Autophagic Synergistic Function Of Extracellular Vesicles And Its Dynamic Imaging In Tumor Cells

Extracellular vesicles(EVs)are the heterogeneous group of lipid bilayer-enclosed nanovesicles secreted by cells.EVs carrying proteins,m RNA/mi RNA,lipids and other materials could transfer from donor cells to recipient cells,which can affect the phenotype and function of recipient cells.EVs are present in biological fluids and involved in multiple physiological and pathological processes.According to their origins,EVs can broadly be divided into exosomes and microvesicles.Exosomes are secreted upon fusion of multivesicular bodies(MVBs)with the cell surface.Microvesicles(also known as plasma-derived extracellular vesicles,PEVs)are formed by directly bud or extend by plasma membrane.Despite their function significance,the basic biology of EVs still remains unknown.During the formation and development of exosomes,the material exchange between MVB and various membrane organelles under different physio/pathological microenvironment needs further explore.The EV function as "messengers" of intercellular material delivery depends on the dynamic release of donor cell and EVs contact with the recipient cell to induce phenotypic changes.In this expanding field,however,much remains unknown regarding the EV dynamic processes,such as release,uptake,and cargo delivery,especially in certain subtypes.The specific research content as follows:Part 1: Autophagy is a cell survival mechanism that functions in response to stressful conditions,but also contributes to cell death or apoptosis.Tumor cells metastasizing through the bloodstream or lymphatic systems must withstand acute shear stress(ASS).We predicted that a compensation pathway to autophagy exists in tumor cells subjected to mechanical stress.We found that ASS promoted autophagosome(AP)accumulation and induced release of EVs containing autophagic components.Furthermore,we found that ASS promoted autophagic vesicles fused with multivesicular body(MVB)to form AP-MVB compartments and then induced autophagic component release into the extracellular space via EVs through the autophagy-MVB-exosome pathway.More importantly,either increasing intracellular autophagosome accumulation or inhibiting autophagic degradation promoted APMVB accumulation but did not induce autophagy-associated protein release via EVs except under ASS,demonstrating the existence of a mechanical stress dependent compensation pathway.Together,these findings revealed that EVs provided an additional protection mechanism for tumor cells and counteracted autophagy to maintain cellular homeostasis under acute shear stress.Part 2: Plasma membrane-derived extracellular vesicles(PEVs)are carriers of biological molecules that perform special cell-cell communications.Nevertheless,the characterization of complicated PEV biology is hampered by the failure of current methods,mainly due to the lack of specific labels and insufficient resolution.Here,we employed atomic force microscopy and scanning ion conductance microscopy,which have three-dimensional nanoscale resolution,to allow label-free visualization of the PEV morphology,release and uptake at single vesicle level.Through single cell topographic analysis,we found that cancer cells have more PEVs than healthy cells.PEVs released by a single tumor cell have diversity form,including classical microvesicles,cluster-like vesicles with different size.Furthermore,we also demonstrated label-free trafficking of PEV release which shown PEV release time was highly correlated with size.Furthermore,we found a new type of PEV that can quickly respond to mechanical stimulation.Importantly,through three-dimensional nanoscale imaging,we visualized the multiform PEV-cell interaction behaviors of individual vesicles,which was challenged in conventional PEV imaging.Collectively,these results revealed the heterogeneity of PEV in terms of morphology and dynamics at the single vesicle level,which enhanced our understanding of EV biology.