Extracellular Vesicle Analysis For Preliminary Diagnosis Of Ovarian Cancer And Brain Glioma And Its Preclinical Applicatio

Objective:Malignant tumors have posed a severe threat to human health.The research on the pathogenesis of cancer and the development of reliable detection techniques are crucial for the accurate early diagnosis of malignant tumors.Compared to conventional tissue biopsy,liquid biopsy provides a less invasive.In this thesis,we put emphasis on the detection of extracellular vesicles（EVs）,by using effective labelling strategies and sensitive detection methods.The biological characteristics of EVs were studied both in vitro and in vivo,so as to understand the influence of EVs on the emergence and progression of diseases,and develop rapid and sensitive methods for disease diagnosis.Briefly,three main subprojects are taken into consideration:firstly,Tracing the biological origin and transport of extracellular vesicles of ovarian cancer in living cells and mice models via high-resolution imaging.Ovarian cancer-derived EVs are small vesicles released by ovarian cancer cells into the extracellular environment.These EVs contain typical tumor-related cargos,such as nucleic acids,proteins and lipids.Particularly,the membrane proteins on the surface of EVs provide critical information on their origin,making it possible to classify vesicle types and to enrich vesicles of specific origin based on these biomarkers.EVs are involved in the intercellular communication,therefore,cancer cells could use EVs as biological messengers to promote their growth.Since EVs are involved in the disease process,high resolution imaging technology will be employed in this thesis to trace biogenesis,intercellular trafficking of EVs in living cells and in mice models which provide valuable information in evaluating the influence of tumor-derived EVs on tumor progression.Secondly,the live tracking of biogenesis and EVs transport were investigated in glioma cells by high resolution fluorescence imaging.Tumor cells and their surrounding tumor microenvironment released a large amount of EVs to promote tumor progression.In addition,bacteria from the gastrointestinal tract had a persistent double-sided effect on glial cells in vivo.In this thesis,high resolution fluorescence microscopy imaging technology will be used to study the biological origin and migration of EVs in living cells and mice models.In addition,the influence of exogenous stimuli on vesicle release was explored.Thirdly,the early diagnosis of ovarian cancer based on the detection of EVs were investigated using Surface-enhanced Raman Scattering（SERS）.Herein,based on label-free SERS and SERS tag techniques,EVs derived from ovarian cancer patients and healthy controls were differentiated.Methods:（1）Based on fluorescence labeling and super-resolution microscope imaging technology,bright,stable and highly sensitive fluorescent dye labeling methods and protein labeling methods were selected by optimizing fluorescent dye labeling strategies.The release of EVs in cells and internalization in different cells were rapidly and sensitively traced by combining these two methods.Visual monitoring of EVs transport process between cells,the distribution in mice and the effect on tumor development and metastasis in mice with cancer can provide evidence for revealing the physiological and pathological functions of EVs in vivo and in vitro.（2）EV_U251,a novel tumor marker derived from human glioblastoma cells,was used as the research object.Super resolution fluorescence imaging of individual vesicles was performed using fluorescent dye and fluorescent protein labeling with high sensitivity and selectivity.At the same time,the effect of EV_E.Colifrom E.Coli on glioblastoma cells and the effect of lipopolysaccharide on the vesicles release of glioblastoma cells was explored.In addition,we performed the bioimaging of mice models to evaluate the distribution of glioma-derived EVs in vivo.（3）Based on SERS detection technology,EVs derived from normal ovarian cells and ovarian cancer cells were distinguished,as well as EVs derived from different cell types of the same cancer,so as to realize the preliminary diagnosis of ovarian cancer.This study provided a reliable research basis for the accurate search of tumor markers and the study of the function of EVs in diseases.Results:（1）The vesicles extracted from the supernatant of NIH:OVCAR3 cells by differential ultracentrifugation were identified as EVs(EV_OVCAR3)by the three main characterizing techniques of EVs（TEM,NTA and WB）.There was no significant difference in morphology,particle size and specific protein expression between PKH67 and Mem Glow ^TM560 labeled EV_OVCAR3and unlabeled EV_OVCAR3,which represents the effectiveness of the labelling strategy.When studied in living cells,EV_OVCAR3was captured and internalized by four type of cells of different origin（mouse monocyte macrophage leukemia RAW264.7,neutrophil granulocyte,human ovarian granulocyte HO23,and human ovarian cancer NIH:OVCAR3）under super resolution fluorescence confocal microscopy.When coincubating lentivirus-constructed HO23 and NIH:OVCAR3 cells,we observed that the two transfected cells released multiple EVs that with overexpressed surface proteins as the parental cells.The vesicles released by the two transfected cells exchanged on the cell membrane of the other cell.For in vivo experiments,we conducted whole-body imaging of nude mice after injecting EV_OVCAR3for 1 h,either via intraperitoneal injection or caudal vein injection.Fluorescence was detected at different organs and lasted for 7 days.Ex vivo fluorescence imaging of different organs after sacrifice showed that the accumulation of EV_OVCAR3-Mem560 was observed in liver,kidney and brain,representing the main metabolism pathways.However,EV_OVCAR3-Mem560could accumulate in all organs,mainly in liver within 24 h.Additionally,the accumulation of EV_OVCAR3in the brain and lungs after tail vein injection was more obvious than that of intraperitoneal injection,while more EV_OVCAR3accumulated in the kidney by intraperitoneal injection than in the caudal vein.However,these two injection methods did not affect the arrival of EV_OVCAR3to ovarian aggregation and the entry of EV_OVCAR3into brain tissue through the blood-brain barrier.After successful modeling of the nude mice with ovarian carcinoma in situ,we injected EV_OVCAR3through the tail vein,and found that compared with the control group,(tumors in nude mice injected with EV_OVCAR3grew and developed faster,which demonstrated the pathological function of tumor derived EVs.（2）After labeling EV_U251with lipid dye PKH67,it was observed that RAW264.7 and human normal astrocyte HA1800 cells could rapidly internalize the vesicles by using high resolution fluorescence microscopy.EV_E.Colico-cultured with U251 showed that glioma cell U251 could also rapidly recognize and capture EV_E.Coli.Subsequently,during the same period,glioma cells stimulated by exogenous lipopolysaccharide produced more vesicles than negative controls.The fluorescence of EV_U251-Mem640 could be detected at different locations in nude mice after 1 h injection in vivo.After 12 hours injection,EV_U251-Mem640 were accumulated on liver,kidney,lung,spleen and brain.（3）Seed growth method was used to synthesize Au NPs with uniform morphology and size,and combined with SERS imaging technology,completely different SERS spectral diagrams were presented through EV_OVCAR3,EV_SKOV3and EV_HO23,and the three were distinguished(EV_OVCAR3,EV_SKOV3and EV_HO23present completely different SERS spectra,so that these three substances can be distinguished).Then,SERS probes were prepared by silver nitrate reduction method.Combined with SERS imaging technology,EV_OVCAR3and EV_HO23were distinguished.Conclusions:（1）Practical EVs can be extracted by differential ultracentrifugation.PKH67 and Mem Glow ^TM560 were used to mark the EVs successfully.Compared with PKH67,Mem Glow ^TM560 dyes are more stable and less prone to agglomeration.In vitro,EV_OVCAR3were captured and internalized by RAW264.7,neutrophils,HO23and NIH:OVCAR3.In addition,EVs can be internalized and exchanged between different cells without any extraction.Physiologic and pathologic EVs have similar transport and uptake mechanisms.EV_OVCAR3can cross the blood-brain barrier into the brain,accumulate in the ovaries and deposit in the liver.EV_OVCAR3can accelerate the growth and progression of ovarian carcinoma in situ,probably due to the function of intercellular communication.（2）RAW264.7 cells and HA1800 cells could rapidly internalize glioma-derived EVs.The cell membrane of glioma cells rapidly recognizes and captures EVs from E.coli.In addition,LPS can stimulate glioma cells to produce more vesicles.In vivo,EV_U251can cross the blood-brain barrier into the brain and have“homing mechanism”.（3）Both label free SERS and SERS tag techniques could be employed to distinguish ovarian cancer cells from normal cells,which indicated potential in early diagnosis of ovarian cancer.