Study On Cell Heterogeneity Based On Exosome Sensing Platform In Single-cell Resolution

For decades,researches have proved that single-cell heterogeneity palyed key role in disease diagnosis and treatment.Single-cell omics studies provide unique information,providing insights into how underlying molecular and structural changes alter cellular behavior,development,and disease processes.On the one hand,emerging single-cell proteomic analysis tools based on microfluidic chips can assess cellular heterogeneity with high throughput,higher sensitivity,and lower cost.On the other hand,exosomes carrying rich information on blasts have become important biomarkers for tumor detection,treatment,and prognosis.However,the exploration of cellular heterogeneity by exosomal proteomics still lacks effective characterization tools and analysis pipelines.Herein,we proposed a single-cell multiplex exosome profiling platform.Furthermore,a robust analytical pipeline that can be used for the characterization of single-cell proteome secretion profiles is presented to explore cellular heterogeneity.The highlights of the study are as follows:A microfluidic platform consisting of a microcavity array chip and an antibody spatially encoded barcode chip was developed for multiplexed single-cell exosome characterization.This platform can encode 20 antibodies and enable characterization of five phenotypic exosomes and two cytokines from more than 1,000 single cells,simultaneously.The prepared antibody spatially encoded barcode chip has low background noise,high antibody capture rate,and excellent uniformity of the patterned antibody,which provides conditions for highsensitivity detection and reduces reagent consumption.The platform was applied to smallvolume clinical samples and successfully distinguished ovarian cancer patients from healthy controls.Besides,the surface-modified PDMS microcavity array chip can capture single cells with high throughput and ensure the normal growth and proliferation of cells.The sensitivity of single-cell exosome detection can be increased to 3 exosome particles per single-cell microcavity using this platform.And intensity distribution within these exosome secreting cells revealed that a very small number of cells can secrete 10 times over average secretion.A standard analysis tool combined with clustering algorithms and heat maps was developed for the analysis of single data.Functional cell subsets of tumor-associated phenotypic exosomes(HSP70+.EPCAM+)were identified in ovarian cells.Using hierarchical clustering and PCA analysis,we found that exosome-secreting cells and cytokine-secreting cells showed functional mutual exclusion,and they were secreted by different functional cell subsets.Besides,correlation analysis showed that IL6 was positively correlated with exosomes of certain phenotypes,confirming that the secretion of IL6 was mediated by exosomes.This thesis provides an important research idea and detection technology platform for high-throughput single-cell exosome detection and cell heterogeneity analysis,which can play an important role in disease pathology research,novel marker screening,drug screening,and precise diagnosis and treatment.It has great application value for both basic research and clinical application.