Research On New Methods For Subtype Analysis Of Breast Cancer-derived Exosomes Based On Homotypic Recognition Ability Of Breast Cancer Cell Membrane Vesicles

Breast cancer has become the most common malignant tumor in the world,which seriously threatens the health of women.With the improvement of early screening and the improvement of systemic treatment,the accurate treatment targeting a specific subtype can effectively improve the prognosis and reduce mortality of breast cancer patients.Therefore,accurate diagnosis of the subtype features and implementation of timely treatment are of great significance to improve the survival rate and quality of life of breast cancer patients.In this thesis,we made use of the homotypic recognition between breast cancer cell membrane vesicles and breast cancer exosomes to obtain the subtype information of breast cancer exosomes and realize sensitive quantitative analysis of breast cancer exosome,which can provide a basis for the classification analysis and real-time monitoring of primary breast cancer.Specifically,breast cancer cell membrane vesicles carrying specific nucleic acid probes could selectively target the breast cancer exosomes from the same source,and deliver nucleic acid probes to the cancer exosomes through membrane fusion.The exosomal RNA acted as endogenous biomarker further triggered the catalytic hairpin self-assembly reaction to obtain amplified electrochemical signals,finally realizing the sensitive determination of breast cancer exosomes.Therefore,we studied the mechanism of homotypic recognition ability of breast cancer cell membrane vesicles,and reported a high-sensitivity method for the homotypic analysis of breast cancer exosomes,which could be applied in subtype analysis of clinical samples and is expected to provide more comprehensive information for the diagnosis and treatment of breast cancer.The specific research contents are as follows:1.Verification of homotypic recognition and fusion of breast cancer cell membrane vesicles and breast cancer exosomesIn this part of the paper,we chose the estrogen receptor positive breast cancer(ER~+BC)cell MCF-7 and the triple negative breast cancer(TNBC)cell MDA-MB-231 as the representative sources to obtain breast cancer membrane vesicles and breast cancer exosomes,and studied the homotypic recognition between breast cancer cell membrane vesicles and breast cancer exosomes.By means of laser confocal microscopy,dynamic light scattering and fluorescence resonance transfer,we proved that the biomimetic vesicles prepared from breast cancer cell membranes could accurately identify breast cancer exosomes with the same subtype features and fused with them,but cannot identify heterotypic breast cancer exosomes.In other words,the MCF-7 derived cancer cell membrane vesicles selectively recognized and fused with MCF-7-derived cancer exosomes,but could not fuse with the triple negative breast cancer cell MDA-MB-231-derived breast cancer exosomes and human normal breast cell MCF-10A cell-derived exosomes.Similarly,MDA-MB-231-derived breast cancer cell membrane vesicles selectively recognized and fused with MDA-MB-231-derived cancer exosomes,but could not fuse with MCF-7-derived breast cancer exosomes and MCF-10A-derived normal exosomes.The work laid the foundation for the subsequent subtype analysis of breast cancer.2.Electrochemical detection of subtype features of breast cancer exosomes based on homotypic recognition ability of cancer cell membrane vesicles.Based on the excellent homotypic recognition and targeting ability of breast cancer cell membrane vesicles derived from MCF-7 and MDA-MB-231,combined with highly sensitive electrochemical detection technology,we further carried out quantitative analysis of specific subtype of breast cancer exosomes.MCF-7-derived breast cancer cell membrane vesicles were shown to recognize the homotypic breast cancer exosomes and made use of micro RNA-375 as an endogenous marker to promote catalytic hairpin assembly reaction,so as to output electrochemical signals for quantificative determination of breast cancer exosomes with a detection limit of557 particles/ml.At the same time,MDA-MB-231-derived membrane vesicles,using potential therapeutic targets PD-L1 m RNA as endogenous biomarkers,showed good specificity in the homotypic analysis of triple-negative breast cancer exosomes with a detection limit of 518 particles/ml.Cross-validation experiments confirmed that homotypic recognition-driven analysis had satisfactory accuracy and selectivity in the identification of molecular subtypes in breast cancer.Therefore,using different breast cancer cell membrane vesicles from different sources to analyze specific sources of breast cancer exosomes is expected to provide more accurate information for breast cancer subtype analysis in clinic.3.Application of homotypic recognition and fusion-driven recognition of breast cancer cell membrane vesicles for subtype analysis of breast cancer in clinical diagnosis.Based on above results of electrochemical analysis of breast cancer exosomes,we applied breast cancer cell membrane vesicles in the homotypic recognition and fusion-driven subtype analysis of breast cancer.The results proved that breast cancer cell membrane vesicles could not only accurately identify breast cancer exosomes with the same subtype in clinical serum samples,but also have positive correlation between electrochemical signals and disease progression,satisfying the demand of the staging of breast cancer.Therefore,breast cancer cell membrane vesicles could be used for subtype analysis of breast cancer patients and accurate monitoring of disease course.According to the subtype characteristics and endogenous RNA differences,our method is expected to provide more comprehensive and biological information for breast cancer molecular subtype and targeted therapy,and meet the demands for classification,staging,treatment and prognosis of breast cancer.