CD9-dependent extracellular vesicles mediate the propagation of ferroptosis
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ABSTRACT:
PROVIDER: PRJNA1055445 | ENA |
REPOSITORIES: ENA
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Project description:Ferroptosis is a form of regulated cell death that is mediated by accumulation of lipid peroxidation. Emerging evidences report dispersion of ferroptosis between neighboring cells. However, the molecular mechanism underlying the propagation of ferroptosis remains unclear. Here we provide evidences that ferroptotic cells elicit cellular projections that produce massive extracellular vesicles (EVs). Proteomic analysis reveals that these EVs are CD9 positive and contain major components of mitogen-activated protein kinase (MAPK) signaling. MAP2K1/2 that is transferred by EVs increases the sensitivity to ferroptosis of target cells. We further show that a MAP2K-interacting transcription factor, peroxisome-proliferator activator receptor gamma (PPARG), activates various components of ferroptosis defense systems. Moreover, perturbation of EV-based transport protects adjacent tissue from ferroptosis in vivo. Our study reveals an EV-dependent transport between ferroptotic cells and surrounding neighbors and establishes that MAPK-responsive PPARG is essential for ferroptosis defense in recipient cells.
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Project description:Cells generate mass amount of EVs during ferroptosis. To evaluate the unique proteins secreted by ferroptitic EVs, and the potential mechnism of ferroptosis propagation through EV transportation,we analysed the proteomics of EVs from U-2 OS cells. U-2 OS cells were treated with DMSO or RSL3 (to induce ferroptosis) and EVs were collected from the culture medium.We further evaluated teh mice kidney derived EV proteomics and identified upregulated proteins in RSL3-treated mice. To elucidate the function of CD9-positive EVs, we generated wild type and CD9-KO HT1080 xenografts and treated the mice with RSL3 and enriched the xenograft EVs and performed proteomics analysis.
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Project description:This study aimed to identify subpopulations of extracellular vesicles (EVs) secreted by HeLa cells, and determine markers which enable to identify them, and which indicate their endosomal or plasma membrane origin. We used CD63 and CD9 as markers of EVs, and followed their intracellular trafficking using the RUSH assay. We used mass spectrometry to identify specific or common proteins in immunoprecipitated GFP+ EVs from HeLa transfected with the RUSH CD63-GFP or CD9-GFP, after a short (3h) or a long (24h) trafficking time from the endoplasmic reticulum.
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Project description:While of growing interest in pancreatic cancer (PC) field, the stromal-tumor cells crosstalk lags behind in terms of biomarkers and therapeutic options improving the clinical armamentarium. Knowledge on cellular communications was drastically enhanced following the discovery of extracellular vesicles (EVs), a powerful process of intercellular exchanges. We previously described a new stromal-tumor cell crosstalk mediated by Cancer-Associated Fibroblasts (CAFs)-derived ANXA6+-EVs, supporting pancreatic cancer cell aggressiveness in hostile areas of PC. In this study, using mass spectrometry analyses to investigate CAFs-derived EVs' cargo, we report that CD9 is a key member of the ANXA6/LRP1/TSP1 complex present in PC-associated CAFs-derived ANXA6+-EVs. We determined that CD9 is expressed by PC-associated CAFs in vivo as well as in vitro following physiopathologic culture conditions. Targeting CD9 impaired CAFs-derived ANXA6+-EVs uptake by pancreatic cancer cells, which consequently decreases their migratory abilities. Signaling pathway arrays highlighted p38/MAPK as activated in pancreatic cancer cells following CAFs-derived ANXA6+/CD9+-EVs uptake. The use of CD9 blocking antibody, p38 siRNA or chemical inhibitors impaired pancreatic cancer cells abilities following incubation with CAFs-derived ANXA6+/CD9+-EVs. Finally, we revealed CD9 expression as an independent poor-prognosis marker in human PC samples. Collectively our data highlight the key role of CD9 in CAFs-derived ANXA6+-EVs internalization by pancreatic cancer cells and the consequent, and mandatory, activation of p38/MAPK pathway to foster their migratory abilities. Measuring the oncogenic CAFs-derived ANXA6+/CD9+-EVs then limiting their action on pancreatic cancer cells abilities might be a promising option for PC stratification and treatment.
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Project description:Clostridioides difficile CD9 Genome sequencing and assembly
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Project description:The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the quantity and type of EVs that are obtained. The aim of this study was therefore to determine the presence of different subpopulations of EVs in plasma and serum. Blood samples were collected from healthy subjects, from which plasma and serum were isolated in parallel. ACD-A or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. We previously developed a method utilising a combination of density cushion and size exclusion chromatography to isolate EVs from plasma with minimal contamination of lipoprotein particles. In the current study, we applied this method to both EDTA-plasma and serum. In addition, EVs were isolated by a combination of density cushion and density gradient or by bead immune capturing (anti-CD63, anti-CD9, and anti-CD81 beads). The subpopulations of EVs were analysed by nanoparticle tracking analysis, Western blot, single particle interferometric reflectance imaging sensing, conventional and nano flow cytometry, magnetic bead enzyme-linked immunosorbent assay, and mass spectrometry.This study shows that a larger number of CD9+ EVs are present in EDTA-plasma compared to ACD-plasma and serum, and the presence of CD41a on theses EVs suggests that they are released from platelets. Furthermore, only a small number of EVs in blood were double positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Together, these findings show that multiple subpopulations of EVs are present in blood including CD9+/CD41a+, CD9+/CD63+/CD41+, CD63+/CD41a+, CD63+/CD9+/CD81−, CD81+/CD9+/CD63−, and CD9+/CD63+/CD81+ and that their presence is dissimilar in EDTA-plasma, ACD-plasma and serum.
2022-05-20 | PXD026863 | Pride
Project description:We developed a column-based CD9 antibody-immobilized HPLC immunoaffinity (CD9-HPLC-IAC) technology for EV isolation from a microliter-scale of serum for downstream proteomic analysis. The CD9-HPLC-IAC method achieved EV isolation from 40 μL of serum in 30 min with a yield of 8.0×109 EVs. Proteomic analysis showed that the common exosomal markers such as CD63, CD81, CD82, Alix, and TSG101 were all identified in EVs. Statistical analysis of EV protein content showed that the top 10 serum proteins in EVs was significantly decreased by using the CD9-HPLC-IAC method compared to the ultracentrifugation (UC) (p=0.001) and size exclusion chromatography (SEC) (p=0.009), and apolipoproteins significantly reduced 4.8-fold compared to the SEC method (p<0.001). The result demonstrated the potential of the CD9-HPLC-IAC method for efficient isolation and proteomic characterization of EVs from a micro-scale volume of serum.
2022-10-14 | PXD027025 | Pride
Project description:Transcriptomes of differentiated cells of the conditionally immortalized mouse podocyte cell line SVI (Schiwek et al., Kidney Int. 66: 91-101, 2004) were determined as described in Warsow et al. (Kidney Int. 84: 104-115, 2013) after application of mechanical stress (Endlich et al., J. Am. Soc. Nephrol. 12: 413-422, 2001) as compared to control conditions. Elevated glomerular pressure represents a high risk for the development of severe kidney diseases and causes an increase of mechanical load to podocytes. In this study we investigated whether mechanical stress alters gene expression in cultured podocytes using gene arrays. We found that tetraspanin CD9 is significantly upregulated in cultured podocytes after mechanical stress. The differential expression of CD9 was confirmed by RT-PCR and Western blot under stretched and unstretched conditions. Furthermore, mechanical stress resulted in a relocalization of CD9. To get an insight into the functional role of CD9, podocytes were transfected with pEGFP-CD9. The expression of CD9 induced the formation of substratum-attached thin arborized protrusions (TAPs). Ca2+ depletion revealed that podocytes over-expressing CD9 possess altered adhesive properties in contrast to the control transfected cells. Finally, elevated CD9 expression increased migration of podocytes in a wound assay. In summary, our results suggest that upregulation of CD9 may play an important role in podocyte morphology, adhesion and migration.
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Project description:Fibroblast-derived extracellular vesicles contain SFRP1 and mediate pulmonary fibrosis
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