Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30–150 nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids.

Introduction: Therapy resistance and metastasis remain the two biggest challenges of breast cancer that result in a majority of cancer deaths. Cancer stem cells (CSCs) are a subpopulation of cancer cells that can initiate new tumors, remain refractory to conventional therapies, and spread throughout the body. In order to effectively block metastasis, the present study aims to develop novel therapeutics that specifically target breast CSCs and spare normal cells. While nanomedicine represents one of the promising future directions, it is necessary to overcome the shortcomings of synthetic nanoparticles, including bio-instability and off-target toxicity. Here, we aim to bio-engineer the naturally produced cell-secreted small vesicles, exosomes (~100 nm diameter), for a novel therapeutic delivery system. Methods: Exosomes from the human mesenchymal stem cells (MSCs) were isolated by differential ultracentrifugation, and characterized by multiple approaches including transmission EM, Immunoblotting and immunofluorescence assays to confirm the size, shape and presence of exosomal markers CD63, CD81 and LAMP2B. Exosomes were engineered to express a peptide ligand on its surface to specifically recognize and target CSCs, and the presence of the ligand was confirmed by western blot, flow cytometry (FCM) and a super resolution confocal laser scanning microscopy (CLSM, Leica SP8). The CSC targeting efficiency of the exosomes was evaluated in vitro by FCM and CLSM. Further, the exosomes were loaded with microRNAs (miR-200, miR-30c), as one of the key factors that regulate breast CSC functions, by following a novel transfection technology. Results: The bio-engineered exosomes specifically target and internalize into the CSCs compared to normal cells with absent expression of the ligand-binding protein. The developed exosomes can effectively deliver candidate miRNAs into the recipient cells and knock down the genes important for functional CSCs. The in vivo targeting of CSCs and therapeutic applications of the exosomes using patient-derived human-in-mouse breast tumor xenograft models are underway. Conclusion: It is expected that exosome-based miRNA therapeutics will have high biocompatibility, low toxicity, high specificity, and promising efficiency in preclinical and clinical applications to reduce breast cancer metastasis and improve patient survival. Citation Format: Golam Kibria, Katelyn E. Lee, Erika K. Ramos, Simo Huang, Clifford V. Harding, Jan Lötvall, Huiping Liu. Cancer stem cell targeted exosomes for the treatment of metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5152. doi:10.1158/1538-7445.AM2017-5152