8d55ab5c1d59f9ea2a5873148ff1d6ec cancers-16-02977.pdf d3fa8637ae1d0d08a168506e2d4667aeeb5b5973 cancers-16-02977.pdf 705290304027f81d8e15ee22e8bb2e9051cd4f335458b8ec573e4a93415c0db1 cancers-16-02977.pdf Title: New Insights into the Exosome-Induced Migration of Uveal Melanoma Cells and the Pre-Metastatic Niche Formation in the Liver Subject: UM is an aggressive intraocular tumor characterized by high plasticity and a propensity to metastasize in the liver. However, the underlying mechanisms governing liver tropism remain poorly understood. Given the emerging significance of exosomes, we sought to investigate the contribution of UM-derived exosomes to specific steps of the metastatic process. Firstly, we isolated exosomes from UM cells sharing a common genetic background and different metastatic properties. A comparison of protein cargo reveals an overrepresentation of proteins related to cytoskeleton remodeling and actin filament-based movement in exosomes derived from the parental cells that may favor the detachment of cells from the primary site. Secondly, we assessed the role of macrophages in reprogramming the HHSCs by exosomes. The activation of HHSCs triggered a pro-inflammatory and pro-fibrotic environment through cytokine production, upregulation of extracellular matrix molecules, and the activation of signaling pathways. Finally, we found that activated HHSCs promote increased adhesion and migration of UM cells. Our findings shed light on the pivotal role of exosomes in pre-metastatic niche construction in the liver. Keywords: uveal melanoma; exosomes; hepatic stellate cells; macrophages; liver metastasis; mesenchymal migration; amoeboid migration Author: Raquel Ramos, Antònia Vinyals, Rafael Campos-Martin, Eduard Cabré, Joan Josep Bech, Javier Vaquero, Ester Gonzalez-Sanchez, Esther Bertran, Josep Ramon Ferreres, Daniel Lorenzo, Carolina G. De La Torre, Isabel Fabregat, Jose Maria Caminal and Àngels Fabra Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed Aug 28 11:24:07 2024 CEST ModDate: Wed Aug 28 11:31:48 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 29 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 36840648 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CXFMNW+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 VVSYYB+EURM10 Type 1 Builtin yes yes yes 16 0 YCNWPD+URWPalladioL-Bold Type 1 Custom yes yes yes 21 0 AUVJYN+URWPalladioL-Roma Type 1 Custom yes yes yes 27 0 YWIUQZ+URWPalladioL-Ital Type 1 Custom yes yes yes 32 0 CUEFWC+VnURWPalladioL Type 1 Custom yes yes yes 37 0 LCJEVJ+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 54 0 RGUFCP+CMSY10 Type 1 Builtin 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PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 1592 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Cell Lines and Cell Culture Conditions Destination: subsection.2.1 Item: Title: Viral Production and Cell Infections Destination: subsection.2.2 Item: Title: Extracellular Vesicles (EVs) Isolation Destination: subsection.2.3 Item: Title: Cryo-Electron Microscopy of EVs Destination: subsection.2.4 Item: Title: EVs Labeling and Cell Uptake Assay Destination: subsection.2.5 Item: Title: Co-Culture Assay Destination: subsection.2.6 Item: Title: Cell Adhesion Assay Destination: subsection.2.7 Item: Title: Migration Assays Destination: subsection.2.8 Item: Title: Proteomic Analysis Destination: subsection.2.9 Children: Item: Title: Protein Extraction and Digestion of EVs Destination: subsubsection.2.9.1 Item: Title: LC-MS/MS Analysis of EVs Destination: subsubsection.2.9.2 Item: Title: Western Blot Analysis Destination: subsubsection.2.9.3 Item: Title: Immunofluorescence Analysis Destination: subsubsection.2.9.4 Item: Title: Rac1 and RhoA Activity Assay Destination: subsubsection.2.9.5 Item: Title: Total RNA Isolation, Reverse Transcription, and Quantitative RT-qPCR Analysis Destination: subsection.2.10 Item: Title: Data Analysis and Biological Functional Network of the EV Protein Cargo Destination: subsection.2.11 Item: Title: Statistical Analysis Destination: subsection.2.12 Item: Title: Results Destination: section.3 Children: Item: Title: Isolation and Characterization of UM Exosomes Destination: subsection.3.1 Item: Title: Differential Proteomic Profiles of Exosomes Derived from UM Metastatic Cells Destination: subsection.3.2 Item: Title: Proteomic Profile and Network of Functional Molecular Interactions of EV Protein Cargo Identify Cell Movement Modes Destination: subsection.3.3 Item: Title: Proteomic Analysis of UM-Derived EVs Reveals a Distinct Integrin Profile in Metastatic and Non-Metastatic Cells Destination: subsection.3.4 Item: Title: Exosome Uptake by Hepatic Stellate Cells In Vitro Destination: subsection.3.5 Item: Title: UM-Derived Exosomes Induce a Pro-Fibrotic and Inflammatory Phenotype in HHSC Destination: subsection.3.6 Item: Title: UM Exosome-Treated LX2/THP-1 Simulates Cell Adhesion and Migration of UM Cells Destination: subsection.3.7 Item: Title: Discussion Destination: section.4 Children: Item: Title: Proteomic Analysis of EVs Secreted by UM-Cells Reveals Key Proteins Related to Migration and Invasion Destination: subsection.4.1 Item: Title: Contribution of EVs Secreted by UM Cells to Pre-Metastatic Niche Formation and Liver Colonization Destination: subsection.4.2 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: New Insights into the Exosome-Induced Migration of Uveal Melanoma Cells and the Pre-Metastatic Niche Formation in the Liver Author: Raquel Ramos, Antònia Vinyals, Rafael Campos-Martin, Eduard Cabré, Joan Josep Bech, Javier Vaquero, Ester Gonzalez-Sanchez, Esther Bertran, Josep Ramon Ferreres, Daniel Lorenzo, Carolina G. De La Torre, Isabel Fabregat, Jose Maria Caminal and Àngels Fabra Subject: UM is an aggressive intraocular tumor characterized by high plasticity and a propensity to metastasize in the liver. However, the underlying mechanisms governing liver tropism remain poorly understood. Given the emerging significance of exosomes, we sought to investigate the contribution of UM-derived exosomes to specific steps of the metastatic process. Firstly, we isolated exosomes from UM cells sharing a common genetic background and different metastatic properties. A comparison of protein cargo reveals an overrepresentation of proteins related to cytoskeleton remodeling and actin filament-based movement in exosomes derived from the parental cells that may favor the detachment of cells from the primary site. Secondly, we assessed the role of macrophages in reprogramming the HHSCs by exosomes. The activation of HHSCs triggered a pro-inflammatory and pro-fibrotic environment through cytokine production, upregulation of extracellular matrix molecules, and the activation of signaling pathways. Finally, we found that activated HHSCs promote increased adhesion and migration of UM cells. Our findings shed light on the pivotal role of exosomes in pre-metastatic niche construction in the liver. 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