Super Resolution Imaging Of Exosomal Tumor Biomarkers And Its Application In Cancer Diagnosis

Exosomes are small extracellular vesicles secreted by almost all cells,with sizes ranging from 40 to 160 nm(average ～100 nm)in diameter.These small vesicles contain various kinds of lipids,proteins as well as nucleic acids,reflecting the status of their original tissue or cells.So far,researchers have revealed that exosomes play an important part in different physiological and pathological processes,like immune suppression and angiogenesis regulation.Besides,since they are enriched in most biological fluids such as urine,blood,and saliva,exosomes have been considered as a potential candidate for drug delivery and disease diagnosis.To further understand the exosomal behaviors and provide a novel insight into exosomeinvolved cell communication and cancer diagnosis,in this paper,we utilize single-molecule localization microscopy(SMLM)for the imaging and dynamic tracking of exosomes and exosomal contents(e.g.mi RNA)in recipient cells.Additionally,a single exosome-based cancer diagnosis technique is developed to explore the potential of exosomes in clinical applications.We believe that this platform provides an avenue to the noninvasive early diagnosis of cancers.We demonstrate the simultaneous dual color SMLM imaging of two kinds of membrane proteins on cancer cell-derived exosomal surface.The successful labeling and imaging of exosomes make it possible to observe the interaction between cancer-derived exosomes and normal cells.Besides,the colocalization of cancer-derived exosomes and lysosomes in recipient cells is observed,which confirms the possibility that exosomes can be transported into lysosomes for degradation.Since exosomes play a vital role in intercellular communications,we anticipate that the presented SMLM-based imaging and tracking of exosomes holds a great potential in the investigation of the mechanism of exosome-mediated cancer metastasis.A highly sensitive exosomal mi RNA labeling strategy is developed using molecular beacons.Then super-resolution tracking of exosomes and exosomal mi RNAs is realized to facilitate our understanding of exosome-mediated cancer metastasis.The motion of exosomes within the intercellular filamentous structures is observed under SMLM.Moreover,the dualcolor SMLM based dynamic tracking reveals the release process of exosomal mi RNAs after uptake by recipient cells.These findings can provide new insights into the pathway of intercellular communication and affirm that SMLM is a powerful technique to track the motion of exosomes and exosomal contents in recipient cells.A quantitative analysis platform is developed to implement a sequential quantification analysis of multiple exosomal surface biomarkers at the single-exosome level,which utilizes DNA-PAINT and machine learning algorithm to automatically analyze the results.As a proof of concept,the profiling of exosomal surface biomarkers is successfully applied to detect pancreatic cancer and breast cancer from unknown samples with high accuracy.The unprecedented sensitivity and sequential quantification ability of DNA-PAINT enables the multiple profiling of surface biomarkers at the single-exosome level,making it qualified for early-disease diagnosis.