Highlights d Human cells release Argonaute 1-4 and major vault protein independently of exosomes d Annexin A1 is a specific marker of microvesicles shed from the plasma membrane d Small extracellular vesicles do not contain DNA d Active secretion of cytosolic DNA occurs through an amphisome-dependent mechanism

SUMMARY Lineage mapping has identified both proliferative and quiescent intestinal stem cells, but the molecular circuitry controlling stem cell quiescence is incompletely understood. By lineage mapping, we show Lrig1, a pan-ErbB inhibitor, marks predominately non-cycling, long-lived stem cells located at the crypt base that, upon injury, proliferate and divide to replenish damaged crypts. Transcriptome profiling of Lrig1+ colonic stem cells differs markedly from highly proliferative, Lgr5+ colonic stem cells; genes up-regulated in the Lrig1+ population include those involved in cell cycle repression and response to oxidative damage. Loss of Apc in Lrig1+ cells leads to intestinal adenomas and genetic ablation of Lrig1 results in heightened ErbB1-3 expression and duodenal adenomas. These results shed light on the relationship between proliferative and quiescent intestinal stem cells, and support a model in which intestinal stem cell quiescence is maintained by calibrated ErbB signaling with loss of a negative regulator predisposing to neoplasia.

Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. To test whether exosomal RNAs also contribute to changes in gene expression in recipient cells, and whether mutant KRAS might regulate the composition of secreted microRNAs (miRNAs), we compared small RNAs of cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status. We show that exosomal profiles are distinct from cellular profiles, and mutant exosomes cluster separately from wild-type KRAS exosomes. miR-10b was selectively increased in wild-type exosomes, while miR-100 was increased in mutant exosomes. Neutral sphingomyelinase inhibition caused accumulation of miR-100 only in mutant cells, suggesting KRAS-dependent miRNA export. In Transwell co-culture experiments, mutant donor cells conferred miR-100-mediated target repression in wild-type-recipient cells. These findings suggest that extracellular miRNAs can function in target cells and uncover a potential new mode of action for mutant KRAS in CRC.DOI: http://dx.doi.org/10.7554/eLife.07197.001

SUMMARY Exomeres are a recently discovered type of extracellular nanoparticle with no known biological function. Herein, we describe a simple ultracentrifugation-based method for separation of exomeres from exosomes. Exomeres are enriched in Argonaute 1–3 and amyloid precursor protein. We identify distinct functions of exomeres mediated by two of their cargo, the β-galactoside α2,6-sialyltransferase 1 (ST6Gal-I) that α2,6- sialylates N-glycans, and the EGFR ligand, amphiregulin (AREG). Functional ST6Gal-I in exomeres can be transferred to cells, resulting in hypersialylation of recipient cell-surface proteins including β1-integrin. AREG-containing exomeres elicit prolonged EGFR and downstream signaling in recipient cells, modulate EGFR trafficking in normal intestinal organoids, and dramatically enhance the growth of colonic tumor organoids. This study provides a simplified method of exomere isolation and demonstrates that exomeres contain and can transfer functional cargo. These findings underscore the heterogeneity of nanoparticles and should accelerate advances in determining the composition and biological functions of exomeres.

SUMMARY Autocrine, paracrine and juxtacrine are recognized modes of action for mammalian EGFR ligands that include EGF, TGF-α (TGFα), amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, epiregulin and epigen. We identify a new mode of EGFR ligand signaling via exosomes. Human breast and colorectal cancer cells release exosomes containing full-length, signaling-competent EGFR ligands. Exosomes isolated from MDCK cells expressing individual full-length EGFR ligands displayed differential activities; AREG exosomes increased invasiveness of recipient breast cancer cells four-fold over TGFα or HB-EGF exosomes and five-fold over equivalent amounts of recombinant AREG. Exosomal AREG displayed significantly greater membrane stability than TGFα or HB-EGF. An average of 24 AREG molecules are packaged within an individual exosome, and AREG exosomes are rapidly internalized by recipient cells. Whether the composition and behavior of exosomes differ between non-transformed and transformed cells is unknown. We show that exosomes from DLD-1 colon cancer cells with a mutant KRAS allele exhibited both higher AREG levels and greater invasive potential than exosomes from isogenically matched, non-transformed cells in which mutant KRAS was eliminated by homologous recombination. We speculate that EGFR ligand signaling via exosomes may contribute to diverse cancer phenomena such as field effect and priming the metastatic niche.