Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non‐Small‐Cell Lung Cancer (NSCLC) SK‐MES‐1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge‐based concentrating methods are more appropriate than pressure‐driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research.

Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum‐containing media. The majority of researchers use serum‐containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non‐sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA‐MB‐231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano‐flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large‐scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.

Small membranous secretions from tumor cells, termed exosomes, contribute significantly to intercellular communication and subsequent reprogramming of the tumor microenvironment. Here, we use optical imaging to determine that exogenously administered fluorescently labeled exosomes derived from highly metastatic murine breast cancer cells distributed predominantly to the lung of syngeneic mice, a frequent site of breast cancer metastasis. At the sites of accumulation, exosomes were taken up by CD45+ bone marrow–derived cells. Subsequent long-term conditioning of naïve mice with exosomes from highly metastatic breast cancer cells revealed the accumulation of myeloid-derived suppressor cells in the lung and liver. This favorable immune suppressive microenvironment was capable of promoting metastatic colonization in the lung and liver, an effect not observed from exosomes derived from nonmetastatic cells and liposome control vesicles. Furthermore, we determined that breast cancer exosomes directly suppressed T-cell proliferation and inhibited NK cell cytotoxicity, and hence likely suppressed the anticancer immune response in premetastatic organs. Together, our findings provide novel insight into the tissue-specific outcomes of breast cancer–derived exosome accumulation and their contribution to immune suppression and promotion of metastases. Cancer Res; 76(23); 6816–27. ©2016 AACR.

Metastatic spread of a cancer to secondary sites is a coordinated, non-random process. Cancer cell-secreted vesicles, especially exosomes, have recently been implicated in the guidance of metastatic dissemination, with specific surface composition determining some aspects of organ-specific localization. Nevertheless, whether the tumor microenvironment influences exosome biodistribution has yet to be investigated. Here, we show that microenvironmental cytokines, particularly CCL2, decorate cancer exosomes via binding to surface glycosaminoglycan side chains of proteoglycans, causing exosome accumulation in specific cell subsets and organs. Exosome retention results in changes in the immune landscape within these organs, coupled with a higher metastatic burden. Strikingly, CCL2-decorated exosomes are directed to a subset of cells that express the CCL2 receptor CCR2, demonstrating that exosome-bound cytokines are a crucial determinant of exosome-cell interactions. In addition to the finding that cytokine-conjugated exosomes are detected in the blood of cancer patients, we discovered that healthy subjects derived exosomes are also associated with cytokines. Although displaying a different profile from exosomes isolated from cancer patients, it further indicates that specific combinations of cytokines bound to exosomes could likewise affect other physiological and disease settings.