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.

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Metastasis, a critical phase of tumor progression, remains a primary challenge in treating cancer and a major cause of cancer mortality. Cell-cell communication via extracellular vesicles (exosomes and microvesicles) between primary tumor cells and the microenvironment of distant organs is crucial for pre-metastatic niche (PMN) formation and metastasis. Here, we review work on the contribution of exosome cargo to cancer progression, the role of exosomes in PMN establishment, and the function of exosomes in organotropic metastasis. We also describe the clinical utility of exosomes.

Highlights d Proteomic profiles of extracellular vesicles and particles (EVPs) from 426 human samples d Identification of pan-EVP markers d Characterization of tumor-derived EVP markers in human tissues and plasma d EVP proteins can be useful for cancer detection and determining cancer type

The protumor functions for autophagy are largely attributed to its ability to promote cancer cell survival in response to stress. This study demonstrates an unexpected connection between autophagy and glucose metabolism that facilitates adhesion-independent growth driven by a strong oncogenic insult—mutationally active Ras.

In order to metastasize, tumor cells must adapt to untoward, stressful microenvironments as they disseminate into the systemic circulation and colonize distant organ sites. Autophagy, a tightly regulated lysosomal self-digestion process that is upregulated during cellular stress, has been demonstrated to suppress primary tumor formation, but how autophagy influences metastasis remains unknown. Autophagy may inhibit metastasis by promoting anti-tumor inflammatory responses or by restricting the expansion of dormant tumor cells into macrometastases. Conversely, self-eating may promote metastasis by enhancing tumor cell fitness in response to environmental stresses, such as anoikis, during metastatic progression. Because autophagy is titratable, it may serve both pro-and anti-metastatic functions depending on the contextual demands placed on tumor cells throughout the metastatic process.

AUTHOR CONTRIBUTIONS G.R. designed the experimental approach, performed the experimental work, analyzed the data, coordinated the project and wrote the manuscript. A.H. performed primary tumour growth and exosome education in vivo studies, cancer cell proliferation in vitro studies, cancer cell culture and exosome isolation, coordinated the project and wrote the manuscript. C.M.K. generated CEMIP overexpression, performed molecular cloning work and genetic manipulation of cancer cells, cancer cell culture and exosome isolation, coordinated the project and wrote the manuscript. I.R.M. performed brain slice ex vivo FACS analysis and exosome education in vivo studies, cancer cell culture and exosome isolation, coordinated the project, wrote and reviewed the manuscript. L.S. performed brain slice ex vivo experimental work, tissue processing and immunostaining, ex vivo and in vivo ImageJ data analysis and quantification, cancer cell invasion in vitro studies, western blot analysis, cancer cell culture and exosome isolation, and contributed to figure panel assembly. D.F. performed density gradient exosome isolation, characterization and analysis, western blot analysis, and cancer cell culture. H.S.K. and P.R.O. performed RNA sequencing data analysis. I.S. performed tissue processing and immunostaining, ex vivo and in vivo ImageJ data analysis and quantification, cancer cell culture and exosome isolation. I.C.S. performed western blot analysis and assisted in analysis of human data.

The tumor promoting functions of autophagy are primarily attributed to its ability to promote cancer cell survival. However, emerging evidence suggests that autophagy plays other roles during tumorigenesis. Here, we uncover that autophagy promotes oncogenic RAS-driven invasion. In epithelial cells transformed with oncogenic RAS, depletion of autophagy-related genes suppresses invasion in three-dimensional culture, decreases cell motility, and reduces pulmonary metastases in vivo. Treatment with conditioned media from autophagy-competent cells rescues the invasive capacity of autophagy-deficient cells, indicating these cells fail to secrete factors required for RAS-driven invasion. Reduced autophagy diminishes the secretion of the pro-migratory cytokine IL6, which is necessary to restore invasion of autophagy-deficient cells. Moreover, autophagy-deficient cells exhibit reduced levels of MMP2 and WNT5A. These results support a previously unrecognized function for autophagy in promoting cancer cell invasion via the coordinate production of multiple secreted factors.