The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution, and functions. By employing asymmetric-flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed “exomeres” (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins and specific pathways, such as glycolysis and mTOR signaling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signaling pathways, respectively. Exo-S, Exo-L, and exomeres each had unique N-glycosylation, protein, lipid, and DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating and addressing the complexities of heterogeneous nanoparticle subpopulations.
We describe the protocol development and optimization of asymmetric flow field-flow fractionation (AF4) technology for separating and characterizing extracellular nanoparticles (ENPs), particularly small extracellular vesicles, known as exosomes, and even smaller novel nanoparticles, known as exomeres. This technique fractionates ENPs based on hydrodynamic sizes and demonstrates a unique capability to separate nanoparticles with sizes ranging from a few nanometers to undefined level of micrometers. ENPs are resolved by two perpendicular flows, channel flow and cross flow, in a flat thin channel with a semi-permissive bottom wall membrane. The AF4 separation method offers several advantages over other isolation methods for ENP analysis, including being label-free, gentle, rapid (< 1 hour), and highly reproducible, and providing efficient recovery of analytes. Most importantly, in contrast to other available techniques, AF4 can separate ENPs at high resolution (1 nm) and provide a large dynamic range of size-based separation. In conjunction with real-time monitors, such as ultraviolet absorbance and dynamic light scattering, and an array of post-separation characterizations, AF4 facilitates the successful separation of distinct subsets of exosomes and the identification of exomeres. Though the whole procedure of cell culture and ENP isolation from the conditioned media by ultracentrifugation can take approximately three days, the AF4 fractionation step takes only one hour to perform. Users of this technology will require expertise in the working principle of AF4 to operate and customize protocol applications. AF4 can contribute to the development of high-quality, exosome- and exomere-based molecular diagnostics and therapeutics.
Intercellular cross talk between cancer cells and stromal and immune cells is essential for tumor progression and metastasis. Extracellular vesicles and particles (EVPs) are a heterogeneous class of secreted messengers that carry bioactive molecules and that have been shown to be crucial for this cell-cell communication. Here, we highlight the multifaceted roles of EVPs in cancer. Functionally, transfer of EVP cargo between cells influences tumor cell growth and invasion, alters immune cell composition and function, and contributes to stromal cell activation. These EVP-mediated changes impact local tumor progression, foster cultivation of pre-metastatic niches at distant organspecific sites, and mediate systemic effects of cancer. Furthermore, we discuss how exploiting the highly selective enrichment of molecules within EVPs has profound implications for advancing diagnostic and prognostic biomarker development and for improving therapy delivery in cancer patients. Altogether, these investigations into the role of EVPs in cancer have led to discoveries that hold great promise for improving cancer patient care and outcome.
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