細胞外囊泡是脂肪細胞通訊中新興的關鍵角色。值得注意的是，脂肪細胞脫落的小細胞外囊泡會刺激黑色素瘤細胞中的脂肪酸氧化和遷移，肥胖症的這些作用會增強。然而，所涉及的囊泡和細胞過程仍然很大程度上未知。1月10日土魯斯大學藥理學與結構生物學研究所團隊在The EMBO journal查看期刊詳情上發表了「Adipocyte extracellular vesicles carry enzymes and fatty acids that stimulate mitochondrial metabolism and remodeling in tumor cells」，文章闡明了將脂肪細胞胞外小泡與代謝重塑和細胞遷移聯繫起來的機制，顯示脂肪細胞囊泡通過提供酶和底物刺激黑色素瘤脂肪酸氧化。在肥胖症中，細胞外囊泡的增強作用取決於增加的脂肪酸運輸，而不是與脂肪酸氧化相關的酶。存儲在癌細胞脂質滴中的這些脂肪酸在通過脂肪吞噬釋放時驅動脂肪酸氧化。線粒體活性的這種增加將線粒體重新分配到遷移細胞的膜突起上，這對於在脂肪細胞囊泡存在下增加細胞遷移是必需的。研究結果提供了關鍵的洞察力，了解細胞外囊泡在脂肪細胞和與肥胖特別相關的腫瘤之間發生的代謝合作中的作用。
Figure 1. Adipocyte EV transfer proteins involved in FA metabolism to melanoma cells. A， 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 wereisolated 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. B 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. C Proteins involved in FAO and oxidative phosphorylation (OXPHOS) that are transferred from adipocytes to melanoma cells via EV are shown in red.
Figure 2. Adipocyte EV-induced FAO is increased by obesity, but this process is not dependent on increased protein transfer.A 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. B ，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. Proteins involved in FAO are indicated by yellow dots.C， 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. 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. D 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.E， 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.F， Analysis of FAO levels in 1205Lu cells exposed to 3T3-F442A EV and treated, or not, with cycloheximide (CHX).
Figure 3. Adipocyte EV transfer FA to melanoma cells to fuel FAO, and this transfer is increased in obesity.A ，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. B， 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. C， 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. D， 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. E， Lipids were extracted from EV secreted by adipocytes from lean (ND) and obese (HFD) mice (left panel) or from human adipose tissue samples from patients with varying BMI (right panel) and fatty acid content was measured. F， 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.
Figure 4. FA transferred from adipocytes to melanoma cells by EV are released from lipid droplets by lipophagy. A， Transmission electron micrographs of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV). B， 1205Lu cells were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 in the presence, or not, of Lalistat 2. Then, live cells were stained with the LysoTracker probe and observed by confocal microscopy. Arrows indicate colocalization. C， 1205Lu were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 and treated, or not, with Lalistat 2. Then, cells were fixed and counterstained with DAPI before observation by confocal microscopy. Quantification of BODIPY FL C16 staining per cell is shown beside. D ，1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Lalistat 2 (Lal). E 1205Lu cells were exposed, or not, to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high-fat diet (HFD) with, or without, Lalistat 2 (Lal). Cells were then fixed, stained with BODIPY, and counterstain with DAPI before observation by confocal microscopy. F，1205Lu cells were exposed, or not, to adipocyte EV from ND or HFD mice with, or without, Lalistat 2 (Lal). Cell motility was then tracked by video microscopy.
Figure 5. Adipocyte EV modify melanoma mitochondrial dynamics, a process that promotes melanoma aggressiveness and is exacerbated by obesity. A ，1205Lu cells exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1, were stained with a MitoTracker probe, fixed, and observed by confocal microscopy. B， Western blot analysis of the indicated mitochondrial fission proteins in melanoma cells treated, or not, with EV from 3T3-F442A adipocytes (3T3-EV). Tubulin (TUB) is used as a loading control. C， 1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1. Cell migration was then evaluated in Boyden chamber assays. D ，1205Lu cells were transfected, or not, with two different siRNA targeted against DRP1 (siDRP1#1 or 2) or an untargeted siRNA (siUT). Top, 48 h after transfection,protein extracts were prepared and DRP1 expression was evaluated by Western blot. Bottom, 36 h post-transfection, cells were exposed to 3T3-F442A EV (3T3-EV),and cell motility was tracked by video microscopy. E ，1205Lu cells were exposed to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high-fat diet (HFD) and immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy.
Figure 6. Lipid droplets are found in membrane protrusions, at proximity to mitochondria, in melanoma cells exposed to adipocyte EV. A, Left panel, indicated melanoma cells exposed to 3T3-F442A EV were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. B, Left panel, melanoma cells exposed to EV secreted by adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high-fat diet (HFD) were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions. C, 1205Lu cells exposed to 3T3-F442A EV were stained with a MitoTracker probe. Then, cells were fixed and stained with BODIPY before observation by confocal microscopy. D ,Transmission electron microscope observations of 1205Lu cells exposed, or not, to 3T3-F442A EV. Mitochondria are colored in pink and lipid droplets are colored in yellow on images on the right. E, Number of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV). F, Area of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV. G, Left panel, 1205Lu melanoma cells were exposed, or not, to 3T3-F442A EV. Then, live cells were stained with LysoTracker and BODIPY probes and observed by confocal microscopy. Right panel, quantification of the percentage of cells presenting lysosome within membrane protrusions.
Figure 7. FA metabolism and mitochondrial dynamics are associated with melanoma aggressiveness. A, TCGA data were analyzed to reveal the effect of high mRNA levels of key FAO enzymes on the overall survival of patients with melanoma. B, BODIPY staining of neutral lipid (from left to right). DAPI was used to counterstain nuclei. C, FAO in the indicated melanoma cell lines was measured. D, TCGA data were analyzed to reveal the effect of high mRNA levels of key actors in mitochondrial dynamics on the overall survival of patients with melanoma.
使用基於SILAC技術的突破性方法，通過EV確定了通過脂肪從脂肪細胞轉移到腫瘤細胞的蛋白質，該方法允許對脂肪細胞蛋白質進行廣泛的標記，以便隨後通過質譜法在腫瘤細胞內進行鑑定。這是首次對通過EV在兩種細胞類型之間轉移的所有蛋白質進行了全面分析，文章還說明了該技術對任何培養的細胞類型研究EV介導的細胞間蛋白質轉運的實用性。使用這種方法，發現在受體黑素瘤細胞中僅檢測到約30％的標記脂肪細胞EV蛋白，而一些高度豐富的EV蛋白則被排除在轉移範圍之外。這個過程表明在脂肪細胞和黑色素瘤細胞之間有高度選擇性的EV介導的蛋白質轉移，這可能是由於某些EV群體沒有被黑色素瘤細胞或某些EV內在化，或者它們攜帶的蛋白質被快速分類以內化後降解。雖然還不完全了解如何在受體細胞中選擇性地內化和/或處EVs，但文章的方法為將來的研究解決這些問題提供了基礎。儘管有這種選擇性轉移，但黑色素瘤細胞還是內在地參與了糧農組織，線粒體呼吸和通過EV產生ATP的脂肪細胞蛋白。重要的是，還確定了黑色素瘤細胞響應脂肪細胞EV誘導的其他細胞過程中的關鍵貢獻者，這有助於調節其促遷移作用，脂質吞噬和線粒體動力學。
除了這種蛋白質轉移，還證明了脂肪細胞EVs還可以將FA傳遞給腫瘤細胞以驅動FAO，該過程的增加增強了肥胖中脂肪細胞EV的作用。先前已經描述了通過EV將代謝底物從腫瘤微環境轉移到癌細胞。確實，CAF衍生的EV將代謝產物轉運至腫瘤細胞，從而觸發了中央碳代謝。文章清楚地表明，脂肪細胞分泌的EV也可能導致FA轉移，僅這些EV就足以重塑黑色素瘤的代謝並促進侵略性。
因此，脂肪細胞EV介導脂肪細胞和黑色素瘤細胞之間的代謝合作，充當穿梭傳遞FAO所需的蛋白質機制和脂質底物的作用。有趣的是，在使用異丙腎上腺素刺激脂肪細胞脂解後，EV分泌和EV FA水平增加。因此，腫瘤誘導的脂解也可能促進這種過程是合理的。在文章之後，未來的研究應集中在癌細胞和脂肪細胞之間發生的雙向串擾對EV的影響。
在這項研究中，集中研究了調節脂肪細胞EV響應的調節黑色素瘤代謝和侵襲性的脂質和蛋白質因子，但重要的是要注意EV還轉運核酸，包括mRNA，microRNA和其他非編碼RNA，也可能參與了其促腫瘤作用。此外，脂肪細胞EV含有脂肪因子，尤其是瘦素，已被證明在黑色素瘤模型中具有促腫瘤作用，並且與黑色素瘤患者淋巴結轉移的風險增加有關。
研究結果也引起了人們對與肥胖相關的代謝併發症的藥物對脂肪細胞與腫瘤細胞之間對話的影響的質疑。同樣，二甲雙胍也影響FA代謝的不同方面，在許多不同的細胞類型中減少脂質的合成並增加FAO的含量，但在臨床前研究中還顯示其具有抗腫瘤特性。因此，要預見這種治療對肥胖癌症患者的影響是具有挑戰性的，未來的研究應集中於評估這一點。
總而言之，脂肪細胞EV提供了FAO對黑素瘤細胞所需的機制（酶）和底物（FA）。這些EVs轉移的脂肪酸被儲存在脂滴中，並通過脂肪吞噬動員，為FAO提供燃料。確定線粒體動力學為連結脂肪細胞誘導的FAO和腫瘤細胞遷移的過程。觀察到線粒體向需要突起的線粒體裂變的細胞突起的重新分布，這增加了由脂肪細胞EV誘導的細胞遷移。在肥胖症中，通過EVs提供更多的FA會放大整個過程。這些結果證明脂肪細胞和腫瘤細胞之間發生代謝合作。此外，文章揭示了這種相互作用中潛在的細胞過程，建議這些途徑可能包括治療肥胖黑色素瘤患者的新型治療靶標。
全文連結:
https://pan.baidu.com/s/1xKmp0MSpFTmUqSGWCCMn-Q
提取碼: vwx6
EVs-Exosomes由蘇大，浙大, 法國居裡研究所數位博士、博後及教授創建。