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• W3000534695 abstract "Article10 January 2020free access Source DataTransparent process Adipocyte extracellular vesicles carry enzymes and fatty acids that stimulate mitochondrial metabolism and remodeling in tumor cells Emily Clement Emily Clement Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Ikrame Lazar Ikrame Lazar Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Camille Attané Camille Attané orcid.org/0000-0002-2073-7291 Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Lorry Carrié Lorry Carrié Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Stéphanie Dauvillier Stéphanie Dauvillier Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Manuelle Ducoux-Petit Manuelle Ducoux-Petit Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author David Esteve David Esteve Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Thomas Menneteau Thomas Menneteau Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Mohamed Moutahir Mohamed Moutahir Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Sophie Le Gonidec Sophie Le Gonidec Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), INSERM, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Stéphane Dalle Stéphane Dalle Department of Dermatology, Centre Hospitalier Lyon Sud, Pierre Bénite Cedex, France Search for more papers by this author Philippe Valet Philippe Valet Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), INSERM, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Odile Burlet-Schiltz Odile Burlet-Schiltz Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Catherine Muller Catherine Muller Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Laurence Nieto Corresponding Author Laurence Nieto [email protected] orcid.org/0000-0002-1006-0168 Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Emily Clement Emily Clement Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Ikrame Lazar Ikrame Lazar Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Camille Attané Camille Attané orcid.org/0000-0002-2073-7291 Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Lorry Carrié Lorry Carrié Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Stéphanie Dauvillier Stéphanie Dauvillier Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Manuelle Ducoux-Petit Manuelle Ducoux-Petit Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author David Esteve David Esteve Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Thomas Menneteau Thomas Menneteau Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Mohamed Moutahir Mohamed Moutahir Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Sophie Le Gonidec Sophie Le Gonidec Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), INSERM, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Stéphane Dalle Stéphane Dalle Department of Dermatology, Centre Hospitalier Lyon Sud, Pierre Bénite Cedex, France Search for more papers by this author Philippe Valet Philippe Valet Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), INSERM, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Odile Burlet-Schiltz Odile Burlet-Schiltz Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Catherine Muller Catherine Muller Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Laurence Nieto Corresponding Author Laurence Nieto [email protected] orcid.org/0000-0002-1006-0168 Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France Search for more papers by this author Author Information Emily Clement1,†,‡, Ikrame Lazar1,†,‡, Camille Attané1, Lorry Carrié1, Stéphanie Dauvillier1, Manuelle Ducoux-Petit1, David Esteve1, Thomas Menneteau1,†,‡, Mohamed Moutahir1, Sophie Le Gonidec2, Stéphane Dalle3, Philippe Valet2, Odile Burlet-Schiltz1, Catherine Muller1 and Laurence Nieto *,1 1Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, UPS, Université de Toulouse, Toulouse, France 2Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), INSERM, UPS, Université de Toulouse, Toulouse, France 3Department of Dermatology, Centre Hospitalier Lyon Sud, Pierre Bénite Cedex, France †Present address: UDEAR, Institut National de la Santé Et de la Recherche Médicale, Université de Toulouse Midi-Pyrénées, Toulouse, France †Present address: INSERM, U1043, CNRS, U5282, UPS, Centre de Physiopathologie de Toulouse-Purpan, Université de Toulouse, Toulouse, France †Present address: Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA †Present address: Department of Structural and Molecular Biology, Institute of Structural and Molecular Biology, University College London, London, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +33 561 17 55 09; Fax: +33 561 17 59 33; E-mail: [email protected] The EMBO Journal (2020)39:e102525https://doi.org/10.15252/embj.2019102525 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Extracellular vesicles are emerging key actors in adipocyte communication. Notably, small extracellular vesicles shed by adipocytes stimulate fatty acid oxidation and migration in melanoma cells and these effects are enhanced in obesity. However, the vesicular actors and cellular processes involved remain largely unknown. Here, we elucidate the mechanisms linking adipocyte extracellular vesicles to metabolic remodeling and cell migration. We show that adipocyte vesicles stimulate melanoma fatty acid oxidation by providing both enzymes and substrates. In obesity, the heightened effect of extracellular vesicles depends on increased transport of fatty acids, not fatty acid oxidation-related enzymes. These fatty acids, stored within lipid droplets in cancer cells, drive fatty acid oxidation upon being released by lipophagy. This increase in mitochondrial activity redistributes mitochondria to membrane protrusions of migrating cells, which is necessary to increase cell migration in the presence of adipocyte vesicles. Our results provide key insights into the role of extracellular vesicles in the metabolic cooperation that takes place between adipocytes and tumors with particular relevance to obesity. Synopsis Extracellular vesicle (EV)-mediated communication between adipocytes and neighbouring cancer cells is known to promote tumour progression, but the underlying mechanism remains ill-defined. Adipocyte-derived extracellular vesicles laden with enzymatic machinery and lipid substrates promote fatty acid oxidation and aggressiveness of melanoma cells, a process that is amplified in obesity. Adipocytes promote melanoma aggressiveness through EV-mediated delivery of fatty acids and enzymes involved in fatty acid oxidation to cancer cells. Obesity boosts the effect of adipocyte-derived EVs on melanoma cells, due to the increased loading of fatty acids, but not enzymes, into EVs. Lipophagy and remodelling of mitochondrial network in melanoma cells promote fatty acid oxidation and cancer cell migration, respectively. Introduction As worldwide obesity rates continue to climb, excess body fat has emerged as a major public health issue, given its associated complications such as cardiovascular diseases, diabetes, and cancer (De Pergola & Silvestris, 2013; Park et al, 2014). Obesity is a recognized factor to increase cancer incidence and progression (Gallagher & LeRoith, 2015). This association occurs in melanoma, a skin cancer that develops from transformed melanocytes, pigment-producing cells that reside at the junction between the epidermis and the dermis. Given their high propensity to invade adjacent tissues, including subcutaneous adipose tissue (AT), and to metastasize to distant organs, melanoma is the most aggressive cutaneous cancer. Studies in murine melanoma models and epidemiological data point to a positive correlation between obesity and melanoma incidence and progression (for a recent review, see Clement et al, 2017). Indeed, although controverted at first (Sergentanis et al, 2013), now, most epidemiological studies indicate that obesity increases the risk of developing melanoma, at least in men (Renehan et al, 2008; Dobbins et al, 2013). Although this association is not always observed in the female population (Olsen et al, 2008; Dobbins et al, 2013), this may be due to confounding factors. Indeed, adjustment for sunlight exposure (Shors et al, 2001; Gallus et al, 2006) or use of hormone replacement therapy and menopausal status (Reeves et al, 2007) reveal positive associations between melanoma risk and obesity in women. On the other hand, the association between obesity and melanoma aggressiveness has been demonstrated in epidemiological studies in both men and women (de Giorgi et al, 2013; Skowron et al, 2015; Stenehjem et al, 2018) and in murine models (Pandey et al, 2012; Jung et al, 2015; Malvi et al, 2016). Seemingly contrarily, recent data from McQuade and collaborators show increased BMI (body mass index) can increase progression-free and overall survival in male patients with metastatic melanoma, but this association only concerns patients treated with immunotherapy or targeted therapy but not chemotherapy, showing that obesity favors response to certain treatments but not the aggressiveness of the disease itself (McQuade et al, 2018). Adipocytes, the main component of AT, reside in many cancer microenvironments and contribute to tumor progression through soluble factors, such as leptin or interleukin 6, and extracellular matrix remodeling (Andarawewa et al, 2005; Dirat et al, 2011; Duong et al, 2017). Recent findings highlight a metabolic cooperation between adipocytes and tumor cells, which is proving to be a key process in their tumor-promoting effects (Nieman et al, 2011; Balaban et al, 2017; Wang et al, 2017; Zhang et al, 2018). Cellular components in the tumor microenvironment are major regulators of tumor metabolism, driving cancer cells to favor certain metabolic pathways (Gouirand et al, 2018). In particular, adipocytes provide a local supply of fatty acids (FA) that can serve as an energy source for tumors. Indeed, tumor secretions trigger lipolysis in neighboring adipocytes (Dirat et al, 2011), and the released FA fuel fatty acid oxidation (FAO) in tumors, which increases tumor aggressiveness (Nieman et al, 2011; Balaban et al, 2017; Wang et al, 2017; Zhang et al, 2018). FAO has recently emerged as a pro-tumoral pathway involved in cancer cell proliferation, stemness, and invasion (Carracedo et al, 2013; Kuo & Ann, 2018), but many of the molecular mechanisms behind these effects remain elusive. Even though adipocytes have a heightened effect on tumor progression in obesity (Nieman et al, 2013; Duong et al, 2017), we still do not understand whether their metabolic cooperation with tumor cells is involved in this process. Until now, studies showed that metabolic exchanges between adipocytes and tumor cells require a close proximity between the two cell types in order for tumors to provoke adipocyte lipolysis. For most types of cancer, this process can only occur at the later stages of cancer progression, when tumors become invasive and penetrate local AT or metastasize to adipocyte-rich environments (Nieman et al, 2011; Wang et al, 2017; Zhang et al, 2018; Laurent et al, 2019). However, whether adipocytes could also influence tumors at distance, for example, during the early stages of disease progression before cancer cells infiltrate surrounding adipose tissue, remains unknown. We predict that extracellular vesicles (EV) are key contributors in such a process. They allow the transfer of biomolecules, including nucleic acids, proteins, and lipids, to distant cells, since they diffuse through tissues and circulate in body fluids (Shah et al, 2018). Previously, we demonstrated that adipocyte EV are key participants in melanoma progression (Lazar et al, 2016). Indeed, adipocytes secrete great quantities of EV that specifically carry proteins involved in lipid metabolism, including FAO enzymes. Melanoma cells internalize these EV, and this increases FAO, which promotes tumor migration and invasion but not proliferation. In obesity, adipocytes secrete more EV and, when used at equal concentrations, these EV elicit a heightened effect on melanoma migration, indicating qualitative changes in EV cargo. We predict these changes likely involve FAO actors, which remain to be identified. Although the increase in tumor aggressiveness in response to adipocyte EV is dependent on FAO, the cellular processes that link this metabolic remodeling to cell migration remain unknown. Here, we reveal a mechanism by which naïve adipocytes (unaltered by tumor cells) influence tumor metabolism. EV secreted by these adipocytes transfer both protein machinery and FA substrates required for FAO. We further show that the heightened effect of adipocyte EV in obesity depends on increased FA levels, whereas FAO-related protein levels remain unchanged. Cytoplasmic lipid droplets store FA transferred from adipocytes to melanoma cells by EV, which are then released by lipophagy to fuel FAO. Finally, we propose a mechanism that links adipocyte-induced FAO to tumor cell migration, mitochondrial dynamics. Results Adipocyte EV transfer proteins involved in FA metabolism to melanoma cells In a previous study, we have shown that both murine and human adipocyte EV increase human melanoma cell migration, invasion and metastasis (Lazar et al, 2016), and clonogenicity (Appendix Fig S1). Although we know that adipocyte EV are enriched in proteins involved in FA metabolism and that they increase melanoma migration through a process dependent on FAO (Lazar et al, 2016), the link remains correlative. So, we developed an experimental workflow using SILAC (Stable Isotope Labeling of Amino Acids in Cell Culture) to perform an unsupervised analysis of adipocyte proteins transferred to recipient tumor cells by EV (Fig 1A). We identified 2107-labeled proteins in adipocyte EV (Table EV1), which equates to approximately 85% of total EV proteins (Fig 1B). In melanoma cells exposed to these EV, we detected 587 proteins containing heavy amino acids, which indicates they were transferred from adipocytes (Table EV1). In cells not exposed to EV, we only aberrantly detected five false-positive “labeled” proteins, which underlines the robustness of this technique. Consequently, we eliminated these proteins from the list of labeled proteins. Our results indicated that approximately 30% of adipocyte EV proteins are effectively transferred to melanoma cells. Many abundant proteins within EV, such as FASN, were not transferred to melanoma cells, indicating a selective transfer and/or uptake of material. Among the proteins effectively transferred from adipocytes to melanoma cells by EV, we identified a large number of proteins involved in vesicle-mediated transport and in energy metabolism, including mitochondrial FAO enzymes (Fig 1C and Table EV1). We also identified proteins involved in FA transport and storage, as well as oxidative phosphorylation (Table EV1). Thus, using an adapted SILAC technique, we conclude that FAO-related proteins are not only present in adipocyte EV, but also efficiently transferred to melanoma cells. Figure 1. Adipocyte EV transfer proteins involved in FA metabolism to melanoma cells Workflow of the SILAC approach. 3T3-F442A cells were seeded and differentiated in the presence of heavy amino acids. After 14 days of differentiation, the EV secreted by the mature labeled adipocytes were isolated and analyzed by mass spectrometry to evaluate the presence of heavy amino acid-containing proteins. These EV were also added to SKMEL28 cells for 12 h, and then, LC-MS/MS analysis was performed to identify heavy amino acid-containing proteins that had been transferred from adipocytes to melanoma cells via EV. Three independent samples (Exp 1–3) of EV secreted by labeled 3T3-F442A cells were analyzed by mass spectrometry (in duplicate injections, Inj1/2). The percentage of proteins bearing at least one peptide containing a heavy amino acid is indicated. Proteins involved in FAO and oxidative phosphorylation (OXPHOS) that are transferred from adipocytes to melanoma cells via EV are shown in red. Download figure Download PowerPoint The heightened effect of adipocyte EV in obesity does not depend on increased FAO protein transfer FAO levels are greater in melanoma cells treated with adipocyte EV from obese mice fed a high-fat diet (HFD), which will be termed hereafter HFD-EV, when compared to those treated with EV secreted by adipocytes from lean mice fed a normal diet (ND-EV) (Fig 2A). To decipher the underlying mechanisms responsible for increased FAO induced by HFD-EV, we performed a comparative quantitative proteomic analysis of adipocyte EV from lean and obese mice. We identified 1,557 proteins, of which 87 and 100 were, respectively, more or less abundant in obese samples (Table EV2). Twenty proteins involved in mitochondrial FAO were present in equal abundance in both samples in this analysis (Fig 2B, Table EV2, and Appendix Fig S2A), which we confirmed by Western blot for the two key FAO enzymes, ECHA (trifunctional enzyme subunit alpha, gene name HADHA) and HCDH (Hydroxyacyl-coenzyme A dehydrogenase, gene name HADH), in murine and human samples (Fig 2C). Moreover, we found that HFD-EV are not preferentially taken up by melanoma cells, when compared to ND-EV (Appendix Fig S2B and C). This suggests that increased transfer of FAO enzymes is not responsible for the heightened effect of adipocyte EV in obesity. In accord, although FAO enzyme levels increased in the presence of primary adipocyte EV (Fig 2D), in align with a transfer of these proteins by EV, as demonstrated in the SILAC experiment (Table EV1 and Fig 1C), we found no further increase in cells treated with HFD-EV (Fig 2D). Moreover, in melanoma cells treated with primary adipocyte EV from lean and obese mice, HADHA and HADH (respectively, coding for ECHA and HCDH) mRNA levels are unchanged, as well as mRNA levels of CPT1A, a rate-limiting enzyme of FAO that controls FA entry in mitochondria (Fig 2E). Finally, cycloheximide, an inhibitor of protein synthesis, has no effect on the increase of FAO induced by adipocyte EV, demonstrating that this process does not depend on endogenous protein synthesis (Fig 2F). Figure 2. Adipocyte EV-induced FAO is increased by obesity, but this process is not dependent on increased protein transfer Two human (SKMEL28 and 1205Lu) and a murine (B16BL6) melanoma cell lines were exposed, or not, to the indicated EV from primary murine adipocytes obtained from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD), and then, FAO was measured (n = 5 for B16BL6, SKMEL28 HFD-EV and 1205Lu ND-EV and n = 6 for other conditions). Volcano plot of mass spectrometry-based quantitative proteomics results showing relative abundance of proteins in primary murine adipocyte EV from obese mice (HFD), as compared to those from lean mice (ND). The dashed lines indicate cutoff values and points colored in gray indicate proteins that display non-significant fold-change by Welch t-test between both conditions (n = 3 for ND, 4 for HFD). Proteins involved in FAO are indicated by yellow dots. Western blot analysis of the indicated FAO enzymes in the EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice (top panel) and from human individuals with varying BMI (normal weight, NW; overweight, OW; and obese, OB) (bottom panel). For each blot, extracts from three independent batches of murine samples or three independent individuals for human samples (1–3) are shown. Flotillin 1 (FLOT1) is used as a loading control. Western blot analysis of the indicated FAO enzymes in melanoma cells treated, or not, with EV from lean (ND) and obese (HFD) mice. Tubulin (TUB) is used as a loading control. RT–qPCR analysis of mRNAs for the indicated genes in 1205Lu cells treated or not with EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice for 48 h. Results are expressed relative to the corresponding value for control cells (n = 3). Analysis of FAO levels in 1205Lu cells exposed to 3T3-F442A EV and treated, or not, with cycloheximide (CHX) (n = 5). Data information: Bars and error bars represent means ± SEM; statistically significant by one-way ANOVA with post hoc Tukey's test, *P < 0.05, **P < 0.01, ***P < 0.001, ns: non-significant. Source data are available online for this figure. Source Data for Figure 2 [embj2019102525-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint Collectively, these results show that the heightened effect of adipocyte EV in obesity is not related to increased levels of FAO enzymes, whether transferred by adipocyte EV or expressed endogenously. Adipocyte EV transfer FA to melanoma cells to fuel FAO, a process increased by obesity Increased FAO requires both the protein machinery necessary to perform the enzymatic processes and the presence of the substrates. So, we postulated that adipocyte EV transfer FA to melanoma cells and that this process may be amplified in obesity, which could account for increased tumor cell FAO (Fig 2A) and consequently aggressiveness. EV secreted by mature 3T3-F442A adipocytes contain high levels of FA compared to their precursors (Fig 3A). To determine whether these FA are subsequently transferred to tumor cells, we used a lipid pulse-chase assay (Fig 3B). We loaded 3T3-F442A adipocytes with BODIPY FL C16 (Appendix Fig S3A) and monitored the EV transfer of this fluorescent FA. We detected fluorescent FA in tumor cells exposed to adipocyte EV (Fig 3C, and Appendix Fig S3B and C). This fluorescent FA serves as a substrate for FAO in melanoma cells, as treatment with the FAO inhibitor, Etomoxir, leads to a further accumulation of fluorescent lipids (Fig 3D). To confirm that EV were responsible for this FA transfer, and not other structures such as lipoproteins or other particles that may have been co-isolated by differential centrifugations, we fractioned the EV-containing 100,000 g pellet by size exclusion chromatography and characterized their size and their FA, triglyceride, and protein content (Appendix Fig S4A–E). Our results show that only fractions containing EV (fractions 6–13) can reproduce lipid droplet accumulation in melanoma cells, but also their pro-migratory effect (Appendix Fig S4F and G). Figure 3. Adipocyte EV transfer FA to melanoma cells to fuel FAO, and this transfer is increased in obesity Lipids were extracted from EV secreted by 3T3-F442A preadipocytes and differentiated 3T3-F442A adipocytes (respectively, preAd-3T3-EV and Ad-3T3-EV), and FA content was measured (n = 6). Workflow of the assay used to evaluate FA transfer by 3T3-F442A adipocyte EV (3T3-EV) to melanoma cells. Mature 3T3-F442A adipocytes were loaded with BODIPY FL C16. Cells were then washed, and fresh medium was added. Seventy-two hours later, conditioned medium was harvested, and 3T3-F442A EV (3T3-EV) were isolated and added to melanoma cells. Indicated melanoma cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 (3T3-FL C16-EV) and, 24 h later, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy. Left panel, indicated cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 and immediately treated, or not, with Etomoxir for 24 h. Then, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy. Right panel, quantification of BODIPY FL C16 staining area per cell (n = 3). Lipids were extracted from EV secreted by adipocytes from lean (ND) and obese (HFD) mice (n = 5) (left panel) or from human adipose tissue samples from patients with varying BMI (right panel) and fatty acid content was measured (n = 15). Indicated cells were exposed, or not, to adipocyte EV from primary adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) for 24 h. Then, cells were fixed, stained with BODIPY, and counterstained with DAPI. Left panel, confocal microscopy observation. Right panel, quantification of BODIPY staining area per cell (n = 5 for SKMEL28 and n = 6 for 1205Lu). Data information: Scale bars represent 20 μm. Bars and error bars represent means ± SEM; statistically significant by unpaired Student's t-test (A, D), or by one-way ANOVA with post hoc Tukey's test (E, F), whereas Spearman's rank correlation was used to evaluate the correlation between FA content in human adipocyte EV and patient BMI (E). *P < 0.05, **P < 0.01. ***P < 0.001, ns: non-significant. Download figure Download PowerPoint EV secreted by primary adipocytes also transfer FA to melanoma cells (Appendix Fig S5A). To determine whether this FA transfer increases in obesity, we evaluated FA content in EV secreted by primary murine and human adipocytes. FA content significantly increased in murine adipocyte EV in obesity (Fig 3E, left panel) and positively correlated with BMI in human samples (Fig 3E, right panel), although triglyceride content remained unchanged in obesity (Appendix Fig S5B), consistent with recent lipidomic data (Flaherty et al, 2019). Since hypertrophic adipocytes internalize less lipids than their smaller counterparts (Frayn, 2001; Hill et al, 2009; Appendix Fig S5C), we could not use the lipid pulse-chase assay described in Fig 3B to compare FA transfer by ND-EV and HFD-EV. Nevertheless, HFD-EV strongly increased the total neutral lipid content in melanoma cells compared to ND-EV, which supports our hypothesis (Fig 3F and Appendix Fig S5D). Thus, these results demonstrate that adipocytes transfer FA to melanoma cells through EV, a process that is amplified by obesity. Transferred FA are stored in lipid droplets and released by lipophagy Although the FA transferred to melanoma cells by adipocyte EV fuel FAO, we also show an increase in neutral lipid storage within cytoplasmic lipid droplets (Fig 3D and F, and Appendix Figs S5D and S6), a process known to prevent lipotoxicity (Listenberger et al, 2003). We therefore examined whether these stored lipids are mobilized to drive FAO. We observed that melanoma cells incubated with adipocyte EV present double membrane structures, characteristic of autophagosomes that contain lipids within their lumen (Figs 4A and EV1A). Thus, we hypothesized that the transferred FA were degraded by lipophagy, an autophagic process that releases FA from lipid droplets (Singh et al, 2009). In accord with this theory, FA transferred by adipocyte EV colocalize with lysosomes (Figs 4B and EV1B). We found that inhibiting autophagy using Bafilomycin A1 prevented the degradation of lipid stores accumulated in response to adipocyte EV (Appendix Fig S7A) and blocked their pro-migratory effect on melanoma cells (Appendix Fig S7B). We obtained similar results using the selective lysosomal acid lipase inhibitor, Lalistat 2 (Rosenbaum et al, 2010; Hamilton et al, 2012). This compound increased colocalization between fluorescent FA transferred by adipocyte EV and lysos" @default.
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• W3000534695 title "Adipocyte extracellular vesicles carry enzymes and fatty acids that stimulate mitochondrial metabolism and remodeling in tumor cells" @default.
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• W3000534695 doi "https://doi.org/10.15252/embj.2019102525" @default.
• W3000534695 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6996584" @default.
• W3000534695 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/31919869" @default.
• W3000534695 hasPublicationYear "2020" @default.
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