cellular states with a withdrawal from the cell cycle, but they can be separated based on the reversibility of the cell cycle arrest, the presence of macromolecular damages, the activated signaling pathways, and the production of a secretome. Arrows figure the interconnection between the states; once senescent, cells cannot reverse their cell fate. From[START_REF] Gorgoulis | Cellular Senescence: Defining a Path Forward[END_REF]).

Figure 4 .

 4 Figure 4. Molecular pathways activated in senescent cells. Senescentassociated cell cycle exit is regulated by two main pathways, p16 INK4A -or p21 CIP1dependent. Both p16 INK4 and p21 CIP1 are cyclin-dependent kinase inhibitors,

  expression is massively regulated at different scales by controlling transcription, mRNA stability, translation, and secretion levels. SASP initiation is not fully understood yet, but diverse signaling pathways can trigger SASP regulation, such as DNA damage response (DDR), p38/MAPK, cGAS/STING, and mTOR (Kumari and Jat 2021). Most converge on activating two major SASP drivers, NF-κB and C/EBPβ. NF-κB and C/EBPβ are potent transcription factors with synergistic inflammatory effects. They accumulate on the chromatin of senescent cells, upregulating the transcription of numerous SASP as IL-1α, IL-6,

Figure 5 .

 5 Figure 5. Multiple functions of senescence-associated secretory phenotype (SASP). The secretome of a senescent cell can be composed of various molecules such as cytokines, chemokines, growth factors, or tissue remodeling modulators. This SASP can act in an autocrine manner, reinforcing the senescent phenotype and the production of SASP via a positive feedback loop. It can also spread the senescent phenotype to neighboring cells in a paracrine manner. Moreover, via the secretion of cytokines, senescent cells can chemoattract immune cells to help with tissue clearance. Senescent cells also promote tissue remodeling through the secretion of metalloproteinases degrading the extracellular matrix or through the release of growth factors stimulating angiogenesis and alleviating fibrosis. From (McHugh and Gil 2018).

Figure 6 .

 6 Figure 6. Pleiotropic roles of senescence.Senescent secretome can influence the microenvironment through different aspects, such as cell proliferation and differentiation, matrix remodeling, or immune reaction. Therefore, senescence is involved in many processes and plays beneficial roles as in embryogenesis, tissue repair upon injury, and tumor suppression. However, senescence is also associated with detrimental processes, promoting tumor progression, chronic inflammation, and age-related diseases. This balance between beneficial and detrimental effects might be time-dependent, as transient SASP is proposed to be salutary, whereas persistent SASP creates a deleterious microenvironment. From (Paramos-de-Carvalho, Jacinto, and Saude 2021).

  exposure limit cancer development, favor immune surveillance, and promote embryogenesis and tissue repair. However, upon chronic damage or impairment of senescence clearance, senescent cells accumulate and become deleterious to the tissue. Persistent SASP production induces persistent inflammation, fibrosis and promotes cancer progression (Munoz-Espin and Serrano 2014).

Figure 8 .

 8 Figure 8. Consensual dichotomic model of senescence. When senescent cells appear in a tissue, they secrete a SASP, stimulating the immune surveillance and the remodeling of the microenvironment. Senescent cells are then cleared from the tissue and this transient presence of senescent cells is beneficial as it promotes tissue regeneration. However, upon persistent damage, disease or ageing, senescent cells accumulate in the tissue, leading to the permanent alteration of the microenvironment with chronic inflammation and fibrosis. Adapted from (Munoz-Espin and Serrano 2014).

Figure 9 .

 9 Figure 9. Knockout of the CDKN2A locus. CDKN2A locus is encoding for two proteins, p16 Ink4a and p19Arf, sharing the DNA sequence of exons 2 and 3 but with an alternative reading frame due to two different first exons. INK4A/ARF knockout mice are depleted for exons 2 and 3. Adapted from (Sherr 2001).

Figure 10 .

 10 Figure 10. Transgenic INK-ATTAC. INK-ATTAC transgenic mouse model has a FKBP-Casp8 cassette under p16 regulation, allowing the production of an inactivated Casp8. Upon administration of AP20187, Casp8 forms an active dimer and induces apoptosis of p16-expressing cells. From (Baker et al. 2011).

Figure 11 .

 11 Figure 11. Transgenic p16-3MR construct. The p16-3MR transgenic mouse model has a 3MR reporter cassette, allowing the identification of p16 expressing cells via luciferase and mRFP synthesis. Moreover, p16 + cells produce a HSV-TK, metabolizing the ganciclovir compound and inducing apoptosis upon ganciclovir treatment. From http://demarialab.com/research.html

Figure 12 .

 12 Figure 12. Transgenic INKBRITE construct. INKBRITE transgenic mouse model has a supersensitive cassette inserted under the endogenous regulation of p16. This construct allows the identification of p16+ cells with GFP fluorescence. From (BioRXiv reference https://doi.org/10.1101/2020.06.10.142893).

Figure 13 .

 13 Figure 13. Senolytic approaches to remove senescent cells. Numerous compounds have been developed to target senescent cells. ABT-263/Navitoclax targets BCL2 family proteins to alleviate apoptosis resistance. Dasatinib induces apoptosis via the pro-apoptotic EFN3 pathway. Quercetin inhibits glycolysis, usually upregulated in senescent cells for SASP production. FOXO4-DRI promotes the nuclear exclusion of p53. Bafilomycin inhibits the V-ATPase, responsible for lysosomal acidification. In addition to these senolytic approaches inducing senescent cell death, new compounds known as senomorphics aim to impair SASP, to alter senescent functions without killing the cells. Adapted from (Hernandez-Segura, Nehme, and Demaria 2018).

  strategies are developed to specifically deliver drugs only to senescent cells and minimize the possible off-targets of senolytics. Nanoparticles have been designed to target senescent cells specifically, by covering them with galacto-oligosaccharides. Galactose-encapsulated nanoparticles are preferentially digested into lysosomes of senescent cells overexpressing the lysosomal enzyme !-galactosidase. Nanoparticles remain intact in normal cells and do not release their cargo, preserving non-senescent cells from cytotoxicity (Munoz-Espin et al. 2018). This promising strategy has been applied recently to ABT-263. Galacto-conjugation of prodrug Navitoclax activated by !galactosidase activity reduces in vivo senescent lung cancer cells. Senolytic activity of ABT-263 is preserved while reducing platelet-induced apoptosis and thrombocytopenia in murine lung cancer model (Gonzalez-Gualda et al. 2020).

Figure 14 .

 14 Figure 14. MG morphology in humans and mice. Women have only one pair of MGs, commonly termed breast, whereas mice possess five pairs of MGs distributed all along the ventral face. At the nulliparous stage, lobules are thinly developed in humans (left) while they are absent in mice (right). At the pregnant stage, MG architectures from both species are similarly developed, with ducts and functional milk alveoli. Adapted from (Carroll et al. 2017).

Figure 15 .

 15 Figure15. MG architecture and cell composition in ducts and alveoli. In the virgin state, MGs are composed of resting ducts, whereas during pregnancy and lactation, MGs are composed of a network of ducts ending with milk-producing alveoli. In the ducts, luminal cells (light pink) face the lumen and are covered by a continuous layer of basal cells (dark pink). This epithelium is enclosed by a basement membrane (brown), which separates the surrounding vascularized stromal compartment composed of adipocytes (yellow), immune cells (purple shades) and fibroblasts (grey). Immune cells such as macrophages (purple) can be located both in the stromal compartment and between the luminal and the basal cell layers. In alveoli, luminal cells fully differentiate into secretory cells, and basal cells adopt a star-like shape to help alveolar contraction. The stromal compartment adapts its size to the alveolar growth by de-differentiation of adipocytes. Designed with Biorender.

Figure 16 .

 16 Figure 16. Model of mammary epithelial stem cell hierarchy. During embryonic development, multipotent stem cells give rise to both basal and luminal lineages. Multipotent stem cell contribution in postnatal development remains more controversial, but evidence supports the hypothesis of unipotent lineage-restricted stem cells in postnatal development and homeostasis. From (Lloyd-Lewis, Harris, et al. 2017).

  5 to E18.5 to the formation of epithelial buds at the position of the future nipples. At birth, each mammary fat pad only contains a rudimentary epithelial tree with ~15 ducts (Figure17)[START_REF] Veltmaat | Mouse embryonic mammogenesis as a model for the molecular regulation of pattern formation[END_REF]. MGs remain quiescent from birth to ~4 weeks old, when puberty-associated hormonal signals orchestrate a massive expansion of the ductal network. Ductal extremities called terminal end buds (TEBs) drive MG pubertal development. They consist of two different cell types, one external layer of cap cells, precursors of basal cells, and multiple internal layers of body cells, which will give rise to ductal luminal cells. Following ovarian secretion of estrogen and progesterone, TEBs will become hyper-proliferative regions, allowing the epithelial elongation in the mammary fat pad. Puberty is considered complete when the branched network of ducts has finished invading the whole mammary fat pad (Figure17)[START_REF] Howlin | Pubertal mammary gland development: insights from mouse models[END_REF].

Figure 17 .

 17 Figure 17. Murine MG developmental cycle through post-natal life. Postnatal MG development begins with a primary duct connected to the nipple. Upon puberty, the ductal network invades the mammary fat pad, and secondary ducts are developed without terminal lobules to reach a resting state in the virgin adult. During pregnancy and lactation, alveolar units are developed and fully

Figure 18 .

 18 Figure18. MG histology during the kinetic of involution. MG involution is separated into two phases based on its reversibility and the capacity to resume lactation. The first reversible phase is characterized by cell death, with dying cells shed in the lumen (H&E staining at Inv1), while alveoli maintain their global architecture (SMA staining at Inv1). At Inv3, the process becomes irreversible, with shrinkage of alveoli and remodeling of the extracellular matrix (SMA and H&E stainings at Inv3). The involution process is considered completed by Inv14, with only small remaining alveoli buddings at the end of ducts. Smooth Muscle Actin (SMA) stains the basal cell layer. Inv0, Inv1, Inv3, Inv7, and Inv14 correspond respectively to involution day 0 (physical pups removal), or 1, 3, 7, or 14 days after the initiation of involution. Adapted from[START_REF] Hitchcock | Dynamic architectural interplay between leucocytes and mammary epithelial cells[END_REF][START_REF] Sutherland | c-myc as a mediator of accelerated apoptosis and involution in mammary glands lacking Socs3[END_REF][START_REF] Kreuzaler | Stat3 controls lysosomal-mediated cell death in vivo[END_REF]).

(

  [START_REF] Stein | Involution of the mouse mammary gland is associated with an immune cascade and an acutephase response, involving LBP, CD14 and STAT3[END_REF]. Mast cells contribute to the second phase of involution, promoting apoptosis of alveolar cells and remodeling of the stromal compartment[START_REF] Lilla | Active plasma kallikrein localizes to mast cells and regulates epithelial cell apoptosis, adipocyte differentiation, and stromal remodeling during mammary gland involution[END_REF]. Notably, macrophages are the main immune cell type participating in MG involution, especially in the irreversible phase. Indeed, depletion of macrophages using the Mafia mouse model, containing a transgeneinducing cell death of CSF1R-expressing cells, dramatically delays the second phase of MG involution[START_REF] O'brien | Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution[END_REF]. Massive macrophage infiltration occurs from Inv3, possibly to promote phagocytosis of apoptotic bodies and cell debris. However, non-professional phagocytes could also fulfill this phagocytic role. Indeed, intact epithelial cells are proposed to engulf dying epithelial cells, contributing to the fast clearance of the tissue[START_REF] Monks | Epithelial cells remove apoptotic epithelial cells during post-lactation involution of the mouse mammary gland[END_REF]. Recently, a new population of tissue-resident CD11b low , CD11c + macrophages has been imaged in the mammary ducts. During pregnancy, they expand to cover alveolar structures, and via dendrite movements, they monitor the epithelium and can react fast to cell death signaling. Therefore, ductal macrophages are well positioned to proceed to milk-producing cell phagocytosis upon early involution[START_REF] Dawson | Tissueresident ductal macrophages survey the mammary epithelium and facilitate tissue remodelling[END_REF]). Then, another population of macrophages infiltrating MGs during the late phase of involution exhibits an expression of the M2 subtype, known to participate in wound healing and tissue repair, in which they promote clearance of debris but also restructuration of the ECM and angiogenesis[START_REF] O'brien | Alternatively activated macrophages and collagen remodeling characterize the postpartum involuting mammary gland across species[END_REF].Finally, the stromal compartment is extensively remodeled throughout involution, and the withdrawal of epithelium is concomitant to the replenishment of adipocytes in the mammary fat pad. During pregnancy and lactation, adipocytes dedifferentiate and delipidate to create a local source of lipids supporting milk production[START_REF] Hovey | Diverse and active roles for adipocytes during mammary gland growth and function[END_REF]. Upon involution, adipocytes fully re-differentiate and undergo hypertrophy due to the uptake of lipids released in the alveolar lumen from milk-producing dying cells. Adipocyte refilling is proposed to participate actively in the proper remodeling of the tissue, as specific depletion of MG white adipose tissue prior to involution impairs MG restructuration(Zwick et al. 2018).

Figure 19 .

 19 Figure 19. MG involution creates a pro-tumor microenvironment favoring the development of human postpartum breast cancer. Postpartum breast cancer is characterized by its poor prognosis, mainly due to the presence of metastasis. Pre-cancerous cells can stochastically appear in the alveolar structures of lactating MG, creating micro lesions. Upon involution, the MG microenvironment is disrupted with a major remodeling of the extracellular matrix, promoting growth, motility, and invasion of tumor cells. Moreover, immune cells recruited during involution secrete numerous cytokines and growth factors, which in turn can activate quiescent tumor cells. From (Schedin 2006).

Figure 20 .

 20 Figure 20. Mammary organoid models. Various organoid models have been developed from mammary epithelial cells to study physiological MG morphogenesis and pathological emergence of breast cancer. From (Srivastava et al. 2020).

  structures and tissue remodeling[START_REF] Watson | Remodeling mechanisms of the mammary gland during involution[END_REF]. To determine the dynamic of SAβGal positive cells during this process, we collected involuting MGs from different time points (Figure1A). At Inv0, fully developed alveoli were the dominant structures in the section (Figure1B, i). At Inv1, dead cells with condensed nuclei were discharged in the lumen of alveoli while alveolar structures remained intact, indicating that the involution process was initiated (Figure.1B, ii). At Inv3, alveolar structures started to shrink and adipocytes refilling becomes evident, indicating the onset of the tissue remodeling phase of involution (Figure1B, iii). Interestingly, SAβGal positive cells started to appear only at Inv3. The intensity of SAβGal staining increased gradually and peaked at Inv7 (Figure1B, iv). The presence of SAβGal positive cells was limited to the alveolar structures whereas ductal regions were negative (Figure1B, iv). Of note, alveolar structures are eventually removed by the end of involution while ductal structures last. SAβGal staining decreased afterwards and by Inv14, we barely detected any blue cells while some of the collapsed alveolar structures remained(Figure 1B, v). The SAβGal staining suggested that there was a transient induction of senescence during the tissue remodeling phase of mammary involution.

Figure 1 .

 1 Figure 1. Transient induction of senescence during mammary gland involution. A. Experimental design highlighting the time points of MGs collect at: mature virgin (8 weeks old) or old virgin (18 months old), pregnant (p18.5), after 10 days of lactation (L10/Inv0), or after 1, 3, 7 and 14 days of involution

Figure 2 .

 2 Figure 2. Alveolar luminal cells are the major senescent cell type. A-D. Representative pictures of co-stainings SAβGal with multiple markers on WT mammary glands from Inv0 and Inv3 (n≥2). (A) SAβGal/Krt5 (basal cells), (B) SAβGal/Krt8 (luminal cells), (C) SAβGal/cleaved caspase 3 (CC3) and (D)

Figure 3 .

 3 Figure 3. Persistent lactogenic hormone prevents the induction of senescence. A. Experimental design of teat sealing of MG #3L and #4L (red), while other MGs are maintained in lactation. MGs were collected 2 or 3 days after sealing and compared with MGs from mice undergoing normal involution (blue). B. Left panel: Whole mount MGs after Carmine staining, magnified 5x, from involuting (top) or sealed (bottom) MGs at day 3; Right panel: SAβGal staining from involuting (top) or sealed (bottom) MGs at day 3, on cryosections, counterstained with FastRed. C. Automatic quantifications of SAβGal staining at day 2 (left) and day 3 (right), from involuting (black) or sealed (red) MGs. (≥5 representative pictures/mouse; n≥2 mice per group). D. qPCR analysis of the indicated genes from MGs collected at day 2 or day 3, from involuting (black), sealed (red) or lactating (white) tissues. Data are represented as mean ± standard deviation. Two-way ANOVAs were performed to analyze the effect of sealing in time on the number of SAβgal + cells (C), or on the expression of genes of interest (D) (normality and equality of variances verified; significant interaction), followed by Tukey adjustments for comparisons. p-values *<0.05, **<0.01, ***<0.001, ****<0.0001.

  , to assess the impact of senescence on physiological involution. Mice were treated with ABT-263 by oral gavage daily, for 3 days prior to harvest at Inv3, Inv4, and Inv5 (Figure4A). Importantly, as ABT-263 is inducing apoptosis of senescent cells, we first assessed the apoptosis level using cleaved-caspase 3 staining comparing control to ABT-263 treated samples. We showed an increase of apoptotic epithelial cells from 11% in control up to 30% in ABT-263 treated MGs at Inv5 (Figure 4B & G). As highlighted with SAβGal staining, ABT-263 treatment efficiently cleared senescent cells. Indeed, we showed that the number of SAβGal positive cells was significantly reduced at all time points in treated MGs, compared to controls (Figure 4C & 4H, Supplementary Figure 4A-C). Therefore, ABT-263 treatment could efficiently eliminate senescent cells via apoptosis during involution. provide evidence supporting an important role of senescence induction in tissue remodeling phase of MG involution.

Figure 4 .

 4 Figure 4. ABT-263 treatment specifically eliminates senescent cells and delays involution. A. Experimental design of in vivo treatment with ABT-263 at 50mg.kg -1 .day -1 or vehicle only. Mice were force-feeded every day for 3 days prior to collect either at Inv3, Inv4 or Inv5. B-F. MG sections from control or ABT-263 treated mice at Inv5, respectively stained for (B) cleaved-caspase 3 (CC3), (C) SAβGal, (D) Carmine, (E) a-SMA, and (F) perilipin. G-J. Quantification of indicated staining. (≥5 representative pictures/mouse; n≥4 mice per group). Data are represented as mean ± standard deviation. Student t-tests (normality and equality of

Figure 5 .

 5 Figure 5. Removing p16 + senescent epithelial cells delays involution-like process in ex vivo organoid model. A. Experimental design. Organoids from transgenic mouse model p16-3MR with integration of 3MR cassette expressing artificial Luciferase/mRFP/HSV-TK reporter, under p16 promoter regulation (upper panel). Administration of ganciclovir 50μg.ml -1 in BOM (GCV) or BOM only (Control), every two days

  characterized. A better comprehension of its physiological functions might bring new insights into the pathological perspective of senescence and how to specifically target the detrimental effects associated with senescence (Munoz-Espin and Serrano 2014).MG involution is a complex process allowing the MG to return to its resting state after lactation. This process has been extensively reviewed and various mechanisms have been shown to occur in regulating MG involution, such as cell death, alveoli shrinkage, remodeling of the ECM, adipocytes refiling and immune cell clearance[START_REF] Watson | Remodeling mechanisms of the mammary gland during involution[END_REF]. With this work, we showed for the first time that senescence occurs physiologically, beyond embryonic development and tissue regeneration, during physiological MG involution. The senescence program occurs more specifically during the irreversible phase of the MG involution, to promote proper tissue remodeling.

  2. A-D. Flow cytometry panels of total cells positive for C12FDG, from MG dissociated at Inv0 (A & B) or Inv5 (C & D), without prior treatment (A & C) or after incubation with bafilomycin (B & D). Annexes Figure 5. A. Whole mount MGs after Carmine staining from wild-type mouse (WT, left) or CDKN2A, CDKN1A double knockout (p16, p21 DKO, right), at involution day 5. B. Whole mount MGs after SAβGal staining, from wild-type mouse (left) or double knock-out (right), at involution day 5. Annexes Figure 6. A. Design of in vivo treatment with ABT-263 at 50mg.kg -1 .day -1 or vehicle only. Mice were force-feeded every day from Inv3 to Inv9 prior to collect at Inv10. B. Fast Red staining from control or ABT-263 treated mice at Inv10. Annexes Figure 7. A. Flow cytometry panel of PDGFRα-GFP + cells from dissociated virgin MGs. B. Pictures of adipocytes derived from isolated PDGFRα-GFP + cells, after 5 days of spontaneous differentiation (SVF medium) or after 3 days in an adipogenic cocktail of dexamethasone, IBMX and insulin, followed by 2 days of recovery in SVF medium. Annexes Figure 9. A-F. Flow cytometry analysis of dissociated MGs from control (A-C) or ABT-263 treated (G-I) MGs at Inv5. Cells were analyzed from the CD45 + population (immune cell fraction). A. and D. SSC/SiglecF from CD45 + population to isolate eosinophils. B. and E. SSC/F4/80 from CD45 + population to isolate total macrophages population. C. and F. CD11c/MHCII from CD45 + ; F4/80 high ; CD11b low population to isolate ductal CD11c high ; MHCII high macrophages. Annexes Figure 10. A. F4/80 staining in MGs from control (Lipo PBS) or from mice injected with liposomes containing clodronate (Lipo Clodronate) at Inv5. B. Whole mount MGs after Carmine staining, magnified 5x, from control (left) or clodronate (right) treated mice at Inv5. C. MG sections from control (left) or clodronate (right) treated mice at Inv5 stained for SMA. D-E. Quantifications of pictures stained with SMA, representing the mean area of alveolar structures (D) and the total coverage of epithelium per picture (E) (≥5 representative pictures/mouse; n=3 per group). Data are represented as mean ± standard deviation. Student t-tests (normality and equality of variances verified) were performed to analyze the effect of clodronate treatment respectively on the area of alveolar structures (D) or remaining epithelium (E).

  -mouse [GBI labs #D55-6]; anti-rabbit [GBI labs #D13-6]; anti-rat [GBI labs #D35-110]; anti-goat [GBI labs #D43-6]. IF: anti-mouse [Diagomics #DkxMu003-D488NHSX]; anti-rabbit [Diagomics #DkxRb003-D594NHSX]; anti-rat [Diagomics #DkxRt003-F488NHSX]; anti-goat [Diagomics #DkxGt003-D488NHSX]; anti-chicken [Diagomics #GtxCk003-D488NHSX].

  shaking 120rpm, in a solution containing 3mg/mL Collagenase A [Roche #10103586001]; 100U/mL Hyaluronidase [Sigma #H3884] in CO2-independent medium [Gibco #18045-054], completed with 5% FBS, 2mM L-glutamine [Sigma Aldrich #G5792] and 100 U/mL of penicillin + 100 μg/mL of streptomycin[Gibco, #15140-122]. Digested tissues were centrifuged 5 min at 1100 rpm and the supernatant was discarded. After a wash with CO2independent medium and a centrifugation, each pellet was resuspended with 2mL of prewarmed PBS + 0.25% trypsin [Dutscher #P10-022100] and 0.1% EDTA Versen [Biochrom #L2113], for 1 min at RT. After a second wash in CO2independent medium + 5% FBS and a centrifugation, each pellet was resuspended in 2ml of CO2-independent medium + 5% FBS containing 5mg/mL of dispase II [Roche #13752000] and 0.1mg/mL of DNAse I [Sigma Aldrich #D4527-40KU], and incubated 5 min at 37°C. After a third wash in CO2independent medium + 5% FBS and a centrifugation, pellets were resuspended in 2mL of cold ammonium chloride solution[Stem Cell Technologies #07800]

  Following suppliers' guidelines, Bafilomycin A1 [Tebu #21910-2060] was resuspended in DMSO to a concentration of 0.1mM and stored at -20°C and C12FDG[Fisher #11590276] was resuspended in DMSO at a concentration of 20mM and stored at -20°C. After mammary cells dissociation, pellets of isolated cells were resuspended into 1mL of CO2-independent medium [Gibco #18045-054]. To neutralize acidic pH of lysosomes, cells were treated with 100nM of Bafilomycin A1 for 1h at 37°C, 5% CO2. Upon use, C12FDG 20mM stock was first diluted with DMSO to a temporary stock of 2mM in fresh culture media, and added to the cell solution at a concentration of 33µM. Cells were incubated 2h at 37°C, 5% CO2 (Eppendorf tubes were manually inverted every 30 min to homogenize the solution). After incubation, cells were washed twice with CO2independent medium and centrifuged 5 min at 1100 rpm. Resuspended pellets were then stained with antibodies compatible with the green FITC-emitted signal (wavelength 488) from C12FDG.

  ): anti-CD45-APC 1:100 [Biolegend #103112, clone 30-F11]; anti-CD31-APC 1:100 [Biolegend #102510, clone MEC13.3]; anti-CD24-BV421 1:50 [Biolegend #101826, clone M1/69]; anti-α6-PE.Cy7 1:50 [Biolegend #313622, clone GoH3]. Gating strategy for mammary cells identification : the two first gates are identical as immune cell profiling gates A. B. (cells and singlets) followed by gatings as shown in Supplementary Figure 2.B. No compensation required.

  manually restraining the animal, a 200-300μl drop of veterinary tissue adhesive [3M Vetbond #1469C] was applied on the nipple and left to dry before the release of the mouse in the cage with the litter. The manipulation was repeated 3h later to ensure a full sealing of the nipple. Maintenance of lactation on counterpart open teats was checked everyday. MGs were harvested 2 or 3 days after teat closure.

  senolytic ABT-263 [Tebu #T2101] (Chang et al. 2016), was initially resuspended in DMSO at 100mg/ml, aliquoted for single doses of 12,5 μl and stored at -20°C. Each ABT-263 dose was then freshly resuspended by adding 237,5μl of vehicle (60% Phosal; 30% PEG400; 10% pure EtOH) upon use. Mice were force-feeded by oral gavage daily with ABT-263 at 50mg/kg for 3 consecutive days before MGs harvesting (Chiche et al. 2017); control mice were force-feeded by oral gavage with 237,5μl of vehicle only supplemented with 12,5 μl DMSO. Volume administrated to each control or treated mouse was 250μl of solution for a mouse of 25g.

  software; regular brightfield images were captured using an Olympus CKX41 microscope, a CoolLED pE-300 white lamp, an Olympus U-LS30-3 Camera and CellSens Entry imaging software; timelapse imaging for organoid morphology was performed using a Zeiss Definite Focus microscope, a Mercury HXP120 Lamp, dual camera sCMOS Hamamatsu ORCA FLASH 4.0 v2.0, an environmental chamber with temperature, CO2 and O2 control and ZEN blue 2012 software.Image quantifications were performed using ImageJ software. FACS profile analyses were performed using Flowjo software. Statistical analyses were performed using GraphPad Prism software; statistical tests and p-values are specified in figure legends. The number of independent biological replicates

FIGURE 1 |

 1 FIGURE 1 | Lactation induction in primary mammary organoids. (A,B) FGF2 pretreatment increases lactation capacity of primary mammary organoids. (A) Scheme depicting the experimental design. BOM, basal organoid medium; LM, lactation medium; FGF2, FGF2 medium. (B) Expression of milk genes Csn2 and Wap in organoids treated with BOM, LM, or FGF2 followed by LM. The values are relative to BOM. The plot shows mean + SD; n = 2. One-way ANOVA, *p < 0.05. (C) Bright-field images and maximum intensity projection images from confocal imaging of whole-mount organoids after treatment with FGF2 only or with FGF2 followed by LM. Yellow-to-brown staining shows F-actin. Scale bars represent 100 µm. (D) Bright-field image and maximum intensity projection images from confocal imaging of whole-mount organoid treated with FGF2 followed by LM. Red, oil red O (lipids); green, F-actin; blue, DAPI (nuclei). Scale bars represent 100 µm.(Continued)

FIGURE 1 |

 1 FIGURE 1 | Continued (E,F) Quantification of b-casein expression in organoids treated with FGF2, or FGF2 followed by LM. (E) RT-qPCR analysis of b-casein gene Csn2. The values are relative to FGF2. The plot shows mean ± SD; n = 3. Unpaired Student's t-test, two tailed, ****p < 0.0001. (F) Western blot analysis of b-casein expression on protein level. The plot shows quantification of band density. The values are relative to FGF2. (G) Immunohistochemical staining of b-casein in organoids treated with FGF2 or FGF2 and then LM at days 6 and 10, respectively. Marked area is shown in higher magnification. Scale bars represent 100 µm.

FIGURE 2 |

 2 FIGURE 2 | Morphology of organoids undergoing long-term lactation. (A) Scheme depicting experimental design. FGF2, FGF2 medium; LM, lactation medium; BOM, basal organoid medium. (B) Bright-field images from time-lapse imaging of organoid morphogenesis under continuous LM treatment (FGF2-LM) or under LM withdrawal and replacement with BOM (FGF2-LM-BOM). Scale bars represent 100 µm. (C,D) Morphometric analysis of organoid area (C) and density (D) from the time-lapse experiment. The plots show mean + SD; n = 2, N = 20 organoids per condition. Two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) Hematoxylin and eosin staining of organoids at different time points of long-term lactation. Scale bars represent 100 µm.

FIGURE 3 |

 3 FIGURE 3 | Milk production during long-term lactation. (A) Immunohistochemical staining of b-casein in organoids during long-term LM treatment or after LM withdrawal (LM-BOM), according to experimental scheme in Figure 2A. Marked area is shown in higher magnification. Scale bars represent 100 µm. (B) Csn2 expression during long-term lactation with continuous lactation medium (FGF2-LM) or with hormonal withdrawal (FGF2-LM-BOM). The plot shows mean + SD; n = 3 for d12 to d18, n = 1 for d20. Two-way ANOVA, ****p < 0.0001. (C) Western blot analysis and band density quantification of b-casein expression in organoids during long-term lactation.

FIGURE 4 |

 4 FIGURE 4 | Lactating organoids retain functional myoepithelial layer. (A) Immunofluorescent staining shows distribution of myoepithelial (keratin 5 positive, green) and luminal cells (keratin 8 positive, red) in organoids treated with FGF2 or FGF2 followed by LM. Hoechst, blue (nuclei). Scale bars represent 100 µm. (B) Immunofluorescent staining shows distribution of myoepithelial (keratin 5 positive, green) and luminal cells (keratin 8 positive, red) in organoids during long-term lactation. Hoechst, blue (nuclei). Scale bars represent 100 µm. (C) Quantification of contracting organoids from movies recorded at indicated time-points. The plot shows mean + SD; n = 2, N = 50 organoids per experiment. Two-way ANOVA, **p < 0.01, ****p < 0.0001.

FIGURE 5 |

 5 FIGURE 5 | Withdrawing hormones induces an involution-like phenotype in lactating organoids. (A) The viability of the lactating and involuting organoids using resazurin assay. The plot shows relative resorufin fluorescence of organoids with continuous LM treatment (FGF2-LM), LM withdrawal and replacement with BOM (FGF2-LM-BOM), and FGF2-LM-BOM organoids treated with 40 µM taxol for 4 days (40 µM taxol) or 70% ethanol for 5 min (70% EtOH) to induce cell death. Values are relative to FGF2-LM. (B-D) Analysis of FGF2-LM-BOM organoids after replating to BOM or FGF2 medium. (B) Quantification of the size of the FGF2-LM-BOM organoids that were replated and cultured with BOM or FGF2 for the number of days as indicated. The plot shows mean + SD; n = 1, N = 25 organoids per condition. Two-way ANOVA, asterisks indicate change in comparison to d0; *p < 0.5, ***p < 0.001, ****p < 0.0001. (C) Quantification of the number of budding FGF2-LM-BOM organoids after replating and culture with BOM or FGF2 for 7 days. (D) Bright-field images showing morphogenesis of FGF2-LM-BOM organoids after replating and culture with BOM or FGF2 for 7 days. Scale bars represent 100 µm. (E,F) RT-qPCR analysis of Mmp2 and Mmp13 expression in organoids during long-term lactation with continuous lactation medium (LM) treatment or with hormonal/LM withdrawal (LM-BOM). The values are relative to FGF2-LM at each time point. The plots show mean + SD; n = 3 for d12-d18, n = 1 for d20. Two-way ANOVA, ⇤ p < 0.05.

  ): e3996. DOI: 10.21769/BioProtoc.3996. A Robust Mammary Organoid System to Model Lactation and Involution-like Processes Elsa Charifou 1 , Jakub Sumbal 1, 2 , Zuzana Koledova 2 , Han Li 1 and Aurélie Chiche 1, * 1 Cellular Plasticity & Disease Modeling -Department of Developmental & Stem Cell Biology, CNRS UMR3738 -Institut Pasteur, 25 rue du Dr Roux, Paris 75015, France; 2 Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 3, Brno 625 00, Czech Republic *For correspondence: aurelie.chiche@pasteur.fr [Abstract] The mammary gland is a highly dynamic tissue that changes throughout reproductive life, including growth during puberty and repetitive cycles of pregnancy and involution. Mammary gland tumors represent the most common cancer diagnosed in women worldwide. Studying the regulatory mechanisms of mammary gland development is essential for understanding how dysregulation can lead to breast cancer initiation and progression. Three-dimensional (3D) mammary organoids offer many exciting possibilities for the study of tissue development and breast cancer. In the present protocol derived from Sumbal et al., we describe a straightforward 3D organoid system for the study of lactation and involution ex vivo. We use primary and passaged mouse mammary organoids stimulated with fibroblast growth factor 2 (FGF2) and prolactin to model the three cycles of mouse mammary gland lactation and involution processes. This 3D organoid model represents a valuable tool to study late postnatal mammary gland development and breast cancer, in particular postpartum-associated breast cancer. Graphic abstract: Mammary gland organoid isolation and culture procedures Keywords: Mouse, Mammary gland, 3D organoid, Ex vivo, Lactation, Involution www.bio-protocol.org/e3996 Bio-protocol 11(08): e3996. DOI:10.21769/BioProtoc.3996

  Bio-protocol 11(08): e3996. DOI:10.21769/BioProtoc.3996 Please cite this article as:Charifou et al., (2021). A Robust Mammary Organoid System to Model Lactation and Involution-like Processes. Bioprotocol 11(8): e3996. DOI: 10.21769/BioProtoc.3996.10. Incubator for cell culture, 37°C, 5% CO2 (e.g., Thermo Scientific, model: HERAcell 150i) 11. Heating plate at 37°C (e.g., Techne DRI-Block DB-2A) 12. Microscope and camera (e.g., Olympus model: CKX41) 13. NanoDrop™ (e.g., Implen Nanophotomoter NP80) 14. Sonicator (e.g., Diagenode Bioruptor Pico) 15. Incubator at 65°C (e.g., Memmert Incubator I) 16. Embedding workstation (e.g., Leica EG1150C) Procedure A. Isolation of mammary primary organoids 1. Dissection of a virgin mouse to harvest mammary glands (see Video 1). Video 1. Mammary gland harvesting. This video was made at Pasteur Institute. according to guidelines from the regulations of Institut Pasteur Animal Care Committees (CETEA). on Animal Care and approved by the French legislation in compliance with European Communities Council Directives (A 75-15-01-3).

  r. Add 6 ml DMEM/F12 and resuspend the solution by performing 5 up-and-down motions with a 10-ml pipette. s. Centrifuge for 10 min at 400 × g. t. Discard the supernatant. Resuspend the pellet in 9 ml DMEM/F12. u. Perform differential centrifugation to separate the mammary epithelium from the stromal fraction: centrifuge the suspension for 15 s at room temperature, 400 × g. Discard the supernatant containing the stromal fraction and resuspend the epithelial pellet in 9 ml DMEM/F12.

Figure 1 .

 1 Figure 1. Key steps of mammary gland collection for organoid isolation and 3D culture. A,B. Images of mouse dissection to access the mammary gland. A. Needles 1-4 represent the points at which to pin the mouse. Needles 5-7 and 8-10 represent the points at which to pin the skin of the mouse. Letters A-F with the blue dotted lines indicate the cuts. B. Green dotted lines denote the mammary gland. The lymph node is denoted in red and must be removed. C. Mammary gland before (left panel) and after (middle panel) mincing with a scalpel. Mammary organoids after transfer to dissociation medium (right panel). D. Example of mammary organoid counting. Left panel: organoids are surrounded by dotted lines. Star represents nerves. Right panel: organoids after embedding in Matrigel ® . Arrow represents the edge of the Matrigel ® dome. Scale bar = 500 μm. E. Freshly isolated primary organoid. Left panel: image of a mammary

5 .

 5 Mimicking involution by hormonal withdrawal a. Pre-heat the BOM at 37°C. b. Aspirate the medium from the wells without touching the Matrigel ® dome and replace with 800 μl fresh BOM.

Figure 2 .

 2 Figure 2. Modeling lactation and involution-like processes in primary mammary organoids. A. Scheme depicting the experimental design. B. Morphology of primary mammary organoids during lactation and involution-like processes. Bright-field images of organoid morphology following morphogenic and lactogenic stimulation and on days 4 or 8 after hormonal withdrawal. Scale bar = 100 μm.

Figure 3 .

 3 Figure 3. Passage of involution-like organoids. A. Scheme depicting the experimental design. PRL: Prolactin; BOM: basal organoid medium. B. Morphology of passaged organoids during the lactation and involution-like processes. Brightfield images of passage 1 (upper panel) and passage 2 (lower panel) organoids following morphogenic and lactogenic stimulation and on days 4 or 8 after hormonal withdrawal. Scale bar = 100 μm.

  Calculate the average of the two counts in 10 μl solution and multiply according to the volume of BOM used to resuspend the pellet to obtain the total number of organoids. . Place a 24-well plate on ice. Calculate the number of wells needed and spread 20-μl Matrigel ® in a round patch on the bottom of each well. Note: Start by placing the tip containing Matrigel ® at the center of a well and expand circularly towards the edges of the well, without touching them.

	The endothelium has a somewhat ragged look in comparison with the smooth-looking organoids. The minor presence of primary nerves and endothelium does not interfere with organoid lactation or involution. iii. Count only the organoids with a diameter greater than 30-50 since the smaller ones may not develop properly. Note: Freshly isolated organoids can be viably frozen in a solution of FBS containing 10% DMSO for long-term storage in liquid nitrogen and later use. B. 3D culture of mammary organoids 1. Embedding in Matrigel ® Reminder: Work inside a laminar flow hood to maintain aseptic conditions. Wash the ice bucket and heating plate thoroughly with 70% EtOH prior to placement in the laminar flow hood. a. Thaw the Matrigel ® on ice or at 4°C. Notes: d. In the meantime, pre-heat a heating plate to 37°C. e. Prepare the organoid suspension in the Matrigel ® : calculate the volume of organoid suspension required to obtain the desired number of organoids. Pipette this volume of suspension into a fresh 1.5-ml tube and centrifuge for 3 min at 400 × g. Note: Adjust the number of organoids per well depending on the type of experiment: 200 organoids per well for morphology and histology, 400 for gene expression, and 1000 for i. Matrigel c. Incubate the 24-well plate in a cell incubator (5% CO2) for 15 min at 37°C. western blotting analysis.

e.

®

solidifies really fast at room temperature. Always keep it on ice before use and during the plating procedure. www.bio-protocol.org/e3996 Bio-protocol 11(08): e3996. DOI:10.21769/BioProtoc.3996 Please cite this article as: Charifou et al., (2021). A Robust Mammary Organoid System to Model Lactation and Involution-like Processes. Bioprotocol 11(8): e3996. DOI: 10.21769/BioProtoc.3996.

ii. Keep in mind that Matrigel ® thawing takes time; therefore, begin thawing prior to the procedure (2 h for a 1-ml aliquot, 6 h for a 10-ml bottle). bf. Carefully remove the supernatant and place the tube on ice. Subsequently, carefully resuspend the pellet in the required volume of cold Matrigel ® (50 μl per well), avoiding bubble formation. Keep on ice.

  This solution is prepared inside a chemical hood and does not need to be filter-sterilized. This solution is prepared inside a chemical hood and does not need to be filter-sterilized.10 μl β-mercaptoethanol per 1 ml RLT lysis buffer (from the RNeasy Micro Kit; this solution can be stored for up to one month at room temperature).8. Phosphatase inhibitor cocktail IINote: This solution is prepared inside a chemical hood or on a bench and does not need to be filter-sterilized.Charifou et al., (2021). A Robust Mammary Organoid System to Model Lactation and Involution-like Processes. Bioprotocol 11(8): e3996. DOI: 10.21769/BioProtoc.3996.

	www.bio-protocol.org/e3996
	1× insulin-transferrin-selenium (ITS)
	100 U/ml penicillin
	100 μg/ml streptomycin
	c. Prepare 3% low gelling temperature agarose in PBS and melt slowly in a microwave for 2 mM glutamine
	1.5-2 min at 1000 W (homogenize every 30 s by hand rotation). in DMEM/F12
	d. Detach the fixed culture using the flat side of a spatula and transfer to a plastic histology 4. Morphogenesis medium
	mold containing melted agarose. Overlay with more agarose. Note: This solution is prepared inside a laminar flow hood under aseptic conditions and does
	e. After solidification of the agarose, unmold the block. Use a scalpel to remove the excess not need to be filter-sterilized.
	agarose surrounding the Matrigel ® dome and place in a plastic embedding cassette for 2.5 nM FGF2 in BOM
	histology. 5. Lactation medium
	f. Proceed to sample dehydration: incubate the embedding cassettes in successive 1-h baths Note: This solution is prepared inside a laminar flow hood under aseptic conditions and does
	of 70% EtOH, 95% EtOH, 100% EtOH (twice), xylene (twice), 50% xylene-50% melted not need to be filter-sterilized.
	paraffin, and 100% melted paraffin. 1 μg/ml prolactin
	1 μg/ml hydrocortisone
	in BOM
	6. 4% PFA
	until further analysis. Note: 4% paraformaldehyde in PBS
	k. Remove the paraffin prior to any staining by successive 5-min baths of xylene (twice), 100% 7. RNA lysis buffer
	EtOH (twice), 95% EtOH, 70% EtOH, and H2O. 1. Dissociation solution Recipes Note: This solution is prepared inside a laminar flow hood under aseptic conditions and does not need to be filter-sterilized. Note: 2 mM imidazole
	2 mg/ml collagenase 1 mM sodium fluoride
	2 mg/ml trypsin 1.15 mM sodium molybdate
	5 μg/ml insulin 1 mM sodium orthovanadate
	50 μg/ml gentamicin 4 mM sodium tartrate dihydrate
	5% FBS in RIPA buffer
	2 mM glutamine 9. Protease inhibitor cocktail I
	in DMEM/F12 Note: This solution is prepared inside a chemical hood or on a bench and does not need to be
	2. BSA solution filter-sterilized.
	500 μM AEBSF hydrochloride
	150 nM aprotinin
	1 μM protease inhibitor E-64

3. Fixation and embedding for histology a. Aspirate the culture medium and rinse the culture twice with 800 μl cold PBS. www.bio-protocol.org/e3996 Bio-protocol 11(08): e3996. DOI:10.21769/BioProtoc.3996 Please cite this article as: Charifou et al., (2021). A Robust Mammary Organoid System to Model Lactation and Involution-like Processes. Bioprotocol 11(8): e3996. DOI: 10.21769/BioProtoc.3996. b. Incubate with 800 μl 4% PFA for 30 min. Following removal of the 4% PFA, wash twice with PBS. Notes: i. Domes should be entirely covered with the solution. Add a greater volume if required.

ii. The fixed cultures can be stored in PBS at 4°C until embedding.

g. Incubate overnight at 65°C in a second bath of 100% melted paraffin.

h. Embed in a histology tissue mold using an embedding workstation.

i. Unmold the paraffin blocks after 24 h of solidification. j. Cut 5-μm sections and spread on microscope slides. Keep the slides at room temperature Note: This solution can be filter-sterilized and reused several times when stored at 4°C. 2.5% BSA in PBS 3. Basal organoid medium (BOM) www.bio-protocol.org/e3996 Bio-protocol 11(08): e3996. DOI:10.21769/BioProtoc.3996 Please cite this article as: Charifou et al., (2021). A Robust Mammary Organoid System to Model Lactation and Involution-like Processes. Bioprotocol 11(8): e3996. DOI: 10.21769/BioProtoc.3996. Note: This solution is prepared inside a laminar flow hood under aseptic conditions and does not need to be filter-sterilized. Bio-protocol 11(08): e3996. DOI:10.21769/BioProtoc.3996 Please cite this article as: 0.5 mM EDTA 1 μM leupeptin hemisulfate in RIPA buffer

Frontiers in Cell and Developmental Biology | www.frontiersin.org

March 2020 | Volume 8 | Article 68

Acknowledgments 6 ACKNOWLEDGMENTS .................................................................. ABSTRACT .................................................................................... INTRODUCTION .......................................................................... ACKNOWLEDGMENTS We are particularly grateful to Katarina Mareckova and Anas Rabata for their excellent technical support, and to Dr. Mari Aoki and Dr. Ulrich Boehm for providing cryosections of Prlr-IRES-Cre;ROSA26-CAGS-GFP mammary glands. We thank the Central Animal Facility of the Institut Pasteur and the Laboratory Animal Breeding and Experimental Facility of the Faculty of Medicine, Acknowledgments Work in the laboratory of HL is funded by the Pasteur, Centre National pour la Recherche the Agence Nationale de la Recherche (ANR-10-LABX-73 and ANR-16-CE13-0017-01), Fondation ARC (PJA 20161205028 and 20181208231), Programme Barrande, and AFM-Telethon Foundation. AC was funded by postdoctoral fellowships from the Revive Consortium. EC was funded by a Ph.D. fellowship from Sorbonne Université. ZK was funded by the Grant Agency of Masaryk University (MUNI/G/1446/2018), Mobility grant by Ministry of Education, and Youth and Sports, and by funds from the Faculty of Medicine MU to the junior researcher (ROZV/28/LF/2020). JS was funded by the P-Pool (Faculty of Medicine MU) and the Grant Agency of Masaryk University (MUNI/A/1565/2018).

This protocol was derived from the original research paper "Primary Mammary Organoid Model of Lactation and Involution" (Sumbal et al., 2020b).

Acknowledgements The authors thank Xiomara Banholzer for help with the organization of the meeting.

FUNDING

Work in the laboratory of HL is funded by the Pasteur, Centre National pour la Recherche the Agence Nationale de la Recherche (ANR-10-LABX-73 and ANR-16-CE13-0017-01), Fondation ARC (PJA 20161205028 and 20181208231), and AFM-Telethon Foundation. Work in the laboratory of ZK was funded by the Grant Agency of Masaryk University (grant no. MUNI/G/1446/2018). AC was funded by the postdoctoral fellowships from the Revive Consortium. JS was funded by the P-Pool (Masaryk University, Faculty of Medicine), Amgen Scholars Europe and Erasmus+ programs and by the Grant Agency of Masaryk University (grant no. MUNI/A/1565/2018). EC was funded by the Ph.D. fellowship from Sorbonne Université. Funding Funding for the meeting was received from The Company of Biologists, Novartis, Roche, Stem Cell, AE Scientific and CAPP.

DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

Abstract

Context and aims of the project

To further characterize the phenotype induced by ABT-263 treatment, we stained MGs with Carmine to reveal their morphology (Figure 4D, Supplementary Figure 4A &B). In control MGs, from Inv3 to Inv5, we observed the expected shrinkage of alveoli clusters, unveiling epithelial ducts, and major adipocytes refilling of fat pad (Figure 4D, Supplementary Figure 4A &B). The ABT-263 treated MGs exhibited the same morphology as controls on Inv3

(Supplementary Figure 4A). Surprisingly, we followed the kinetic of regression of treated MGs and noticed a potential morphological defect of involution at Inv4 and Inv5, with enlarged alveoli clusters (Figure 4D, Supplementary Figure 4B).

To refine the phenotype induced by senescent cells removal, we stained MG histological sections with α-smooth-muscle actin (SMA), a specific marker of the mammary basal cells. This label allowed us to quantify the area of alveoli during the kinetic of involution. From Inv3 to Inv5, we showed an expected regression of alveoli size (Figure 4E, Supplementary Figure 4A &B). This shrinkage correlated with a diminution of the percentage of epithelium remaining in the tissue, from 35% of coverage per picture area at Inv3 to only 6% at Inv5 (Figure 4I). In contrast, in ABT-treated MGs, alveoli remained enlarged from Inv3 to Inv5, with more than 17% of epithelium occupying the fat pad at Inv5 (Figure 4E & I). Therefore, the kinetic of alveoli regression is slower in the ABT-263 treated MGs comparing to control.

Moreover, we characterized adipocytes refilling using adipocyte intra-membrane perilipin staining. In controls, conversely to epithelium dynamic, adipocytes were small and numerous at Inv3 and enlarged up to cover 45% of picture area by Inv5 (Figure 4F, Supplementary Figure 4A &B). In treated MGs, the area covered by mammary adipocytes was smaller at Inv5 compared to control sections (Figure 4F, Supplementary Figure 4 A & B). Therefore, the adipocytes refilling process was impaired upon ABT-263 treatment.

Taken together, our results showed that elimination of senescent cells by ABT-263 impedes the MG involution, specifically the tissue remodeling process, evidenced by slower alveoli regression and less adipocyte refilling. These results

Supplementary Figures

ETHICS STATEMENT

The

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2020.00068/ full#supplementary-material

Conflict of Interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Competing interests

The authors declare that they have no competing interests.

Ethics

The animal study was reviewed and approved by French legislation in compliance with European Communities Council Directives (A 75-15-01-3) and the regulations of the Institut Pasteur Animal Care Committees (CETEA).

Authors' Contributions EC, GAT, MB-A, BH and AVK wrote the manuscript. All authors approved the final manuscript.

Declarations

Ethics MB-A, BH and AVK are Editorial board members of the Journal of Mammary Gland Biology and Neoplasia.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.

Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Abstract

Cellular senescence is a biological stress response characterized by a stable cell cycle arrest. Nonetheless, cells remain metabolically active and acquire a senescence-associated secretory phenotype (SASP), a complex secretome composed of cytokines, chemokines, growth factors, and extracellular matrix remodeling modulators. Senescence is associated with various pathological processes, such as tumorigenesis and aging. However, it is unknown when, where and how senescence contributes to physiological processes. To answer this question, we took advantage of the mammary gland (MG), an organ with remarkable plasticity throughout postnatal development. The MG involution is one of the major mammalian cell death and tissue remodeling events, when milkproducing epithelial cells are removed, and the MG returns to its near pregestation state, resting for further pregnancy. During my Ph.D., we showed that senescence was transiently induced during the irreversible phase of involution.

The senescent program occurred specifically in the alveolar milk-producing luminal cells and correlated with the expression of the cell cycle inhibitor p16. In parallel, we established a novel organoid system to mimic MG gestation, lactation, and involution. In this ex-vivo model, we also highlighted the presence of senescent cells strictly during the involution-like process. To assess the biological impact of senescence in vivo, we used a teat sealing method to uncouple the reversible and irreversible phases of involution. We unveiled a close association between the withdrawal of lactogenic hormones occurring in the second phase of involution and the induction of the senescence program. To further define the physiological roles of senescence during involution, we treated mice with ABT-263, a senolytic compound inducing apoptosis of senescent cells. Interestingly, we observed an impaired tissue remodeling upon senescence elimination, with larger remaining alveolar structures and delayed adipocyte refilling. Moreover, in organoids from transgenic p16-3MR mice, we successfully removed senescent cells with ganciclovir and delayed the involution-like process. Taken together, both in vivo and ex-vivo models suggest an essential role of senescence in modulating the tissue remodeling phase of MG involution. Importantly, the involution process is intimately associated with postpartum breast cancer (PPBC), a cancer diagnosed within 10 years following delivery with a poor prognosis. Investigating how senescence impacts the microenvironment during the involution process might provide major insights to understand PPBC.