c1e634eba45473c7d17ba45a903da4ee pmc_34638272.pdf 863ed5417285534071800b6f0e95cee24c9527bb pmc_34638272.pdf 5cbadfeff89b78e164dbb9da58e7e4deabeb2f7b0757c62b54a848a1d545e0fd pmc_34638272.pdf Title: Heterogeneity of Melanoma Cell Responses to Sleep Apnea-Derived Plasma Exosomes and to Intermittent Hypoxia Subject: Obstructive sleep apnea (OSA) is associated with increased cutaneous melanoma incidence and adverse outcomes. Exosomes are secreted by most cells, and play a role in OSA-associated tumor progression and metastasis. We aimed to study the effects of plasma exosomes from OSA patients before and after adherent treatment with continuous positive airway pressure (CPAP) on melanoma cells lines, and also to identify exosomal miRNAs from melanoma cells exposed to intermittent hypoxia (IH) or normoxia. Plasma-derived exosomes were isolated from moderate-to-severe OSA patients before (V1) and after (V2) adherent CPAP treatment for one year. Exosomes were co-incubated with three3 different melanoma cell lines (CRL 1424; CRL 1619; CRL 1675) that are characterized by genotypes involving different mutations in BRAF, STK11, CDKN2A, and PTEN genes to assess the effect of exosomes on cell proliferation and migration, as well as on pAMK activity in the presence or absence of a chemical activator. Subsequently, CRL-1424 and CRL-1675 cells were exposed to intermittent hypoxia (IH) and normoxia, and exosomal miRNAs were identified followed by GO and KEG pathways and gene networks. The exosomes from these IH-exposed melanoma cells were also administered to THP1 macrophages to examine changes in M1 and M2 polarity markers. Plasma exosomes from V1 increased CRL-1424 melanoma cell proliferation and migration compared to V2, but not the other two cell lines. Exposure to CRL-1424 exosomes reduced pAMPK/tAMPK in V1 compared to V2, and treatment with AMPK activator reversed the effects. Unique exosomal miRNAs profiles were identified for CRL-1424 and CRL-1675 in IH compared to normoxia, with six miRNAs being regulated and several KEGG pathways were identified. Two M1 markers (CXCL10 and IL6) were significantly increased in monocytes when treated with exosomes from IH-exposed CRL-1424 and CRL-1625 cells. Our findings suggest that exosomes from untreated OSA patients increase CRL-1424 melanoma malignant properties, an effect that is not observed in two other melanoma cell lines. Exosomal cargo from CRL-1424 cells showed a unique miRNA signature compared to CRL-1675 cells after IH exposures, suggesting that melanoma cells are differentially susceptible to IH, even if they retain similar effects on immune cell polarity. It is postulated that mutations in STK-11 gene encoding for the serine/threonine kinase family that acts as a tumor suppressor may underlie susceptibility to IH-induced metabolic dysfunction, as illustrated by CRL-1424 cells. Keywords: sleep apnea; positive airway pressure; CPAP; exosomes; melanoma; miRNAs; AMPK; CRL-1424; CRL-1619; CRL-1675; intermittent hypoxia Author: Abdelnaby Khalyfa, Wojciech Trzepizur, Alex Gileles-Hillel, Zhuanhong Qiao, David Sanz-Rubio, José M. Marin, Miguel A. Martinez-Garcia, Francisco Campos-Rodriguez, Isaac Almendros, Ramon Farre, Manuel Sanchez-de-la-Torre, Francisco García-Río and David Gozal Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sun Sep 26 04:45:14 2021 CEST ModDate: Sun Sep 26 04:50:29 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 25 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3271063 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- SAULST+URWPalladioL-Ital Type 1 Custom yes yes no 10 0 YNNQWF+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 XQRMXF+URWPalladioL-Roma Type 1 Custom yes yes no 19 0 YSPQJI+VnURWPalladioL-Bold Type 1 Custom yes yes no 23 0 DNNUWA+VnURWPalladioL Type 1 Custom yes yes no 28 0 LCJEVJ+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 37 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom 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WinAnsi yes yes no 297 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 301 0 ABCDEE+Palatino Linotype,BoldItalic TrueType WinAnsi yes yes no 305 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-03-22 02:42:42 CET RepresentationInformation: pmc_34638272.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-01-11 05:22:45 CET Size: 3271063 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 635 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Human Subjects Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Cell Lines and Culture Reagents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Exosome Isolation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Children: Item: Title: Plasma Exosomes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Cell Culture Exosomes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Exosome Uptake by Melanoma Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Exosome Size Determination and Quantification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Proliferation and Migration Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: p-AMPK and p-AMPK Activator Western Blots Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Cell Cultures and Intermittent Hypoxia Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: qRT-PCR Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Exosomal miRNA Isolation from Melanoma Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Children: Item: Title: miRNAs Microarray Processing and Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Target Gene Prediction and Function Enrichment Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: THP-1 Monocytes and Differentiation to Macrophages Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Data Analyses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Children: Item: Title: Human Subjects Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Exosome Characterization and Uptake Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Cell Proliferation and Migration Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Effect of Exosomes on AMPK Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Effect of Intermittent Hypoxia on Exosomes Released by Melanoma Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: Melanoma Gene Ontology and KEGG Pathways Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Effects of Exosomes Derived from Melanoma Cells on Naïve THP1 Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Info: Title: Heterogeneity of Melanoma Cell Responses to Sleep Apnea-Derived Plasma Exosomes and to Intermittent Hypoxia Author: Abdelnaby Khalyfa, Wojciech Trzepizur, Alex Gileles-Hillel, Zhuanhong Qiao, David Sanz-Rubio, José M. Marin, Miguel A. Martinez-Garcia, Francisco Campos-Rodriguez, Isaac Almendros, Ramon Farre, Manuel Sanchez-de-la-Torre, Francisco García-Río and David Gozal Subject: Obstructive sleep apnea (OSA) is associated with increased cutaneous melanoma incidence and adverse outcomes. Exosomes are secreted by most cells, and play a role in OSA-associated tumor progression and metastasis. We aimed to study the effects of plasma exosomes from OSA patients before and after adherent treatment with continuous positive airway pressure (CPAP) on melanoma cells lines, and also to identify exosomal miRNAs from melanoma cells exposed to intermittent hypoxia (IH) or normoxia. Plasma-derived exosomes were isolated from moderate-to-severe OSA patients before (V1) and after (V2) adherent CPAP treatment for one year. Exosomes were co-incubated with three3 different melanoma cell lines (CRL 1424; CRL 1619; CRL 1675) that are characterized by genotypes involving different mutations in BRAF, STK11, CDKN2A, and PTEN genes to assess the effect of exosomes on cell proliferation and migration, as well as on pAMK activity in the presence or absence of a chemical activator. Subsequently, CRL-1424 and CRL-1675 cells were exposed to intermittent hypoxia (IH) and normoxia, and exosomal miRNAs were identified followed by GO and KEG pathways and gene networks. The exosomes from these IH-exposed melanoma cells were also administered to THP1 macrophages to examine changes in M1 and M2 polarity markers. Plasma exosomes from V1 increased CRL-1424 melanoma cell proliferation and migration compared to V2, but not the other two cell lines. Exposure to CRL-1424 exosomes reduced pAMPK/tAMPK in V1 compared to V2, and treatment with AMPK activator reversed the effects. Unique exosomal miRNAs profiles were identified for CRL-1424 and CRL-1675 in IH compared to normoxia, with six miRNAs being regulated and several KEGG pathways were identified. Two M1 markers (CXCL10 and IL6) were significantly increased in monocytes when treated with exosomes from IH-exposed CRL-1424 and CRL-1625 cells. Our findings suggest that exosomes from untreated OSA patients increase CRL-1424 melanoma malignant properties, an effect that is not observed in two other melanoma cell lines. Exosomal cargo from CRL-1424 cells showed a unique miRNA signature compared to CRL-1675 cells after IH exposures, suggesting that melanoma cells are differentially susceptible to IH, even if they retain similar effects on immune cell polarity. It is postulated that mutations in STK-11 gene encoding for the serine/threonine kinase family that acts as a tumor suppressor may underlie susceptibility to IH-induced metabolic dysfunction, as illustrated by CRL-1424 cells. Keywords: sleep apnea; positive airway pressure; CPAP; exosomes; melanoma; miRNAs; AMPK; CRL-1424; CRL-1619; CRL-1675; intermittent hypoxia Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sun Sep 26 04:45:14 CEST 2021 ModDate: Sun Sep 26 04:50:29 CEST 2021 ID: 0x60bfcda66b69b1b771c6bba431af15e3, 0x60bfcda66b69b1b771c6bba431af15e3 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: QMDDXF+CMSY10 FontSubset: true FirstChar: 0 LastChar: 14 FontDescriptor: FontName: QMDDXF+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: XQRMXF+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 239 FontDescriptor: FontName: XQRMXF+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: 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