The Mechanism Study And Dynamic Visualization Of Extracellular Vesicle MiRNA Mediated Regulation Of Tumor Angiogenesis In Oral Squamous Cell Carcinoma

Part I EGFR regulates angiogenesis by promoting extracellular vesicle-mediated miRNA-delivery in oral squamous cell carcinomaObjective: oral squamous cell carcinoma(OSCC)is one of the most common malignant tumors of the oral and maxillofacial region.The abnormal activation of EGFR signaling pathway is one of the most important features of OSCC,and is closely associated with the malignant phenotypes and poor prognosis.The purpose of this study is to investigate the roles of EGFR in regulating extracellular vesicle(EV)secretion and tumor angiogenesis in OSCC.Methods: The differences in the expression profiles of miRNA among normal oral mucosa cells(HIOECs),OSCC cells,and EGFR-overexpressing OSCC cells as well as their derived EVs were analyzed by the high-throughput sequencing technology.The characteristics of OSCC cell-secreted EVs were evaluated by nanoparticle tracking analysis and transmission electron microscopy.The correlation between EGFR activation and tumor progression was investigated on clinical specimens.The effects of EV-mediated miRNA-delivery from OSCC cells to endothelial cells(ECs)were studied with co-culture experiment.The regulation of EV production by EGFR activation and its effects on tumor angiogenesis in OSCC was verified in vitro and in vivo.Results: EGFR activation in OSCC not only promoted the production of EVs,but also increased the enrichment of angiogenic miRNA into EVs.However,there were no significant differences in the physical and chemical characteristics of EVs derived from OSCC cells and HIOECs in terms of size distribution,morphology,and zeta potential.EVs derived from OSCC cells could be efficiently taken up by ECs in a time-and concentration-dependent manner,and up-regulated the level of multiple angiogenic miRNA in ECs through the horizontal delivery.EV production regulated by EGFR significantly promoted the growth of OSCC by activating tumor angiogenesis.Conclusion: EGFR activates tumor angiogenesis through EV-dependent horizontal delivery of angiogenic miRNAs from tumor cells to ECs,leading to the maliganant progression of OSCC.Part II Establishment of the single particle tracking platform based on quantum dot labeling of extracellular vesicles and the carried miRNAObjective: The drawbacks of traditional labeling strategies and visualization platforms have severely hindered the in-depth understanding of EVs’ biological behaviors and functions.This study aims to develop a reliable and controllable strategy to label EVs with quantum dots(QDs),and to establish a single particle tracking platform for dissecting the dynamic behaviors of EVs at the subcellular level.Methods: The membrane biotinylation of different subtypes of EVs was realized and optimized by utilizing the self-assemble of phospholipid bilayer.QD labeling of EVs was achieved through the specific binding between streptavidin-modified QDs and biotin,and the superiority of which was evaluated from the aspects of optical properties and biocompatibility.The multi-color and multi-component labeling of EVs was performed by optimizing the electroporation technology and combining it with the membrane biotinylation-based QD labeling strategy.A universal single particle tracking(SPT)platform was developed based on a spinning disk confocal microscope,and its feasibility in tracking the dynamic behaviors of EVs was verified.Results: By optimizing the culture concentration and period with DSPE-PEG-Biotin,we successfully achieved efficient biotinylation of different subtypes of EVs regardless of their size distribution or cell origin.The site-specific and quantitative labeling of EVs with QDs was realized through further adjustment of the incubation concentration of SA-QDs.This strategy not only possesses unprecedented advantages such as high efficiency,good specificity,superior photostability,and also exhibits excellent biocompatibility,with little influence on the inherent characteristics of EVs.Fluorescently labeled miRNA were efficiently loaded into the EVs,achieving simultaneous multi-color labeling of different components of EVs.A single particle tracking platform,which can fully meet the requirement of high temporal-spatial resolution,was successfully established based on QD-labeled EVs.Conclusion: By virtue of an advanced labeling strategy using QDs,this study establishes a versatile QD-based single particle tracking platform,thus providing a powerful tool for dissecting the dynamic process of EV-mediated miRNA delivery.Part III Quantum dot-based single particle tracking dissects the internalization and miRNA-release dynamics of oral squamous cell carcinoma-derived small extracellular vesicles in endothelial cellsPurpose: OSCC-derived EVs may promote tumor angiogenesis through horizontal delivery of angiogenic miRNA from tumor cells to ECs.However,the detailed information and molecular underpinings of this process is still to be elucidated.This study aims to dissect the dynamic behaviors of OSCC-derived EVs in ECs by taking advantage of the QD-based single particle tracking platform.Methods: OSCC-derived EVs labeled by QDs were co-cultured with ECs,and the dynamic interaction between them was visualized and recorded in situ and in real-time through single particle tracking.The characteristics of the internalization and transportation of EVs by ECs,and the specific steps of subsequent miRNA-release were analyzed by trajectory reconstruction and motion analysis based on the acquired video files.The key regulatory molecules in this dynamic process were investigated by the treatment with specific inhibitors.Those results were further verified by real-time quantitative PCR,western blotting and cell function experiments.Results: ECs efficiently internalized OSCC-derived EVs through clathrin-mediated endocytosis in a time-and temperature-dependent manner,and subsequently transported them to the perinuclear region through a typical "slow-fast-slow" threestage pattern,which was mediated by different cytoskeleton.Stage 1 was a slow directional movement,while stage 2 was a fast directional movement,and stage 3 was a slow confined movement.Finally,the internalized EVs rapidly released their miRNA possibly through their interaction with acidic endosomes.In addition,targeting acidification effectively blocked the miRNA-delivery by EVs,thereby exerting an inhibitory effect on their-induced angiogenesis.Conclusion: OSCC-derived s EVs delivered their cargos by targeting acidic endosomes after the endocytosis and transportation by ECs,thereby regulating the function of recipient cells.These findings not only help to supplement the pathogenesis of angiogenesis in OSCC,but also provide a template for the dynamic analysis of EVmediated intercellular communication.