ミーティングID：854 5449 5200
|Google Classroom コード||smwn3h2|
|講演者||Dr. Richard J. Simpson|
|講演タイトル||"Contribution of extracellular vesicles (EVs) to the epithelial-mesenchymal (EMT) program: functional insights into cancer progression"
Epithelial-mesenchymal transition is an evolutionary-conserved morphogenic process crucial for embryogenesis, wound healing, and malignant progression. EMT is defined by loss of epithelial characteristics and acquisition of a mesenchymal phenotype. While the EMT process has been defined by signal transduction networks and transcriptional factors that activate mesenchymal-associated gene expression, the contribution of extracellular vesicles such as exosomes and shed microvesicles (sMVs; also referred to as microparticles, microvesicles, and ectosomes) are emerging as important mediators of the EMT process. Here, I present a comparative proteomic and functional analysis of Exos and sMVs in the context of EMT using the Madin-Darby canine kidney (MDCK) cells transformed with oncogenic H-RasG12V (21D1 cells) as an in vitro EMT model. Morphofunctional characteristics of oncogenic H-Ras-transformed MDCK cells were examined: phase contrast microscopy revealed MDCK cells possess a highly polarized, cobblestone-like morphology, whereas 21D1 cells possess mesenchymal properties/ spindle shape morphology. Immunofluorescence microscopy and immunoblot analysis revealed attenuation of epithelia cell marker proteins (CDH1, ZO-1) in 21D1 cells and augmentation of mesenchymal marker proteins (VIM, FN1 and CDH2). Wound healing (scratch) assay, anchorage-independent growth (soft agar) / anoikis-resistance assay, Transwell-migration and Transwell collagen-Matrigel™ invasion assay confirmed MDCK cells acquire migration and invasion capability upon H-Ras transformation (key characteristic of EMT phenotype). Milligram amounts of purified exosomes and sMVs, necessary for biochemical/ functional analyses, were obtained from ~2,000 mL MDCK/ 21D1 cell culture media (continuous culture CELLine™ Bioreactor device) followed by a purification strategy using sequential differential ultracentrifugation and buoyant density gradient centrifugation (iodixanol, OptiPrep™).
GeLC-MS/MS (label-free quantitation) revealed sMVs and Exos from MDCK/ 21D1 cells have distinct proteome signatures: for example, TGM2 ternary complex (TGM2:FN1:ITGB1), RBPs and centralspindlin complex (KIF23:Racgap1) and mitochondrial proteins selectively traffic to sMVs, whereas MMP14, TGFR1/2, TGFBI, VIM and YBX1 traffic only to Exos.
A comprehensive examination of proteins common to both MDCK and 21D1 cell-derived sMVs or Exos revealed proteins that distinguish vesicle subtypes (i.e., their basic building blocks), which might be useful for EV-based liquid-biopsy disease diagnostics. For example, annexins (ANXA-3/4/6/7/9/10/11/13) syntenin-1, CDs-9/44/55/59/81/83/99/151/276, CDC42, PDCD6, PDCD10, PDCD61P (Alix), PLCD3, TSG101, TSPANs (3,4, 8, 9, 14,15), integrins (ITGA1, A2, A6, B1, B3) were found only in Exos; centralspindlin complex proteins, heparin sulphate proteoglycan-2, and heat shock proteins HSPA9 (hsp70)/ D1(hsp60)/ 90B1/ and A5 were restricted to only sMVs.
H-RasG12V transformation of MDCK cells conferred cell migration and invasion capabilities in 21D1 cell-derived Exos and sMVs. Intriguingly, 21D1-sMVs/-Exos promote cell migration and confer anchorage-independent growth properties in parental MDCK cells and NIH3T3 fibroblasts upon co-culture by targeting distinct signalling pathways to promote cancer progression.
In summary, these findings provide new insights into distinguishing features of EV subtypes (sMVs/exosomes) and their potential role in the EMT process and cancer progression.
|共催||グローバルイノベーション研究院 ライフサイエンス分野 池袋ユニット
|お問い合わせ窓口||グローバルイノベーション研究院 農学研究院 天竺桂 弘子
e-mail: h_tabuno ( ここに@ を入れてください） cc.tuat.ac.jp