Cristian Saquel

Christine Viaris De Lesegno

Carlos Urena-Martin

Graça Raposo

Christophe Lamaze
email: christophe.lamaze@curie.fr

Ilse Katz

Qianru He

Jorge Rodríguez-Antolín

Felipe A Court
email: felipe.court@umayor.cl

Romina J Catalan

Rodrigo Lopez-Leal

Ramon A Ramirez

David Necuñir

Ursula Wyneken

Caveolin-1 and mechanical stress control the release of a pro-metastatic subpopulation of small extracellular vesicles

grew up the most, both personally and scientifically. I was able to develop a project from start to finish, including a publication later on, so I consider it as a test run for the PhD, a successful one. It is in part, thanks to Felipe that I met Christophe. You trusted me to come to Paris in a collaboration project not only once, but twice, and thanks to this I was able to start my own PhD project with Christophe. To both of you, Juan Antonio and Felipe, you have my eternal gratitude and I'm looking forward to meeting you back in Chile soon.

I would be remiss not to mention the extraordinary support from my friends, mainly Angel and Andrés in Chile and Spain, Trini, Nati and Erik, in Paris, who have become like a second family to me in this academic pursuit. The PvMcrew Gold, Gun, and Wild, for those frustrating but fun weekend and late-night gaming sessions.

Finally, I'd like to thank my family, being this far away from my relatives in these challenging years was not easy. To my mother,

Sigue abriendo los caminos el surco de tu destino.

La alegría de sembrar no te la pueden quitar, la alegría de sembrar es tuya, de nadie más.

-La pala, Víctor Jara

This dream could not have come true without all the people that I met throughout this journey.

First I would like to thank my supervisor Christophe, who trusted me to come from Chile and work in this exciting project. Every project comes with complications and roadblocks, and I feel privileged to have had the support of Christophe in these times, whether it was by trusting in my proposals to overcome the issues or by his willingness to arrange meetings with different experts that could help me. Of all that I have learned under his direction, I believe what stood the most for me was the importance of finding joy in what one does. I am tremendously grateful for the invaluable knowledge and guidance I have received from him during these past four years.

"The people that you work with are, when you get down to it, your very best friends." -M.S.

By being more than 11,000 kilometers away from home, across the equator and the Atlantic Ocean, one realizes that your colleagues are in reality more than just colleagues, they are the people that you see every single day, who you talk with, take a coffee together after lunch, or get together after work. For this I would like to thank all my colleagues, in no particular order, my office mates Changting and Satish, for the daily conversations about everything including life, science, cultural exchanges, and so on. It's good to have people that you get along with sitting right next to you.

I'm thankful of Christine, for always being there greeting me with a big smile whenever I had any question or needed help with something, you really are a pillar and the heart of the lab, don't ever change. On the other hand, I want to thank Cedric as well, who did not normally greet you with a smile when you needed something but rather a "what is it this time…?" kind of look. But we all know that behind that façade he is the sweetest guy, and the second pillar of the lab. Looking forward to the next fun/interesting idea we can try to work on together, that doesn't fail miserably like the EV expansion microscopy or EV lattice imaging… maybe EVs are the problem… I'd like to thank Vibha and Pedro for their always helpful comments and suggestions in the lab meetings. I also want to thank the newest members of the lab, Camille, who has made our bond stronger by setting up after work meet ups and acting as a catalyzer of friendships within the unit. Celine, who has helped me the most in the past few months, I really appreciate your willingness and motivation to get involved in my project, even when you've learned that working with EVs comes with more fails than successes.

"I live by one rule: No office romances, no way. Very messy, inappropriate… no. But, I live by another rule: Just do it… Nike." -MS Last but not least in our lab, I'd like to thank my colleague, friend and wife, Pamela, because without her I wouldn't be here. We started this adventure together, and I don't think I'd have been able to conclude it without her by my side. You have been my pillar of sanity, motivation, responsibility and support, I can't thank you enough. We were both students in the university courses, we both did our master's thesis in the same lab, and now we both work in the same lab, here. I couldn't have wished for a better story. I don't forget about the people that left the lab. First and foremost, Carlos, who was finishing his time in the lab as I was arriving, in the few months we were together at the lab we built a friendship that still lasts. You taught me many things about how everything in the lab worked, and even helped me experimentally. I hope to see you again, maybe soon in Spain… Manon, always kind and willing to show you exactly how to do things the correct way. Joanna, very knowledgeable and resourceful, thanks for the long discussions about topics I had little experience back then. I'd also like to thank Dr. Ludger Johannes and his team for the valuable input during meetings and other instances. In particular, I'd like to thank Ewan for his friendliness and for always being willing to discuss my project and never leaving without a really good suggestion or advice. Christian, who always offered good technical advice on topics where I needed help, and for all the fun cooking discussions we had. Massi and Estelle for their kindness. Valérie, who taught me all I needed to know once I arrived to the lab and with who despite our language barriers I can still laugh. Alison who left to start her own group but when she was here she never failed to make me laugh, even when times were not great. Debarpan, Akhil, Jon, Leishemba, Zhi-Qian and Justine for random talks and laughs in the hallways.

"And I knew exactly what to do, but in a much more real sense I had no idea what to do." -MS

Whenever I felt stuck in the project I'd meet with experts from different places so in this part I'd like to take the time to thank all the people that have helped me as advisors, contributors or collaborators.

In no particular order, I'd like to express my heartfelt appreciation to Dr. Graça Raposo for her valuable input throughout the four years guiding me towards a more nicely built work. Her expertise is only comparable to the enthusiasm and friendliness she showed me every time we met. Another key advisor was Dr. Etienne Morel, who also followed me throughout the past four years lending guidance towards filling the empty spots I had in my story. I have the luck of having him be part of my thesis jury. I'd like to thank Dr. Clotilde Théry an advisor and now member of the thesis defense jury, for her always pertinent comments and suggestions during our meetings, her advice was invaluable and highly appreciated. I'd like to extend my gratitude towards Dr. Guillaume van Niel, a key advisor that helped me shape the direction of my project and provided the tools to prove one of my hypothesis. In this same collaboration I met Dr. Frederik Verweij, who helped me put into practice tools he worked on with Dr. van Niel. Not only did he help me technically but we also had fruitful discussions about the results we were seeing while performing the experiment. And again, I'm lucky enough to have him be part of my thesis Jury. I'd like to thank Dr. Jacky Goetz and Dr. Vincent Hyenne, for their enthusiasm about my results and willingness to collaborate after my thesis, to further strengthen the story. I also want to extend my thanks to Dr. Fatima Mechta-Grigoriu and Dr. Rana Mhaidly for their motivation to get involved in my project and trying to establish the groundwork for future collaborations between our labs.

"The only time I set the bar low is for limbo. Always keep the bar raised no matter what." -MS

In the early stages I consulted regularly with Dr. Gregory Lavieu, his expertise in EV uptake helped me generate well-controlled, reproducible and robust results. His high standards for experimental designs and appropriate controls stuck with me afterwards and were a great lesson in this journey. I'd also like to thank Emeline who was always happy to help me in this same topic. I'd like to take the time to thank the people who indirectly help with our work. Mainly the laverie team, member of the various facilities of the Curie, and the HR and administrative department, specially Myriam, Yannick and Yasmina.

I would not be here if it wasn't for my scientific background. I express my appreciation to two major influences I had before the start of my PhD, and that shaped, and prepared me for the journey ahead. First, I'd like to thank Dr. Juan Valdes from my alma mater Universidad Andres Bello. I had the fortune to have you as the director of my career as well as the director of my master's thesis. You taught me all the basics of research as well as scientific communication, with much patience. So for this I thank you. And secondly, I want to thank Dr. Felipe Court, with whom I worked as a research assistant for a couple of years after my masters and before coming to France. Those were the years where I

THESIS SUMMARY

Extracellular vesicles (EVs) are lipid-enclosed vesicles that are released by all cells studied to date and present in all human bodily fluids. EVs contain genetic material and proteins that are able to be transferred to and generate an effect in other cells. Caveolin-1 (Cav1) is a key component of the small invagination of the plasma membrane called caveolae, where it functions as mechano-sensors and membrane tension buffering device. Cav1 facilitates the migration and invasion of tumor cells and its expression is often increased in the late stages of cancer. Interestingly, high levels of Cav1 have been found in EVs of patients with advanced cancer. Given the importance of mechanical forces in the microenvironment of cancer cells, we hypothesized that caveolae and/or Cav1 may represent key players in the regulation of EV biology and cancer progression under mechanical strain. To test this hypothesis, we subjected different cancer cell lines, having (WT) or deleted for Cav1 expression (KO) to 2D or 3D systems of mechanical stress. EVs were purified from these cells and analyzed by nanoparticle tracking analysis.

We found a striking increase in the release of EVs after mechanical stress both in 2D and 3D models. This increase was strictly dependent on the presence of Cav1 and correlated with enhanced fusion of multivesicular bodies to the plasma membrane, indicating that the population of EVs increased upon mechanical stress corresponding to exosomes. We observed that Cav1 was enriched in EVs after mechanical strain. The increase of EVs observed after mechanical stress was drastically reduced upon downregulation of the endosomal sorting complex ESCRT-0, further confirming their exosomal nature. Lipidomic analysis revealed differences in the lipid composition of EVs in Cav1 -/-cells and after mechanical stress, suggesting a difference in the properties of these EVs. Finally, EVs from mechanically stressed cells were shown to promote enhanced migration and invasion phenotype in triple negative breast cancer cells, whereas EVs from Cav1 depleted cells did not. These data allow us to conclude that mechanical stress is associated with increased release of EVs and the acquisition of metastatic traits in receiving cells in vitro, with Cav1 being a key player of this process. In parallel, we also studied the involvement of Cav1 in the uptake of EVs, a key process in the EV mediated cellular communication context. We used different protein markers to follow and visualize the uptake of EVs. EV uptake was strongly decreased in Cav1 -/-cells as compared to WT receiving cells. We also made the observation that Cav1 but not caveolae was required for this uptake of EVs by cells and that the uptake efficiency was correlated with the level of Cav1 expression in receiving cells. Overall, our work has revealed a new role for Cav1 in cell-to-cell communication and the propagation of metastatic phenotypes through the mechanical control of EV production and dynamics.

INTRODUCTION Chapter 1: Extracellular Vesicles

Extracellular vesicles (EVs) are small membranous particles present in all bodily fluids and released to the extracellular space by all cell types studied thus far. EVs can range in size from 30 to 1000 nanometers and are classified based on their biogenesis, size, and content (Théry et al., 2018). They are known to play important roles in intercellular communication, immune regulation, and disease pathogenesis [START_REF] Kugeratski | Exosomes as mediators of immune regulation and immunotherapy in cancer[END_REF].

Historical perspective

What could be considered as the beginning of the exploration of the biology of EVs is a series of observations in two articles published one by Erwin Chargaff in 1945 and another by Erwin Chargaff together with Randolph West in 1946, where they describe a very particulate sediment that is obtained after centrifugation at 31,000 g of blood samples with high clotting potential [START_REF] Chargaff | The Coagulation of Blood[END_REF][START_REF] Chargaff | The biological significance of the thromboplastic protein of blood[END_REF]. Two decades later, Wolf observed small particles in human plasma that were derived from platelets and proposed that they were involved in blood clotting, calling them "platelet dust" [START_REF] Wolf | The Nature and Significance of Platelet Products in Human Plasma[END_REF]. H. Clarke Anderson, on the other hand, identified small vesicles in the matrix of epiphyseal cartilage that were associated with calcification, which were called "matrix vesicles" [START_REF] Anderson | Vesicles associated with calcification in the matrix of epiphyseal cartilage[END_REF]. These early observations laid the foundation for further studies on the role of EVs in physiological and pathological processes. Later on, Aaronson and colleagues were able to visualize and isolate a heterogeneous population of extracellular particles from Ochromonas danica, a flagellated alga, using centrifugation; it is Aaronson who first used the term "extracellular vesicles'' [START_REF] Aaronson | Ultrastructure of intracellular and extracellular vesicles, membranes, and myelin figures produced by Ochromonas danica[END_REF]. The first insight about one of the biogenesis pathways of EVs comes from the work of Eladio Nunez and collaborators. They were the first to observe the presence of vesicle-containing endosomal structures in the bat thyroid gland that they called them multivesicular bodies (MVBs). They suggested that the fusion of the membrane of the MVBs with the plasma membrane of the cell leads to the release of the small vesicles that now we know as intraluminal vesicles (ILVs) [START_REF] Nunez | Secretory processes in follicular cells of the bat thyroid. III. The occurrence of extracellular vesicles and colloid droplets during arousal from hibernation[END_REF]. The 1980s were a prolific decade for EV research, Clifford Harding and colleagues used electron microscopy to study the process of transferrin receptor trafficking in reticulocytes and observed the release ILVs from MVBs fused with the plasma membrane [START_REF] Harding | Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes[END_REF]. In parallel, Bin-Tao Pan and Rose Johnstone also observed the release of small vesicles from sheep reticulocytes and proposed that these vesicles were involved in the shedding of membrane proteins (Pan & Johnstone, 1983). Later and for the first time, Johnstone coined the term "exosome" to specifically describe these small vesicles derived from the endocytic pathway [START_REF] Johnstone | Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes)[END_REF] (Figure 1).

Figure 1. Timeline of major milestones in EV research

From the very first observations made by E. Chargaff and R. West to the functional relevance breakthrough and the formation of national and international societies, this timeline depicts the growing interest in the EV field throughout time [START_REF] Couch | A brief history of nearly EV-erything -The rise and rise of extracellular vesicles[END_REF].

In 1996, EVs from immune cells were shown to contain various proteins, including MHC class II molecules, therefore demonstrating that EVs are capable of antigen presentation. This report was a ground-breaking moment, as it revealed that EVs have the potential to serve functional purposes in various biological processes, including in disease [START_REF] Raposo | B lymphocytes secrete antigenpresenting vesicles[END_REF]. Since then, the growing interest in EV research has led to an in-depth exploration of the composition and content of the vesicles as well as their physiological role.

Early studies showed that EVs carry a diverse range of proteins, including receptors, enzymes, and cytoskeletal proteins [START_REF] Théry | Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles[END_REF].

The presence of specific proteins in EVs can provide insights into the cellular origin of the vesicles and their potential functions. In addition to proteins, EVs carry a specific subset of lipids, including phosphatidylserine, sphingomyelin, and cholesterol [START_REF] Subra | Exosome lipidomics unravels lipid sorting at the level of multivesicular bodies[END_REF]. The lipid composition of EVs can also vary depending on the cell type and physiological or pathological state of the parental cell (Figure 2). For example, cancer-derived EVs have been shown to have a distinct lipid profile compared to EVs derived from normal cells [START_REF] Skotland | Lipids in exosomes: Current knowledge and the way forward[END_REF]. Interestingly, when comparing the lipid profile of small EVs to the lipid composition of the parental cells, there is a notable enrichment of certain lipid species, such as sphingolipids, phosphatidylserine and cholesterol in the EV population, on the other hand, phosphatidylcholine and phosphatidylinositol are found to be much more abundant in the total lipid composition of the parental cells compared to the EVs [START_REF] Skotland | Lipids in Extracellular Vesicles: What Can Be Learned about Membrane Structure and Function?[END_REF] (Figure 2).

Figure 2. Enrichment of lipid species in cells and EVs

Enrichment of lipid classes in cells or exosomes calculated as mol% of lipids in these samples. (LPE) lysoPE, (LPI) lysoPI, (CHOL) cholesterol. GM2, Gb3, GD1, GM1, GM3, LacCer, and HexCer are all glycosphingolipids (GSLs) [START_REF] Llorente | Molecular lipidomics of exosomes released by PC-3 prostate cancer cells[END_REF].

EVs were found to carry various types of nucleic acids with functional capabilities, including messenger RNA (mRNA), microRNA (miRNA), and long non-coding RNA (lncRNA) [START_REF] Ratajczak | Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: Evidence for horizontal transfer of mRNA and protein delivery[END_REF][START_REF] Skog | Glioblastoma microvesicles transport RNA and protein that promote tumor growth and provide diagnostic biomarkers[END_REF][START_REF] Valadi | Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells[END_REF] (Figure 3).

Figure 3. Overall composition and cargo of EVs

Schematic representation of the different components of EVs, including families of proteins, lipids, and nucleic acids. Among the proteins we can find tetraspanins, including CD63, CD81, Lamp1 and CD9. Typical species of lipids found in EVs include sphingomyelin, phosphatidylserine, cholesterol and ceramide, among others. And for nucleic acids, we can typically find miRNAs, mRNAs, and other noncoding RNAs (Colombo et al., 2014).

The presence of specific nucleic acids in EVs can provide insights into the gene expression profile of the parental cell and may have implications for disease diagnosis and monitoring. Overall, the specific cargo carried by EVs is highly dependent not only on the cell type that secretes the EVs but also on the physiological or pathological state of the parental cell.

EV subtypes and biogenesis

Based on their biogenesis, EVs can be classified into several types, including exomeres, exosomes, microvesicles, and apoptotic bodies (Figure 4).

Exomeres are the newest members of the EV family and the smallest type of EVs, with a diameter of less than 50 nm. They lack an external membrane structure yet are still able to carry lipids, proteins and nucleic acids. The biogenesis of exomeres has not been described to date and its hypothesized that exomeres are rather a heterogeneous group of complexes that are purified together because of their biophysical properties rather than a single type of entity [START_REF] Tosar | Exomeres and supermeres: Monolithic or diverse?[END_REF][START_REF] Zhang | Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation[END_REF].

Figure 4. Extracellular vesicle subtypes

Schematic representation of the classification of EVs according to their biogenesis and size. Depicted we can see exomeres, the newly discovered EV subtype, exosomes, that are released after the fusion of MVBs to the plasma membrane, and large EVs which are mainly released by shedding of the plasma membrane, which include microvesicles, ectosomes, microparticles, oncosomes and apoptotic bodies [START_REF] Cocozza | SnapShot: Extracellular Vesicles[END_REF].

Microvesicles are formed by the outward budding of the plasma membrane and are released into the extracellular space; they are larger than exosomes, with a diameter of 100-1000 nm [START_REF] Van Niel | Shedding light on the cell biology of extracellular vesicles[END_REF]. Given their heterogeneity, microvesicles can be formed by multiple mechanisms including the endosomal sorting complex required for transport (ESCRT) machinery, ceramide-dependent production, bleb scission, among others (Bianco et al., 2009;[START_REF] Sedgwick | Extracellular microvesicles and invadopodia mediate nonoverlapping modes of tumor cell invasion[END_REF][START_REF] Wang | Plasma membrane-derived extracellular microvesicles mediate non-canonical intercellular NOTCH signaling[END_REF].

Apoptotic bodies are large EVs with a diameter of over 1000 nm that are released by cells undergoing programmed cell death (apoptosis). They are formed by the fragmentation of the apoptotic cell and contain cellular debris, including organelles and nucleic acids [START_REF] Hristov | Apoptotic bodies from endothelial cells enhance the number and initiate the differentiation of human endothelial progenitor cells in vitro[END_REF].

Exosomes are another type of EVs with a diameter of roughly 50-150 nm that are formed by the inward budding of the endosomal membrane, resulting in the formation of MVBs. The MVBs can then fuse with the plasma membrane, releasing the ILVs into the extracellular space as exosomes (Théry et al., 2002). The biogenesis of MVBs can be divided into ESCRT-dependent and ESCRT-independent pathways, the former being the most extensively described. The ESCRT-dependent pathway is a complex multi-step process that begins with the recognition and recruitment of ubiquitinated cargo proteins by the ESCRT-0 complex, composed of a heterodimer of HRS and STAM subunits which contain ubiquitin-binding domains, to the early endosomal membrane [START_REF] Raiborg | The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins[END_REF]. In humans, there are two isoforms of STAM, STAM1 and STAM2. Downregulation of ESCRT-0, either by HRS or STAM depletion, shows impairment in the recruitment of other ESCRT members that results in defects in the endosomal membrane invagination, ILV formation, and ultimately impaired exosome secretion (Colombo et al., 2013;[START_REF] Wenzel | Concerted ESCRT and clathrin recruitment waves define the timing and morphology of intraluminal vesicle formation[END_REF]. Next, the ESCRT-I complex, which is a heterotetramer of TSG101, MVB12, VPS28, and VPS37, is recruited to the endosomal membrane by ESCRT-0 [START_REF] Hurley | Membrane budding and scission by the ESCRT machinery: it's all in the neck[END_REF].

ESCRT-I contributes to the clustering of ubiquitinated cargo to the early endosome and acts as a bridge between ESCRT-0 and ESCRT-II [START_REF] Katzmann | Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I[END_REF]. The ESCRT-I complex recruits the ESCRT-II complex, which is a heterotetramer of two VPS22 subunits, one VPS25, and one VPS36 subunit [START_REF] Carlton | Parallels between cytokinesis and retroviral budding: A role for the ESCRT machinery[END_REF]. The ESCRT-II complex also has ubiquitin binding domains but more importantly will stabilize the ESCRT-III complex involved in membrane scission [START_REF] Alam | Ubiquitin interactions of NZF zinc fingers[END_REF]. ESCRT-III is composed of CHMP2A/B, CHMP3, CHMP4A/B/C, and CHMP6. The main role of this complex is to constrict the inward budding membrane and recruit the ESCRTassociated AAA-ATPase VPS4 that catalyzes the disassembly of the ESCRT-III complex and drives membrane scission and ILV formation [START_REF] Hurley | Membrane budding and scission by the ESCRT machinery: it's all in the neck[END_REF][START_REF] Wollert | Membrane scission by the ESCRT-III complex[END_REF]. The ESCRT pathway has additional but mechanistically similar roles in membrane abscission during cytokinesis, viral budding, membrane repair, and in autophagy [START_REF] Carlton | Parallels between cytokinesis and retroviral budding: A role for the ESCRT machinery[END_REF][START_REF] Fujii | Functional role of Alix in HIV-1 replication[END_REF][START_REF] Jimenez | ESCRT machinery is required for plasma membrane repair[END_REF][START_REF] Rusten | How do ESCRT proteins control autophagy[END_REF].

Independently of ESCRT proteins, cells can generate MVBs and secrete

exosomes in different ways involving other protein species or lipids, although the composition, shape, and size may differ in many cases.

Several reports have shown that tetraspanins, which are protein scaffolds embedded in the membranes through four transmembrane domains, contribute to ILV cargo sorting. Cells depleted of key subunits of all ESCRT complexes were still able to form MVBs [START_REF] Stuffers | Multivesicular endosome biogenesis in the absence of ESCRTs[END_REF]. Furthermore, in melanocytes, it has been shown that PMEL, a protein essential for the formation of melanosome amyloid fibrils, is sorted into ILVs independently of the ESCRT machinery through a process that relies on cholesterol-and sphingolipid-rich microdomains in a CD63 dependent manner [START_REF] Van Niel | The tetraspanin CD63 regulates ESCRT-independent and -dependent endosomal sorting during melanogenesis[END_REF]. Tetraspanin-6 has been suggested to interact with syntenin, a PDZdomain-containing protein, and induce an ESCRT-independent formation of ILVs that leads to release of a specific subpopulation of exosomes containing amyloid precursor protein [START_REF] Guix | Tetraspanin 6: A pivotal protein of the multiple vesicular body determining exosome release and lysosomal degradation of amyloid precursor protein fragments[END_REF].

Ceramide, a lipid produced by hydrolysis of sphingomyelin by nSMase2 sphingomyelinase has been shown to play a role in exosome biogenesis, through the promotion of ceramide-rich microdomains with a spherical curvature of the endosomal membrane, leading to the inward budding of ILVs into the lumen of MVBs in oligodendroglial cells without the need for ESCRT function [START_REF] Trajkovic | Ceramide triggers budding of exosome vesicles into multivesicular endosomes[END_REF]. Moreover, the nSMase2-ceramide pathway promotes the secretion of exosomes with a specific packaging of miRNAs with a functional significance in tumor invasion [START_REF] Singh | Exosomemediated transfer of miR-10b promotes cell invasion in breast cancer[END_REF]. The detailed mechanism of how ceramide is able to induce the formation of ILVs and release of exosomes is not fully understood but it is proposed that ceramide can recruit tetraspanins that help in the budding of the plasma membrane or induce curvatures and scission of the MVB membrane [START_REF] Trajkovic | Ceramide triggers budding of exosome vesicles into multivesicular endosomes[END_REF] (Figure 5). The ESCRT accessory protein ALIX has been demonstrated to interact with syntenin and play a role in exosome biogenesis and exosomal sorting of syndecans, transmembrane proteoglycans thought to act as coreceptors, independently of ubiquitination [START_REF] Baietti | Syndecan-syntenin-ALIX regulates the biogenesis of exosomes[END_REF]. The membrane scaffolding protein flotillins have been shown to control specific exosomal cargo sorting, whereby activation of the small GTPase Rab31 leads to its binding to flotillins and epidermal growth factor receptor (EGFR) sorting into ILVs via lipid microdomains [START_REF] Wei | RAB31 marks and controls an ESCRT-independent exosome pathway[END_REF]. Caveolin-1 (Cav1), a key protein in the formation of the characteristic small invaginations, called caveolae, in the plasma membrane has been described to be sorted into ILVs and released in exosomes (Hayer et al., 2010). Ubiquitinated Cav1 regulates sorting of extracellular matrix (ECM) cargoes into exosomes such as tenascin-C and fibronectin [START_REF] Albacete-Albacete | ECM deposition is driven by caveolin-1-dependent regulation of exosomal biogenesis and cargo sorting[END_REF]. Additionally, Cav1 can regulate the specific loading of miRNAs into ILVs through its interaction with the RNA binding protein hnRNPK [START_REF] Robinson | Caveolin-1-driven membrane remodelling regulates hnRNPKmediated exosomal microRNA sorting in cancer[END_REF]. The intricate relationship between Cav1 and EVs as well as their physiological implications of Cav1 bearing EVs will be further explored in detail below.

EVs in pathology, focus on cancer

EVs have gained significant attention in recent years due to their involvement in various physiological and pathological processes, including the development and progression of neurologic, cardiovascular diseases, inflammation, and cancer [START_REF] Yáñez-Mó | Biological properties of extracellular vesicles and their physiological functions[END_REF] (Figure 6).

Figure 6. Schematic representation of different roles for circulating EVs in diseases

Circulating EVs have the potential to facilitate intercellular communication by transferring bioactive molecules like proteins and RNA. This process can have significant impacts on various systemic processes, including immune function, inflammation, and disease-and organ-specific functions [START_REF] Shah | Circulating Extracellular Vesicles in Human Disease[END_REF].

EVs have been found to be involved in the pathogenesis of various neurological disorders, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. EVs can carry misfolded proteins, such as betaamyloid and alpha-synuclein, which are involved in Alzheimer's and Parkinson's diseases, respectively [START_REF] Dinkins | Exosome reduction in vivo is associated with lower amyloid plaque load in the 5XFAD mouse model of Alzheimer's disease[END_REF][START_REF] Herman | CSF-derived extracellular vesicles from patients with Parkinson's disease induce symptoms and pathology[END_REF].

In addition, EVs can modulate neuroinflammatory responses by carrying proinflammatory molecules, such as interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNFα) [START_REF] Spiers | Neuroinflammatory Modulation of Extracellular Vesicle Biogenesis and Cargo Loading[END_REF].

The role of EVs in cardiovascular diseases, such as atherosclerosis, myocardial infarction, and heart failure, has been extensively studied. EVs derived from endothelial cells, platelets, and immune cells can modulate vascular inflammation and thrombosis, contributing to the progression of cardiovascular diseases [START_REF] Oggero | The Contrasting Role of Extracellular Vesicles in Vascular Inflammation and Tissue Repair[END_REF][START_REF] Zarà | Biology and Role of Extracellular Vesicles (EVs) in the Pathogenesis of Thrombosis[END_REF].

Extracellular vesicles are actively involved in promoting and regulating inflammatory processes in various conditions, including rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease. EVs can transfer bioactive molecules, such as cytokines, chemokines, and microRNAs, between cells, thereby modulating immune responses and contributing to the perpetuation of chronic inflammatory diseases [START_REF] Buzas | Emerging role of extracellular vesicles in inflammatory diseases[END_REF]. For example, EVs can induce the differentiation of monocytes into proinflammatory macrophages, promoting the production of pro-inflammatory cytokines and the destruction of tissues [START_REF] Vasina | Microparticles from apoptotic platelets promote resident macrophage differentiation[END_REF].

In the context of cancer, EVs have been implicated in multiple aspects of tumor progression and metastasis, such as promoting cell proliferation, invasion, angiogenesis, and immune evasion. Interestingly, tumor cells have been shown to release an increased amount of EVs compared to healthy cells [START_REF] Silva | Analysis of exosome release and its prognostic value in human colorectal cancer[END_REF][START_REF] Vasconcelos | Extracellular vesicles as a novel source of biomarkers in liquid biopsies for monitoring cancer progression and drug resistance[END_REF]) (Figure 7). Cancer progression is often characterized by alterations in the ECM, which can be modulated by EVs. EVs can enhance directional cell movement within the ECM by interacting with components like fibronectin. These components bind to integrins found on the surface of EVs, creating a substrate that facilitates cell-EV interactions, encourages cell attachment and increases cell movement speed (Sung et al., 2015). In rat pancreatic adenocarcinoma, it was demonstrated that CD151 and tetraspanin-8, two tetraspanins found in EVs, contribute to ECM degradation, driving cells to a motile phenotype [START_REF] Yue | The tetraspanins CD151 and Tspan8 are essential exosome components for the crosstalk between cancer initiating cells and their surrounding[END_REF]. Matrix metalloproteinases (MMPs) are membrane-associated proteins that are secreted and are required for ECM remodeling. MMPs have been observed to be associated to EVs, specifically in the case of skin cancer cells that are able to promote invasion by secreting exosomes containing MMP-MT1 and MMP2 that mediate invadopodia biogenesis, stability and activity in matrix degradation [START_REF] Hoshino | Exosome secretion is enhanced by invadopodia and drives invasive behavior[END_REF].

Tumor-derived EVs can travel to distant secondary sites to play a crucial role in initiating pre-metastatic niche formation, the process of preparing a microenvironment in specific organs for the colonization of circulating tumor cells (Figure 7). The pre-metastatic niche remodeling is mediated by EVs by establishing a supportive and receptive niche that promotes tumor cell colonization and metastasis. EVs derived from melanoma cells can instruct bone marrow progenitors to adopt a pro-metastatic by reprogramming them to exhibit a pro-vasculogenic phenotype and inducing increased vascular permeability at pre-metastatic locations (Peinado et al., 2012). Pancreatic ductal adenocarcinoma-derived EVs are uptaken by Kupffer cells, which leads to the secretion of transforming growth factor β (TGF-β) and increased fibronectin production by hepatic stellate cells, creating a fibrotic microenvironment [START_REF] Costa-Silva | Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver[END_REF]. In a breast cancer model, miR-105 is specifically expressed and secreted by highly metastatic cells and can be transferred to endothelial cells via exosomes. Overexpressed miR-105 in tumors is transferred to endothelial cells where it destabilizes the tight junctions and alters the integrity of endothelial barriers, thereby facilitating distant metastases in the lung and brain [START_REF] Zhou | Article Vascular Endothelial Barriers to Promote Metastasis[END_REF]. EVs, specifically microvesicles, released by CD105+ cancer stem cells trigger angiogenesis and promote lung premetastatic niche formation on normal human endothelial cells, resulting in a significant increase in lung metastasis [START_REF] Grange | Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche[END_REF]. Ral GTPases A and B in breast cancer cells are responsible for the organotropic targeting of pro-metastatic EVs towards lungs, where the presence of the adhesion molecules MCAM/CD146 in EVs where found to be crucial for this tropism [START_REF] Ghoroghi | Ral GTPases promote breast cancer metastasis by controlling biogenesis and organ targeting of exosomes[END_REF]. Furthermore, integrin patterns in EVs from lung-tropic models were distinct; integrins α6β4 and α6β1 were associated with lung metastasis, whereas integrin αvβ5 was linked to liver metastasis (Hoshino et al., 2015).

EVs can also serve as means of communication between both cancerous and non-cancerous cells, such as epithelial cells, cancer-associated fibroblasts (CAFs), endothelial cells, and immune cells, where they can play a crucial role in reprogramming cells and tissues to promote cancer progression. In melanoma cells, exosomes containing the immune checkpoint inhibitor PD-L1 have been found to contribute to immunosuppression by inhibiting the function of immune T cells (Chen et al., 2018). In another study, exosomes from prostate cancer cells were shown to induce an adenosine-mediated immunosuppressive effect, which led to the suppression of dendritic cell function by inhibiting TNFαand IL-12 production through the induction of the expression of CD73 [START_REF] Salimu | Dominant immunosuppression of dendritic cell function by prostatecancer-derived exosomes[END_REF]. Tumor-secreted EVs have been described to promote the activation of CAFs in in vivo experiments where the population of fibroblasts expressing CD140a, an activated fibroblast marker, were increased after EV injection in a miRNA-125b dependent manner [START_REF] Vu | Tumor-secreted extracellular vesicles promote the activation of cancer-associated fibroblasts via the transfer of microRNA-125b[END_REF]. Similarly, it has been shown that exosomes from various types of cancer cells can trigger the differentiation of fibroblasts into myofibroblasts [START_REF] Webber | Cancer exosomes trigger fibroblast to myofibroblast differentiation[END_REF]. This transformation is known to further contribute to the formation of an environment that supports tumor growth and metastasis by increasing contractile activity, vascularization and angiogenesis [START_REF] Hinz | Alpha-smooth muscle actin expression upregulates fibroblast contractile activity[END_REF][START_REF] Orimo | Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion[END_REF].

Interestingly, the caveolae protein Cav1 is significantly abundant in tumorderived EVs and has been proposed to serve as a biomarker for cancer progression (Campos et al., 2019;Logozzi et al., 2009;[START_REF] Tahir | Preoperative serum caveolin-1 as a prognostic marker for recurrence in a radical prostatectomy cohort[END_REF].

The presence of Cav1 in tumor-derived extracellular vesicles not only serves as a potential biomarker for the progression of various cancers but also promotes malignancy. Cav1 was found to be highly expressed in various melanoma cell lines and it was observed that Cav1 silencing led to reduced tumor growth and angiogenesis [START_REF] Felicetti | Caveolin-1 tumor-promoting role in human melanoma[END_REF]. Cav1 bearing EVs were shown to stimulate in vitro anchorage independence, migration, and invasion through paracrine and autocrine mechanisms. Notably, they are capable of transferring metastatic properties from the donor melanoma cell to the less aggressive recipient cell line. In another study, the proteome of the EVs from various melanoma cell lines revealed that their specific protein compositions are dependent on the aggressive nature of the parental cells, with Cav1 being one of the varying proteins [START_REF] Lazar | Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines[END_REF]. In metastatic breast cancer, cells were observed to release Cav1-loaded EVs, which in turn, upon uptake by non-metastatic receiving cells, prompted the acquisition of pro-metastatic traits. Such traits included a marked increase in invasion and migration capabilities, in a Cav1 dependent manner. This effect was suggested to be conferred, at least partially, by the presence of specific cell adhesion-related proteins unique to EVs released from Cav1 expressing cells (Campos et al., 2018). Indeed, Cav1 was found to be required for exosomal sorting of specific ECM proteins such as tenascin-c, promoting local tumor microenvironment remodeling for enhanced tumor invasion and the generation of distant ECM-enriched stromal niches in vivo [START_REF] Albacete-Albacete | ECM deposition is driven by caveolin-1-dependent regulation of exosomal biogenesis and cargo sorting[END_REF].

In summary, the identification of the caveolar protein Cav1 within the protein composition of EVs originating from cancer cells and its role in various cancer models implicates it as a significant factor involved in cancer progression.

Further study of its role in EV dynamics can therefore offer valuable insights into the mechanisms of cancer progression. This is the main topic of the present work.

Chapter 2: Caveolae

Structure and formation

Caveolae were first observed in 1953 in endothelial cells and two years later the term caveola intracellularis or "intracellular cave" was suggested [START_REF] Palade | Fine structure of blood[END_REF][START_REF] Yamada | The fine structure of the gall bladder epithelium of the mouse[END_REF]. Caveolae are specialized, cup-shaped invaginations of the plasma membrane with a diameter of 60-80 nm that are present in most cell types with a few exceptions such as red blood cells, platelets, immune cells, and some neuronal cell types. Caveolae research traversed from the early years of electron microscopy to the discovery of the essential protein components [START_REF] Echarri | Caveolae[END_REF]. The structural and functional integrity of caveolae is attributed to two groups of proteins known as caveolins and cavins. The caveolin family of proteins is composed of the homologues Cav1, caveolin-2 (Cav2), and the muscle specific caveolin-3 (Cav3) [START_REF] Kurzchalia | VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles[END_REF][START_REF] Rothberg | Caveolin, a protein component of caveolae membrane coats[END_REF][START_REF] Scherer | Identification, sequence, and expression of caveolin-2 defines a caveolin gene family[END_REF][START_REF] Tang | Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle[END_REF][START_REF] Way | M-caveolin, a muscle-specific caveolinrelated protein[END_REF]. The cavin protein family is composed of four members, cavin-1, cavin-2, cavin-3 and the muscle-specific cavin-4 (Kovtun et al., 2015).

Particularly, Cav1 together with cavin-1 (initially called polymerase I and transcript-release factor, PTRF) in non-muscle cells and Cav3 together with cavin-4 in muscle cells are strictly required for caveolae formation. The interaction between caveolins and cavins is crucial because the absence of either protein leads to a loss in the formation of caveolae (Drab et al., 2001;Hill et al., 2008) (Figure 8). Cav1 is a 21 kDa protein that has distinct domains, in the N-terminal region (residues 1-101) there is the signature motif (SM), an oligomerization domain (OM), a pin motif (PM) and a scaffolding domain (SD), in the central region (residues 102-134) there is the intramembrane domain (IMD), and in the Cterminal region (residues 135-178) there are three palmitoylation sites.

Together, these regions control Cav1 trafficking and are overall key elements in the biogenesis and functions of caveolae [START_REF] Machleidt | Multiple domains in caveolin-1 control its intracellular traffic[END_REF]Ohi & Kenworthy, 2022;[START_REF] Porta | Molecular architecture of the human caveolin-1 complex[END_REF]. Recent cryo-electron microscopy data revealed that the Cav1 complex is preferably composed of 11 protomers forming a flat disk stabilized by numerous interactions between the protomers that are embedded in the plasma membrane. Furthermore, structural analysis revealed that a single caveola is composed of 12 faces, each formed by one Cav1 8S disc complex, meaning that in total, a single caveolae contains roughly 132 copies of Cav1 protomers [START_REF] Porta | Molecular architecture of the human caveolin-1 complex[END_REF] (Figure 9). Importantly, phosphorylation and ubiquitination sites can be found on the cytoplasmic side of Cav1 (Hayer et al., 2010a;[START_REF] Schlegel | Caveolin-1 Binding to Endoplasmic Reticulum Membranes and Entry into the Regulated Secretory Pathway Are Regulated by[END_REF]. For the formation of caveolae, first, it is necessary for Cav1 to oligomerize into what are called 8S complexes; this first step occurs at endoplasmic reticulum exit sites shortly after synthesis (Hayer et al., 2010b). The formation of the 8S complexes is dependent on the oligomerization domain, scaffolding domain and signature motif (Hayer et al., 2010b;[START_REF] Porta | Molecular architecture of the human caveolin-1 complex[END_REF]. Later, Cav1 is transported from the Golgi apparatus to the plasma membrane in caveolinenriched vesicles, where different species of lipids, including cholesterol play a crucial role in the oligomerization of Cav1 (Hayer et al., 2010b;Pol et al., 2005). At the plasma membrane, Cav1 is known to recruit specific species of lipids such as sphingolipids and phospholipids, therefore, generating particular lipid nanodomains within the plasma membrane (Krishna & Sengupta, 2019;Prakash et al., 2021;Sonnino & Prinetti, 2009). Once Cav1 oligomers reach the plasma membrane, cavins are recruited to form an outer filamentous coat that assists in the transition from a Cav1 oligomer flat disk to a curved structure characteristic of caveolae (Hill et al., 2008;[START_REF] Ludwig | Architecture of the caveolar coat complex[END_REF]. All four cavin proteins share sequence homology and possess conserved secondary helical structure elements (HR1 and HR2), which have been found to be important in the recruitment of phosphatidylserine and PIP2 and subsequent caveolae formation [START_REF] Burgener | Purification and characterization of a major phosphatidylserine-binding phosphoprotein from human platelets[END_REF]Hill et al., 2008;[START_REF] Kovtun | Structural insights into the organization of the cavin membrane coat complex[END_REF]. Similarly, to Cav1, cavins form oligomers composed of 3 cavin subunits that will further polymerize to form the caveolar coat [START_REF] Gambin | Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae[END_REF]) (Figure 10). In conjunction with these two primary components of caveolae, there exists other accessory proteins working synergistically, including the GTPase dynamin2 which associates with caveolae to stabilize it at the plasma membrane, the caveolar "neck" protein EHD2 whose role is mainly attributed to the stabilization of caveolae through their interaction with the actin cytoskeleton, pacsin2/syndapin II, and pacsin3/syndapin III (Lamaze et al., 2017;[START_REF] Larsson | Dynamin2 functions as an accessory protein to reduce the rate of caveola internalization[END_REF][START_REF] Seemann | Deciphering caveolar functions by syndapin III KOmediated impairment of caveolar invagination[END_REF][START_REF] Senju | Essential role of PACSIN2/syndapin-II in caveolae membrane sculpting[END_REF][START_REF] Stoeber | Oligomers of the ATPase EHD2 confine caveolae to the plasma membrane through association with actin[END_REF].

Caveolae have been shown to have distinct and multifunctional roles in various cellular processes including endocytosis, cholesterol transport, signal transduction, and mechanosensing, among others [START_REF] Cheng | Caveolae: One Function or Many?[END_REF].

Additionally, they have been shown to play roles in the pathogenesis of several diseases, such as cancer, heart disease, and neurological disorders. These roles will be reviewed in the following paragraphs.

Caveolae and lipid regulation

Caveolae play a pivotal role in regulating lipid homeostasis. These distinct regions of the plasma membrane are rich in cholesterol and sphingolipids and compartmentalize cellular processes by serving as organizing centers for the assembly of signaling molecules [START_REF] Thomas | Caveolae structure and function: Caveolae Review Series[END_REF].

Cav1, 2, and 3 have a binding domain for cholesterol, suggesting an intricate involvement in cholesterol dynamics. As already mentioned, the association of caveolin proteins with cholesterol is critical for the formation and stability of caveolae structures (Pol et al., 2005). Moreover, Cav1 is an important regulator of cholesterol levels within various subcellular compartments. For example, in fibroblasts taken from mice lacking Cav1, the build-up of cholesterol within mitochondria is linked to impaired functioning of this organelle [START_REF] Graf | The Class B , Type I Scavenger Receptor Promotes the Selective Uptake of High Density Lipoprotein Cholesterol Ethers into Caveolae *[END_REF]. In humans, a stop codon mutation in Cav1, that leads to the absence of Cav1 expression and no caveolae, leads to severe depletion of adipose tissue accompanied with insulin resistance and dyslipidemia [START_REF] Kim | Association of a homozygous nonsense caveolin-1 mutation with Berardinelli-Seip congenital lipodystrophy[END_REF].

Caveolae and caveolin proteins are also directly involved in lipid transport and storage. For example, Cav1 has been shown to bind fatty acids and is suggested to be part of the machinery for intracellular transport of lipids, especially long-chain fatty acids (Pohl et al., 2002;[START_REF] Trigatti | Identification of caveolin-1 as a fatty acid binding protein[END_REF]. The expression of Cav1 in human embryonic kidney (HEK) cells was found to aid in the absorption of fatty acids by cells, with cholesterol playing a role as well [START_REF] Meshulam | Role of caveolin-1 and cholesterol in transmembrane fatty acid movement[END_REF]. In adipocytes, where caveolae are abundantly found, they are essential sites for the regulation of lipolysis, a process whereby triglycerides stored inside fat cells are broken down to produce glycerol and free fatty acids [START_REF] Cohen | Lipid Droplet Formation[END_REF]. In adipocytes Cav1 is required to maintain the cholesterol content of lipid droplets, conversely, cholesterol induces the translocation of caveolins to lipid droplets where they bind together. Moreover, adipocytes from Cav1 knockout mice show decreased levels of free cholesterol, modified lipid and protein composition in lipid droplets [START_REF] Blouin | Lipid droplet analysis in caveolin-deficient adipocytes: alterations in surface phospholipid composition and maturation defects[END_REF][START_REF] Lay | Cholesterolinduced caveolin targeting to lipid droplets in adipocytes: A role for caveolar endocytosis[END_REF]. Cavin1 deficiency has also been related to lipid-related defects. Cavin1 depleted mice show increased levels of insulin and triglycerides in their bloodstream, have significantly reduced amounts of fat tissue, along with a lower tolerance for glucose, therefore displaying a clear lipodystrophic phenotype [START_REF] Liu | Deletion of Cavin/PTRF causes global loss of caveolae, dyslipidemia and glucose intolerance[END_REF].

Recent evidence has revealed the nanoscale lipid profile of caveolae. Using de novo assembly of caveolae on a caveolae deficient cellular model it was observed that Cav1 alone is able to cluster phosphatidylserine, PIP3, and cholesterol, cavin1 alone recruits mostly PIP2, PIP3, and phosphatidic acid, revealing that Cav1 and cavin1 have the ability to cluster different lipids at the plasma membrane. Finally, the expression of both proteins led to a unique lipid profile composed mainly of PIP2, phosphatidylserine and cholesterol [START_REF] Zhou | Caveolin-1 and cavin1 act synergistically to generate a unique lipid environment in caveolae[END_REF] (Figure 11). (Sonnino & Prinetti, 2009). B. Schematic comparison of the lipid profiles associated with Cav1 (left), cavin1 (middle), or caveolae (right). Colors of square boxes indicate lipid species; the size of the box indicates the level of co-clustering with the indicated proteins (faded boxes indicate statistically insignificant association) [START_REF] Zhou | Caveolin-1 and cavin1 act synergistically to generate a unique lipid environment in caveolae[END_REF].

Given the crucial roles of caveolae in regulating lipid metabolism and trafficking, alterations in caveolae structure or function can have profound effects on cellular lipid homeostasis and contribute thereby to several pathophysiological conditions like lipid disorders, cardiovascular disease, and cancer.

Caveolae in cell trafficking

Initially, it was believed that caveolae functioned as carriers for endocytosis.

This assumption was based on their prominent presence in the plasma membrane and their morphological resemblance to clathrin-coated pits. Some cargoes that have been characterized to be endocytosed by caveolae include simian virus 40, Shiga and cholera toxins and albumin. These cargoes however can be endocytosed by alternative means [START_REF] Ghitescu | Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: receptor-mediated transcytosis[END_REF][START_REF] Norkin | The caveolae-mediated sv40 entry pathway bypasses the golgi complex en route to the endoplasmic reticulum[END_REF][START_REF] Richards | Inhibitors of COP-mediated transport and cholera toxin action inhibit simian virus 40 infection[END_REF][START_REF] Schubert | Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo[END_REF]. Once caveolae undergo endocytosis, they can be observed colocalized with early endosomes and other endosomal structures [START_REF] Shvets | Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids[END_REF]. Recently, researchers in the field have come to a consensus that the primary function of caveolae is not related to endocytosis [START_REF] Parton | Caveolae: The FAQs[END_REF].

Interestingly, increasing evidence related EV uptake to Cav1-mediated endocytosis. Exosomes derived from Epstein-Barr virus infected cells were found to be internalized by epithelial acceptor cells in a Cav1 dependent pathway, that lead to the colocalization of the EVs with Rab5 and Rab7 positive endosomes [START_REF] Nanbo | Exosomes derived from Epstein-Barr virus-infected cells are internalized via caveola-dependent endocytosis and promote phenotypic modulation in target cells[END_REF]. Pharmacological inhibition of Cav1 with nystatin or filipin sterol binding agents, has been shown to lead to a reduction in EV uptake in lung cancer and pancreatic cells [START_REF] Javeed | Pancreatic Cancer-Derived Exosomes Cause Paraneoplastic β-cell Dysfunction[END_REF][START_REF] Wei | Exosomes derived from gemcitabine-resistant cells transfer malignant phenotypic traits via delivery of miRNA-222-3p[END_REF]. Contrary to this, there is also evidence showing that Cav1 is a negative regulator of exosome uptake, where knockout of Cav1 in fibroblasts induces an increased uptake of EVs [START_REF] Svensson | Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid raft-mediated endocytosis negatively regulated by caveolin-1[END_REF]). It appears that the role of Cav1 in the uptake of EVs is still under debate.

Further research is needed to fully understand the mechanisms and factors that determine the involvement of Cav1 in EV uptake.

Caveolae as mechanosensing and mechanotransducing domains

Cells have to face different stresses in both normal and abnormal conditions, such as changes in volume, stretching, compression or shear stresses, which have the potential to increase tension at the plasma membrane. A lipid bilayer is incapable of supporting major increases in membrane area, however, considerable stretching in cell length or significant swelling are frequently observed and reported without compromise to cell viability. Therefore, a rapid underlying mechanosensitive mechanism regulating membrane tension must be in place.

The first suggestions that caveolae could be mechanosensitive were postulated in 1975, where caveolae from frog skeletal muscle fibers were observed to "open" after stretching, an effect also documented in stretched smooth muscle from the sea slug Aplysia californica, this suggested that caveolae could serve as "safety valves" that would open and flatten to protect the plasma membrane from rupturing [START_REF] Dulhunty | The relative contributions of the folds and caveolae to the surface membrane of frog skeletal muscle fibres at different sarcomere lengths[END_REF][START_REF] Prescott | The sarcolemma of Aplysia smooth muscle in freeze-fracture preparations[END_REF]). Nevertheless, this potential role of caveolae was not pursued at the time given that further research generated contradictory results, showing that caveolae were rather semi-permanent structures in the plasma membrane, with no differences in quantity between mechanical stress and resting conditions [START_REF] Gabella | Effect of stretch and contraction on caveolae of smooth muscle cells[END_REF][START_REF] Poulos | Ultrarapid freezing reveals that skeletal muscle caveolae are semipermanent structures[END_REF].

It was not until 2011, where it was established for the first time the role of caveolae as mechanosensors that flatten upon increase of PM tension, and thereby buffer membrane tension increase. This rapid flattening of caveolae happens in an ATP and actin-independent manner. Furthermore, the interaction between Cav1 and cavin is lost after mechanical stress. On the other hand, when stress is relieved, caveolae undergo a complete reassembly process that is dependent on actin and ATP (Sinha et al., 2011) (Figure 12). The mechano-protective role of caveolae has subsequently been observed in vivo. Loss of cavin-1 in zebrafish revealed a severe disorganization of the cellular membrane that protects the skeletal muscle, notochord lesions and severe locomotor defects. Furthermore, strenuous muscle activity led to sarcolemmal damage and notochord cell collapse in the absence of caveolae [START_REF] Garcia | Sheath Cell Invasion and Trans-differentiation Repair Mechanical Damage Caused by Loss of Caveolae in the Zebrafish Notochord[END_REF][START_REF] Lim | Caveolae Protect Notochord Cells against Catastrophic Mechanical Failure during Development[END_REF][START_REF] Lo | The caveolin-cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle[END_REF].

Recent evidence has proven that indeed this flattening of caveolae can regulate intracellular signaling through its components (Torrino et al., 2018) (Figure 13). Myotubes derived from patients with mutations in the CAV3 gene demonstrate a notable reduction in the quantity of caveolae at the plasma membrane. As a consequence, these mutant myotubes exhibit an abnormal reaction to mechanical stress, as they are unable to effectively mitigate the heightened membrane tension caused by such stress. This deficiency subsequently triggers persistent over activation of the IL6/STAT3 signaling pathway. Reconstituting functional caveolae through the reexpression of Cav3 reversed these defects (Dewulf et al., 2019). Upon caveolae flattening, Cav3 can also interact with BRCA1, a protein that help repair damaged DNA, regulating its expression levels, subcellular location, and function, there playing a key role in the cellular protection against stress induced DNA damage [START_REF] Mcmahon | Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response[END_REF]. On the other hand, Cav1 has been shown to interact with various signaling molecules, such as the endothelial nitric oxide synthase (eNOS), EGF, and TFG-β receptors among others [START_REF] Bucci | In vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation[END_REF][START_REF] Engelman | Genes encoding human caveolin-1 and -2 are co-localized to the D7S522 locus (7q31.1), a known fragile site (FRA7G) that is frequently deleted in human cancers[END_REF][START_REF] Strippoli | Transition and Fibrosis During Peritoneal Dialysis[END_REF]. In a study in mice, the effect of blood flow-induced shear stress was evaluated, when comparing WT with Cav1 -/-mice. It was found that the absence of Cav1 lead to an impaired shear stress regulation of vessel diameter both in vivo and in vitro. Furthermore, they found that Cav1 is necessary for flow-induced eNOS activation. However, reconstitution of Cav1 in the endothelium of the Cav1 -/-mice rescued this impairment [START_REF] Yu | Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels[END_REF].

In smooth muscle cells, it was shown that mechanical strain induced the colocalization of PI3-K with Cav1 at focal adhesion sites. In addition, the presence of Cav1 is necessary for integrin-mediated PI3-K/Akt activation during cyclic stretch of cells in vitro and in a model of elevated tensile force in vivo. On the contrary, this activation of Akt was not present in Cav1 knockout mice [START_REF] Sedding | Caveolin-1 facilitates mechanosensitive protein kinase B (Akt) signaling in vitro and in vivo[END_REF].

The disassembly of caveolae has been shown to play a critical role in mechanoprotection, as evidenced by the abnormal response to mechanical stress observed in cells with absent or reduced caveolae quantity.

Understanding the mechanosensing and mechanotransducing roles of caveolae and Cav1 may have implications for developing therapies targeting disorders and conditions associated with altered response to mechanical stress.

Caveolae in disease

Caveolae related diseases are inherited diseases that arise from mutations in the caveolin genes, primarily CAV1 and CAV3. Gene mutations or mislocalization or deficiency of the encoded proteins, lead to a broad range of disorders affecting multiple organs and systems including lipodystrophy, musculopathies and cancer [START_REF] Yin | Caveolin proteins: a molecular insight into disease[END_REF].

Lipodystrophy is a rare and heterogeneous genetic disorder that is characterized by a loss of adipose tissue or its abnormal accumulation.

Several clinical studies have identified lipodystrophy-associated mutations in the CAV1 gene. For example, a mutation in patients that results in a heterozygous frameshift mutation in CAV1 has been associated with partial atypical lipodystrophy and hypertriglyceridemia [START_REF] Cao | Heterozygous CAV1 frameshift mutations (MIM 601047) in patients with atypical partial lipodystrophy and hypertriglyceridemia[END_REF]. A stop codon mutation in CAV1 in human patients leads to depletion of adipose tissue, hypercholesterolemia, insulin resistance, dyslipidemia, and hypercalcemia [START_REF] Kim | Association of a homozygous nonsense caveolin-1 mutation with Berardinelli-Seip congenital lipodystrophy[END_REF]. Similarly, loss of Cavin1 in mice induces elevated levels of triglycerides in the bloodstream, a significant decrease in adipose tissue mass, impaired glucose tolerance, and high levels of insulin in the blood [START_REF] Liu | Deletion of Cavin/PTRF causes global loss of caveolae, dyslipidemia and glucose intolerance[END_REF].

Several studies have reported an association between caveolar dysfunction and musculopathies [START_REF] Gazzerro | Caveolinopathies: from the biology of caveolin-3 to human diseases[END_REF]. A loss of Cav3 in mice induces an exclusion of the dystrophin-glycoprotein complex, a critical regulator of muscle function, from lipid raft domains, and the development of T-tubules mainly in a longitudinal orientation, which is typical of immature muscle cells [START_REF] Galbiati | Transgenic overexpression of caveolin-3 in skeletal muscle fibers induces a Duchenne-like muscular dystrophy phenotype[END_REF]. The numerous Cav3 mutations lead to the expression of non-functional Cav3 protein, unable to oligomerize and form caveolae in striated muscle cells. Thus, patients bearing these mutations suffer from a broad spectrum of muscle dystrophies including limb-girdle muscular dystrophy (LGMD), hyperckemia (HCK) and rippling muscle disease (RMD) [START_REF] Minetti | Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy[END_REF]. On the other hand, overexpression of Cav3 was shown to induce a phenotype similar to Duchenne muscular dystrophy (DMD). It induces a significant increase in the abundance of caveolae in skeletal muscle, hypertrophic, immature, and necrotic muscle fibers, and a decrease in the expression of skeletal muscle β-dystroglycan, an important protein involved in muscle stability [START_REF] Pradhan | A Role for Caveolin-3 in the Pathogenesis of Muscular Dystrophies[END_REF].

The role of caveolae in cancer has remained a controversial topic for decades, with the function of Cav1 being the most studied to date in this complex pathology. Cav1 has been described both as a tumor suppressor gene as well as an oncogene. This contradictory role has been suggested to be dependent on the type and stage of cancer [START_REF] Williams | Caveolin-1 in oncogenic transformation, cancer, and metastasis[END_REF] (Figure 14).

Figure 14. The dual role of caveolae in cancer

In breast cancer, Cav1 has been described with a dual role. On one hand, Cav1 acts as a tumor suppressor by inhibiting breast cancer cell proliferation, autophagy, invasion, and migration while promoting apoptosis (Left). On the other hand, Cav1 can also act as a tumor promoter in breast cancer by stimulating breast cancer cell proliferation, autophagy, invasion, migration, and metastasis, while inhibiting apoptosis and anoikis (Right) [START_REF] Qian | Caveolin-1: a multifaceted driver of breast cancer progression and its application in clinical treatment[END_REF].

Several studies have demonstrated the tumor suppressor role of Cav1. The genes encoding for Cav1 and Cav2 are localized in a locus that is normally deleted in various human cancers, including but not restricted to squamous cell carcinomas of the head and neck, prostate cancers, renal cell carcinomas, ovarian adenocarcinomas, colon carcinomas, and breast cancers, suggesting that the genes contained in this area, including the caveolins isoforms are important for tumor suppression [START_REF] Engelman | Genes encoding human caveolin-1 and -2 are co-localized to the D7S522 locus (7q31.1), a known fragile site (FRA7G) that is frequently deleted in human cancers[END_REF]. The presence of a specific mutation in the transmembrane domain of Cav1, where proline 132 is changed to leucine (P132L), has adverse consequences on the formation of caveolae within the body [START_REF] Han | Structural analysis of the P132L disease mutation in caveolin-1 reveals its role in the assembly of oligomeric complexes[END_REF].

This mutation has been associated with various disease conditions, with a particular emphasis on aggressive forms of breast cancer, where 9-16% of patient cases present this mutation [START_REF] Bonuccelli | Caveolin-1 (P132L), a common breast cancer mutation, confers mammary cell invasiveness and defines a novel stem cell/metastasis-associated gene signature[END_REF][START_REF] Shatz | Caveolin-1 mutants P132L and Y14F are dominant negative regulators of invasion, migration and aggregation in H1299 lung cancer cells[END_REF]. In fibroblasts, the downregulation of Cav1 displays unrestricted growth.

They have the ability to develop tumors in mice with weakened immune systems, and they exhibit excessive activation of the Ras-p42/44 mitogenactivated protein (MAP) kinase cascade [START_REF] Galbiati | Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade[END_REF]. A recent study has discovered a new mechanism in which Cav1 plays a role in inhibiting tumor progression. This mechanism involves the suppression of the unfolded protein response (UPR) signaling pathway, a key pathway needed to adapt and respond to endoplasmic reticulum stress conditions, promoting cell survival. The study found that Cav1's ability to suppress the UPR signaling is dependent on the presence of serine-80, an amino acid that is involved in Cav1 secretion and its localization to the endoplasmic reticulum [START_REF] Díaz | Caveolin-1 suppresses tumor formation through the inhibition of the unfolded protein response[END_REF]. Alternatively, the presence of Cav1 could lead to an enhanced cell migration and invasion phenotype. In fact, an earlier study from the same group demonstrated that increased expression of Cav1 is associated with increased migration, invasion and anchorage-independent growth, therefore enhanced malignancy in a model of endometrial cancer cells [START_REF] Diaz-Valdivia | Enhanced caveolin-1 expression increases migration, anchorageindependent growth and invasion of endometrial adenocarcinoma cells[END_REF]. Accordingly, multiple reports have characterized the pro-metastatic effect of Cav1 in different models of cancer. In metastatic cancer cells, Cav1 stimulates the activation of Rab5, leading to increased Rac1 activity, which ultimately promotes enhanced migration and invasion of tumor cells (Díaz et al., 2014). The tyrosine (Tyr14) phosphorylation of Cav1 has been found to be important for the stimulation of Rho activation which leads to enhanced invasion and migration in various breast cancer cells lines [START_REF] Joshi | Phosphorylated caveolin-1 regulates Rho/ROCK-dependent focal adhesion dynamics and tumor cell migration and invasion[END_REF].

Furthermore, tyr14 was identified as a substrate for Src kinase, a known oncoprotein associated with enhanced tumor cell migration, invasion, survival and focal adhesion signalling [START_REF] Wong | Tyrosine phosphorylation of tumor cell caveolin-1: impact on cancer progression[END_REF]. In Hs578T cells, Cav1 overexpression induces an increased formation of colonies in soft agar and a decreased apoptosis [START_REF] Wu | Suppression of staurosporine-mediated apoptosis in Hs578T breast cells through inhibition of neutral-sphingomyelinase by caveolin-1[END_REF]. Similarly, MCF7 cells that have been transfected with human CAV1 plasmid show improved cell survival by the suppression of anoikis, a programmed cell death that happens when the connection between a cell and the ECM and neighboring cells, which is facilitated by integrins, is disturbed [START_REF] Ravid | Caveolin-1 inhibits cell detachment-induced p53 activation and anoikis by upregulation of insulin-like growth factor-I receptors and signaling[END_REF].

Finally, enhanced expression of Cav1 has been reported in metastatic cancers. Cav1 upregulates the expression of the fucosyltransferase pofut1, resulting in the activation of the Notch pathway, leading to the generation of a more aggressive glycosylation phenotype in hepatocellular carcinoma, which promotes invasion and metastasis [START_REF] Zhang | Caveolin-1 promotes invasion and metastasis by upregulating Pofut1 expression in mouse hepatocellular carcinoma[END_REF]. When MDA-MB-231 cells are submitted to a fluid-induced low shear stress environment, similar to the shear stress metastatic cells have to undergo while migrating in the circulatory system, the expression of Cav1 mRNA and protein is increased.

This enhanced expression of Cav1 provides these cells with resistance to anoikis. The mechanism by which Cav1 impairs anoikis resistance is through the inactivation of caspase-8, a key enzyme involved in programmed cell death [START_REF] Chen | Shear stress enhances anoikis resistance of cancer cells through ROS and NO suppressed degeneration of Caveolin-1[END_REF].

The specific molecular mechanisms underlying this contradictory reported behavior are still not fully understood and require further investigation. To date, the consensus is that the role of Cav1 in cancer may depend on the stage of the cancer, with clear differences between early and late phenotypes (Campos et al., 2019). The specific context of each cancer type is another important aspect to consider as well as the intricate interactions between caveolin-1 and other proteins in order to fully comprehend its dual role in the progression of such a complex pathology as cancer.

Chapter 3: Mechanics of cancer progression

One important aspect of cancer research is understanding the mechanics of cancer. These particular terms refer to the physical properties and behaviors of cancer cells and their surrounding environment that facilitate cancer growth, invasion, and metastasis. The research in this field has revealed how alterations in cellular mechanics, such as changes in cell-cell interactions, adhesion to extracellular matrix, and mechanical properties, contribute to the development and progression of cancer.

ECM remodeling

Cancer cells reside within a complex ecosystem known as the tumor microenvironment (TME), comprising various components such as neighboring cells, blood vessels, and ECM (Northcott et al., 2018). The ECM is often altered in cancer, with increased stiffness or cross-linking that can provide rigidity and anchorage for invading cancer cells [START_REF] Paszek | Tensional homeostasis and the malignant phenotype[END_REF][START_REF] Yu | Forcing form and function: Biomechanical regulation of tumor evolution[END_REF].

ECM remodeling involves two main processes: degradation and synthesis.

Cancer cells can secrete enzymes called MMPs, which include collagenases, gelatinases, stromelysins, among others, that degrade ECM components, such as collagen and fibronectin (Table 1). Their activity is tightly regulated in healthy tissues but becomes dysregulated in cancer, leading to excessive ECM degradation [START_REF] Niland | Matrix metalloproteinases shape the tumor microenvironment in cancer progression[END_REF]. These enzymes can break down the ECM barriers, allowing cancer cells to invade surrounding tissues and migrate to distant sites. The increased production and activity of MMPs by cancer cells contribute to the invasive and metastatic potential of tumors [START_REF] Westermarck | Regulation of matrix metalloproteinase expression in tumor invasion[END_REF]. The dysregulated expression of MMPs in cancer is influenced by various factors. Several signaling pathways, such as the TGF-β pathway and the vascular endothelial growth factor (VEGF) pathway, can upregulate the production of MMPs in cancer cells leading to increased metastatic phenotypes [START_REF] Gomes | TGF-b 1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1 / 2 in highly invasive breast cancer cells[END_REF][START_REF] Hiratsuka | MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis[END_REF] (Figure 15). Table 1. Classification and function of matrix metalloproteinases (MMPs) [START_REF] Groblewska | The role of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in the development of esophageal cancer[END_REF].

In addition to degradation, cancer cells can also actively synthesize ECM components. They can produce and deposit molecules, such as fibronectin, collagen, and proteoglycans, which can influence the mechanical properties of the ECM [START_REF] Huijbers | A role for fibrillar collagen deposition and the collagen internalization receptor endo180 in glioma invasion[END_REF][START_REF] Kaplan | VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche[END_REF]. These newly synthesized ECM molecules can further modify the structure and composition of the ECM, again, promoting tumor growth and metastasis. Indeed, the presence of a higher concentration of proteoglycans in the ECM has been associated with a more aggressive tumor phenotype and an increased stiffness of the TME [START_REF] Varga | Brevican, neurocan, tenascin-C and versican are mainly responsible for the invasiveness of low-grade astrocytoma[END_REF]. Fibronectin is a glycoprotein that plays a role in cell adhesion, migration, and tissue organization. Cancer cells can upregulate the production of fibronectin in surrounding CAFs, leading to the deposition of fibronectin-rich ECM around tumors. This deposition can facilitate cell adhesion, support angiogenesis, and promote interactions between cancer cells and the ECM that drive tumor growth and metastasis [START_REF] Wang | Fibronectin: How its aberrant expression in tumors may improve therapeutic targeting[END_REF]. Collagen fibers form a scaffold-like structure that gives strength and stability to tissues. In cancer, the remodeling of collagen fibers is essential for tumor invasion and metastasis [START_REF] Lu | The extracellular matrix: A dynamic niche in cancer progression[END_REF] (Figure 15). increases matrix stiffness around the tumor. Increased matrix stiffness promotes the interaction between ECM components and cell-surface receptors on tumor cells that triggers mechanosignaling. C. CAFs and tumor cells secrete ECM-degrading proteases, including MMPs. ECM degradation generates bioactive molecules that induce pro-tumorigenic ECM signaling that promotes tumor proliferation, migration, invasion and angiogenesis (Modified from [START_REF] Winkler | Concepts of extracellular matrix remodelling in tumour progression and metastasis[END_REF].

Metastasis

As tumors progress and metastasize, the changes that occur are not limited to biochemical signaling or genetic expression. The physical environment undergoes significant alterations at each stage of tumor progression; hyperplasia, invasion, dissemination and colonization at secondary sites [START_REF] Gensbittel | Mechanical Adaptability of Tumor Cells in Metastasis[END_REF]Northcott et al., 2018) (Figure 16).

Increase of cell contractility, uncontrolled growth of cells, and abnormal ECM

results in an increase of stiffness in in situ carcinomas (ISC) that has already been observed in various tumors, including breast, pancreatic, and colorectal cancers [START_REF] Brauchle | Biomechanical and biomolecular characterization of extracellular matrix structures in human colon carcinomas[END_REF][START_REF] Butcher | A tense situation: forcing tumour progression[END_REF][START_REF] Levental | Matrix crosslinking forces tumor progression by enhancing integrin signaling[END_REF].

Cancer cells thrive in these conditions in different ways, for example, through the assembly of filopodia, lamellipodia and invadosomes that enhance migration and invasion [START_REF] Wolf | Physical limits of cell migration: Control by ECM space and nuclear deformation and tuning by proteolysis and traction force[END_REF]. Stiff substrates have the ability to trigger a mesenchymal/basal-like invasive phenotype in tumor cells through increased contractile force mediated by integrins and the formation of mature focal adhesions [START_REF] Mekhdjian | Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix[END_REF].

It is important to acknowledge that many mechanotransduction pathways are regulated by oncogenes or tumor suppressor genes [START_REF] Broders-Bondon | Mechanotransduction in tumor progression: The dark side of the force[END_REF]. Therefore, the profound changes that cells undergo in these modified mechanical environments can induce cellular transformations comparable to genetic mutations [START_REF] Yu | Forcing form and function: Biomechanical regulation of tumor evolution[END_REF]. Disturbances in this system can impact many cellular processes like cell differentiation, cell viability, proliferation, adhesion, migration, among others, all of which contribute to the promotion of cellular invasion. C. The growth of the tumor within the surrounding stromal tissue leads to compression of both the tumor and the neighboring tissue. The increased stiffness of the ECM intensifies the resistance of the stromal tissue to compression and further amplifies the solid stress. D. A gradient of pressure within the surrounding tissue leads to the movement of fluid from the central region of the tumor to its outer edges, facilitating the spread of cancer cells to distant locations (metastasis). After escaping from the primary tumor, cancer cells migrate, guiding their movement towards blood vessels. During this process, tumor cells experience significant shear stresses as they pass between endothelial cells during intravasation and extravasation, as well as while traveling through the bloodstream to potential secondary tumor sites (Northcott et al., 2018).

When ISC becomes invasive and cancerous cells start migrating, moving through the circulatory system, and infiltrating other tissues and organs, they will experience other types of mechanical strains, including solid forces and fluid forces that generate shear stress. It can be shear stress generated by the blood flow along the surface of cells, known as hemodynamic shear stress, but also the stress induced by solid forces encountered when tumor cells come into contact with endothelial cells during the process of intravasation and extravasation from the blood vessels [START_REF] Wirtz | The physics of cancer: the role of physical interactions and mechanical forces in metastasis[END_REF].

Hemodynamic flow forces can favor the arrest, adhesion, and extravasation of circulating tumor cells (CTCs) in vivo. In a model of zebrafish, blood flowinduced endothelial cell remodeling is a crucial mechanism facilitating the extravasation of CTCs to promote metastasis [START_REF] Follain | Hemodynamic Forces Tune the Arrest, Adhesion, and Extravasation of Circulating Tumor Cells[END_REF].

Interestingly, tumor cells are more resistant to shear stress than normal cells [START_REF] Mitchell | Lamin A/C deficiency reduces circulating tumor cell resistance to fluid shear stress[END_REF]. Normal levels of shear stress in the body, ranging from approximately 5 to 30 dynes/cm2 (0.5-3 Pa), have been found to stimulate the movement and attachment of tumor cells [START_REF] Ma | Hemodynamic shear stress stimulates migration and extravasation of tumor cells by elevating cellular oxidative level[END_REF][START_REF] Xiong | Involvement of caveolin-1 in low shear stress-induced breast cancer cell motility and adhesion: Roles of FAK/Src and ROCK/p-MLC pathways[END_REF].

Nevertheless, shear stress levels similar to those experienced during exercise, approximately 60 dynes/cm2 (6 Pa), have been shown to induce cell death in tumor cells [START_REF] Regmi | High Shear Stresses under Exercise Condition Destroy Circulating Tumor Cells in a Microfluidic System[END_REF].

The activation of mechano-signaling pathways can promote tumor progression by enhancing cancer cell survival. Low shear stress can induce the activation of mechanical signaling pathways that promote tumor growth and migration. Studies have demonstrated that low shear stress conditions activate multiple signaling pathways in a Cav1-dependent manner, including AKT/Src and Rho-associated protein kinase (ROCK)/p-Myosin Light Chain (MLC) pathways, leading to enhanced cell motility and adhesion [START_REF] Xiong | Involvement of caveolin-1 in low shear stress-induced breast cancer cell motility and adhesion: Roles of FAK/Src and ROCK/p-MLC pathways[END_REF]. Furthermore, shear stress was found able to induce an epithelialmesenchymal transition in circulating tumor cells by activating Jun N-terminal Kinase (JNK) signaling, therefore increasing their survival rate during hematogenous dissemination [START_REF] Xin | Fluid shear stress induces emt of circulating tumor cells via jnk signaling in favor of their survival during hematogenous dissemination[END_REF].

Therefore, for a tumor cell to survive and thrive during its journey from the primary ISC to a secondary site, it must constantly adjust and adapt to the different mechanical stresses it encounters.

EV dynamics

The ECM has been demonstrated to impact the secretion of EVs from surrounding cells through its stiffness and mechanical properties. Several studies have highlighted the significance of ECM-induced mechanical force on EV biogenesis, with evidence suggesting that these mechanical forces can influence regulatory pathways [START_REF] Liu | Exosomemediated transduction of mechanical force regulates prostate cancer migration via microRNA[END_REF][START_REF] Patwardhan | Biomaterials ECM stiffness-tuned exosomes drive breast cancer motility through[END_REF][START_REF] Schwager | Cell-Cell Mechanical Communication in Cancer[END_REF][START_REF] Xiao | Matrix stiffness mediates pancreatic cancer chemoresistance through induction of exosome hypersecretion in a cancer associated fibroblasts-tumor organoid biomimetic model[END_REF].

For example, it was discovered that a stiff ECM induced the release of a specific population of EVs with particular miRNAs. These EVs were found capable of regulating prostate cancer cell motility and migration, a finding that implies that mechanical force can modulate cancer metastasis [START_REF] Liu | Exosomemediated transduction of mechanical force regulates prostate cancer migration via microRNA[END_REF]. Moreover, in a model of breast cancer it was shown that stiff ECMs promote exosome secretion in a YAP/TAZ pathway-dependent manner.

These exosomes are able to induce alterations in cell shape, attachment, and protrusion kinetics that can enhance cellular motility and invasiveness [START_REF] Patwardhan | Biomaterials ECM stiffness-tuned exosomes drive breast cancer motility through[END_REF].

Another type of mechanical stress that has been well established as a regulator of EV dynamics is shear stress. This type of mechanical stress has been found to be crucial for platelets to release microvesicles where integrins serve as mechanical sensors and downstream signal transducers [START_REF] Pang | Shear-induced integrin signaling in platelet phosphatidylserine exposure, microvesicle release, and coagulation[END_REF]. Endothelial cell-derived EVs have been shown to present a very distinct miRNA cargo in shear stressed cells. Interestingly, these EVs are able to modulate the function of endothelial cells, including their angiogenesis, migration, and inflammatory response [START_REF] Chung | Fluid Shear Stress Regulates the Landscape of microRNAs in Endothelial Cell-Derived Small Extracellular Vesicles and Modulates the Function of Endothelial Cells[END_REF]. Shear stress can also affect other aspects of EV dynamics, endothelial cells were subjected to low or high shear stress and their ability to uptake EVs was analyzed.

Interestingly, low and oscillating shear stress promoted the uptake of EVs in vitro and in vivo, respectively [START_REF] Qin | Bioactive Materials Uptake of oxidative stress-mediated extracellular vesicles by vascular endothelial cells under low magnitude shear stress[END_REF]. Shear as well as cyclic stretch has been shown to induce an increased release of EVs in different cell models. The amount of EVs released after these types of mechanical stress have been described to range from 10 to 100 times the amount of EVs released in resting conditions [START_REF] Guo | Stimulating Extracellular Vesicles Production from Engineered Tissues by Mechanical Forces[END_REF], this is of particular interest, as it reveals that it may not only be the nature of the EVs that is affected upon mechanical stress, but also the amount of EVs secreted, suggesting a potential combined effect exerted on EV receiving cells.

Cyclic stretch is another type of mechanical strain that cells have to endure in normal conditions, in tissues that are cyclically stretched due to breathing, heart beating or peristalsis of the gut. It has been described that cyclic stretch is able to induce the release of EVs with the ability to suppress IL-1β production and pyroptosis, an inflammatory cell death, through the blockade of NF-κB signaling in human monocytes and macrophages [START_REF] Wang | Cyclic stretch force induces periodontal ligament cells to secrete exosomes that suppress IL-1β production through the inhibition of the NF-κB signaling pathway in macrophages[END_REF].

When cells are stretched due to changes in osmotic pressure or when mice are experiencing increased pressure in their hearts, it triggers the release of exosomes that are rich in the angiotensin II type 1 receptor. These exosomes are then released from the heart and can affect how blood vessels respond to neurohormonal stimulation [START_REF] Pironti | Circulating Exosomes Induced by Cardiac Pressure Overload Contain Functional Angiotensin II Type 1 Receptors[END_REF].

HYPOTHESIS AND OBJECTIVES

So far, we have discussed topics related to EVs, caveolae, cancer, and the role of cell mechanics in these processes. We have explored the different mechanisms by which EVs are secreted and their diverse functions in carrying various molecular cargoes such as nucleic acids, proteins, and lipids, and delivering them to their target cells. One of the key conclusions is that EVs play a crucial role in intercellular communication and cell-to-cell signaling across different physiological contexts.

Additionally, we have also discussed the importance of caveolae in cellular and tissue homeostasis, and in disease development, including cancer, where we explored the contradictory role of Cav1 in cancer progression. Moreover, we have examined the mechanical properties of caveolae in mechanoprotection and signaling.

Finally, we delved into the field of cell mechanics, including mechanics of cancer and EV dynamics. Even though there is an increasing interest in the topics listed above, there are still enormous gaps in knowledge in certain areas. Understanding the role of mechanical stress as a regulator of these processes is critical for identifying new therapeutic targets and developing interventions for cancer. Therefore, the overall aim of my thesis was to investigate the potential role of caveolae in EV biology in a context of cancer and enhanced mechanical strains.

For this I have pursued three main objectives:

1)

To investigate the effect of different types of mechanical stress on EV biology, including secretion, uptake, composition, and physiological relevance from a cancer perspective,

2)

To explore the role of caveolae and its components in the mechanically-induced changes in EV biology,

3)

To shed light into the molecular mechanism by which caveolae may regulate these effects.

RESULTS

Introduction

Extracellular vesicles (EVs) are small membranous particles present in all bodily fluids and released to the extracellular space by all cell types studied thus far (Théry et al., 2018). EVs have gained significant attention in recent years due to their involvement in various physiological and pathological processes, including cancer [START_REF] Yáñez-Mó | Biological properties of extracellular vesicles and their physiological functions[END_REF]. In the context of cancer, EVs have been implicated in multiple aspects of tumor progression and metastasis, such as promoting cell proliferation, invasion, angiogenesis, and immune evasion. Interestingly, tumor cells have been shown to release higher amounts of EVs compared to healthy cells. Tumor-derived EVs can affect the local tumor microenvironment, travel to distant secondary sites or serve as means of communication between both cancerous and non-cancerous cells, promoting cancer progression (Chen et al., 2018;Peinado et al., 2012;Sung et al., 2015).

Caveolae are specialized, 60-80 nm cup-shaped invaginations of the plasma membrane that are present in most cell types (Lamaze et al., 2017;[START_REF] Palade | Fine structure of blood[END_REF].

Caveolins together with cavins are required for the structural and functional integrity of caveolae (Kovtun et al., 2015). Mutations or abnormal expression of caveolae components have been associated with various disorders, including cancer (Le [START_REF] Lay | Getting rid of caveolins: phenotypes of caveolin-deficient animals[END_REF]. The caveolae protein caveolin-1 (Cav1) is more abundant in tumorderived EVs and has been proposed as a biomarker for cancer progression (Campos et al., 2019;[START_REF] Lamaze | Caveolae and cancer: A new mechanical perspective[END_REF]. In 2011, a new function of caveolae was established as mechano-sensors and mechano-transducers that play an essential role in cell mechano-protection (Dewulf et al., 2019;Sinha et al., 2011;Torrino et al., 2018).

As tumors progress and metastasize, their physical microenvironment undergoes significant alterations. During its metastatic journey from a primary site to a secondary site, tumor cells must constantly adapt and respond to the different mechanical stresses they encounter.

In this study, we explored the role of caveolae in EV dynamics in cancer cells subjected to mechanical strains. We observed a drastic increase in the amount of EVs released after mechanical stress. The increase of EV release required Cav1 and the endosomal sorting complex endosomal sorting complex required for transport (ESCRT)-0 proteins, indicating their exosomal origin. This specific subpopulation of Cav1-positive EVs contained a particular lipid signature and was able to induce the acquisition of metastatic traits in receiving cells, including increased migration and invasion in vitro. We have therefore unveiled a novel mechanism by which cancerous cells take advantage of the mechanical properties of caveolae to adapt to the mechanical environment of tumors and to promote the secretion of pro-metastatic EVs.

Results

Mechanical stress enhances the release of small EVs.

In order to investigate the effect of mechanical stress on the release of small EVs, we first applied a hypo-osmotic shock, where cells are subjected to a short and acute hypo-osmotic medium that induces the flattening of caveolae (Sinha et al., 2011) (Figure S1A and S2B). EVs were purified by ultracentrifugation from WT HeLa cells under resting or osmotic shock conditions. The size distribution measured by nanoparticle tracking analysis (NTA) shows that most secreted EVs are ranging from 50-200 nm (Figure 1A). We observed a drastic increase in EV release in cells subjected to hypo-osmotic shock with a 3.5-fold increase in the number of secreted EVs detected by NTA (Figure 1B). We used electron microscopy (EM) to assess the purity and integrity of released EVs. EM images revealed the presence of EVs with a characteristic "donut" shape albeit with a smaller diameter (~80 nm mean size) than the values obtained through NTA measurement (~128 nm mean size). The substantial reduction in observed EV size is a known artifact of the preparation method used to visualize EVs in TEM (Wu et al., 2015) (Figure 1C and2D). We confirmed by western blot the presence of bona fide EV markers, including CD63 and CD9 [START_REF] Mathieu | Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9[END_REF]. Also, the endoplasmic reticulum (ER) protein, calnexin commonly used as a negative marker was detected in cell extracts but not in EVs as expected. The two main components of caveolae, Cav1 and cavin1 were detected in WT HeLa EVs, with an enrichment of both proteins (~2.5-fold increase) in EVs from HeLa cells subjected to hypo-osmotic shock compared to EVs from resting cells (Figure 1E).

Cav1 is required for the enhanced release of small EVs induced by mechanical stress.

Considering the role of caveolae in mechano-sensing, we examined their potential involvement in the increase of EV release induced by mechanical stress. Remarkably, in HeLa cells stably knocked out for Cav1 expression, we no longer observed the increase in the number of secreted EVs induced by hypo-osmotic (Figure 2A). To rule out that this effect was specific of the two-dimensional hypo-osmotic shock model, we tested a three-dimensional osmotically induced mechanical compression in multicellular spheroids grown in an agarose bed with high molecular weight dextran (100 kDa), a high molecular weight sugar that is neutral for mammalian cell metabolism (Figure S1D andS1E) [START_REF] Dolega | Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression[END_REF]. We also found that this different type of mechanical strain caused the disassembly of caveolae without compromising cell viability (Figure S1E-G). Dextran compression of spheroids induced a 2-fold significant increase in the number of secreted EVs detected by NTA as compared to resting spheroids. Similarly to the results observed with hypo-osmotic shock, the absence of Cav1 prevented the increase in secreted EVs (Figure 2B).

Cavin1 is required for caveolae morphogenesis and stability at the plasma membrane.

In the absence of cavin1, caveolae are not formed and only Cav1 is present at the plasma membrane albeit at a lower expression level (Drab et al., 2001;Hill et al., 2008). In HeLa cells stably knocked out for cavin1, we could still measure an increased number of secreted EVs after mechanical stress, yet the increase was significantly less than in WT HeLa cells (Figure 2A and2B). Because of the co-transcriptional regulation of cavin1 and Cav1 expression, cavin1 depleted cells are known to express lower amounts of Cav1 (Hill et al., 2008). We therefore re-expressed Cav1 in Cavin1 - /-HeLa cells to restore basal levels of Cav1. Under this condition, we could measure a level of EVs similar to the one induced by mechanical stress in WT HeLa cells (Figure S2D). These data indicate that Cav1 but not caveolae are required for the increase of EV release by mechanical stress.

We could extend this mechanism to other cell types, including Hs578t and MDA-MB-231 triple negative breast cancer cell lines. In these cells, hypo-osmotic shock or dextran compression induced an increase in the number EVs detected through NTA as compared to control cells, and this increase was also dependent on the presence of Cav1 (Figure S2A-C).

Different mechanisms could lead to an increase of EV production, including enhanced secretion of EVs by producing cells or impaired reuptake. We addressed the latter possibility by selectively measuring the release of EVs, using a CD63-pHluorin construct previously described [START_REF] Verweij | Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling[END_REF]. CD63-pHluorin is a pH sensitive plasmid that becomes fluorescent once the pH becomes neutral inside endosomal acidic compartments such as multivesicular bodies (MVB) that contain intraluminal vesicles (ILVs). This construct allows therefore to visualize the fusion of MVBs at the plasma membrane and the release of ILVs as exosomes to the extracellular medium in live cells. In line with the EV secretion results measured by NTA, the exosomal release activity of HeLa cells was increased after osmotic shock (Figure 2C). The increase in fusion activity after mechanical stress was absent in HeLa Cav1 -/-cells, confirming that the increased EV detection is due to an increase in EV release from producing cells. Altogether, this data reveals a novel Cav1-dependent mechanism by which hypo-osmotic shock and 3D mechanical compression induce a drastic increase in the release of small EVs i.e. exosomes from cells.

The mechanical release of small EVs requires the endosomal ESCRT-0 complex.

We next explored the mechanism by which the MVB pathway is affected by mechanical stress or Cav1 depletion. First, we analyzed by EM the characteristics of MVBs in Hs578t cell spheroids in resting conditions, and after 5 min, 1 day or 5 days of dextran compression (Figure 3 A). While the diameter of MVBs observed after mechanical compression did not significantly change over time (Figure 3B), the overall number of MVB-like structures in compressed Hs578t spheroids was significantly reduced as compared to resting spheroids for all tested compression times (Figure 3C). In parallel, in HeLa cells subjected to osmotic shock, we observed the presence of enlarged CD63-positive endosomal-like structures that were decorated with Cav1 (Figure S1C, inserts).

We next evaluated the protein levels of the components of the ESCRT pathway, one of the key machineries controlling MVB biogenesis and exosome production. Signal transducing adaptor molecule (STAM) binds to hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) to form the ESCRT-0 at the early endosome [START_REF] Migliano | Biophysical and molecular mechanisms of ESCRT functions, and their implications for disease[END_REF]. We found by western blot that HRS and STAM were significantly downregulated to approximately 45% expression in the absence of Cav1. We also found that Lamp1, a lysosomal marker, and LC3b a marker of autophagy, were both upregulated in Cav1 depleted cells, further confirming the alteration in the endosomal pathways (Figure 4A and4B). Next, we tested the effect of ESCRT-0 downregulation on the mechanical increase of EV release. The treatment of WT HeLa cells with siRNA targeting HRS resulted in a strong downregulation of HRS (>90%) and a milder downregulation of STAM (~50%) (Figure 4C). We next purified EVs from HRS downregulated cells subjected to hypo-osmotic shock. We observed a significant reduction in the secreted EVs detection through NTA in HeLa cells treated with siRNA against HRS, confirming the established role of HRS in EV biogenesis (Colombo et al., 2013, Colombo et al., 2014). Interestingly, we observed that mechanical stress did not increase the number of secreted EVs by HeLa cells downregulated for HRS, indicating that the increased EV release after mechanical stress is also dependent on ESCRT-0, pointing towards a co-regulation of this effect by both Cav1 and ESCRT-0 (Figure 4D).

Small EVs from mechanically stressed cells present a distinct lipid signature.

Caveolae organize lipid nanodomains at the plasma membrane that are enriched in cholesterol and sphingolipids, and have been described to play a central role in regulating lipid homeostasis (Krishna & Sengupta, 2019;Prakash et al., 2021;Sonnino & Prinetti, 2009). We evaluated whether mechanical stress and Cav1 depletion would affect the lipid composition of EVs. For this we performed lipidomic analysis of EVs purified from WT or Cav1 -/-HeLa cells under resting conditions or after exposure to mechanical stress. Indeed, mass spectrometry analysis revealed differences in the lipid composition between EVs from WT and Cav1 depleted HeLa cells, as well as differences between EVs from resting or mechanically stressed HeLa cells. Mechanically stressed cells release small EVs that promote enhanced migration and invasion of cancer cells.

Mechanical

We examined the potential impact of EV uptake in cancer biology through cell proliferation and invasion assays. Cell proliferation was investigated using a woundhealing assay [START_REF] Ilina | Mechanisms of collective cell migration at a glance[END_REF]. We found that EVs from mechanically stressed cells promoted faster wound closure in WT Hs578t cells compared to EVs from resting cells (Figure 6A-C). Importantly, we observed that Cav1 was required, as EVs secreted from Cav1-deleted cells failed to enhance wound closure in both resting and mechanical stress conditions. We also analyzed the invasive potential of these EVs by pre-incubating recipient cells with the EVs for 24 hours and then analyzing their invasiveness using a thin layer of matrigel, which primarily consists of collagen type IV, entactin, heparan sulfate proteoglycan, and laminin. We observed a similar effect, where EVs from mechanically stressed cells enhanced the invasion capabilities of Hs578t cells through the ECM, while EVs from resting cells also increased the invasion, albeit to a lesser extent. The absence of Cav1 in secreting cells completely prevented the enhanced invasion ability conferred by the EVs (Figure 6D).

In summary, these data indicate that EVs secreted by HeLa cells in response to mechanical stress enhanced the migratory and invasion capabilities of Hs578t cells through the ECM in a Cav1 dependent manner, thereby increasing their tumorigenic potential. In contrast, EVs from resting HeLa cells do not confer these properties to Hs578t cells.

Discussion

In this study, we characterized EVs whose release is specifically increased after mechanical stress, and identified Cav1, the primary constituent of caveolae, as a key player in this process. EVs released from mechanically stressed cancer cells displayed distinct lipid composition differences and promoted enhanced in vitro migration and invasion. These effects were dependent on the presence of Cav1.

The average size distribution of EVs purified from HeLa WT, Cav1 -/-, and cavin1 -/- ranged up to ~350 nm, with over 80% comprising vesicles smaller than 200 nm, falling within the typical exosome size range. The exosomal nature of these vesicles was confirmed through monitoring MVB fusion to the plasma membrane using the pHluorin-CD63 construct and by the dependence on a functional ESCRT-0 complex.

In addition to universally accepted EV markers such as CD63 and CD9 [START_REF] Keerthikumar | ExoCarta: A Web-Based Compendium of Exosomal Cargo[END_REF][START_REF] Mathieu | Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9[END_REF], we also detected the presence of the two main caveolar proteins, Cav1 and cavin1, in the EV samples from WT HeLa cells. This finding is consistent with prior studies reporting the presence of Cav1 in EVs from cancerous cells (Campos et al., 2018;Logozzi et al., 2009).

The molecular mechanisms responsible for the increase in EV release during mechanical stress remain unclear. We discovered that both Cav1 and the ESCRT-0 complex were essential for the mechanically induced increase in EV release. Previous studies have suggested a potential interaction between Cav1 and the ESCRT machinery. Thus, in HeLa cells, depleting HRS reduced the ubiquitination-dependent targeting of Cav1 to ILVs within MVBs. In this study, it was also proposed that overexpressing Cav1 leads to the accumulation of Cav1 in endosomal structures (Hayer et al., 2010a).

Given the critical role of caveolae in mechano-sensing and mechano-protection, we decided to test our hypothesis in cancer cell lines with either Cav1 expression or depletion, considering the mechanical challenges that cancer cells encounter during their progression from a growing tumor to metastasis (Northcott et al., 2018). It is known that Cav1 acts as an enhancer of metastatic traits, promoting cell migration, invasion and anoikis resistance (Díaz et al., 2014;[START_REF] Urra | Caveolin-1-enhanced motility and focal adhesion turnover require tyrosine-14 but not accumulation to the rear in metastatic cancer cells[END_REF][START_REF] Wang | Caveolin-1 contributes to anoikis resistance in human gastric cancer SGC-7901 cells via regulating Srcdependent EGFR-ITGB1 signaling[END_REF]).

Yet the exact mechanism by which it exerts these effects is still poorly understood.

Recently, the presence of Cav1 in EVs from breast cancer cells was associated with increased migration and invasion of T47-D epithelial and MDA-MB-231 recipient cells (Campos et al., 2018). Considering this, we assessed the metastatic potential of EVs released from mechanically stressed or resting WT and Cav1-depleted cells. We observed increased migration and invasion of Hs578t cells after incubation with EVs from HeLa cells in resting conditions, and this effect was further enhanced when incubated with EVs secreted under mechanical stress. Again, this depended on the presence of Cav1 in donor cells. Since Hs578t cells contain endogenous Cav1, it suggests that the enhanced migration and invasion effects of EVs may not be solely due to the presence of Cav1 within the EVs themselves. Instead, it may be linked to changes in the overall composition of the EV subpopulation induced by mechanical stress, where Cav1 could play a role.

We assessed the potential components of the EVs responsible for mediating these effects, taking into account the established role of Cav1 in lipid clustering at the plasma membrane and lipid nanodomain assembly (Pohl et al., 2002;Pol et al., 2005).

Mechanical forces can alter lipid distribution in reconstituted membranes, as seen in giant unilamellar vesicles (GUV), where increased tension triggers lipid phase separation [START_REF] Colom | A fluorescent membrane tension probe[END_REF]. We performed lipidomics to investigate whether Cav1 and mechanical stress could recruit distinct lipids to the membrane of MVBs and, consequently, EVs. Our lipidomic assays revealed significant differences in the lipid proportion and composition of EVs, both between WT and Cav1 depleted cells, as well as between resting and mechanically stressed donor cells. In WT cells, mechanical stress led to a slight increase in the content of globoside Gb4 and ganglioside GM2 in EVs. Notably, Cav1 depletion in donor cells prevented the incorporation of these lipids in EVs. Gb4 has been demonstrated to stimulate the Raf-MEK-ERK MAPK signaling pathway, leading to increased cancer cell proliferation [START_REF] Park | Globoside promotes activation of ERK by interaction with the epidermal growth factor receptor[END_REF]. GM2 role in cancer progression is well-documented, as it is highly expressed in various human tumors and functions as a pro-cancer signaling regulator [START_REF] Sasaki | Gangliosides as Signaling Regulators in Cancer[END_REF]. In this context, we cannot rule out potential changes in the protein composition of EVs.

Differential EV protein composition can influence various aspects of EV biology, including their impact on receiving cells. This can occur through a protein cargo carrying molecules that activate pro-metastatic signaling pathways or by inducing differential EV uptake by receiving cells, as seen with the clustering of integrins in EVs that facilitate surface docking and internalization (Hoshino et al., 2015). The potential changes in the protein cargo of EVs remain to be defined.

Our data establish a connection between the response of caveolae to mechanical stress and EV production. We and others have shown that upon mechanical stress, caveolae immediately flatten out to counteract membrane tension increase and prevent rupture (Sinha et al., 2011). This flattening is immediately followed by caveolae disassembly, releasing caveolae coat components into the cytoplasm and non-caveolar Cav1 at the plasma membrane. This process is linked to mechanotransduction (Dewulf et al., 2019;Nassoy & Lamaze, 2012;Torrino et al., 2018). Our discovery that Cav1, rather than caveolae, is responsible for EV production under mechanical stress aligns with Cav1's presence in both MVBs and EVs. Under mechanical stress, caveolae flatten out and release non-caveolar Cav1, which is subsequently endocytosed into the endosomal network, engulfed in ILVs, and eventually secreted in EVs. Cells lacking Cav1 fail to respond to mechanical stress and do not enhance EV production, whereas cells lacking cavin1 can produce EVs, albeit at a reduced level. These data indicate a correlation between the level of EV production under mechanical stress and the quantity of Cav1 present in MVBs. This was further validated by the increase of EV production when Cav1 was overexpressed in HeLa cells. In support of these findings, EM observation of MVBs revealed a significant enrichment of Cav1 in mechanically stressed cells (Figure S3).

In conclusion, our findings emphasize the importance of Cav1 in regulating the dynamics of EVs in the context of the constant mechanical strains that cancer cells must endure. This mechanism provides an explanation for how cancer cells can utilize their mechanical environment to further promote cancer progression.

(FFCR), Fondation ARC pour la recherche sur le cancer (Grant # ARCDOC42022010004536), and Becas Chile.

Materials and Methods

Cell lines

HeLa, Hs578t, MDA-MB-231 cells along with the Cav1 -/-and cavin1 -/-sub lines were cultured in Dulbecco's modified Eagle's medium (DMEM-Glutamax, Gibco), with 10% FBS (FBS, Gibco), 100U/ml penicillin and 100 μg/ml streptomycin (Gibco). Cell lines were grown at 37°C, under 5% CO2. Cell spheroids were prepared following the classical agarose cushion protocol. First, 50 µl of agarose 1,5% w/v in PBS (ultrapure agarose, Invitrogen) and 50 µl per well were dispensed in 96-well plate and incubated for 10-15 min for polymerization at room temperature. Then, cells were seeded on agarose cushion at 105 cells per well. Spheroid formation usually takes between 24 and 48 h. Cav1 -/-and cavin1 -/-cells were obtained by CRISPR-Cas9 gene editing.

Antibodies and plasmids

Primary antibodies used include rabbit anti-cav1 (BD Transduction Laboratories, cat.

no. 610059; 1:2,000 for WB and 1:200 for IF), mouse anti-Hrs (Abcam, cat. no.

ab56468; 1:2,000 for WB), rabbit anti-STAM1/2 (Abcam, cat. no. ab76061; 1:2,500 for WB), mouse anti-CD9 (clone MM2/57; Sigma, cat. no. CBL162; 1:1,000 for WB), mouse anti-α-tubulin (clone B512; Sigma, cat. no. T5168; 1:5,000 for WB), rabbit anti-LC3B (Abcam, cat. no. ab51520; 1:3,000 for WB), mouse anti-CD63 (Santa Cruz, cat.

no. sc-5275; 1:2,000 for WB and 1:100 for IF), mouse anti-calnexin (BD transduction laboratories. cat. no. 610523; 1:2,000 for WB), rabbit anti-PTRF (Proteintech. cat. no. The pCMV-CD63-pHluorin construct (Addgene, #130901) was transfected in HeLa cells using the lipofectamine LTX reagent (Invitrogen) protocol, with 1 µg of plasmid DNA. Cells were imaged 48 hours after transfection.

RNA interference

For hrs knockdown we used the following: Control siRNA (Dharmacon, Thermo Fisher, cat. no. SI03650325; 5′-AATTCTCCGAACGTGTCACGT-3′), the hrs pool of four siRNA SMARTpool ON-TARGETplus HGS siRNA (Thermo Fisher, L-016835-00-0005; 5′-GAGGUAAACGUCCGUAACA-3′, 5′-GCACGUCUUUCCAGAAUUC-3′, 5′-AAAGAACUGUGGCCAGACA-3′ and 5′-GAACCCACACGUCGCCUUG-3′).

Transfection was done using Lipofectamine RNAiMAX according to the manufacturer's protocol (Thermo Fisher), siRNA was used at 20 nM. Depletion efficiency was assessed by immunoblotting.

Mechanical stress models

To induce swelling of cells in 2D cultures we used hypo-osmotic shock. Hypo-osmotic shock was performed on cells by using the corresponding growth medium diluted appropriately in deionized water (1:9 dilution for 30 mOsm hypo-osmotic shock).

To induce compression of multicellular spheroids, we used osmotic stress as described by Cappello's team [START_REF] Dolega | Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression[END_REF]. Shortly, we prepared hyperosmotic medium by adding high molecular weight dextran to reach a final concentration of 160 g/ml (2X solution). We used 2 MDa dextran (Sigma Aldrich, 95771) to avoid the penetration of the polymer in the cellular spheroid. Dextran containing medium was added on spheroid at 80 g/ml final concentration. For longterm compression experiments, dextran containing culture medium is renewed after 3 days.

EV isolation

For the hypo-osmotic shock experiments, 6 × 10 6 of cells were seeded per T75 culture flask. After 24 h, cells were washed 1 X with PBS (Gibco) and 12 ml of serum free iso or hypo-osmotic culture medium. Medium was changed after 5 min to iso-osmotic, serum free, culture medium and incubated for the corresponding time periods. For the osmotic induced compression experiments, spheroids were washed with 1X PBS (Gibco) and 100 µl of serum free medium containing or not dextran. After treatments with mechanical stimulations (Hypo-osmotic shock or osmotic induced compression) culture supernatant was collected and was subjected to serial centrifugations (2,000 g for 10 min, 11,000 g for 30 min at 4°C), followed by ultracentrifugation at 100,000 g

for 90 min at 4°C (45Ti or TLA110 rotors, Beckman Coulter). The pellet containing EVs was washed in cold 1x PBS (Gibco) and ultracentrifuged again at 100,000 g for 90 min at 4°C.

EV characterization by Nanoparticle Tracking Analysis

NTA was performed using ZetaView PMX-120 (Particle Metrix) equipped with a 488 nm laser, with software version 8.05.02 to measure the concentration and distribution size of particles by evaluating the Brownian motion in a light scattering system. The samples were diluted in 1X PBS (Gibco) to obtain a concentration of 10 6 to 10 9 particles/ml per field.

Western blotting

Cell and EV extracts were separated by SDS-PAGE, transferred to a nitrocellulose, blocked in 1x PBS containing 3% BSA or 5% nonfat milk according to the antibody used. Membranes were probed overnight at 4°C. Bound antibodies were developed using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific)

and the ChemiDoc Touch Imaging System (BioRad). Protein bands were quantified by densitometric analysis using the ImageJ.

Electron microscopy

For imaging cell spheroids, epon embedding was used to preserve the integrity of cell structures for electron microscopy (EM). Spheroids were fixed sequentially for 1 h at room temperature with 1.25% glutaraldehyde in 0.1 M Cacodylate and then overnight at 4 °C. Cells were washed extensively with 0.1 M Cacodylate, pH 7.2. Post-fixation was performed for 1 h at room temperature with 1% OsO4 in 0.1 M Cacodylate, pH 7.2. Spheroids were dehydrated through a graded-concentration series of ethanol (50, 70, 90, then 100%, each for 10 min at RT). Embedding was finally performed in LX112 resin. Cells were infiltrated with an increasing ratio of LX112: ethanol solution (1:2, 1:1 and 2:1) and finally with pure LX112. Samples in resin were polymerized overnight at 60°C. Semi-thin 500 nm sections were sliced using a Leica UCT ultra microtome and mounted onto microscopic glass slides and dried on a hot plate. Semithin sections were stained for 30 s on a hot plate with a mix of Azure B and basic fuchsin in sodium tetraborate [START_REF] Morikawa | A simple, one-step polychromatic staining method for epoxy-embedded semithin tissue sections[END_REF]. Sections were then mounted in DPX for microscopy and covered with coverslips. Micrographs were acquired on an Upright Wide field Leica DM6000b Microscope equipped with a color CoolSNAP HQ2 camera.

Ultrathin 65 nm sections were sliced using a Leica UCT ultra microtome and mounted on nickel formvar/carbon-coated grids for observations. Contrast was obtained by incubation of the sections for 10 min in 4% uranyl acetate followed by 1 min in lead citrate. Caveolae were identified based on their ultrastructural features. The length of plasma membranes observed were measured using ImageJ software and the number of the structures observed was reported to µm of membrane.

For imaging EVs: EVs pellets were resuspended in 50-100 µl of 1x PBS, 5 µl were deposited on a coated-side of a formvar-carbon grid and left for 20 minutes to adhere.

Grids were then transferred on 2% PFA droplets for 20 minutes fixation and then washed 10 times on filtered distilled water droplets. Grids were incubated for 10 minutes in 0.4 % uranyl acetate in 2 % methylcellulose droplets on ice and in the dark.

Grids were removed from the droplet with a stainless steel loop and excess liquid was removed by pushing the loop on a Whatman filter. Grids are then air dried for 30 minutes on the loops.

Electron micrographs were acquired on a Tecnai Spirit electron microscope (FEI, Eindhoven, The Netherlands) equipped with a 4k CCD camera (EMSIS GmbH, Münster, Germany).

Immunofluorescence

After the treatments, cells were fixed with 4% PFA (EMS) for 15 min at room temperature. Cells were incubated for 1h in a blocking solution: 1x PBS with 0.1% triton and 0.3% BSA. Then primary and secondary antibodies were successively incubated during 1 h each at RT in 1x PBS containing 0.1% triton and 0.1% BSA.

Coverslips were then mounted on microscope slides with Fluoromount G (Invitrogen).

Images were acquired on a Zeiss LSM 780 confocal microscope with a 63x/1.46 Oil objective. At least 10 cells per replicate were imaged.

Live TIRF microscopy

Cells were grown in 35-mm imaging plates (FluoroDish WPI; IBIDI). An inverted Eclipse Ti-E (Nikon) full motorized with PFS 2 (Perfect Focus System) to reduce the drift was used with a 100 ×CFI PlanApoTIRF, oil, 1.49/0.12-mm objective (Nikon), images were acquired with Metamorph (Molecular Devices). A cage incubator (Life Imaging Services) was used to keep the imaging chamber at 37°C and 5% CO2. To visualize fusion events, 1-3 min videos were acquired in different areas to analyze at least 20 cells per condition.

Sample preparation for lipidomics

For lipidomics analysis, 20 ug of protein corresponding to around 109 EV particles were spiked with 1.65 μL of internal standard lipid mixture containing 500 pmol of Chol-d6, 100 pmol of Chol-16:0-d7, 100 pmol of DAG 17:0-17:0, 50 pmol of triglycerides 17:0-17:0-17:0, 100 pmol of SM 18:1;2-12:0, 30 pmol of Cer 18:1;2-12:0, 30 pmol of GalCer 18:1;2-12:0, 50 pmol of LacCer 18:1;2-12:0, 300 pmol of PC 17:0-17:0, 50 pmol of PE 17:0-17:0, 50 pmol of PI 16:0-16:0, 50 pmol of PS 17:0-17:0, 30 pmol of PG 17:0-17:0, 30 pmol of PA 17:0-17:0, 40 pmol of Gb3 18:1;2-16:0(d9), 25 pmol of GM3 18:1;2-18:0-d5, 25 pmol of GM2 18:1;2-18:0-d9, 25 pmol of GM1 18:1;2-18:0-d5, 30 pmol of lysophosphatidic acid (LPA) 17:0, 30 pmol of lysophosphatidylcholine (LPC) 12:0, 30 pmol of lysophosphatidylethanolamine (LPE) 17:1, and 30 pmol of LPS 17:1 and subjected to lipid extraction at 4°C, as described elsewhere (78). Briefly, the sample was dissolved in 200 μL of 155 mM ammonium bicarbonate and then extracted with 1 mL of chloroform-methanol (10:1) for 2 hours. The lower organic phase was collected, and the aqueous phase was re-extracted with 1 mL of chloroform-methanol were detected as deprotonated ions in the 2:1 extract in negative ion mode after polarity switch in FTMS by scanning m/z = 1,100-1,650 Da, at Rm/z=200= 280,000

with lock mass activated at a common background (m/z = 1,175.7768) for 30 s. Every scan is the average of two micro-scans, AGC was set to 1E6 and IT was set to 50 ms.

PA, PI, PS, LPA and LPS were detected as deprotonated ions in the 2:1 extract in negative ion mode in FTMS by scanning m/z = 520-1,100 Da, at Rm/z=200 = 280,000 with lock mass activated at a common background (m/z = 529.4626) for 30 s. Every scan is the average of two micro-scans, AGC was set to 1E6 and IT was set to 50 ms.

All data were acquired in centroid mode.

All data were analysed with the lipid identification software, LipidXplorer (Herzog et al.

Genome Biology 2011, 12:R8). Tolerance for MS identification was set to 2 ppm. Data post-processing and normalization to internal standards were done manually.

Migration and invasion assays

Migration assays of Hs578t cells were performed by seeding 300,000 cells in each well of a 24-well plate 24 h before the experiment. The cells were incubated with 1 μg/ml of EVs 37 °C for 4 h. After incubation, a scratch was done in the middle of each well with a pipette tip, cells were washed 1 time with PBS and incubated with FBSfree culture medium. Cell migration was monitored using time-lapse microscopy (IncuCyte Live Cell Analysis Systems, 4x objective lens, Sartorius) with an interval of 3 h during 21 h. Image analysis was performed with ImageJ using a wound healing size tool [START_REF] Suarez-Arnedo | An image J plugin for the high throughput image analysis of in vitro scratch wound healing assays[END_REF].

Invasion assays were performed in 8 μm pore size 24-well inserts (Greiner bio-one) pretreated with Matrigel (BD Biosciences) according to the manufacturer's instructions. Briefly, the inserts were coated with Matrigel and were left in a 37°C

incubator for 30 minutes before seeding. Then, 300,000 Hs578t cells previously treated with 1 μg/ml of EVs during 24 h were resuspended in serum-free medium and added to the top of each chamber insert, and medium with FBS as a chemo-attractant was added to the bottom chamber. After 24 h, inserts were removed, washed and cells that migrated to the lower side of the inserts were stained with 0.1% toluidine blue, washed, and counted in an inverted microscope.

Statistical analysis

All analyses were performed using GraphPad Prism version 6.0 to 8.0, (GraphPad Software). Two-tailed (paired or unpaired) Student's t-tests were used when comparing only two conditions. For more than two conditions, the Kruskal-Wallis test, ordinary one-way ANOVA or two-way ANOVA were used. Significantly different comparisons of means are marked on the graphs with asterisks. Error bars denote SEM or SD. Toxicology, 32 [START_REF] Jns ; Spadari | In vivo distribution and activity of aphidicolin on dividing and quiescent cells[END_REF]

Figures Legends

Further investigating the involvement of Cav1 in the dynamics of EVs

Effect of cancer-derived EVs on CAF-mediated immunosuppression

Among the various components of the TEM, CAFs have emerged as key players in cancer progression. CAFs are a type of activated fibroblast that interact with cancer cells [START_REF] Gascard | Carcinoma-associated fibroblasts: orchestrating the composition of malignancy[END_REF]. CAFs act as master regulators of the TME, through the release of soluble factors, regulation of extracellular matrix composition and involvement in cell-to-cell communication, all contributing to the progression of cancer [START_REF] Kalluri | The biology and function of fibroblasts in cancer[END_REF].

CAFs are involved in the secretion of a range of growth factors such as TGFβ, fibroblast growth factor (FGF), Platelet-derived growth factor (PDGF), and hepatocyte growth factor (HGF), along with cytokines and chemokines that activate cancer cell growth [START_REF] Mhaidly | Fibroblast heterogeneity in tumor micro-environment: Role in immunosuppression and new therapies[END_REF]. They also promote angiogenesis, alter ECM properties, as well as enhance the invasiveness and migration of cancer cells by producing proteases such as MMPs and cathepsins which remodel the ECM [START_REF] Xouri | Origin and function of tumor stroma fibroblasts[END_REF].

CAFs also induce epithelial-mesenchymal transition (EMT), which is essential in promoting cancer stem cell-like features and often leads to increased metastasis [START_REF] Jia | Cancer-associated Fibroblasts induce epithelial-mesenchymal transition via the Transglutaminase 2dependent IL-6/IL6R/STAT3 axis in Hepatocellular Carcinoma[END_REF]).

CAF's contribution to cancer cell invasion and metastasis is further facilitated

by their ability to communicate with immune cells. They modulate the recruitment, differentiation, and function of immune cells present in the TME, promoting an immunosuppressive environment that favors cancer progression [START_REF] Mhaidly | Fibroblast heterogeneity in tumor micro-environment: Role in immunosuppression and new therapies[END_REF].

Recent studies have shown that cancer cells can transfer EVs to CAFs, and vice versa, thereby facilitating communication [START_REF] Naito | Intercellular crosstalk between cancer cells and cancer-associated fibroblasts via extracellular vesicles[END_REF]. These EVs can carry bioactive molecules that modulate the behavior and function of recipient cells (Figure 17). EVs from CAFs have been shown to influence breast cancer cell behavior through the transfer of proteins and miRNA to promote EMT and facilitate metastasis by promoting phenotypes such as increased migration, proliferation and invasion [START_REF] Chen | Aberrant low expression of p85α in stromal fibroblasts promotes breast cancer cell metastasis through exosome-mediated paracrine Wnt10b[END_REF][START_REF] Liu | MiR-3613-3p from carcinomaassociated fibroblasts exosomes promoted breast cancer cell proliferation and metastasis by regulating SOCS2 expression[END_REF][START_REF] Luga | Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration[END_REF]. Conversely, EVs derived from cancer cells can promote the activation and transformation of CAFs into a more supportive phenotype for tumor growth and metastasis.

Extensive research has led to the characterization and comparison of four subtypes of CAFs in breast cancer, with the identification of a specific subpopulation of CAFs, which express the fibroblast activation protein (FAP) marker, that are linked to immunosuppression and resistance to immunotherapy [START_REF] Feig | Targeting CXCL12 from FAP-expressing carcinomaassociated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer[END_REF]. This specific subset of CAFs, now called CAF-S1, were shown to increase attraction, survival, and population of CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) in the tumor microenvironment through the release of CXCL12 and upregulation of different molecules including OF40L, JAM2, PD-L2, CD73, DPP4 and B7H3, leading to immunosuppression [START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF] (Figure 18). Furthermore, using single-cell RNA sequencing on CAF-S1, they found clusters of myofibroblasts that express high levels of extracellular matrix proteins and modulators of the immune system, including the immunosuppressive cytokine TGF-β. These particular CAFs create a localized immunosuppressive environment that enables tumor growth and metastasis [START_REF] Kieffer | Single-Cell Analysis Reveals Fibroblast Clusters Linked to Immunotherapy Resistance in Cancer[END_REF]. 19B). This increased immunosuppressive phenotype of CAFs observed after pre-treatment with mechanically stressed cells is in line with previous studies. In a liver cancer model, it was showed that EVs released from cancer cells subjected to shear stress, were able to activate CAFs by upregulating the expression of FAP, and alpha-smooth muscle actin (α-SMA) through the activation of insuling growth factor (IGF) pathway [START_REF] Feng | Fluid Shear Stress-Induced Exosomes from Liver Cancer Cells Promote Activation of Cancer-Associated Fibroblasts via IGF2-PI3K Axis[END_REF]. These data demonstrate that mechanically stressed-induced EVs from cancer cells are more efficient to activate CAFs than EVs from cancer cells in resting conditions. Additionally, these EVs were also found to induce endothelial-to-mesenchymal transition and convert endothelial cells to CAFs through the TGF-β/Smad pathway in gastric cancer in vivo [START_REF] Li | Exosomal miR-21-5p derived from gastric cancer promotes peritoneal metastasis via mesothelial-to-mesenchymal transition[END_REF]. The role of cancer derived EVs in CAF activation has also been reported in breast cancer models. EVs released by breast cancer cells and containing microRNA miR-105 had the ability to induce a specific metabolic program in CAFs, which included enhancing glucose and glutamine metabolism to provide nutrients to adjacent cancer cells [START_REF] Yan | Cancer-cell-secreted exosomal miR-105 promotes tumour growth through the MYCdependent metabolic reprogramming of stromal cells[END_REF]. Furthermore, triple negative breast cancer cell-derived EVs can convert normal fibroblasts into CAFs, promoting tumor growth in vivo [START_REF] Baroni | Exosome-mediated delivery of miR-9 induces cancerassociated fibroblast-like properties in human breast fibroblasts[END_REF].

Counterintuitively, our data shows that EVs from breast cancer cells do not enhance the immunosuppressive effect of CAFs over T cells. This may be explained by the fact that our model for the evaluation of immunosuppression involves the use of the already activated and immunosuppressive CAFs-S1 purified from breast cancer samples. Moreover, our co-culture model is devoid of cancer cells, therefore, any element of the secretome of cancer cells that may contribute to the CAF mediated immunosuppression is absent. We speculate that using a subpopulation of CAF that is not immunosuppressive per se, as is the case of CAF-S4 for example [START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF], we might find that breast cancer EVs act as expected and activate these CAFs towards a more immunosuppressive phenotype.

Overall, our results indicate that EVs from cancer cells, particularly those derived from mechanically stressed cells, can influence the pre-disposed immunosuppressive phenotype of CAFs. Moreover, Cav1 may contribute to the modulation of this effect, although other factors may also be involved.

Further research is needed to fully understand the mechanisms underlying these findings and explore their potential implications in cancer immunotherapy.

Role of Cav1 in EV uptake by receiving cells

EVs are now well known to be essential mediators of intercellular communication, delivering a plethora of bioactive molecules to recipient cells, including proteins and genetic material [START_REF] Tkach | Communication by Extracellular Vesicles: Where We Are and Where We Need to Go[END_REF]. The biological outcomes of this cell-to-cell communication is determined by the efficient uptake of EVs by recipient cells.

This intricate process is thought to involve various pathways and sequential steps, including docking and signaling, EV-plasma membrane fusion or intact EV uptake, and EV degradation or fusion inside endosomes to finally release its contents in the cell (Figure 20). Understanding these mechanisms enables us to dissect the intricate interplay between EVs and recipient cells, providing critical insights into their physiological and pathological implications [START_REF] Russell | Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop[END_REF]. The initial step of EV uptake involves the interaction between surface molecules on EVs and specific receptors or ligands on the membrane of the recipient cell. This docking process enables the attachment and binding of EVs to the recipient cells, followed by internalization. Experiments based on treatment of EVs with proteinase K showed a notable decrease in their uptake by cancer cells, indicating the crucial role of proteins in EV uptake [START_REF] Escrevente | Interaction and uptake of exosomes by ovarian cancer cells[END_REF]. Different types of proteins have been identified as key regulators of EV uptake. Tetraspanins, which are abundant in EVs, have been suggested to interact with various receptors on recipient cells.

Tetraspanins facilitate EV binding and uptake and mediate the targeted delivery of EVs to specific cells or tissues. For instance, the tspan8-ITGα4 complex found on cancer EVs binds to CD54 in recipient cells specifically directing EVs to endothelial and pancreatic cells, while EVs carrying the tetraspanins CD9 and CD81, in conjunction with CD11a, CD54 and phosphatidylserine are able to specifically bind to dendritic cells expressing αvβ3 integrin [START_REF] Morelli | Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells[END_REF][START_REF] Rana | Toward tailored exosomes: the exosomal tetraspanin web contributes to target cell selection[END_REF]. Notably, tetraspanins interact with a variety of receptors on recipient cells, including integrins.

Activated T cells and dendritic cells can capture EVs through expression of

the integrin LFA-1 (Nolte-'t [START_REF] Nolte-'t Hoen | Activated T cells recruit exosomes secreted by dendritic cells via LFA-1[END_REF][START_REF] Segura | CD8+ dendritic cells use LFA-1 to capture MHC-peptide complexes from exosomes in vivo[END_REF]. Inhibition of αvβ3 integrin binding, using a specific inhibitor, disrupted tumor EV adhesion and uptake by epithelial cells [START_REF] Altei | Inhibition of αvβ3 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles[END_REF]. Intriguingly, this integrin was found to be present on the EVs rather than on the recipient cell surface, adding a layer of complexity to the process. Depletion of integrins ITGβ4 and ITGβ5 in EV-secreting cells resulted in a sharp decrease of the tropism of these EVs towards the lung and liver, respectively. EVs containing ITGβ5, when administered to cells, resulted in an upregulation of several prometastatic S100 genes which were not upregulated in ITGβ5-depleted EVs (Hoshino et al., 2015). These findings suggest that downstream signaling pathways may be triggered after EV binding to the plasma membrane.

EV-bound proteins activate signaling cascades within recipient cells, triggering intracellular events that modulate the cellular response to EV uptake. These signaling events can involve the recruitment of adaptor proteins, activation of downstream kinases, and modulation of cytoskeletal dynamics. EVs have been shown to carry components of the Wnt signaling pathway, such as Wnt protein and Frizzled receptor, which can activate the Wnt signaling pathway in recipient cells upon EV uptake, leading to diverse cellular responses [START_REF] Korkut | Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless[END_REF]. Studies have demonstrated that glioblastoma-derived EVs activate ERK 1/2 and HSP27 downstream signaling. This activated signaling pathway serves as a positive feedback loop that enhances the uptake of EVs [START_REF] Svensson | Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid raft-mediated endocytosis negatively regulated by caveolin-1[END_REF].

Following docking, EVs can directly fuse with the recipient cell plasma membrane, merging their lipid bilayers and delivering their cargo directly into the cytoplasm. This process relies on the interaction between specific fusogenic proteins on the EV and recipient cell membranes. Alternatively, EVs can be internalized via endocytic pathways, ultimately leading to the formation of endosomes. Once within the endosomes, EVs can fuse with the endosomal membrane, releasing their contents into the cytoplasm [START_REF] Mulcahy | Routes and mechanisms of extracellular vesicle uptake[END_REF].

When EVs are not able to fuse directly with the plasma membrane of recipient cells, several processes are responsible for EV uptake, including phagocytosis, macropinocytosis, clathrin-mediated endocytosis, and caveolinmediated endocytosis [START_REF] Delenclos | Investigation of Endocytic Pathways for the Internalization of Exosome-Associated Oligomeric Alpha-Synuclein[END_REF]. Studies have shown that pharmacological inhibition of the caveolar protein Cav1 leads to a reduction in EV uptake in lung cancer and pancreatic cells [START_REF] Javeed | Pancreatic Cancer-Derived Exosomes Cause Paraneoplastic β-cell Dysfunction[END_REF][START_REF] Wei | Exosomes derived from gemcitabine-resistant cells transfer malignant phenotypic traits via delivery of miRNA-222-3p[END_REF]. Interestingly, in a model of ischemia, hippocampal neurons upregulate the expression of Cav1 to capture EVs and generate a neuroprotective effect, mitigating ischemia-induced apoptosis [START_REF] Yue | Neurons can upregulate Cav-1 to increase intake of endothelial cells-derived extracellular vesicles that attenuate apoptosis via miR-1290[END_REF]. Often, Cav1-dependent EV uptake has been called caveolae-dependent endocytosis by some researchers, without extensive exploration into the mechanism of this endocytosis pathway [START_REF] Nanbo | Exosomes derived from Epstein-Barr virus-infected cells are internalized via caveola-dependent endocytosis and promote phenotypic modulation in target cells[END_REF]. However, contradictory results have been reported regarding the role of Cav1 in EV uptake. One study found that Cav1 negatively regulates EV uptake by suppressing ERK1/2 signaling, and downregulating Cav1 actually increased EV uptake [START_REF] Svensson | Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid raft-mediated endocytosis negatively regulated by caveolin-1[END_REF]. These discrepancies suggest that specific proteins or molecules on the surface of EVs might be necessary for their uptake, indicating the possibility of targeted or specific uptake by recipient cells. It is important to note that the often use of non-Cav1 specific pharmacological inhibitors, such as filipin (a cholesterol sequestering probe), dynasore (a dynamin inhibitor), or nystatin (a sterol binding agent), may provide results that reflect the impairment of membrane integrity rather than the effect of Cav1 deficiency on EV uptake.

Therefore, our objective was to investigate the role of caveolae and /or Cav1 in EV uptake in cancer cells using genome edited cells. The following experiments were performed in collaboration with Dr. Gregory Lavieu.

To test the role of Cav1 in EV uptake we used two different engineered HeLa cells lines as EV donors, one stably expressing NanoLuc luciferase-tagged Hsp70. Hsp70 is a known EV cargo that in combination with the high sensitivity engineered NanoLuc reporter in EVs allows to monitor EV uptake in recipient cells with a high signal-to-noise ratio as already reported [START_REF] Bonsergent | Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells[END_REF], and one cell line stably expressing a GFP-tagged Hsp70 that allows us to follow the uptake of GFP positive-EVs by immunofluorescence [START_REF] Bonsergent | Content release of extracellular vesicles in a cell-free extract[END_REF]. As receiving cells, we used the previously used WT HeLa cells. We measured the levels of Cav1 and cavin1 in the receiving WT HeLa cells, and western blot analysis showed that Cav1 was expressed at very low levels. We took advantage of this low expression to investigate the effect of different levels of caveolar components on EV uptake. For this, we overexpressed Cav1 and/or cavin1 in these cells (Figure 21A). Remarkably, we observed that overexpression of Cav1 alone or with cavin1 increased EV uptake significantly. Overexpression of cavin1 in receiving cells had no effect on EV uptake (Figure 21B).

It is worth noting that the kinetics of EV uptake followed an inverse exponential-like function, with a constant EV uptake rate (~1% spontaneous rate per 1 h) that persists for 6 to 7 hours of incubation, until it reaches a plateau thereafter. This kinetics are in line with previous results based on the same construct to monitor EV uptake [START_REF] Bonsergent | Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells[END_REF].

The highest amount of internalized EVs was observed when Cav1 was overexpressed alone. Cavin1 being required for caveolae morphogenesis and stabilization at the plasma membrane, suggesting that the increase of EV uptake by receiving cells is likely due to an increase in the pool of noncaveolar Cav1, rather than an increase in the overall number of caveolae (Figure 21B).

To verify this hypothesis, we evaluated the uptake of EVs by cavin1 -/-MEF cells expressing high or low levels of Cav1. In the absence of cavin1, these cells cannot assemble caveolae. We found that the uptake of HeLa derived

EVs was significantly increased in receiving MEF cells with higher levels of Cav1 (Figure 21C). Thus, it confirms that the efficiency of uptake is correlated with the level of non-caveolar Cav1 present in receiving cells.

Finally, we measured the uptake of GFP-tagged EVs in Cav1 -/-or cavin1 -/- receiving HeLa cells. In line with our previous results, we observed that depletion of Cav1 significantly reduces the uptake of EVs compared to WT cells, further confirming the crucial role of Cav1 in this process. Cavin1 depletion reduced slightly the uptake of EVs, yet this reduction was not significant. Considering that downregulation of cavin1, which is cotranscriptionally regulated with Cav1, leads to downregulation of Cav1 as well, we believe that it explains the reduced uptake of EVs in cavin1 depleted cells (Hill et al., 2008) (Figure 21D).

Caveolae have long been associated with endocytosis, not only of EVs but also of other molecules [START_REF] Pelkmans | Secrets of caveolae-and lipid raft-mediated endocytosis revealed by mammalian viruses[END_REF]. There has however been a recent change of paradigm based on several studies showing that caveolae are quite static at the plasma membrane and do not efficiently endocytose cargo. Also, it has become evident that there are no specific cargoes that depend strictly on caveolae for endocytosis. Caveolae endocytosis would mainly serve as a means to control their density at the plasma membrane. The consensus to date is that endocytosis is not their primary function [START_REF] Parton | Caveolae: The FAQs[END_REF]. Our results therefore confirm this assumption as the uptake of EVs does not rely on caveolae, but on the level of non-caveolar Cav1.

Outside of caveolae, Cav1 has been shown to form oligomerized microdomains called Cav1 scaffolds. These stable structures can be present in different sizes (S1A, S1B, and S2) and are proposed to have signaling properties similar to the ones associated with Cav1, including signaling and endocytosis [START_REF] Khater | Superresolution modularity analysis shows polyhedral caveolin-1 oligomers combine to form scaffolds and caveolae[END_REF][START_REF] Lajoie | Lattices, rafts, and scaffolds: domain regulation of receptor signaling at the plasma membrane[END_REF]. It has been proposed that these Cav1 scaffolds could modulate clathrin-independent endocytosis through an indirect mechanism by modifying the lipid composition of raft nanodomains [START_REF] Lajoie | Regulation of raft-dependent endocytosis[END_REF]. Furthermore, cells lacking Cav1 or cavin-1 show an upregulated clathrin-independent carriers/GPI-AP enriched early endosomal compartment (CLIC/GEEC) endocytic pathway, conversely, upregulating Cav1 or Cav3 results in the inhibition of the CLIC/GEEC endocytic pathway [START_REF] Chaudhary | Endocytic crosstalk: cavins, caveolins, and caveolae regulate clathrin-independent endocytosis[END_REF]. We cannot rule out the possibility that these pathways might be involved in EV uptake.

Cav1 has been shown to recruit specific species of lipids in the plasma membrane, including phosphatidylserine, sphingomyelin and certain phospholipids, creating particular lipid nanodomains (Prakash et al., 2021;Sonnino & Prinetti, 2009). These nanodomains at the plasma membrane can differentially cluster transmembrane proteins, proteins with specific binding motifs for cholesterol, or proteins with preferential affinities towards certain features like acyl chains [START_REF] Harayama | Beyond Fluidity: The Role of Lipid Unsaturation in Membrane Function[END_REF][START_REF] Sezgin | The mystery of membrane organization: composition, regulation and roles of lipid rafts[END_REF].

We can hypothesize that the lipid-protein nanodomains formed by Cav1 scaffolds could cluster specific species of proteins in the recipient cell membrane that can interact with surface molecules on the EV favoring docking, fusion, or clathrin-independent endocytosis, resulting in enhanced uptake. Additional experiments are needed to test this hypothesis.

Altogether, our results reveal the critical role played by Cav1 in the enhancement of the uptake of EVs by receiving cells. The kinetics of EV uptake and the limited role of cavin1 suggest a more nuanced regulation of this process, which may involve other factors that require further investigation.

MATERIALS AND METHODS

Cell lines

HeLa, MEF and MDA-MB-231 cells and sublines were cultured in Dulbecco's modified Eagle's medium (DMEM Glutamax, Gibco), with 10% FBS (FBS, Gibco), and 100U/ml penicillin and 100 μg/ml streptomycin (Gibco). Cell lines were grown at 37°C, under 5% CO2.

Antibodies and plasmids

Primary antibodies used include rabbit anti-cav1 (BD

Mechanical stress models

To induce swelling of cells in 2D cultures we used hypo-osmotic shock. Hypoosmotic shock was performed on cells by using the corresponding growth medium diluted appropriately in deionized water (1:9 dilution for 30 mOsm hypo-osmotic shock).

EV isolation

For the hypo-osmotic shock experiments, 6 × 10 6 of cells were seeded per T75 culture flask. After 24 h, cells were washed 1 X with PBS (Gibco) and 12 ml of serum free iso or hypo-osmotic culture media. Media was changed after 5 min to iso-osmotic, serum free, culture media and incubated for the corresponding time periods. Culture supernatant was collected and was subjected to serial centrifugations (2,000 g for 10 min, 11,000 g for 30 min at 4°C), followed by ultracentrifugation at 100,000 g for 90 min at 4°C (45Ti or TLA110 rotors, Beckman Coulter). The pellet containing EVs was washed in cold 1x PBS (Gibco) and ultracentrifuged again at 100,000 g for 90 min at 4°C.

EV characterization by NTA

NTA was performed using ZetaView PMX-120 (Particle Metrix) equipped with a 488 nm laser, with software version 8.05.02 to measure the concentration and distribution size of particles by evaluating the Brownian motion in a light scattering system. The samples were diluted in 1X PBS (Gibco) to obtain a concentration of 10 6 to 10 9 particles/ml per field.

CAF immunosuppressive phenotype characterization

Fresh human samples (tumors and juxta-tumors) were obtained from the operating room, after the specimen's macroscopic examination and selection of areas of interest for diagnosis by a pathologist. BC samples were cut into small fragments (around 1 mm3) and digested in CO2-independent medium (Gibco, #18045-054) supplemented with 5% fetal bovine serum (FBS, PAA, #A11-151), 2 mg/ml collagenase I (Sigma-Aldrich, #C0130), 2 mg/ml hyaluronidase (Sigma-Aldrich, #H3506) and 25 μg/ml DNase I (Roche, #11284932001) for 45 min at 37°C with shaking (160 rpm). After tissue digestion, cells were filtered using a cell strainer (40 μm, Fischer Scientific, #223635447) and resuspended in PBS (Gibco, #14190) supplemented with 2 mM EDTA (Gibco, #15575) and 1% Human serum (BioWest, #S4190-100) to a final concentration at approximately 5x105 cells in 50 μl. EVs were added on top of the CAFs in serum-free media for a final concentration of 1 μg protein/mL. After 24 hours, CAFs were washed with 1x PBS (Gibco) and X00,000 CD4+ CD25+ T cells were seeded on top. After 24 hours of coculture, cells were prepared for flow cytometry analyses.

Western blotting

Cell extracts were separated by SDS-PAGE, transferred to nitrocellulose, blocked in 1x PBS containing 3% BSA or 5% nonfat milk according to the antibody used. Membranes were probed overnight at 4°C. Bound antibodies were developed using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific) and the ChemiDoc Touch Imaging System (BioRad). Protein bands were quantified by densitometric analysis using the ImageJ.

Uptake assays

For the NanoLuc-Hsp70 uptake assay, acceptor cells were seeded 24 h before the uptake experiment, at 20,000 cells per well in a 96-well plate.

NLuc-Hsp70 EV input was added in serum-free DMEM for a final concentration of 1 μg protein/mL. Cells were incubated with EVs at 4 °C for 0-16 h. After incubation, cells were washed with PBS and then lysed in lysis buffer, prior to transferring in white 96-well plates. Finally, 50 μL of Nano-GloTM reagent (Promega, Wisconsin, USA) was added on each well and luminescence activity was read using iD3 SpectraMax microplate reader (Molecular Devices, California, USA) or Centro LB 960 microplate luminometer (Berthold, Germany).

For the GFP-Hsp70 uptake assay, acceptor cells were seeded 24 h before the uptake experiment, at 100,000 cells per well in a 24-well plate on the top of coverslips. Acceptor cells were incubated with GFP-Hsp70 EVs at a final concentration of 1 μg protein/mL for up to 16 h at 37 °C. Then cells were washed with PBS, fixed 15 min RT with PBS 4% Paraformaldehyde and permeabilized 30 min RT in PBS 1% bovine serum albumin (BSA) 0.1%

Triton-X-100. Immunofluorescence was performed as already described.

Image analysis and colocalization quantification were performed using ImageJ software (NIH, Maryland, USA).

Immunofluorescence

After the treatments, cells were fixed with 4% paraformaldehyde (EMS) for 15 min at room temperature. Cells were incubated for 1h in a blocking solution: 1x PBS with 0.1% triton and 0.3% BSA. Then primary and secondary antibodies were successively incubated during 1 h each at RT in 1x PBS containing 0.1% triton and 0.1% BSA. Coverslips were then mounted on microscope slides with Fluoromount G (Invitrogen). Images were acquired on a Zeiss LSM 780 confocal microscope with a 63x/1.46 Oil objective. At least 10 cells per replicate were imaged.

Statistical analysis

All analyses were performed using GraphPad Prism version 6.0 to 8.0, (GraphPad Software). Two-tailed (paired or unpaired) Student's t-tests were used when comparing only two conditions. For more than two conditions, the Kruskal-Wallis test, ordinary one-way ANOVA or two-way ANOVA were used.

Significantly different comparisons of means are marked on the graphs with asterisks. Error bars denote SEM or SD.

DISCUSSION

Mechanical strains and EV secretion

We were able to demonstrate in a robust manner the effect of mechanical stress on EV secretion. Both 2D osmotic shock and 3D compression models of mechanical strain, as demonstrated in our study, caused an increase in the secretion of EVs from HeLa, Hs578t and MDA-MB-231 human cancer cells, moreover, we show for the first time that this mechanically induced increase in EV secretion is Cav1 dependent.

Various systems have been identified to stimulate EV secretion, molecular disruption in the forms of small molecule modulators or inhibition of lysosomal pathways, as well as external factors and inducers such as starvation or hypoxia have been shown to increase EV secretion (Figure 22) [START_REF] Wang | Engineering Extracellular Vesicles as Delivery Systems in Therapeutic Applications[END_REF]. Physical perturbations have also been shown to stimulate EV secretion, with shear stress being a reliable and scalable method. For instance, cells grown on 3D scaffolds and subjected to fluid shear stress (0.5-6 mL/min direct flow) showed up to a 100-fold increase in the number of EVs detected by NTA after incubation in a bioreactor. However, analysis of EV cargo indicates that total protein content can decrease after mechanical stress [START_REF] Guo | Stimulating Extracellular Vesicles Production from Engineered Tissues by Mechanical Forces[END_REF][START_REF] Patel | Enhanced extracellular vesicle production and ethanol-mediated vascularization bioactivity via a 3D-printed scaffold-perfusion bioreactor system[END_REF]. Another model of mechanical stress, cell stretching, has been shown to increase the secretion of a subpopulation of EVs with an altered miRNA cargo and differential activity on recipient cells. This subpopulation has been observed to promote the induction of proliferation and myogenic differentiation on myoblasts, or to inhibit bone marrow macrophage osteoclast differentiation via inhibition of signaling pathways [START_REF] Mullen | Mechanical strain drives exosome production, function, and miRNA cargo in C2C12 muscle progenitor cells[END_REF][START_REF] Xiao | Exosomes derived from cyclic mechanical stretchexposed bone marrow mesenchymal stem cells inhibit RANKL-induced osteoclastogenesis through the NF-κB signaling pathway[END_REF].

One reason for the recent increased interest in boosting EV secretion is their potential as therapeutic agents. In fact, in the last few years, multiple novel EVbased therapies have been developed as an alternate strategy to cell therapies for treating diverse medical conditions. These therapies offer unique advantages, including lower immunogenicity, improved safety and regulatory aspects, and the ability to serve as readily available products. However, to achieve industrial-scale production of EVs, poor yield and scalability must be resolved [START_REF] Debbi | Boosting extracellular vesicle secretion[END_REF]. To this end, several reports have already characterized the therapeutic properties of large scale mechanical stress induced production of EVs. Stem cells grown on bioreactors have been reported to secrete EVs with altered miRNA and cytokine cargo that are able to confer specific therapeutic properties to receiving cells such as increased angiogenesis, neurogenesis and axonal sprouting [START_REF] Cha | Efficient scalable production of therapeutic microvesicles derived from human mesenchymal stem cells[END_REF][START_REF] Guo | Stimulating Extracellular Vesicles Production from Engineered Tissues by Mechanical Forces[END_REF]. Furthermore, EVs secreted by natural killer cells grown in bioreactors were functionally active in killing melanoma and liver cancer cells in both 2D and 3D culture conditions in vitro, as well as suppressing melanoma growth in vivo (Wu et al., 2022). Despite the advances in understanding the effects of mechanical stress on EV secretion, the underlying mechanism of this increase remains poorly understood. Although there are several documented effects of mechanical perturbations, alterations in the endosomal pathway, changes in membrane properties, and alterations in the cytoskeleton dynamics, the exact mechanism of EV secretion under mechanical stress remains elusive.

Of note, some reports have highlighted the potential relationship between increased EV secretion and the Yes-associated protein (YAP) pathway. The YAP pathway is a key signaling pathway involved in mechanotransduction, translating mechanical cues into cellular responses. Activation of the YAP pathway has been linked to various cellular processes, including proliferation, differentiation and has been implicated in various diseases, including cancer [START_REF] Cunningham | The Hippo pathway in cancer: YAP/TAZ and TEAD as therapeutic targets in cancer[END_REF].

A recent study found that breast cancer cells grown in stiff substrates present a 2.5-fold increase in EV secretion compared to cells grown in soft substrates,

showing that this effect is dependent on the transcriptional co-activator YAP, which is regulated by the Hippo signaling pathway and is known for its role in mechanotransduction and transcriptional activation of genes. By inhibiting YAP interaction with TEAD and its translocation to the nucleus using the pharmacologic inhibitor Verteporfin it was observed that the increased EV secretion was absent. Moreover, these EVs were found to confer pro-migratory and invasiveness traits to receiving cells [START_REF] Patwardhan | Biomaterials ECM stiffness-tuned exosomes drive breast cancer motility through[END_REF]. Another study that used shear forces to induce increased EV secretion found that upon mechanical stress YAP is robustly translocated to the nucleus, and inhibition of this translocation with Verteporfin reduced the number of secreted EVs [START_REF] Guo | Stimulating Extracellular Vesicles Production from Engineered Tissues by Mechanical Forces[END_REF].

In all these studies, the increased EV secretion was evaluated after long times of exposure to mechanical strains (1-3 days), whereas in our models of mechanical strain, we observed that the increased EV secretion happens in as short time as 1 hour after as evaluated by NTA. Furthermore, the increased MVB-PM fusion activity observed in our experiments suggest an increase in the secretion of exosomes just minutes after the mechanical stress. Considering this, we don't believe the translocation of YAP and subsequent gene transcription regulation is involved in this rapid reaction. Nevertheless, we could hypothesize that the YAP dependent regulation of increased EV secretion could act as a secondary "line of defense" to sustained mechanical strains the cell could be subjected to.

Recently, the interaction between the YAP pathway and caveolae has been described to be important to respond to mechanical strains. YAP has been shown to be key mediators of the gene expression of both Cav1 and cavin1 proteins, without YAP, the cell is not able to form caveolae at the plasma membrane. Moreover, the activation of YAP is triggered by the caveolarmediated response to shear stress (Figure 23) [START_REF] Rausch | The Hippo Pathway Regulates Caveolae Expression and Mediates Flow Response via Caveolae[END_REF]. Analyzing the potential involvement of the hippo pathway in our models of mechanical stress could be of interest.

Caveolae-free Cav1, at the plasma membrane and beyond

We show that upon disassembly of caveolae, triggered by mechanical stress, a population of Cav1 translocates from the plasma membrane towards endosomal compartments. At the same time, we demonstrated that both Cav1 and the ESCRT-0 complex are strictly required for the increased EV release after mechanical stress. Yet the precise mechanism by which Cav1

translocates to the MVB and acts together with ESCRT-0 is not yet fully understood.

Phosphorylation of Cav1 has been identified as a critical regulatory mechanism that affects various cellular processes. Among the different phosphorylation events, the specific phosphorylation of tyrosine 14 (Y14) on Cav1 has received significant attention due to its functional implications in multiple cellular functions. The phosphorylation of Y14 in Cav1 has been associated with cell migration, dynamics of focal adhesions, disassembly of caveolae, and subsequent endocytosis, among other processes [START_REF] Joshi | Phosphorylated caveolin-1 regulates Rho/ROCK-dependent focal adhesion dynamics and tumor cell migration and invasion[END_REF].

Interestingly, Y14 phosphorylation of Cav1 has been observed in models of mechanical strain, as is the case of cyclic stretching, where this phosphorylation plays a crucial role in activating downstream signaling pathways and promoting the expression of caveolar genes, resulting in an increase in caveolae numbers at the plasma membrane [START_REF] Joshi | Phosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulation[END_REF][START_REF] Zhang | Caveolin-1 phosphorylation is required for stretch-induced EGFR and Akt activation in mesangial cells[END_REF]. Furthermore, when exposed to shear flow, Cav1 is phosphorylated at Y14 and is involved in facilitating actin reorganization [START_REF] Radel | Integrin mechanotransduction stimulates caveolin-1 phosphorylation and recruitment of Csk to mediate actin reorganization[END_REF]. In our models of mechanical strain, we did not observe an increase in Y14 phosphorylation. We hypothesize that this discrepancy is due to the type of mechanical strain utilized in our models of 2D osmotic shock and 3D compression, though achieving similar caveolae disassembly outcomes, may trigger distinct downstream events leading to altered Y14 phosphorylation dynamics. Additionally, the cell type studied may also play a role in contributing to the observed differences in Cav1 phosphorylation dynamics following mechanical strain.

This dynamic regulation of caveolae turnover through Y14 phosphorylation provides cells with the ability to modulate the abundance of caveolae on the plasma membrane, thereby influencing intracellular trafficking and downstream signaling. Importantly, Y14 phosphorylation of Cav1 has been implicated in key aspects of tumor dynamics, including enhanced growth, motility, adhesion, and increased metastatic potential in cancer cells [START_REF] Goetz | Concerted regulation of focal adhesion dynamics by galectin-3 and tyrosine-phosphorylated caveolin-1[END_REF][START_REF] Joshi | Phosphorylated caveolin-1 regulates Rho/ROCK-dependent focal adhesion dynamics and tumor cell migration and invasion[END_REF][START_REF] Ortiz | Extracellular matrix-specific Caveolin-1 phosphorylation on tyrosine 14 is linked to augmented melanoma metastasis but not tumorigenesis[END_REF].

In ILVs in endolysomal structures, therefore, serving as a signal for its degradation via the proteasome pathway. Mutation of these specific N-terminal ubiquitination sites resulted in enhanced stability of Cav1 (Hayer et al., 2010).

Another study shed light on the involvement of ubiquitination in the sorting of cargo into ILVs within MVBs. It was found that ubiquitination of Cav1 in the Nterminal region acts as a signal for its sorting into the ILVs. Again, mutations in the ubiquitination sites affected the incorporation of Cav1 into the MVBs and altered the sorting of cargo into the ILVs, highlighting the significance of ubiquitination in intracellular trafficking and signaling [START_REF] Kirchner | Ubiquitination of the N-terminal region of caveolin-1 regulates endosomal sorting by the VCP/p97 AAA-ATPase[END_REF].

Furthermore, at the ILVs, Cav1 can aid in the sorting of specific cargo, including adhesion proteins and ECM components, to be released in exosomes [START_REF] Albacete-Albacete | ECM deposition is driven by caveolin-1-dependent regulation of exosomal biogenesis and cargo sorting[END_REF]Campos et al., 2018).

In our experimental investigation, we aimed to explore the translocation mechanism of Cav1 from the plasma membrane to endosomal compartments in response to mechanical stress. Given the importance of phosphorylation and ubiquitination modifications in regulating Cav1 dynamics, we hypothesized that these post-translational modifications could play a role in this translocation process. To investigate this, our initial approach involved assessing the levels of Cav1 Y14 phosphorylation and ubiquitination following osmotic shock.

Western blot analysis of both Y14 phosphorylation or ubiquitination of Cav1 revealed no differences at 5 or 10 minutes after mechanical stress (Figure 24).

It is important to note that both phosphorylation and ubiquitination modifications are highly dynamic events. Therefore, capturing these modifications can be challenging, making it critical to use appropriate methods and controls for their accurate detection and interpretation. To overcome these challenges associated with the dynamic nature of phosphorylation and ubiquitination modifications, we devised a strategy to address the functional consequences of these modifications on Cav1.

Specifically, we decided to express a Y14 phosphorylation mutant (Cav1-Y14F)

or an ubiquitin mutant (Cav1-K*R) form of Cav1 in Cav1 depleted cells (Hayer et al., 2010;[START_REF] Joshi | Phosphorylated caveolin-1 regulates Rho/ROCK-dependent focal adhesion dynamics and tumor cell migration and invasion[END_REF]. Following the reexpression, we subjected the cells to mechanical stress and quantitatively assessed the EV release using NTA. This experimental setup allowed us to investigate the impact of specific phosphorylation or ubiquitination modifications of Cav1 on EV release under mechanical stress conditions.

Interestingly, expression of the phosphorylation or ubiquitination mutant did not restore the cavin1 protein levels as compared to WT HeLa cells, suggesting reduced levels of caveolae at the plasma membrane (Figure 25A). We observed that when we expressed the Cav1 mutants in Cav1 -/-cells, the total number of secreted EVs detected by NTA were reduced drastically, showing a 3-fold reduction as compared to WT and Cav1 -/-HeLa cells in resting conditions and under mechanical stress (Figure 25B). The nature of this extraordinary reduction in EV release when cells were transfected with the mutant forms of Cav1 raises more questions than answers.

First, could the transfection of cells itself be affecting the release of EVs? We used a lipid based transfection reagent, this type of transfection can affect the lipid content of cells and their plasma membranes, therefore, affecting their topology and potential reaction to mechanical stress. Furthermore, transfecting cells can affect their viability, for example, long exposures to lipid based transfection reagents can induce reduced cell proliferation, toxicity due to the positively charged lipids used, and eventually, increased cell death [START_REF] Li | Lipid-based nanoparticles for nucleic acid delivery[END_REF].

As part of a post-doctoral project, we plan to further investigate the potential role of ubiquitination and phosphorylation of Cav1 in the enhanced EV secretion after mechanical stress. For example, another strategy to evaluate the involvement of ubiquitination on this process is to abolish the ubiquitination process from its start, by the use of ubiquitin-like modifier activating enzyme 1 (UBA1) inhibitors like TAK-234. This inhibitor stably binds to UBA1 and the ubiquitin substrate, preventing UBA1 from releasing ubiquitin, therefore, inhibiting all further downstream ubiquitin activation. If used in a timely manner, without provoking the over accumulation of ubiquitinated cargo that can become toxic to the cell, we will be able to evaluate if ubiquitination is a process needed for the increased EV release after mechanical stress [START_REF] Hyer | A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment[END_REF].

Exocytosis and endocytosis: A tense relationship

Regulating the physical properties of cells, including their shape and volume, is crucial for maintaining cellular homeostasis and enabling various physiological processes. Indeed, changes in membrane tension have been observed in various physiological contexts.

For example, during cell migration, the plasma membrane undergoes dynamic changes in tension to facilitate cell shape changes and movement [START_REF] Sheetz | Modulation of membrane dynamics and cell motility by membrane tension[END_REF]. This tension regulation ensures that the cells can move efficiently through tissues or substrates and respond appropriately to external physical cues [START_REF] Sens | Membrane tension and cytoskeleton organization in cell motility[END_REF]. Additionally, in processes such as cell division, changes in membrane tension are essential for proper cell shape changes during cytokinesis [START_REF] Reichl | The stress and strain of cytokinesis[END_REF].

Endocytosis and exocytosis events have emerged as crucial regulators of plasma membrane tension homeostasis. These processes work together to dynamically adjust the plasma membrane area, ensuring the maintenance of tension equilibrium at the plasma membrane. During endocytosis, vesicles bud inwards from the plasma membrane, leading to a reduction in the overall plasma membrane area and an increase in plasma membrane tension.

Conversely, during exocytosis, intracellular vesicles fuse with the plasma membrane, resulting in an increase in plasma membrane area and a decrease in plasma membrane tension. Consequently, changes in plasma membrane tension reciprocally influence exocytosis and endocytosis events. Generally, higher tension levels facilitate exocytosis, enabling the delivery of additional lipids and proteins to expand the surface area of the membrane (and conversely blocks endocytosis) [START_REF] Dai | The secretion-coupled endocytosis correlates with membrane tension changes in RBL 2H3 cells[END_REF]. On the other hand, lower tension levels promote endocytosis, removing excess membrane from the surface [START_REF] Apodaca | Modulation of membrane traffic by mechanical stimuli[END_REF][START_REF] Gauthier | Temporary increase in plasma membrane tension coordinates the activation of exocytosis and contraction during cell spreading[END_REF][START_REF] Mao | Endocytosis and exocytosis protect cells against severe membrane tension variations[END_REF].

Increases in membrane tension by hypo-osmotic shock has been characterized

to induce a two-phase reaction. Initially, when there is an increase in plasma membrane tension, cells respond by utilizing reservoirs in the form of invaginations, such as caveolae, and ruffles to expand the surface area of the membrane. Subsequently, there is a secondary reaction characterized by myosin contraction accompanied by a significant increase in vesicle exocytosis.

Importantly, the initial phase has been described to last no more than 1 minute, while the second phase can last more than 15 minutes [START_REF] Gauthier | Plasma membrane area increases with spread area by exocytosis of a GPI-anchored protein compartment[END_REF][START_REF] Gauthier | Temporary increase in plasma membrane tension coordinates the activation of exocytosis and contraction during cell spreading[END_REF].

In our experiments using hypo-osmotic shock, we identified two major events.

Firstly, we have observed that caveolae, which act as plasma membrane reservoirs, quickly sense the heightened plasma membrane tension and flatten out. This allows cells to utilize these reservoirs to increase the surface area and alleviate the increased tension (phase 1) (Figure 26A). Notably, this flattening of caveolae leads to an enrichment of non-caveolar Cav1 or Cav1 scaffolds at the plasma membrane, which have shown to be much more diffusive at the plasma membrane than caveolae, based on unpublished data from our lab.

Consequently, these Cav1 scaffolds can be subjected to endocytosis, as supported by our findings of enriched Cav1 in endosomal compartments after hypo-osmotic shock (Figure 26B).

Secondly, we have demonstrated that the surge in plasma membrane tension following hypo-osmotic shock induces an increase in the fusion of MVBs with the plasma membrane. This, in turn, leads to a sustained increase in the secretion of EVs (phase 2). Altogether, the phenotype that we observe upon hypo-osmotic shock in our experiments fits perfectly with the two-phase model of response to hypo-osmotic shock (Figure 26C). It is important to note that the results obtained from our experiments using hypoosmotic shock represent extreme conditions of membrane tension changes.

Cells in physiological conditions are unlikely to experience such drastic changes in tension. Instead, it is likely that there is a delicate balance between high and low membrane tension promoting cycles of exocytosis and endocytosis. The interplay between these processes allows cells to finely regulate membrane tension according to their physiological needs and as a result the production of sEVS. Further investigation is needed to explore the intricate dynamics of membrane tension regulation in normal cellular processes and how imbalances in this regulation may contribute to various physiological and pathological conditions, such as cancer.

CONCLUSIONS AND PERSPECTIVES

To conclude, while caveolae have historically been associated with numerous cellular functions, increasing evidence since the seminal discovery in 2011

regarding their role in cell mechanics suggests that this may be their primary function. Recent evidence has shown that Cav1 can also exist as oligomers outside of caveolae, referred to as non-caveolar Cav1 or Cav1 scaffolds [START_REF] Khater | Superresolution modularity analysis shows polyhedral caveolin-1 oligomers combine to form scaffolds and caveolae[END_REF][START_REF] Wong | Single molecule network analysis identifies structural changes to caveolae and scaffolds due to mutation of the caveolin-1 scaffolding domain[END_REF]. This paradigm shift has highlighted the need to differentiate the functions of caveolae from those of non-caveolar Cav1. Our

Introduction

The nervous system function is critically dependent on glial cells, which regulate several neuronal functions. In both the central and peripheral nervous systems (CNS and PNS, respectively), glial cells modulate nerve conduction velocity, provide metabolic support to axons and synapses, and respond to tissue injury by activating mechanisms aimed to restore homeostasis [START_REF] Jäkel | Glial cells and their function in the adult brain: a journey through the history of their ablation[END_REF][START_REF] Barros | Glia in brain energy metabolism: a perspective[END_REF]. In the PNS, regeneration of injured axons depends on the reprogramming of Schwann cells from a differentiated phenotype (dSC) to a repair Schwann cell phenotype (rSC), specialized for clearing myelin debris, recruiting macrophages, and guiding regenerating axons to their targets by secretion of neurotrophic factors and contact-mediated signaling events [START_REF] Chen | Role of macrophages in Wallerian degeneration and axonal regeneration after peripheral nerve injury[END_REF][START_REF] Gomez-Sanchez | Schwann cell autophagy, myelinophagy, initiates myelin clearance from injured nerves[END_REF][START_REF] Jessen | The repair Schwann cell and its function in regenerating nerves[END_REF][START_REF] Szepesi | Bidirectional microglia-neuron communication in health and disease[END_REF][START_REF] Jessen | Repair Schwann cell update: adaptive reprogramming, EMT, and stemness in regenerating nerves[END_REF]. In addition, neuronal responses can be modulated by the horizontal transfer of RNA and proteins by means of extracellular vesicles [START_REF] Rajendran | Emerging roles of extracellular vesicles in the nervous system[END_REF]. Astrocyte-derived extracellular vesicles regulate synaptic activity by miRNA transfer to neurons [START_REF] Chaudhuri | TNFα and IL-1β modify the miRNA cargo of astrocyte shed extracellular vesicles to regulate neurotrophic signaling in neurons article[END_REF], and microgliaderived extracellular vesicles stimulate neurotransmission [START_REF] Antonucci | Microvesicles released from microglia stimulate synaptic activity via enhanced sphingolipid metabolism[END_REF]. We have demonstrated that rSCderived exosomes enhance axonal regeneration in vitro and in vivo [START_REF] Lopez-Verrilli | Schwann cell-derived exosomes enhance axonal regeneration in the peripheral nervous system[END_REF], an effect dependent on the reprogramming of dSC into a repair phenotype [START_REF] López-Leal | Schwann cell reprogramming into repair cells increases miRNA-21 expression in exosomes promoting axonal growth[END_REF]. Exosomes correspond to small extracellular vesicles (EVs) or nano-vesicles (50-120 nm) released after the fusion of multivesicular bodies (MVBs) with the plasma membrane (Théry et al., 2002). During their formation, exosomes are loaded with proteins, lipids, mRNA, and non-coding RNAs [START_REF] Jeppesen | Reassessment of exosome composition[END_REF]. Exosomes are enriched in specific repertoires of miRNAs, showing an asymmetric distribution when compared to their cells of origin [START_REF] Valadi | Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells[END_REF]Villarroya-Beltri et al., 2013). Secretion of exosomes is regulated by several mechanisms, including the phenotypic state of the cell, as well as activation by non-cell autonomous mechanisms [START_REF] Mathieu | Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication[END_REF]. In the nervous system, exosome secretion by oligodendrocytes is enhanced by the neurotransmitter glutamate released by neurons [START_REF] Frühbeis | Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication[END_REF], suggesting that nerve activity might modulate exosome secretion by glial cells. Interestingly, nerve activity enhances axonal regeneration and functional recovery in the PNS (Haastert-Talini and Grothe, 2013). The electrical stimulation of the sciatic nerves of the transected mice increases axonal regeneration across the injured site by 30-50% [START_REF] Singh | Accelerated axon outgrowth, guidance, and target reinnervation across nerve transection gaps following a brief electrical stimulation paradigm: laboratory investigation[END_REF], and in a rat model of femoral nerve regeneration, brief periods of electrical stimulation enhance axonal regeneration and muscle reinnervation in a TTX-dependent manner [START_REF] Al-Majed | Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration[END_REF]. Nevertheless, whether neuronal activity modulates the release of pro-regenerative exosomes by rSC after nerve injury has not been investigated.

Using in vitro models of neuronal and glial cultures, we demonstrate that sensory neurons activate small EV secretion by rSC through an activity-dependent release of ATP, acting over P2Y receptors in rSCs. Strikingly, activation of this signaling pathway not only enhances the release of rSC EVs but also modifies their miRNA content, increasing the EV expression of the pro-regenerative miRNA-21. Taken together, our results identify a signaling mechanism by which neurons modulate the quantity and quality of glial-derived EVs, enhancing their regenerative capacity.

Methods

Schwann cell primary culture SC primary cultures were obtained from newborn Sprague Dawley (SD) rat sciatic nerves as previously described (Wilby et al., 1999;[START_REF] De Gregorio | Purification of exosomes from primary schwann cells, rna extraction, and nextgeneration sequencing of exosomal RNAs[END_REF]. Briefly, the perineurium was removed, and the nerve was dissociated in 0.05% trypsin/1% collagenase type I solution. Cells were plated on laminin (40 ng/mL; Millipore)-treated flasks in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 10% penicillin-streptomycin (Invitrogen). The following day, cells were treated with 10 mM cytosine arabinoside (Sigma). After 1 week in culture, contaminant fibroblasts were eliminated by complement-mediated cell lysis using an anti-CD90 antibody (Invitrogen) and rabbit complement (Sigma). SCs were maintained in DMEM-10%, FBS-1%, penicillinstreptomycin supplemented with 2 µM forskolin (Millipore), and 20 mg/ml bovine pituitary extract (Invitrogen). SCs were then frozen in 10% DMSO and 90% DMEM and passaged up to five times. For each independent biological replicate, two newborn SD rat sciatic nerves from the same rat were used for the preparation of the cell culture.

Schwann cell stimuli, EV purification, and analysis

SC primary cultures supplemented with DMEM containing 2 mM forskolin, 20 mg/ml bovine pituitary extract, and 10% EV-free FBS (obtained by serum ultracentrifugation at 100,000 g for 2 h) (SC media) were stimulated with different reagents, such as 50 µM ATP (Sigma), 2 µM ionomycin (Sigma), 300 µM suramin, 10 µM BBG, and 100 mM EDTA (Sigma), for 5 min. Then the media was replaced with fresh SC media to eliminate all traces of reagents. The cells were incubated for different time points (1, 3, 6, 8, 16, and 24 h), and the supernatant was subjected to serial centrifugations (2,000 g for 10 min and 11,000 g for 30 min at 4 • C), followed by ultracentrifugation at 100,000 g for 60 min at 4 • C (T865 rotor, OTD Combi . /fncel. .

Sorvall ultracentrifuge, Dupont

). The pellet containing EVs was washed in cold 0.1 M phosphate buffer saline, pH 7.4 (PBS), and ultracentrifuged again at 100,000 g for 60 min at 4 • C. In each EV preparation, the concentration of total proteins was quantified by NanoOrange protein quantitation kit (Invitrogen) and stored at -20 • C for later use. For the analysis of EVs, the pellets were resuspended in 1 ml of PBS, and the concentration and the particle size distribution were analyzed under a Nanosight NS3000 (Malvern).

DRG explants culture and axonal elongation assay

Dorsal root ganglia (DRG) were obtained from day 16 SD rat embryos as previously described [START_REF] López-Leal | In vitro analysis of the role of Schwann cells on axonal degeneration and regeneration using sensory neurons from dorsal root ganglia[END_REF]. Briefly, E16 rat embryos were decapitated, and the limbs and organs were removed. The spinal cord with DRG was dissected and placed on coverslips or glass plates coated with poly-L-lysine (100 ng/mL; Sigma) and collagen (50 µg/mL; Invitrogen). DRG were maintained in Neurobasal medium (Invitrogen) supplemented with 2% B27 (Invitrogen), 2 mM L-glutamine, 50 ng/mL human nerve growth factor (NGF; Invitrogen), and 1% penicillin-streptomycin. In all experiments, DRG were treated with 2.5 µM 5-?uoro-2 ′ -deoxyuridine (Sigma) and 3.75 µM aphidicolin (Sigma) to inhibit the proliferation of SCs by inhibiting the action of DNA polymerase [START_REF] Jns ; Spadari | In vivo distribution and activity of aphidicolin on dividing and quiescent cells[END_REF]Wallace and Johnson, 1989). Axonal elongation was evaluated by plating DRG explants, and the next day, PBS (control) or EVs from SC in different conditions were added daily for 4 days. Axonal elongation was measured in mm 2 using the Image J software. For each independent biological replicate, a pool of 12-15 SD rat embryos was used for the preparation of the DRG cell culture.

mRNA and miRNA qPCR

The expression levels of mRNA were evaluated by plating DRG explants, PBS (control), or EVs from SC in different conditions, and were added daily for 4 days. Then, the pool of DRG was homogenized in TRIzol reagent (Life technologies) for the extraction of total RNA by the Trizol method. The aqueous phase was precipitated with 1 volume of isopropanol, 0.1 volumes of 3 M sodium acetate, and 10 µg of glycogen overnight at -20 • C. cDNA synthesis was performed with 5x iScript Reverse Transcription Supermix (BioRad). RNA levels of each sample were evaluated by real-time PCR using 5x HOT FIREpol Evagreen qPCR Mix Plus (Medibena). The mixture was run in a real-time PCR thermal cycler (CFX96 Touch Real-Time PCR Detection System, BioRad). Thermal cycling parameters were 15 min at 95 Ct method of normalizing. The miRNA levels of miRNA-21 were analyzed in SCs, and their EVs were treated with or without 50 µM ATP. SC and SC-derived EVs were homogenized in Qiazol reagent (Qiagen) for the extraction of total RNA based on the miRNeasy Mini kit (Qiagen) protocol. MicroRNA qPCR for miRNA-21 was performed using the MystiCq MicroRNA Quantitation System (Sigma) according to the manufacturer's protocol; briefly, miRNA cDNA of 20 ng of RNA was synthesized using the MystiCq microRNA cDNA synthesis mix (Qiagen). The miRNA levels of each sample were determined by realtime PCR using MystiCq microRNA SYBR Green qPCR ReadyMix (Qiagen). The mixture was run in a real-time PCR thermal cycler (Lightcycler System; Roche Diagnosis Corp). Thermal cycling parameters were 120 s at 95 • C, followed by 40 cycles of 5 s at 95 • C, and 30 s at 60 • C. At the end of the program, a melting curve analysis was performed at 60 • C for 30 s, followed by a cooling step at 37 • C for 30 s. Each sample was also run with primers for a Cel-miR39 spikein RNA (Qiagen) introduced in the cDNA synthesis step. Primers for rat miRNA-21 (rno-miR-21-5p; MIRAP00047) were ordered from Sigma. For relative comparison of miRNA-21 levels, the Ct value was analyzed with the Ct method of normalizing.

Functional assays

For the gain and loss of function assays, we used miRNA mimics and inhibitors (Exiqon) for miRNA-21 from Rattus norvegicus. First, DRG explants were transfected with lipofectamine 2000 (Thermo Fisher) with the miRNA-21 mimic 1 day after plating the DRG, and axonal elongation was quantified 4 days post-transfection. For the loss-of-function assay, SCs were transfected with lipofectamine 2000 with 50 nM LNA-enhanced antisense miRNA inhibitor of miRNA-21 or a negative control A inhibitor (YI00199006) (Exiqon) in Optimem media (Gibco). The media was replaced after 4 h with SC media. Then, EVs were purified by ultracentrifugation from SCs incubated with or without 50 µM ATP after 8 h, and DRG explants were treated daily with 0.5 µg of these EVs. Axonal elongation was measured on day 4 after treatment.

. /fncel. .

Extracellular vesicle internalization assay

SCs were transduced with a lentivirus coding for CMV-Palm-eGFP [START_REF] Lai | Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters[END_REF]. EVs were purified from SC incubated with or without 50 µM ATP for 8 h by differential ultracentrifugation and quantified by NanoOrange protein assay. DRG were treated with 5 µg of EVs and were fixed 4 h later.

Co-culture of schwann cells and sensory neurons

SCs were transduced with a lentivirus coding for CMV-Palm-eGFP [START_REF] Lai | Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters[END_REF]. Twenty-four hours after the transduction process, SCs were trypsinized and seeded over DRG plated 7 days before and left for 3 days before the experiments were performed. Co-cultured cells were treated with apyrase 1.5 U/mL (Sigma) or 0.5 µM TTX, and the EV release from the SC and internalization by the axon were measured in a Leica TCS SP8 spectral confocal microscope, measuring the colocalization of the GFP-tagged EVs with the axon NFM signal.

Immunofluorescence

The culture medium was removed from SC or DRG cultures, washed three times with PBS, and fixed with 4% PFA for 15 min at RT. Blocking and permeabilization were done with 0.1% Triton X-100 and 2% fish skin gelatin in PBS for 2 h at RT. Then, the cells were incubated with the primary antibody in 0.1% Triton X-100 and 1% fish skin gelatin in PBS and incubated overnight at 4 • C. Then, the primary antibody was removed, the cells were washed three times with PBS, and the secondary antibody diluted in 0.1% Triton X-100 and 1% fish skin gelatin in PBS was incubated for 2 h at RT. Afterward, the secondary antibody was removed, and the cells were washed two times with PBS and one time with DAPI stain in PBS (100 ng/ml). Then, the cells were washed once more in PBS, and coverslips were mounted with fluoromount-G (EMS).

Statistical analysis

All data are presented as mean ± SEM from the indicated number of experiments. Statistical analysis was performed using Student's t-test, linear regression, or ANOVA with Tukey's multiple comparison tests.

Results

Neuronal activity increases extracellular vesicle release by rSC and their transfer to axons in an ATP-dependent manner

As activity-dependent ATP secretion by peripheral neurons has been associated with the activation of signaling pathways in SCs [START_REF] Negro | ATP released by injured neurons activates schwann cells[END_REF][START_REF] Rodella | Schwann cells are activated by ATP released from neurons in an in vitro cellular model of Miller Fisher syndrome[END_REF], we investigated the effect of nerve activity on small EV release by SCs and their transfer to axons. To this end, we used sensory neurons from dorsal root ganglia (DRG), which have been previously demonstrated to have spontaneous activity in vitro [START_REF] Matsuka | Altered ATP release and metabolism in dorsal root ganglia of neuropathic rats[END_REF][START_REF] Mutafova-Yambolieva | The purinergic neurotransmitter revisited: a single substance or multiple players?[END_REF]. Indeed, purified DRG neurons in vitro release ATP to the culture medium, which is decreased more than 2-fold after blocking action potentials using the voltage-dependent sodium channel blocker tetrodotoxin (TTX, Figure 1A). We then evaluated whether this TTX-dependent ATP secretion induces a change in the release and uptake of SC-derived EVs. rSCs expressing GFP-labeled EVs were seeded over DRG neurons, and the GFP signal associated with axons was analyzed using a custom-developed particle detection method from confocalderived images (Supplementary Figure 1 and Methods section). In neurons co-cultured with rSC for 72 h, extensive GFPpositive puncta colocalized with axons immunolabeled against neurofilament protein (Figures 1B,C). After blocking neuronal activity using TTX, rSC-derived GFP signal associated with axons decreases by several folds (Figures 1B,C), and hydrolyzing extracellular ATP using apyrase diminishes the amount of rSC-derived EVs in axons by a magnitude similar to that of TTX (Figures 1B,C). Importantly, TTX or apyrase does not inhibit EV release by rSC (Supplementary Figure 2), suggesting that spontaneous neuronal activity enhances EV release by rSC through a mechanism associated with activity-dependent ATP release.

ATP enhances the pro-growth capacities of rSC-derived extracellular vesicles

As rSC-derived EV secretion can be modulated by activitydependent ATP release from sensory neurons, we next used purified rSC in order to study the dynamics of this phenomenon and their effect on axonal growth. To this end, rSCs were stimulated with ATP, and the number of released EVs was quantified using nanoparticle tracking analysis (NTA) after differential ultracentrifugation of the conditioned media (see Methods section). In control rSC cultures, EV content in the culture medium reaches a plateau after 3 h. In contrast, EVs from ATP-stimulated rSCs continue to accumulate after 3 h, reaching a 3-fold increase compared to control at 16 h poststimulation (Figure 2A). Ionomycin, which transiently increases intracellular Ca 2+ in rSC (not shown), has no noticeable effect on EV secretion (Figure 2A). We then study if this ATPdependent increase in EV accumulation is associated with changes in the rate of EV release by rSC. For this, rSCs were treated with vehicle, ATP, or ionomycin, and 1-h conditioned media was collected at different time points post-stimulation. As expected, in control conditions, the rate of EV release was constant for up to 24 h, and stimulation with ionomycin led to an increase in EV release only in the first hour (Figure 2B), which probably corresponds to the ready-releasable pool of MVBs present in rSCs. Remarkably, an acute 5-min ATP treatment leads to a long-lasting 3-fold increase in the rate of EVs released by rSC until 8 h post-treatment (Figure 2B). Associated with this increase in EV release, rSC at 8 h post-ATP treatment showed an increase in the presence of enlarged clusters of the MVB marker CD63 compared to vehicle-treated cells (Figures 2C,D). Importantly, changes in the rSC EV secretion rate triggered by ATP were not accompanied by major alterations in the EV size distribution as revealed by NTA (Figure 2E).

As we have previously demonstrated that rSC-derived exosomes increase axonal growth and regeneration of sensory neurons [START_REF] Lopez-Verrilli | Schwann cell-derived exosomes enhance axonal regeneration in the peripheral nervous system[END_REF], we tested the pro-growth effect of EVs obtained from control and ATP-stimulated rSC. We first tested the effect using EVs secreted from equal volumes of conditioned media of control and ATP-treated rSC. DRG devoid of SC were daily treated with vehicle, EVs from control, or ATP-treated rSC, and the axonal extension was evaluated (Figure 2F). EVs derived from HEK cells were used as an additional control. As expected, control rSCderived EVs enhanced axonal regeneration when compared to vehicle or HEK-derived EVs (Figure 2F). Interestingly, EVs derived from ATP-rSC showed an even higher pro-regenerative effect (Figures 2F,G). Since ATP-treated rSCs released a higher number of EVs (Figures 2A,B), we performed the same assays using comparable amounts of EVs for the different conditions (assessed by protein content or EV number analyzed by NTA, see Supplementary Table 1). Remarkably, when normalized, ATPtreated rSC-derived EVs still possess a higher pro-growth capacity than control rSC or HEK-derived EVs (Figures 2H,I). Importantly, EVs from control or ATP-treated rSC were similarly internalized by DRG axons (Supplementary Figure 3). Taken together, these results demonstrate that ATP enhances the rate of EV release by rSCs and modifies the pro-growth capacity of these EVs.

ATP-dependent enhancement of the pro-growth capacity of rSC extracellular vesicles is associated with the activation of P Y receptors in SCs

Extracellular ATP can activate metabotropic or ionotropic purinergic receptors, both expressed by SCs (del Puerto et al., 2013). Therefore, we used a pharmacological strategy to evaluate the pathway by which ATP enhances rSC-EV secretion rates and their pro-growth properties. First, we analyzed the effect of the P2X7 and P2Y receptor antagonists BBG and suramin, respectively, on EV release rate. Although both BBG and suramin do not change the basal release rate of rSC-EVs, the ATP-induced increase of EV release was completely abrogated by suramin (Figure 3A). Next, we analyzed the effect of purinergic receptor inhibition on the pro-growth effect of EVs secreted by ATP-treated rSC. Indeed, EVs from suramin-treated ATP-rSC lose their pro-growth activity over sensory neurons (Figures 3B,C). Together, these data show that ATP activates P2Y metabotropic receptors in Schwann cells, leading to an increase in the rate of EV release, as well as an enhancement in their pro-growth effect over peripheral neurons. Considering the inhibitory effect of suramin over the rate of EV secretion and their pro-growth capacity in ATP-treated rSC, we next evaluated the effect of purinergic inhibition in a co-culture system as described above. In agreement with the previous results, suramin strongly inhibited the transfer of GFP-labeled EVs from SCs (Figures 3D,E). Taken together, these results support our hypothesis that neuron-derived ATP released under spontaneous activity acts through P2Y receptors in rSC, and not P2X, to induce the release and transfer of EVs from rSC to axons.

The enhanced growth e ect of extracellular vesicles from ATP-activated rSC depends on the increased expression of miRNA-

We have already demonstrated that miRNA contained in rSC exosomes enhances axonal growth [START_REF] López-Leal | Schwann cell reprogramming into repair cells increases miRNA-21 expression in exosomes promoting axonal growth[END_REF]. Therefore, we hypothesized that miRNAs could be implicated in the pro-growth effect of ATP-treated SCs. We first used ultraviolet (UV) treatment to photochemically damage RNA molecules contained in rSC EVs [START_REF] Eldh | Exosomes communicate protective messages during oxidative stress; possible role of exosomal Shuttle RNA[END_REF][START_REF] Yin | Caveolin proteins: a molecular insight into disease[END_REF]. We observed that UV exposure completely abolished the increase in axonal growth of ATP-rSC-derived EVs (Figures 4A,B). We also previously demonstrated that miRNA-21 contained in rSC exosomes mediates their pro-growth effect over sensory neurons [START_REF] López-Leal | Schwann cell reprogramming into repair cells increases miRNA-21 expression in exosomes promoting axonal growth[END_REF]. Importantly, miRNA-21 contained in rSC-EVs showed a 1.7-fold increase in ATP-treated rSC compared to non-treated rSC (Figure 4C). miRNA-21 targets various genes involved in the regulation of neurite elongation and axonal growth, such as PTEN, SPRY2, and TIMP3 [START_REF] Duraikannu | Beyond trophic factors: exploiting the intrinsic regenerative properties of adult neurons[END_REF][START_REF] Jamsuwan | Simultaneous knockdown of Sprouty2 and PTEN promotes axon elongation of adult sensory neurons[END_REF][START_REF] Borger | How miRNAs regulate schwann cells during peripheral nerve regeneration-A systemic review[END_REF]; therefore, we evaluated whether ATP-rSCderived EVs regulate the expression of these genes in DRG neurons. Indeed, we observed a 70% decrease in the expression of PTEN, a 15% decrease in the expression of SPRY2, and a 30% decrease in the expression of TIMP3 in neurons treated with ATP-rSC-derived EVs compared to neurons treated with control rSC-derived EVs (Figure 4D). To evaluate the participation of miRNA-21 in axonal growth, we first transfected DRG neurons with a miRNA-21 oligonucleotide mimic. Indeed, miRNA-21 overexpression in DRG increases axonal growth when compared to the non-targeting miRNA-39-3p (Figures 4E,F). We next assessed the participation of rSC miRNA-21-containing EVs in the elongation of axons. For this, a miRNA-21 inhibitor was transfected into rSCs, and secreted EVs were used for growth assays. Depleting miRNA-21 from ATP-stimulated rSCs causes a decrease in growth enhancement provided by the secreted EVs (Figures 4G,H). In summary, these results demonstrate that ATP increases the presence of miRNA-21 in rSC-derived EVs, which is responsible for their pro-growth effect on DRG neurons.

Discussion

The role of EVs in intercellular communication and delivery of active molecules has been widely described in the nervous system [START_REF] Lopez-Verrilli | Schwann cell-derived exosomes enhance axonal regeneration in the peripheral nervous system[END_REF][START_REF] Prada | Glia-to-neuron transfer of miRNAs via extracellular vesicles: a new mechanism underlying inflammation-induced synaptic alterations[END_REF]. Furthermore, exosome release from glial cells has been linked to synaptic activity and neurotransmitter release from neurons in the central nervous system [START_REF] Frühbeis | Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication[END_REF]. In this work, we have been able to thoroughly characterize the mechanism by which neurons are able, through the release of ATP, to signal SCs into modifying their sEV dynamics by not only increasing their secretion rate but also changing their miRNA cargo into a specific phenotype that is beneficial for neurons and their neurite growth.

Our previous studies demonstrated that SC-derived exosomes induce an increase in the axonal elongation of sensory neurons [START_REF] Lopez-Verrilli | Schwann cell-derived exosomes enhance axonal regeneration in the peripheral nervous system[END_REF], but the mechanism that causes this increased neurite growth remained a mystery. Recent studies have shown that ATP is released by degenerating cerebellar granular neurons and spinal cord motor neurons. Moreover, ATP is capable of triggering Ca 2+ spikes, cyclic AMP production, and activating the ERK/CREB pathway in SC co-cultured with these neurons [START_REF] Negro | ATP released by injured neurons activates schwann cells[END_REF][START_REF] Rodella | Schwann cells are activated by ATP released from neurons in an in vitro cellular model of Miller Fisher syndrome[END_REF]. It is important to note that SCs express ionotropic and metabotropic purinergic ATP receptors, including P2X7, P2Y1, and P2Y2 [START_REF] Fields | ATP: an extracellular signaling molecule between neurons and glia[END_REF]. The involvement of these purinoreceptors in EV dynamics has been described in various models. It has been shown that the activation of the P2X7 receptor is able to induce the release of large and small EVs in microglia (Bianco et al., 2009;[START_REF] Asai | Depletion of microglia and inhibition of exosome synthesis halt tau propagation[END_REF]. Furthermore, the microglial-EVs released upon ATP stimulation have been associated with various phenotypes in neurological diseases, such as traumatic brain injury or multiple sclerosis [START_REF] Matute | P2X7 receptor blockade prevents ATP excitotoxicity in oligodendrocytes and ameliorates experimental autoimmune encephalomyelitis[END_REF]Verderio et al., 2012). On the other hand, the P2Y family of receptors has been described to have a positive role in platelet-EV formation and release, in a multi-step process involving an increase in intracellular calcium concentration, decrease in intracellular cAMP concentration, and activation of a signaling cascade including PI3K, which leads to the activation and phosphorylation of various target proteins that eventually lead to the increased platelet-EV formation [START_REF] Gasecka | Role of p2y receptors in platelet extracellular vesicle release[END_REF]. Interestingly, we show that the increased release of SCderived EVs induced by ATP is not dependent on P2X receptors but rather dependent on the P2Y family of receptors and lasts for several hours, which suggest that there is a long-lasting effect, consistent with the activation of intracellular pathways. Changes in the RNA cargo of EVs have been reported in several cell types after induction by certain stimuli. Cardiomyocytes subjected to ischemia change the miRNA cargo of their EVs promoting cardiac angiogenesis [START_REF] Ribeiro-Rodrigues | Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis[END_REF]. TNFα and IL-1β have been shown to modify a specific set of miRNAs in EVs derived from astrocytes to diminish the activity of target neurons [START_REF] Chaudhuri | TNFα and IL-1β modify the miRNA cargo of astrocyte shed extracellular vesicles to regulate neurotrophic signaling in neurons article[END_REF]. To date, only a handful of studies have demonstrated that ATP is able to impact EV composition. It has been shown that microglia-derived EVs present a distinct protein content after a prolonged ATP exposure compared to EVs from non-stimulated cells [START_REF] Drago | ATP modifies the proteome of extracellular vesicles released by microglia and influences their action on astrocytes[END_REF]. Furthermore, EVs from ATP-stimulated microglia have a stronger impact on the activation state of recipient cells [START_REF] Drago | ATP modifies the proteome of extracellular vesicles released by microglia and influences their action on astrocytes[END_REF].

Even though the mechanism by which ATP, through P2Y receptors, leads to a change in the composition of EV miRNAs remains to be explored, different sorting mechanisms for the asymmetric miRNA expression in EVs have been described, including those controlled by the recognition of specific miRNA motifs by loading proteins, as well as regulation by the cellular levels of the target transcripts (Villarroya-Beltri et al., 2013;[START_REF] Santangelo | The RNA-binding protein SYNCRIP is a component of the hepatocyte exosomal machinery controlling MicroRNA sorting[END_REF].

Among the miRNAs that are more abundant in ATPstimulated SC-derived EVs, we found miRNA-21. miRNAs have already been shown to be involved in the regulation of neuron growth. During the early stages of brain development, miRNA-29c is expressed and promotes neurite outgrowth by decreasing PTEN expression (Zou et al., 2015). We have previously described that SC reprogramming into repair cells modifies the exosomal cargo, promoting neurite growth in receiving neurons. Furthermore, this effect is dependent on the selective loading of miRNA-21 into the secreted exosomes [START_REF] López-Leal | Schwann cell reprogramming into repair cells increases miRNA-21 expression in exosomes promoting axonal growth[END_REF]. Interestingly, PTEN and SPRY2 are mRNA targets of miRNA-21, and DRG treated with EVs derived from ATP-stimulated SC show a decrease in the levels of PTEN and SPRY2. We hypothesize that this decrease in PTEN and SPRY2 expression may be partially responsible for the axonal elongation effect seen on EV-treated DRG. Interestingly, this effect has already been reported in the growth of sensory neurons in vitro and in vivo regeneration after spinal cord injury, where miRNA-21 promotes neurite outgrowth by regulating PTEN and SPRY2 [START_REF] Jiang | MicroRNA-21 promotes neurite outgrowth by regulating PDCD4 in a rat model of spinal cord injury[END_REF][START_REF] Jamsuwan | Simultaneous knockdown of Sprouty2 and PTEN promotes axon elongation of adult sensory neurons[END_REF].

It has been demonstrated that nerve stimulation activates pro-regenerating programs in both neurons and glial cells (Willand et al., 2016), including an increase in the secondary messenger cAMP in peripheral neurons [START_REF] Aglah | cAMP promotes neurite outgrowth and extension through protein kinase A but independently of Erk activation in cultured rat motoneurons[END_REF][START_REF] Udina | Electrical stimulation of intact peripheral sensory axons in rats promotes outgrowth of their central projections[END_REF] and the upregulation of neurotrophic factors by neurons and SCs [START_REF] Al-Majed | Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration[END_REF][START_REF] Huang | Electrical stimulation induces calcium-dependent release of NGF from cultured Schwann cells[END_REF][START_REF] Sharma | Electrical stimulation and testosterone differentially enhance expression of regenerationassociated genes[END_REF]Wan et al., 2010). In the PNS, neurons release ATP during the firing of action potentials, and after nerve injury, this ATP then activates purinergic receptors in SCs, which lead to Ca 2+ spikes in these cells and cause the activation of ERK/CREB, a signaling pathway with a key role in peripheral nerve regeneration [START_REF] Negro | ATP released by injured neurons activates schwann cells[END_REF]. Our work establishes an unexpected mechanism by which neurons can modulate axonal elongation in a non-cell autonomous fashion, which involves the modulation of the miRNA cargo of SC-derived EVs. Overall, our data provide evidence about new checkpoints that could serve to develop strategies to enhance axonal elongation in conditions where regeneration is greatly impaired.

SUPPLEMENTARY FIGURE

Particle transfer detection protocol. (A) Images were taken using a size of , × pixels, and each stack was taken every . µm with a speed of Hz. Background levels were normalized using the software LAS X based on the control image corresponding to not infected (GFP-palm) SC and DRG co-cultures incubated with the primary anti-GFP and secondary antibody Alexa . The value of filter settings was , which allowed the total subtraction of the non-specific signal. Each image was deconvolved in D in the software LASX using a total of iterations, under the blind method, and then the images were analyzed using IMARIS . . software. Scale bar, µm. (B) For each channel, a surface was created using the voxel number algorithm. The SC surface reconstruction (green channel) was performed with a detail of . µm and a threshold setting of . , and subsequently, a mask that contains the voxels outside of this surface was used for analysis. The axon surface analysis (red channel) was performed with a detail of . µm and a threshold setting of , and a mask of this was created for analysis. The voxels outside the surface mask of the green channel were colocalized with the surface of the red channel mask, adjusting the threshold to a value of for all images. The colocalization mask was analyzed with the spot detection tool, detecting particles between and nm. The particle density was determined by taking the number of spots and dividing for the axonal volume. Scale bar, µm.

SUPPLEMENTARY FIGURE

TTX and apyrase treatments do not a ect exosome release from rSC. rSCs were treated with ATP, TTX, and/or apyrase, and the conditioned media from each condition was centrifuged to purify sEVs. Samples were measured by NTA to analyze the release of particles in h. In all quantifications, average and SEM values of at least three independent experiments are shown ( * p < . ).

SUPPLEMENTARY FIGURE

Internalization of rSC exosomes by DRG neurons. Exosomes were isolated from control or ATP-treated rSCs transfected with CMV-palm-eGFP to visualize EVs. (A) DRG explants were treated with µg of EVs for h, washed, and immunostained against neurofilament heavy chain (NF-H, red) and GFP (green). Scale bar, µm. (B) The axon-exosome colocalization percentage was obtained from deconvolved z-stack confocal images by measuring the eGFP mean staining area colocalized with the NF-H staining. Scale bar, µm. In all quantifications, average and SEM values of at least three independent experiments are shown.

SUPPLEMENTARY TABLE

Protein quantification by NanoOrange and particle count by Nanosight.

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  RÉSUMÉ DE LA THÈSE Les vésicules extracellulaires (VE) sont des vésicules lipidiques qui sont libérées par toutes les cellules étudiées à ce jour et présentes dans tous les fluides corporels humains. Les VE contiennent du matériel génétique et des protéines qui peuvent être transférés à d'autres cellules et produire un effet sur celles-ci. La cavéoline-1 (Cav1) est un composant clé des petites invagination de la membrane plasmique appelées cavéoles, où elles fonctionnent comme mécano-capteurs et régulateur de la tension membranaire. La Cav1 facilite la migration et l'invasion des cellules tumorales et son expression est souvent augmentée dans les stades avancés du cancer. Des niveaux élevés de Cav1 ont ainsi été rapportés dans les VE de patients atteints d'un cancer avancé. Étant donné l'importance des forces mécaniques dans le micro-environnement des cellules cancéreuses, nous avons émis l'hypothèse que les cavéoles et/ou Cav1 pourraient être des acteurs clés dans la régulation de la biologie des VE et la progression du cancer sous contraintes mécaniques. Pour tester cette hypothèse, nous avons soumis différentes lignées cancéreuses exprimant (WT) ou délétées pour l'expression de Cav1 (KO) à des systèmes de stress mécanique en 2D et 3D. Les VE ont été purifiées et analysées par suivi des nanoparticules (NTA). Nous avons trouvé une augmentation conséquente de la libération des VE après un stress mécanique à la fois dans les modèles 2D et 3D. Cette augmentation était strictement dépendante de la présence de Cav1 et corrélée à une fusion accrue des corps multivésiculaires avec la membrane plasmique, indiquant que la population de VE augmentée lors d'un stress mécanique est constituée d'exosomes. Nous avons observé que Cav1 était enrichie dans les VE après une contrainte mécanique. L'augmentation des VE suite à un stress mécanique est considérablement réduite suite à la régulation négative du complexe de tri endosomal ESCRT-0, confirmant la nature exosomale de ces VE. L'analyse lipidomique a révélé des différences dans la composition lipidique des VE provenant de cellules Cav1KO et après un stress mécanique, suggèrant une différence dans les propriétés de ces VE. Enfin, il a été démontré que les EVs provenant de cellules soumises à un stress mécanique favorisent un phénotype de migration et d'invasion accru, dépendant de Cav1, dans les cellules cancéreuses. Ces résultats nous permettent de conclure que le stress mécanique est associé à la libération accrue de VE et à l'acquisition de traits métastatiques dans les cellules réceptrices in vitro, Cav1 étant un acteur clé de ce processus. En parallèle, nous avons également étudié l'implication de Cav1 dans l'endocytose des EVs, un processus clé dans le contexte de la communication cellulaire médiée par les EVs. Nous avons utilisé différents marqueurs protéiques pour suivre et visualiser la capture des VE dans les cellules réceptrices. La capture des VE était fortement diminuée dans les cellules réceptrices Cav1 KO par rapport aux cellules WT. Nous avons également fait l'observation que Cav1, et non les cavéoles, était nécessaire à la capture des VE par les cellules, et que l'efficacité de capture était corrélée au niveau d'expression de Cav1 dans les cellules réceptrices. Dans l'ensemble, nos travaux ont révélé un rôle nouveau de Cav1 dans la communication intercellulaire médiée par les VE et la propagation des phénotypes métastatiques par le contrôle mécanique de la sécrétion et la dynamique des VE.

  Abbreviations

Figure 5 .

 5 Figure 5. Biogenesis of extracellular vesicles Schematic representation of the different pathways involved in the biogenesis of exosomes and microvesicles. 1. Membrane-bound proteins are recruited in microdomains of the MVB (here called MVE) membrane (bottom) or at the plasma membrane (top). 2. Other molecules such as proteins and nucleic acids are recruited to be sorted into ILVs (bottom) or microvesicles (top). 3. These clustered microdomains together with other proteins induce the budding of the plasma membrane towards the inside of the MVB (bottom) or outside of the cell (top), and posterior scission to form ILVs (bottom) or microvesicles, accordingly (top) (van Niel et al., 2018).

Figure 7 .

 7 Figure 7. EV mediated pro-metastatic effects Tumor-derived EVs affect the local tumor microenvironment by inducing enhanced migration, invasiveness potential, ECM remodeling and motility among other phenotypes (left). Tumor-derived EVs can also affect distal sites by establishing premetastatic niches through the induction of increased vascular permeability, ECM remodeling or recruitment of bone marrow-derived cells (Tkach & Théry, 2016).

Figure 8 .

 8 Figure 8. Caveolar components Schematic representation of the structural components of caveolae. A. Caveolin monomer and oligomer structure (See figure 9 for more details). B. Cavin monomer and trimer structure (See figure 10 for more details). C. Interaction between caveolins and cavins triggers the assembly of the budded caveolae (Lamaze et al., 2017).

Figure 9 .

 9 Figure 9. Cav1 structure A. Top view and B. side view of the 11-unit oligomer of Cav1 that forms the 8S complex. C. Top view and D. side view of the Cav1 monomer with its structural features, including the signature motif (SM), oligomerization domain (OM), pin motif (PM), scaffolding domain (SD), and intramembrane domain (IMD) (Modified from Porta et al., 2022).

Figure 10 .

 10 Figure 10. Cavins protein family structure Schematic representation of cavins structure and complex formation. A-B. Cavins can form a trimeric homo-oligomer complex of cavin1 or hetero-oligomers complexes where two Cavin1 subunits can bind either a single Cavin2 or Cavin3 subunit. Cavin2 and Cavin3 cannot interact with each other. C. Cavin oligomeric particles can assemble; D. and these can associate with PS and PIP-containing membranes. E. Cavins are only stably associated with caveolae in conjunction with caveolin (Modified from Kovtun et al., 2014).

Figure 11 .

 11 Figure 11. Lipid profile of caveolae A. Schematic representation of the lipid species enriched in caveolae. Caveolae are highly enriched in cholesterol among other species, including sphingolipids and

Figure 12 .

 12 Figure12. Caveolae flattening after exposure to mechanical stress Schematic representation of the process of flattening of caveolae after mechanical stress. At steady state, Cav1 and cavin together form budded caveolae. Upon tension enhancing cues such as swelling or stretching of the plasma membrane, budded caveolae immediately flatten out(Sinha et al., 2011).

Figure 13 .

 13 Figure 13. Consequences of caveolae mechanical response on signaling Schematic representation of possible signaling events triggered by mechanical stress-dependent caveolae flattening. At steady state (left), Cav1 and cavin1 are at the plasma membrane in the caveolar compartment, after mechanical stress (right), Cav1 is released from the caveolae and free cavin1 can translocate to the nucleus to promote gene transcription (Nassoy & Lamaze, 2012).

Figure 15 .

 15 Figure 15. Mechanisms of ECM remodeling A. Secreted factors activate stromal cells, which differentiate into CAFs leading to the secretion and deposition of ECM components along with the secretion of ECM components from cancer cells. B. Cross-linking and alignment of collagen fibers

Figure 16 .

 16 Figure 16. Mechanics of cancer Schematic representation of the mechanical stimuli on tumor cells throughout cancer progression. A. Normal tissue environment presenting balanced forces between cells and ECM. B. The tumor microenvironment consists of cancer cells exhibiting increased tension, enveloped by an ECM that becomes progressively stiffer, and

  stress led to the specific enrichment of lipid species in EVs including phosphatidylcholinephosphatidylinositol. In contrast, mechanical stress led to a decrease in the cholesterol and ceramide content of EVs. Depletion of Cav1 in cells induced an increase in the presence of lactosylceramide (LacCer), and a decrease in ceramide, and the gangliosides Gb4 and GM2 in EVs (Figure5A and 5B). These differences in lipid composition of EVs induced by mechanical stress led us to hypothesized that EVs released by mechanical stress could convey distinct specific functions on receiving cells.

  (2:1) for 1 hour. The lower organic phase was collected and evaporated in a SpeedVac vacuum concentrator(Thermo Fisher Scientific). Lipid extracts were dissolved in 100 μL of infusion mixture consisting of 7.5 mM ammonium acetate dissolved in propanol/chloroform/methanol (4:1:2 vol/vol). Samples were analyzed by direct infusion in a Q Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences). A total of 5 μL of sample was infused with gas pressure and voltage set to 1.25 psi and 0.95 kV, respectively. DAG, TAG and CE were detected as ammonium adducts and PC, PC O-and SM were detected as protonated ions in the 10:1 extract, by positive ion mode fourier transform mass spectrometry (FTMS) by scanning m/z = 580-1,000 Da, Rm/z =200= 280,000 with lock mass activated at a common background (m/z = 680.4802) for 30 s. Every scan is the average of two microscans, automatic gain control (AGC) was set to 1E6 and maximum ion injection time (IT) was set to 200 ms. Cer and hexosylceramide (HexCer) were detected as acetate adducts and PE, PE O-, PG, LPC and LPE were detected as deprotonated ions in the 10:1 extract, after polarity switch by negative ion mode FTMS by scanning m/z = 520-1,050 Da, at Rm/z = 200 = 280,000 with lock mass activated at a common background (m/z = 529.4626) for 30 s. Every scan is the average of two microscans, AGC was set to 1E6 and IT was set to 50 ms. Hex2Cer, Gb3 and Gb4 were detected as protonated ions in the 2:1 extract in positive ion mode FTMS by scanning m/z = 800-1,600 Da, at Rm/z=200= 280,000 with lock mass activated at a common background (m/z = 1,194.8179) for 30 s. GM1, GM2, and GM3

Figure 1 :Figure 4 :

 14 Figure 1: Characterization of EVs from mechanically stressed cells.

Figure 5 :

 5 Figure 5: Lipid signature of EVs from mechanically stressed cells.

Figure 6 :

 6 Figure 6: Migration and invasion of Hs578t cells treated with EVs.

Figure 6 :

 6 Figure 6: Migration and invasion of Hs578t cells treated with EVs.

Figure 17 .

 17 Figure17. EV-mediated crosstalk between cancer cells and cancer-associated fibroblasts. Schematic representation of the intercellular crosstalk between cancer cells and CAFs via EVs. Cancer cells release bioactive molecules in EVs that are taken up by surrounding CAFs. Such molecules include TGF-β, ITGB4, p53, mRNAs and miRNAs. The uptake of cancer-derived EVs leads to specific changes in the recipient cells, including the induction of mitophagy and glycolysis, the development of myofibroblast features, alterations in the extracellular matrix, and the expression of inflammatory genes. Cancer-derived EVs also induce distinct CAF subtypes and contribute to the heterogeneity of CAFs. On the other hand, CAFs themselves utilize EVs to transfer various functional molecules to cancer cells. These functional molecules include CD81, CD9, FAP, miRNAs, lncRNAs, transposable RNAs, and metabolites. The transfer of these molecules from CAFs to cancer cells leads to the acquisition of more aggressive phenotypes by cancer cells[START_REF] Naito | Intercellular crosstalk between cancer cells and cancer-associated fibroblasts via extracellular vesicles[END_REF].

Figure 18 .

 18 Figure 18. Schematic representation of CAF subtypes and T cell recruitment. A. CAF heterogeneity (CAF-S1-4) in breast cancer and their markers. B. CAF-S1enriched breast cancer show high FOXP3 + and low CD8 + T cells compared with CAF-S4-enriched breast cancer. C. CAF-S1 promotes an immunosuppressive environment by attracting CD4 + CD25 + T cells, retention of T cells, promoting their survival and differentiation into CD25 + FOXP3 + T cells[START_REF] Costa | Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer[END_REF].

Figure 19 .

 19 Figure 19. Effect of mechanical stress on breast cancer EV release and CAF immunosuppressive capabilities. A. Quantification of EVs released by WT, Cav1 -/-and Cav1 rescue MDA-MB-231 cells after osmotic shock, graph shows total particle number secreted measured by NTA. B. Detection by flow cytometry of CD4+ CD25+ FOXP3+ T cells after incubation with CAFs pretreated with EVs from MDA-MB-231 cells WT, Cav1 -/-or Cav1 rescue, in resting conditions or after mechanical stress. *p < 0.05, **p <0.01.

Figure 20 .

 20 Figure 20. Potential outcomes of EV-plasma membrane docking at the recipient cell. Schematic representation of EV-cell interaction. 1. After EV-PM docking, signaling pathways can be activated following interactions from the membrane contact. 2. EV-PM fusion can occur, leading to the release of the EV cargo directly into the recipient cell cytosol. 3. Endocytosis through different mechanisms can lead to the uptake of whole EVs. 4. Once inside endosomal structures, EVs can be degraded by fusion of endosomes with lysosomes. 5. Fusion of the EV membrane with the endosomal membrane can lead to the release of the EV cargo to the cytosol (Modified from Bonsergent et al., 2021).

Figure 21 .

 21 Figure 21. EV uptake by cells expressing different levels of caveolar proteins. A. Western Blot analysis of Cav1 in donor, WT, Cav1 -/-or cavin1 -/-HeLa cells. B. Percentage uptake of Nanoluc-Hsp70 EVs by HeLa cells overexpressing Cav1 and/or cavin1, measured by luminescence. C. Percentage uptake of Nanoluc-Hsp70 EVs by cavin1 -/-MEF cells with low or high levels of Cav1, measured by luminescence. D. Particle density of GFP-Hsp70 EVs uptaken by Cav1 -/-or cavin1 -/- HeLa cells. *p < 0.05.

Figure 22 .

 22 Figure 22. Different types of approaches described to date for boosting EV secretion. Schematic representation of the different methods for increasing EV secretion for clinical use including physical signals such as mechanical loading, 3D culture, ultrasound and electrical stimulation, molecular interference, environmental factors, external inducers like liposomes, and other environmental factors like hypoxia, starvation, and hyperglycemia (Wang et al., 2023).

Figure 23 .

 23 Figure 23. Interaction between the Hippo pathway and caveolae mechanics. Schematic representation of regulation caveolae abundance by the Hippo pathway by the YAP/TAZ-TEAD mediated regulation of caveolar genes. At the same time, caveolae facilitate the mechanical stress mediated signaling via the Hippo pathway (Rausch et al., 2019).

  addition to phosphorylation, another important post-translational modification that plays a crucial role in regulating the function and localization of Cav1 is ubiquitination. Ubiquitination is a process that involves the covalent attachment of ubiquitin molecules to a substrate protein, typically marking it for degradation or sorting to specific subcellular structures. Multiple studies have highlighted the importance of ubiquitination in regulating the intracellular localization, stability, and function of Cav1. Ubiquitination of Cav1 can occur at any of its 20 ubiquitination sites. One important site of Cav1 ubiquitination is the N-terminal region, which has been demonstrated to serve as a translocation signal for Cav1 to relocate towards

Figure 24 .

 24 Figure 24. Phosphorylation and ubiquitination of Cav1 after mechanical stress. A. (Left) Western Blot analysis of phospho-Cav1 (Y14) and total Cav1 in WT HeLa cells. (Right) Protein signal quantification graphs normalized to the stain free gel. B. (Left) Immunoprecipitation of Cav1 and western Blot analysis of Cav1 and ubiquitin in WT HeLa cells. (Right) Protein signal quantification.

Figure 25 .

 25 Figure 25. Phosphorylation and ubiquitination of Cav1 importance in EV release after mechanical stress. A. Western Blot analysis of Cav1, cavin1 and tubulin in WT, Cav1 -/-HeLa cells an Cav1 -/-HeLa cells transfected with an ubiquitin mutant (K*R) or phosphorylation mutant (Y14F) Cav1. B. Quantification of EVs released by WT, Cav1 -/-HeLa cells and Cav1 -/- HeLa cells expressing the Cav1 mutants in response to hypo-osmotic shock. Graph shows the number of total secreted EVs.

Figure 26 .

 26 Figure 26. Schematic representation of the two-phase tension buffering model. A. Increase in plasma membrane tension triggers phase 1 where the cell utilizes the reservoirs of plasma membrane to increase the surface area of the membrane, as reflected by the rapid flattening of caveolae. Caveolae flattening generates the production of Cav1 scaffolds. B. Cav1 scaffolds are endocytosed towards endosomal compartments (i.e. MVBs). C. There is an increase in exocytosis events as reflected in the increase in MVB-PM fusion and secretion of EVs.

  study offers new insights into the regulation of EV dynamics by Cav1. We demonstrate that upon being released from caveolae at the plasma membrane, non-caveolar Cav1 controls EV secretion through ESCRT-0 and increased MVB-PM fusion, leading to exosome secretion. Additionally, our findings underscore the physiological importance of this enhanced EV release mediated by Cav1, as evidenced by changes in the composition and cargo of secreted EVs and the acquisition of increased metastatic traits by recipient cells.Nevertheless, further research is required to fully elucidate the intracellular mechanisms underlying this Cav1-dependent effect. Moreover, our study also revealed that the uptake of EVs by recipient cells is dependent on Cav1 and independent of caveolae. Overall, our comprehensive investigation has yielded compelling and robust evidence elucidating the intricate mechanisms underlying altered EV dynamics within the mechanical environments of the cell. By delving into this relatively unexplored domain of significant importance, we have greatly contributed to the existing knowledge base. This work not only expands our understanding of EV 8. ANNEX Investigating the cell-to-cell communication through EVs in the nervous system The following research article describing an intricate process of crosstalk through EVs between neurons and glial cells was in part developed before the start of my PhD project in the team of Felipe Court (Center for Integrative Biology, Universidad Mayor. Santiago, Chile). It was continued and finished for publication in collaboration after I had begun my thesis in my current lab. Saquel, C., Catalan, R. J., Lopez-Leal, R., Ramirez, R. A., Necuñir, D., Wyneken, U., Lamaze, C., & Court, F. A. (2022). Neuronal activity-dependent ATP enhances the pro-growth effect of repair Schwann cell extracellular vesicles by increasing their miRNA-21 loading.

FIGURE

  FIGUREsEV transfer from rSC to axons in co-culture is inhibited upon ATP depletion and action potential inhibition. The involvement of neuronal ATP and spontaneous activity in the transfer of EVs from rSC to axons was analyzed in a co-culture system. (A) DRG neurons from primary culture were seeded in white bottom culture plates and left to grow for days; ATP release from neurons was analyzed by luminescence with the ATPlite detection kit in a luminescence spectrometer. (B) DRG neurons co-cultured with rSC expressing CD -GFP to visualize EVs were analyzed for EV transfer by immunofluorescence and detection of GFP signal in axons. Scale bar, µm. (C) Colocalized particles were quantified as described in Supplementary Figure and plotted as particle density per volume of axons. In all quantifications, the average and SEM values of at least three independent experiments are shown (*p < . ).

FIGUREE

  FIGURE E ect of ATP in the release of sEVs from rSC and their e ect on axonal elongation. The e ect of ATP ( µM) on rSC sEV was analyzed by nanoparticle tracking analysis (NTA) and immunofluorescence. (A,B) Conditioned media from di erent time points after ATP treatment were measured by NTA to analyze the accumulation of particles over time and their rate of release by rSC. (C,D) CD -transduced rSC were quantified for GFP puncta (arrowheads) after ATP treatment. (E) Size distribution of EVs in the di erent conditions generated by NTA. (F,G) The e ect of sEV from rSC treated with or without ATP ( µM) on neurons was analyzed by daily treatment of DRG with these sEVs. sEVs from the same volume of conditioned media from rSC stimulated with or without ATP were used to treat DRG daily. (H,I) sEVs normalized by quantity to . µg of protein from rSC stimulated with or without ATP were used to treat DRG daily. The elongation e ect on DRG was visualized on day by immunofluorescence using neurofilament medium (NFM) immunostaining in axons. Scale bar, , µm. In all quantifications, average and SEM of at least three independent experiments are shown ( # p < , ; **p < . ; *p < . ).

FIGUREP

  FIGURE P Y family of receptors is involved in the increased sEV release and axonal elongation e ect of ATP on rSC. To analyze to which receptor ATP binds to exert its e ect on rSC we pharmacologically inhibited P X or P Y receptors and analyzed the sEV released by NTA and their e ect on axonal growth in DRG after ATP treatment. (A) Conditioned media from h after ATP and drug treatment were measured by NTA to analyze the rate of release of sEV from rSC. (B,C) The e ect of sEV from rSC treated with or without ATP in the presence of BBG or suramin on neurons was analyzed by daily treatment of DRG with . µg of sEV. The elongation e ect on DRG was visualized on day by immunofluorescence using neurofilament medium (NFM) immunostaining in axons. Scale bar, , µm. (D,E) Co-cultured DRG neurons with rSC expressing CD -GFP to visualize EVs were analyzed for EV transfer by immunofluorescence after incubation with BBG or suramin and detection of GFP signal in axons. Scale bar, µm. In all quantifications, average and SEM of at least three independent experiments are shown (***p < , ; **p < . ; *p < . ).

FIGURE

  FIGUREmiRNA-is a key participant in the axonal elongation e ect of rSC-derived sEV on DRG neurons. (A,B) RNA participation in the axonal elongation e ect of sEV over DRG neurons was first evaluated by UV irradiation. Axonal growth was analyzed on day after adding rSC-derived sEV daily from SC treated with or without ATP to the DRG neurons. For RNA inactivation, EVs were previously UV irradiated. The axonal elongation of DRG induced by sEV was visualized by immunofluorescence using neurofilament medium (NFM) immunostaining in axons. Scale bar, , µm. (C) miRNA-content in rSC-derived sEVs was assessed by qPCR in sEV samples from control or ATP-stimulated rSCs. (D) Expression of PTEN, SPRY , and TIMP was measured by qPCR from DRG neurons after treatment with sEV derived from control or ATP-treated rSC. (E,F) The involvement of miRNA-was assessed by transfection of miRNA-inhibitors or mimics in rSC. miRNA-was directly transfected in DRG neurons, and the axonal growth area was analyzed days after transfection, and the miRNA mimic mir--p with no known rat mRNA target was used as a control. The increased axonal growth induced by transfection of miRNA-was visualized by immunofluorescence using neurofilament medium (NFM) immunostaining in axons. Scale bar, , µm. (G,H) SCs were transfected with miRNA-inhibitors, sEVs were purified, DRG were treated daily with these sEVs, and the axonal growth area was analyzed days after transfection. The e ect of the inhibitors on the sEVs and their axonal elongation e ect were visualized by immunofluorescence using neurofilament medium (NFM) immunostaining in axons. Scale bar: , µm. In all quantifications, average and SEM of at least three independent experiments are shown ( # p < , ; **p < . ; *p < . ).

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  • C, followed by 40 cycles of 15 s at 95 • C, 20 s at 60 • C, and 20 s at 72 • C. At the end of the program, melting curve analysis was performed from 60 to 95 • C. The following primers were used: PTEN:

				Forward AAGGACGGACTGGTGTAA
	and		Reverse	CCTGAGTTGGAGGAGTAGAT,
	SPRTY2:		Forward	GCAGGATACGCGCTTGGG
	and	Reverse	CCTCACAGCGGCTCAACTC,	TIMP3:
	Forward	CTGGAGCCTTGGGCACTG	and	Reverse
	CGGATCACGATGTCGGAGT,	and	GAPDH:
	Forward TCCCTCAAGATTGTCAGCAA and Reverse
	AGATCCACAACGGATACATT. For relative comparison, the
	Ct value was analyzed with the

Frontiers in Cellular Neuroscience frontiersin.org

ACKNOWLEDGEMENTS

"…An office is for not dying. An office is a place to live life to the fullest. To the max. To… an office is a place where dreams come true." -Michael Scott.

Acknowledgements

The authors greatly acknowledge the Cell and Tissue Imaging (PICT-IBiSA), Institut Curie, member of the French National Research Infractucture France-BioImaging (ANR10-INBS-04). This work was supported by the Fondation pour la Recherche Médicale (FRM N° DGE20111123020), the Cancerople-IdF (n°2012-2-EML-04-IC-1), InCA (Cancer National Institute, n° 2011-1-LABEL-IC-4), member of the French National Research Infrastructure France-BioImaging (ANR10-INSB-04), and the CurieCoreTech Metabolomics and Lipidomics platform. The authors would like to thank Dr. Clotilde Théry and Dr. Guillaume van Niel for the fruitful discussions and advice during the development of the present work. This work was performed as part of a doctoral thesis supported by grants from Fonds France Canada pour la Recherche

Funding

This work was supported by grants from ECOS-ANID N • C17S03, Geroscience Center for Brain Health and Metabolism, FONDAP N • 15150012, and Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) N • 1150766 (to FC).

Data availability statement

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

biology but also lays the groundwork for further exploration into the potential therapeutic applications of modulating EV dynamics in various physiological and pathological contexts. In sum, our research fills a critical gap in the field and provides a stepping stone for future investigations in this captivating area of research.

Ethics statement

The animal study was reviewed and approved by CBB, Universidad Mayor, Santiago, Chile.

Author contributions

CS: planning and executing experiments and writing the manuscript. RC, RL-L, RR, and DN: planning and executing experiments. UW, CL, and FC: planning experiments. All authors contributed to the article, reviewed the final manuscript and approved the submitted version.

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.

Publisher's note

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ fncel.2022.943506/full#supplementary-material ABSTRACT Extracellular vesicles (EVs) are released by most cell types and present in all human bodily fluids. EVs contain genetic material and proteins that can be transferred to and generate an effect in other cells. Caveolin-1 (Cav1) is a key component of the small invagination of the plasma membrane called caveolae, where it functions as mechano-sensors and membrane tension buffering device. Cav1 expression is often increased in the late stages of cancer. Interestingly, high levels of Cav1 have been found in EVs of patients with advanced cancer. Given the importance of mechanical forces in the microenvironment of cancer cells, we hypothesized that caveolae and/or Cav1 may represent key players in the regulation of EV biology and cancer progression under mechanical strain. To test this hypothesis, we subjected different cancer cell lines, having (WT) or deleted for Cav1 expression (KO) to 2D or 3D systems of mechanical stress. We found a striking increase in the release of EVs after mechanical stress both in 2D and 3D models. This increase was strictly dependent on the presence of Cav1 and correlated with enhanced fusion of multivesicular bodies to the plasma membrane. Lipidomic analysis revealed differences in the lipid composition of EVs in Cav1 -/-cells and after mechanical stress, suggesting a difference in the properties of these EVs. Finally, EVs from mechanically stressed cells were shown to promote enhanced migration and invasion phenotype in breast cancer cells, whereas EVs from Cav1 depleted cells did not. These data allow us to conclude that mechanical stress is associated with increased release of EVs and the acquisition of metastatic traits in receiving cells in vitro, with Cav1 being a key player of this process.

MOTS CLÉS

Cavéoles, vésicules extracellulaires, stress mécanique, cancer RÉSUMÉ Les vésicules extracellulaires (VE) sont libérées par toutes les cellules étudiées à ce jour et présentes dans tous les fluides corporels humains. Les VE contiennent du matériel génétique et des protéines qui peuvent être transférés à d'autres cellules et produire un effet sur celles-ci. La cavéoline-1 (Cav1) est un composant clé des petites invagination de la membrane plasmique appelées cavéoles, où elles fonctionnent comme mécano-capteurs et régulateur de la tension membranaire. La expression de Cav1 est souvent augmentée dans les stades avancés du cancer. Des niveaux élevés de Cav1 ont ainsi été rapportés dans les VE de patients atteints d'un cancer avancé. Étant donné l'importance des forces mécaniques dans le microenvironnement des cellules cancéreuses, nous avons émis l'hypothèse que les cavéoles et/ou Cav1 pourraient être des acteurs clés dans la régulation de la biologie des VE et la progression du cancer sous contraintes mécaniques. Pour tester cette hypothèse, nous avons soumis différentes lignées cancéreuses exprimant (WT) ou délétées pour l'expression de Cav1 (KO) à des systèmes de stress mécanique en 2D et 3D. Nous avons trouvé une augmentation conséquente de la libération des VE après un stress mécanique à la fois dans les modèles 2D et 3D. Cette augmentation était strictement dépendante de la présence de Cav1 et corrélée à une fusion accrue des corps multivésiculaires avec la membrane plasmique, indiquant que la population de VE augmentée lors d'un stress mécanique est constituée d'exosomes. Nous avons observé que Cav1 était enrichie dans les VE après une contrainte mécanique. L'analyse lipidomique a révélé des différences dans la composition lipidique des VE provenant de cellules Cav1KO et après un stress mécanique, suggèrant une différence dans les propriétés de ces VE. Enfin, il a été démontré que les EVs provenant de cellules soumises à un stress mécanique favorisent un phénotype de migration et d'invasion accru, dépendant de Cav1, dans les cellules cancéreuses. Ces résultats nous permettent de conclure que le stress mécanique est associé à la libération accrue de VE et à l'acquisition de traits métastatiques dans les cellules réceptrices in vitro, Cav1 étant un acteur clé de ce processus.

KEYWORDS

Caveolae, extracellular vesicles, mechanical stress, cancer